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Sample records for receptor gpcr superfamily

  1. Multiple Receptor Subtypes for the CGRP Super-Family

    Directory of Open Access Journals (Sweden)

    R. Quirion

    2001-01-01

    Full Text Available Molecular evidence for the existence of multiple receptors for CGRP has been rather difficult to obtain. Over 10 years after suggesting the existence of at least two classes (CGRP1 and CGRP2 of CGRP receptors on the basis of pharmacological data[1], molecular data on the CGRP2 receptor subtype are still lacking as well as potent and selective antagonists. The situation is somewhat different for the functional CGRP1 subtype which is likely composed of diverse subunits CRLR, RAMP1 and possibly RCP[2]. Moreover, BIBN 4096BS was recently reported as the first nonpeptide highly potent CGRP1 receptor antagonist[3]. However, in situ hybridization and receptor autoradiographic data have clearly shown the existence of major mismatches (e.g., cerebellum between the discrete localization of CRLR, RAMP1, and specific CGRP binding sites supporting the existence of CGRP receptor subtypes. Functional studies have also provided evidence in that regard (for a recent review: [4]. Accordingly, additional studies aiming at cloning additional CGRP receptors are certainly warranted. Similarly, recent evidence from various laboratories including ours suggests the existence of more than one class (CRLR and RAMP2 of adrenomedullin receptors at least in the rat brain. In contrast, most evidence suggests the existence of a single class of amylin receptors. In brief, it appears that multiple receptors or receptor complexes do exist for CGRP and related peptides but their composition is apparently unique among the GPCR super-family and additional data are needed to fully establish the molecular organization of each subtype. Supported by CIHR of Canada.

  2. GPCR & company: databases and servers for GPCRs and interacting partners.

    Science.gov (United States)

    Kowalsman, Noga; Niv, Masha Y

    2014-01-01

    G-protein-coupled receptors (GPCRs) are a large superfamily of membrane receptors that are involved in a wide range of signaling pathways. To fulfill their tasks, GPCRs interact with a variety of partners, including small molecules, lipids and proteins. They are accompanied by different proteins during all phases of their life cycle. Therefore, GPCR interactions with their partners are of great interest in basic cell-signaling research and in drug discovery.Due to the rapid development of computers and internet communication, knowledge and data can be easily shared within the worldwide research community via freely available databases and servers. These provide an abundance of biological, chemical and pharmacological information.This chapter describes the available web resources for investigating GPCR interactions. We review about 40 freely available databases and servers, and provide a few sentences about the essence and the data they supply. For simplification, the databases and servers were grouped under the following topics: general GPCR-ligand interactions; particular families of GPCRs and their ligands; GPCR oligomerization; GPCR interactions with intracellular partners; and structural information on GPCRs. In conclusion, a multitude of useful tools are currently available. Summary tables are provided to ease navigation between the numerous and partially overlapping resources. Suggestions for future enhancements of the online tools include the addition of links from general to specialized databases and enabling usage of user-supplied template for GPCR structural modeling.

  3. Advances in GPCR modeling evaluated by the GPCR Dock 2013 assessment

    DEFF Research Database (Denmark)

    Kufareva, Irina; Katritch, Vsevolod; Biggin, Phil

    2014-01-01

    Despite tremendous successes of GPCR crystallography, the receptors with available structures represent only a small fraction of human GPCRs. An important role of the modeling community is to maximize structural insights for the remaining receptors and complexes. The community-wide GPCR Dock asse...

  4. G protein-coupled receptor transmembrane binding pockets and their applications in GPCR research and drug discovery: a survey.

    Science.gov (United States)

    Kratochwil, Nicole A; Gatti-McArthur, Silvia; Hoener, Marius C; Lindemann, Lothar; Christ, Andreas D; Green, Luke G; Guba, Wolfgang; Martin, Rainer E; Malherbe, Pari; Porter, Richard H P; Slack, Jay P; Winnig, Marcel; Dehmlow, Henrietta; Grether, Uwe; Hertel, Cornelia; Narquizian, Robert; Panousis, Constantinos G; Kolczewski, Sabine; Steward, Lucinda

    2011-01-01

    G protein-coupled receptors (GPCRs) share a common architecture consisting of seven transmembrane (TM) domains. Various lines of evidence suggest that this fold provides a generic binding pocket within the TM region for hosting agonists, antagonists, and allosteric modulators. Hence, an automated method was developed that allows a fast analysis and comparison of these generic ligand binding pockets across the entire GPCR family by providing the relevant information for all GPCRs in the same format. This methodology compiles amino acids lining the TM binding pocket including parts of the ECL2 loop in a so-called 1D ligand binding pocket vector and translates these 1D vectors in a second step into 3D receptor pharmacophore models. It aims to support various aspects of GPCR drug discovery in the pharmaceutical industry. Applications of pharmacophore similarity analysis of these 1D LPVs include definition of receptor subfamilies, prediction of species differences within subfamilies in regard to in vitro pharmacology and identification of nearest neighbors for GPCRs of interest to generate starting points for GPCR lead identification programs. These aspects of GPCR research are exemplified in the field of melanopsins, trace amine-associated receptors and somatostatin receptor subtype 5. In addition, it is demonstrated how 3D pharmacophore models of the LPVs can support the prediction of amino acids involved in ligand recognition, the understanding of mutational data in a 3D context and the elucidation of binding modes for GPCR ligands and their evaluation. Furthermore, guidance through 3D receptor pharmacophore modeling for the synthesis of subtype-specific GPCR ligands will be reported. Illustrative examples are taken from the GPCR family class C, metabotropic glutamate receptors 1 and 5 and sweet taste receptors, and from the GPCR class A, e.g. nicotinic acid and 5-hydroxytryptamine 5A receptor. © 2011 Bentham Science Publishers

  5. GPCR homomers and heteromers: a better choice as targets for drug development than GPCR monomers?

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    Casadó, Vicent; Cortés, Antoni; Mallol, Josefa; Pérez-Capote, Kamil; Ferré, Sergi; Lluis, Carmen; Franco, Rafael; Canela, Enric I

    2009-11-01

    G protein-coupled receptors (GPCR) are targeted by many therapeutic drugs marketed to fight against a variety of diseases. Selection of novel lead compounds are based on pharmacological parameters obtained assuming that GPCR are monomers. However, many GPCR are expressed as dimers/oligomers. Therefore, drug development may consider GPCR as homo- and hetero-oligomers. A two-state dimer receptor model is now available to understand GPCR operation and to interpret data obtained from drugs interacting with dimers, and even from mixtures of monomers and dimers. Heteromers are distinct entities and therefore a given drug is expected to have different affinities and different efficacies depending on the heteromer. All these concepts would lead to broaden the therapeutic potential of drugs targeting GPCRs, including receptor heteromer-selective drugs with a lower incidence of side effects, or to identify novel pharmacological profiles using cell models expressing receptor heteromers.

  6. Dissecting signaling and functions of adhesion G protein-coupled receptors

    DEFF Research Database (Denmark)

    Araç, Demet; Aust, Gabriela; Calebiro, Davide

    2012-01-01

    G protein-coupled receptors (GPCRs) comprise an expanded superfamily of receptors in the human genome. Adhesion class G protein-coupled receptors (adhesion-GPCRs) form the second largest class of GPCRs. Despite the abundance, size, molecular structure, and functions in facilitating cell and matrix...... contacts in a variety of organ systems, adhesion-GPCRs are by far the most poorly understood GPCR class. Adhesion-GPCRs possess a unique molecular structure, with extended N-termini containing various adhesion domains. In addition, many adhesion-GPCRs are autoproteolytically cleaved into an N......-terminal fragment (NTF, NT, α-subunit) and C-terminal fragment (CTF, CT, β-subunit) at a conserved GPCR autoproteolysis-inducing (GAIN) domain that contains a GPCR proteolysis site (GPS). These two features distinguish adhesion-GPCRs from other GPCR classes. Though active research on adhesion-GPCRs in diverse areas...

  7. Receptor oligomerization in family B1 of G-protein-coupled receptors

    DEFF Research Database (Denmark)

    Roed, Sarah Norklit; Ørgaard, Anne; Jørgensen, Rasmus

    2012-01-01

    , the glucagon receptor, and the receptors for parathyroid hormone (PTHR1 and PTHR2). The dysregulation of several family B1 receptors is involved in diseases, such as diabetes, chronic inflammation, and osteoporosis which underlines the pathophysiological importance of this GPCR subfamily. In spite of this......, investigation of family B1 receptor oligomerization and especially its pharmacological importance is still at an early stage. Even though GPCR oligomerization is a well-established phenomenon, there is a need for more investigations providing a direct link between these interactions and receptor functionality......The superfamily of the seven transmembrane G-protein-coupled receptors (7TM/GPCRs) is the largest family of membrane-associated receptors. GPCRs are involved in the pathophysiology of numerous human diseases, and they constitute an estimated 30-40% of all drug targets. During the last two decades...

  8. Transient receptor potential channel superfamily: Role in lower urinary tract function.

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    Ogawa, Teruyuki; Imamura, Tetsuya; Nakazawa, Masaki; Hiragata, Shiro; Nagai, Takashi; Minagawa, Tomonori; Yokoyama, Hitoshi; Ishikawa, Masakuni; Domen, Takahisa; Ishizuka, Osamu

    2015-11-01

    Lower urinary tract symptoms associated with neurogenic bladder and overactive bladder syndrome are mediated in part by members of the transient receptor potential channel superfamily. The best studied member of this superfamily is the vanilloid receptor. Other transient receptor potential channels, such as the melastatin receptor and the ankyrin receptor, are also active in the pathogenesis of lower urinary tract dysfunction. However, the detailed mechanisms by which the transient receptor potential channels contribute to lower urinary tract symptoms are still not clear, and the therapeutic benefits of modulating transient receptor potential channel activity have not been proved in the clinical setting. In the present review, to better understand the pathophysiology and therapeutic potential for lower urinary tract symptoms, we summarize the presence and role of different members of the transient receptor potential channel superfamily in the lower urinary tract. © 2015 The Japanese Urological Association.

  9. Membrane-mediated oligomerization of G protein coupled receptors and its implications for GPCR function

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    Stefan Gahbauer

    2016-10-01

    Full Text Available The dimerization or even oligomerization of G protein coupled receptors (GPCRs causes ongoing, controversial debates about its functional role and the coupled biophysical, biochemical or biomedical implications. A continously growing number of studies hints to a relation between oligomerization and function of GPCRs and strengthens the assumption that receptor assembly plays a key role in the regulation of protein function. Additionally, progress in the structural analysis of GPCR-G protein and GPCR-ligand interactions allows to distinguish between actively functional and non-signalling complexes. Recent findings further suggest that the surrounding membrane, i.e. its lipid composition may modulate the preferred dimerization interface and as a result the abundance of distinct dimeric conformations. In this review, the association of GPCRs and the role of the membrane in oligomerization will be discussed. An overview of the different reported oligomeric interfaces is provided and their capability for signaling discussed. The currently available data is summarized with regard to the formation of GPCR oligomers, their structures and dependency on the membrane microenvironment as well as the coupling of oligomerization to receptor function.

  10. Efficient silkworm expression of human GPCR (nociceptin receptor) by a Bombyx mori bacmid DNA system

    International Nuclear Information System (INIS)

    Kajikawa, Mizuho; Sasaki, Kaori; Wakimoto, Yoshitaro; Toyooka, Masaru; Motohashi, Tomoko; Shimojima, Tsukasa; Takeda, Shigeki; Park, Enoch Y.; Maenaka, Katsumi

    2009-01-01

    Guanine nucleotide-binding protein (G protein) coupled receptors (GPCRs) are frequently expressed by a baculovirus expression vector system (BEVS). We recently established a novel BEVS using the bacmid system of Bombyx mori nucleopolyhedrovirus (BmNPV), which is directly applicable for protein expression in silkworms. Here, we report the first example of GPCR expression in silkworms by the simple injection of BmNPV bacmid DNA. Human nociceptin receptor, an inhibitory GPCR, and its fusion protein with inhibitory G protein alpha subunit (G i α) were both successfully expressed in the fat bodies of silkworm larvae as well as in the BmNPV viral fraction. Its yield was much higher than that from Sf9 cells. The microsomal fractions including the nociceptin receptor fusion, which are easily prepared by only centrifugation steps, exhibited [ 35 S]GTPγS-binding activity upon specific stimulation by nociceptin. Therefore, this rapid method is easy-to-use and has a high expression level, and thus will be an important tool for human GPCR production.

  11. GPCR-SSFE: A comprehensive database of G-protein-coupled receptor template predictions and homology models

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    Kreuchwig Annika

    2011-05-01

    Full Text Available Abstract Background G protein-coupled receptors (GPCRs transduce a wide variety of extracellular signals to within the cell and therefore have a key role in regulating cell activity and physiological function. GPCR malfunction is responsible for a wide range of diseases including cancer, diabetes and hyperthyroidism and a large proportion of drugs on the market target these receptors. The three dimensional structure of GPCRs is important for elucidating the molecular mechanisms underlying these diseases and for performing structure-based drug design. Although structural data are restricted to only a handful of GPCRs, homology models can be used as a proxy for those receptors not having crystal structures. However, many researchers working on GPCRs are not experienced homology modellers and are therefore unable to benefit from the information that can be gleaned from such three-dimensional models. Here, we present a comprehensive database called the GPCR-SSFE, which provides initial homology models of the transmembrane helices for a large variety of family A GPCRs. Description Extending on our previous theoretical work, we have developed an automated pipeline for GPCR homology modelling and applied it to a large set of family A GPCR sequences. Our pipeline is a fragment-based approach that exploits available family A crystal structures. The GPCR-SSFE database stores the template predictions, sequence alignments, identified sequence and structure motifs and homology models for 5025 family A GPCRs. Users are able to browse the GPCR dataset according to their pharmacological classification or search for results using a UniProt entry name. It is also possible for a user to submit a GPCR sequence that is not contained in the database for analysis and homology model building. The models can be viewed using a Jmol applet and are also available for download along with the alignments. Conclusions The data provided by GPCR-SSFE are useful for investigating

  12. GPCR-SSFE: a comprehensive database of G-protein-coupled receptor template predictions and homology models.

    Science.gov (United States)

    Worth, Catherine L; Kreuchwig, Annika; Kleinau, Gunnar; Krause, Gerd

    2011-05-23

    G protein-coupled receptors (GPCRs) transduce a wide variety of extracellular signals to within the cell and therefore have a key role in regulating cell activity and physiological function. GPCR malfunction is responsible for a wide range of diseases including cancer, diabetes and hyperthyroidism and a large proportion of drugs on the market target these receptors. The three dimensional structure of GPCRs is important for elucidating the molecular mechanisms underlying these diseases and for performing structure-based drug design. Although structural data are restricted to only a handful of GPCRs, homology models can be used as a proxy for those receptors not having crystal structures. However, many researchers working on GPCRs are not experienced homology modellers and are therefore unable to benefit from the information that can be gleaned from such three-dimensional models. Here, we present a comprehensive database called the GPCR-SSFE, which provides initial homology models of the transmembrane helices for a large variety of family A GPCRs. Extending on our previous theoretical work, we have developed an automated pipeline for GPCR homology modelling and applied it to a large set of family A GPCR sequences. Our pipeline is a fragment-based approach that exploits available family A crystal structures. The GPCR-SSFE database stores the template predictions, sequence alignments, identified sequence and structure motifs and homology models for 5025 family A GPCRs. Users are able to browse the GPCR dataset according to their pharmacological classification or search for results using a UniProt entry name. It is also possible for a user to submit a GPCR sequence that is not contained in the database for analysis and homology model building. The models can be viewed using a Jmol applet and are also available for download along with the alignments. The data provided by GPCR-SSFE are useful for investigating general and detailed sequence-structure-function relationships

  13. Impact of G protein-coupled receptor heteromers in endocrine systems.

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    Jonas, K C; Hanyaloglu, A C

    2017-07-05

    The fine-tuning of endocrine homeostasis is regulated by dynamic receptor mediated processes. The superfamily of G protein-coupled receptors (GPCRs) have diverse roles in the modulation of all endocrine axes, thus understanding the mechanisms underpinning their functionality is paramount for treatment of endocrinopathies. Evidence over the last 20 years has highlighted homo and heteromerization as a key mode of mediating GPCR functional diversity. This review will discuss the concept of GPCR heteromerization and its relevance to endocrine function, detailing in vitro and in vivo evidence, and exploring current and potential pharmacological strategies for specific targeting of GPCR heteromers in endocrine heath and disease. Crown Copyright © 2017. Published by Elsevier B.V. All rights reserved.

  14. Efficient silkworm expression of human GPCR (nociceptin receptor) by a Bombyx mori bacmid DNA system

    Energy Technology Data Exchange (ETDEWEB)

    Kajikawa, Mizuho; Sasaki, Kaori [Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582 (Japan); Wakimoto, Yoshitaro; Toyooka, Masaru [Department of Chemistry and Chemical Biology, Graduate School of Engineering, Gunma University, 1-5-1 Tenjin-cho, Kiryu, Gunma 376-8515 (Japan); Motohashi, Tomoko; Shimojima, Tsukasa [National Institute of Genetics, 1111 Yata, Mishima, Shizuoka 411-8540 (Japan); Takeda, Shigeki [Department of Chemistry and Chemical Biology, Graduate School of Engineering, Gunma University, 1-5-1 Tenjin-cho, Kiryu, Gunma 376-8515 (Japan); Park, Enoch Y. [Laboratory of Biotechnology, Integrated Bioscience Section, Graduate School of Science and Technology, Shizuoka University, 836 Oya, Suruga-ku, Shizuoka, Shizuoka 422-8529 (Japan); Maenaka, Katsumi, E-mail: kmaenaka-umin@umin.net [Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582 (Japan)

    2009-07-31

    Guanine nucleotide-binding protein (G protein) coupled receptors (GPCRs) are frequently expressed by a baculovirus expression vector system (BEVS). We recently established a novel BEVS using the bacmid system of Bombyx mori nucleopolyhedrovirus (BmNPV), which is directly applicable for protein expression in silkworms. Here, we report the first example of GPCR expression in silkworms by the simple injection of BmNPV bacmid DNA. Human nociceptin receptor, an inhibitory GPCR, and its fusion protein with inhibitory G protein alpha subunit (G{sub i}{alpha}) were both successfully expressed in the fat bodies of silkworm larvae as well as in the BmNPV viral fraction. Its yield was much higher than that from Sf9 cells. The microsomal fractions including the nociceptin receptor fusion, which are easily prepared by only centrifugation steps, exhibited [{sup 35}S]GTP{gamma}S-binding activity upon specific stimulation by nociceptin. Therefore, this rapid method is easy-to-use and has a high expression level, and thus will be an important tool for human GPCR production.

  15. Nuclear Receptors in atherosclerosis: a superfamily with many 'Goodfellas'

    NARCIS (Netherlands)

    Kurakula, Kondababu; Hamers, Anouk A. J.; de Waard, Vivian; de Vries, Carlie J. M.

    2013-01-01

    Nuclear Receptors form a superfamily of 48 transcription factors that exhibit a plethora of functions in steroid hormone signaling, regulation of metabolism, circadian rhythm and cellular differentiation. In this review, we describe our current knowledge on the role of Nuclear Receptors in

  16. GPCR crystal structures: Medicinal chemistry in the pocket.

    Science.gov (United States)

    Shonberg, Jeremy; Kling, Ralf C; Gmeiner, Peter; Löber, Stefan

    2015-07-15

    Recent breakthroughs in GPCR structural biology have significantly increased our understanding of drug action at these therapeutically relevant receptors, and this will undoubtedly lead to the design of better therapeutics. In recent years, crystal structures of GPCRs from classes A, B, C and F have been solved, unveiling a precise snapshot of ligand-receptor interactions. Furthermore, some receptors have been crystallized in different functional states in complex with antagonists, partial agonists, full agonists, biased agonists and allosteric modulators, providing further insight into the mechanisms of ligand-induced GPCR activation. It is now obvious that there is enormous diversity in the size, shape and position of the ligand binding pockets in GPCRs. In this review, we summarise the current state of solved GPCR structures, with a particular focus on ligand-receptor interactions in the binding pocket, and how this can contribute to the design of GPCR ligands with better affinity, subtype selectivity or efficacy. Copyright © 2015 Elsevier Ltd. All rights reserved.

  17. Role of spatial inhomogenity in GPCR dimerisation predicted by receptor association-diffusion models

    Science.gov (United States)

    Deshpande, Sneha A.; Pawar, Aiswarya B.; Dighe, Anish; Athale, Chaitanya A.; Sengupta, Durba

    2017-06-01

    G protein-coupled receptor (GPCR) association is an emerging paradigm with far reaching implications in the regulation of signalling pathways and therapeutic interventions. Recent super resolution microscopy studies have revealed that receptor dimer steady state exhibits sub-second dynamics. In particular the GPCRs, muscarinic acetylcholine receptor M1 (M1MR) and formyl peptide receptor (FPR), have been demonstrated to exhibit a fast association/dissociation kinetics, independent of ligand binding. In this work, we have developed a spatial kinetic Monte Carlo model to investigate receptor homo-dimerisation at a single receptor resolution. Experimentally measured association/dissociation kinetic parameters and diffusion coefficients were used as inputs to the model. To test the effect of membrane spatial heterogeneity on the simulated steady state, simulations were compared to experimental statistics of dimerisation. In the simplest case the receptors are assumed to be diffusing in a spatially homogeneous environment, while spatial heterogeneity is modelled to result from crowding, membrane micro-domains and cytoskeletal compartmentalisation or ‘corrals’. We show that a simple association-diffusion model is sufficient to reproduce M1MR association statistics, but fails to reproduce FPR statistics despite comparable kinetic constants. A parameter sensitivity analysis is required to reproduce the association statistics of FPR. The model reveals the complex interplay between cytoskeletal components and their influence on receptor association kinetics within the features of the membrane landscape. These results constitute an important step towards understanding the factors modulating GPCR organisation.

  18. GPCR-SSFE 2.0-a fragment-based molecular modeling web tool for Class A G-protein coupled receptors.

    Science.gov (United States)

    Worth, Catherine L; Kreuchwig, Franziska; Tiemann, Johanna K S; Kreuchwig, Annika; Ritschel, Michele; Kleinau, Gunnar; Hildebrand, Peter W; Krause, Gerd

    2017-07-03

    G-protein coupled receptors (GPCRs) are key players in signal transduction and therefore a large proportion of pharmaceutical drugs target these receptors. Structural data of GPCRs are sparse yet important for elucidating the molecular basis of GPCR-related diseases and for performing structure-based drug design. To ameliorate this problem, GPCR-SSFE 2.0 (http://www.ssfa-7tmr.de/ssfe2/), an intuitive web server dedicated to providing three-dimensional Class A GPCR homology models has been developed. The updated web server includes 27 inactive template structures and incorporates various new functionalities. Uniquely, it uses a fingerprint correlation scoring strategy for identifying the optimal templates, which we demonstrate captures structural features that sequence similarity alone is unable to do. Template selection is carried out separately for each helix, allowing both single-template models and fragment-based models to be built. Additionally, GPCR-SSFE 2.0 stores a comprehensive set of pre-calculated and downloadable homology models and also incorporates interactive loop modeling using the tool SL2, allowing knowledge-based input by the user to guide the selection process. For visual analysis, the NGL viewer is embedded into the result pages. Finally, blind-testing using two recently published structures shows that GPCR-SSFE 2.0 performs comparably or better than other state-of-the art GPCR modeling web servers. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  19. Targeting of the tumor necrosis factor receptor superfamily for cancer immunotherapy

    NARCIS (Netherlands)

    Bremer, Edwin

    2013-01-01

    The tumor necrosis factor (TNF) ligand and cognate TNF receptor superfamilies constitute an important regulatory axis that is pivotal for immune homeostasis and correct execution of immune responses. TNF ligands and receptors are involved in diverse biological processes ranging from the selective

  20. Novel evolutionary lineages of the invertebrate oxytocin/vasopressin superfamily peptides and their receptors in the common octopus (Octopus vulgaris)

    Science.gov (United States)

    Kanda, Atsuhiro; Satake, Honoo; Kawada, Tsuyoshi; Minakata, Hiroyuki

    2004-01-01

    The common octopus, Octopus vulgaris, is the first invertebrate species that was shown to possess two oxytocin/vasopressin (OT/VP) superfamily peptides, octopressin (OP) and cephalotocin (CT). Previously, we cloned a GPCR (G-protein-coupled receptor) specific to CT [CTR1 (CT receptor 1)]. In the present study, we have identified an additional CTR, CTR2, and a novel OP receptor, OPR. Both CTR2 and OPR include domains and motifs typical of GPCRs, and the intron– exon structures are in accord with those of OT/VP receptor genes. CTR2 and OPR expressed in Xenopus oocytes induced calcium-mediated inward chloride current in a CT- and OP-specific manner respectively. Several regions and residues, which are requisite for binding of the vertebrate OT/VP receptor family with their ligands, are highly conserved in CTRs, but not in OPR. These different sequences between CTRs and OPR, as well as the amino acid residues of OP and CT at positions 2–5, were presumed to play crucial roles in the binding selectivity to their receptors, whereas the difference in the polarity of OT/VP family peptide residues at position 8 confers OT and VP with the binding specificity in vertebrates. CTR2 mRNA was present in various peripheral tissues, and OPR mRNA was detected in both the nervous system and peripheral tissues. Our findings suggest that the CT and OP genes, similar to the OT/VP family, evolved through duplication, but the ligand–receptor selectivity were established through different evolutionary lineages from those of their vertebrate counterparts. PMID:15504101

  1. Quantitative Measurement of GPCR Endocytosis via Pulse-Chase Covalent Labeling.

    Directory of Open Access Journals (Sweden)

    Hidetoshi Kumagai

    Full Text Available G protein-coupled receptors (GPCRs play a critical role in many physiological systems and represent one of the largest families of signal-transducing receptors. The number of GPCRs at the cell surface regulates cellular responsiveness to their cognate ligands, and the number of GPCRs, in turn, is dynamically controlled by receptor endocytosis. Recent studies have demonstrated that GPCR endocytosis, in addition to affecting receptor desensitization and resensitization, contributes to acute G protein-mediated signaling. Thus, endocytic GPCR behavior has a significant impact on various aspects of physiology. In this study, we developed a novel GPCR internalization assay to facilitate characterization of endocytic GPCR behavior. We genetically engineered chimeric GPCRs by fusing HaloTag (a catalytically inactive derivative of a bacterial hydrolase to the N-terminal end of the receptor (HT-GPCR. HaloTag has the ability to form a stable covalent bond with synthetic HaloTag ligands that contain fluorophores or a high-affinity handle (such as biotin and the HaloTag reactive linker. We selectively labeled HT-GPCRs at the cell surface with a HaloTag PEG ligand, and this pulse-chase covalent labeling allowed us to directly monitor the relative number of internalized GPCRs after agonist stimulation. Because the endocytic activities of GPCR ligands are not necessarily correlated with their agonistic activities, applying this novel methodology to orphan GPCRs, or even to already characterized GPCRs, will increase the likelihood of identifying currently unknown ligands that have been missed by conventional pharmacological assays.

  2. Self-Checking Cell-Based Assays for GPCR Desensitization and Resensitization.

    Science.gov (United States)

    Fisher, Gregory W; Fuhrman, Margaret H; Adler, Sally A; Szent-Gyorgyi, Christopher; Waggoner, Alan S; Jarvik, Jonathan W

    2014-09-01

    G protein-coupled receptors (GPCRs) play stimulatory or modulatory roles in numerous physiological states and processes, including growth and development, vision, taste and olfaction, behavior and learning, emotion and mood, inflammation, and autonomic functions such as blood pressure, heart rate, and digestion. GPCRs constitute the largest protein superfamily in the human and are the largest target class for prescription drugs, yet most are poorly characterized, and of the more than 350 nonolfactory human GPCRs, over 100 are orphans for which no endogenous ligand has yet been convincingly identified. We here describe new live-cell assays that use recombinant GPCRs to quantify two general features of GPCR cell biology-receptor desensitization and resensitization. The assays employ a fluorogen-activating protein (FAP) reporter that reversibly complexes with either of two soluble organic molecules (fluorogens) whose fluorescence is strongly enhanced when complexed with the FAP. Both assays require no wash or cleanup steps and are readily performed in microwell plates, making them adaptable to high-throughput drug discovery applications. © 2014 Society for Laboratory Automation and Screening.

  3. The essential role of G protein-coupled receptor (GPCR) signaling in regulating T cell immunity.

    Science.gov (United States)

    Wang, Dashan

    2018-06-01

    The aim of this paper is to clarify the critical role of GPCR signaling in T cell immunity. The G protein-coupled receptors (GPCRs) are the most common targets in current pharmaceutical industry, and represent the largest and most versatile family of cell surface communicating molecules. GPCRs can be activated by a diverse array of ligands including neurotransmitters, chemokines as well as sensory stimuli. Therefore, GPCRs are involved in many key cellular and physiological processes, such as sense of light, taste and smell, neurotransmission, metabolism, endocrine and exocrine secretion. In recent years, GPCRs have been found to play an important role in immune system. T cell is an important type of immune cell, which plays a central role in cell-mediated immunity. A variety of GPCRs and their signaling mediators (RGS proteins, GRKs and β-arrestin) have been found to express in T cells and involved T cell-mediated immunity. We will summarize the role of GPCR signaling and their regulatory molecules in T cell activation, homeostasis and function in this article. GPCR signaling plays an important role in T cell activation, homeostasis and function. GPCR signaling is critical in regulating T cell immunity.

  4. Conformational biosensors reveal GPCR signalling from endosomes

    DEFF Research Database (Denmark)

    Irannejad, R; Tomshine, Jin C; Tomshine, Jon R

    2013-01-01

    A long-held tenet of molecular pharmacology is that canonical signal transduction mediated by G-protein-coupled receptor (GPCR) coupling to heterotrimeric G proteins is confined to the plasma membrane. Evidence supporting this traditional view is based on analytical methods that provide limited...... or no subcellular resolution. It has been subsequently proposed that signalling by internalized GPCRs is restricted to G-protein-independent mechanisms such as scaffolding by arrestins, or GPCR activation elicits a discrete form of persistent G protein signalling, or that internalized GPCRs can indeed contribute...... in the early endosome membrane, and that internalized receptors contribute to the overall cellular cyclic AMP response within several minutes after agonist application. These findings provide direct support for the hypothesis that canonical GPCR signalling occurs from endosomes as well as the plasma membrane...

  5. Conformational entropic maps of functional coupling domains in GPCR activation: A case study with beta2 adrenergic receptor

    Science.gov (United States)

    Liu, Fan; Abrol, Ravinder; Goddard, William, III; Dougherty, Dennis

    2014-03-01

    Entropic effect in GPCR activation is poorly understood. Based on the recent solved structures, researchers in the GPCR structural biology field have proposed several ``local activating switches'' that consisted of a few number of conserved residues, but have long ignored the collective dynamical effect (conformational entropy) of a domain comprised of an ensemble of residues. A new paradigm has been proposed recently that a GPCR can be viewed as a composition of several functional coupling domains, each of which undergoes order-to-disorder or disorder-to-order transitions upon activation. Here we identified and studied these functional coupling domains by comparing the local entropy changes of each residue between the inactive and active states of the β2 adrenergic receptor from computational simulation. We found that agonist and G-protein binding increases the heterogeneity of the entropy distribution in the receptor. This new activation paradigm and computational entropy analysis scheme provides novel ways to design functionally modified mutant and identify new allosteric sites for GPCRs. The authors thank NIH and Sanofi for funding this project.

  6. Structural Elements in the Gαs and Gαq C Termini That Mediate Selective G Protein-coupled Receptor (GPCR) Signaling.

    Science.gov (United States)

    Semack, Ansley; Sandhu, Manbir; Malik, Rabia U; Vaidehi, Nagarajan; Sivaramakrishnan, Sivaraj

    2016-08-19

    Although the importance of the C terminus of the α subunit of the heterotrimeric G protein in G protein-coupled receptor (GPCR)-G protein pairing is well established, the structural basis of selective interactions remains unknown. Here, we combine live cell FRET-based measurements and molecular dynamics simulations of the interaction between the GPCR and a peptide derived from the C terminus of the Gα subunit (Gα peptide) to dissect the molecular mechanisms of G protein selectivity. We observe a direct link between Gα peptide binding and stabilization of the GPCR conformational ensemble. We find that cognate and non-cognate Gα peptides show deep and shallow binding, respectively, and in distinct orientations within the GPCR. Binding of the cognate Gα peptide stabilizes the agonist-bound GPCR conformational ensemble resulting in favorable binding energy and lower flexibility of the agonist-GPCR pair. We identify three hot spot residues (Gαs/Gαq-Gln-384/Leu-349, Gln-390/Glu-355, and Glu-392/Asn-357) that contribute to selective interactions between the β2-adrenergic receptor (β2-AR)-Gαs and V1A receptor (V1AR)-Gαq The Gαs and Gαq peptides adopt different orientations in β2-AR and V1AR, respectively. The β2-AR/Gαs peptide interface is dominated by electrostatic interactions, whereas the V1AR/Gαq peptide interactions are predominantly hydrophobic. Interestingly, our study reveals a role for both favorable and unfavorable interactions in G protein selection. Residue Glu-355 in Gαq prevents this peptide from interacting strongly with β2-AR. Mutagenesis to the Gαs counterpart (E355Q) imparts a cognate-like interaction. Overall, our study highlights the synergy in molecular dynamics and FRET-based approaches to dissect the structural basis of selective G protein interactions. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  7. Barcoding of GPCR trafficking and signaling through the various trafficking roadmaps by compartmentalized signaling networks.

    Science.gov (United States)

    Bahouth, Suleiman W; Nooh, Mohammed M

    2017-08-01

    Proper signaling by G protein coupled receptors (GPCR) is dependent on the specific repertoire of transducing, enzymatic and regulatory kinases and phosphatases that shape its signaling output. Activation and signaling of the GPCR through its cognate G protein is impacted by G protein-coupled receptor kinase (GRK)-imprinted "barcodes" that recruit β-arrestins to regulate subsequent desensitization, biased signaling and endocytosis of the GPCR. The outcome of agonist-internalized GPCR in endosomes is also regulated by sequence motifs or "barcodes" within the GPCR that mediate its recycling to the plasma membrane or retention and eventual degradation as well as its subsequent signaling in endosomes. Given the vast number of diverse sequences in GPCR, several trafficking mechanisms for endosomal GPCR have been described. The majority of recycling GPCR, are sorted out of endosomes in a "sequence-dependent pathway" anchored around a type-1 PDZ-binding module found in their C-tails. For a subset of these GPCR, a second "barcode" imprinted onto specific GPCR serine/threonine residues by compartmentalized kinase networks was required for their efficient recycling through the "sequence-dependent pathway". Mutating the serine/threonine residues involved, produced dramatic effects on GPCR trafficking, indicating that they played a major role in setting the trafficking itinerary of these GPCR. While endosomal SNX27, retromer/WASH complexes and actin were required for efficient sorting and budding of all these GPCR, additional proteins were required for GPCR sorting via the second "barcode". Here we will review recent developments in GPCR trafficking in general and the human β 1 -adrenergic receptor in particular across the various trafficking roadmaps. In addition, we will discuss the role of GPCR trafficking in regulating endosomal GPCR signaling, which promote biochemical and physiological effects that are distinct from those generated by the GPCR signal transduction

  8. Prediction of GPCR-Ligand Binding Using Machine Learning Algorithms

    Directory of Open Access Journals (Sweden)

    Sangmin Seo

    2018-01-01

    Full Text Available We propose a novel method that predicts binding of G-protein coupled receptors (GPCRs and ligands. The proposed method uses hub and cycle structures of ligands and amino acid motif sequences of GPCRs, rather than the 3D structure of a receptor or similarity of receptors or ligands. The experimental results show that these new features can be effective in predicting GPCR-ligand binding (average area under the curve [AUC] of 0.944, because they are thought to include hidden properties of good ligand-receptor binding. Using the proposed method, we were able to identify novel ligand-GPCR bindings, some of which are supported by several studies.

  9. Structure-Based Sequence Alignment of the Transmembrane Domains of All Human GPCRs: Phylogenetic, Structural and Functional Implications

    Science.gov (United States)

    Cvicek, Vaclav; Goddard, William A.; Abrol, Ravinder

    2016-01-01

    The understanding of G-protein coupled receptors (GPCRs) is undergoing a revolution due to increased information about their signaling and the experimental determination of structures for more than 25 receptors. The availability of at least one receptor structure for each of the GPCR classes, well separated in sequence space, enables an integrated superfamily-wide analysis to identify signatures involving the role of conserved residues, conserved contacts, and downstream signaling in the context of receptor structures. In this study, we align the transmembrane (TM) domains of all experimental GPCR structures to maximize the conserved inter-helical contacts. The resulting superfamily-wide GpcR Sequence-Structure (GRoSS) alignment of the TM domains for all human GPCR sequences is sufficient to generate a phylogenetic tree that correctly distinguishes all different GPCR classes, suggesting that the class-level differences in the GPCR superfamily are encoded at least partly in the TM domains. The inter-helical contacts conserved across all GPCR classes describe the evolutionarily conserved GPCR structural fold. The corresponding structural alignment of the inactive and active conformations, available for a few GPCRs, identifies activation hot-spot residues in the TM domains that get rewired upon activation. Many GPCR mutations, known to alter receptor signaling and cause disease, are located at these conserved contact and activation hot-spot residue positions. The GRoSS alignment places the chemosensory receptor subfamilies for bitter taste (TAS2R) and pheromones (Vomeronasal, VN1R) in the rhodopsin family, known to contain the chemosensory olfactory receptor subfamily. The GRoSS alignment also enables the quantification of the structural variability in the TM regions of experimental structures, useful for homology modeling and structure prediction of receptors. Furthermore, this alignment identifies structurally and functionally important residues in all human GPCRs

  10. G protein-coupled receptor (GPCR) signaling via heterotrimeric G proteins from endosomes.

    Science.gov (United States)

    Tsvetanova, Nikoleta G; Irannejad, Roshanak; von Zastrow, Mark

    2015-03-13

    Some G protein-coupled receptors (GPCRs), in addition to activating heterotrimeric G proteins in the plasma membrane, appear to elicit a "second wave" of G protein activation after ligand-induced internalization. We briefly summarize evidence supporting this view and then discuss what is presently known about the functional significance of GPCR-G protein activation in endosomes. Endosomal activation can shape the cellular response temporally by prolonging its overall duration, and may shape the response spatially by moving the location of intracellular second messenger production relative to effectors. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  11. Glycosylation of KSHV Encoded vGPCR Functions in Its Signaling and Tumorigenicity

    Directory of Open Access Journals (Sweden)

    Hui Wu

    2015-03-01

    Full Text Available Kaposi’s sarcoma-associated herpesvirus (KSHV is a tumor virus and the etiologic agent of Kaposi’s Sarcoma (KS. KSHV G protein-coupled receptor (vGPCR is an oncogene that is implicated in malignancies associated with KHSV infection. In this study, we show that vGPCR undergoes extensive N-linked glycosylation within the extracellular domains, specifically asparagines 18, 22, 31 and 202. An immunofluorescence assay demonstrates that N-linked glycosylation are necessary for vGPCR trafficking to the cellular membrane. Employing vGPCR mutants whose glycosylation sites were ablated, we show that these vGPCR mutants failed to activate downstream signaling in cultured cells and were severely impaired to induce tumor formation in the xenograph nude mouse model. These findings support the conclusion that glycosylation is critical for vGPCR tumorigenesis and imply that chemokine regulation at the plasma membrane is crucial for vGPCR mediated signaling.

  12. Cellular and molecular biology of orphan G protein-coupled receptors.

    Science.gov (United States)

    Oh, Da Young; Kim, Kyungjin; Kwon, Hyuk Bang; Seong, Jae Young

    2006-01-01

    The superfamily of G protein-coupled receptors (GPCRs) is the largest and most diverse group of membrane-spanning proteins. It plays a variety of roles in pathophysiological processes by transmitting extracellular signals to cells via heterotrimeric G proteins. Completion of the human genome project revealed the presence of approximately 168 genes encoding established nonsensory GPCRs, as well as 207 genes predicted to encode novel GPCRs for which the natural ligands remained to be identified, the so-called orphan GPCRs. Eighty-six of these orphans have now been paired to novel or previously known molecules, and 121 remain to be deorphaned. A better understanding of the GPCR structures and classification; knowledge of the receptor activation mechanism, either dependent on or independent of an agonist; increased understanding of the control of GPCR-mediated signal transduction; and development of appropriate ligand screening systems may improve the probability of discovering novel ligands for the remaining orphan GPCRs.

  13. Targeting GPCR-Gβγ-GRK2 signaling as a novel strategy for treating cardiorenal pathologies.

    Science.gov (United States)

    Rudomanova, Valeria; Blaxall, Burns C

    2017-08-01

    The pathologic crosstalk between the heart and kidney is known as cardiorenal syndrome (CRS). While the specific mechanisms underlying this crosstalk remain poorly understood, CRS is associated with exacerbated dysfunction of either or both organs and reduced survival. Maladaptive fibrotic remodeling is a key component of both heart and kidney failure pathogenesis and progression. G-protein coupled receptor (GPCR) signaling is a crucial regulator of cardiovascular and renal function. Chronic/pathologic GPCR signaling elicits the interaction of the G-protein Gβγ subunit with GPCR kinase 2 (GRK2), targeting the receptor for internalization, scaffolding to pathologic signals, and receptor degradation. Targeting this pathologic Gβγ-GRK2 interaction has been suggested as a possible strategy for the treatment of HF. In the current review, we discuss recent updates in understanding the role of GPCR-Gβγ-GRK2 signaling as a crucial mediator of maladaptive organ remodeling detected in HF and kidney dysfunction, with specific attention to small molecule-mediated inhibition of pathologic Gβγ-GRK2 interactions. Further, we explore the potential of GPCR-Gβγ-GRK2 signaling as a possible therapeutic target for cardiorenal pathologies. Copyright © 2017 Elsevier B.V. All rights reserved.

  14. Functional Consequences of Glucagon-like Peptide-1 Receptor Cross-talk and Trafficking

    DEFF Research Database (Denmark)

    Roed, Sarah Noerklit; Nøhr, Anne Cathrine; Wismann, Pernille

    2015-01-01

    The signaling capacity of seven-transmembrane/G-protein-coupled receptors (7TM/GPCRs) can be regulated through ligand-mediated receptor trafficking. Classically, the recycling of internalized receptors is associated with resensitization, whereas receptor degradation terminates signaling. We have......) and glucagon (GCGR) receptors. The interaction and cross-talk between coexpressed receptors is a wide phenomenon of the 7TM/GPCR superfamily. Numerous reports show functional consequences for signaling and trafficking of the involved receptors. On the basis of the high structural similarity and tissue...... coexpression, we here investigated the potential cross-talk between GLP-1R and GIPR or GCGR in both trafficking and signaling pathways. Using a real-time time-resolved FRET-based internalization assay, we show that GLP-1R, GIPR, and GCGR internalize with differential properties. Remarkably, upon coexpression...

  15. Multiplexed profiling of GPCR activities by combining split TEV assays and EXT-based barcoded readouts.

    Science.gov (United States)

    Galinski, Sabrina; Wichert, Sven P; Rossner, Moritz J; Wehr, Michael C

    2018-05-25

    G protein-coupled receptors (GPCRs) are the largest class of cell surface receptors and are implicated in the physiological regulation of many biological processes. The high diversity of GPCRs and their physiological functions make them primary targets for therapeutic drugs. For the generation of novel compounds, however, selectivity towards a given target is a critical issue in drug development as structural similarities between members of GPCR subfamilies exist. Therefore, the activities of multiple GPCRs that are both closely and distantly related to assess compound selectivity need to be tested simultaneously. Here, we present a cell-based multiplexed GPCR activity assay, termed GPCRprofiler, which uses a β-arrestin recruitment strategy and combines split TEV protein-protein interaction and EXT-based barcode technologies. This approach enables simultaneous measurements of receptor activities of multiple GPCR-ligand combinations by applying massively parallelized reporter assays. In proof-of-principle experiments covering 19 different GPCRs, both the specificity of endogenous agonists and the polypharmacological effects of two known antipsychotics on GPCR activities were demonstrated. Technically, normalization of barcode reporters across individual assays allows quantitative pharmacological assays in a parallelized manner. In summary, the GPCRprofiler technique constitutes a flexible and scalable approach, which enables simultaneous profiling of compound actions on multiple receptor activities in living cells.

  16. Quantification of GPCR internalization by single-molecule microscopy in living cells.

    NARCIS (Netherlands)

    Serge, A.; Keijzer, S. de; Hemert, F. Van; Hickman, M.R.; Hereld, D.; Spaink, H.P.; Schmidt, T.; Snaar-Jagalska, B.E.

    2011-01-01

    Receptor internalization upon ligand stimulation is a key component of a cell's response and allows a cell to correctly sense its environment. Novel fluorescent methods have enabled the direct visualization of the agonist-stimulated G-protein-coupled receptors (GPCR) trafficking in living cells.

  17. GPCR-autoantibodies in chronic heart failure.

    Science.gov (United States)

    Boivin-Jahns, Valerie; Jahns, Roland

    2018-06-01

    Chronic heart failure (CHF) is a syndrome characterized by shortness of breath, fluid retention, and a progressive reduction in cardiac function. More than 60% of the cases are ischemic in origin (i.e., due to myo-cardial infarction) and about 30% are caused by non-ischemic myocardial damage (i.e., due to genetic or non-genetic causes like myocardial inflammation). Because of alterations in both cellular and humoral immunity patients with non-ischemic CHF often develop abnormal or misled immune responses, including cross-reacting antibodies and/or autoantibodies to various cardiac anti-gens. Non-ischemic myo-cardial damage was found to progress to CHF particularly, when associated (a) with the generation of autoantibodies directed against distinct myocyte membrane proteins critically involved in cardiac function - like G-protein coup-led membrane receptors (GPCRs), or (b) with virus persistence in the myocardium. This article will review current knowledge on the pathophysiological relevance of GPCR-autoreactivity in CHF by giving an overview on the so far available evidence from pre-clinical, clinical and epidemiological studies on the CHF-inducing potential of GPCR-autoantibodies and thereon based novel therapeutic approaches in GPCR autoantibody-associated CHF.

  18. GPCR-Bench: A Benchmarking Set and Practitioners' Guide for G Protein-Coupled Receptor Docking.

    Science.gov (United States)

    Weiss, Dahlia R; Bortolato, Andrea; Tehan, Benjamin; Mason, Jonathan S

    2016-04-25

    Virtual screening is routinely used to discover new ligands and in particular new ligand chemotypes for G protein-coupled receptors (GPCRs). To prepare for a virtual screen, we often tailor a docking protocol that will enable us to select the best candidates for further screening. To aid this, we created GPCR-Bench, a publically available docking benchmarking set in the spirit of the DUD and DUD-E reference data sets for validation studies, containing 25 nonredundant high-resolution GPCR costructures with an accompanying set of diverse ligands and computational decoy molecules for each target. Benchmarking sets are often used to compare docking protocols; however, it is important to evaluate docking methods not by "retrospective" hit rates but by the actual likelihood that they will produce novel prospective hits. Therefore, docking protocols must not only rank active molecules highly but also produce good poses that a chemist will select for purchase and screening. Currently, no simple objective machine-scriptable function exists that can do this; instead, docking hit lists must be subjectively examined in a consistent way to compare between docking methods. We present here a case study highlighting considerations we feel are of importance when evaluating a method, intended to be useful as a practitioners' guide.

  19. Evidence of G-protein-coupled receptor and substrate transporter heteromerization at a single molecule level.

    Science.gov (United States)

    Fischer, Jana; Kleinau, Gunnar; Rutz, Claudia; Zwanziger, Denise; Khajavi, Noushafarin; Müller, Anne; Rehders, Maren; Brix, Klaudia; Worth, Catherine L; Führer, Dagmar; Krude, Heiko; Wiesner, Burkhard; Schülein, Ralf; Biebermann, Heike

    2018-06-01

    G-protein-coupled receptors (GPCRs) can constitute complexes with non-GPCR integral membrane proteins, while such interaction has not been demonstrated at a single molecule level so far. We here investigated the potential interaction between the thyrotropin receptor (TSHR) and the monocarboxylate transporter 8 (MCT8), a member of the major facilitator superfamily (MFS), using fluorescence cross-correlation spectroscopy (FCCS). Both the proteins are expressed endogenously on the basolateral plasma membrane of the thyrocytes and are involved in stimulation of thyroid hormone production and release. Indeed, we demonstrate strong interaction between both the proteins which causes a suppressed activation of G q/11 by TSH-stimulated TSHR. Thus, we provide not only evidence for a novel interaction between the TSHR and MCT8, but could also prove this interaction on a single molecule level. Moreover, this interaction forces biased signaling at the TSHR. These results are of general interest for both the GPCR and the MFS research fields.

  20. Onco-GPCR signaling and dysregulated expression of microRNAs in human cancer.

    Science.gov (United States)

    Nohata, Nijiro; Goto, Yusuke; Gutkind, J Silvio

    2017-01-01

    The G-protein-coupled receptor (GPCR) family is the largest family of cell-surface receptors involved in signal transduction. Aberrant expression of GPCRs and G proteins are frequently associated with prevalent human diseases, including cancer. In fact, GPCRs represent the therapeutic targets of more than a quarter of the clinical drugs currently on the market. MiRNAs (miRNAs) are also aberrantly expressed in many human cancers, and they have significant roles in the initiation, development and metastasis of human malignancies. Recent studies have revealed that dysregulation of miRNAs and their target genes expression are associated with cancer progression. The emerging information suggests that miRNAs play an important role in the fine tuning of many signaling pathways, including GPCR signaling. We summarize our current knowledge of the individual functions of miRNAs regulated by GPCRs and GPCR signaling-associated molecules, and miRNAs that regulate the expression and activity of GPCRs, their endogenous ligands and their coupled heterotrimeric G proteins in human cancer.

  1. Structure of the [delta]-opioid receptor bound to naltrindole

    Energy Technology Data Exchange (ETDEWEB)

    Granier, Sébastien; Manglik, Aashish; Kruse, Andrew C.; Kobilka, Tong Sun; Thian, Foon Sun; Weis, William I.; Kobilka, Brian K. (Stanford-MED)

    2012-07-11

    The opioid receptor family comprises three members, the {mu}-, {delta}- and {kappa}-opioid receptors, which respond to classical opioid alkaloids such as morphine and heroin as well as to endogenous peptide ligands like endorphins. They belong to the G-protein-coupled receptor (GPCR) superfamily, and are excellent therapeutic targets for pain control. The {delta}-opioid receptor ({delta}-OR) has a role in analgesia, as well as in other neurological functions that remain poorly understood. The structures of the {mu}-OR and {kappa}-OR have recently been solved. Here we report the crystal structure of the mouse {delta}-OR, bound to the subtype-selective antagonist naltrindole. Together with the structures of the {mu}-OR and {kappa}-OR, the {delta}-OR structure provides insights into conserved elements of opioid ligand recognition while also revealing structural features associated with ligand-subtype selectivity. The binding pocket of opioid receptors can be divided into two distinct regions. Whereas the lower part of this pocket is highly conserved among opioid receptors, the upper part contains divergent residues that confer subtype selectivity. This provides a structural explanation and validation for the 'message-address' model of opioid receptor pharmacology, in which distinct 'message' (efficacy) and 'address' (selectivity) determinants are contained within a single ligand. Comparison of the address region of the {delta}-OR with other GPCRs reveals that this structural organization may be a more general phenomenon, extending to other GPCR families as well.

  2. Trends in GPCR drug discovery: new agents, targets and indications.

    Science.gov (United States)

    Hauser, Alexander S; Attwood, Misty M; Rask-Andersen, Mathias; Schiöth, Helgi B; Gloriam, David E

    2017-12-01

    G protein-coupled receptors (GPCRs) are the most intensively studied drug targets, mostly due to their substantial involvement in human pathophysiology and their pharmacological tractability. Here, we report an up-to-date analysis of all GPCR drugs and agents in clinical trials, which reveals current trends across molecule types, drug targets and therapeutic indications, including showing that 475 drugs (~34% of all drugs approved by the US Food and Drug Administration (FDA)) act at 108 unique GPCRs. Approximately 321 agents are currently in clinical trials, of which ~20% target 66 potentially novel GPCR targets without an approved drug, and the number of biological drugs, allosteric modulators and biased agonists has increased. The major disease indications for GPCR modulators show a shift towards diabetes, obesity and Alzheimer disease, although several central nervous system disorders are also highly represented. The 224 (56%) non-olfactory GPCRs that have not yet been explored in clinical trials have broad untapped therapeutic potential, particularly in genetic and immune system disorders. Finally, we provide an interactive online resource to analyse and infer trends in GPCR drug discovery.

  3. Genome-wide identification of nuclear receptor (NR) superfamily genes in the copepod Tigriopus japonicus.

    Science.gov (United States)

    Hwang, Dae-Sik; Lee, Bo-Young; Kim, Hui-Su; Lee, Min Chul; Kyung, Do-Hyun; Om, Ae-Son; Rhee, Jae-Sung; Lee, Jae-Seong

    2014-11-18

    Nuclear receptors (NRs) are a large superfamily of proteins defined by a DNA-binding domain (DBD) and a ligand-binding domain (LBD). They function as transcriptional regulators to control expression of genes involved in development, homeostasis, and metabolism. The number of NRs differs from species to species, because of gene duplications and/or lineage-specific gene losses during metazoan evolution. Many NRs in arthropods interact with the ecdysteroid hormone and are involved in ecdysone-mediated signaling in arthropods. The nuclear receptor superfamily complement has been reported in several arthropods, including crustaceans, but not in copepods. We identified the entire NR repertoire of the copepod Tigriopus japonicus, which is an important marine model species for ecotoxicology and environmental genomics. Using whole genome and transcriptome sequences, we identified a total of 31 nuclear receptors in the genome of T. japonicus. Nomenclature of the nuclear receptors was determined based on the sequence similarities of the DNA-binding domain (DBD) and ligand-binding domain (LBD). The 7 subfamilies of NRs separate into five major clades (subfamilies NR1, NR2, NR3, NR4, and NR5/6). Although the repertoire of NR members in, T. japonicus was similar to that reported for other arthropods, there was an expansion of the NR1 subfamily in Tigriopus japonicus. The twelve unique nuclear receptors identified in T. japonicus are members of NR1L. This expansion may be a unique lineage-specific feature of crustaceans. Interestingly, E78 and HR83, which are present in other arthropods, were absent from the genomes of T. japonicus and two congeneric copepod species (T. japonicus and Tigriopus californicus), suggesting copepod lineage-specific gene loss. We identified all NR receptors present in the copepod, T. japonicus. Knowledge of the copepod nuclear receptor repertoire will contribute to a better understanding of copepod- and crustacean-specific NR evolution.

  4. TNF and TNF Receptor Superfamily Members in HIV infection: New Cellular Targets for Therapy?

    Directory of Open Access Journals (Sweden)

    Amit Kumar

    2013-01-01

    Full Text Available Tumor necrosis factor (TNF and TNF receptors (TNFR superfamily members are engaged in diverse cellular phenomena such as cellular proliferation, morphogenesis, apoptosis, inflammation, and immune regulation. Their role in regulating viral infections has been well documented. Viruses have evolved with numerous strategies to interfere with TNF-mediated signaling indicating the importance of TNF and TNFR superfamily in viral pathogenesis. Recent research reports suggest that TNF and TNFRs play an important role in the pathogenesis of HIV. TNFR signaling modulates HIV replication and HIV proteins interfere with TNF/TNFR pathways. Since immune activation and inflammation are the hallmark of HIV infection, the use of TNF inhibitors can have significant impact on HIV disease progression. In this review, we will describe how HIV infection is modulated by signaling mediated through members of TNF and TNFR superfamily and in turn how these latter could be targeted by HIV proteins. Finally, we will discuss the emerging therapeutics options based on modulation of TNF activity that could ultimately lead to the cure of HIV-infected patients.

  5. Integrated Approaches for Genome-wide Interrogation of the Druggable Non-olfactory G Protein-coupled Receptor Superfamily.

    Science.gov (United States)

    Roth, Bryan L; Kroeze, Wesley K

    2015-08-07

    G-protein-coupled receptors (GPCRs) are frequent and fruitful targets for drug discovery and development, as well as being off-targets for the side effects of a variety of medications. Much of the druggable non-olfactory human GPCR-ome remains under-interrogated, and we present here various approaches that we and others have used to shine light into these previously dark corners of the human genome. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  6. FLIPR assays of intracellular calcium in GPCR drug discovery

    DEFF Research Database (Denmark)

    Hansen, Kasper Bø; Bräuner-Osborne, Hans

    2009-01-01

    Fluorescent dyes sensitive to changes in intracellular calcium have become increasingly popular in G protein-coupled receptor (GPCR) drug discovery for several reasons. First of all, the assays using the dyes are easy to perform and are of low cost compared to other assays. Second, most non...

  7. The GPCR membrane receptor, DopEcR, mediates the actions of both dopamine and ecdysone to control sex pheromone perception in an insect

    Directory of Open Access Journals (Sweden)

    Antoine eAbrieux

    2014-09-01

    Full Text Available Olfactory information mediating sexual behavior is crucial for reproduction in many animals, including insects. In male moths, the macroglomerular complex of the primary olfactory center, the antennal lobe (AL is specialized in the treatment of information on the female-emitted sex pheromone. Evidence is accumulating that modulation of behavioral pheromone responses occurs through neuronal plasticity via the action of hormones and/or catecholamines. We recently showed that a G-protein-coupled receptor (GPCR, AipsDopEcR, with its homologue known in Drosophila for its double affinity to the main insect steroid hormone 20-hydroxyecdysone (20E, and dopamine (DA, present in the ALs, is involved in the behavioral response to pheromone in the moth, Agrotis ipsilon. Here we tested the role of AipsDopEcR as compared to nuclear 20E receptors in central pheromone processing combining receptor inhibition with intracellular recordings of AL neurons. We show that the sensitivity of AL neurons for the pheromone in males decreases strongly after AipsDopEcR-dsRNA injection but also after inhibition of nuclear 20E receptors. Moreover we tested the involvement of 20E and DA in the receptor-mediated behavioral modulation in wind tunnel experiments, using ligand applications and receptor inhibition treatments. We show that both ligands are necessary and act on AipsDopEcR-mediated behavior. Altogether these results indicate that the GPCR membrane receptor, AipsDopEcR, controls sex pheromone perception through the action of both 20E and DA in the central nervous system, probably in concert with 20E action through nuclear receptors.

  8. Functional characterization of bursicon receptor and genome-wide analysis for identification of genes affected by bursicon receptor RNAi

    Science.gov (United States)

    Bai, Hua; Palli, Subba R.

    2010-01-01

    Bursicon is an insect neuropeptide hormone that is secreted from the central nervous system into the hemolymph and initiates cuticle tanning. The receptor for bursicon is encoded by the rickets (rk) gene and belongs to the G protein-coupled receptor (GPCR) superfamily. The bursicon and its receptor regulate cuticle tanning as well as wing expansion after adult eclosion. However, the molecular action of bursicon signaling remains unclear. We utilized RNA interference (RNAi) and microarray to study the function of the bursicon receptor (Tcrk) in the model insect, Tribolium castaneum. The data included here showed that in addition to cuticle tanning and wing expansion reported previously, Tcrk is also required for development and expansion of integumentary structures and adult eclosion. Using custom microarrays, we identified 24 genes that are differentially expressed between Tcrk RNAi and control insects. Knockdown in the expression of one of these genes, TC004091, resulted in the arrest of adult eclosion. Identification of genes that are involved in bursicon receptor mediated biological processes will provide tools for future studies on mechanisms of bursicon action. PMID:20457145

  9. High Content Analysis of Compositional Heterogeneities to Study GPCR Oligomerization

    DEFF Research Database (Denmark)

    Walsh, Samuel McEwen

    In this thesis I demonstrate how the natural compositional heterogeneities of synthetic and living cell model systems can be used to quantitate the mechanics of G-protein coupled receptor (GPCR) oligomerization with Förster resonance energy transfer (FRET). The thesis is structured around three a...

  10. An orphan viral TNF receptor superfamily member identified in lymphocystis disease virus.

    Science.gov (United States)

    Pontejo, Sergio M; Sánchez, Carolina; Martín, Rocío; Mulero, Victoriano; Alcami, Antonio; Alejo, Alí

    2013-06-07

    Lymphocystis disease virus (LCDV) is a large icosahedral dsDNA-containing virus of the Lymphocystivirus genus within the Iridoviridae family that can cause disease in more than 140 marine and freshwater fish species. While several isolates have been charcaterized and classified into distinct genotypes the complete genomic sequence is currently only available from two species, the LCDV-1, isolated from flounder (Platichtys flesus) in Europe and the LCDV-C, isolated from Japanese cultured flounder (Paralichthys olivaceus) in China. Analysis of the genome of LCDV-C showed it to encode a protein named LDVICp016 with similarities to the Tumour necrosis factor receptor (TNFR) superfamily with immunomodulatory potential. We have expressed and purified the recombinant protein LDVICp016 and screened for potential interaction partners using surface plasmon resonance. Commercially available human and mouse members of the TNF superfamily (TNFSF), along with a representative set of fish-derived TNFSF were tested.We have found the LDVICp016 protein to be secreted and we have identified a second viral TNFR encoded by ORF 095 of the same virus. None of the 42 tested proteins were found to interact with LDVICp016. We show that LDVICp016 is a secreted protein belonging to the TNF receptor family that may be part of a larger gene family in Lymphocystiviruses. While the ligand of this protein remains unknown, possibly due to the species specific nature of this interaction, further investigations into the potential role of this protein in the blockade of immune responses in its fish host are required.

  11. Pharmacogenomics of GPCR Drug Targets

    DEFF Research Database (Denmark)

    Hauser, Alexander Sebastian; Chavali, Sreenivas; Masuho, Ikuo

    2018-01-01

    Natural genetic variation in the human genome is a cause of individual differences in responses to medications and is an underappreciated burden on public health. Although 108 G-protein-coupled receptors (GPCRs) are the targets of 475 (∼34%) Food and Drug Administration (FDA)-approved drugs...... and account for a global sales volume of over 180 billion US dollars annually, the prevalence of genetic variation among GPCRs targeted by drugs is unknown. By analyzing data from 68,496 individuals, we find that GPCRs targeted by drugs show genetic variation within functional regions such as drug......- and effector-binding sites in the human population. We experimentally show that certain variants of μ-opioid and Cholecystokinin-A receptors could lead to altered or adverse drug response. By analyzing UK National Health Service drug prescription and sales data, we suggest that characterizing GPCR variants...

  12. Feeding and the rhodopsin family g-protein coupled receptors in nematodes and arthropods.

    Science.gov (United States)

    Cardoso, João C R; Félix, Rute C; Fonseca, Vera G; Power, Deborah M

    2012-01-01

    In vertebrates, receptors of the rhodopsin G-protein coupled superfamily (GPCRs) play an important role in the regulation of feeding and energy homeostasis and are activated by peptide hormones produced in the brain-gut axis. These peptides regulate appetite and energy expenditure by promoting or inhibiting food intake. Sequence and function homologs of human GPCRs involved in feeding exist in the nematode roundworm, Caenorhabditis elegans (C. elegans), and the arthropod fruit fly, Drosophila melanogaster (D. melanogaster), suggesting that the mechanisms that regulate food intake emerged early and have been conserved during metazoan radiation. Nematodes and arthropods are the most diverse and successful animal phyla on Earth. They can survive in a vast diversity of environments and have acquired distinct life styles and feeding strategies. The aim of the present review is to investigate if this diversity has affected the evolution of invertebrate GPCRs. Homologs of the C. elegans and D. melanogaster rhodopsin receptors were characterized in the genome of other nematodes and arthropods and receptor evolution compared. With the exception of bombesin receptors (BBR) that are absent from nematodes, a similar gene complement was found. In arthropods, rhodopsin GPCR evolution is characterized by species-specific gene duplications and deletions and in nematodes by gene expansions in species with a free-living stage and gene deletions in representatives of obligate parasitic taxa. Based upon variation in GPCR gene number and potentially divergent functions within phyla we hypothesize that life style and feeding diversity practiced by nematodes and arthropods was one factor that contributed to rhodopsin GPCR gene evolution. Understanding how the regulation of food intake has evolved in invertebrates will contribute to the development of novel drugs to control nematodes and arthropods and the pests and diseases that use them as vectors.

  13. Genetic polymorphisms of tumour necrosis factor receptor superfamily 1b and fas ligand are associated with clinical efficacy and/or acute severe infusion reactions to infliximab in Crohn's disease

    DEFF Research Database (Denmark)

    Steenholdt, C; Enevold, C; Ainsworth, M A

    2012-01-01

    Single nucleotide polymorphisms (SNPs) in TNF receptor superfamily (TNFRSF) 1A and 1B, and Fas ligand (FASLG) genes, have been associated with responsiveness to infliximab (IFX) in Crohn's disease.......Single nucleotide polymorphisms (SNPs) in TNF receptor superfamily (TNFRSF) 1A and 1B, and Fas ligand (FASLG) genes, have been associated with responsiveness to infliximab (IFX) in Crohn's disease....

  14. Feeding and the rhodopsin family G-Protein Coupled Receptors (GPCRs in nematodes and arthropods

    Directory of Open Access Journals (Sweden)

    Joao Carlos dos Reis Cardoso

    2012-12-01

    Full Text Available In vertebrates, receptors of the rhodopsin G-protein coupled superfamily (GPCRs play an important role in the regulation of feeding and energy homeostasis and are activated by peptide hormones produced in the brain-gut axis. These peptides regulate appetite and energy expenditure by promoting or inhibiting food intake. Sequence and function homologues of human GPCRs involved in feeding exist in the nematode roundworm, Caenorhabditis elegans (C. elegans and the arthropod fruit fly, Drosophila melanogaster (D. melanogaster, suggesting that the mechanisms that regulate food intake emerged early and have been conserved during metazoan radiation. Nematodes and arthropods are the most diverse and successful animal phyla on Earth. They can survive in a vast diversity of environments and have acquired distinct life styles and feeding strategies. The aim of the present review is to investigate if this diversity has affected the evolution of invertebrate GPCRs. Homologues of the C. elegans and D. melanogaster rhodopsin receptors were characterized in the genome of other nematodes and arthropods and receptor evolution compared. With the exception of bombesin receptors (BBR that are absent from nematodes, a similar gene complement was found. In arthropods, rhodopsin GPCR evolution is characterized by species-specific gene duplications and deletions and in nematodes by gene expansions in species with a free-living stage and gene deletions in representatives of obligate parasitic taxa. Based upon variation in GPCR gene number and potentially divergent functions within phyla we hypothesize that life style and feeding diversity practiced by nematodes and arthropods was one factor that contributed to rhodopsin GPCR gene evolution. Understanding how the regulation of food intake has evolved in invertebrates will contribute to the development of novel drugs to control nematodes and arthropods and the pests and diseases that use them as vectors.

  15. Regulation of GPCR-mediated smooth muscle contraction : implications for asthma and pulmonary hypertension

    NARCIS (Netherlands)

    Wright, D B; Tripathi, S; Sikarwar, A; Santosh, K T; Perez-Zoghbi, J; Ojo, O O; Irechukwu, N; Ward, J P T; Schaafsma, D

    Contractile G-protein-coupled receptors (GPCRs) have emerged as key regulators of smooth muscle contraction, both under healthy and diseased conditions. This brief review will discuss some key topics and novel insights regarding GPCR-mediated airway and vascular smooth muscle contraction as

  16. METHODS FOR RECOMBINANT EXPRESSION AND FUNCTIONAL CHARACTERIZATION OF HUMAN CANNABINOID RECEPTOR CB2

    Directory of Open Access Journals (Sweden)

    Alexei A. Yeliseev

    2013-03-01

    Full Text Available Cannabinoid receptor CB2 is a seven transmembrane-domain integral membrane protein that belongs to a large superfamily of G protein-coupled receptors (GPCR. CB2 is a part of the endocannabinoid system that plays vital role in regulation of immune response, inflammation, pain sensitivity, obesity and other physiological responses. Information about the structure and mechanisms of functioning of this receptor in cell membranes is essential for the rational development of specific pharmaceuticals. Here we review the methodology for recombinant expression, purification, stabilization and biochemical characterization of CB2 suitable for preparation of multi-milligram quantities of functionally active receptor. The biotechnological protocols include expression of the recombinant CB2 in E. coli cells as a fusion with the maltose binding protein, stabilization with a high affinity ligand and a derivative of cholesterol in detergent micelles, efficient purification by tandem affinity chromatography, and reconstitution of the receptor into lipid bilayers. The purified recombinant CB2 receptor is amenable to functional and structural studies including nuclear magnetic resonance spectroscopy and a wide range of biochemical and biophysical techniques.

  17. Trends in GPCR drug discovery: new agents, targets and indications

    DEFF Research Database (Denmark)

    Hauser, Alexander Sebastian; Gloriam, David E.; Attwood, Misty M.

    2017-01-01

    current trends across molecule types, drug targets and therapeutic indications, including showing that 475 drugs (~34% of all drugs approved by the US Food and Drug Administration (FDA)) act at 108 unique GPCRs. Approximately 321 agents are currently in clinical trials, of which ~20% target 66 potentially...... are also highly represented. The 224 (56%) non-olfactory GPCRs that have not yet been explored in clinical trials have broad untapped therapeutic potential, particularly in genetic and immune system disorders. Finally, we provide an interactive online resource to analyse and infer trends in GPCR drug......G protein-coupled receptors (GPCRs) are the most intensively studied drug targets, mostly due to their substantial involvement in human pathophysiology and their pharmacological tractability. Here, we report an up-to-date analysis of all GPCR drugs and agents in clinical trials, which reveals...

  18. Seeking Ligand Bias: Assessing GPCR Coupling to Beta-Arrestins for Drug Discovery

    OpenAIRE

    Bohn, Laura M.; McDonald, Patricia H.

    2010-01-01

    G protein-coupled receptors (GPCR) are the major site of action for endogenous hormones and neurotransmitters. Early drug discovery efforts focused on determining whether ligands could engage G protein coupling and subsequently activate or inhibit cognate “second messengers.” Gone are those simple days as we now realize that receptors can also couple βarrestins. As we delve into the complexity of ligand-directed signaling and receptosome scaffolds, we are faced with what may seem like endless...

  19. Superfamily of G-protein coupled receptors (GPCRs – extraordinary and outstanding success of evolution

    Directory of Open Access Journals (Sweden)

    Kazimierz Kochman

    2014-10-01

    Full Text Available The G protein-coupled receptors (GPCRs are considered as very diverse and also surprisingly successful structures during the whole evolutionary process, being capable of transducing the different forms of “information” within the cell and also between cells, such as different peptides, lipids, proteins, nucleotides, nucleosides, organic odorants and photons. Complex studies as well as two-dimensional crystallization of rhodopsin, their paradigm, led to the creation of a useful model having a common central core, consisting of seven transmembrane helical domains, which undergoes appropriate structural modification during activation and signal transduction. After the complete delineation of the human genome, which is the apogee of human scientific civilization and culture, it was possible to identify more than 800 human GPCR sequences and in parallel analyze 342 unique functional nonolfactory human GPCR sequences with phylogenetic analyses. These results support, with high bootstrap values, the existence of five main families, named by the authors glutamate, rhodopsin, adhesion, frizzle/taste2, and secretin, forming the GRAFS classification system. Positions of the GPCRs in chromosomal paralogous regions indicate the importance of tetraploidizations or local gene duplication events during their creation. Some families of GPCRs show, however, very little or no similarity in the sequence of amino acid chains. They utilize an enormous number of different domains to bind ligands and to activate the appropriate G-proteins. The delicate tuning of their coupling to G proteins is further regulated by splicing, RNA editing and phosphorylation. A number of GPCRs may also form homodimers or heterodimers with structurally different GPCRs and also with membrane-bound proteins having one transmembrane domain. It should also be stressed that not all GPCRs are strictly faithful to G proteins because growing evidence indicates that they can interact directly

  20. MicroRNA-34a promotes genomic instability by a broad suppression of genome maintenance mechanisms downstream of the oncogene KSHV-vGPCR.

    Science.gov (United States)

    Krause, Claudia J; Popp, Oliver; Thirunarayanan, Nanthakumar; Dittmar, Gunnar; Lipp, Martin; Müller, Gerd

    2016-03-01

    The Kaposi's sarcoma-associated herpesvirus (KSHV)-encoded chemokine receptor vGPCR acts as an oncogene in Kaposi's sarcomagenesis. Until now, the molecular mechanisms by which the vGPCR contributes to tumor development remain incompletely understood. Here, we show that the KSHV-vGPCR contributes to tumor progression through microRNA (miR)-34a-mediated induction of genomic instability. Large-scale analyses on the DNA, gene and protein level of cell lines derived from a mouse model of vGPCR-driven tumorigenesis revealed that a vGPCR-induced upregulation of miR-34a resulted in a broad suppression of genome maintenance genes. A knockdown of either the vGPCR or miR-34a largely restored the expression of these genes and confirmed miR-34a as a downstream effector of the KSHV-vGPCR that compromises genome maintenance mechanisms. This novel, protumorigenic role of miR-34a questions the use of miR-34a mimetics in cancer therapy as they could impair genome stability.

  1. Expression of TGF-beta superfamily growth factors, their receptors, the associated SMADs and antagonists in five isolated size-matched populations of pre-antral follicles from normal human ovaries

    DEFF Research Database (Denmark)

    Kristensen, Stine Gry; Andersen, Kasper; Clement, Christian Alexandro

    2014-01-01

    In mammals, members of the transforming growth factor-beta (TGF-β) superfamily are known to have key roles in the regulation of follicular growth and development. The aim of the study was to evaluate the expression of TGF-β superfamily growth factors, their receptors, downstream SMAD signalling m...... growth. Moreover, the presence of multiple TGF-β/BMP antagonists imply that certain growth factors are subjected to local regulation on different levels which address another important level of intraovarian regulation of follicle development in humans.......In mammals, members of the transforming growth factor-beta (TGF-β) superfamily are known to have key roles in the regulation of follicular growth and development. The aim of the study was to evaluate the expression of TGF-β superfamily growth factors, their receptors, downstream SMAD signalling...... molecules and TGF- β/BMP antagonists during early human folliculogenesis.Human preantral follicles were enzymatically isolated from surplus ovarian tissue obtained from women having ovarian cortical tissue frozen for fertility preservation. A total of 348 human preantral follicles, ranging from 40 to 200 µm...

  2. Inhibitory effects of two G protein-coupled receptor kinases on the cell surface expression and signaling of the human adrenomedullin receptor

    Energy Technology Data Exchange (ETDEWEB)

    Kuwasako, Kenji, E-mail: kuwasako@med.miyazaki-u.ac.jp [Frontier Science Research Center, University of Miyazaki, Miyazaki, 889-1692 (Japan); Sekiguchi, Toshio [Noto Marine Laboratory, Division of Marine Environmental Studies, Institute of Nature and Environmental Technology, Kanazawa University, Ishikawa, 927-0553 (Japan); Nagata, Sayaka [Division of Circulatory and Body Fluid Regulation, Faculty of Medicine, University of Miyazaki, Miyazaki, 889-1692 (Japan); Jiang, Danfeng; Hayashi, Hidetaka [Frontier Science Research Center, University of Miyazaki, Miyazaki, 889-1692 (Japan); Murakami, Manabu [Department of Pharmacology, Hirosaki University, Graduate School of Medicine, Hirosaki, 036-8562 (Japan); Hattori, Yuichi [Department of Molecular and Medical Pharmacology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama, 930-0194 (Japan); Kitamura, Kazuo [Division of Circulatory and Body Fluid Regulation, Faculty of Medicine, University of Miyazaki, Miyazaki, 889-1692 (Japan); Kato, Johji [Frontier Science Research Center, University of Miyazaki, Miyazaki, 889-1692 (Japan)

    2016-02-19

    Receptor activity-modifying protein 2 (RAMP2) enables the calcitonin receptor-like receptor (CLR, a family B GPCR) to form the type 1 adrenomedullin receptor (AM{sub 1} receptor). Here, we investigated the effects of the five non-visual GPCR kinases (GRKs 2 through 6) on the cell surface expression of the human (h)AM{sub 1} receptor by cotransfecting each of these GRKs into HEK-293 cells that stably expressed hRAMP2. Flow cytometric analysis revealed that when coexpressed with GRK4 or GRK5, the cell surface expression of the AM{sub 1} receptor was markedly decreased prior to stimulation with AM, thereby attenuating both the specific [{sup 125}I]AM binding and AM-induced cAMP production. These inhibitory effects of both GRKs were abolished by the replacement of the cytoplasmic C-terminal tail (C-tail) of CLR with that of the calcitonin receptor (a family B GPCR) or β{sub 2}-adrenergic receptor (a family A GPCR). Among the sequentially truncated CLR C-tail mutants, those lacking the five residues 449–453 (Ser-Phe-Ser-Asn-Ser) abolished the inhibition of the cell surface expression of CLR via the overexpression of GRK4 or GRK5. Thus, we provided new insight into the function of GRKs in agonist-unstimulated GPCR trafficking using a recombinant AM{sub 1} receptor and further determined the region of the CLR C-tail responsible for this GRK function. - Highlights: • We discovered a novel function of GRKs in GPCR trafficking using human CLR/RAMP2. • GRKs 4 and 5 markedly inhibited the cell surface expression of human CLR/RAMP2. • Both GRKs exhibited highly significant receptor signaling inhibition. • Five residues of the C-terminal tail of CLR govern this function of GRKs.

  3. Inhibitory effects of two G protein-coupled receptor kinases on the cell surface expression and signaling of the human adrenomedullin receptor

    International Nuclear Information System (INIS)

    Kuwasako, Kenji; Sekiguchi, Toshio; Nagata, Sayaka; Jiang, Danfeng; Hayashi, Hidetaka; Murakami, Manabu; Hattori, Yuichi; Kitamura, Kazuo; Kato, Johji

    2016-01-01

    Receptor activity-modifying protein 2 (RAMP2) enables the calcitonin receptor-like receptor (CLR, a family B GPCR) to form the type 1 adrenomedullin receptor (AM_1 receptor). Here, we investigated the effects of the five non-visual GPCR kinases (GRKs 2 through 6) on the cell surface expression of the human (h)AM_1 receptor by cotransfecting each of these GRKs into HEK-293 cells that stably expressed hRAMP2. Flow cytometric analysis revealed that when coexpressed with GRK4 or GRK5, the cell surface expression of the AM_1 receptor was markedly decreased prior to stimulation with AM, thereby attenuating both the specific ["1"2"5I]AM binding and AM-induced cAMP production. These inhibitory effects of both GRKs were abolished by the replacement of the cytoplasmic C-terminal tail (C-tail) of CLR with that of the calcitonin receptor (a family B GPCR) or β_2-adrenergic receptor (a family A GPCR). Among the sequentially truncated CLR C-tail mutants, those lacking the five residues 449–453 (Ser-Phe-Ser-Asn-Ser) abolished the inhibition of the cell surface expression of CLR via the overexpression of GRK4 or GRK5. Thus, we provided new insight into the function of GRKs in agonist-unstimulated GPCR trafficking using a recombinant AM_1 receptor and further determined the region of the CLR C-tail responsible for this GRK function. - Highlights: • We discovered a novel function of GRKs in GPCR trafficking using human CLR/RAMP2. • GRKs 4 and 5 markedly inhibited the cell surface expression of human CLR/RAMP2. • Both GRKs exhibited highly significant receptor signaling inhibition. • Five residues of the C-terminal tail of CLR govern this function of GRKs.

  4. A new inhibitor of the ?-arrestin/AP2 endocytic complex reveals interplay between GPCR internalization and signalling

    OpenAIRE

    Beautrait, Alexandre; Paradis, Justine S.; Zimmerman, Brandon; Giubilaro, Jenna; Nikolajev, Ljiljana; Armando, Sylvain; Kobayashi, Hiroyuki; Yamani, Lama; Namkung, Yoon; Heydenreich, Franziska M.; Khoury, Etienne; Audet, Martin; Roux, Philippe P.; Veprintsev, Dmitry B.; Laporte, St?phane A.

    2017-01-01

    In addition to G protein-coupled receptor (GPCR) desensitization and endocytosis, ?-arrestin recruitment to ligand-stimulated GPCRs promotes non-canonical signalling cascades. Distinguishing the respective contributions of ?-arrestin recruitment to the receptor and ?-arrestin-promoted endocytosis in propagating receptor signalling has been limited by the lack of selective analytical tools. Here, using a combination of virtual screening and cell-based assays, we have identified a small molecul...

  5. A knockout mutation of a constitutive GPCR in Tetrahymena decreases both G-protein activity and chemoattraction.

    Directory of Open Access Journals (Sweden)

    Thomas J Lampert

    Full Text Available Although G-protein coupled receptors (GPCRs are a common element in many chemosensory transduction pathways in eukaryotic cells, no GPCR or regulated G-protein activity has yet been shown in any ciliate. To study the possible role for a GPCR in the chemoresponses of the ciliate Tetrahymena, we have generated a number of macronuclear gene knockouts of putative GPCRs found in the Tetrahymena Genome database. One of these knockout mutants, called G6, is a complete knockout of a gene that we call GPCR6 (TTHERM_00925490. Based on sequence comparisons, the Gpcr6p protein belongs to the Rhodopsin Family of GPCRs. Notably, Gpcr6p shares highest amino acid sequence homologies to GPCRs from Paramecium and several plants. One of the phenotypes of the G6 mutant is a decreased responsiveness to the depolarizing ions Ba²⁺ and K⁺, suggesting a decrease in basal excitability (decrease in Ca²⁺ channel activity. The other major phenotype of G6 is a loss of chemoattraction to lysophosphatidic acid (LPA and proteose peptone (PP, two known chemoattractants in Tetrahymena. Using microsomal [³⁵S]GTPγS binding assays, we found that wild-type (CU427 have a prominent basal G-protein activity. This activity is decreased to the same level by pertussis toxin (a G-protein inhibitor, addition of chemoattractants, or the G6 mutant. Since the basal G-protein activity is decreased by the GPCR6 knockout, it is likely that this gene codes for a constitutively active GPCR in Tetrahymena. We propose that chemoattractants like LPA and PP cause attraction in Tetrahymena by decreasing the basal G-protein stimulating activity of Gpcr6p. This leads to decreased excitability in wild-type and longer runs of smooth forward swimming (less interrupted by direction changes towards the attractant. Therefore, these attractants may work as inverse agonists through the constitutively active Gpcr6p coupled to a pertussis-sensitive G-protein.

  6. Ligand binding modes from low resolution GPCR models and mutagenesis: chicken bitter taste receptor as a test-case.

    Science.gov (United States)

    Di Pizio, Antonella; Kruetzfeldt, Louisa-Marie; Cheled-Shoval, Shira; Meyerhof, Wolfgang; Behrens, Maik; Niv, Masha Y

    2017-08-15

    Bitter taste is one of the basic taste modalities, warning against consuming potential poisons. Bitter compounds activate members of the bitter taste receptor (Tas2r) subfamily of G protein-coupled receptors (GPCRs). The number of functional Tas2rs is species-dependent. Chickens represent an intriguing minimalistic model, because they detect the bitter taste of structurally different molecules with merely three bitter taste receptor subtypes. We investigated the binding modes of several known agonists of a representative chicken bitter taste receptor, ggTas2r1. Because of low sequence similarity between ggTas2r1 and crystallized GPCRs (~10% identity, ~30% similarity at most), the combination of computational approaches with site-directed mutagenesis was used to characterize the agonist-bound conformation of ggTas2r1 binding site between TMs 3, 5, 6 and 7. We found that the ligand interactions with N93 in TM3 and/or N247 in TM5, combined with hydrophobic contacts, are typically involved in agonist recognition. Next, the ggTas2r1 structural model was successfully used to identify three quinine analogues (epiquinidine, ethylhydrocupreine, quinidine) as new ggTas2r1 agonists. The integrated approach validated here may be applicable to additional cases where the sequence identity of the GPCR of interest and the existing experimental structures is low.

  7. Structural Probing and Molecular Modeling of the A₃ Adenosine Receptor: A Focus on Agonist Binding.

    Science.gov (United States)

    Ciancetta, Antonella; Jacobson, Kenneth A

    2017-03-11

    Adenosine is an endogenous modulator exerting its functions through the activation of four adenosine receptor (AR) subtypes, termed A₁, A 2A , A 2B and A₃, which belong to the G protein-coupled receptor (GPCR) superfamily. The human A₃AR (hA₃AR) subtype is implicated in several cytoprotective functions. Therefore, hA₃AR modulators, and in particular agonists, are sought for their potential application as anti-inflammatory, anticancer, and cardioprotective agents. Structure-based molecular modeling techniques have been applied over the years to rationalize the structure-activity relationships (SARs) of newly emerged A₃AR ligands, guide the subsequent lead optimization, and interpret site-directed mutagenesis (SDM) data from a molecular perspective. In this review, we showcase selected modeling-based and guided strategies that were applied to elucidate the binding of agonists to the A₃AR and discuss the challenges associated with an accurate prediction of the receptor extracellular vestibule through homology modeling from the available X-ray templates.

  8. Interrogating the Spatiotemporal Landscape of Neuromodulatory GPCR Signaling by Real-Time Imaging of cAMP in Intact Neurons and Circuits

    Directory of Open Access Journals (Sweden)

    Brian S. Muntean

    2018-01-01

    Full Text Available Summary: Modulation of neuronal circuits is key to information processing in the brain. The majority of neuromodulators exert their effects by activating G-protein-coupled receptors (GPCRs that control the production of second messengers directly impacting cellular physiology. How numerous GPCRs integrate neuromodulatory inputs while accommodating diversity of incoming signals is poorly understood. In this study, we develop an in vivo tool and analytical suite for analyzing GPCR responses by monitoring the dynamics of a key second messenger, cyclic AMP (cAMP, with excellent quantitative and spatiotemporal resolution in various neurons. Using this imaging approach in combination with CRISPR/Cas9 editing and optogenetics, we interrogate neuromodulatory mechanisms of defined populations of neurons in an intact mesolimbic reward circuit and describe how individual inputs generate discrete second-messenger signatures in a cell- and receptor-specific fashion. This offers a resource for studying native neuronal GPCR signaling in real time. : Muntean et al. develop an in vivo reagent to study processing of neurotransmitter GPCR signals by monitoring real-time dynamics of cAMP responses. They demonstrate application of this approach, in combination with CRISPR/Cas9 gene editing and optogenetics, to interrogate the functional organization of a striatal circuit. Keywords: cAMP, GPCR, neuromodulation, dopamine, striatum, imaging, optogenetics

  9. Molecular evolution of the insect chemoreceptor gene superfamily in Drosophila melanogaster

    Science.gov (United States)

    Robertson, Hugh M.; Warr, Coral G.; Carlson, John R.

    2003-01-01

    The insect chemoreceptor superfamily in Drosophila melanogaster is predicted to consist of 62 odorant receptor (Or) and 68 gustatory receptor (Gr) proteins, encoded by families of 60 Or and 60 Gr genes through alternative splicing. We include two previously undescribed Or genes and two previously undescribed Gr genes; two previously predicted Or genes are shown to be alternative splice forms. Three polymorphic pseudogenes and one highly defective pseudogene are recognized. Phylogenetic analysis reveals deep branches connecting multiple highly divergent clades within the Gr family, and the Or family appears to be a single highly expanded lineage within the superfamily. The genes are spread throughout the Drosophila genome, with some relatively recently diverged genes still clustered in the genome. The Gr5a gene on the X chromosome, which encodes a receptor for the sugar trehalose, has transposed from one such tandem cluster of six genes at cytological location 64, as has Gr61a, and all eight of these receptors might bind sugars. Analysis of intron evolution suggests that the common ancestor consisted of a long N-terminal exon encoding transmembrane domains 1-5 followed by three exons encoding transmembrane domains 6-7. As many as 57 additional introns have been acquired idiosyncratically during the evolution of the superfamily, whereas the ancestral introns and some of the older idiosyncratic introns have been lost at least 48 times independently. Altogether, these patterns of molecular evolution suggest that this is an ancient superfamily of chemoreceptors, probably dating back at least to the origin of the arthropods. PMID:14608037

  10. The G protein-coupled receptor subset of the dog genome is more similar to that in humans than rodents.

    Science.gov (United States)

    Haitina, Tatjana; Fredriksson, Robert; Foord, Steven M; Schiöth, Helgi B; Gloriam, David E

    2009-01-15

    The dog is an important model organism and it is considered to be closer to humans than rodents regarding metabolism and responses to drugs. The close relationship between humans and dogs over many centuries has lead to the diversity of the canine species, important genetic discoveries and an appreciation of the effects of old age in another species. The superfamily of G protein-coupled receptors (GPCRs) is one of the largest gene families in most mammals and the most exploited in terms of drug discovery. An accurate comparison of the GPCR repertoires in dog and human is valuable for the prediction of functional similarities and differences between the species. We searched the dog genome for non-olfactory GPCRs and obtained 353 full-length GPCR gene sequences, 18 incomplete sequences and 13 pseudogenes. We established relationships between human, dog, rat and mouse GPCRs resolving orthologous pairs and species-specific duplicates. We found that 12 dog GPCR genes are missing in humans while 24 human GPCR genes are not part of the dog GPCR repertoire. There is a higher number of orthologous pairs between dog and human that are conserved as compared with either mouse or rat. In almost all cases the differences observed between the dog and human genomes coincide with other variations in the rodent species. Several GPCR gene expansions characteristic for rodents are not found in dog. The repertoire of dog non-olfactory GPCRs is more similar to the repertoire in humans as compared with the one in rodents. The comparison of the dog, human and rodent repertoires revealed several examples of species-specific gene duplications and deletions. This information is useful in the selection of model organisms for pharmacological experiments.

  11. The G protein-coupled receptor subset of the dog genome is more similar to that in humans than rodents

    Directory of Open Access Journals (Sweden)

    Schiöth Helgi B

    2009-01-01

    Full Text Available Abstract Background The dog is an important model organism and it is considered to be closer to humans than rodents regarding metabolism and responses to drugs. The close relationship between humans and dogs over many centuries has lead to the diversity of the canine species, important genetic discoveries and an appreciation of the effects of old age in another species. The superfamily of G protein-coupled receptors (GPCRs is one of the largest gene families in most mammals and the most exploited in terms of drug discovery. An accurate comparison of the GPCR repertoires in dog and human is valuable for the prediction of functional similarities and differences between the species. Results We searched the dog genome for non-olfactory GPCRs and obtained 353 full-length GPCR gene sequences, 18 incomplete sequences and 13 pseudogenes. We established relationships between human, dog, rat and mouse GPCRs resolving orthologous pairs and species-specific duplicates. We found that 12 dog GPCR genes are missing in humans while 24 human GPCR genes are not part of the dog GPCR repertoire. There is a higher number of orthologous pairs between dog and human that are conserved as compared with either mouse or rat. In almost all cases the differences observed between the dog and human genomes coincide with other variations in the rodent species. Several GPCR gene expansions characteristic for rodents are not found in dog. Conclusion The repertoire of dog non-olfactory GPCRs is more similar to the repertoire in humans as compared with the one in rodents. The comparison of the dog, human and rodent repertoires revealed several examples of species-specific gene duplications and deletions. This information is useful in the selection of model organisms for pharmacological experiments.

  12. Molecular and Functional Characterization of Mouse S5D-SRCRB: A New Group B Member of the Scavenger Receptor Cysteine-Rich Superfamily

    DEFF Research Database (Denmark)

    Miró-Julià, Cristina; Roselló, Sandra; Martínez, Vanesa G

    2011-01-01

    The scavenger receptor cysteine-rich superfamily (SRCR-SF) members are transmembrane and/or secreted receptors exhibiting one or several repeats of a cysteine-rich protein module of ∼100 aa, named scavenger receptor cysteine-rich (SRCR). Two types of SRCR domains (A or B) have been reported, which...... differ in the number of coding exons and intradomain cysteines. Although no unifying function has been reported for SRCR-SF members, recognition of pathogen-associated molecular patterns (PAMPs) was recently shown for some of them. In this article, we report the structural and functional characterization...

  13. Oligomerization of G protein-coupled receptors: computational methods.

    Science.gov (United States)

    Selent, J; Kaczor, A A

    2011-01-01

    Recent research has unveiled the complexity of mechanisms involved in G protein-coupled receptor (GPCR) functioning in which receptor dimerization/oligomerization may play an important role. Although the first high-resolution X-ray structure for a likely functional chemokine receptor dimer has been deposited in the Protein Data Bank, the interactions and mechanisms of dimer formation are not yet fully understood. In this respect, computational methods play a key role for predicting accurate GPCR complexes. This review outlines computational approaches focusing on sequence- and structure-based methodologies as well as discusses their advantages and limitations. Sequence-based approaches that search for possible protein-protein interfaces in GPCR complexes have been applied with success in several studies, but did not yield always consistent results. Structure-based methodologies are a potent complement to sequence-based approaches. For instance, protein-protein docking is a valuable method especially when guided by experimental constraints. Some disadvantages like limited receptor flexibility and non-consideration of the membrane environment have to be taken into account. Molecular dynamics simulation can overcome these drawbacks giving a detailed description of conformational changes in a native-like membrane. Successful prediction of GPCR complexes using computational approaches combined with experimental efforts may help to understand the role of dimeric/oligomeric GPCR complexes for fine-tuning receptor signaling. Moreover, since such GPCR complexes have attracted interest as potential drug target for diverse diseases, unveiling molecular determinants of dimerization/oligomerization can provide important implications for drug discovery.

  14. Pathophysiological consequences of receptor mistraffic: Tales from the platelet P2Y12 receptor.

    Science.gov (United States)

    Cunningham, Margaret R; Aungraheeta, Riyaad; Mundell, Stuart J

    2017-07-05

    Genetic variations in G protein-coupled receptor (GPCR) genes can disrupt receptor function in a wide variety of human genetic diseases, including platelet bleeding disorders. Platelets are critical for haemostasis with inappropriate platelet activation leading to the development of arterial thrombosis, which can result in heart attack and stroke whilst decreased platelet activity is associated with an increased risk of bleeding. GPCRs expressed on the surface of platelets play key roles in regulating platelet activity and therefore function. Receptors include purinergic receptors (P2Y 1 and P2Y 12 ), proteinase-activated receptor (PAR1 and PAR4) and thromboxane receptors (TPα), among others. Pharmacological blockade of these receptors forms a powerful therapeutic tool in the treatment and prevention of arterial thrombosis. With the advance of genomic technologies, there has been a substantial increase in the identification of naturally occurring rare and common GPCR variants. These variants include single-nucleotide polymorphisms (SNPs) and insertion or deletions that have the potential to alter GPCR expression or function. A number of defects in platelet GPCRs that disrupt receptor function have now been characterized in patients with mild bleeding disorders. This review will focus on rare, function-disrupting variants of platelet GPCRs with particular emphasis upon mutations in the P2Y 12 receptor gene that affect receptor traffic to modulate platelet function. Further this review will outline how the identification and characterization of function-disrupting GPCR mutations provides an essential link in translating our detailed understanding of receptor traffic and function in cell line studies into relevant human biological systems. Copyright © 2017. Published by Elsevier B.V.

  15. GPCR-I-TASSER: A Hybrid Approach to G Protein-Coupled Receptor Structure Modeling and the Application to the Human Genome.

    Science.gov (United States)

    Zhang, Jian; Yang, Jianyi; Jang, Richard; Zhang, Yang

    2015-08-04

    Experimental structure determination remains difficult for G protein-coupled receptors (GPCRs). We propose a new hybrid protocol to construct GPCR structure models that integrates experimental mutagenesis data with ab initio transmembrane (TM) helix assembly simulations. The method was tested on 24 known GPCRs where the ab initio TM-helix assembly procedure constructed the correct fold for 20 cases. When combined with weak homology and sparse mutagenesis restraints, the method generated correct folds for all the tested cases with an average Cα root-mean-square deviation 2.4 Å in the TM regions. The new hybrid protocol was applied to model all 1,026 GPCRs in the human genome, where 923 have a high confidence score and are expected to have correct folds; these contain many pharmaceutically important families with no previously solved structures, including Trace amine, Prostanoids, Releasing hormones, Melanocortins, Vasopressin, and Neuropeptide Y receptors. The results demonstrate new progress on genome-wide structure modeling of TM proteins. Copyright © 2015 Elsevier Ltd. All rights reserved.

  16. Regulation of G Protein-Coupled Receptors by Ubiquitination

    Directory of Open Access Journals (Sweden)

    Kamila Skieterska

    2017-04-01

    Full Text Available G protein-coupled receptors (GPCRs comprise the largest family of membrane receptors that control many cellular processes and consequently often serve as drug targets. These receptors undergo a strict regulation by mechanisms such as internalization and desensitization, which are strongly influenced by posttranslational modifications. Ubiquitination is a posttranslational modification with a broad range of functions that is currently gaining increased appreciation as a regulator of GPCR activity. The role of ubiquitination in directing GPCRs for lysosomal degradation has already been well-established. Furthermore, this modification can also play a role in targeting membrane and endoplasmic reticulum-associated receptors to the proteasome. Most recently, ubiquitination was also shown to be involved in GPCR signaling. In this review, we present current knowledge on the molecular basis of GPCR regulation by ubiquitination, and highlight the importance of E3 ubiquitin ligases, deubiquitinating enzymes and β-arrestins. Finally, we discuss classical and newly-discovered functions of ubiquitination in controlling GPCR activity.

  17. The systematic annotation of the three main GPCR families in Reactome.

    Science.gov (United States)

    Jassal, Bijay; Jupe, Steven; Caudy, Michael; Birney, Ewan; Stein, Lincoln; Hermjakob, Henning; D'Eustachio, Peter

    2010-07-29

    Reactome is an open-source, freely available database of human biological pathways and processes. A major goal of our work is to provide an integrated view of cellular signalling processes that spans from ligand-receptor interactions to molecular readouts at the level of metabolic and transcriptional events. To this end, we have built the first catalogue of all human G protein-coupled receptors (GPCRs) known to bind endogenous or natural ligands. The UniProt database has records for 797 proteins classified as GPCRs and sorted into families A/1, B/2 and C/3 on the basis of amino acid sequence. To these records we have added details from the IUPHAR database and our own manual curation of relevant literature to create reactions in which 563 GPCRs bind ligands and also interact with specific G-proteins to initiate signalling cascades. We believe the remaining 234 GPCRs are true orphans. The Reactome GPCR pathway can be viewed as a detailed interactive diagram and can be exported in many forms. It provides a template for the orthology-based inference of GPCR reactions for diverse model organism species, and can be overlaid with protein-protein interaction and gene expression datasets to facilitate overrepresentation studies and other forms of pathway analysis. Database URL: http://www.reactome.org.

  18. Mutations in the C-terminal region affect subcellular localization of crucian carp herpesvirus (CaHV) GPCR.

    Science.gov (United States)

    Wang, Jun; Gui, Lang; Chen, Zong-Yan; Zhang, Qi-Ya

    2016-08-01

    G protein-coupled receptors (GPCRs) are known as seven transmembrane domain receptors and consequently can mediate diverse biological functions via regulation of their subcellular localization. Crucian carp herpesvirus (CaHV) was recently isolated from infected fish with acute gill hemorrhage. CaHV GPCR of 349 amino acids (aa) was identified based on amino acid identity. A series of variants with truncation/deletion/substitution mutation in the C-terminal (aa 315-349) were constructed and expressed in fathead minnow (FHM) cells. The roles of three key C-terminal regions in subcellular localization of CaHV GPCR were determined. Lysine-315 (K-315) directed the aggregation of the protein preferentially at the nuclear side. Predicted N-myristoylation site (GGGWTR, aa 335-340) was responsible for punctate distribution in periplasm or throughout the cytoplasm. Predicted phosphorylation site (SSR, aa 327-329) and GGGWTR together determined the punctate distribution in cytoplasm. Detection of organelles localization by specific markers showed that the protein retaining K-315 colocalized with the Golgi apparatus. These experiments provided first evidence that different mutations of CaHV GPCR C-terminals have different affects on the subcellular localization of fish herpesvirus-encoded GPCRs. The study provided valuable information and new insights into the precise interactions between herpesvirus and fish cells, and could also provide useful targets for antiviral agents in aquaculture.

  19. G Protein-Coupled Receptors in Cancer

    Directory of Open Access Journals (Sweden)

    Rachel Bar-Shavit

    2016-08-01

    Full Text Available Despite the fact that G protein-coupled receptors (GPCRs are the largest signal-conveying receptor family and mediate many physiological processes, their role in tumor biology is underappreciated. Numerous lines of evidence now associate GPCRs and their downstream signaling targets in cancer growth and development. Indeed, GPCRs control many features of tumorigenesis, including immune cell-mediated functions, proliferation, invasion and survival at the secondary site. Technological advances have further substantiated GPCR modifications in human tumors. Among these are point mutations, gene overexpression, GPCR silencing by promoter methylation and the number of gene copies. At this point, it is imperative to elucidate specific signaling pathways of “cancer driver” GPCRs. Emerging data on GPCR biology point to functional selectivity and “biased agonism”; hence, there is a diminishing enthusiasm for the concept of “one drug per GPCR target” and increasing interest in the identification of several drug options. Therefore, determining the appropriate context-dependent conformation of a functional GPCR as well as the contribution of GPCR alterations to cancer development remain significant challenges for the discovery of dominant cancer genes and the development of targeted therapeutics.

  20. Criteria for confirming sequence periodicity identified by Fourier transform analysis: application to GCR2, a candidate plant GPCR?

    Science.gov (United States)

    Illingworth, Christopher J R; Parkes, Kevin E; Snell, Christopher R; Mullineaux, Philip M; Reynolds, Christopher A

    2008-03-01

    Methods to determine periodicity in protein sequences are useful for inferring function. Fourier transformation is one approach but care is required to ensure the periodicity is genuine. Here we have shown that empirically-derived statistical tables can be used as a measure of significance. Genuine protein sequences data rather than randomly generated sequences were used as the statistical backdrop. The method has been applied to G-protein coupled receptor (GPCR) sequences, by Fourier transformation of hydrophobicity values, codon frequencies and the extent of over-representation of codon pairs; the latter being related to translational step times. Genuine periodicity was observed in the hydrophobicity whereas the apparent periodicity (as inferred from previously reported measures) in the translation step times was not validated statistically. GCR2 has recently been proposed as the plant GPCR receptor for the hormone abscisic acid. It has homology to the Lanthionine synthetase C-like family of proteins, an observation confirmed by fold recognition. Application of the Fourier transform algorithm to the GCR2 family revealed strongly predicted seven fold periodicity in hydrophobicity, suggesting why GCR2 has been reported to be a GPCR, despite negative indications in most transmembrane prediction algorithms. The underlying multiple sequence alignment, also required for the Fourier transform analysis of periodicity, indicated that the hydrophobic regions around the 7 GXXG motifs commence near the C-terminal end of each of the 7 inner helices of the alpha-toroid and continue to the N-terminal region of the helix. The results clearly explain why GCR2 has been understandably but erroneously predicted to be a GPCR.

  1. A molecular pharmacologist's guide to G protein-coupled receptor crystallography

    NARCIS (Netherlands)

    Piscitelli, Chayne L.; Kean, James; de Graaf, C.; Deupi, Xavier

    2015-01-01

    G protein-coupled receptor (GPCR) structural biology has progressed dramatically in the last decade. There are now over 120 GPCR crystal structures deposited in the Protein Data Bank of 32 different receptors from families scattered across the phylogenetic tree, including class B, C, and Frizzled

  2. Calcitonin and calcitonin receptor-like receptors: common themes with family B GPCRs?

    Science.gov (United States)

    Barwell, James; Gingell, Joseph J; Watkins, Harriet A; Archbold, Julia K; Poyner, David R; Hay, Debbie L

    2012-05-01

    The calcitonin receptor (CTR) and calcitonin receptor-like receptor (CLR) are two of the 15 human family B (or Secretin-like) GPCRs. CTR and CLR are of considerable biological interest as their pharmacology is moulded by interactions with receptor activity-modifying proteins. They also have therapeutic relevance for many conditions, such as osteoporosis, diabetes, obesity, lymphatic insufficiency, migraine and cardiovascular disease. In light of recent advances in understanding ligand docking and receptor activation in both the family as a whole and in CLR and CTR specifically, this review reflects how applicable general family B GPCR themes are to these two idiosyncratic receptors. We review the main functional domains of the receptors; the N-terminal extracellular domain, the juxtamembrane domain and ligand interface, the transmembrane domain and the intracellular C-terminal domain. Structural and functional findings from the CLR and CTR along with other family B GPCRs are critically appraised to gain insight into how these domains may function. The ability for CTR and CLR to interact with receptor activity-modifying proteins adds another level of sophistication to these receptor systems but means careful consideration is needed when trying to apply generic GPCR principles. This review encapsulates current thinking in the realm of family B GPCR research by highlighting both conflicting and recurring themes and how such findings relate to two unusual but important receptors, CTR and CLR. © 2011 The Authors. British Journal of Pharmacology © 2011 The British Pharmacological Society.

  3. iGPCR-drug: a web server for predicting interaction between GPCRs and drugs in cellular networking.

    Directory of Open Access Journals (Sweden)

    Xuan Xiao

    Full Text Available Involved in many diseases such as cancer, diabetes, neurodegenerative, inflammatory and respiratory disorders, G-protein-coupled receptors (GPCRs are among the most frequent targets of therapeutic drugs. It is time-consuming and expensive to determine whether a drug and a GPCR are to interact with each other in a cellular network purely by means of experimental techniques. Although some computational methods were developed in this regard based on the knowledge of the 3D (dimensional structure of protein, unfortunately their usage is quite limited because the 3D structures for most GPCRs are still unknown. To overcome the situation, a sequence-based classifier, called "iGPCR-drug", was developed to predict the interactions between GPCRs and drugs in cellular networking. In the predictor, the drug compound is formulated by a 2D (dimensional fingerprint via a 256D vector, GPCR by the PseAAC (pseudo amino acid composition generated with the grey model theory, and the prediction engine is operated by the fuzzy K-nearest neighbour algorithm. Moreover, a user-friendly web-server for iGPCR-drug was established at http://www.jci-bioinfo.cn/iGPCR-Drug/. For the convenience of most experimental scientists, a step-by-step guide is provided on how to use the web-server to get the desired results without the need to follow the complicated math equations presented in this paper just for its integrity. The overall success rate achieved by iGPCR-drug via the jackknife test was 85.5%, which is remarkably higher than the rate by the existing peer method developed in 2010 although no web server was ever established for it. It is anticipated that iGPCR-Drug may become a useful high throughput tool for both basic research and drug development, and that the approach presented here can also be extended to study other drug - target interaction networks.

  4. Structural Probing and Molecular Modeling of the A3 Adenosine Receptor: A Focus on Agonist Binding

    Science.gov (United States)

    Ciancetta, Antonella; Jacobson, Kenneth A.

    2017-01-01

    Adenosine is an endogenous modulator exerting its functions through the activation of four adenosine receptor (AR) subtypes, termed A1, A2A, A2B and A3, which belong to the G protein-coupled receptor (GPCR) superfamily. The human A3AR (hA3AR) subtype is implicated in several cytoprotective functions. Therefore, hA3AR modulators, and in particular agonists, are sought for their potential application as anti-inflammatory, anticancer, and cardioprotective agents. Structure-based molecular modeling techniques have been applied over the years to rationalize the structure-activity relationships (SARs) of newly emerged A3AR ligands, guide the subsequent lead optimization, and interpret site-directed mutagenesis (SDM) data from a molecular perspective. In this review, we showcase selected modeling-based and guided strategies that were applied to elucidate the binding of agonists to the A3AR and discuss the challenges associated with an accurate prediction of the receptor extracellular vestibule through homology modeling from the available X-ray templates. PMID:28287473

  5. Structural Probing and Molecular Modeling of the A3 Adenosine Receptor: A Focus on Agonist Binding

    Directory of Open Access Journals (Sweden)

    Antonella Ciancetta

    2017-03-01

    Full Text Available Adenosine is an endogenous modulator exerting its functions through the activation of four adenosine receptor (AR subtypes, termed A1, A2A, A2B and A3, which belong to the G protein-coupled receptor (GPCR superfamily. The human A3AR (hA3AR subtype is implicated in several cytoprotective functions. Therefore, hA3AR modulators, and in particular agonists, are sought for their potential application as anti-inflammatory, anticancer, and cardioprotective agents. Structure-based molecular modeling techniques have been applied over the years to rationalize the structure–activity relationships (SARs of newly emerged A3AR ligands, guide the subsequent lead optimization, and interpret site-directed mutagenesis (SDM data from a molecular perspective. In this review, we showcase selected modeling-based and guided strategies that were applied to elucidate the binding of agonists to the A3AR and discuss the challenges associated with an accurate prediction of the receptor extracellular vestibule through homology modeling from the available X-ray templates.

  6. Platforms for the identification of GPCR targets, and of orthosteric and allosteric modulators.

    Science.gov (United States)

    Franco, Rafael; Canela, Enric I; Casado, Vicent; Ferre, Sergi

    2010-04-01

    The review provides a summary of old and new approaches for GPCR target identification and for the screening of molecules acting on GPCR targets. The new findings in the field are presented as well as an opinion about how these developments may help GPCR drug discovery. Importance in the field: GPCRs have been the most useful family of proteins in terms of targets for drug discovery. The expectations for GPCR target identification and discovery of new drugs acting on 'old' or 'new' GPCR targets are very high. Given the fact that the pace at which new 'GPCR drugs' appear in the market is decreasing and since the new developments in the field are not being translated into drug discovery there is a need to review the field from a critical perspective. To overcome the limitation of the old approaches used in GPCR target identification and drugs discovery new approaches are required. In particular successful approaches in GPCR drug discovery should take into account that the real GPCR targets for a given disease are not GPCR monomers but GPCR heteromers. The reader will gain an overview of the strategies currently used and their pros and cons. The reader will also understand that new strategies may help in accelerating the access of GPCR into the market, and also notice that successful strategies should take advantage of the new findings in the field of GPCRs.

  7. Rare platelet GPCR variants: what can we learn?

    Science.gov (United States)

    Nisar, S P; Jones, M L; Cunningham, M R; Mumford, A D; Mundell, S J

    2015-07-01

    Platelet-expressed GPCRs are critical regulators of platelet function. Pharmacological blockade of these receptors forms a powerful therapeutic tool in the treatment and prevention of arterial thrombosis associated with coronary atherosclerosis and ischaemic stroke. However, anti-thrombotic drug therapy is associated with high inter-patient variability in therapeutic response and adverse bleeding side effects. In order to optimize the use of existing anti-platelet drugs and to develop new therapies, more detailed knowledge is required relating to the molecular mechanisms that regulate GPCR and therefore platelet function. One approach has been to identify rare, function-disrupting mutations within key platelet proteins in patients with bleeding disorders. In this review, we describe how an integrated functional genomics strategy has contributed important structure-function information about platelet GPCRs with specific emphasis upon purinergic and thromboxane A2 receptors. We also discuss the potential implications these findings have for pharmacotherapy and for understanding the molecular basis of mild bleeding disorders. © 2014 The British Pharmacological Society.

  8. Molecular modeling of ligand-receptor interactions in the OR5 olfactory receptor.

    Science.gov (United States)

    Singer, M S; Shepherd, G M

    1994-06-02

    Olfactory receptors belong to the superfamily of seven transmembrane domain, G protein-coupled receptors. In order to begin analysis of mechanisms of receptor activation, a computer model of the OR5 olfactory receptor has been constructed and compared with other members of this superfamily. We have tested docking of the odor molecule lyral, which is known to activate the OR5 receptor. The results point to specific ligand-binding residues on helices III through VII that form a binding pocket in the receptor. Some of these residues occupy sequence positions identical to ligand-binding residues conserved among other superfamily members. The results provide new insights into possible molecular mechanisms of odor recognition and suggest hypotheses to guide future experimental studies using site-directed mutagenesis.

  9. The GTPase Rab43 Controls the Anterograde ER-Golgi Trafficking and Sorting of GPCRs

    Directory of Open Access Journals (Sweden)

    Chunman Li

    2017-10-01

    Full Text Available G-protein-coupled receptors (GPCRs constitute the largest superfamily of cell-surface signaling proteins. However, mechanisms underlying their surface targeting and sorting are poorly understood. Here, we screen the Rab family of small GTPases in the surface transport of multiple GPCRs. We find that manipulation of Rab43 function significantly alters the surface presentation and signaling of all GPCRs studied without affecting non-GPCR membrane proteins. Rab43 specifically regulates the transport of nascent GPCRs from the endoplasmic reticulum (ER to the Golgi. More interestingly, Rab43 directly interacts with GPCRs in an activation-dependent fashion. The Rab43-binding domain identified in the receptors effectively converts non-GPCR membrane protein transport into a Rab43-dependent pathway. These data reveal a crucial role for Rab43 in anterograde ER-Golgi transport of nascent GPCRs, as well as the ER sorting of GPCR members by virtue of its ability to interact directly.

  10. Gastrin-releasing peptide receptors in the central nervous system: role in brain function and as a drug target

    Directory of Open Access Journals (Sweden)

    Rafael eRoesler

    2012-12-01

    Full Text Available Neuropeptides acting on specific cell membrane receptors of the G protein-coupled receptor (GPCR superfamily regulate a range of important aspects of nervous and neuroendocrine function. Gastrin-releasing peptide (GRP is a mammalian neuropeptide that binds to the GRP receptor (GRPR, BB2. Increasing evidence indicates that GRPR-mediated signaling in the central nervous system (CNS plays an important role in regulating brain function, including aspects related to emotional responses, social interaction, memory, and feeding behavior. In addition, some alterations in GRP or GRPR expression or function have been described in patients with neurodegenerative, neurodevelopmental, and psychiatric disorders, as well as in brain tumors. Findings from preclinical models are consistent with the view that the GRPR might play a role in brain disorders, and raise the possibility that GRPR agonists might ameliorate cognitive and social deficits associated with neurological diseases, while antagonists may reduce anxiety and inhibit the growth of some types of brain cancer. Further preclinical and translational studies evaluating the potential therapeutic effects of GRPR ligands are warranted.

  11. Chemogenomic analysis of G-protein coupled receptors and their ligands deciphers locks and keys governing diverse aspects of signalling.

    Directory of Open Access Journals (Sweden)

    Jörg D Wichard

    Full Text Available Understanding the molecular mechanism of signalling in the important super-family of G-protein-coupled receptors (GPCRs is causally related to questions of how and where these receptors can be activated or inhibited. In this context, it is of great interest to unravel the common molecular features of GPCRs as well as those related to an active or inactive state or to subtype specific G-protein coupling. In our underlying chemogenomics study, we analyse for the first time the statistical link between the properties of G-protein-coupled receptors and GPCR ligands. The technique of mutual information (MI is able to reveal statistical inter-dependence between variations in amino acid residues on the one hand and variations in ligand molecular descriptors on the other. Although this MI analysis uses novel information that differs from the results of known site-directed mutagenesis studies or published GPCR crystal structures, the method is capable of identifying the well-known common ligand binding region of GPCRs between the upper part of the seven transmembrane helices and the second extracellular loop. The analysis shows amino acid positions that are sensitive to either stimulating (agonistic or inhibitory (antagonistic ligand effects or both. It appears that amino acid positions for antagonistic and agonistic effects are both concentrated around the extracellular region, but selective agonistic effects are cumulated between transmembrane helices (TMHs 2, 3, and ECL2, while selective residues for antagonistic effects are located at the top of helices 5 and 6. Above all, the MI analysis provides detailed indications about amino acids located in the transmembrane region of these receptors that determine G-protein signalling pathway preferences.

  12. Modeling structure of G protein-coupled receptors in huan genome

    KAUST Repository

    Zhang, Yang

    2016-01-26

    G protein-coupled receptors (or GPCRs) are integral transmembrane proteins responsible to various cellular signal transductions. Human GPCR proteins are encoded by 5% of human genes but account for the targets of 40% of the FDA approved drugs. Due to difficulties in crystallization, experimental structure determination remains extremely difficult for human GPCRs, which have been a major barrier in modern structure-based drug discovery. We proposed a new hybrid protocol, GPCR-I-TASSER, to construct GPCR structure models by integrating experimental mutagenesis data with ab initio transmembrane-helix assembly simulations, assisted by the predicted transmembrane-helix interaction networks. The method was tested in recent community-wide GPCRDock experiments and constructed models with a root mean square deviation 1.26 Å for Dopamine-3 and 2.08 Å for Chemokine-4 receptors in the transmembrane domain regions, which were significantly closer to the native than the best templates available in the PDB. GPCR-I-TASSER has been applied to model all 1,026 putative GPCRs in the human genome, where 923 are found to have correct folds based on the confidence score analysis and mutagenesis data comparison. The successfully modeled GPCRs contain many pharmaceutically important families that do not have previously solved structures, including Trace amine, Prostanoids, Releasing hormones, Melanocortins, Vasopressin and Neuropeptide Y receptors. All the human GPCR models have been made publicly available through the GPCR-HGmod database at http://zhanglab.ccmb.med.umich.edu/GPCR-HGmod/ The results demonstrate new progress on genome-wide structure modeling of transmembrane proteins which should bring useful impact on the effort of GPCR-targeted drug discovery.

  13. GPCRdb: an information system for G protein-coupled receptors.

    Science.gov (United States)

    Isberg, Vignir; Mordalski, Stefan; Munk, Christian; Rataj, Krzysztof; Harpsøe, Kasper; Hauser, Alexander S; Vroling, Bas; Bojarski, Andrzej J; Vriend, Gert; Gloriam, David E

    2016-01-04

    Recent developments in G protein-coupled receptor (GPCR) structural biology and pharmacology have greatly enhanced our knowledge of receptor structure-function relations, and have helped improve the scientific foundation for drug design studies. The GPCR database, GPCRdb, serves a dual role in disseminating and enabling new scientific developments by providing reference data, analysis tools and interactive diagrams. This paper highlights new features in the fifth major GPCRdb release: (i) GPCR crystal structure browsing, superposition and display of ligand interactions; (ii) direct deposition by users of point mutations and their effects on ligand binding; (iii) refined snake and helix box residue diagram looks; and (iii) phylogenetic trees with receptor classification colour schemes. Under the hood, the entire GPCRdb front- and back-ends have been re-coded within one infrastructure, ensuring a smooth browsing experience and development. GPCRdb is available at http://www.gpcrdb.org/ and it's open source code at https://bitbucket.org/gpcr/protwis. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  14. New O-superfamily conotoxins from Conus striatus inhabited near Chinese Hainan Island

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Conotoxins are short peptide-toxins with specific targets and large diversity.They are useful in analgesia,neuroprotection,detection of some kinds of deseases,and receptor and ion channel study.In order to explore the conotoxin resourses of Chinese oceans,rapid amplification of 3' cDNA ends (RACE) method was utilized to systemically analyze the O-superfamily conotoxin content of Conus striatus inhabited near Chinese Hainan Island.Six new O-superfamily conopeptides were identified,one of which is highly homologous to MVIIA,an N-type calcium channel antagonist.

  15. In silico modeling techniques for predicting the tertiary structure of human H4 receptor.

    Science.gov (United States)

    Zaid, Hilal; Raiyn, Jamal; Osman, Midhat; Falah, Mizied; Srouji, Samer; Rayan, Anwar

    2016-01-01

    First cloned in 2000, the human Histamine H4 Receptor (hH4R) is the last member of the histamine receptors family discovered so far, it belongs to the GPCR super-family and is involved in a wide variety of immunological and inflammatory responses. Potential hH4R antagonists are proposed to have therapeutic potential for the treatment of allergies, inflammation, asthma and colitis. So far, no hH4R ligands have been successfully introduced to the pharmaceutical market, which creates a strong demand for new selective ligands to be developed. in silico techniques and structural based modeling are likely to facilitate the achievement of this goal. In this review paper we attempt to cover the fundamental concepts of hH4R structure modeling and its implementations in drug discovery and development, especially those that have been experimentally tested and to highlight some ideas that are currently being discussed on the dynamic nature of hH4R and GPCRs, in regards to computerized techniques for 3-D structure modeling.

  16. Allosteric modulation of G-protein coupled receptors

    DEFF Research Database (Denmark)

    Jensen, Anders A.; Spalding, Tracy A

    2004-01-01

    are believed to activate (agonists) or inhibit (competitive antagonists) receptor signalling by binding the receptor at the same site as the endogenous agonist, the orthosteric site. In contrast, allosteric ligands modulate receptor function by binding to different regions in the receptor, allosteric sites....... In recent years, combinatorial chemistry and high throughput screening have helped identify several allosteric GPCR modulators with novel structures, several of which already have become valuable pharmacological tools and may be candidates for clinical testing in the near future. This mini review outlines...... the current status and perspectives of allosteric modulation of GPCR function with emphasis on the pharmacology of endogenous and synthesised modulators, their receptor interactions and the therapeutic prospects of allosteric ligands compared to orthosteric ligands....

  17. Novel Therapeutic GPCRs for Psychiatric Disorders

    Directory of Open Access Journals (Sweden)

    Hidetoshi Komatsu

    2015-06-01

    Full Text Available G protein-coupled receptors (GPCRs are the most common targets of the neuropharmacological drugs in the central nervous system (CNS. GPCRs are activated by manifold neurotransmitters, and their activation in turn evokes slow synaptic transmission. They are deeply involved in multiple neurological and psychiatric disorders such as Parkinson’s disease and schizophrenia. In the brain, the striatum is strongly innervated by the ventral tegmental area (VTA and plays a central role in manifestation of psychiatric disorders. Recently, anatomical and comprehensive transcriptome analysis of the non-odorant GPCR superfamily revealed that the orphan GPCRs GPR88, GPR6, and GPR52, as well as dopamine D1 and D2 receptors and the adenosine A2a receptor, are the most highly enriched in the rodent striatum. Genetically engineered animal models and molecular biological studies have suggested that these striatally enriched GPCRs have a potential to be therapeutic psychiatric receptors. This review summarizes the current understanding of the therapeutic GPCR candidates for psychiatric disorders.

  18. G-Protein Coupled Receptors: Surface Display and Biosensor Technology

    Science.gov (United States)

    McMurchie, Edward; Leifert, Wayne

    Signal transduction by G-protein coupled receptors (GPCRs) underpins a multitude of physiological processes. Ligand recognition by the receptor leads to the activation of a generic molecular switch involving heterotrimeric G-proteins and guanine nucleotides. With growing interest and commercial investment in GPCRs in areas such as drug targets, orphan receptors, high-throughput screening of drugs and biosensors, greater attention will focus on assay development to allow for miniaturization, ultrahigh-throughput and, eventually, microarray/biochip assay formats that will require nanotechnology-based approaches. Stable, robust, cell-free signaling assemblies comprising receptor and appropriate molecular switching components will form the basis of future GPCR/G-protein platforms, which should be able to be adapted to such applications as microarrays and biosensors. This chapter focuses on cell-free GPCR assay nanotechnologies and describes some molecular biological approaches for the construction of more sophisticated, surface-immobilized, homogeneous, functional GPCR sensors. The latter points should greatly extend the range of applications to which technologies based on GPCRs could be applied.

  19. Inhibition of Ebola and Marburg Virus Entry by G Protein-Coupled Receptor Antagonists.

    Science.gov (United States)

    Cheng, Han; Lear-Rooney, Calli M; Johansen, Lisa; Varhegyi, Elizabeth; Chen, Zheng W; Olinger, Gene G; Rong, Lijun

    2015-10-01

    Filoviruses, consisting of Ebola virus (EBOV) and Marburg virus (MARV), are among the most lethal infectious threats to mankind. Infections by these viruses can cause severe hemorrhagic fevers in humans and nonhuman primates with high mortality rates. Since there is currently no vaccine or antiviral therapy approved for humans, there is an urgent need to develop prophylactic and therapeutic options for use during filoviral outbreaks and bioterrorist attacks. One of the ideal targets against filoviral infection and diseases is at the entry step, which is mediated by the filoviral glycoprotein (GP). In this report, we screened a chemical library of small molecules and identified numerous inhibitors, which are known G protein-coupled receptor (GPCR) antagonists targeting different GPCRs, including histamine receptors, 5-HT (serotonin) receptors, muscarinic acetylcholine receptor, and adrenergic receptor. These inhibitors can effectively block replication of both infectious EBOV and MARV, indicating a broad antiviral activity of the GPCR antagonists. The time-of-addition experiment and microscopic studies suggest that GPCR antagonists block filoviral entry at a step following the initial attachment but prior to viral/cell membrane fusion. These results strongly suggest that GPCRs play a critical role in filoviral entry and GPCR antagonists can be developed as an effective anti-EBOV/MARV therapy. Infection of Ebola virus and Marburg virus can cause severe illness in humans with a high mortality rate, and currently there is no FDA-approved vaccine or therapeutic treatment available. The 2013-2015 epidemic in West Africa underscores a lack of our understanding in the infection and pathogenesis of these viruses and the urgency of drug discovery and development. In this study, we have identified numerous inhibitors that are known G protein-coupled receptor (GPCR) antagonists targeting different GPCRs. These inhibitors can effectively block replication of both infectious

  20. Amplification of the angiogenic signal through the activation of the TSC/mTOR/HIF axis by the KSHV vGPCR in Kaposi's sarcoma.

    Directory of Open Access Journals (Sweden)

    Bruno C Jham

    2011-04-01

    Full Text Available Kaposi's sarcoma (KS is a vascular neoplasm characterized by the dysregulated expression of angiogenic and inflammatory cytokines. The driving force of the KS lesion, the KSHV-infected spindle cell, secretes elevated levels of vascular endothelial growth factor (VEGF, essential for KS development. However, the origin of VEGF in this tumor remains unclear.Here we report that the KSHV G protein-coupled receptor (vGPCR upregulates VEGF in KS through an intricate paracrine mechanism. The cytokines secreted by the few vGPCR-expressing tumor cells activate in neighboring cells multiple pathways (including AKT, ERK, p38 and IKKβ that, in turn, converge on TSC1/2, promoting mTOR activation, HIF upregulation, and VEGF secretion. Conditioned media from vGPCR-expressing cells lead to an mTOR-dependent increase in HIF-1α and HIF-2α protein levels and VEGF upregulation. In a mouse allograft model for KS, specific inhibition of the paracrine activation of mTOR in non-vGPCR-expressing cells was sufficient to inhibit HIF upregulation in these cells, and abolished the ability of the vGPCR-expressing cells to promote tumor formation in vivo. Similarly, pharmacologic inhibition of HIF in this model blocked VEGF secretion and also lead to tumor regression.Our findings provide a compelling explanation for how the few tumor cells expressing vGPCR can contribute to the dramatic amplification of VEGF secretion in KS, and further provide a molecular mechanism for how cytokine dysregulation in KS fuels angiogenesis and tumor development. These data further suggest that activation of HIF by vGPCR may be a vulnerable target for the treatment of patients with KS.

  1. The N-terminal region of the dopamine D2 receptor, a rhodopsin-like GPCR, regulates correct integration into the plasma membrane and endocytic routes

    Science.gov (United States)

    Cho, DI; Min, C; Jung, KS; Cheong, SY; Zheng, M; Cheong, SJ; Oak, MH; Cheong, JH; Lee, BK; Kim, KM

    2012-01-01

    BACKGROUND AND PURPOSE Functional roles of the N-terminal region of rhodopsin-like GPCR family remain unclear. Using dopamine D2 and D3 receptors as a model system, we probed the roles of the N-terminal region in the signalling, intracellular trafficking of receptor proteins, and explored the critical factors that determine the functionality of the N-terminal region. EXPERIMENTAL APPROACH The N-terminal region of the D2 receptor was gradually shortened or switched with that of the D3 receptor or a non-specific sequence (FLAG), or potential N-terminal glycosylation sites were mutated. Effects of these manipulations on surface expression, internalization, post-endocytic behaviours and signalling were determined. KEY RESULTS Shortening the N-terminal region of the D2 receptor enhanced receptor internalization and impaired surface expression and signalling; ligand binding, desensitization and down-regulation were not affected but their association with a particular microdomain, caveolae, was disrupted. Replacement of critical residues within the N-terminal region with the FLAG epitope failed to restore surface expression but partially restored the altered internalization and signalling. When the N-terminal regions were switched between D2 and D3 receptors, cell surface expression pattern of each receptor was switched. Mutations of potential N-terminal glycosylation sites inhibited surface expression but enhanced internalization of D2 receptors. CONCLUSIONS AND IMPLICATIONS Shortening of N-terminus or mutation of glycosylation sites located within the N-terminus enhanced receptor internalization but impaired the surface expression of D2 receptors. The N-terminal region of the D2 receptor, in a sequence-specific manner, controls the receptor's conformation and integration into the plasma membrane, which determine its subcellular localization, intracellular trafficking and signalling properties. PMID:22117524

  2. GOMoDo: A GPCRs online modeling and docking webserver.

    Directory of Open Access Journals (Sweden)

    Massimo Sandal

    Full Text Available G-protein coupled receptors (GPCRs are a superfamily of cell signaling membrane proteins that include >750 members in the human genome alone. They are the largest family of drug targets. The vast diversity and relevance of GPCRs contrasts with the paucity of structures available: only 21 unique GPCR structures have been experimentally determined as of the beginning of 2013. User-friendly modeling and small molecule docking tools are thus in great demand. While both GPCR structural predictions and docking servers exist separately, with GOMoDo (GPCR Online Modeling and Docking, we provide a web server to seamlessly model GPCR structures and dock ligands to the models in a single consistent pipeline. GOMoDo can automatically perform template choice, homology modeling and either blind or information-driven docking by combining together proven, state of the art bioinformatic tools. The web server gives the user the possibility of guiding the whole procedure. The GOMoDo server is freely accessible at http://molsim.sci.univr.it/gomodo.

  3. High content screening for G protein-coupled receptors using cell-based protein translocation assays

    DEFF Research Database (Denmark)

    Grånäs, Charlotta; Lundholt, Betina Kerstin; Heydorn, Arne

    2005-01-01

    G protein-coupled receptors (GPCRs) have been one of the most productive classes of drug targets for several decades, and new technologies for GPCR-based discovery promise to keep this field active for years to come. While molecular screens for GPCR receptor agonist- and antagonist-based drugs...... will continue to be valuable discovery tools, the most exciting developments in the field involve cell-based assays for GPCR function. Some cell-based discovery strategies, such as the use of beta-arrestin as a surrogate marker for GPCR function, have already been reduced to practice, and have been used...... as valuable discovery tools for several years. The application of high content cell-based screening to GPCR discovery has opened up additional possibilities, such as direct tracking of GPCRs, G proteins and other signaling pathway components using intracellular translocation assays. These assays provide...

  4. F4/80: the macrophage-specific adhesion-GPCR and its role in immunoregulation.

    Science.gov (United States)

    Lin, Hsi-Hsien; Stacey, Martin; Stein-Streilein, Joan; Gordon, Siamon

    2010-01-01

    As a macrophage-restricted reagent, the generation and application of the F4/80 mAb has greatly benefited the phenotypic characterization of mouse tissue macrophages for three decades. Following the molecular identification of the F4/80 antigen as an EGF-TM7 member of the adhesion-GPCR family, great interest was ignited to understand its cell type-specific expression pattern as well as its functional role in macrophage biology. Recent studies have shown that the F4/80 gene is regulated by a novel set of transcription factors that recognized a unique promoter sequence. Gene targeting experiments have produced two F4/80 knock out animal models and showed that F4/80 is not required for normal macrophage development. Nevertheless, the F4/80 receptor was found to be necessary for the induction of efferent CD8+ regulatory T cells responsible for peripheral immune tolerance. The identification of cellular ligands for F4/80 and delineation of its signaling pathway remain elusive but are critical to understand the in vivo role of this macrophage-specific adhesion-GPCR.

  5. VPAC receptors: structure, molecular pharmacology and interaction with accessory proteins.

    Science.gov (United States)

    Couvineau, Alain; Laburthe, Marc

    2012-05-01

    The vasoactive intestinal peptide (VIP) is a neuropeptide with wide distribution in both central and peripheral nervous systems, where it plays important regulatory role in many physiological processes. VIP displays a large biological functions including regulation of exocrine secretions, hormone release, fetal development, immune responses, etc. VIP appears to exert beneficial effect in neuro-degenerative and inflammatory diseases. The mechanism of action of VIP implicates two subtypes of receptors (VPAC1 and VPAC2), which are members of class B receptors belonging to the super-family of GPCR. This article reviews the current knowledge regarding the structure and molecular pharmacology of VPAC receptors. The structure-function relationship of VPAC1 receptor has been extensively studied, allowing to understand the molecular basis for receptor affinity, specificity, desensitization and coupling to adenylyl cyclase. Those studies have clearly demonstrated the crucial role of the N-terminal ectodomain (N-ted) of VPAC1 receptor in VIP recognition. By using different approaches including directed mutagenesis, photoaffinity labelling, NMR, molecular modelling and molecular dynamic simulation, it has been shown that the VIP molecule interacts with the N-ted of VPAC1 receptor, which is itself structured as a 'Sushi' domain. VPAC1 receptor also interacts with a few accessory proteins that play a role in cell signalling of receptors. Recent advances in the structural characterization of VPAC receptor and more generally of class B GPCRs will lead to the design of new molecules, which could have considerable interest for the treatment of inflammatory and neuro-degenerative diseases. © 2011 The Authors. British Journal of Pharmacology © 2011 The British Pharmacological Society.

  6. Arrestin scaffolds NHERF1 to the P2Y12 receptor to regulate receptor internalization.

    Science.gov (United States)

    Nisar, Shaista P; Cunningham, Margaret; Saxena, Kunal; Pope, Robert J; Kelly, Eamonn; Mundell, Stuart J

    2012-07-13

    We have recently shown in a patient with mild bleeding that the PDZ-binding motif of the platelet G protein-coupled P2Y(12) receptor (P2Y(12)R) is required for effective receptor traffic in human platelets. In this study we show for the first time that the PDZ motif-binding protein NHERF1 exerts a major role in potentiating G protein-coupled receptor (GPCR) internalization. NHERF1 interacts with the C-tail of the P2Y(12)R and unlike many other GPCRs, NHERF1 interaction is required for effective P2Y(12)R internalization. In vitro and prior to agonist stimulation P2Y(12)R/NHERF1 interaction requires the intact PDZ binding motif of this receptor. Interestingly on receptor stimulation NHERF1 no longer interacts directly with the receptor but instead binds to the receptor via the endocytic scaffolding protein arrestin. These findings suggest a novel model by which arrestin can serve as an adaptor to promote NHERF1 interaction with a GPCR to facilitate effective NHERF1-dependent receptor internalization.

  7. TRPC4α and TRPC4β Similarly Affect Neonatal Cardiomyocyte Survival during Chronic GPCR Stimulation.

    Directory of Open Access Journals (Sweden)

    Nadine Kirschmer

    Full Text Available The Transient Receptor Potential Channel Subunit 4 (TRPC4 has been considered as a crucial Ca2+ component in cardiomyocytes promoting structural and functional remodeling in the course of pathological cardiac hypertrophy. TRPC4 assembles as homo or hetero-tetramer in the plasma membrane, allowing a non-selective Na+ and Ca2+ influx. Gαq protein-coupled receptor (GPCR stimulation is known to increase TRPC4 channel activity and a TRPC4-mediated Ca2+ influx which has been regarded as ideal Ca2+ source for calcineurin and subsequent nuclear factor of activated T-cells (NFAT activation. Functional properties of TRPC4 are also based on the expression of the TRPC4 splice variants TRPC4α and TRPC4β. Aim of the present study was to analyze cytosolic Ca2+ signals, signaling, hypertrophy and vitality of cardiomyocytes in dependence on the expression level of either TRPC4α or TRPC4β. The analysis of Ca2+ transients in neonatal rat cardiomyocytes (NRCs showed that TRPC4α and TRPC4β affected Ca2+ cycling in beating cardiomyocytes with both splice variants inducing an elevation of the Ca2+ transient amplitude at baseline and TRPC4β increasing the Ca2+ peak during angiotensin II (Ang II stimulation. NRCs infected with TRPC4β (Ad-C4β also responded with a sustained Ca2+ influx when treated with Ang II under non-pacing conditions. Consistent with the Ca2+ data, NRCs infected with TRPC4α (Ad-C4α showed an elevated calcineurin/NFAT activity and a baseline hypertrophic phenotype but did not further develop hypertrophy during chronic Ang II/phenylephrine stimulation. Down-regulation of endogenous TRPC4α reversed these effects, resulting in less hypertrophy of NRCs at baseline but a markedly increased hypertrophic enlargement after chronic agonist stimulation. Ad-C4β NRCs did not exhibit baseline calcineurin/NFAT activity or hypertrophy but responded with an increased calcineurin/NFAT activity after GPCR stimulation. However, this effect was not

  8. Regulation of G protein-coupled receptor signalling: focus on the cardiovascular system and regulator of G protein signalling proteins

    NARCIS (Netherlands)

    Hendriks-Balk, Mariëlle C.; Peters, Stephan L. M.; Michel, Martin C.; Alewijnse, Astrid E.

    2008-01-01

    G protein-coupled receptors (GPCRs) are involved in many biological processes. Therefore, GPCR function is tightly controlled both at receptor level and at the level of signalling components. Well-known mechanisms by which GPCR function can be regulated comprise desensitization/resensitization

  9. A novel inhibitor of α9α10 nicotinic acetylcholine receptors from Conus vexillum delineates a new conotoxin superfamily.

    Directory of Open Access Journals (Sweden)

    Sulan Luo

    Full Text Available Conotoxins (CTxs selectively target a range of ion channels and receptors, making them widely used tools for probing nervous system function. Conotoxins have been previously grouped into superfamilies according to signal sequence and into families based on their cysteine framework and biological target. Here we describe the cloning and characterization of a new conotoxin, from Conus vexillum, named αB-conotoxin VxXXIVA. The peptide does not belong to any previously described conotoxin superfamily and its arrangement of Cys residues is unique among conopeptides. Moreover, in contrast to previously characterized conopeptide toxins, which are expressed initially as prepropeptide precursors with a signal sequence, a ''pro'' region, and the toxin-encoding region, the precursor sequence of αB-VxXXIVA lacks a ''pro'' region. The predicted 40-residue mature peptide, which contains four Cys, was synthesized in each of the three possible disulfide arrangements. Investigation of the mechanism of action of αB-VxXXIVA revealed that the peptide is a nicotinic acetylcholine receptor (nAChR antagonist with greatest potency against the α9α10 subtype. (1H nuclear magnetic resonance (NMR spectra indicated that all three αB-VxXXIVA isomers were poorly structured in aqueous solution. This was consistent with circular dichroism (CD results which showed that the peptides were unstructured in buffer, but adopted partially helical conformations in aqueous trifluoroethanol (TFE solution. The α9α10 nAChR is an important target for the development of analgesics and cancer chemotherapeutics, and αB-VxXXIVA represents a novel ligand with which to probe the structure and function of this protein.

  10. Optodynamic simulation of β-adrenergic receptor signalling.

    Science.gov (United States)

    Siuda, Edward R; McCall, Jordan G; Al-Hasani, Ream; Shin, Gunchul; Il Park, Sung; Schmidt, Martin J; Anderson, Sonya L; Planer, William J; Rogers, John A; Bruchas, Michael R

    2015-09-28

    Optogenetics has provided a revolutionary approach to dissecting biological phenomena. However, the generation and use of optically active GPCRs in these contexts is limited and it is unclear how well an opsin-chimera GPCR might mimic endogenous receptor activity. Here we show that a chimeric rhodopsin/β2 adrenergic receptor (opto-β2AR) is similar in dynamics to endogenous β2AR in terms of: cAMP generation, MAP kinase activation and receptor internalization. In addition, we develop and characterize a novel toolset of optically active, functionally selective GPCRs that can bias intracellular signalling cascades towards either G-protein or arrestin-mediated cAMP and MAP kinase pathways. Finally, we show how photoactivation of opto-β2AR in vivo modulates neuronal activity and induces anxiety-like behavioural states in both fiber-tethered and wireless, freely moving animals when expressed in brain regions known to contain β2ARs. These new GPCR approaches enhance the utility of optogenetics and allow for discrete spatiotemporal control of GPCR signalling in vitro and in vivo.

  11. Imaging of persistent cAMP signaling by internalized G protein-coupled receptors.

    Science.gov (United States)

    Calebiro, Davide; Nikolaev, Viacheslav O; Lohse, Martin J

    2010-07-01

    G protein-coupled receptors (GPCRs) are the largest family of plasma membrane receptors. They mediate the effects of several endogenous cues and serve as important pharmacological targets. Although many biochemical events involved in GPCR signaling have been characterized in great detail, little is known about their spatiotemporal dynamics in living cells. The recent advent of optical methods based on fluorescent resonance energy transfer allows, for the first time, to directly monitor GPCR signaling in living cells. Utilizing these methods, it has been recently possible to show that the receptors for two protein/peptide hormones, the TSH and the parathyroid hormone, continue signaling to cAMP after their internalization into endosomes. This type of intracellular signaling is persistent and apparently triggers specific cellular outcomes. Here, we review these recent data and explain the optical methods used for such studies. Based on these findings, we propose a revision of the current model of the GPCR-cAMP signaling pathway to accommodate receptor signaling at endosomes.

  12. Superfamily of genes encoding G protein-coupled receptors in the diamondback moth Plutella xylostella (Lepidoptera: Plutellidae).

    Science.gov (United States)

    Wu, S-F; Yu, H-Y; Jiang, T-T; Gao, C-F; Shen, J-L

    2015-08-01

    G protein-coupled receptors (GPCRs) are the largest and most versatile superfamily of cell membrane proteins, which mediate various physiological processes including reproduction, development and behaviour. The diamondback moth, Plutella xylostella (Lepidoptera: Plutellidae), is one of the most notorious insect pests, preferentially feeding on cruciferous plants. P. xylostella is not only one of the world's most widespread lepidopteran insects, but has also developed resistance to nearly all classes of insecticides. Although the mechanisms of insecticide resistance have been studied extensively in many insect species, few investigations have been carried out on GPCRs in P. xylostella. In the present study, we identified 95 putative GPCRs in the P. xylostella genome. The identified GPCRs were compared with their homologues in Bombyx mori and Drosophila melanogaster. Our results suggest that GPCRs in different insect species may have evolved by a birth-and-death process. One of the differences among compared insects is the duplication of short neuropeptide F receptor and adipokinetic hormone receptors in P. xylostella and B. mori. Another divergence is the decrease in quantity and diversity of the stress-tolerance gene, Mth, in P. xylostella. The evolution by the birth-and-death process is probably involved in adaptation to the feeding behaviour, reproduction and stress responses of P. xylostella. Some of the genes identified in the present study could be potential targets for the development of novel pesticides. © 2015 The Royal Entomological Society.

  13. CD147 Immunoglobulin Superfamily Receptor Function and Role in Pathology

    OpenAIRE

    Iacono, Kathryn T.; Brown, Amy L.; Greene, Mark I.; Saouaf, Sandra J.

    2007-01-01

    The immunoglobulin superfamily member CD147 plays an important role in fetal, neuronal, lymphocyte and extracellular matrix development. Here we review the current understanding of CD147 expression and protein interactions with regard to CD147 function and its role in pathologic conditions including heart disease, Alzheimer’s disease, stroke and cancer. A model linking hypoxic conditions found within the tumor microenvironment to up-regulation of CD147 expression and tumor progression is intr...

  14. Muscarinic supersensitivity and impaired receptor desensitization in G protein-coupled receptor kinase 5-deficient mice.

    Science.gov (United States)

    Gainetdinov, R R; Bohn, L M; Walker, J K; Laporte, S A; Macrae, A D; Caron, M G; Lefkowitz, R J; Premont, R T

    1999-12-01

    G protein-coupled receptor kinase 5 (GRK5) is a member of a family of enzymes that phosphorylate activated G protein-coupled receptors (GPCR). To address the physiological importance of GRK5-mediated regulation of GPCRs, mice bearing targeted deletion of the GRK5 gene (GRK5-KO) were generated. GRK5-KO mice exhibited mild spontaneous hypothermia as well as pronounced behavioral supersensitivity upon challenge with the nonselective muscarinic agonist oxotremorine. Classical cholinergic responses such as hypothermia, hypoactivity, tremor, and salivation were enhanced in GRK5-KO animals. The antinociceptive effect of oxotremorine was also potentiated and prolonged. Muscarinic receptors in brains from GRK5-KO mice resisted oxotremorine-induced desensitization, as assessed by oxotremorine-stimulated [5S]GTPgammaS binding. These data demonstrate that elimination of GRK5 results in cholinergic supersensitivity and impaired muscarinic receptor desensitization and suggest that a deficit of GPCR desensitization may be an underlying cause of behavioral supersensitivity.

  15. The Biased G-Protein-Coupled Receptor Agonism Bridges the Gap between the Insulin Receptor and the Metabolic Syndrome

    Science.gov (United States)

    Liauchonak, Iryna; Dawoud, Fady; Riat, Yatin; Sambi, Manpreet; Jain, Justin; Kalaydina, Regina-Veronicka; Mendonza, Nicole; Bajwa, Komal

    2018-01-01

    Insulin signaling, as mediated through the insulin receptor (IR), plays a critical role in metabolism. Aberrations in this signaling cascade lead to several pathologies, the majority of which are classified under the umbrella term “metabolic syndrome”. Although many of these pathologies are associated with insulin resistance, the exact mechanisms are not well understood. One area of current interest is the possibility of G-protein-coupled receptors (GPCRs) influencing or regulating IR signaling. This concept is particularly significant, because GPCRs have been shown to participate in cross-talk with the IR. More importantly, GPCR signaling has also been shown to preferentially regulate specific downstream signaling targets through GPCR agonist bias. A novel study recently demonstrated that this GPCR-biased agonism influences the activity of the IR without the presence of insulin. Although GPCR-IR cross-talk has previously been established, the notion that GPCRs can regulate the activation of the IR is particularly significant in relation to metabolic syndrome and other pathologies that develop as a result of alterations in IR signaling. As such, we aim to provide an overview of the physiological and pathophysiological roles of the IR within metabolic syndrome and its related pathologies, including cardiovascular health, gut microflora composition, gastrointestinal tract functioning, polycystic ovarian syndrome, pancreatic cancer, and neurodegenerative disorders. Furthermore, we propose that the GPCR-biased agonism may perhaps mediate some of the downstream signaling effects that further exacerbate these diseases for which the mechanisms are currently not well understood. PMID:29462993

  16. G protein-coupled receptor mutations and human genetic disease.

    Science.gov (United States)

    Thompson, Miles D; Hendy, Geoffrey N; Percy, Maire E; Bichet, Daniel G; Cole, David E C

    2014-01-01

    Genetic variations in G protein-coupled receptor genes (GPCRs) disrupt GPCR function in a wide variety of human genetic diseases. In vitro strategies and animal models have been used to identify the molecular pathologies underlying naturally occurring GPCR mutations. Inactive, overactive, or constitutively active receptors have been identified that result in pathology. These receptor variants may alter ligand binding, G protein coupling, receptor desensitization and receptor recycling. Receptor systems discussed include rhodopsin, thyrotropin, parathyroid hormone, melanocortin, follicle-stimulating hormone (FSH), luteinizing hormone, gonadotropin-releasing hormone (GNRHR), adrenocorticotropic hormone, vasopressin, endothelin-β, purinergic, and the G protein associated with asthma (GPRA or neuropeptide S receptor 1 (NPSR1)). The role of activating and inactivating calcium-sensing receptor (CaSR) mutations is discussed in detail with respect to familial hypocalciuric hypercalcemia (FHH) and autosomal dominant hypocalemia (ADH). The CASR mutations have been associated with epilepsy. Diseases caused by the genetic disruption of GPCR functions are discussed in the context of their potential to be selectively targeted by drugs that rescue altered receptors. Examples of drugs developed as a result of targeting GPCRs mutated in disease include: calcimimetics and calcilytics, therapeutics targeting melanocortin receptors in obesity, interventions that alter GNRHR loss from the cell surface in idiopathic hypogonadotropic hypogonadism and novel drugs that might rescue the P2RY12 receptor congenital bleeding phenotype. De-orphanization projects have identified novel disease-associated receptors, such as NPSR1 and GPR35. The identification of variants in these receptors provides genetic reagents useful in drug screens. Discussion of the variety of GPCRs that are disrupted in monogenic Mendelian disorders provides the basis for examining the significance of common

  17. NCS-1 associates with adenosine A2A receptors and modulates receptor function

    Directory of Open Access Journals (Sweden)

    Gemma eNavarro

    2012-04-01

    Full Text Available Modulation of G protein-coupled receptor (GPCR signalling by local changes in intracellular calcium concentration is an established function of Calmodulin which is known to interact with many GPCRs. Less is known about the functional role of the closely related neuronal EF-hand Ca2+-sensor proteins that frequently associate with calmodulin targets with different functional outcome. In the present study we aimed to investigate if a target of calmodulin – the A2A adenosine receptor, is able to associate with two other neuronal calcium binding proteins, namely NCS-1 and caldendrin. Using bioluminescence resonance energy transfer and co-immunoprecipitation experiments we show the existence of A2A - NCS-1 complexes in living cells whereas caldendrin did not associate with A2A receptors under the conditions tested. Interestingly, NCS-1 binding modulated downstream A2A receptor intracellular signalling in a Ca2+-dependent manner. Taken together this study provides further evidence that neuronal Ca2+-sensor proteins play an important role in modulation of GPCR signalling.

  18. Gs-coupled GPCR signalling in AgRP neurons triggers sustained increase in food intake.

    Science.gov (United States)

    Nakajima, Ken-ichiro; Cui, Zhenzhong; Li, Chia; Meister, Jaroslawna; Cui, Yinghong; Fu, Ou; Smith, Adam S; Jain, Shalini; Lowell, Bradford B; Krashes, Michael J; Wess, Jürgen

    2016-01-08

    Agouti-related peptide (AgRP) neurons of the hypothalamus play a key role in regulating food intake and body weight, by releasing three different orexigenic molecules: AgRP; GABA; and neuropeptide Y. AgRP neurons express various G protein-coupled receptors (GPCRs) with different coupling properties, including Gs-linked GPCRs. At present, the potential role of Gs-coupled GPCRs in regulating the activity of AgRP neurons remains unknown. Here we show that the activation of Gs-coupled receptors expressed by AgRP neurons leads to a robust and sustained increase in food intake. We also provide detailed mechanistic data linking the stimulation of this class of receptors to the observed feeding phenotype. Moreover, we show that this pathway is clearly distinct from other GPCR signalling cascades that are operative in AgRP neurons. Our data suggest that drugs able to inhibit this signalling pathway may become useful for the treatment of obesity.

  19. RECEPTOR SUPERFAMILY OF TUMOR NECROSIS FACTOR Α, AND HSP90 HEAT SHOCK PROTEIN: A MOLECULAR BASIS FOR INTERACTIONS

    Directory of Open Access Journals (Sweden)

    N. V. Ryazantseva

    2011-01-01

    Full Text Available Abstract.  A  study  was  performed  aiming  to  investigate  interactions  between  TNFα  receptor  (TNF1 superfamily and heat shock protein Hsp90, using a Jurkat tumor cell line. The tumor cells cultured in presence of Hsp90 inhibitor (17-AAG showed increased numbers of cells, presenting surface TNFR1 and FasR, which facilitate  triggering  of  programmed  cell  death.  It  was  also  revealed  that  Hsp90  blockage  under  the  in  vitro conditions causes a decrease in FasL, while not affecting TNFα and sTNFR1 production by the tumor cells. (Med. Immunol., 2011, vol. 13, N 2-3, pp 247-252 

  20. An automated system for the analysis of G protein-coupled receptor transmembrane binding pockets: alignment, receptor-based pharmacophores, and their application.

    Science.gov (United States)

    Kratochwil, Nicole A; Malherbe, Pari; Lindemann, Lothar; Ebeling, Martin; Hoener, Marius C; Mühlemann, Andreas; Porter, Richard H P; Stahl, Martin; Gerber, Paul R

    2005-01-01

    G protein-coupled receptors (GPCRs) share a common architecture consisting of seven transmembrane (TM) domains. Various lines of evidence suggest that this fold provides a generic binding pocket within the TM region for hosting agonists, antagonists, and allosteric modulators. Here, a comprehensive and automated method allowing fast analysis and comparison of these putative binding pockets across the entire GPCR family is presented. The method relies on a robust alignment algorithm based on conservation indices, focusing on pharmacophore-like relationships between amino acids. Analysis of conservation patterns across the GPCR family and alignment to the rhodopsin X-ray structure allows the extraction of the amino acids lining the TM binding pocket in a so-called ligand binding pocket vector (LPV). In a second step, LPVs are translated to simple 3D receptor pharmacophore models, where each amino acid is represented by a single spherical pharmacophore feature and all atomic detail is omitted. Applications of the method include the assessment of selectivity issues, support of mutagenesis studies, and the derivation of rules for focused screening to identify chemical starting points in early drug discovery projects. Because of the coarseness of this 3D receptor pharmacophore model, however, meaningful scoring and ranking procedures of large sets of molecules are not justified. The LPV analysis of the trace amine-associated receptor family and its experimental validation is discussed as an example. The value of the 3D receptor model is demonstrated for a class C GPCR family, the metabotropic glutamate receptors.

  1. Evidence for functional pre-coupled complexes of receptor heteromers and adenylyl cyclase.

    Science.gov (United States)

    Navarro, Gemma; Cordomí, Arnau; Casadó-Anguera, Verónica; Moreno, Estefanía; Cai, Ning-Sheng; Cortés, Antoni; Canela, Enric I; Dessauer, Carmen W; Casadó, Vicent; Pardo, Leonardo; Lluís, Carme; Ferré, Sergi

    2018-03-28

    G protein-coupled receptors (GPCRs), G proteins and adenylyl cyclase (AC) comprise one of the most studied transmembrane cell signaling pathways. However, it is unknown whether the ligand-dependent interactions between these signaling molecules are based on random collisions or the rearrangement of pre-coupled elements in a macromolecular complex. Furthermore, it remains controversial whether a GPCR homodimer coupled to a single heterotrimeric G protein constitutes a common functional unit. Using a peptide-based approach, we here report evidence for the existence of functional pre-coupled complexes of heteromers of adenosine A 2A receptor and dopamine D 2 receptor homodimers coupled to their cognate Gs and Gi proteins and to subtype 5 AC. We also demonstrate that this macromolecular complex provides the necessary frame for the canonical Gs-Gi interactions at the AC level, sustaining the ability of a Gi-coupled GPCR to counteract AC activation mediated by a Gs-coupled GPCR.

  2. Function of the cytoplasmic tail of human calcitonin receptor-like receptor in complex with receptor activity-modifying protein 2

    Energy Technology Data Exchange (ETDEWEB)

    Kuwasako, Kenji, E-mail: kuwasako@fc.miyazaki-u.ac.jp [Frontier Science Research Center, University of Miyazaki, 5200 Kihara, Kiyotake, Miyazaki 889-1692 (Japan); Kitamura, Kazuo; Nagata, Sayaka; Hikosaka, Tomomi [Division of Circulation and Body Fluid Regulation, Faculty of Medicine, University of Miyazaki, 5200 Kihara, Kiyotake, Miyazaki 889-1692 (Japan); Kato, Johji [Frontier Science Research Center, University of Miyazaki, 5200 Kihara, Kiyotake, Miyazaki 889-1692 (Japan)

    2010-02-12

    Receptor activity-modifying protein 2 (RAMP2) enables calcitonin receptor-like receptor (CRLR) to form an adrenomedullin (AM)-specific receptor. Here we investigated the function of the cytoplasmic C-terminal tail (C-tail) of human (h)CRLR by co-transfecting its C-terminal mutants into HEK-293 cells stably expressing hRAMP2. Deleting the C-tail from CRLR disrupted AM-evoked cAMP production or receptor internalization, but did not affect [{sup 125}I]AM binding. We found that CRLR residues 428-439 are required for AM-evoked cAMP production, though deleting this region had little effect on receptor internalization. Moreover, pretreatment with pertussis toxin (100 ng/mL) led to significant increases in AM-induced cAMP production via wild-type CRLR/RAMP2 complexes. This effect was canceled by deleting CRLR residues 454-457, suggesting Gi couples to this region. Flow cytometric analysis revealed that CRLR truncation mutants lacking residues in the Ser/Thr-rich region extending from Ser{sup 449} to Ser{sup 467} were unable to undergo AM-induced receptor internalization and, in contrast to the effect on wild-type CRLR, overexpression of GPCR kinases-2, -3 and -4 failed to promote internalization of CRLR mutants lacking residues 449-467. Thus, the hCRLR C-tail is crucial for AM-evoked cAMP production and internalization of the CRLR/RAMP2, while the receptor internalization is dependent on the aforementioned GPCR kinases, but not Gs coupling.

  3. Diversity and impact of rare variants in genes encoding the platelet G protein-coupled receptors.

    Science.gov (United States)

    Jones, Matthew L; Norman, Jane E; Morgan, Neil V; Mundell, Stuart J; Lordkipanidzé, Marie; Lowe, Gillian C; Daly, Martina E; Simpson, Michael A; Drake, Sian; Watson, Steve P; Mumford, Andrew D

    2015-04-01

    Platelet responses to activating agonists are influenced by common population variants within or near G protein-coupled receptor (GPCR) genes that affect receptor activity. However, the impact of rare GPCR gene variants is unknown. We describe the rare single nucleotide variants (SNVs) in the coding and splice regions of 18 GPCR genes in 7,595 exomes from the 1,000-genomes and Exome Sequencing Project databases and in 31 cases with inherited platelet function disorders (IPFDs). In the population databases, the GPCR gene target regions contained 740 SNVs (318 synonymous, 410 missense, 7 stop gain and 6 splice region) of which 70 % had global minor allele frequency (MAF) < 0.05 %. Functional annotation using six computational algorithms, experimental evidence and structural data identified 156/740 (21 %) SNVs as potentially damaging to GPCR function, most commonly in regions encoding the transmembrane and C-terminal intracellular receptor domains. In 31 index cases with IPFDs (Gi-pathway defect n=15; secretion defect n=11; thromboxane pathway defect n=3 and complex defect n=2) there were 256 SNVs in the target regions of 15 stimulatory platelet GPCRs (34 unique; 12 with MAF< 1 % and 22 with MAF≥ 1 %). These included rare variants predicting R122H, P258T and V207A substitutions in the P2Y12 receptor that were annotated as potentially damaging, but only partially explained the platelet function defects in each case. Our data highlight that potentially damaging variants in platelet GPCR genes have low individual frequencies, but are collectively abundant in the population. Potentially damaging variants are also present in pedigrees with IPFDs and may contribute to complex laboratory phenotypes.

  4. Pulmonary artery hypertension in childhood: The transforming growth factor-β superfamily-related genes

    Directory of Open Access Journals (Sweden)

    Shi-Min Yuan

    2018-04-01

    Full Text Available Pulmonary artery hypertension (PAH is very rare in childhood, and it can be divided into heritable, idiopathic drug- and toxin-induced and other disease (connective tissue disease, human immunodeficiency virus infection, portal hypertension, congenital heart disease, or schistosomiasis-associated types. PAH could not be interpreted solely by pathophysiological theories. The impact of the transforming growth factor-β superfamily-related genes on the development of PAH in children remains to be clarified. Pertinent literature on the transforming growth factor-β superfamily-related genes in relation to PAH in children published after the year 2000 was reviewed and analyzed. Bone morphogenetic protein receptor type II gene mutation promotes cell division or prevents cell death, resulting in an overgrowth of cells in small arteries throughout the lungs. About 20% of individuals with a bone morphogenetic protein receptor type II gene mutation develop symptomatic PAH. In heritable PAH, bone morphogenetic protein receptor type II mutations may be absent; while mutations of other genes, such as type I receptor activin receptor-like kinase 1 and the type III receptor endoglin (both associated with hereditary hemorrhagic telangiectasia, caveolin-1 and KCNK3, the gene encoding potassium channel subfamily K, member 3, can be detected, instead. Gene mutations, environmental changes and acquired adjustment, etc. may explain the development of PAH. The researches on PAH rat model and familial PAH members may facilitate the elucidations of the mechanisms and further provide theories for prophylaxis and treatment of PAH. Key Words: bone morphogenetic proteins, mutation, pulmonary hypertension

  5. Functional understanding of the diverse exon-intron structures of human GPCR genes.

    Science.gov (United States)

    Hammond, Dorothy A; Olman, Victor; Xu, Ying

    2014-02-01

    The GPCR genes have a variety of exon-intron structures even though their proteins are all structurally homologous. We have examined all human GPCR genes with at least two functional protein isoforms, totaling 199, aiming to gain an understanding of what may have contributed to the large diversity of the exon-intron structures of the GPCR genes. The 199 genes have a total of 808 known protein splicing isoforms with experimentally verified functions. Our analysis reveals that 1301 (80.6%) adjacent exon-exon pairs out of the total of 1,613 in the 199 genes have either exactly one exon skipped or the intron in-between retained in at least one of the 808 protein splicing isoforms. This observation has a statistical significance p-value of 2.051762 * e(-09), assuming that the observed splicing isoforms are independent of the exon-intron structures. Our interpretation of this observation is that the exon boundaries of the GPCR genes are not randomly determined; instead they may be selected to facilitate specific alternative splicing for functional purposes.

  6. Sphingosine-1-phosphate receptors: Zooming in on ligand-induced intracellular trafficking and its functional implications

    NARCIS (Netherlands)

    Verzijl, Dennis; Peters, Stephan L. M.; Alewijnse, Astrid E.

    2010-01-01

    Regulatory processes including receptor phosphorylation and intracellular trafficking, also referred to as receptor internalization, are important processes to terminate G protein-coupled receptor (GPCR) signaling. Compelling evidence now indicates that internalization of a receptor is not

  7. Historical perspectives on tumor necrosis factor and its superfamily: 25 years later, a golden journey.

    Science.gov (United States)

    Aggarwal, Bharat B; Gupta, Subash C; Kim, Ji Hye

    2012-01-19

    Although activity that induced tumor regression was observed and termed tumor necrosis factor (TNF) as early as the 1960s, the true identity of TNF was not clear until 1984, when Aggarwal and coworkers reported, for the first time, the isolation of 2 cytotoxic factors: one, derived from macrophages (molecular mass 17 kDa), was named TNF, and the second, derived from lymphocytes (20 kDa), was named lymphotoxin. Because the 2 cytotoxic factors exhibited 50% amino acid sequence homology and bound to the same receptor, they came to be called TNF-α and TNF-β. Identification of the protein sequences led to cloning of their cDNA. Based on sequence homology to TNF-α, now a total of 19 members of the TNF superfamily have been identified, along with 29 interacting receptors, and several molecules that interact with the cytoplasmic domain of these receptors. The roles of the TNF superfamily in inflammation, apoptosis, proliferation, invasion, angiogenesis, metastasis, and morphogenesis have been documented. Their roles in immunologic, cardiovascular, neurologic, pulmonary, and metabolic diseases are becoming apparent. TNF superfamily members are active targets for drug development, as indicated by the recent approval and expanding market of TNF blockers used to treat rheumatoid arthritis, psoriasis, Crohns disease, and osteoporosis, with a total market of more than US $20 billion. As we learn more about this family, more therapeutics will probably emerge. In this review, we summarize the initial discovery of TNF-α, and the insights gained regarding the roles of this molecule and its related family members in normal physiology and disease.

  8. Constitutive dimerization of the G-protein coupled receptor, neurotensin receptor 1, reconstituted into phospholipid bilayers.

    Science.gov (United States)

    Harding, Peter J; Attrill, Helen; Boehringer, Jonas; Ross, Simon; Wadhams, George H; Smith, Eleanor; Armitage, Judith P; Watts, Anthony

    2009-02-01

    Neurotensin receptor 1 (NTS1), a Family A G-protein coupled receptor (GPCR), was expressed in Escherichia coli as a fusion with the fluorescent proteins eCFP or eYFP. A fluorophore-tagged receptor was used to study the multimerization of NTS1 in detergent solution and in brain polar lipid bilayers, using fluorescence resonance energy transfer (FRET). A detergent-solubilized receptor was unable to form FRET-competent complexes at concentrations of up to 200 nM, suggesting that the receptor is monomeric in this environment. When reconstituted into a model membrane system at low receptor density, the observed FRET was independent of agonist binding, suggesting constitutive multimer formation. In competition studies, decreased FRET in the presence of untagged NTS1 excludes the possibility of fluorescent protein-induced interactions. A simulation of the experimental data indicates that NTS1 exists predominantly as a homodimer, rather than as higher-order multimers. These observations suggest that, in common with several other Family A GPCRs, NTS1 forms a constitutive dimer in lipid bilayers, stabilized through receptor-receptor interactions in the absence of other cellular signaling components. Therefore, this work demonstrates that well-characterized model membrane systems are useful tools for the study of GPCR multimerization, allowing fine control over system composition and complexity, provided that rigorous control experiments are performed.

  9. Crystal structure of the PAC1R extracellular domain unifies a consensus fold for hormone recognition by class B G-protein coupled receptors.

    Directory of Open Access Journals (Sweden)

    Shiva Kumar

    Full Text Available Pituitary adenylate cyclase activating polypeptide (PACAP is a member of the PACAP/glucagon family of peptide hormones, which controls many physiological functions in the immune, nervous, endocrine, and muscular systems. It activates adenylate cyclase by binding to its receptor, PAC1R, a member of class B G-protein coupled receptors (GPCR. Crystal structures of a number of Class B GPCR extracellular domains (ECD bound to their respective peptide hormones have revealed a consensus mechanism of hormone binding. However, the mechanism of how PACAP binds to its receptor remains controversial as an NMR structure of the PAC1R ECD/PACAP complex reveals a different topology of the ECD and a distinct mode of ligand recognition. Here we report a 1.9 Å crystal structure of the PAC1R ECD, which adopts the same fold as commonly observed for other members of Class B GPCR. Binding studies and cell-based assays with alanine-scanned peptides and mutated receptor support a model that PAC1R uses the same conserved fold of Class B GPCR ECD for PACAP binding, thus unifying the consensus mechanism of hormone binding for this family of receptors.

  10. G Protein-coupled Receptor Kinases of the GRK4 Protein Subfamily Phosphorylate Inactive G Protein-coupled Receptors (GPCRs).

    Science.gov (United States)

    Li, Lingyong; Homan, Kristoff T; Vishnivetskiy, Sergey A; Manglik, Aashish; Tesmer, John J G; Gurevich, Vsevolod V; Gurevich, Eugenia V

    2015-04-24

    G protein-coupled receptor (GPCR) kinases (GRKs) play a key role in homologous desensitization of GPCRs. It is widely assumed that most GRKs selectively phosphorylate only active GPCRs. Here, we show that although this seems to be the case for the GRK2/3 subfamily, GRK5/6 effectively phosphorylate inactive forms of several GPCRs, including β2-adrenergic and M2 muscarinic receptors, which are commonly used as representative models for GPCRs. Agonist-independent GPCR phosphorylation cannot be explained by constitutive activity of the receptor or membrane association of the GRK, suggesting that it is an inherent ability of GRK5/6. Importantly, phosphorylation of the inactive β2-adrenergic receptor enhanced its interactions with arrestins. Arrestin-3 was able to discriminate between phosphorylation of the same receptor by GRK2 and GRK5, demonstrating preference for the latter. Arrestin recruitment to inactive phosphorylated GPCRs suggests that not only agonist activation but also the complement of GRKs in the cell regulate formation of the arrestin-receptor complex and thereby G protein-independent signaling. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  11. The G protein Gi1 exhibits basal coupling but not preassembly with G protein-coupled receptors.

    Science.gov (United States)

    Bondar, Alexey; Lazar, Josef

    2017-06-09

    The G i/o protein family transduces signals from a diverse group of G protein-coupled receptors (GPCRs). The observed specificity of G i/o -GPCR coupling and the high rate of G i/o signal transduction have been hypothesized to be enabled by the existence of stable associates between G i/o proteins and their cognate GPCRs in the inactive state (G i/o -GPCR preassembly). To test this hypothesis, we applied the recently developed technique of two-photon polarization microscopy (2PPM) to Gα i1 subunits labeled with fluorescent proteins and four GPCRs: the α 2A -adrenergic receptor, GABA B , cannabinoid receptor type 1 (CB 1 R), and dopamine receptor type 2. Our experiments with non-dissociating mutants of fluorescently labeled Gα i1 subunits (exhibiting impaired dissociation from activated GPCRs) showed that 2PPM is capable of detecting GPCR-G protein interactions. 2PPM experiments with non-mutated fluorescently labeled Gα i1 subunits and α 2A -adrenergic receptor, GABA B , or dopamine receptor type 2 receptors did not reveal any interaction between the G i1 protein and the non-stimulated GPCRs. In contrast, non-stimulated CB 1 R exhibited an interaction with the G i1 protein. Further experiments revealed that this interaction is caused solely by CB 1 R basal activity; no preassembly between CB 1 R and the G i1 protein could be observed. Our results demonstrate that four diverse GPCRs do not preassemble with non-active G i1 However, we also show that basal GPCR activity allows interactions between non-stimulated GPCRs and G i1 (basal coupling). These findings suggest that G i1 interacts only with active GPCRs and that the well known high speed of GPCR signal transduction does not require preassembly between G proteins and GPCRs. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  12. Balancing focused combinatorial libraries based on multiple GPCR ligands

    Science.gov (United States)

    Soltanshahi, Farhad; Mansley, Tamsin E.; Choi, Sun; Clark, Robert D.

    2006-08-01

    G-Protein coupled receptors (GPCRs) are important targets for drug discovery, and combinatorial chemistry is an important tool for pharmaceutical development. The absence of detailed structural information, however, limits the kinds of combinatorial design techniques that can be applied to GPCR targets. This is particularly problematic given the current emphasis on focused combinatorial libraries. By linking an incremental construction method (OptDesign) to the very fast shape-matching capability of ChemSpace, we have created an efficient method for designing targeted sublibraries that are topomerically similar to known actives. Multi-objective scoring allows consideration of multiple queries (actives) simultaneously. This can lead to a distribution of products skewed towards one particular query structure, however, particularly when the ligands of interest are quite dissimilar to one another. A novel pivoting technique is described which makes it possible to generate promising designs even under those circumstances. The approach is illustrated by application to some serotonergic agonists and chemokine antagonists.

  13. Fighting obesity with a sugar-based library: discovery of novel MCH-1R antagonists by a new computational-VAST approach for exploration of GPCR binding sites.

    Science.gov (United States)

    Heifetz, Alexander; Barker, Oliver; Verquin, Geraldine; Wimmer, Norbert; Meutermans, Wim; Pal, Sandeep; Law, Richard J; Whittaker, Mark

    2013-05-24

    Obesity is an increasingly common disease. While antagonism of the melanin-concentrating hormone-1 receptor (MCH-1R) has been widely reported as a promising therapeutic avenue for obesity treatment, no MCH-1R antagonists have reached the market. Discovery and optimization of new chemical matter targeting MCH-1R is hindered by reduced HTS success rates and a lack of structural information about the MCH-1R binding site. X-ray crystallography and NMR, the major experimental sources of structural information, are very slow processes for membrane proteins and are not currently feasible for every GPCR or GPCR-ligand complex. This situation significantly limits the ability of these methods to impact the drug discovery process for GPCR targets in "real-time", and hence, there is an urgent need for other practical and cost-efficient alternatives. We present here a conceptually pioneering approach that integrates GPCR modeling with design, synthesis, and screening of a diverse library of sugar-based compounds from the VAST technology (versatile assembly on stable templates) to provide structural insights on the MCH-1R binding site. This approach creates a cost-efficient new avenue for structure-based drug discovery (SBDD) against GPCR targets. In our work, a primary VAST hit was used to construct a high-quality MCH-1R model. Following model validation, a structure-based virtual screen yielded a 14% hit rate and 10 novel chemotypes of potent MCH-1R antagonists, including EOAI3367472 (IC50 = 131 nM) and EOAI3367474 (IC50 = 213 nM).

  14. Reducing the n-gram feature space of class C GPCRs to subtype-discriminating patterns

    Directory of Open Access Journals (Sweden)

    König Caroline

    2014-12-01

    Full Text Available G protein-coupled receptors (GPCRs are a large and heterogeneous superfamily of receptors that are key cell players for their role as extracellular signal transmitters. Class C GPCRs, in particular, are of great interest in pharmacology. The lack of knowledge about their full 3-D structure prompts the use of their primary amino acid sequences for the construction of robust classifiers, capable of discriminating their different subtypes. In this paper, we investigate the use of feature selection techniques to build Support Vector Machine (SVM-based classification models from selected receptor subsequences described as n-grams. We show that this approach to classification is useful for finding class C GPCR subtype-specific motifs.

  15. Dimers of G-Protein Coupled Receptors as Versatile Storage and Response Units

    Directory of Open Access Journals (Sweden)

    Michael S. Parker

    2014-03-01

    Full Text Available The status and use of transmembrane, extracellular and intracellular domains in oligomerization of heptahelical G-protein coupled receptors (GPCRs are reviewed and for transmembrane assemblies also supplemented by new experimental evidence. The transmembrane-linked GPCR oligomers typically have as the minimal unit an asymmetric ~180 kDa pentamer consisting of receptor homodimer or heterodimer and a G-protein αβγ subunit heterotrimer. With neuropeptide Y (NPY receptors, this assembly is converted to ~90 kDa receptor monomer-Gα complex by receptor and Gα agonists, and dimers/heteropentamers are depleted by neutralization of Gαi subunits by pertussis toxin. Employing gradient centrifugation, quantification and other characterization of GPCR dimers at the level of physically isolated and identified heteropentamers is feasible with labeled agonists that do not dissociate upon solubilization. This is demonstrated with three neuropeptide Y (NPY receptors and could apply to many receptors that use large peptidic agonists.

  16. G protein-coupled receptor internalization assays in the high-content screening format.

    Science.gov (United States)

    Haasen, Dorothea; Schnapp, Andreas; Valler, Martin J; Heilker, Ralf

    2006-01-01

    High-content screening (HCS), a combination of fluorescence microscopic imaging and automated image analysis, has become a frequently applied tool to study test compound effects in cellular disease-modeling systems. This chapter describes the measurement of G protein-coupled receptor (GPCR) internalization in the HCS format using a high-throughput, confocal cellular imaging device. GPCRs are the most successful group of therapeutic targets on the pharmaceutical market. Accordingly, the search for compounds that interfere with GPCR function in a specific and selective way is a major focus of the pharmaceutical industry today. This chapter describes methods for the ligand-induced internalization of GPCRs labeled previously with either a fluorophore-conjugated ligand or an antibody directed against an N-terminal tag of the GPCR. Both labeling techniques produce robust assay formats. Complementary to other functional GPCR drug discovery assays, internalization assays enable a pharmacological analysis of test compounds. We conclude that GPCR internalization assays represent a valuable medium/high-throughput screening format to determine the cellular activity of GPCR ligands.

  17. Quaternary structure of a G-protein-coupled receptor heterotetramer in complex with Gi and Gs.

    Science.gov (United States)

    Navarro, Gemma; Cordomí, Arnau; Zelman-Femiak, Monika; Brugarolas, Marc; Moreno, Estefania; Aguinaga, David; Perez-Benito, Laura; Cortés, Antoni; Casadó, Vicent; Mallol, Josefa; Canela, Enric I; Lluís, Carme; Pardo, Leonardo; García-Sáez, Ana J; McCormick, Peter J; Franco, Rafael

    2016-04-05

    G-protein-coupled receptors (GPCRs), in the form of monomers or homodimers that bind heterotrimeric G proteins, are fundamental in the transfer of extracellular stimuli to intracellular signaling pathways. Different GPCRs may also interact to form heteromers that are novel signaling units. Despite the exponential growth in the number of solved GPCR crystal structures, the structural properties of heteromers remain unknown. We used single-particle tracking experiments in cells expressing functional adenosine A1-A2A receptors fused to fluorescent proteins to show the loss of Brownian movement of the A1 receptor in the presence of the A2A receptor, and a preponderance of cell surface 2:2 receptor heteromers (dimer of dimers). Using computer modeling, aided by bioluminescence resonance energy transfer assays to monitor receptor homomerization and heteromerization and G-protein coupling, we predict the interacting interfaces and propose a quaternary structure of the GPCR tetramer in complex with two G proteins. The combination of results points to a molecular architecture formed by a rhombus-shaped heterotetramer, which is bound to two different interacting heterotrimeric G proteins (Gi and Gs). These novel results constitute an important advance in understanding the molecular intricacies involved in GPCR function.

  18. Expression of fatty acid sensing G-protein coupled receptors in peripartal Holstein cows.

    Science.gov (United States)

    Agrawal, Alea; Alharthi, Abdulrahman; Vailati-Riboni, Mario; Zhou, Zheng; Loor, Juan J

    2017-01-01

    G-protein coupled receptors (GPCR), also referred as Free Fatty Acid Receptors (FFAR), are widely studied within human medicine as drug targets for metabolic disorders. To combat metabolic disorders prevalent in dairy cows during the transition period, which co-occur with negative energy balance and changes to lipid and glucose metabolism, it may be helpful to identify locations and roles of FFAR and other members of the GPCR family in bovine tissues. Quantitative RT-PCR (qPCR) of subcutaneous adipose, liver, and PMNL samples during the transition period (-10, +7, and +20 or +30 d) were used for expression profiling of medium- (MCFA) and long-chain fatty acid (LCFA) receptors GPR120 and GPR40 , MCFA receptor GPR84 , and niacin receptor HCAR2/3 . Adipose samples were obtained from cows with either high (HI; BCS ≥ 3.75) or low (LO; BCS ≤ 3.25) body condition score (BCS) to examine whether FFAR expression is correlated with this indicator of health and body reserves. Supplementation of rumen-protected methionine (MET), which may improve immune function and production postpartum, was also compared with unsupplemented control (CON) cows for liver and blood polymorphonuclear leukocytes (PMNL) samples. In adipose tissue, GPR84 and GPR120 were differentially expressed over time, while GPR40 was not expressed; in PMNL, GPR40 was differentially expressed over time and between MET vs. CON, GPR84 expression differed only between dietary groups, and GPR120 was not expressed; in liver, GPCR were either not expressed or barely detectable. The data indicate that there is likely not a direct role in liver for the selected GPCR during the transition period, but they do play variable roles in adipose and PMN. In future, these receptors may prove useful targets and/or markers for peripartal metabolism and immunity.

  19. Free fatty acids-sensing G protein-coupled receptors in drug targeting and therapeutics.

    Science.gov (United States)

    Yonezawa, Tomo; Kurata, Riho; Yoshida, Kaori; Murayama, Masanori A; Cui, Xiaofeng; Hasegawa, Akihiko

    2013-01-01

    G protein-coupled receptor (GPCR) (also known as seven-transmembrane domain receptor) superfamily represents the largest protein family in the human genome. These receptors respond to various physiological ligands such as photons, odors, pheromones, hormones, ions, and small molecules including amines, amino acids to large peptides and steroids. Thus, GPCRs are involved in many diseases and the target of around half of all conventional drugs. The physiological roles of free fatty acids (FFAs), in particular, long-chain FFAs, are important for the development of many metabolic disease including obesity, diabetes, and atherosclerosis. In the past half decade, deorphanization of several GPCRs has revealed that GPR40, GPR41, GPR43, GPR84 and GPR120 sense concentration of extracellular FFAs with various carbon chain lengths. GPR40 and GPR120 are activated by medium- and long-chain FFAs. GPR84 is activated by medium- chain, but not long-chain, FFAs. GPR41 and GPR43 are activated by short-chain FFAs. GPR40 is highly expressed in pancreatic beta cells and plays a crucial role in FFAs-induced insulin secretion. GPR120 is mainly expressed in enteroendocrine cells and plays an important role for FFAs-induced glucagon-like peptide-1. GPR43 is abundant in leukocytes and adipose tissue, whilst GPR41 is highly expressed in adipose tissue, the pancreas and leukocytes. GPR84 is expressed in leukocytes and monocyte/macrophage. This review aims to shed light on the physiological roles and development of drugs targeting these receptors.

  20. Functional and Structural Overview of G-Protein-Coupled Receptors Comprehensively Obtained from Genome Sequences

    Directory of Open Access Journals (Sweden)

    Makiko Suwa

    2011-04-01

    Full Text Available An understanding of the functional mechanisms of G-protein-coupled receptors (GPCRs is very important for GPCR-related drug design. We have developed an integrated GPCR database (SEVENS http://sevens.cbrc.jp/ that includes 64,090 reliable GPCR genes comprehensively identified from 56 eukaryote genome sequences, and overviewed the sequences and structure spaces of the GPCRs. In vertebrates, the number of receptors for biological amines, peptides, etc. is conserved in most species, whereas the number of chemosensory receptors for odorant, pheromone, etc. significantly differs among species. The latter receptors tend to be single exon type or a few exon type and show a high ratio in the numbers of GPCRs, whereas some families, such as Class B and Class C receptors, have long lengths due to the presence of many exons. Statistical analyses of amino acid residues reveal that most of the conserved residues in Class A GPCRs are found in the cytoplasmic half regions of transmembrane (TM helices, while residues characteristic to each subfamily found on the extracellular half regions. The 69 of Protein Data Bank (PDB entries of complete or fragmentary structures could be mapped on the TM/loop regions of Class A GPCRs covering 14 subfamilies.

  1. Serotonin Signaling in Schistosoma mansoni: A Serotonin–Activated G Protein-Coupled Receptor Controls Parasite Movement

    Science.gov (United States)

    Rashid, Mohammed; Ribeiro, Paula

    2014-01-01

    Serotonin is an important neuroactive substance in all the parasitic helminths. In Schistosoma mansoni, serotonin is strongly myoexcitatory; it potentiates contraction of the body wall muscles and stimulates motor activity. This is considered to be a critical mechanism of motor control in the parasite, but the mode of action of serotonin is poorly understood. Here we provide the first molecular evidence of a functional serotonin receptor (Sm5HTR) in S. mansoni. The schistosome receptor belongs to the G protein-coupled receptor (GPCR) superfamily and is distantly related to serotonergic type 7 (5HT7) receptors from other species. Functional expression studies in transfected HEK 293 cells showed that Sm5HTR is a specific serotonin receptor and it signals through an increase in intracellular cAMP, consistent with a 5HT7 signaling mechanism. Immunolocalization studies with a specific anti-Sm5HTR antibody revealed that the receptor is abundantly distributed in the worm's nervous system, including the cerebral ganglia and main nerve cords of the central nervous system and the peripheral innervation of the body wall muscles and tegument. RNA interference (RNAi) was performed both in schistosomulae and adult worms to test whether the receptor is required for parasite motility. The RNAi-suppressed adults and larvae were markedly hypoactive compared to the corresponding controls and they were also resistant to exogenous serotonin treatment. These results show that Sm5HTR is at least one of the receptors responsible for the motor effects of serotonin in S. mansoni. The fact that Sm5HTR is expressed in nerve tissue further suggests that serotonin stimulates movement via this receptor by modulating neuronal output to the musculature. Together, the evidence identifies Sm5HTR as an important neuronal protein and a key component of the motor control apparatus in S. mansoni. PMID:24453972

  2. Five Drosophila Genomes Reveal Nonneutral Evolution and the Signature of Host Specialization in the Chemoreceptor Superfamily

    OpenAIRE

    McBride, Carolyn S.; Arguello, J. Roman

    2007-01-01

    The insect chemoreceptor superfamily comprises the olfactory receptor (Or) and gustatory receptor (Gr) multigene families. These families give insects the ability to smell and taste chemicals in the environment and are thus rich resources for linking molecular evolutionary and ecological processes. Although dramatic differences in family size among distant species and high divergence among paralogs have led to the belief that the two families evolve rapidly, a lack of evolutionary data over s...

  3. Receptor downregulation and desensitization enhance the information processing ability of signalling receptors

    Directory of Open Access Journals (Sweden)

    Resat Haluk

    2007-11-01

    Full Text Available Abstract Background In addition to initiating signaling events, the activation of cell surface receptors also triggers regulatory processes that restrict the duration of signaling. Acute attenuation of signaling can be accomplished either via ligand-induced internalization of receptors (endocytic downregulation or via ligand-induced receptor desensitization. These phenomena have traditionally been viewed in the context of adaptation wherein the receptor system enters a refractory state in the presence of sustained ligand stimuli and thereby prevents the cell from over-responding to the ligand. Here we use the epidermal growth factor receptor (EGFR and G-protein coupled receptors (GPCR as model systems to respectively examine the effects of downregulation and desensitization on the ability of signaling receptors to decode time-varying ligand stimuli. Results Using a mathematical model, we show that downregulation and desensitization mechanisms can lead to tight and efficient input-output coupling thereby ensuring synchronous processing of ligand inputs. Frequency response analysis indicates that upstream elements of the EGFR and GPCR networks behave like low-pass filters with the system being able to faithfully transduce inputs below a critical frequency. Receptor downregulation and desensitization increase the filter bandwidth thereby enabling the receptor systems to decode inputs in a wider frequency range. Further, system-theoretic analysis reveals that the receptor systems are analogous to classical mechanical over-damped systems. This analogy enables us to metaphorically describe downregulation and desensitization as phenomena that make the systems more resilient in responding to ligand perturbations thereby improving the stability of the system resting state. Conclusion Our findings suggest that in addition to serving as mechanisms for adaptation, receptor downregulation and desensitization can play a critical role in temporal information

  4. G Protein-Coupled Receptor-G-Protein βγ-Subunit Signaling Mediates Renal Dysfunction and Fibrosis in Heart Failure.

    Science.gov (United States)

    Kamal, Fadia A; Travers, Joshua G; Schafer, Allison E; Ma, Qing; Devarajan, Prasad; Blaxall, Burns C

    2017-01-01

    Development of CKD secondary to chronic heart failure (CHF), known as cardiorenal syndrome type 2 (CRS2), clinically associates with organ failure and reduced survival. Heart and kidney damage in CRS2 results predominantly from chronic stimulation of G protein-coupled receptors (GPCRs), including adrenergic and endothelin (ET) receptors, after elevated neurohormonal signaling of the sympathetic nervous system and the downstream ET system, respectively. Although we and others have shown that chronic GPCR stimulation and the consequent upregulated interaction between the G-protein βγ-subunit (Gβγ), GPCR-kinase 2, and β-arrestin are central to various cardiovascular diseases, the role of such alterations in kidney diseases remains largely unknown. We investigated the possible salutary effect of renal GPCR-Gβγ inhibition in CKD developed in a clinically relevant murine model of nonischemic hypertrophic CHF, transverse aortic constriction (TAC). By 12 weeks after TAC, mice developed CKD secondary to CHF associated with elevated renal GPCR-Gβγ signaling and ET system expression. Notably, systemic pharmacologic Gβγ inhibition by gallein, which we previously showed alleviates CHF in this model, attenuated these pathologic renal changes. To investigate a direct effect of gallein on the kidney, we used a bilateral ischemia-reperfusion AKI mouse model, in which gallein attenuated renal dysfunction, tissue damage, fibrosis, inflammation, and ET system activation. Furthermore, in vitro studies showed a key role for ET receptor-Gβγ signaling in pathologic fibroblast activation. Overall, our data support a direct role for GPCR-Gβγ in AKI and suggest GPCR-Gβγ inhibition as a novel therapeutic approach for treating CRS2 and AKI. Copyright © 2016 by the American Society of Nephrology.

  5. Pan-Cancer Analyses of the Nuclear Receptor Superfamily

    Directory of Open Access Journals (Sweden)

    Mark D. Long

    2015-12-01

    Full Text Available Nuclear receptors (NR act as an integrated conduit for environmental and hormonal signals to govern genomic responses, which relate to cell fate decisions. We review how their integrated actions with each other, shared co-factors and other transcription factors are disrupted in cancer. Steroid hormone nuclear receptors are oncogenic drivers in breast and prostate cancer and blockade of signaling is a major therapeutic goal. By contrast to blockade of receptors, in other cancers enhanced receptor function is attractive, as illustrated initially with targeting of retinoic acid receptors in leukemia. In the post-genomic era large consortia, such as The Cancer Genome Atlas, have developed a remarkable volume of genomic data with which to examine multiple aspects of nuclear receptor status in a pan-cancer manner. Therefore to extend the review of NR function we have also undertaken bioinformatics analyses of NR expression in over 3000 tumors, spread across six different tumor types (bladder, breast, colon, head and neck, liver and prostate. Specifically, to ask how the NR expression was distorted (altered expression, mutation and CNV we have applied bootstrapping approaches to simulate data for comparison, and also compared these NR findings to 12 other transcription factor families. Nuclear receptors were uniquely and uniformly downregulated across all six tumor types, more than predicted by chance. These approaches also revealed that each tumor type had a specific NR expression profile but these were most similar between breast and prostate cancer. Some NRs were down-regulated in at least five tumor types (e.g., NR3C2/MR and NR5A2/LRH-1 whereas others were uniquely down-regulated in one tumor (e.g., NR1B3/RARG. The downregulation was not driven by copy number variation or mutation and epigenetic mechanisms maybe responsible for the altered nuclear receptor expression.

  6. Involvement of a putative intercellular signal-recognizing G protein-coupled receptor in the engulfment of Salmonella by the protozoan Tetrahymena

    Directory of Open Access Journals (Sweden)

    P.N. Agbedanu

    2013-07-01

    Full Text Available In an effort to investigate the molecular basis of protozoa engulfment-mediated hypervirulence of Salmonella in cattle, we evaluated protozoan G protein-coupled receptors (GPCRs as transducers of Salmonella engulfment by the model protozoan Tetrahymena. Our laboratory previously demonstrated that non-pathogenic protozoa (including Tetrahymena engulf Salmonella and then exacerbate its virulence in cattle, but the mechanistic details of the phenomenon are not fully understood. GPCRs were investigated since these receptors facilitate phagocytosis of particulates by Tetrahymena, and a GPCR apparently modulates bacterial engulfment for the pathogenic protozoan Entamoeba histolytica. A database search identified three putative Tetrahymena GPCRs, based on sequence homologies and predicted transmembrane domains, that were the focus of this study. Salmonella engulfment by Tetrahymena was assessed in the presence of suramin, a non-specific GPCR inhibitor. Salmonella engulfment was also assessed in Tetrahymena in which expression of putative GPCRs was knocked-down using RNAi. A candidate GPCR was then expressed in a heterologous yeast expression system for further characterization. Our results revealed that Tetrahymena were less efficient at engulfing Salmonella in the presence of suramin. Engulfment was reduced concordantly with a reduction in the density of protozoa. RNAi-based studies revealed that knock-down of one the Tetrahymena GPCRs caused diminished engulfment of Salmonella. Tetrahymena lysates activated this receptor in the heterologous expression system. These data demonstrate that the Tetrahymena receptor is a putative GPCR that facilitates bacterial engulfment by Tetrahymena. Activation of the putative GPCR seemed to be related to protozoan cell density, suggesting that its cognate ligand is an intercellular signaling molecule.

  7. Recent Progress in Understanding Subtype Specific Regulation of NMDA Receptors by G Protein Coupled Receptors (GPCRs

    Directory of Open Access Journals (Sweden)

    Kai Yang

    2014-02-01

    Full Text Available G Protein Coupled Receptors (GPCRs are the largest family of receptors whose ligands constitute nearly a third of prescription drugs in the market. They are widely involved in diverse physiological functions including learning and memory. NMDA receptors (NMDARs, which belong to the ionotropic glutamate receptor family, are likewise ubiquitously expressed in the central nervous system (CNS and play a pivotal role in learning and memory. Despite its critical contribution to physiological and pathophysiological processes, few pharmacological interventions aimed directly at regulating NMDAR function have been developed to date. However, it is well established that NMDAR function is precisely regulated by cellular signalling cascades recruited downstream of G protein coupled receptor (GPCR stimulation. Accordingly, the downstream regulation of NMDARs likely represents an important determinant of outcome following treatment with neuropsychiatric agents that target selected GPCRs. Importantly, the functional consequence of such regulation on NMDAR function varies, based not only on the identity of the GPCR, but also on the cell type in which relevant receptors are expressed. Indeed, the mechanisms responsible for regulating NMDARs by GPCRs involve numerous intracellular signalling molecules and regulatory proteins that vary from one cell type to another. In the present article, we highlight recent findings from studies that have uncovered novel mechanisms by which selected GPCRs regulate NMDAR function and consequently NMDAR-dependent plasticity.

  8. A Molecular Mechanism for Sequential Activation of a G Protein-Coupled Receptor

    DEFF Research Database (Denmark)

    Grundmann, Manuel; Tikhonova, Irina G; Hudson, Brian D

    2016-01-01

    Ligands targeting G protein-coupled receptors (GPCRs) are currently classified as either orthosteric, allosteric, or dualsteric/bitopic. Here, we introduce a new pharmacological concept for GPCR functional modulation: sequential receptor activation. A hallmark feature of this is a stepwise ligand...

  9. Exploring G Protein-Coupled Receptors (GPCRs) Ligand Space via Cheminformatics Approaches: Impact on Rational Drug Design

    Science.gov (United States)

    Basith, Shaherin; Cui, Minghua; Macalino, Stephani J. Y.; Park, Jongmi; Clavio, Nina A. B.; Kang, Soosung; Choi, Sun

    2018-01-01

    The primary goal of rational drug discovery is the identification of selective ligands which act on single or multiple drug targets to achieve the desired clinical outcome through the exploration of total chemical space. To identify such desired compounds, computational approaches are necessary in predicting their drug-like properties. G Protein-Coupled Receptors (GPCRs) represent one of the largest and most important integral membrane protein families. These receptors serve as increasingly attractive drug targets due to their relevance in the treatment of various diseases, such as inflammatory disorders, metabolic imbalances, cardiac disorders, cancer, monogenic disorders, etc. In the last decade, multitudes of three-dimensional (3D) structures were solved for diverse GPCRs, thus referring to this period as the “golden age for GPCR structural biology.” Moreover, accumulation of data about the chemical properties of GPCR ligands has garnered much interest toward the exploration of GPCR chemical space. Due to the steady increase in the structural, ligand, and functional data of GPCRs, several cheminformatics approaches have been implemented in its drug discovery pipeline. In this review, we mainly focus on the cheminformatics-based paradigms in GPCR drug discovery. We provide a comprehensive view on the ligand– and structure-based cheminformatics approaches which are best illustrated via GPCR case studies. Furthermore, an appropriate combination of ligand-based knowledge with structure-based ones, i.e., integrated approach, which is emerging as a promising strategy for cheminformatics-based GPCR drug design is also discussed. PMID:29593527

  10. A new inhibitor of the β-arrestin/AP2 endocytic complex reveals interplay between GPCR internalization and signalling

    Science.gov (United States)

    Beautrait, Alexandre; Paradis, Justine S.; Zimmerman, Brandon; Giubilaro, Jenna; Nikolajev, Ljiljana; Armando, Sylvain; Kobayashi, Hiroyuki; Yamani, Lama; Namkung, Yoon; Heydenreich, Franziska M.; Khoury, Etienne; Audet, Martin; Roux, Philippe P.; Veprintsev, Dmitry B.; Laporte, Stéphane A.; Bouvier, Michel

    2017-04-01

    In addition to G protein-coupled receptor (GPCR) desensitization and endocytosis, β-arrestin recruitment to ligand-stimulated GPCRs promotes non-canonical signalling cascades. Distinguishing the respective contributions of β-arrestin recruitment to the receptor and β-arrestin-promoted endocytosis in propagating receptor signalling has been limited by the lack of selective analytical tools. Here, using a combination of virtual screening and cell-based assays, we have identified a small molecule that selectively inhibits the interaction between β-arrestin and the β2-adaptin subunit of the clathrin adaptor protein AP2 without interfering with the formation of receptor/β-arrestin complexes. This selective β-arrestin/β2-adaptin inhibitor (Barbadin) blocks agonist-promoted endocytosis of the prototypical β2-adrenergic (β2AR), V2-vasopressin (V2R) and angiotensin-II type-1 (AT1R) receptors, but does not affect β-arrestin-independent (transferrin) or AP2-independent (endothelin-A) receptor internalization. Interestingly, Barbadin fully blocks V2R-stimulated ERK1/2 activation and blunts cAMP accumulation promoted by both V2R and β2AR, supporting the concept of β-arrestin/AP2-dependent signalling for both G protein-dependent and -independent pathways.

  11. Insulin signaling inhibits the 5-HT2C receptor in choroid plexus via MAP kinase

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    Guan Kunliang

    2003-06-01

    Full Text Available Abstract Background G protein-coupled receptors (GPCRs interact with heterotrimeric GTP-binding proteins (G proteins to modulate acute changes in intracellular messenger levels and ion channel activity. In contrast, long-term changes in cellular growth, proliferation and differentiation are often mediated by tyrosine kinase receptors and certain GPCRs by activation of mitogen-activated protein (MAP kinases. Complex interactions occur between these signaling pathways, but the specific mechanisms of such regulatory events are not well-understood. In particular it is not clear whether GPCRs are modulated by tyrosine kinase receptor-MAP kinase pathways. Results Here we describe tyrosine kinase receptor regulation of a GPCR via MAP kinase. Insulin reduced the activity of the 5-HT2C receptor in choroid plexus cells which was blocked by the MAP kinase kinase (MEK inhibitor, PD 098059. We demonstrate that the inhibitory effect of insulin and insulin-like growth factor type 1 (IGF-1 on the 5-HT2C receptor is dependent on tyrosine kinase, RAS and MAP kinase. The effect may be receptor-specific: insulin had no effect on another GPCR that shares the same G protein signaling pathway as the 5-HT2C receptor. This effect is also direct: activated MAP kinase mimicked the effect of insulin, and removing a putative MAP kinase site from the 5-HT2C receptor abolished the effect of insulin. Conclusion These results show that insulin signaling can inhibit 5-HT2C receptor activity and suggest that MAP kinase may play a direct role in regulating the function of a specific GPCR.

  12. Synthesis of 99mTc-oxybutynin for M3-receptor-mediated imaging of urinary bladder

    International Nuclear Information System (INIS)

    Moustapha, M.E.; Benha University, Benha; Motaleb, M.A.; Ibrahim, I.T.

    2011-01-01

    Radiolabeling of oxybutynin, a muscarinic acetylcholine (mACh) receptor antagonist agent with 99m Tc is of considerable interest for imaging of urinary bladder. This study is aimed to optimize radiolabeling yield of oxybutynin with 99m Tc using SnCl 2 x 2H 2 O as a reducing agent with respect to factors that affect the reaction conditions such as oxybutynin amount, stannous chloride amount, reaction time and pH of the reaction mixture. In vitro stability of the radiolabeled complex was checked and it was found to be stable for up to 8 h. 99m Tc-oxybutynin was injected via subcutaneous and intravenous administration routes into normal Sprague-Dawley rats. Biodistribution studies have revealed that 99m Tc-oxybutynin exhibits high affinity and specificity for the muscarinic M 3 subtype located on the smooth muscle of urinary bladder relative to the M 1 and M 2 subtypes of the G protein coupled receptor (GPCR) superfamily. In vivo uptake of subcutaneous 99m Tc-oxybutynin in urinary bladder was 19.6 ± 0.42% ID at 0.5 h, whereas in intravenous administration route the accumulation in the urinary bladder was found to be 9.4 ± 0.31% ID at 0.5 h post injection. Administration of cold oxybutynin effectively blocked urinary bladder uptake and further confirms the high specificity of this complex for the M 3 receptor. (author)

  13. Comparative sequence and structural analyses of G-protein-coupled receptor crystal structures and implications for molecular models.

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    Catherine L Worth

    Full Text Available BACKGROUND: Up until recently the only available experimental (high resolution structure of a G-protein-coupled receptor (GPCR was that of bovine rhodopsin. In the past few years the determination of GPCR structures has accelerated with three new receptors, as well as squid rhodopsin, being successfully crystallized. All share a common molecular architecture of seven transmembrane helices and can therefore serve as templates for building molecular models of homologous GPCRs. However, despite the common general architecture of these structures key differences do exist between them. The choice of which experimental GPCR structure(s to use for building a comparative model of a particular GPCR is unclear and without detailed structural and sequence analyses, could be arbitrary. The aim of this study is therefore to perform a systematic and detailed analysis of sequence-structure relationships of known GPCR structures. METHODOLOGY: We analyzed in detail conserved and unique sequence motifs and structural features in experimentally-determined GPCR structures. Deeper insight into specific and important structural features of GPCRs as well as valuable information for template selection has been gained. Using key features a workflow has been formulated for identifying the most appropriate template(s for building homology models of GPCRs of unknown structure. This workflow was applied to a set of 14 human family A GPCRs suggesting for each the most appropriate template(s for building a comparative molecular model. CONCLUSIONS: The available crystal structures represent only a subset of all possible structural variation in family A GPCRs. Some GPCRs have structural features that are distributed over different crystal structures or which are not present in the templates suggesting that homology models should be built using multiple templates. This study provides a systematic analysis of GPCR crystal structures and a consistent method for identifying

  14. Comparative sequence and structural analyses of G-protein-coupled receptor crystal structures and implications for molecular models.

    Science.gov (United States)

    Worth, Catherine L; Kleinau, Gunnar; Krause, Gerd

    2009-09-16

    Up until recently the only available experimental (high resolution) structure of a G-protein-coupled receptor (GPCR) was that of bovine rhodopsin. In the past few years the determination of GPCR structures has accelerated with three new receptors, as well as squid rhodopsin, being successfully crystallized. All share a common molecular architecture of seven transmembrane helices and can therefore serve as templates for building molecular models of homologous GPCRs. However, despite the common general architecture of these structures key differences do exist between them. The choice of which experimental GPCR structure(s) to use for building a comparative model of a particular GPCR is unclear and without detailed structural and sequence analyses, could be arbitrary. The aim of this study is therefore to perform a systematic and detailed analysis of sequence-structure relationships of known GPCR structures. We analyzed in detail conserved and unique sequence motifs and structural features in experimentally-determined GPCR structures. Deeper insight into specific and important structural features of GPCRs as well as valuable information for template selection has been gained. Using key features a workflow has been formulated for identifying the most appropriate template(s) for building homology models of GPCRs of unknown structure. This workflow was applied to a set of 14 human family A GPCRs suggesting for each the most appropriate template(s) for building a comparative molecular model. The available crystal structures represent only a subset of all possible structural variation in family A GPCRs. Some GPCRs have structural features that are distributed over different crystal structures or which are not present in the templates suggesting that homology models should be built using multiple templates. This study provides a systematic analysis of GPCR crystal structures and a consistent method for identifying suitable templates for GPCR homology modelling that will

  15. Monitoring β-arrestin recruitment via β-lactamase enzyme fragment complementation: purification of peptide E as a low-affinity ligand for mammalian bombesin receptors.

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    Yuichi Ikeda

    Full Text Available Identification of cognate ligands for G protein-coupled receptors (GPCRs provides a starting point for understanding novel regulatory mechanisms. Although GPCR ligands have typically been evaluated through the activation of heterotrimeric G proteins, recent studies have shown that GPCRs signal not only through G proteins but also through β-arrestins. As such, monitoring β-arrestin signaling instead of G protein signaling will increase the likelihood of identifying currently unknown ligands, including β-arrestin-biased agonists. Here, we developed a cell-based assay for monitoring ligand-dependent GPCR-β-arrestin interaction via β-lactamase enzyme fragment complementation. Inter alia, β-lactamase is a superior reporter enzyme because of its cell-permeable fluorescent substrate. This substrate makes the assay non-destructive and compatible with fluorescence-activated cell sorting (FACS. In a reporter cell, complementary fragments of β-lactamase (α and ω were fused to β-arrestin 2 and GPCR, respectively. Ligand stimulation initiated the interaction of these chimeric proteins (β-arrestin-α and GPCR-ω, and this inducible interaction was measured through reconstituted β-lactamase activity. Utilizing this system, we screened various mammalian tissue extracts for agonistic activities on human bombesin receptor subtype 3 (hBRS3. We purified peptide E as a low-affinity ligand for hBRS3, which was also found to be an agonist for the other two mammalian bombesin receptors such as gastrin-releasing peptide receptor (GRPR and neuromedin B receptor (NMBR. Successful purification of peptide E has validated the robustness of this assay. We conclude that our newly developed system will facilitate the discovery of GPCR ligands.

  16. Role of post-translational modifications on structure, function and pharmacology of class C G protein-coupled receptors

    DEFF Research Database (Denmark)

    Nørskov-Lauritsen, Lenea; Bräuner-Osborne, Hans

    2015-01-01

    taste receptors (T1R1-3), one calcium-sensing (CaS) receptor, one GPCR, class C, group 6, subtype A (GPRC6) receptor, and seven orphan receptors. G protein-coupled receptors undergo a number of post-translational modifications, which regulate their structure, function and/or pharmacology. Here, we...

  17. GPCRM: a homology modeling web service with triple membrane-fitted quality assessment of GPCR models.

    Science.gov (United States)

    Miszta, Przemyslaw; Pasznik, Pawel; Jakowiecki, Jakub; Sztyler, Agnieszka; Latek, Dorota; Filipek, Slawomir

    2018-05-21

    Due to the involvement of G protein-coupled receptors (GPCRs) in most of the physiological and pathological processes in humans they have been attracting a lot of attention from pharmaceutical industry as well as from scientific community. Therefore, the need for new, high quality structures of GPCRs is enormous. The updated homology modeling service GPCRM (http://gpcrm.biomodellab.eu/) meets those expectations by greatly reducing the execution time of submissions (from days to hours/minutes) with nearly the same average quality of obtained models. Additionally, due to three different scoring functions (Rosetta, Rosetta-MP, BCL::Score) it is possible to select accurate models for the required purposes: the structure of the binding site, the transmembrane domain or the overall shape of the receptor. Currently, no other web service for GPCR modeling provides this possibility. GPCRM is continually upgraded in a semi-automatic way and the number of template structures has increased from 20 in 2013 to over 90 including structures the same receptor with different ligands which can influence the structure not only in the on/off manner. Two types of protein viewers can be used for visual inspection of obtained models. The extended sortable tables with available templates provide links to external databases and display ligand-receptor interactions in visual form.

  18. Applications of molecular replacement to G protein-coupled receptors

    International Nuclear Information System (INIS)

    Kruse, Andrew C.; Manglik, Aashish; Kobilka, Brian K.; Weis, William I.

    2013-01-01

    The use of molecular replacement in solving the structures of G protein-coupled receptors is discussed, with specific examples being described in detail. G protein-coupled receptors (GPCRs) are a large class of integral membrane proteins involved in regulating virtually every aspect of human physiology. Despite their profound importance in human health and disease, structural information regarding GPCRs has been extremely limited until recently. With the advent of a variety of new biochemical and crystallographic techniques, the structural biology of GPCRs has advanced rapidly, offering key molecular insights into GPCR activation and signal transduction. To date, almost all GPCR structures have been solved using molecular-replacement techniques. Here, the unique aspects of molecular replacement as applied to individual GPCRs and to signaling complexes of these important proteins are discussed

  19. A constitutively activating mutation alters the dynamics and energetics of a key conformational change in a ligand-free G protein-coupled receptor.

    Science.gov (United States)

    Tsukamoto, Hisao; Farrens, David L

    2013-09-27

    G protein-coupled receptors (GPCRs) undergo dynamic transitions between active and inactive conformations. Usually, these conversions are triggered when the receptor detects an external signal, but some so-called constitutively activating mutations, or CAMs, induce a GPCR to bind and activate G proteins in the absence of external stimulation, in ways still not fully understood. Here, we investigated how a CAM alters the structure of a GPCR and the dynamics involved as the receptor transitions between different conformations. Our approach used site-directed fluorescence labeling (SDFL) spectroscopy to compare opsin, the ligand-free form of the GPCR rhodopsin, with opsin containing the CAM M257Y, focusing specifically on key movements that occur in the sixth transmembrane helix (TM6) during GPCR activation. The site-directed fluorescence labeling data indicate opsin is constrained to an inactive conformation both in detergent micelles and lipid membranes, but when it contains the M257Y CAM, opsin is more dynamic and can interact with a G protein mimetic. Further study of these receptors using tryptophan-induced quenching (TrIQ) methods indicates that in detergent, the CAM significantly increases the population of receptors in the active state, but not in lipids. Subsequent Arrhenius analysis of the TrIQ data suggests that, both in detergent and lipids, the CAM lowers the energy barrier for TM6 movement, a key transition required for conversion between the inactive and active conformations. Together, these data suggest that the lowered energy barrier is a primary effect of the CAM on the receptor dynamics and energetics.

  20. The Cannabinoid Receptor CB1 Modulates the Signaling Properties of the Lysophosphatidylinositol Receptor GPR55*

    Science.gov (United States)

    Kargl, Julia; Balenga, Nariman; Parzmair, Gerald P.; Brown, Andrew J.; Heinemann, Akos; Waldhoer, Maria

    2012-01-01

    The G protein-coupled receptor (GPCR) 55 (GPR55) and the cannabinoid receptor 1 (CB1R) are co-expressed in many tissues, predominantly in the central nervous system. Seven transmembrane spanning (7TM) receptors/GPCRs can form homo- and heteromers and initiate distinct signaling pathways. Recently, several synthetic CB1 receptor inverse agonists/antagonists, such as SR141716A, AM251, and AM281, were reported to activate GPR55. Of these, SR141716A was marketed as a promising anti-obesity drug, but was withdrawn from the market because of severe side effects. Here, we tested whether GPR55 and CB1 receptors are capable of (i) forming heteromers and (ii) whether such heteromers could exhibit novel signaling patterns. We show that GPR55 and CB1 receptors alter each others signaling properties in human embryonic kidney (HEK293) cells. We demonstrate that the co-expression of FLAG-CB1 receptors in cells stably expressing HA-GPR55 specifically inhibits GPR55-mediated transcription factor activation, such as nuclear factor of activated T-cells and serum response element, as well as extracellular signal-regulated kinases (ERK1/2) activation. GPR55 and CB1 receptors can form heteromers, but the internalization of both receptors is not affected. In addition, we observe that the presence of GPR55 enhances CB1R-mediated ERK1/2 and nuclear factor of activated T-cell activation. Our data provide the first evidence that GPR55 can form heteromers with another 7TM/GPCR and that this interaction with the CB1 receptor has functional consequences in vitro. The GPR55-CB1R heteromer may play an important physiological and/or pathophysiological role in tissues endogenously co-expressing both receptors. PMID:23161546

  1. Family C 7TM receptor dimerization and activation

    DEFF Research Database (Denmark)

    Bonde, Marie Mi; Sheikh, Søren P; Hansen, Jakob Lerche

    2006-01-01

    The family C seven transmembrane (7TM) receptors constitutes a small and especially well characterized subfamily of the large 7TM receptor superfamily. Approximately 50% of current prescription drugs target 7TM receptors, this biologically important family represents the largest class of drug...... to be fully defined. This review presents the biochemical support for family C 7TM receptor dimerization and discusses its importance for receptor biosynthesis, surface expression, ligand binding and activation, since lessons learnt here may well be applicable to the whole superfamily of 7TM receptors.......-targets today. It is well established that family C 7TM receptors form homo- or hetero-dimers on the cell surface of living cells. The large extra-cellular domains (ECD) have been crystallized as a dimer in the presence and absence of agonist. Upon agonist binding, the dimeric ECD undergoes large conformational...

  2. Structure modeling of all identified G protein-coupled receptors in the human genome.

    Science.gov (United States)

    Zhang, Yang; Devries, Mark E; Skolnick, Jeffrey

    2006-02-01

    G protein-coupled receptors (GPCRs), encoded by about 5% of human genes, comprise the largest family of integral membrane proteins and act as cell surface receptors responsible for the transduction of endogenous signal into a cellular response. Although tertiary structural information is crucial for function annotation and drug design, there are few experimentally determined GPCR structures. To address this issue, we employ the recently developed threading assembly refinement (TASSER) method to generate structure predictions for all 907 putative GPCRs in the human genome. Unlike traditional homology modeling approaches, TASSER modeling does not require solved homologous template structures; moreover, it often refines the structures closer to native. These features are essential for the comprehensive modeling of all human GPCRs when close homologous templates are absent. Based on a benchmarked confidence score, approximately 820 predicted models should have the correct folds. The majority of GPCR models share the characteristic seven-transmembrane helix topology, but 45 ORFs are predicted to have different structures. This is due to GPCR fragments that are predominantly from extracellular or intracellular domains as well as database annotation errors. Our preliminary validation includes the automated modeling of bovine rhodopsin, the only solved GPCR in the Protein Data Bank. With homologous templates excluded, the final model built by TASSER has a global C(alpha) root-mean-squared deviation from native of 4.6 angstroms, with a root-mean-squared deviation in the transmembrane helix region of 2.1 angstroms. Models of several representative GPCRs are compared with mutagenesis and affinity labeling data, and consistent agreement is demonstrated. Structure clustering of the predicted models shows that GPCRs with similar structures tend to belong to a similar functional class even when their sequences are diverse. These results demonstrate the usefulness and robustness

  3. Structure modeling of all identified G protein-coupled receptors in the human genome.

    Directory of Open Access Journals (Sweden)

    Yang Zhang

    2006-02-01

    Full Text Available G protein-coupled receptors (GPCRs, encoded by about 5% of human genes, comprise the largest family of integral membrane proteins and act as cell surface receptors responsible for the transduction of endogenous signal into a cellular response. Although tertiary structural information is crucial for function annotation and drug design, there are few experimentally determined GPCR structures. To address this issue, we employ the recently developed threading assembly refinement (TASSER method to generate structure predictions for all 907 putative GPCRs in the human genome. Unlike traditional homology modeling approaches, TASSER modeling does not require solved homologous template structures; moreover, it often refines the structures closer to native. These features are essential for the comprehensive modeling of all human GPCRs when close homologous templates are absent. Based on a benchmarked confidence score, approximately 820 predicted models should have the correct folds. The majority of GPCR models share the characteristic seven-transmembrane helix topology, but 45 ORFs are predicted to have different structures. This is due to GPCR fragments that are predominantly from extracellular or intracellular domains as well as database annotation errors. Our preliminary validation includes the automated modeling of bovine rhodopsin, the only solved GPCR in the Protein Data Bank. With homologous templates excluded, the final model built by TASSER has a global C(alpha root-mean-squared deviation from native of 4.6 angstroms, with a root-mean-squared deviation in the transmembrane helix region of 2.1 angstroms. Models of several representative GPCRs are compared with mutagenesis and affinity labeling data, and consistent agreement is demonstrated. Structure clustering of the predicted models shows that GPCRs with similar structures tend to belong to a similar functional class even when their sequences are diverse. These results demonstrate the usefulness

  4. A monomeric G protein-coupled receptor isolated in a high-density lipoprotein particle efficiently activates its G protein

    DEFF Research Database (Denmark)

    Whorton, Matthew R; Bokoch, Michael P; Rasmussen, Søren Gøgsig Faarup

    2007-01-01

    G protein-coupled receptors (GPCRs) respond to a diverse array of ligands, mediating cellular responses to hormones and neurotransmitters, as well as the senses of smell and taste. The structures of the GPCR rhodopsin and several G proteins have been determined by x-ray crystallography, yet...... the organization of the signaling complex between GPCRs and G proteins is poorly understood. The observations that some GPCRs are obligate heterodimers, and that many GPCRs form both homo- and heterodimers, has led to speculation that GPCR dimers may be required for efficient activation of G proteins. However......, technical limitations have precluded a definitive analysis of G protein coupling to monomeric GPCRs in a biochemically defined and membrane-bound system. Here we demonstrate that a prototypical GPCR, the beta2-adrenergic receptor (beta2AR), can be incorporated into a reconstituted high-density lipoprotein...

  5. Combining on-chip synthesis of a focused combinatorial library with computational target prediction reveals imidazopyridine GPCR ligands.

    Science.gov (United States)

    Reutlinger, Michael; Rodrigues, Tiago; Schneider, Petra; Schneider, Gisbert

    2014-01-07

    Using the example of the Ugi three-component reaction we report a fast and efficient microfluidic-assisted entry into the imidazopyridine scaffold, where building block prioritization was coupled to a new computational method for predicting ligand-target associations. We identified an innovative GPCR-modulating combinatorial chemotype featuring ligand-efficient adenosine A1/2B and adrenergic α1A/B receptor antagonists. Our results suggest the tight integration of microfluidics-assisted synthesis with computer-based target prediction as a viable approach to rapidly generate bioactivity-focused combinatorial compound libraries with high success rates. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. Behavioral control by striatal adenosine A2A -dopamine D2 receptor heteromers.

    Science.gov (United States)

    Taura, J; Valle-León, M; Sahlholm, K; Watanabe, M; Van Craenenbroeck, K; Fernández-Dueñas, V; Ferré, S; Ciruela, F

    2018-04-01

    G protein-coupled receptors (GPCR) exhibit the ability to form receptor complexes that include molecularly different GPCR (ie, GPCR heteromers), which endow them with singular functional and pharmacological characteristics. The relative expression of GPCR heteromers remains a matter of intense debate. Recent studies support that adenosine A 2A receptors (A 2A R) and dopamine D 2 receptors (D 2 R) predominantly form A 2A R-D 2 R heteromers in the striatum. The aim of the present study was evaluating the behavioral effects of pharmacological manipulation and genetic blockade of A 2A R and D 2 R within the frame of such a predominant striatal heteromeric population. First, in order to avoid possible strain-related differences, a new D 2 R-deficient mouse with the same genetic background (CD-1) than the A 2A R knock-out mouse was generated. Locomotor activity, pre-pulse inhibition (PPI) and drug-induced catalepsy were then evaluated in wild-type, A 2A R and D 2 R knock-out mice, with and without the concomitant administration of either the D 2 R agonist sumanirole or the A 2A R antagonist SCH442416. SCH442416-mediated locomotor effects were demonstrated to be dependent on D 2 R signaling. Similarly, a significant dependence on A 2A R signaling was observed for PPI and for haloperidol-induced catalepsy. The results could be explained by the existence of one main population of striatal postsynaptic A 2A R-D 2 R heteromers, which may constitute a relevant target for the treatment of Parkinson's disease and other neuropsychiatric disorders. © 2017 John Wiley & Sons Ltd and International Behavioural and Neural Genetics Society.

  7. Neuro-psychopharmacological perspective of Orphan receptors of Rhodopsin (class A) family of G protein-coupled receptors.

    Science.gov (United States)

    Khan, Muhammad Zahid; He, Ling

    2017-04-01

    In the central nervous system (CNS), G protein-coupled receptors (GPCRs) are the most fruitful targets for neuropsychopharmacological drug development. Rhodopsin (class A) is the most studied class of GPCR and includes orphan receptors for which the endogenous ligand is not known or is unclear. Characterization of orphan GPCRs has proven to be challenging, and the production pace of GPCR-based drugs has been incredibly slow. Determination of the functions of these receptors may provide unexpected insight into physiological and neuropathological processes. Advances in various methods and techniques to investigate orphan receptors including in situ hybridization and knockdown/knockout (KD/KO) showed extensive expression of these receptors in the mammalian brain and unmasked their physiological and neuropathological roles. Due to these rapid progress and development, orphan GPCRs are rising as a new and promising class of drug targets for neurodegenerative diseases and psychiatric disorders. This review presents a neuropsychopharmacological perspective of 26 orphan receptors of rhodopsin (class A) family, namely GPR3, GPR6, GPR12, GPR17, GPR26, GPR35, GPR39, GPR48, GPR49, GPR50, GPR52, GPR55, GPR61, GPR62, GPR63, GPR68, GPR75, GPR78, GPR83, GPR84, GPR85, GPR88, GPR153, GPR162, GPR171, and TAAR6. We discussed the expression of these receptors in mammalian brain and their physiological roles. Furthermore, we have briefly highlighted their roles in neurodegenerative diseases and psychiatric disorders including Alzheimer's disease, Parkinson's disease, neuroinflammation, inflammatory pain, bipolar and schizophrenic disorders, epilepsy, anxiety, and depression.

  8. G-protein-coupled receptors: new approaches to maximise the impact of GPCRS in drug discovery.

    Science.gov (United States)

    Davey, John

    2004-04-01

    IBC's Drug Discovery Technology Series is a group of conferences highlighting technological advances and applications in niche areas of the drug discovery pipeline. This 2-day meeting focused on G-protein-coupled receptors (GPCRs), probably the most important and certainly the most valuable class of targets for drug discovery. The meeting was chaired by J Beesley (Vice President, European Business Development for LifeSpan Biosciences, Seattle, USA) and included 17 presentations on various aspects of GPCR activity, drug screens and therapeutic analyses. Keynote Addresses covered two of the emerging areas in GPCR regulation; receptor dimerisation (G Milligan, Professor of Molecular Pharmacology and Biochemistry, University of Glasgow, UK) and proteins that interact with GPCRs (J Bockaert, Laboratory of Functional Genomics, CNRS Montpellier, France). A third Keynote Address from W Thomsen (Director of GPCR Drug Screening, Arena Pharmaceuticals, USA) discussed Arena's general approach to drug discovery and illustrated this with reference to the development of an agonist with potential efficacy in Type II diabetes.

  9. A single amino acid residue controls Ca2+ signaling by an octopamine receptor from Drosophila melanogaster.

    Science.gov (United States)

    Hoff, Max; Balfanz, Sabine; Ehling, Petra; Gensch, Thomas; Baumann, Arnd

    2011-07-01

    Rhythmic activity of cells and cellular networks plays an important role in physiology. In the nervous system oscillations of electrical activity and/or second messenger concentrations are important to synchronize neuronal activity. At the molecular level, rhythmic activity can be initiated by different routes. We have recently shown that an octopamine-activated G-protein-coupled receptor (GPCR; DmOctα1Rb, CG3856) from Drosophila initiates Ca(2+) oscillations. Here, we have unraveled the molecular basis of cellular Ca(2+) signaling controlled by the DmOctα1Rb receptor using a combination of pharmacological intervention, site-directed mutagenesis, and functional cellular Ca(2+) imaging on heterologously expressed receptors. Phosphorylation of a single amino acid residue in the third intracellular loop of the GPCR by PKC is necessary and sufficient to desensitize the receptor. From its desensitized state, DmOctα1Rb is resensitized by dephosphorylation, and a new Ca(2+) signal occurs on octopamine stimulation. Our findings show that transient changes of the receptor's surface profile have a strong effect on its physiological signaling properties. We expect that the detailed knowledge of DmOctα1Rb-dependent signal transduction fosters the identification of specific drugs that can be used for GPCR-mediated pest control, since octopamine serves important physiological and behavioral functions in arthropods.

  10. Crystal structure of the β2 adrenergic receptor-Gs protein complex

    DEFF Research Database (Denmark)

    Rasmussen, Søren Gøgsig Faarup; DeVree, Brian T; Zou, Yaozhong

    2011-01-01

    -occupied receptor. The β(2) adrenergic receptor (β(2)AR) activation of Gs, the stimulatory G protein for adenylyl cyclase, has long been a model system for GPCR signalling. Here we present the crystal structure of the active state ternary complex composed of agonist-occupied monomeric β(2)AR and nucleotide-free Gs...

  11. A Broad G Protein-Coupled Receptor Internalization Assay that Combines SNAP-Tag Labeling, Diffusion-Enhanced Resonance Energy Transfer, and a Highly Emissive Terbium Cryptate.

    Science.gov (United States)

    Levoye, Angélique; Zwier, Jurriaan M; Jaracz-Ros, Agnieszka; Klipfel, Laurence; Cottet, Martin; Maurel, Damien; Bdioui, Sara; Balabanian, Karl; Prézeau, Laurent; Trinquet, Eric; Durroux, Thierry; Bachelerie, Françoise

    2015-01-01

    Although G protein-coupled receptor (GPCR) internalization has long been considered as a major aspect of the desensitization process that tunes ligand responsiveness, internalization is also involved in receptor resensitization and signaling, as well as the ligand scavenging function of some atypical receptors. Internalization thus contributes to the diversity of GPCR-dependent signaling, and its dynamics and quantification in living cells has generated considerable interest. We developed a robust and sensitive assay to follow and quantify ligand-induced and constitutive-induced GPCR internalization but also receptor recycling in living cells. This assay is based on diffusion-enhanced resonance energy transfer (DERET) between cell surface GPCRs labeled with a luminescent terbium cryptate donor and a fluorescein acceptor present in the culture medium. GPCR internalization results in a quantifiable reduction of energy transfer. This method yields a high signal-to-noise ratio due to time-resolved measurements. For various GPCRs belonging to different classes, we demonstrated that constitutive and ligand-induced internalization could be monitored as a function of time and ligand concentration, thus allowing accurate quantitative determination of kinetics of receptor internalization but also half-maximal effective or inhibitory concentrations of compounds. In addition to its selectivity and sensitivity, we provided evidence that DERET-based internalization assay is particularly suitable for characterizing biased ligands. Furthermore, the determination of a Z'-factor value of 0.45 indicates the quality and suitability of DERET-based internalization assay for high-throughput screening (HTS) of compounds that may modulate GPCRs internalization.

  12. Crystal structure of the β2 adrenergic receptor-Gs protein complex

    Energy Technology Data Exchange (ETDEWEB)

    Rasmussen, Søren G.F.; DeVree, Brian T; Zou, Yaozhong; Kruse, Andrew C; Chung, Ka Young; Kobilka, Tong Sun; Thian, Foon Sun; Chae, Pil Seok; Pardon, Els; Calinski, Diane; Mathiesen, Jesper M; Shah, Syed T.A.; Lyons, Joseph A; Caffrey, Martin; Gellman, Samuel H; Steyaert, Jan; Skiniotis, Georgios; Weis, William I; Sunahara, Roger K; Kobilka, Brian K [Brussels; (Trinity); (Michigan); (Stanford-MED); (Michigan-Med); (UW)

    2011-12-07

    G protein-coupled receptors (GPCRs) are responsible for the majority of cellular responses to hormones and neurotransmitters as well as the senses of sight, olfaction and taste. The paradigm of GPCR signalling is the activation of a heterotrimeric GTP binding protein (G protein) by an agonist-occupied receptor. The β2 adrenergic receptor2AR) activation of Gs, the stimulatory G protein for adenylyl cyclase, has long been a model system for GPCR signalling. Here we present the crystal structure of the active state ternary complex composed of agonist-occupied monomeric β2AR and nucleotide-free Gs heterotrimer. The principal interactions between the β2AR and Gs involve the amino- and carboxy-terminal α-helices of Gs, with conformational changes propagating to the nucleotide-binding pocket. The largest conformational changes in the β2AR include a 14Å outward movement at the cytoplasmic end of transmembrane segment 6 (TM6) and an α-helical extension of the cytoplasmic end of TM5. The most surprising observation is a major displacement of the α-helical domain of Gαs relative to the Ras-like GTPase domain. This crystal structure represents the first high-resolution view of transmembrane signalling by a GPCR.

  13. Role of G-protein-coupled receptor-related genes in insecticide resistance of the mosquito, Culex quinquefasciatus.

    Science.gov (United States)

    Li, Ting; Liu, Lena; Zhang, Lee; Liu, Nannan

    2014-09-29

    G-protein-coupled receptors regulate signal transduction pathways and play diverse and pivotal roles in the physiology of insects, however, the precise function of GPCRs in insecticide resistance remains unclear. Using quantitative RT-PCR and functional genomic methods, we, for the first time, explored the function of GPCRs and GPCR-related genes in insecticide resistance of mosquitoes, Culex quinquefasciatus. A comparison of the expression of 115 GPCR-related genes at a whole genome level between resistant and susceptible Culex mosquitoes identified one and three GPCR-related genes that were up-regulated in highly resistant Culex mosquito strains, HAmCq(G8) and MAmCq(G6), respectively. To characterize the function of these up-regulated GPCR-related genes in resistance, the up-regulated GPCR-related genes were knockdown in HAmCq(G8) and MAmCq(G6) using RNAi technique. Knockdown of these four GPCR-related genes not only decreased resistance of the mosquitoes to permethrin but also repressed the expression of four insecticide resistance-related P450 genes, suggesting the role of GPCR-related genes in resistance is involved in the regulation of resistance P450 gene expression. This results help in understanding of molecular regulation of resistance development in Cx. quinquefasciatus.

  14. Phosphatidylcholine Transfer Protein Interacts with Thioesterase Superfamily Member 2 to Attenuate Insulin Signaling

    OpenAIRE

    Ersoy, Baran A.; Tarun, Akansha; D’Aquino, Katharine; Hancer, Nancy J.; Ukomadu, Chinweike; White, Morris F.; Michel, Thomas; Manning, Brendan D.; Cohen, David E.

    2013-01-01

    Phosphatidylcholine transfer protein (PC-TP) is a phospholipid-binding protein that is enriched in liver and that interacts with thioesterase superfamily member 2 (THEM2). Mice lacking either protein exhibit improved hepatic glucose homeostasis and are resistant to diet-induced diabetes. Insulin receptor substrate 2 (IRS2) and mammalian target of rapamycin complex 1 (mTORC1) are key effectors of insulin signaling, which is attenuated in diabetes. We found that PC-TP inhibited IRS2, as evidenc...

  15. Structural basis for activation of G-protein-coupled receptors

    DEFF Research Database (Denmark)

    Gether, Ulrik; Asmar, Fazila; Meinild, Anne Kristine

    2002-01-01

    into conformational changes accompanying GPCR activation and the underlying molecular mechanism governing transition of the receptor between its active and inactive states. Using the beta2-adrenergic receptor as a model system we have obtained evidence for an evolutionary conserved activation mechanism where...... changes and receptor activation. At the current stage we are exploring the possibility of reaching this goal by direct in situ labeling of the beta2-adrenergic receptor in Xenopus laevis oocytes with conformationally sensitive fluorescent probes and parallel detection of receptor activation by co...

  16. Receptor Activity-modifying Protein-directed G Protein Signaling Specificity for the Calcitonin Gene-related Peptide Family of Receptors.

    Science.gov (United States)

    Weston, Cathryn; Winfield, Ian; Harris, Matthew; Hodgson, Rose; Shah, Archna; Dowell, Simon J; Mobarec, Juan Carlos; Woodlock, David A; Reynolds, Christopher A; Poyner, David R; Watkins, Harriet A; Ladds, Graham

    2016-10-14

    The calcitonin gene-related peptide (CGRP) family of G protein-coupled receptors (GPCRs) is formed through the association of the calcitonin receptor-like receptor (CLR) and one of three receptor activity-modifying proteins (RAMPs). Binding of one of the three peptide ligands, CGRP, adrenomedullin (AM), and intermedin/adrenomedullin 2 (AM2), is well known to result in a Gα s -mediated increase in cAMP. Here we used modified yeast strains that couple receptor activation to cell growth, via chimeric yeast/Gα subunits, and HEK-293 cells to characterize the effect of different RAMP and ligand combinations on this pathway. We not only demonstrate functional couplings to both Gα s and Gα q but also identify a Gα i component to CLR signaling in both yeast and HEK-293 cells, which is absent in HEK-293S cells. We show that the CGRP family of receptors displays both ligand- and RAMP-dependent signaling bias among the Gα s , Gα i , and Gα q/11 pathways. The results are discussed in the context of RAMP interactions probed through molecular modeling and molecular dynamics simulations of the RAMP-GPCR-G protein complexes. This study further highlights the importance of RAMPs to CLR pharmacology and to bias in general, as well as identifying the importance of choosing an appropriate model system for the study of GPCR pharmacology. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  17. A broad G protein-coupled receptor internalization assay that combines SNAP-tag labeling, diffusion-enhanced resonance energy transfer, and a highly emissive terbium cryptate acceptor

    Directory of Open Access Journals (Sweden)

    Angélique eLEVOYE

    2015-11-01

    Full Text Available Although G protein-coupled receptor (GPCR internalization has long been considered a major aspect of the desensitization process that tunes ligand responsiveness, internalization is also involved in receptor resensitization and signaling, as well as the ligand scavenging function of some atypical receptors. Internalization thus contributes to the diversity of GPCR-dependent signaling, and its dynamics and quantification in living cells has generated considerable interest. We developed a robust and sensitive assay to follow and quantify ligand-induced and constitutive GPCR internalization but also receptor recycling in living cells. This assay is based on diffusion-enhanced resonance energy transfer (DERET between cell surface GPCRs labeled with a luminescent terbium cryptate donor and a fluorescein acceptor present in the culture medium. GPCR internalization results in a quantifiable reduction of energy transfer. This method yields a high signal-to-noise ratio due to time-resolved measurements. For various GPCRs belonging to different classes, we demonstrated that constitutive and ligand-induced internalization could be monitored as a function of time and ligand concentration, thus allowing accurate quantitative determination of kinetics of receptor internalization but also half-maximal effective or inhibitory concentrations of compounds. In addition to its selectivity and sensitivity, we provided evidence that DERET-based internalization assay is particularly suitable for characterizing biased ligands. Furthermore, the determination of a Z’-factor value of 0.45 indicates the quality and suitability of DERET-based internalization assay for high-throughput screening (HTS of compounds that may modulate GPCRs internalization.

  18. GABA(B) receptor phosphorylation regulates KCTD12-induced K+ current desensitization

    Czech Academy of Sciences Publication Activity Database

    Adelfinger, L.; Tureček, Rostislav; Ivankova, K.; Jensen, A. A.; Moss, S. J.; Gassmann, M.; Bettler, B.

    2014-01-01

    Roč. 91, č. 3 (2014), s. 369-379 ISSN 0006-2952 Institutional support: RVO:68378041 Keywords : GABA-B * G-protein coupled receptor * GPCR Subject RIV: FH - Neurology Impact factor: 5.009, year: 2014

  19. Gpr161 anchoring of PKA consolidates GPCR and cAMP signaling.

    Science.gov (United States)

    Bachmann, Verena A; Mayrhofer, Johanna E; Ilouz, Ronit; Tschaikner, Philipp; Raffeiner, Philipp; Röck, Ruth; Courcelles, Mathieu; Apelt, Federico; Lu, Tsan-Wen; Baillie, George S; Thibault, Pierre; Aanstad, Pia; Stelzl, Ulrich; Taylor, Susan S; Stefan, Eduard

    2016-07-12

    Scaffolding proteins organize the information flow from activated G protein-coupled receptors (GPCRs) to intracellular effector cascades both spatially and temporally. By this means, signaling scaffolds, such as A-kinase anchoring proteins (AKAPs), compartmentalize kinase activity and ensure substrate selectivity. Using a phosphoproteomics approach we identified a physical and functional connection between protein kinase A (PKA) and Gpr161 (an orphan GPCR) signaling. We show that Gpr161 functions as a selective high-affinity AKAP for type I PKA regulatory subunits (RI). Using cell-based reporters to map protein-protein interactions, we discovered that RI binds directly and selectively to a hydrophobic protein-protein interaction interface in the cytoplasmic carboxyl-terminal tail of Gpr161. Furthermore, our data demonstrate that a binary complex between Gpr161 and RI promotes the compartmentalization of Gpr161 to the plasma membrane. Moreover, we show that Gpr161, functioning as an AKAP, recruits PKA RI to primary cilia in zebrafish embryos. We also show that Gpr161 is a target of PKA phosphorylation, and that mutation of the PKA phosphorylation site affects ciliary receptor localization. Thus, we propose that Gpr161 is itself an AKAP and that the cAMP-sensing Gpr161:PKA complex acts as cilium-compartmentalized signalosome, a concept that now needs to be considered in the analyzing, interpreting, and pharmaceutical targeting of PKA-associated functions.

  20. Immunoglobulin superfamily members encoded by viruses and their multiple roles in immune evasion.

    Science.gov (United States)

    Farré, Domènec; Martínez-Vicente, Pablo; Engel, Pablo; Angulo, Ana

    2017-05-01

    Pathogens have developed a plethora of strategies to undermine host immune defenses in order to guarantee their survival. For large DNA viruses, these immune evasion mechanisms frequently rely on the expression of genes acquired from host genomes. Horizontally transferred genes include members of the immunoglobulin superfamily, whose products constitute the most diverse group of proteins of vertebrate genomes. Their promiscuous immunoglobulin domains, which comprise the building blocks of these molecules, are involved in a large variety of functions mediated by ligand-binding interactions. The flexible structural nature of the immunoglobulin domains makes them appealing targets for viral capture due to their capacity to generate high functional diversity. Here, we present an up-to-date review of immunoglobulin superfamily gene homologs encoded by herpesviruses, poxviruses, and adenoviruses, that include CD200, CD47, Fc receptors, interleukin-1 receptor 2, interleukin-18 binding protein, CD80, carcinoembryonic antigen-related cell adhesion molecules, and signaling lymphocyte activation molecules. We discuss their distinct structural attributes, binding properties, and functions, shaped by evolutionary pressures to disarm specific immune pathways. We include several novel genes identified from extensive genome database surveys. An understanding of the properties and modes of action of these viral proteins may guide the development of novel immune-modulatory therapeutic tools. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  1. Chemogenomic discovery of allosteric antagonists at the GPRC6A receptor

    DEFF Research Database (Denmark)

    Gloriam, David E.; Wellendorph, Petrine; Johansen, Lars Dan

    2011-01-01

    and pharmacological character: (1) chemogenomic lead identification through the first, to our knowledge, ligand inference between two different GPCR families, Families A and C; and (2) the discovery of the most selective GPRC6A allosteric antagonists discovered to date. The unprecedented inference of...... pharmacological activity across GPCR families provides proof-of-concept for in silico approaches against Family C targets based on Family A templates, greatly expanding the prospects of successful drug design and discovery. The antagonists were tested against a panel of seven Family A and C G protein-coupled receptors...

  2. Structural Studies of Nicotinic Acetylcholine Receptors

    DEFF Research Database (Denmark)

    Shahsavar, Azadeh; Gajhede, Michael; Kastrup, Jette

    2016-01-01

    Nicotinic acetylcholine receptors (nAChRs) are members of the pentameric ligand-gated ion channel superfamily that play important roles in control of neurotransmitter release in the central and peripheral nervous system. These receptors are important therapeutic targets for development of drugs...

  3. Development of a human vasopressin V1a-receptor antagonist from an evolutionary-related insect neuropeptide

    DEFF Research Database (Denmark)

    Di Giglio, Maria Giulia; Muttenthaler, Markus; Harpsøe, Kasper

    2017-01-01

    Characterisation of G protein-coupled receptors (GPCR) relies on the availability of a toolbox of ligands that selectively modulate different functional states of the receptors. To uncover such molecules, we explored a unique strategy for ligand discovery that takes advantage of the evolutionary...

  4. A monoclonal antibody for G protein-coupled receptor crystallography

    DEFF Research Database (Denmark)

    Day, Peter W; Rasmussen, Søren Gøgsig Faarup; Parnot, Charles

    2007-01-01

    G protein-coupled receptors (GPCRs) constitute the largest family of signaling proteins in mammals, mediating responses to hormones, neurotransmitters, and senses of sight, smell and taste. Mechanistic insight into GPCR signal transduction is limited by a paucity of high-resolution structural inf...

  5. Modeling structure of G protein-coupled receptors in huan genome

    KAUST Repository

    Zhang, Yang

    2016-01-01

    G protein-coupled receptors (or GPCRs) are integral transmembrane proteins responsible to various cellular signal transductions. Human GPCR proteins are encoded by 5% of human genes but account for the targets of 40% of the FDA approved drugs. Due

  6. Integrin and GPCR Crosstalk in the Regulation of ASM Contraction Signaling in Asthma.

    Science.gov (United States)

    Teoh, Chun Ming; Tam, John Kit Chung; Tran, Thai

    2012-01-01

    Airway hyperresponsiveness (AHR) is one of the cardinal features of asthma. Contraction of airway smooth muscle (ASM) cells that line the airway wall is thought to influence aspects of AHR, resulting in excessive narrowing or occlusion of the airway. ASM contraction is primarily controlled by agonists that bind G protein-coupled receptor (GPCR), which are expressed on ASM. Integrins also play a role in regulating ASM contraction signaling. As therapies for asthma are based on symptom relief, better understanding of the crosstalk between GPCRs and integrins holds good promise for the design of more effective therapies that target the underlying cellular and molecular mechanism that governs AHR. In this paper, we will review current knowledge about integrins and GPCRs in their regulation of ASM contraction signaling and discuss the emerging concept of crosstalk between the two and the implication of this crosstalk on the development of agents that target AHR.

  7. The orphan adhesion-GPCR GPR126 is required for embryonic development in the mouse.

    Directory of Open Access Journals (Sweden)

    Helen Waller-Evans

    2010-11-01

    Full Text Available Adhesion-GPCRs provide essential cell-cell and cell-matrix interactions in development, and have been implicated in inherited human diseases like Usher Syndrome and bilateral frontoparietal polymicrogyria. They are the second largest subfamily of seven-transmembrane spanning proteins in vertebrates, but the function of most of these receptors is still not understood. The orphan Adhesion-GPCR GPR126 has recently been shown to play an essential role in the myelination of peripheral nerves in zebrafish. In parallel, whole-genome association studies have implicated variation at the GPR126 locus as a determinant of body height in the human population. The physiological function of GPR126 in mammals is still unknown. We describe a targeted mutation of GPR126 in the mouse, and show that GPR126 is required for embryonic viability and cardiovascular development.

  8. Application of GPCR Structures for Modelling of Free Fatty Acid Receptors.

    Science.gov (United States)

    Tikhonova, Irina G

    2017-01-01

    Five G protein-coupled receptors (GPCRs) have been identified to be activated by free fatty acids (FFA). Among them, FFA1 (GPR40) and FFA4 (GPR120) bind long-chain fatty acids, FFA2 (GPR43) and FFA3 (GPR41) bind short-chain fatty acids and GPR84 binds medium-chain fatty acids. Free fatty acid receptors have now emerged as potential targets for the treatment of diabetes, obesity and immune diseases. The recent progress in crystallography of GPCRs has now enabled the elucidation of the structure of FFA1 and provided reliable templates for homology modelling of other FFA receptors. Analysis of the crystal structure and improved homology models, along with mutagenesis data and structure activity, highlighted an unusual arginine charge-pairing interaction in FFA1-3 for receptor modulation, distinct structural features for ligand binding to FFA1 and FFA4 and an arginine of the second extracellular loop as a possible anchoring point for FFA at GPR84. Structural data will be helpful for searching novel small-molecule modulators at the FFA receptors.

  9. The activation mechanisms of G protein-coupled receptors : the case of the adenosine A2B and HCA2/3 receptors

    NARCIS (Netherlands)

    Liu, R.

    2016-01-01

    Identifying and elucidating the functions and activation of GPCRs will provide opportunities for novel drug discovery. We confirmed that a yeast system with an extended library of G proteins is very well suited for the study of GPCR activation, G protein coupling profiles, receptor-G protein binding

  10. Molecular Recognition of Corticotropin releasing Factor by Its G protein-coupled Receptor CRFR1

    Energy Technology Data Exchange (ETDEWEB)

    Pioszak, Augen A.; Parker, Naomi R.; Suino-Powell, Kelly; Xu, H. Eric (Van Andel)

    2009-01-15

    The bimolecular interaction between corticotropin-releasing factor (CRF), a neuropeptide, and its type 1 receptor (CRFR1), a class B G-protein-coupled receptor (GPCR), is crucial for activation of the hypothalamic-pituitary-adrenal axis in response to stress, and has been a target of intense drug design for the treatment of anxiety, depression, and related disorders. As a class B GPCR, CRFR1 contains an N-terminal extracellular domain (ECD) that provides the primary ligand binding determinants. Here we present three crystal structures of the human CRFR1 ECD, one in a ligand-free form and two in distinct CRF-bound states. The CRFR1 ECD adopts the alpha-beta-betaalpha fold observed for other class B GPCR ECDs, but the N-terminal alpha-helix is significantly shorter and does not contact CRF. CRF adopts a continuous alpha-helix that docks in a hydrophobic surface of the ECD that is distinct from the peptide-binding site of other class B GPCRs, thereby providing a basis for the specificity of ligand recognition between CRFR1 and other class B GPCRs. The binding of CRF is accompanied by clamp-like conformational changes of two loops of the receptor that anchor the CRF C terminus, including the C-terminal amide group. These structural studies provide a molecular framework for understanding peptide binding and specificity by the CRF receptors as well as a template for designing potent and selective CRFR1 antagonists for therapeutic applications.

  11. Evolutionarily conserved substrate substructures for automated annotation of enzyme superfamilies.

    Directory of Open Access Journals (Sweden)

    Ranyee A Chiang

    2008-08-01

    Full Text Available The evolution of enzymes affects how well a species can adapt to new environmental conditions. During enzyme evolution, certain aspects of molecular function are conserved while other aspects can vary. Aspects of function that are more difficult to change or that need to be reused in multiple contexts are often conserved, while those that vary may indicate functions that are more easily changed or that are no longer required. In analogy to the study of conservation patterns in enzyme sequences and structures, we have examined the patterns of conservation and variation in enzyme function by analyzing graph isomorphisms among enzyme substrates of a large number of enzyme superfamilies. This systematic analysis of substrate substructures establishes the conservation patterns that typify individual superfamilies. Specifically, we determined the chemical substructures that are conserved among all known substrates of a superfamily and the substructures that are reacting in these substrates and then examined the relationship between the two. Across the 42 superfamilies that were analyzed, substantial variation was found in how much of the conserved substructure is reacting, suggesting that superfamilies may not be easily grouped into discrete and separable categories. Instead, our results suggest that many superfamilies may need to be treated individually for analyses of evolution, function prediction, and guiding enzyme engineering strategies. Annotating superfamilies with these conserved and reacting substructure patterns provides information that is orthogonal to information provided by studies of conservation in superfamily sequences and structures, thereby improving the precision with which we can predict the functions of enzymes of unknown function and direct studies in enzyme engineering. Because the method is automated, it is suitable for large-scale characterization and comparison of fundamental functional capabilities of both characterized

  12. Evolutionarily conserved substrate substructures for automated annotation of enzyme superfamilies.

    Science.gov (United States)

    Chiang, Ranyee A; Sali, Andrej; Babbitt, Patricia C

    2008-08-01

    The evolution of enzymes affects how well a species can adapt to new environmental conditions. During enzyme evolution, certain aspects of molecular function are conserved while other aspects can vary. Aspects of function that are more difficult to change or that need to be reused in multiple contexts are often conserved, while those that vary may indicate functions that are more easily changed or that are no longer required. In analogy to the study of conservation patterns in enzyme sequences and structures, we have examined the patterns of conservation and variation in enzyme function by analyzing graph isomorphisms among enzyme substrates of a large number of enzyme superfamilies. This systematic analysis of substrate substructures establishes the conservation patterns that typify individual superfamilies. Specifically, we determined the chemical substructures that are conserved among all known substrates of a superfamily and the substructures that are reacting in these substrates and then examined the relationship between the two. Across the 42 superfamilies that were analyzed, substantial variation was found in how much of the conserved substructure is reacting, suggesting that superfamilies may not be easily grouped into discrete and separable categories. Instead, our results suggest that many superfamilies may need to be treated individually for analyses of evolution, function prediction, and guiding enzyme engineering strategies. Annotating superfamilies with these conserved and reacting substructure patterns provides information that is orthogonal to information provided by studies of conservation in superfamily sequences and structures, thereby improving the precision with which we can predict the functions of enzymes of unknown function and direct studies in enzyme engineering. Because the method is automated, it is suitable for large-scale characterization and comparison of fundamental functional capabilities of both characterized and uncharacterized

  13. Regulation of P450-mediated permethrin resistance in Culex quinquefasciatus by the GPCR/Gαs/AC/cAMP/PKA signaling cascade.

    Science.gov (United States)

    Li, Ting; Liu, Nannan

    2017-12-01

    This study explores the role of G-protein-coupled receptor-intracellular signaling in the development of P450-mediated insecticide resistance in mosquitoes, Culex quinquefasciatus , focusing on the essential function of the GPCRs and their downstream effectors of Gs alpha subunit protein (Gαs) and adenylyl cyclase (ACs) in P450-mediated insecticide resistance of Culex mosquitoes. Our RNAi-mediated functional study showed that knockdown of Gαs caused the decreased expression of the downstream effectors of ACs and PKAs in the GPCR signaling pathway and resistance P450 genes, whereas knockdown of ACs decreased the expression of PKAs and resistance P450 genes. Knockdown of either Gαs or ACs resulted in an increased susceptibility of mosquitoes to permethrin. These results add significantly to our understanding of the molecular basis of resistance P450 gene regulation through GPCR/Gαs/AC/cAMP-PKA signaling pathways in the insecticide resistance of mosquitoes. The temporal and spatial dynamic analyses of GPCRs, Gαs, ACs, PKAs, and P450s in two insecticide resistant mosquito strains revealed that all the GPCR signaling pathway components tested, namely GPCRs, Gαs, ACs and PKAs, were most highly expressed in the brain for both resistant strains, suggesting the role played by these genes in signaling transduction and regulation. The resistance P450 genes were mainly expressed in the brain, midgut and malpighian tubules (MTs), suggesting their critical function in the central nervous system and importance for detoxification. The temporal dynamics analysis for the gene expression showed a diverse expression profile during mosquito development, indicating their initially functional importance in response to exposure to insecticides during their life stages.

  14. Orphan receptor GPR179 forms macromolecular complexes with components of metabotropic signaling cascade in retina ON-bipolar neurons.

    Science.gov (United States)

    Orlandi, Cesare; Cao, Yan; Martemyanov, Kirill A

    2013-10-29

    In the mammalian retina, synaptic transmission between light-excited rod photoreceptors and downstream ON-bipolar neurons is indispensable for dim vision, and disruption of this process leads to congenital stationary night blindness in human patients. The ON-bipolar neurons use the metabotropic signaling cascade, initiated by the mGluR6 receptor, to generate depolarizing responses to light-induced changes in neurotransmitter glutamate release from the photoreceptor axonal terminals. Evidence for the identity of the components involved in transducing these signals is growing rapidly. Recently, the orphan receptor, GPR179, a member of the G protein-coupled receptor (GPCR) superfamily, has been shown to be indispensable for the synaptic responses of ON-bipolar cells. In our study, we investigated the interaction of GPR179 with principle components of the signal transduction cascade. We used immunoprecipitation and proximity ligation assays in transfected cells and native retinas to characterize the protein-protein interactions involving GPR179. The influence of cascade components on GPR179 localization was examined through immunohistochemical staining of the retinas from genetic mouse models. We demonstrated that, in mouse retinas, GPR179 forms physical complexes with the main components of the metabotropic cascade, recruiting mGluR6, TRPM1, and the RGS proteins. Elimination of mGluR6 or RGS proteins, but not TRPM1, detrimentally affects postsynaptic targeting or GPR179 expression. These observations suggest that the mGluR6 signaling cascade is scaffolded as a macromolecular complex in which the interactions between the components ensure the optimal spatiotemporal characteristics of signal transduction.

  15. Quantitative GPCR and ion channel transcriptomics in primary alveolar macrophages and macrophage surrogates

    Directory of Open Access Journals (Sweden)

    Groot-Kormelink Paul J

    2012-10-01

    Full Text Available Abstract Background Alveolar macrophages are one of the first lines of defence against invading pathogens and play a central role in modulating both the innate and acquired immune systems. By responding to endogenous stimuli within the lung, alveolar macrophages contribute towards the regulation of the local inflammatory microenvironment, the initiation of wound healing and the pathogenesis of viral and bacterial infections. Despite the availability of protocols for isolating primary alveolar macrophages from the lung these cells remain recalcitrant to expansion in-vitro and therefore surrogate cell types, such as monocyte derived macrophages and phorbol ester-differentiated cell lines (e.g. U937, THP-1, HL60 are frequently used to model macrophage function. Methods The availability of high throughput gene expression technologies for accurate quantification of transcript levels enables the re-evaluation of these surrogate cell types for use as cellular models of the alveolar macrophage. Utilising high-throughput TaqMan arrays and focussing on dynamically regulated families of integral membrane proteins, we explore the similarities and differences in G-protein coupled receptor (GPCR and ion channel expression in alveolar macrophages and their widely used surrogates. Results The complete non-sensory GPCR and ion channel transcriptome is described for primary alveolar macrophages and macrophage surrogates. The expression of numerous GPCRs and ion channels whose expression were hitherto not described in human alveolar macrophages are compared across primary macrophages and commonly used macrophage cell models. Several membrane proteins known to have critical roles in regulating macrophage function, including CXCR6, CCR8 and TRPV4, were found to be highly expressed in macrophages but not expressed in PMA-differentiated surrogates. Conclusions The data described in this report provides insight into the appropriate choice of cell models for

  16. Post-endocytotic Deubiquitination and Degradation of the MetabotropicAminobutyric Acid Receptor by the Ubiquitin-specific Protease 14

    Czech Academy of Sciences Publication Activity Database

    Lahaie, N.; Králíková, Michaela; Prezeau, L.; Blahoš, Jaroslav; Bouvier, M.

    2016-01-01

    Roč. 291, č. 13 (2016), s. 7156-7170 ISSN 0021-9258 R&D Projects: GA ČR GAP303/12/2408 Institutional support: RVO:68378050 Keywords : bioluminescence resonance energy transfer (BRET) * biosensor * deubiquitylation * G protein-coupled receptor (GPCR) * GABA receptor * protein degradation * receptor endocytosis * ubiquitination Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 4.125, year: 2016

  17. Receptor-G Protein Interaction Studied by Bioluminescence Resonance Energy Transfer: Lessons From Protease-Activated Receptor 1

    Directory of Open Access Journals (Sweden)

    Mohammed Akli eAYOUB

    2012-06-01

    Full Text Available Since its development, the bioluminescence resonance energy transfer (BRET approach has been extensively applied to study G protein-coupled receptors (GPCRs in real time and in live cells. One of the major aspects of GPCRs investigated in considerable details is their physical coupling to the heterotrimeric G proteins. As a result, new concepts have emerged, but few questions are still a matter of debate illustrating the complexity of GPCR-G protein interactions and coupling. Here, we summarized the recent advances on our understanding of GPCR-G protein coupling based on BRET approaches and supported by other FRET-based studies. We essentially focused on our recent studies in which we addressed the concept of preassembly versus the agonist-dependent interaction between the protease-activated receptor 1 (PAR1 and its cognate G proteins. We discussed the concept of agonist-induced conformational changes within the preassembled PAR1-G protein complexes as well as the critical question how the multiple coupling of PAR1 with two different G proteins, Gi1 and G12, but also -arrestin 1, can be regulated.

  18. Interaction of Protease-Activated Receptor 2 with G Proteins and Beta-Arrestin 1 Studied by Bioluminescence Resonance Energy Transfer

    Directory of Open Access Journals (Sweden)

    Mohammed Akli eAyoub

    2013-12-01

    Full Text Available G protein-coupled receptors (GPCRs are well recognized as being able to activate several signaling pathways through the activation of different G proteins as well as other signaling proteins such as beta-arrestins. Therefore, understanding how such multiple GPCR-mediated signaling can be integrated constitute an important aspect. Here, we applied bioluminescence resonance energy transfer (BRET to shed more light on the G protein coupling profile of trypsin receptor, or protease-activated receptor 2 (PAR2, and its interaction with beta-arrestin1. Using YFP and Rluc fusion constructs expressed in COS-7 cells, BRET data revealed a pre-assembly of PAR2 with both Galphai1 and Galphao and a rapid and transient activation of these G proteins upon receptor activation. In contrast, no preassembly of PAR2 with Galpha12 could be detected and their physical association can be measured with a very slow and sustained kinetics similar to that of beta-arrestin1 recruitment. These data demonstrate the coupling of PAR2 with Galphai1, Galphao and Galpha12 in COS-7 cells with differences in the kinetics of GPCR-G protein coupling, a parameter that very likely influences the cellular response. Moreover, this further illustrates that preassembly or agonist-induced G protein interaction depends on receptor-G protein pairs indicating another level of complexity and regulation of the signaling of GPCR-G protein complexes and its multiplicity.

  19. Main trends of karyotype evolution in the superfamily Chalcidoidea (Hymenoptera

    Directory of Open Access Journals (Sweden)

    Vladimir Gokhman

    2009-08-01

    Full Text Available An overview of karyotype evolution in the superfamily Chalcidoidea is given. Structural types of chromosome sets in the superfamily are listed. Main pathways of karyotypic change in the Chalcidoidea are outlined. The chromosome set containing eleven subtelo- or acrocentrics is considered as an ancestral karyotype for the superfamily. Multiple independent reductions in n values through chromosomal fusions presumably occurred in various groups of chalcid families.

  20. 3-Iodothyronamine, a Novel Endogenous Modulator of Transient Receptor Potential Melastatin 8?

    Directory of Open Access Journals (Sweden)

    Noushafarin Khajavi

    2017-08-01

    Full Text Available The decarboxylated and deiodinated thyroid hormone (TH derivative, 3-iodothyronamine (3-T1AM, is suggested to be involved in energy metabolism and thermoregulation. G protein-coupled receptors (GPCRs are known as the main targets for 3-T1AM; however, transient receptor potential channels (TRPs were also recently identified as new targets of 3-T1AM. This article reviews the current knowledge of a putative novel role of 3-T1AM in the modulation of TRPs. Specifically, the TRP melastatin 8 (TRPM8 was identified as a target of 3-T1AM in different cell types including neoplastic cells, whereby 3-T1AM significantly increased cytosolic Ca2+ through TRPM8 activation. Similarly, the β-adrenergic receptor is involved in 3-T1AM-induced Ca2+ influx. Therefore, it has been suggested that 3-T1AM-induced Ca2+ mobilization might be due to β-adrenergic receptor/TRPM8 channel interaction, which adds to the complexity of GPCR regulation by TRPs. It has been revealed that TRPM8 activation leads to a decline in TRPV1 activity, which may be of therapeutic benefit in clinical circumstances such as treatment of TRPV1-mediated inflammatory hyperalgesia, colitis, and dry eye syndrome. This review also summarizes the inverse association between changes in TRPM8 and TRPV1 activity after 3-T1AM stimulation. This finding prompted further detailed investigations of the interplay between 3-T1AM and the GPCR/TRPM8 axis and indicated the probability of additional GPCR/TRP constellations that are modulated by this TH derivative.

  1. Nuclear receptors from the ctenophore Mnemiopsis leidyi lack a zinc-finger DNA-binding domain: lineage-specific loss or ancestral condition in the emergence of the nuclear receptor superfamily?

    Directory of Open Access Journals (Sweden)

    Reitzel Adam M

    2011-02-01

    Full Text Available Abstract Background Nuclear receptors (NRs are an ancient superfamily of metazoan transcription factors that play critical roles in regulation of reproduction, development, and energetic homeostasis. Although the evolutionary relationships among NRs are well-described in two prominent clades of animals (deuterostomes and protostomes, comparatively little information has been reported on the diversity of NRs in early diverging metazoans. Here, we identified NRs from the phylum Ctenophora and used a phylogenomic approach to explore the emergence of the NR superfamily in the animal kingdom. In addition, to gain insight into conserved or novel functions, we examined NR expression during ctenophore development. Results We report the first described NRs from the phylum Ctenophora: two from Mnemiopsis leidyi and one from Pleurobrachia pileus. All ctenophore NRs contained a ligand-binding domain and grouped with NRs from the subfamily NR2A (HNF4. Surprisingly, all the ctenophore NRs lacked the highly conserved DNA-binding domain (DBD. NRs from Mnemiopsis were expressed in different regions of developing ctenophores. One was broadly expressed in the endoderm during gastrulation. The second was initially expressed in the ectoderm during gastrulation, in regions corresponding to the future tentacles; subsequent expression was restricted to the apical organ. Phylogenetic analyses of NRs from ctenophores, sponges, cnidarians, and a placozoan support the hypothesis that expansion of the superfamily occurred in a step-wise fashion, with initial radiations in NR family 2, followed by representatives of NR families 3, 6, and 1/4 originating prior to the appearance of the bilaterian ancestor. Conclusions Our study provides the first description of NRs from ctenophores, including the full complement from Mnemiopsis. Ctenophores have the least diverse NR complement of any animal phylum with representatives that cluster with only one subfamily (NR2A. Ctenophores and

  2. Identification of the bacteria-binding peptide domain on salivary agglutinin (gp-340/DMBT1), a member of the scavenger receptor cysteine-rich superfamily

    DEFF Research Database (Denmark)

    Bikker, Floris J; Ligtenberg, Antoon J M; Nazmi, Kamran

    2002-01-01

    Salivary agglutinin is encoded by DMBT1 and identical to gp-340, a member of the scavenger receptor cysteine-rich (SRCR) superfamily. Salivary agglutinin/DMBT1 is known for its Streptococcus mutans agglutinating properties. This 300-400 kDa glycoprotein is composed of conserved peptide motifs: 14...... containing exclusively SRCR and SID domains that binds to S. mutans. To define more closely the S. mutans-binding domain, consensus-based peptides of the SRCR domains and SIDs were designed and synthesized. Only one of the SRCR peptides, designated SRCRP2, and none of the SID peptides bound to S. mutans....... Strikingly, this peptide was also able to induce agglutination of S. mutans and a number of other bacteria. The repeated presence of this peptide in the native molecule endows agglutinin/DMBT1 with a general bacterial binding feature with a multivalent character. Moreover, our studies demonstrate...

  3. Phosphorylation of G Protein-Coupled Receptors: From the Barcode Hypothesis to the Flute Model.

    Science.gov (United States)

    Yang, Zhao; Yang, Fan; Zhang, Daolai; Liu, Zhixin; Lin, Amy; Liu, Chuan; Xiao, Peng; Yu, Xiao; Sun, Jin-Peng

    2017-09-01

    Seven transmembrane G protein-coupled receptors (GPCRs) are often phosphorylated at the C terminus and on intracellular loops in response to various extracellular stimuli. Phosphorylation of GPCRs by GPCR kinases and certain other kinases can promote the recruitment of arrestin molecules. The arrestins critically regulate GPCR functions not only by mediating receptor desensitization and internalization, but also by redirecting signaling to G protein-independent pathways via interactions with numerous downstream effector molecules. Accumulating evidence over the past decade has given rise to the phospho-barcode hypothesis, which states that ligand-specific phosphorylation patterns of a receptor direct its distinct functional outcomes. Our recent work using unnatural amino acid incorporation and fluorine-19 nuclear magnetic resonance ( 19 F-NMR) spectroscopy led to the flute model, which provides preliminary insight into the receptor phospho-coding mechanism, by which receptor phosphorylation patterns are recognized by an array of phosphate-binding pockets on arrestin and are translated into distinct conformations. These selective conformations are recognized by various effector molecules downstream of arrestin. The phospho-barcoding mechanism enables arrestin to recognize a wide range of phosphorylation patterns of GPCRs, contributing to their diverse functions. Copyright © 2017 by The Author(s).

  4. An improved classification of G-protein-coupled receptors using sequence-derived features

    Directory of Open Access Journals (Sweden)

    Peng Zhen-Ling

    2010-08-01

    Full Text Available Abstract Background G-protein-coupled receptors (GPCRs play a key role in diverse physiological processes and are the targets of almost two-thirds of the marketed drugs. The 3 D structures of GPCRs are largely unavailable; however, a large number of GPCR primary sequences are known. To facilitate the identification and characterization of novel receptors, it is therefore very valuable to develop a computational method to accurately predict GPCRs from the protein primary sequences. Results We propose a new method called PCA-GPCR, to predict GPCRs using a comprehensive set of 1497 sequence-derived features. The principal component analysis is first employed to reduce the dimension of the feature space to 32. Then, the resulting 32-dimensional feature vectors are fed into a simple yet powerful classification algorithm, called intimate sorting, to predict GPCRs at five levels. The prediction at the first level determines whether a protein is a GPCR or a non-GPCR. If it is predicted to be a GPCR, then it will be further predicted into certain family, subfamily, sub-subfamily and subtype by the classifiers at the second, third, fourth, and fifth levels, respectively. To train the classifiers applied at five levels, a non-redundant dataset is carefully constructed, which contains 3178, 1589, 4772, 4924, and 2741 protein sequences at the respective levels. Jackknife tests on this training dataset show that the overall accuracies of PCA-GPCR at five levels (from the first to the fifth can achieve up to 99.5%, 88.8%, 80.47%, 80.3%, and 92.34%, respectively. We further perform predictions on a dataset of 1238 GPCRs at the second level, and on another two datasets of 167 and 566 GPCRs respectively at the fourth level. The overall prediction accuracies of our method are consistently higher than those of the existing methods to be compared. Conclusions The comprehensive set of 1497 features is believed to be capable of capturing information about amino acid

  5. Phylogenomic analysis of the GIY-YIG nuclease superfamily

    Directory of Open Access Journals (Sweden)

    Bujnicki Janusz M

    2006-04-01

    Full Text Available Abstract Background The GIY-YIG domain was initially identified in homing endonucleases and later in other selfish mobile genetic elements (including restriction enzymes and non-LTR retrotransposons and in enzymes involved in DNA repair and recombination. However, to date no systematic search for novel members of the GIY-YIG superfamily or comparative analysis of these enzymes has been reported. Results We carried out database searches to identify all members of known GIY-YIG nuclease families. Multiple sequence alignments together with predicted secondary structures of identified families were represented as Hidden Markov Models (HMM and compared by the HHsearch method to the uncharacterized protein families gathered in the COG, KOG, and PFAM databases. This analysis allowed for extending the GIY-YIG superfamily to include members of COG3680 and a number of proteins not classified in COGs and to predict that these proteins may function as nucleases, potentially involved in DNA recombination and/or repair. Finally, all old and new members of the GIY-YIG superfamily were compared and analyzed to infer the phylogenetic tree. Conclusion An evolutionary classification of the GIY-YIG superfamily is presented for the very first time, along with the structural annotation of all (subfamilies. It provides a comprehensive picture of sequence-structure-function relationships in this superfamily of nucleases, which will help to design experiments to study the mechanism of action of known members (especially the uncharacterized ones and will facilitate the prediction of function for the newly discovered ones.

  6. Emerging Paradigm of Intracellular Targeting of G Protein-Coupled Receptors.

    Science.gov (United States)

    Chaturvedi, Madhu; Schilling, Justin; Beautrait, Alexandre; Bouvier, Michel; Benovic, Jeffrey L; Shukla, Arun K

    2018-05-04

    G protein-coupled receptors (GPCRs) recognize a diverse array of extracellular stimuli, and they mediate a broad repertoire of signaling events involved in human physiology. Although the major effort on targeting GPCRs has typically been focused on their extracellular surface, a series of recent developments now unfold the possibility of targeting them from the intracellular side as well. Allosteric modulators binding to the cytoplasmic surface of GPCRs have now been described, and their structural mechanisms are elucidated by high-resolution crystal structures. Furthermore, pepducins, aptamers, and intrabodies targeting the intracellular face of GPCRs have also been successfully utilized to modulate receptor signaling. Moreover, small molecule compounds, aptamers, and synthetic intrabodies targeting β-arrestins have also been discovered to modulate GPCR endocytosis and signaling. Here, we discuss the emerging paradigm of intracellular targeting of GPCRs, and outline the current challenges, potential opportunities, and future outlook in this particular area of GPCR biology. Copyright © 2018 Elsevier Ltd. All rights reserved.

  7. Two different groups of signal sequence in M-superfamily conotoxins.

    Science.gov (United States)

    Wang, Qi; Jiang, Hui; Han, Yu-Hong; Yuan, Duo-Duo; Chi, Cheng-Wu

    2008-04-01

    M-superfamily conotoxins can be divided into four branches (M-1, M-2, M-3 and M-4) according to the number of amino acid residues in the third Cys loop. In general, it is widely accepted that the conotoxin signal peptides of each superfamily are strictly conserved. Recently, we cloned six cDNAs of novel M-superfamily conotoxins from Conus leopardus, Conus marmoreus and Conus quercinus, belonging to either M-1 or M-3 branch. These conotoxins, judging from the putative peptide sequences deducted from cDNAs, are rich in acidic residues and share highly conserved signal and pro-peptide region. However, they are quite different from the reported conotoxins of M-2 and M-4 branches even in their signal peptides, which in general are considered highly conserved for each superfamily of conotoxins. The signal sequences of M-1 and M-3 conotoxins composed of 24 residues start with MLKMGVVL-, while those of M-2 and M-4 conotoxins composed of 25 residues start with MMSKLGVL-. It is another example that different types of signal peptides can exist within a superfamily besides the I-conotoxin superfamily. In addition to the different disulfide connectivity of M-1 conotoxins from that of M-4 or M-2 conotoxins, the sequence alignment, preferential Cys codon usage and phylogenetic tree analysis suggest that M-1 and M-3 conotoxins have much closer relationship, being different from the conotoxins of other two branches (M-4 and M-2) of M-superfamily.

  8. Pathophysiology of GPCR Homo- and Heterodimerization: Special Emphasis on Somatostatin Receptors

    Directory of Open Access Journals (Sweden)

    Rishi K. Somvanshi

    2012-04-01

    Full Text Available G-protein coupled receptors (GPCRs are cell surface proteins responsible for translating >80% of extracellular reception to intracellular signals. The extracellular information in the form of neurotransmitters, peptides, ions, odorants etc is converted to intracellular signals via a wide variety of effector molecules activating distinct downstream signaling pathways. All GPCRs share common structural features including an extracellular N-terminal, seven-transmembrane domains (TMs linked by extracellular/intracellular loops and the C-terminal tail. Recent studies have shown that most GPCRs function as dimers (homo- and/or heterodimers or even higher order of oligomers. Protein-protein interaction among GPCRs and other receptor proteins play a critical role in the modulation of receptor pharmacology and functions. Although ~50% of the current drugs available in the market target GPCRs, still many GPCRs remain unexplored as potential therapeutic targets, opening immense possibility to discover the role of GPCRs in pathophysiological conditions. This review explores the existing information and future possibilities of GPCRs as tools in clinical pharmacology and is specifically focused for the role of somatostatin receptors (SSTRs in pathophysiology of diseases and as the potential candidate for drug discovery.

  9. Impedance analysis of GPCR-mediated changes in endothelial barrier function: overview and fundamental considerations for stable and reproducible measurements.

    Science.gov (United States)

    Stolwijk, Judith A; Matrougui, Khalid; Renken, Christian W; Trebak, Mohamed

    2015-10-01

    The past 20 years has seen significant growth in using impedance-based assays to understand the molecular underpinning of endothelial and epithelial barrier function in response to physiological agonists and pharmacological and toxicological compounds. Most studies on barrier function use G protein-coupled receptor (GPCR) agonists which couple to fast and transient changes in barrier properties. The power of impedance-based techniques such as electric cell-substrate impedance sensing (ECIS) resides in its ability to detect minute changes in cell layer integrity label-free and in real-time ranging from seconds to days. We provide a comprehensive overview of the biophysical principles, applications, and recent developments in impedance-based methodologies. Despite extensive application of impedance analysis in endothelial barrier research, little attention has been paid to data analysis and critical experimental variables, which are both essential for signal stability and reproducibility. We describe the rationale behind common ECIS data presentation and interpretation and illustrate practical guidelines to improve signal intensity by adapting technical parameters such as electrode layout, monitoring frequency, or parameter (resistance versus impedance magnitude). Moreover, we discuss the impact of experimental parameters, including cell source, liquid handling, and agonist preparation on signal intensity and kinetics. Our discussions are supported by experimental data obtained from human microvascular endothelial cells challenged with three GPCR agonists, thrombin, histamine, and sphingosine-1-phosphate.

  10. Glucagon-like peptide-1 and its class B G Protein-coupled receptors: A long march to therapeutic successes.

    NARCIS (Netherlands)

    de Graaf, C.; Donnely, D.; Wootten, D.; Lau, J.; Sexton, P.M.; Miller, L.J.; Ahn, J.M.; Liao, J.; Fletcher, M.M.; Yang, D.; Brown, A.J.; Zhou, C.; Deng, J.; Wang, M.W.

    2016-01-01

    Theglucagon-likepeptide (GLP)-1receptor (GLP-1R) is a class B G protein-coupled receptor (GPCR) that mediates the action of GLP-1, a peptide hormone secretedfromthreemajor tissues inhumans,enteroendocrine L cells in the distal intestine, a cells in the pancreas, and the central nervous system, which

  11. Maturing of the nuclear receptor family.

    Science.gov (United States)

    Lazar, Mitchell A

    2017-04-03

    Members of the nuclear receptor (NR) superfamily of ligand-regulated transcription factors play important roles in reproduction, development, and physiology. In humans, genetic mutations in NRs are causes of rare diseases, while hormones and drugs that target NRs are in widespread therapeutic use. The present issue of the JCI includes a series of Review articles focused on specific NRs and their wide range of biological functions. Here I reflect on the past, present, and potential future highlights of research on the NR superfamily.

  12. Insight into partial agonism by observing multiple equilibria for ligand-bound and Gs-mimetic nanobody-bound β1-adrenergic receptor.

    Science.gov (United States)

    Solt, Andras S; Bostock, Mark J; Shrestha, Binesh; Kumar, Prashant; Warne, Tony; Tate, Christopher G; Nietlispach, Daniel

    2017-11-27

    A complex conformational energy landscape determines G-protein-coupled receptor (GPCR) signalling via intracellular binding partners (IBPs), e.g., G s and β-arrestin. Using 13 C methyl methionine NMR for the β 1 -adrenergic receptor, we identify ligand efficacy-dependent equilibria between an inactive and pre-active state and, in complex with G s -mimetic nanobody, between more and less active ternary complexes. Formation of a basal activity complex through ligand-free nanobody-receptor interaction reveals structural differences on the cytoplasmic receptor side compared to the full agonist-bound nanobody-coupled form, suggesting that ligand-induced variations in G-protein interaction underpin partial agonism. Significant differences in receptor dynamics are observed ranging from rigid nanobody-coupled states to extensive μs-to-ms timescale dynamics when bound to a full agonist. We suggest that the mobility of the full agonist-bound form primes the GPCR to couple to IBPs. On formation of the ternary complex, ligand efficacy determines the quality of the interaction between the rigidified receptor and an IBP and consequently the signalling level.

  13. Low multiple electrode aggregometry platelet responses are not associated with non-synonymous variants in G-protein coupled receptor genes.

    Science.gov (United States)

    Norman, Jane E; Lee, Kurtis R; Walker, Mary E; Murden, Sherina L; Harris, Jessica; Mundell, Stuart; J Murphy, Gavin; Mumford, Andrew D

    2015-10-01

    Multiple electrode aggregometry (MEA) improves prediction of thrombosis and bleeding in cardiac patients. However, the causes of inter-individual variation in MEA results are incompletely understood. We explore whether low MEA results are associated with platelet G-protein coupled receptor (GPCR) gene variants. The effects of P2Y12 receptor (P2Y12), thromboxane A2 receptor (TPα) and protease-activated receptor 1 (PAR1) dysfunction on the MEA ADP-test, ASPI-test and TRAP-test were determined using receptor antagonists. Cardiac surgery patients with pre-operative MEA results suggesting GPCR dysfunction were selected for P2Y12 (P2RY12), TPα (TBXA2R) and PAR1 (F2R) sequencing. In control blood samples, P2Y12, TPα or PAR1 antagonists markedly reduced ADP-test, ASPI-test and TRAP-test results respectively. In the 636 patients from a cohort of 2388 cardiac surgery patients who were not receiving aspirin or a P2Y12 blocker, the median ADP-test result was 75.1 U (range 4.8-153.2), ASPI-test 83.7 U (1.4-157.3) and TRAP-test 117.7 U (2.4-194.1), indicating a broad range of results unexplained by anti-platelet drugs. In 238 consenting patients with unexplained low MEA results, three P2RY12 variants occurred in 70/107 (65%) with suspected P2Y12 dysfunction and four TBXA2R variants occurred in 19/22 (86%) with suspected TPα dysfunction although the later group was too small to draw meaningful conclusions about variant frequency. All the variants were synonymous and unlikely to cause GPCR dysfunction. There were no F2R variants in the 109 cases with suspected PAR1 dysfunction. MEA results suggesting isolated platelet GPCR dysfunction were common in cardiac surgery patients, but were not associated with non-synonymous variants in P2RY12 or F2R. Copyright © 2015 Elsevier Ltd. All rights reserved.

  14. Chronic regulation of colonic epithelial secretory function by activation of G protein-coupled receptors.

    LENUS (Irish Health Repository)

    Toumi, F

    2011-02-01

    Enteric neurotransmitters that act at G protein-coupled receptors (GPCRs) are well known to acutely promote epithelial Cl(-) and fluid secretion. Here we examined if acute GPCR activation might have more long-term consequences for epithelial secretory function.

  15. Heterotrimeric G Protein-coupled Receptor Signaling in Yeast Mating Pheromone Response.

    Science.gov (United States)

    Alvaro, Christopher G; Thorner, Jeremy

    2016-04-08

    The DNAs encoding the receptors that respond to the peptide mating pheromones of the budding yeastSaccharomyces cerevisiaewere isolated in 1985, and were the very first genes for agonist-binding heterotrimeric G protein-coupled receptors (GPCRs) to be cloned in any organism. Now, over 30 years later, this yeast and its receptors continue to provide a pathfinding experimental paradigm for investigating GPCR-initiated signaling and its regulation, as described in this retrospective overview. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  16. Structural basis for chemokine recognition and activation of a viral G protein-coupled receptor

    Energy Technology Data Exchange (ETDEWEB)

    Burg, John S.; Ingram, Jessica R.; Venkatakrishnan, A.J.; Jude, Kevin M.; Dukkipati, Abhiram; Feinberg, Evan N.; Angelini, Alessandro; Waghray, Deepa; Dror, Ron O.; Ploegh, Hidde L.; Garcia, K. Christopher (Stanford); (Stanford-MED); (Whitehead); (MIT)

    2015-03-05

    Chemokines are small proteins that function as immune modulators through activation of chemokine G protein-coupled receptors (GPCRs). Several viruses also encode chemokines and chemokine receptors to subvert the host immune response. How protein ligands activate GPCRs remains unknown. We report the crystal structure at 2.9 angstrom resolution of the human cytomegalovirus GPCR US28 in complex with the chemokine domain of human CX3CL1 (fractalkine). The globular body of CX3CL1 is perched on top of the US28 extracellular vestibule, whereas its amino terminus projects into the central core of US28. The transmembrane helices of US28 adopt an active-state-like conformation. Atomic-level simulations suggest that the agonist-independent activity of US28 may be due to an amino acid network evolved in the viral GPCR to destabilize the receptor’s inactive state.

  17. G protein-coupled receptor modulation with pepducins: moving closer to the clinic

    DEFF Research Database (Denmark)

    Dimond, Patricia; Carlson, Kenneth; Bouvier, Michel

    2011-01-01

    At the 2nd Pepducin Science Symposium held in Cambridge, Massachusetts, on November 4–5, 2010, investigators working in G protein–coupled receptor (GPCR) research convened to discuss progress since last year's inaugural conference. This year's symposium focused on increasing knowledge of the stru...

  18. Sequential inter- and intrasubunit rearrangements during activation of dimeric metabotropic glutamate receptor 1

    Czech Academy of Sciences Publication Activity Database

    Hlaváčková, Veronika; Zabel, U.; Franková, Daniela; Batz, J.; Hoffmann, C.; Prezeau, L.; Pin, J. P.; Blahoš, Jaroslav; Lohse, M. J.

    2012-01-01

    Roč. 5, č. 237 (2012), ra59 ISSN 1937-9145 R&D Projects: GA ČR GA303/08/1591; GA MŠk(CZ) LC06063; GA ČR GAP303/12/2408 Institutional research plan: CEZ:AV0Z50520514 Keywords : G-protein coupled receptor * metabotropic glutamate receptor 1 * class C GPCR Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 7.648, year: 2012

  19. Fatty acids activate a chimera of the clofibric acid-activated receptor and the glucocorticoid receptor.

    Science.gov (United States)

    Göttlicher, M; Widmark, E; Li, Q; Gustafsson, J A

    1992-01-01

    Peroxisome proliferators such as clofibric acid, nafenopin, and WY-14,643 have been shown to activate PPAR (peroxisome proliferator-activated receptor), a member of the steroid nuclear receptor superfamily. We have cloned the cDNA from the rat that is homologous to that from the mouse [Issemann, I. & Green, S. (1990) Nature (London) 347, 645-650], which encodes a 97% similar protein with a particularly well-conserved putative ligand-binding domain. To search for physiologically occurring activators, we established a transcriptional transactivation assay by stably expressing in CHO cells a chimera of rat PPAR and the human glucocorticoid receptor that activates expression of the placental alkaline phosphatase reporter gene under the control of the mouse mammary tumor virus promoter. Testing of compounds related to lipid metabolism or peroxisomal proliferation revealed that 150 microM concentrations of arachidonic or linoleic acid but not of dehydroepiandrosterone, cholesterol, or 25-hydroxy-cholesterol, activate the receptor chimera. In addition, saturated fatty acids induce the reporter gene. Shortening the chain length to n = 6 or introduction of an omega-terminal carboxylic group abolished the activation potential of the fatty acid. In conclusion, the present results indicate that fatty acids can regulate gene expression mediated by a member of the steroid nuclear receptor superfamily. Images PMID:1316614

  20. New functions and signaling mechanisms for the class of adhesion G protein-coupled receptors

    DEFF Research Database (Denmark)

    Liebscher, Ines; Ackley, Brian; Araç, Demet

    2014-01-01

    The class of adhesion G protein-coupled receptors (aGPCRs), with 33 human homologs, is the second largest family of GPCRs. In addition to a seven-transmembrane α-helix-a structural feature of all GPCRs-the class of aGPCRs is characterized by the presence of a large N-terminal extracellular region....... In addition, all aGPCRs but one (GPR123) contain a GPCR autoproteolysis-inducing (GAIN) domain that mediates autoproteolytic cleavage at the GPCR autoproteolysis site motif to generate N- and a C-terminal fragments (NTF and CTF, respectively) during protein maturation. Subsequently, the NTF and CTF...

  1. The current state of GPCR-based drug discovery to treat metabolic disease.

    Science.gov (United States)

    Sloop, Kyle W; Emmerson, Paul J; Statnick, Michael A; Willard, Francis S

    2018-02-02

    One approach of modern drug discovery is to identify agents that enhance or diminish signal transduction cascades in various cell types and tissues by modulating the activity of GPCRs. This strategy has resulted in the development of new medicines to treat many conditions, including cardiovascular disease, psychiatric disorders, HIV/AIDS, certain forms of cancer and Type 2 diabetes mellitus (T2DM). These successes justify further pursuit of GPCRs as disease targets and provide key learning that should help guide identifying future therapeutic agents. This report reviews the current landscape of GPCR drug discovery with emphasis on efforts aimed at developing new molecules for treating T2DM and obesity. We analyse historical efforts to generate GPCR-based drugs to treat metabolic disease in terms of causal factors leading to success and failure in this endeavour. © 2018 The British Pharmacological Society.

  2. MetaSINEs: Broad Distribution of a Novel SINE Superfamily in Animals.

    Science.gov (United States)

    Nishihara, Hidenori; Plazzi, Federico; Passamonti, Marco; Okada, Norihiro

    2016-02-12

    SINEs (short interspersed elements) are transposable elements that typically originate independently in each taxonomic clade (order/family). However, some SINE families share a highly similar central sequence and are thus categorized as a SINE superfamily. Although only four SINE superfamilies (CORE-SINEs, V-SINEs, DeuSINEs, and Ceph-SINEs) have been reported so far, it is expected that new SINE superfamilies would be discovered by deep exploration of new SINEs in metazoan genomes. Here we describe 15 SINEs, among which 13 are novel, that have a similar 66-bp central region and therefore constitute a new SINE superfamily, MetaSINEs. MetaSINEs are distributed from fish to cnidarians, suggesting their common evolutionary origin at least 640 Ma. Because the 3' tails of MetaSINEs are variable, these SINEs most likely survived by changing their partner long interspersed elements for retrotransposition during evolution. Furthermore, we examined the presence of members of other SINE superfamilies in bivalve genomes and characterized eight new SINEs belonging to the CORE-SINEs, V-SINEs, and DeuSINEs, in addition to the MetaSINEs. The broad distribution of bivalve SINEs suggests that at least three SINEs originated in the common ancestor of Bivalvia. Our comparative analysis of the central domains of the SINEs revealed that, in each superfamily, only a restricted region is shared among all of its members. Because the functions of the central domains of the SINE superfamilies remain unknown, such structural information of SINE superfamilies will be useful for future experimental and comparative analyses to reveal why they have been retained in metazoan genomes during evolution. © The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

  3. A G protein-coupled receptor (GPCR) in red: live cell imaging of the kappa opioid receptor-tdTomato fusion protein (KOPR-tdT) in neuronal cells

    Science.gov (United States)

    Huang, Peng; Chiu, Yi-Ting; Chen, Chongguang; Wang, Yujun; Liu-Chen, Lee-Yuan

    2013-01-01

    Introduction In contrast to green fluorescent protein and variants (GFPs), red fluorescent proteins (RFPs) have rarely been employed for generation of GPCR fusion proteins, likely because of formation of aggregates and cell toxicity of some RFPs. Among all the RFPs available, tdTomato (tdT), one of the non-aggregating RFP, has the highest brightness score (about 3 times that of eGFP) and unsurpassed photostability. Methods We fused tdT to the KOPR C-terminus. The KOPR-tdT cDNA construct was transfected into Neuro2A mouse neuroblastoma cell line (Neuro2A cells) and rat cortical primary neurons for characterization of pharmacological properties and imaging studies on KOPR trafficking. Results KOPR-tdT retained KOPR properties (cell surface expression, ligand binding, agonist-induced signaling and internalization) when expressed in Neuro2A cells and rat primary cortical neurons. Live cell imaging of KOPR-tdT enables visualization of time course of agonist-induced internalization of KOPR in real time for 60 min, without photobleaching and apparent cell toxicity. U50,488H-induced KOPR internalization occurred as early as 4 min and plateaued at about 30 min. A unique pattern of internalized KOPR in processes of primary neurons was induced by U50,488H. Discussion tdT is an alternative to, or even a better tool than, GFPs for fusing to GPCR for trafficking studies, because tdT has higher brightness and thus better resolution and less photobleaching problems due to reduced laser power used. It also has advantages associated with its longer-wavelength emission including spectral separation from autofluorescence and GFPs, reduced cell toxicity the laser may impose, and greater tissue penetration. These advantages of tdT over GPFs may be critical for live cell imaging studies of GPCRs in vitro and for studying GPCRs in vivo because of their low abundance. PMID:23856011

  4. The aldo-keto reductase superfamily homepage.

    Science.gov (United States)

    Hyndman, David; Bauman, David R; Heredia, Vladi V; Penning, Trevor M

    2003-02-01

    The aldo-keto reductases (AKRs) are one of the three enzyme superfamilies that perform oxidoreduction on a wide variety of natural and foreign substrates. A systematic nomenclature for the AKR superfamily was adopted in 1996 and was updated in September 2000 (visit www.med.upenn.edu/akr). Investigators have been diligent in submitting sequences of functional proteins to the Web site. With the new additions, the superfamily contains 114 proteins expressed in prokaryotes and eukaryotes that are distributed over 14 families (AKR1-AKR14). The AKR1 family contains the aldose reductases, the aldehyde reductases, the hydroxysteroid dehydrogenases and steroid 5beta-reductases, and is the largest. Other families of interest include AKR6, which includes potassium channel beta-subunits, and AKR7 the aflatoxin aldehyde reductases. Two new families include AKR13 (yeast aldose reductase) and AKR14 (Escherichia coli aldehyde reductase). Crystal structures of many AKRs and their complexes with ligands are available in the PDB and accessible through the Web site. Each structure has the characteristic (alpha/beta)(8)-barrel motif of the superfamily, a conserved cofactor binding site and a catalytic tetrad, and variable loop structures that define substrate specificity. Although the majority of AKRs are monomeric proteins of about 320 amino acids in length, the AKR2, AKR6 and AKR7 family may form multimers. To expand the nomenclature to accommodate multimers, we recommend that the composition and stoichiometry be listed. For example, AKR7A1:AKR7A4 (1:3) would designate a tetramer of the composition indicated. The current nomenclature is recognized by the Human Genome Project (HUGO) and the Web site provides a link to genomic information including chromosomal localization, gene boundaries, human ESTs and SNPs and much more.

  5. Impact of cell type and epitope tagging on heterologous expression of G protein-coupled receptor: a systematic study on angiotensin type II receptor.

    Directory of Open Access Journals (Sweden)

    Lili Jiang

    Full Text Available Despite heterologous expression of epitope-tagged GPCR is widely adopted for functional characterization, there is lacking of systematic analysis of the impact of expression host and epitope tag on GPCR expression. Angiotensin type II (AT2 receptor displays agonist-dependent and -independent activities, coupling to a spectrum of signaling molecules. However, consensus has not been reached on the subcellular distributions, signaling cascades and receptor-mediated actions. To examine the contributions of host cell and epitope tag on receptor expression and activity, epitope-tagged AT2 receptor variants were transiently or stably expressed in HEK293, CHO-K1 and PC12 cells. The epitope-tagged AT2 receptor variants were detected both on the cell membrane and in the perinuclear region. In transiently transfected HEK293 cells, Myc-AT2 existed predominantly as monomer. Additionally, a ladder of ubiquitinated AT2 receptor proteins was detected. By contrast, stably expressed epitope-tagged AT2 receptor variants existed as both monomer and high molecular weight complexes, and the latter was enriched in cell surface. Glycosylation promoted cell surface expression of Myc-AT2 but had no effect on AT2-GFP in HEK293 cells. In cells that stably expressed Myc-AT2, serum starvation induced apoptosis in CHO-K1 cells but not in HEK293 or PC12 cells. Instead, HEK293 and PC12 cells stably expressing Myc-AT2 exhibited partial cell cycle arrest with cells accumulating at G1 and S phases, respectively. Taken together, these results suggest that expression levels, subcellular distributions and ligand-independent constitutive activities of AT2 receptor were cell type-dependent while posttranslational processing of nascent AT2 receptor protein was modulated by epitope tag and mode of expression.

  6. Selective Allosteric Antagonists for the G Protein-Coupled Receptor GPRC6A Based on the 2-Phenylindole Privileged Structure Scaffold

    DEFF Research Database (Denmark)

    Johansson, Henrik; Boesgaard, Michael Worch; Nørskov-Lauritsen, Lenea

    2015-01-01

    G protein-coupled receptors (GPCRs) represent a biological target class of fundamental importance in drug therapy. The GPRC6A receptor is a newly deorphanized class C GPCR that we recently reported for the first allosteric antagonists based on the 2-arylindole privileged structure scaffold (e.g., 1...

  7. Using paleogenomics to study the evolution of gene families: origin and duplication history of the relaxin family hormones and their receptors.

    Directory of Open Access Journals (Sweden)

    Sergey Yegorov

    Full Text Available Recent progress in the analysis of whole genome sequencing data has resulted in the emergence of paleogenomics, a field devoted to the reconstruction of ancestral genomes. Ancestral karyotype reconstructions have been used primarily to illustrate the dynamic nature of genome evolution. In this paper, we demonstrate how they can also be used to study individual gene families by examining the evolutionary history of relaxin hormones (RLN/INSL and relaxin family peptide receptors (RXFP. Relaxin family hormones are members of the insulin superfamily, and are implicated in the regulation of a variety of primarily reproductive and neuroendocrine processes. Their receptors are G-protein coupled receptors (GPCR's and include members of two distinct evolutionary groups, an unusual characteristic. Although several studies have tried to elucidate the origins of the relaxin peptide family, the evolutionary origin of their receptors and the mechanisms driving the diversification of the RLN/INSL-RXFP signaling systems in non-placental vertebrates has remained elusive. Here we show that the numerous vertebrate RLN/INSL and RXFP genes are products of an ancestral receptor-ligand system that originally consisted of three genes, two of which apparently trace their origins to invertebrates. Subsequently, diversification of the system was driven primarily by whole genome duplications (WGD, 2R and 3R followed by almost complete retention of the ligand duplicates in most vertebrates but massive loss of receptor genes in tetrapods. Interestingly, the majority of 3R duplicates retained in teleosts are potentially involved in neuroendocrine regulation. Furthermore, we infer that the ancestral AncRxfp3/4 receptor may have been syntenically linked to the AncRln-like ligand in the pre-2R genome, and show that syntenic linkages among ligands and receptors have changed dynamically in different lineages. This study ultimately shows the broad utility, with some caveats, of

  8. IL-33 stimulates expression of the GPR84 (EX33) fatty acid receptor gene and of cytokine and chemokine genes in human adipocytes.

    Science.gov (United States)

    Zaibi, Mohamed S; Kępczyńska, Małgorzata A; Harikumar, Parvathy; Alomar, Suliman Y; Trayhurn, Paul

    2018-05-15

    Expression of GPCR fatty acid sensor/receptor genes in adipocytes is modulated by inflammatory mediators, particularly IL-1β. In this study we examined whether the IL-1 gene superfamily member, IL-33, also regulates expression of the fatty acid receptor genes in adipocytes. Human fat cells, differentiated from preadipocytes, were incubated with IL-33 at three different dose levels for 3 or 24 h and mRNA measured by qPCR. Treatment with IL-33 induced a dose-dependent increase in GPR84 mRNA at 3 h, the level with the highest dose being 13.7-fold greater than in controls. Stimulation of GPR84 expression was transitory; the mRNA level was not elevated at 24 h. In contrast to GPR84, IL-33 had no effect on GPR120 expression. IL-33 markedly stimulated expression of the IL1B, CCL2, IL6, CXCL2 and CSF3 genes, but there was no effect on ADIPOQ expression. The largest effect was on CSF3, the mRNA level of which increased 183-fold over controls at 3 h with the highest dose of IL-33; there was a parallel increase in the secretion of G-CSF protein into the medium. It is concluded that in human adipocytes IL-33, which is synthesised in adipose tissue, has a strong stimulatory effect on the expression of cytokine and chemokine genes, particularly CSF3, and on the expression of GPR84, a pro-inflammatory fatty acid receptor. Copyright © 2018 Elsevier Ltd. All rights reserved.

  9. G-protein-coupled receptor structures were not built in a day.

    Science.gov (United States)

    Blois, Tracy M; Bowie, James U

    2009-07-01

    Among the most exciting recent developments in structural biology is the structure determination of G-protein-coupled receptors (GPCRs), which comprise the largest class of membrane proteins in mammalian cells and have enormous importance for disease and drug development. The GPCR structures are perhaps the most visible examples of a nascent revolution in membrane protein structure determination. Like other major milestones in science, however, such as the sequencing of the human genome, these achievements were built on a hidden foundation of technological developments. Here, we describe some of the methods that are fueling the membrane protein structure revolution and have enabled the determination of the current GPCR structures, along with new techniques that may lead to future structures.

  10. Structural mutations of C-domains in members of the Ig superfamily. Consequences for the interactions between the T cell antigen receptor and the zeta 2 homodimer

    DEFF Research Database (Denmark)

    Geisler, C; Rubin, B; Caspar-Bauguil, S

    1992-01-01

    Several molecules belonging to the Ig superfamily are expressed together with noncovalently associated subunits. This applies for membrane-bound IgM and IgD, some of the FcR, and the Ti dimers of the TCR. The interactions between members of the Ig superfamily and their associated subunits are sti...

  11. PDZ domain-mediated interactions of G protein-coupled receptors with postsynaptic density protein 95

    DEFF Research Database (Denmark)

    Møller, Thor C; Wirth, Volker F; Roberts, Nina Ingerslev

    2013-01-01

    G protein-coupled receptors (GPCRs) constitute the largest family of membrane proteins in the human genome. Their signaling is regulated by scaffold proteins containing PDZ domains, but although these interactions are important for GPCR function, they are still poorly understood. We here present...

  12. MetaSINEs: Broad Distribution of a Novel SINE Superfamily in Animals

    OpenAIRE

    Nishihara, Hidenori; Plazzi, Federico; Passamonti, Marco; Okada, Norihiro

    2016-01-01

    SINEs (short interspersed elements) are transposable elements that typically originate independently in each taxonomic clade (order/family). However, some SINE families share a highly similar central sequence and are thus categorized as a SINE superfamily. Although only four SINE superfamilies (CORE-SINEs, V-SINEs, DeuSINEs, and Ceph-SINEs) have been reported so far, it is expected that new SINE superfamilies would be discovered by deep exploration of new SINEs in metazoan genomes. Here we de...

  13. Computerized modeling techniques predict the 3D structure of H₄R: facts and fiction.

    Science.gov (United States)

    Zaid, Hilal; Ismael-Shanak, Siba; Michaeli, Amit; Rayan, Anwar

    2012-01-01

    The functional characterization of proteins presents a daily challenge r biochemical, medical and computational sciences, especially when the structures are undetermined empirically, as in the case of the Histamine H4 Receptor (H₄R). H₄R is a member of the GPCR superfamily that plays a vital role in immune and inflammatory responses. To date, the concept of GPCRs modeling is highlighted in textbooks and pharmaceutical pamphlets, and this group of proteins has been the subject of almost 3500 publications in the scientific literature. The dynamic nature of determining the GPCRs structure was elucidated through elegant and creative modeling methodologies, implemented by many groups around the world. H₄R which belongs to the GPCR family was cloned in 2000; understandably, its biological activity was reported only 65 times in pubmed. Here we attempt to cover the fundamental concepts of H₄R structure modeling and its implementation in drug discovery, especially those that have been experimentally tested and to highlight some ideas that are currently being discussed on the dynamic nature of H₄R and GPCRs computerized techniques for 3D structure modeling.

  14. 5-HT2C Receptor Structures Reveal the Structural Basis of GPCR Polypharmacology

    DEFF Research Database (Denmark)

    Peng, Yao; Mccorvy, John D.; Harpsøe, Kasper

    2018-01-01

    Drugs frequently require interactions with multiple targets—via a process known as polypharmacology—to achieve their therapeutic actions. Currently, drugs targeting several serotonin receptors, including the 5-HT2C receptor, are useful for treating obesity, drug abuse, and schizophrenia. The comp...... the structural basis of polypharmacology at canonical GPCRs and illustrates how understanding characteristic patterns of ligand-receptor interaction and activation may ultimately facilitate drug design at multiple GPCRs....

  15. Keanekaragaman Jenis Kupu-Kupu Superfamili Papilionoidae di Banyuwindu, Limbangan Kendal

    Directory of Open Access Journals (Sweden)

    Ratna Oqtafiana

    2013-03-01

    Full Text Available Kupu-kupu turut memberi andil dalam mempertahankan keseimbangan ekosistem dan memperkaya keanekaragaman hayati. Tujuan dari penelitian ini adalah untuk mengetahui keanekaragaman jenis kupu-kupu superfamili Papilionoidae di Dukuh Banyuwindu Desa Limbangan Kecamatan Limbangan Kabupaten Kendal khususnya di habitat hutan sekunder, permukiman, Daerah Aliran Sungai (DAS dan persawahan.Populasi dalam penelitian ini adalah semua jenis kupu-kupu superfamili Papilionoidae yang ada di Banyuwindu, Limbangan Kendal. Sampel penelitian ini adalah jenis kupu-kupu superfamili Papilionoidae yang teramati di Banyuwindu Limbangan Kendal khususnya di habitat hutan sekunder, permukiman, DAS dan persawahan. Penelitian dilakukan dengan metode Indeks Point Abudance (IPA atau metode titik hitung.Hasil penelitian ditemukan sebanyak 62 jenis kupu-kupu superfamili Papilionoidae yang terdiri dari 737 individu yang tergolong kedalam empat famili yaitu Papilionidae, Pieridae, Lycaenidae dan Nymphalidae. Hasil analisis indeks keanekaragaman jenis berkisar antara 2,74-3,09, indeks kemerataan jenis berkisar antara 0,86-0,87 dan memiliki dominansi berkisar antara 0,07-0,09. Indeks keanekaragaman jenis dan indeks kemerataan jenis tertinggi tercatat pada habitat permukiman yaitu 3,09 dan 0,87 dan memiliki dominansi 0,07 sedangkan terendah tercatat pada habitat persawahan yaitu 2,74 dan 0,86 dan memiliki dominansi 0,07.Butterfly also contribute in maintaining the ecological balance and enrich biodiversity. The aim of this research was to determine the diversity of butterflies’ superfamily Papilionoidae in Banyuwindu Hamlet Limbangan Sub district Kendal Regency, especially in the secondary forest habitat, settlements, river flow area (RFA and rice field. The population in this research were all kinds of butterflies’ Papilionoidae superfamily in Banyuwindu, Limbangan Kendal. The sample was kind of butterfly superfamily Papilionoidae that observed in Banyuwindu Limbangan Kendal

  16. Designer TGFβ superfamily ligands with diversified functionality.

    Directory of Open Access Journals (Sweden)

    George P Allendorph

    Full Text Available Transforming Growth Factor--beta (TGFβ superfamily ligands, including Activins, Growth and Differentiation Factors (GDFs, and Bone Morphogenetic Proteins (BMPs, are excellent targets for protein-based therapeutics because of their pervasiveness in numerous developmental and cellular processes. We developed a strategy termed RASCH (Random Assembly of Segmental Chimera and Heteromer, to engineer chemically-refoldable TGFβ superfamily ligands with unique signaling properties. One of these engineered ligands, AB208, created from Activin-βA and BMP-2 sequences, exhibits the refolding characteristics of BMP-2 while possessing Activin-like signaling attributes. Further, we find several additional ligands, AB204, AB211, and AB215, which initiate the intracellular Smad1-mediated signaling pathways more strongly than BMP-2 but show no sensitivity to the natural BMP antagonist Noggin unlike natural BMP-2. In another design, incorporation of a short N-terminal segment from BMP-2 was sufficient to enable chemical refolding of BMP-9, without which was never produced nor refolded. Our studies show that the RASCH strategy enables us to expand the functional repertoire of TGFβ superfamily ligands through development of novel chimeric TGFβ ligands with diverse biological and clinical values.

  17. Using random forests for assistance in the curation of G-protein coupled receptor databases.

    Science.gov (United States)

    Shkurin, Aleksei; Vellido, Alfredo

    2017-08-18

    Biology is experiencing a gradual but fast transformation from a laboratory-centred science towards a data-centred one. As such, it requires robust data engineering and the use of quantitative data analysis methods as part of database curation. This paper focuses on G protein-coupled receptors, a large and heterogeneous super-family of cell membrane proteins of interest to biology in general. One of its families, Class C, is of particular interest to pharmacology and drug design. This family is quite heterogeneous on its own, and the discrimination of its several sub-families is a challenging problem. In the absence of known crystal structure, such discrimination must rely on their primary amino acid sequences. We are interested not as much in achieving maximum sub-family discrimination accuracy using quantitative methods, but in exploring sequence misclassification behavior. Specifically, we are interested in isolating those sequences showing consistent misclassification, that is, sequences that are very often misclassified and almost always to the same wrong sub-family. Random forests are used for this analysis due to their ensemble nature, which makes them naturally suited to gauge the consistency of misclassification. This consistency is here defined through the voting scheme of their base tree classifiers. Detailed consistency results for the random forest ensemble classification were obtained for all receptors and for all data transformations of their unaligned primary sequences. Shortlists of the most consistently misclassified receptors for each subfamily and transformation, as well as an overall shortlist including those cases that were consistently misclassified across transformations, were obtained. The latter should be referred to experts for further investigation as a data curation task. The automatic discrimination of the Class C sub-families of G protein-coupled receptors from their unaligned primary sequences shows clear limits. This study has

  18. Understanding the Added Value of G-Protein-Coupled Receptor Heteromers

    Directory of Open Access Journals (Sweden)

    Nuria Franco

    2014-01-01

    Full Text Available G-protein-coupled receptors (GPCRs constitute the most populated family of proteins within the human genome. Since the early sixties work on GPCRs and on GPCR-mediated signaling has led to a number of awards, the most recent being the Nobel Prize in Chemistry for 2012. The future of GPCRs research is surely based on their capacity for heteromerization. Receptor heteromers offer a series of challenges that will help in providing success in academic/basic research and translation into more effective and safer drugs.

  19. Dietary modification of metabolic pathways via nuclear hormone receptors.

    Science.gov (United States)

    Caiozzi, Gianella; Wong, Brian S; Ricketts, Marie-Louise

    2012-10-01

    Nuclear hormone receptors (NHRs), as ligand-dependent transcription factors, have emerged as important mediators in the control of whole body metabolism. Because of the promiscuous nature of several members of this superfamily that have been found to bind ligand with lower affinity than the classical steroid NHRs, they consequently display a broader ligand selectivity. This promiscuous nature has facilitated various bioactive dietary components being able to act as agonist ligands for certain members of the NHR superfamily. By binding to these NHRs, bioactive dietary components are able to mediate changes in various metabolic pathways, including, glucose, cholesterol and triglyceride homeostasis among others. This review will provide a general overview of the nuclear hormone receptors that have been shown to be activated by dietary components. The physiological consequences of such receptor activation by these dietary components will then be discussed in more detail. Copyright © 2012 John Wiley & Sons, Ltd.

  20. The macrophage scavenger receptor CD163 functions as an innate immune sensor for bacteria

    NARCIS (Netherlands)

    Fabriek, Babs O.; van Bruggen, Robin; Deng, Dong Mei; Ligtenberg, Antoon J. M.; Nazmi, Kamran; Schornagel, Karin; Vloet, Rianka P. M.; Dijkstra, Christine D.; van den Berg, Timo K.

    2009-01-01

    The plasma membrane glycoprotein receptor CD163 is a member of the scavenger receptor cystein-rich (SRCR) superfamily class B that is highly expressed on resident tissue macrophages in vivo. Previously, the molecule has been shown to act as a receptor for hemoglobin-haptoglobin complexes and to

  1. The peripheral cannabinoid receptor Cb2, frequently expressed on AML blasts, either induces a neutrophilic differentiation block or confers abnormal migration properties in a ligand-dependent manner

    NARCIS (Netherlands)

    M. Alberich-Jorda (Meritxell); N. Rayman (Nazik); M. Tas (Marjolein); S.E. Verbakel (Sandra); N. Battista (Natalia); K. van Lom (Kirsten); B. Löwenberg (Bob); M. Maccarrone (Mauro); H.R. Delwel (Ruud)

    2004-01-01

    textabstractCb2, the gene encoding the peripheral cannabinoid receptor, is located in a common virus integration site and is overex-pressed in retrovirally induced murine myeloid leukemias. Here we show that this G protein-coupled receptor (GPCR) is also aberrantly expressed in a

  2. Inference of functional properties from large-scale analysis of enzyme superfamilies.

    Science.gov (United States)

    Brown, Shoshana D; Babbitt, Patricia C

    2012-01-02

    As increasingly large amounts of data from genome and other sequencing projects become available, new approaches are needed to determine the functions of the proteins these genes encode. We show how large-scale computational analysis can help to address this challenge by linking functional information to sequence and structural similarities using protein similarity networks. Network analyses using three functionally diverse enzyme superfamilies illustrate the use of these approaches for facile updating and comparison of available structures for a large superfamily, for creation of functional hypotheses for metagenomic sequences, and to summarize the limits of our functional knowledge about even well studied superfamilies.

  3. GPCR engineering yields high-resolution structural insights into beta2-adrenergic receptor function

    DEFF Research Database (Denmark)

    Rosenbaum, Daniel M; Cherezov, Vadim; Hanson, Michael A

    2007-01-01

    The beta2-adrenergic receptor (beta2AR) is a well-studied prototype for heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors (GPCRs) that respond to diffusible hormones and neurotransmitters. To overcome the structural flexibility of the beta2AR and to facilitate its cr...

  4. Trace amine-associated receptor 1-Family archetype or iconoclast?

    Science.gov (United States)

    Grandy, David K

    2007-12-01

    Interest has recently been rekindled in receptors that are activated by low molecular weight, noncatecholic, biogenic amines that are typically found as trace constituents of various vertebrate and invertebrate tissues and fluids. The timing of this resurgent focus on receptors activated by the "trace amines" (TA) beta-phenylethylamine (PEA), tyramine (TYR), octopamine (OCT), synephrine (SYN), and tryptamine (TRYP) is the direct result of 2 publications that appeared in 2001 describing the cloning of a novel G protein-coupled receptor (GPCR) referred to by their discoverers Borowsky et al. as TA1 and Bunzow et al. as TA receptor 1 (TAR1). When heterologously expressed in Xenopus laevis oocytes and various eukaryotic cell lines, recombinant rodent and human TAR dose-dependently couple to the stimulation of adenosine 3',5'-monophosphate (cAMP) production. Structure-activity profiling based on this functional response has revealed that in addition to the TA, other biologically active compounds containing a 2-carbon aliphatic side chain linking an amino group to at least 1 benzene ring are potent and efficacious TA receptor agonists with amphetamine (AMPH), methamphetamine, 3-iodothyronamine, thyronamine, and dopamine (DA) among the most notable. Almost 100 years after the search for TAR began, numerous TA1/TAR1-related sequences, now called TA-associated receptors (TAAR), have been identified in the genome of every species of vertebrate examined to date. Consequently, even though heterologously expressed TAAR1 fits the pharmacological criteria established for a bona fide TAR, a major challenge for those working in the field is to discern the in vivo pharmacology and physiology of each purported member of this extended family of GPCR. Only then will it be possible to establish whether TAAR1 is the family archetype or an iconoclast.

  5. Navigating the conformational landscape of G protein-coupled receptor kinases during allosteric activation.

    Science.gov (United States)

    Yao, Xin-Qiu; Cato, M Claire; Labudde, Emily; Beyett, Tyler S; Tesmer, John J G; Grant, Barry J

    2017-09-29

    G protein-coupled receptors (GPCRs) are essential for transferring extracellular signals into carefully choreographed intracellular responses controlling diverse aspects of cell physiology. The duration of GPCR-mediated signaling is primarily regulated via GPCR kinase (GRK)-mediated phosphorylation of activated receptors. Although many GRK structures have been reported, the mechanisms underlying GRK activation are not well-understood, in part because it is unknown how these structures map to the conformational landscape available to this enzyme family. Unlike most other AGC kinases, GRKs rely on their interaction with GPCRs for activation and not phosphorylation. Here, we used principal component analysis of available GRK and protein kinase A crystal structures to identify their dominant domain motions and to provide a framework that helps evaluate how close each GRK structure is to being a catalytically competent state. Our results indicated that disruption of an interface formed between the large lobe of the kinase domain and the regulator of G protein signaling homology domain (RHD) is highly correlated with establishment of the active conformation. By introducing point mutations in the GRK5 RHD-kinase domain interface, we show with both in silico and in vitro experiments that perturbation of this interface leads to higher phosphorylation activity. Navigation of the conformational landscape defined by this bioinformatics-based study is likely common to all GPCR-activated GRKs. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  6. Mechanism of the G-protein mimetic nanobody binding to a muscarinic G-protein-coupled receptor.

    Science.gov (United States)

    Miao, Yinglong; McCammon, J Andrew

    2018-03-20

    Protein-protein binding is key in cellular signaling processes. Molecular dynamics (MD) simulations of protein-protein binding, however, are challenging due to limited timescales. In particular, binding of the medically important G-protein-coupled receptors (GPCRs) with intracellular signaling proteins has not been simulated with MD to date. Here, we report a successful simulation of the binding of a G-protein mimetic nanobody to the M 2 muscarinic GPCR using the robust Gaussian accelerated MD (GaMD) method. Through long-timescale GaMD simulations over 4,500 ns, the nanobody was observed to bind the receptor intracellular G-protein-coupling site, with a minimum rmsd of 2.48 Å in the nanobody core domain compared with the X-ray structure. Binding of the nanobody allosterically closed the orthosteric ligand-binding pocket, being consistent with the recent experimental finding. In the absence of nanobody binding, the receptor orthosteric pocket sampled open and fully open conformations. The GaMD simulations revealed two low-energy intermediate states during nanobody binding to the M 2 receptor. The flexible receptor intracellular loops contribute remarkable electrostatic, polar, and hydrophobic residue interactions in recognition and binding of the nanobody. These simulations provided important insights into the mechanism of GPCR-nanobody binding and demonstrated the applicability of GaMD in modeling dynamic protein-protein interactions.

  7. A G-protein-coupled chemokine receptor: A putative insertion site for a multi-pathogen recombinant capripoxvirus vaccine strategy.

    Science.gov (United States)

    Cêtre-Sossah, Catherine; Dickmu, Simon; Kwiatek, Olivier; Albina, Emmanuel

    2017-09-01

    Capripoxviruses (CaPVs) have been shown to be ideal viral vectors for the development of recombinant multivalent vaccines to enable delivery of immunogenic genes from ruminant pathogens. So far, the viral thymidine kinase (TK) gene is the only gene used to generate recombinants. A putative non-essential gene encoding a G-protein-coupled chemokine receptor subfamily homologue (GPCR) was targeted as an additional insertion site. Peste des petits ruminants (PPR) was chosen as a disease model. A new recombinant CaPV expressing the viral attachment hemagglutinin (H) of the PPR virus (PPRV) in the GPCR insertion site (rKS1-HPPR-GPCR) was generated in the backbone North African isolate KS1 strain of lumpy skin disease virus (LSDV). Comparison with the recombinant CaPV expressing the H of PPRV in the TK gene (rKS1-HPPR-TK) shown to induce protection against both PPR and LSD in both sheep and goats was assessed. The suitability of the GPCR gene to be a putative additional insertion site in the CaPV genome is evaluated and discussed. Copyright © 2017 Elsevier B.V. All rights reserved.

  8. Diversification of a single ancestral gene into a successful toxin superfamily in highly venomous Australian funnel-web spiders.

    Science.gov (United States)

    Pineda, Sandy S; Sollod, Brianna L; Wilson, David; Darling, Aaron; Sunagar, Kartik; Undheim, Eivind A B; Kely, Laurence; Antunes, Agostinho; Fry, Bryan G; King, Glenn F

    2014-03-05

    Spiders have evolved pharmacologically complex venoms that serve to rapidly subdue prey and deter predators. The major toxic factors in most spider venoms are small, disulfide-rich peptides. While there is abundant evidence that snake venoms evolved by recruitment of genes encoding normal body proteins followed by extensive gene duplication accompanied by explosive structural and functional diversification, the evolutionary trajectory of spider-venom peptides is less clear. Here we present evidence of a spider-toxin superfamily encoding a high degree of sequence and functional diversity that has evolved via accelerated duplication and diversification of a single ancestral gene. The peptides within this toxin superfamily are translated as prepropeptides that are posttranslationally processed to yield the mature toxin. The N-terminal signal sequence, as well as the protease recognition site at the junction of the propeptide and mature toxin are conserved, whereas the remainder of the propeptide and mature toxin sequences are variable. All toxin transcripts within this superfamily exhibit a striking cysteine codon bias. We show that different pharmacological classes of toxins within this peptide superfamily evolved under different evolutionary selection pressures. Overall, this study reinforces the hypothesis that spiders use a combinatorial peptide library strategy to evolve a complex cocktail of peptide toxins that target neuronal receptors and ion channels in prey and predators. We show that the ω-hexatoxins that target insect voltage-gated calcium channels evolved under the influence of positive Darwinian selection in an episodic fashion, whereas the κ-hexatoxins that target insect calcium-activated potassium channels appear to be under negative selection. A majority of the diversifying sites in the ω-hexatoxins are concentrated on the molecular surface of the toxins, thereby facilitating neofunctionalisation leading to new toxin pharmacology.

  9. Inference of Functional Properties from Large-scale Analysis of Enzyme Superfamilies*

    Science.gov (United States)

    Brown, Shoshana D.; Babbitt, Patricia C.

    2012-01-01

    As increasingly large amounts of data from genome and other sequencing projects become available, new approaches are needed to determine the functions of the proteins these genes encode. We show how large-scale computational analysis can help to address this challenge by linking functional information to sequence and structural similarities using protein similarity networks. Network analyses using three functionally diverse enzyme superfamilies illustrate the use of these approaches for facile updating and comparison of available structures for a large superfamily, for creation of functional hypotheses for metagenomic sequences, and to summarize the limits of our functional knowledge about even well studied superfamilies. PMID:22069325

  10. Virus-encoded chemokine receptors--putative novel antiviral drug targets

    DEFF Research Database (Denmark)

    Rosenkilde, Mette M

    2005-01-01

    Large DNA viruses, in particular herpes- and poxviruses, have evolved proteins that serve as mimics or decoys for endogenous proteins in the host. The chemokines and their receptors serve key functions in both innate and adaptive immunity through control of leukocyte trafficking, and have...... receptors belong to the superfamily of G-protein coupled 7TM receptors that per se are excellent drug targets. At present, non-peptide antagonists have been developed against many chemokine receptors. The potentials of the virus-encoded chemokine receptors as drug targets--ie. as novel antiviral strategies...

  11. Allosteric mechanisms within the adenosine A2A-dopamine D2 receptor heterotetramer

    Science.gov (United States)

    Ferré, Sergi; Bonaventura, Jordi; Tomasi, Dardo; Navarro, Gemma; Moreno, Estefanía; Cortés, Antonio; Lluís, Carme; Casadó, Vicent; Volkow, Nora D.

    2017-01-01

    The structure constituted by a G protein coupled receptor (GPCR) homodimer and a G protein provides a main functional unit and oligomeric entities can be viewed as multiples of dimers. For GPCR heteromers, experimental evidence supports a tetrameric structure, comprised of two different homodimers, each able to signal with its preferred G protein. GPCR homomers and heteromers can act as the conduit of allosteric interactions between orthosteric ligands. The well-known agonist/agonist allosteric interaction in the adenosine A2A receptor (A2AR)-dopamine D2 receptor (D2R) heteromer, by which A2AR agonists decrease the affinity of D2R agonists, gave the first rationale for the use of A2AR antagonists in Parkinson’s disease. We review new pharmacological findings that can be explained in the frame of a tetrameric structure of the A2AR-D2R heteromer: first, ligand-independent allosteric modulations by the D2R that result in changes of the binding properties of A2AR ligands; second, differential modulation of the intrinsic efficacy of D2R ligands for G protein-dependent and independent signaling; third, the canonical antagonistic Gs-Gi interaction within the frame of the heteromer; and fourth, the ability of A2AR antagonists, including caffeine, to also exert the same allosteric modulations of D2R ligands than A2AR agonists, while A2AR agonists and antagonists counteract each other’s effects. These findings can have important clinical implications when evaluating the use of A2AR antagonists. They also call for the need of monitoring caffeine intake when evaluating the effect of D2R ligands, when used as therapeutic agents in neuropsychiatric disorders or as probes in imaging studies. PMID:26051403

  12. The VPAC1 receptor: structure and function of a class B GPCR prototype

    Directory of Open Access Journals (Sweden)

    Alain eCouvineau

    2012-11-01

    Full Text Available The class B G protein-coupled receptors (GPCRs represents a small sub-family encompassing 15 members, and are very promising targets for the development of drugs to treat many diseases such as chronic inflammation, neurodegeneration, diabetes, stress and osteoporosis. The VPAC1 receptor which is an archetype of the class B GPCRs binds Vasoactive Intestinal Peptide (VIP, a neuropeptide widely distributed in central and peripheral nervous system modulating many physiological processes including regulation of exocrine secretions, hormone release, foetal development, immune response... VIP appears to exert beneficial effect in neuro-degenerative and inflammatory diseases. This article reviews the current knowledge regarding the structure and molecular pharmacology of VPAC1 receptors. Over the past decade, structure-function relationship studies have demonstrated that the N-terminal ectodomain (N-ted of VPAC1 plays a pivotal role in VIP recognition. The use of different approaches such as directed mutagenesis, photoaffinity labeling, Nuclear Magnetic Resonance (NMR, molecular modeling and molecular dynamic simulation has led to demonstrate that: i the central and C-terminal part of the VIP molecule interacts with the N-ted of VPAC1 receptor which is itself structured as a « Sushi » domain; ii the N-terminal end of the VIP molecule interacts with the first transmembrane domain of the receptor where three residues (K143, T144 and T147 play an important role in VPAC1 interaction with the first histidine residue of VIP.

  13. Large-Scale Analysis Exploring Evolution of Catalytic Machineries and Mechanisms in Enzyme Superfamilies.

    Science.gov (United States)

    Furnham, Nicholas; Dawson, Natalie L; Rahman, Syed A; Thornton, Janet M; Orengo, Christine A

    2016-01-29

    Enzymes, as biological catalysts, form the basis of all forms of life. How these proteins have evolved their functions remains a fundamental question in biology. Over 100 years of detailed biochemistry studies, combined with the large volumes of sequence and protein structural data now available, means that we are able to perform large-scale analyses to address this question. Using a range of computational tools and resources, we have compiled information on all experimentally annotated changes in enzyme function within 379 structurally defined protein domain superfamilies, linking the changes observed in functions during evolution to changes in reaction chemistry. Many superfamilies show changes in function at some level, although one function often dominates one superfamily. We use quantitative measures of changes in reaction chemistry to reveal the various types of chemical changes occurring during evolution and to exemplify these by detailed examples. Additionally, we use structural information of the enzymes active site to examine how different superfamilies have changed their catalytic machinery during evolution. Some superfamilies have changed the reactions they perform without changing catalytic machinery. In others, large changes of enzyme function, in terms of both overall chemistry and substrate specificity, have been brought about by significant changes in catalytic machinery. Interestingly, in some superfamilies, relatives perform similar functions but with different catalytic machineries. This analysis highlights characteristics of functional evolution across a wide range of superfamilies, providing insights that will be useful in predicting the function of uncharacterised sequences and the design of new synthetic enzymes. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

  14. The Multiple Faces of Prostaglandin E2 G-Protein Coupled Receptor Signaling during the Dendritic Cell Life Cycle

    Directory of Open Access Journals (Sweden)

    Alessandra Cambi

    2013-03-01

    Full Text Available Many processes regulating immune responses are initiated by G-protein coupled receptors (GPCRs and report biochemical changes in the microenvironment. Dendritic cells (DCs are the most potent antigen-presenting cells and crucial for the regulation of innate and adaptive immune responses. The lipid mediator Prostaglandin E2 (PGE2 via four GPCR subtypes (EP1-4 critically regulates DC generation, maturation and migration. The role of PGE2 signaling in DC biology was unraveled by the characterization of EP receptor subtype expression in DC progenitor cells and DCs, the identification of the signaling pathways initiated by these GPCR subtypes and the classification of DC responses to PGE2 at different stages of differentiation. Here, we review the advances in PGE2 signaling in DCs and describe the efforts still to be made to understand the spatio-temporal fine-tuning of PGE2 responses by DCs.

  15. Regulation of G-protein coupled receptor traffic by an evolutionary conserved hydrophobic signal.

    Science.gov (United States)

    Angelotti, Tim; Daunt, David; Shcherbakova, Olga G; Kobilka, Brian; Hurt, Carl M

    2010-04-01

    Plasma membrane (PM) expression of G-protein coupled receptors (GPCRs) is required for activation by extracellular ligands; however, mechanisms that regulate PM expression of GPCRs are poorly understood. For some GPCRs, such as alpha2c-adrenergic receptors (alpha(2c)-ARs), heterologous expression in non-native cells results in limited PM expression and extensive endoplasmic reticulum (ER) retention. Recently, ER export/retentions signals have been proposed to regulate cellular trafficking of several GPCRs. By utilizing a chimeric alpha(2a)/alpha(2c)-AR strategy, we identified an evolutionary conserved hydrophobic sequence (ALAAALAAAAA) in the extracellular amino terminal region that is responsible in part for alpha(2c)-AR subtype-specific trafficking. To our knowledge, this is the first luminal ER retention signal reported for a GPCR. Removal or disruption of the ER retention signal dramatically increased PM expression and decreased ER retention. Conversely, transplantation of this hydrophobic sequence into alpha(2a)-ARs reduced their PM expression and increased ER retention. This evolutionary conserved hydrophobic trafficking signal within alpha(2c)-ARs serves as a regulator of GPCR trafficking.

  16. Multitarget-directed tricyclic pyridazinones as G protein-coupled receptor ligands and cholinesterase inhibitors.

    Science.gov (United States)

    Pau, Amedeo; Catto, Marco; Pinna, Giovanni; Frau, Simona; Murineddu, Gabriele; Asproni, Battistina; Curzu, Maria M; Pisani, Leonardo; Leonetti, Francesco; Loza, Maria Isabel; Brea, José; Pinna, Gérard A; Carotti, Angelo

    2015-06-01

    By following a multitarget ligand design approach, a library of 47 compounds was prepared, and they were tested as binders of selected G protein-coupled receptors (GPCRs) and inhibitors of acetyl and/or butyryl cholinesterase. The newly designed ligands feature pyridazinone-based tricyclic scaffolds connected through alkyl chains of variable length to proper amine moieties (e.g., substituted piperazines or piperidines) for GPCR and cholinesterase (ChE) molecular recognition. The compounds were tested at three different GPCRs, namely serotoninergic 5-HT1A, adrenergic α1A, and dopaminergic D2 receptors. Our main goal was the discovery of compounds that exhibit, in addition to ChE inhibition, antagonist activity at 5-HT1A because of its involvement in neuronal deficits typical of Alzheimer's and other neurodegenerative diseases. Ligands with nanomolar affinity for the tested GPCRs were discovered, but most of them behaved as dual antagonists of α1A and 5-HT1A receptors. Nevertheless, several compounds displaying this GPCR affinity profile also showed moderate to good inhibition of AChE and BChE, thus deserving further investigations to exploit the therapeutic potential of such unusual biological profiles. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. Tracking G-protein-coupled receptor activation using genetically encoded infrared probes.

    Science.gov (United States)

    Ye, Shixin; Zaitseva, Ekaterina; Caltabiano, Gianluigi; Schertler, Gebhard F X; Sakmar, Thomas P; Deupi, Xavier; Vogel, Reiner

    2010-04-29

    Rhodopsin is a prototypical heptahelical family A G-protein-coupled receptor (GPCR) responsible for dim-light vision. Light isomerizes rhodopsin's retinal chromophore and triggers concerted movements of transmembrane helices, including an outward tilting of helix 6 (H6) and a smaller movement of H5, to create a site for G-protein binding and activation. However, the precise temporal sequence and mechanism underlying these helix rearrangements is unclear. We used site-directed non-natural amino acid mutagenesis to engineer rhodopsin with p-azido-l-phenylalanine residues incorporated at selected sites, and monitored the azido vibrational signatures using infrared spectroscopy as rhodopsin proceeded along its activation pathway. Here we report significant changes in electrostatic environments of the azido probes even in the inactive photoproduct Meta I, well before the active receptor state was formed. These early changes suggest a significant rotation of H6 and movement of the cytoplasmic part of H5 away from H3. Subsequently, a large outward tilt of H6 leads to opening of the cytoplasmic surface to form the active receptor photoproduct Meta II. Thus, our results reveal early conformational changes that precede larger rigid-body helix movements, and provide a basis to interpret recent GPCR crystal structures and to understand conformational sub-states observed during the activation of other GPCRs.

  18. The interleukin-4 receptor: signal transduction by a hematopoietin receptor.

    Science.gov (United States)

    Keegan, A D; Pierce, J H

    1994-02-01

    Over the last several years, the receptors for numerous cytokines have been molecularly characterized. Analysis of their amino acid sequences shows that some of these receptors bear certain motifs in their extracellular domains that define a family of receptors called the Hematopoietin receptor superfamily. Significant advances in characterizing the structure, function, and mechanisms of signal transduction have been made for several members of this family. The purpose of this review is to discuss the recent advances made for one of the family members, the interleukin (IL) 4 receptor. Other receptor systems have recently been reviewed elsewhere. The IL-4 receptor consists of, at the minimum, the cloned 140 kDa IL-4-binding chain with the potential for associating with other chains. The IL-4 receptor transduces its signal by activating a tyrosine kinase that phosphorylates cellular substrates, including the receptor itself, and the 170 kDa substrate called 4PS. Phosphorylated 4PS interacts with the SH2 domain of the enzyme PI-3'-kinase and increases its enzymatic activity. These early events in the IL-4 receptor initiated signaling pathway may trigger a series of signals that will ultimately lead to an IL-4 specific biologic outcome.

  19. Diversity, classification and function of the plant protein kinase superfamily

    OpenAIRE

    Lehti-Shiu, Melissa D.; Shiu, Shin-Han

    2012-01-01

    Eukaryotic protein kinases belong to a large superfamily with hundreds to thousands of copies and are components of essentially all cellular functions. The goals of this study are to classify protein kinases from 25 plant species and to assess their evolutionary history in conjunction with consideration of their molecular functions. The protein kinase superfamily has expanded in the flowering plant lineage, in part through recent duplications. As a result, the flowering plant protein kinase r...

  20. Alteration in contractile G-protein coupled receptor expression by moist snuff and nicotine in rat cerebral arteries

    DEFF Research Database (Denmark)

    Sandhu, Hardip; Xu, Cang-Bao; Edvinsson, Lars

    2011-01-01

    The cardiovascular risk for users of use of Swedish snus/American snuff (moist tobacco) has been debated for a long time. The present study was designed to examine the effects of water- or lipid-soluble (DMSO-soluble) snus and nicotine, the most important substance in tobacco, on the expression...... kinases (MAPK). However, the effects of moist tobacco on the expression of GPCR are less studied. Rat middle cerebral arteries were isolated and organ cultured in serum-free medium for 24h in the presence of water-soluble snus (WSS), DMSO-soluble snus (DSS), or nicotine. The dose of snus and nicotine...... was kept at plasma level of snus users (25ng nicotine/ml). A high dose (250ng nicotine/ml) was also included due to the previous results showing alteration in the GPCR expression by nicotine at this concentration. Contractile responses to the ET(B) receptor agonist sarafotoxin 6c, 5-HT(1B) receptor agonist...

  1. Identification of G Protein-Coupled Receptors (GPCRs in Primary Cilia and Their Possible Involvement in Body Weight Control.

    Directory of Open Access Journals (Sweden)

    Yoshihiro Omori

    Full Text Available Primary cilia are sensory organelles that harbor various receptors such as G protein-coupled receptors (GPCRs. We analyzed subcellular localization of 138 non-odorant GPCRs. We transfected GPCR expression vectors into NIH3T3 cells, induced ciliogenesis by serum starvation, and observed subcellular localization of GPCRs by immunofluorescent staining. We found that several GPCRs whose ligands are involved in feeding behavior, including prolactin-releasing hormone receptor (PRLHR, neuropeptide FF receptor 1 (NPFFR1, and neuromedin U receptor 1 (NMUR1, localized to the primary cilia. In addition, we found that a short form of dopamine receptor D2 (DRD2S is efficiently transported to the primary cilia, while a long form of dopamine receptor D2 (DRD2L is rarely transported to the primary cilia. Using an anti-Prlhr antibody, we found that Prlhr localized to the cilia on the surface of the third ventricle in the vicinity of the hypothalamic periventricular nucleus. We generated the Npy2r-Cre transgenic mouse line in which Cre-recombinase is expressed under the control of the promoter of Npy2r encoding a ciliary GPCR. By mating Npy2r-Cre mice with Ift80 flox mice, we generated Ift80 conditional knockout (CKO mice in which Npy2r-positive cilia were diminished in number. We found that Ift80 CKO mice exhibited a body weight increase. Our results suggest that Npy2r-positive cilia are important for body weight control.

  2. Identification of G Protein-Coupled Receptors (GPCRs) in Primary Cilia and Their Possible Involvement in Body Weight Control.

    Science.gov (United States)

    Omori, Yoshihiro; Chaya, Taro; Yoshida, Satoyo; Irie, Shoichi; Tsujii, Toshinori; Furukawa, Takahisa

    2015-01-01

    Primary cilia are sensory organelles that harbor various receptors such as G protein-coupled receptors (GPCRs). We analyzed subcellular localization of 138 non-odorant GPCRs. We transfected GPCR expression vectors into NIH3T3 cells, induced ciliogenesis by serum starvation, and observed subcellular localization of GPCRs by immunofluorescent staining. We found that several GPCRs whose ligands are involved in feeding behavior, including prolactin-releasing hormone receptor (PRLHR), neuropeptide FF receptor 1 (NPFFR1), and neuromedin U receptor 1 (NMUR1), localized to the primary cilia. In addition, we found that a short form of dopamine receptor D2 (DRD2S) is efficiently transported to the primary cilia, while a long form of dopamine receptor D2 (DRD2L) is rarely transported to the primary cilia. Using an anti-Prlhr antibody, we found that Prlhr localized to the cilia on the surface of the third ventricle in the vicinity of the hypothalamic periventricular nucleus. We generated the Npy2r-Cre transgenic mouse line in which Cre-recombinase is expressed under the control of the promoter of Npy2r encoding a ciliary GPCR. By mating Npy2r-Cre mice with Ift80 flox mice, we generated Ift80 conditional knockout (CKO) mice in which Npy2r-positive cilia were diminished in number. We found that Ift80 CKO mice exhibited a body weight increase. Our results suggest that Npy2r-positive cilia are important for body weight control.

  3. Bioinformatic prediction of G protein-coupled receptor encoding sequences from the transcriptome of the foreleg, including the Haller's organ, of the cattle tick, Rhipicephalus australis.

    Directory of Open Access Journals (Sweden)

    Sergio Munoz

    Full Text Available The cattle tick of Australia, Rhipicephalus australis, is a vector for microbial parasites that cause serious bovine diseases. The Haller's organ, located in the tick's forelegs, is crucial for host detection and mating. To facilitate the development of new technologies for better control of this agricultural pest, we aimed to sequence and annotate the transcriptome of the R. australis forelegs and associated tissues, including the Haller's organ. As G protein-coupled receptors (GPCRs are an important family of eukaryotic proteins studied as pharmaceutical targets in humans, we prioritized the identification and classification of the GPCRs expressed in the foreleg tissues. The two forelegs from adult R. australis were excised, RNA extracted, and pyrosequenced with 454 technology. Reads were assembled into unigenes and annotated by sequence similarity. Python scripts were written to find open reading frames (ORFs from each unigene. These ORFs were analyzed by different GPCR prediction approaches based on sequence alignments, support vector machines, hidden Markov models, and principal component analysis. GPCRs consistently predicted by multiple methods were further studied by phylogenetic analysis and 3D homology modeling. From 4,782 assembled unigenes, 40,907 possible ORFs were predicted. Using Blastp, Pfam, GPCRpred, TMHMM, and PCA-GPCR, a basic set of 46 GPCR candidates were compiled and a phylogenetic tree was constructed. With further screening of tertiary structures predicted by RaptorX, 6 likely GPCRs emerged and the strongest candidate was classified by PCA-GPCR to be a GABAB receptor.

  4. Towards structural models of molecular recognition in olfactory receptors.

    Science.gov (United States)

    Afshar, M; Hubbard, R E; Demaille, J

    1998-02-01

    The G protein coupled receptors (GPCR) are an important class of proteins that act as signal transducers through the cytoplasmic membrane. Understanding the structure and activation mechanism of these proteins is crucial for understanding many different aspects of cellular signalling. The olfactory receptors correspond to the largest family of GPCRs. Very little is known about how the structures of the receptors govern the specificity of interaction which enables identification of particular odorant molecules. In this paper, we review recent developments in two areas of molecular modelling: methods for modelling the configuration of trans-membrane helices and methods for automatic docking of ligands into receptor structures. We then show how a subset of these methods can be combined to construct a model of a rat odorant receptor interacting with lyral for which experimental data are available. This modelling can help us make progress towards elucidating the specificity of interactions between receptors and odorant molecules.

  5. CADM1 is essential for KSHV-encoded vGPCR-and vFLIP-mediated chronic NF-κB activation.

    Directory of Open Access Journals (Sweden)

    Richard Hunte

    2018-04-01

    Full Text Available Approximately 12% of all human cancers worldwide are caused by infections with oncogenic viruses. Kaposi's sarcoma herpesvirus/human herpesvirus 8 (KSHV/HHV8 is one of the oncogenic viruses responsible for human cancers, including Kaposi's sarcoma (KS, Primary Effusion Lymphoma (PEL, and the lymphoproliferative disorder multicentric Castleman's disease (MCD. Chronic inflammation mediated by KSHV infection plays a decisive role in the development and survival of these cancers. NF-κB, a family of transcription factors regulating inflammation, cell survival, and proliferation, is persistently activated in KSHV-infected cells. The KSHV latent and lytic expressing oncogenes involved in NF-κB activation are vFLIP/K13 and vGPCR, respectively. However, the mechanisms by which NF-κB is activated by vFLIP and vGPCR are poorly understood. In this study, we have found that a host molecule, Cell Adhesion Molecule 1 (CADM1, is robustly upregulated in KSHV-infected PBMCs and KSHV-associated PEL cells. Further investigation determined that both vFLIP and vGPCR interacted with CADM1. The PDZ binding motif localized at the carboxyl terminus of CADM1 is essential for both vGPCR and vFLIP to maintain chronic NF-κB activation. Membrane lipid raft associated CADM1 interaction with vFLIP is critical for the initiation of IKK kinase complex and NF-κB activation in the PEL cells. In addition, CADM1 played essential roles in the survival of KSHV-associated PEL cells. These data indicate that CADM1 plays key roles in the activation of NF-κB pathways during latent and lytic phases of the KSHV life cycle and the survival of KSHV-infected cells.

  6. Pharmacological profiling an abundantly expressed schistosome serotonergic GPCR identifies nuciferine as a potent antagonist

    Directory of Open Access Journals (Sweden)

    John D. Chan

    2016-12-01

    Full Text Available 5-hydroxytryptamine (5-HT is a key regulator of muscle contraction in parasitic flatworms. In Schistosoma mansoni, the myoexcitatory action of 5-HT is effected through activation of a serotonergic GPCR (Sm.5HTRL, prioritizing pharmacological characterization of this target for anthelmintic drug discovery. Here, we have examined the effects of several aporphine alkaloids on the signaling activity of a heterologously expressed Sm.5HTRL construct using a cAMP biosensor assay. Four structurally related natural products – nuciferine, D-glaucine, boldine and bulbocapnine – were demonstrated to block Sm.5HTRL evoked cAMP generation with the potency of GPCR blockade correlating well with the ability of each drug to inhibit contractility of schistosomule larvae. Nuciferine was also effective at inhibiting both basal and 5-HT evoked motility of adult schistosomes. These data advance our understanding of structure-affinity relationships at Sm.5HTRL, and demonstrate the effectiveness of Sm.5HTRL antagonists as hypomotility-evoking drugs across different parasite life cycle stages.

  7. Melatonin Receptor Genes in Vertebrates

    Directory of Open Access Journals (Sweden)

    Hua Dong Yin

    2013-05-01

    Full Text Available Melatonin receptors are members of the G protein-coupled receptor (GPCR family. Three genes for melatonin receptors have been cloned. The MT1 (or Mel1a or MTNR1A and MT2 (or Mel1b or MTNR1B receptor subtypes are present in humans and other mammals, while an additional melatonin receptor subtype, Mel1c (or MTNR1C, has been identified in fish, amphibians and birds. Another melatonin related orphan receptor, GPR50, which does not bind melatonin, is found exclusively in mammals. The hormone melatonin is secreted primarily by the pineal gland, with highest levels occurring during the dark period of a circadian cycle. This hormone acts systemically in numerous organs. In the brain, it is involved in the regulation of various neural and endocrine processes, and it readjusts the circadian pacemaker, the suprachiasmatic nucleus. This article reviews recent studies of gene organization, expression, evolution and mutations of melatonin receptor genes of vertebrates. Gene polymorphisms reveal that numerous mutations are associated with diseases and disorders. The phylogenetic analysis of receptor genes indicates that GPR50 is an outgroup to all other melatonin receptor sequences. GPR50 may have separated from a melatonin receptor ancestor before the split between MTNR1C and the MTNR1A/B ancestor.

  8. Orphan G protein receptor GPR55 as an emerging target in cancer therapy and management

    Directory of Open Access Journals (Sweden)

    Leyva-Illades D

    2013-07-01

    Full Text Available Dinorah Leyva-Illades,1–3 Sharon DeMorrow1–3 1Digestive Disease Research Center, Scott and White Hospital, Temple, TX, USA; 2Department of Internal MedicineTexas A&M Health Science Center, Temple, TX, USA; 3Research Service, Central Texas Veterans Health Care System, Temple, TX, USA Abstract: G protein-coupled receptors (GPCRs modulate a vast array of cellular processes. The current review gives an overview of the general characteristics of GPCRs and their role in physiological conditions. In addition, it describes the current knowledge of the physiological and pathophysiological functions of GPR55, an orphan GPCR, and how it can be exploited as a therapeutic target to combat various cancers. Keywords: GPR55, cancer, GPCR, endocannabinoids

  9. Regulation of Epithelial Morphogenesis by the G-Protein Coupled Receptor Mist and its Ligand Fog*

    Science.gov (United States)

    Manning, Alyssa J.; Peters, Kimberly A.; Peifer, Mark; Rogers, Stephen L.

    2014-01-01

    Epithelial morphogenesis is essential for shaping organs and tissues and for establishment of the three embryonic germ layers during gastrulation. Studies of gastrulation in Drosophila have provided insight into how epithelial morphogenesis is governed by developmental patterning mechanisms. We developed an assay to recapitulate morphogenetic shape changes in individual cultured cells, and used RNAi-based screening to identify Mist, a Drosophila G protein-coupled receptor (GPCR) that transduces signals from the secreted ligand Folded gastrulation (Fog) in cultured cells. Mist functioned in Fog-dependent embryonic morphogenesis, and the transcription factor Snail regulated expression of mist in zygotes. Our data revealed how a cell fate transcriptional program acts through a ligand-GPCR pair to stimulate epithelial morphogenetic shape changes. PMID:24222713

  10. Development of novel ligands for peptide GPCRs.

    Science.gov (United States)

    Moran, Brian M; McKillop, Aine M; O'Harte, Finbarr Pm

    2016-12-01

    Incretin based glucagon-like peptide-1 receptor (GLP-1R) agonists which target a G-protein coupled receptor (GPCR) are currently used in the treatment of type 2 diabetes. This review focuses on GPCRs from pancreatic β-cells, including GLP-1, glucose-dependent insulinotropic polypeptide (GIP), glucagon, somatostatin, pancreatic polypeptide (PP), cholecystokinin (CCK), peptide YY (PYY), oxyntomodulin (OXM) and ghrelin receptors. In addition, fatty acids GPCRs are thought to have an increasing role in regulating peptide secretions namely short fatty acids GPCR (GPR41, GPR43), medium chain fatty acid GPCR (GPR84), long chain fatty acid GPCR (GPR40, GPR120) and cannabinoid-like GPCR (GPR55, GPR119). Several pre-clinical and clinical trials are currently ongoing in peptide GPCR based therapies, including dual and triple agonist peptides which activate two or more GPCRs simultaneously. Copyright © 2016 Elsevier Ltd. All rights reserved.

  11. Pivotal role of extended linker 2 in the activation of Gα by G protein-coupled receptor.

    Science.gov (United States)

    Huang, Jianyun; Sun, Yutong; Zhang, J Jillian; Huang, Xin-Yun

    2015-01-02

    G protein-coupled receptors (GPCRs) relay extracellular signals mainly to heterotrimeric G-proteins (Gαβγ) and they are the most successful drug targets. The mechanisms of G-protein activation by GPCRs are not well understood. Previous studies have revealed a signal relay route from a GPCR via the C-terminal α5-helix of Gα to the guanine nucleotide-binding pocket. Recent structural and biophysical studies uncover a role for the opening or rotating of the α-helical domain of Gα during the activation of Gα by a GPCR. Here we show that β-adrenergic receptors activate eight Gαs mutant proteins (from a screen of 66 Gαs mutants) that are unable to bind Gβγ subunits in cells. Five of these eight mutants are in the αF/Linker 2/β2 hinge region (extended Linker 2) that connects the Ras-like GTPase domain and the α-helical domain of Gαs. This extended Linker 2 is the target site of a natural product inhibitor of Gq. Our data show that the extended Linker 2 is critical for Gα activation by GPCRs. We propose that a GPCR via its intracellular loop 2 directly interacts with the β2/β3 loop of Gα to communicate to Linker 2, resulting in the opening and closing of the α-helical domain and the release of GDP during G-protein activation. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  12. The Epstein-Barr virus BILF1 gene encodes a G protein-coupled receptor that inhibits phosphorylation of RNA-dependent protein kinase

    NARCIS (Netherlands)

    Beisser, P.S.; Verzijl, D.; Gruijthuijsen, Y.K.; Beuken, E.V.; Smit, M.J.; Leurs, R.; Bruggeman, C.A.; Vink, C.

    2005-01-01

    Epstein-Barr vires (EBV) infection is associated with many lymphoproliferative diseases, such as infectious mononucleosis and Burkitt's lymphoma. Consequently, EBV is one of the most extensively studied herpesvirases. Surprisingly, a putative G protein-coupled receptor (GPCR) gene of EBV, BILF1, has

  13. Identification and functional comparison of seven-transmembrane G-protein-coupled BILF1 receptors in recently discovered nonhuman primate lymphocryptoviruses

    DEFF Research Database (Denmark)

    Spiess, Katja; Fares, Suzan; Sparre-Ulrich, Alexander H

    2015-01-01

    Coevolution of herpesviruses with their respective host has resulted in a delicate balance between virus-encoded immune evasion mechanisms and host antiviral immunity. BILF1 encoded by human Epstein-Barr virus (EBV) is a 7-transmembrane (7TM) G-protein-coupled receptor (GPCR) with multiple immuno...

  14. Conopeptide ρ-TIA Defines a New Allosteric Site on the Extracellular Surface of the α1B-Adrenoceptor*♦

    Science.gov (United States)

    Ragnarsson, Lotten; Wang, Ching-I Anderson; Andersson, Åsa; Fajarningsih, Dewi; Monks, Thea; Brust, Andreas; Rosengren, K. Johan; Lewis, Richard J.

    2013-01-01

    The G protein-coupled receptor (GPCR) superfamily is an important drug target that includes over 1000 membrane receptors that functionally couple extracellular stimuli to intracellular effectors. Despite the potential of extracellular surface (ECS) residues in GPCRs to interact with subtype-specific allosteric modulators, few ECS pharmacophores for class A receptors have been identified. Using the turkey β1-adrenergic receptor crystal structure, we modeled the α1B-adrenoceptor (α1B-AR) to help identify the allosteric site for ρ-conopeptide TIA, an inverse agonist at this receptor. Combining mutational radioligand binding and inositol 1-phosphate signaling studies, together with molecular docking simulations using a refined NMR structure of ρ-TIA, we identified 14 residues on the ECS of the α1B-AR that influenced ρ-TIA binding. Double mutant cycle analysis and docking confirmed that ρ-TIA binding was dominated by a salt bridge and cation-π between Arg-4-ρ-TIA and Asp-327 and Phe-330, respectively, and a T-stacking-π interaction between Trp-3-ρ-TIA and Phe-330. Water-bridging hydrogen bonds between Asn-2-ρ-TIA and Val-197, Trp-3-ρ-TIA and Ser-318, and the positively charged N terminus and Glu-186, were also identified. These interactions reveal that peptide binding to the ECS on transmembrane helix 6 (TMH6) and TMH7 at the base of extracellular loop 3 (ECL3) is sufficient to allosterically inhibit agonist signaling at a GPCR. The ligand-accessible ECS residues identified provide the first view of an allosteric inhibitor pharmacophore for α1-adrenoceptors and mechanistic insight and a new set of structural constraints for the design of allosteric antagonists at related GPCRs. PMID:23184947

  15. Conopeptide ρ-TIA defines a new allosteric site on the extracellular surface of the α1B-adrenoceptor.

    Science.gov (United States)

    Ragnarsson, Lotten; Wang, Ching-I Anderson; Andersson, Åsa; Fajarningsih, Dewi; Monks, Thea; Brust, Andreas; Rosengren, K Johan; Lewis, Richard J

    2013-01-18

    The G protein-coupled receptor (GPCR) superfamily is an important drug target that includes over 1000 membrane receptors that functionally couple extracellular stimuli to intracellular effectors. Despite the potential of extracellular surface (ECS) residues in GPCRs to interact with subtype-specific allosteric modulators, few ECS pharmacophores for class A receptors have been identified. Using the turkey β(1)-adrenergic receptor crystal structure, we modeled the α(1B)-adrenoceptor (α(1B)-AR) to help identify the allosteric site for ρ-conopeptide TIA, an inverse agonist at this receptor. Combining mutational radioligand binding and inositol 1-phosphate signaling studies, together with molecular docking simulations using a refined NMR structure of ρ-TIA, we identified 14 residues on the ECS of the α(1B)-AR that influenced ρ-TIA binding. Double mutant cycle analysis and docking confirmed that ρ-TIA binding was dominated by a salt bridge and cation-π between Arg-4-ρ-TIA and Asp-327 and Phe-330, respectively, and a T-stacking-π interaction between Trp-3-ρ-TIA and Phe-330. Water-bridging hydrogen bonds between Asn-2-ρ-TIA and Val-197, Trp-3-ρ-TIA and Ser-318, and the positively charged N terminus and Glu-186, were also identified. These interactions reveal that peptide binding to the ECS on transmembrane helix 6 (TMH6) and TMH7 at the base of extracellular loop 3 (ECL3) is sufficient to allosterically inhibit agonist signaling at a GPCR. The ligand-accessible ECS residues identified provide the first view of an allosteric inhibitor pharmacophore for α(1)-adrenoceptors and mechanistic insight and a new set of structural constraints for the design of allosteric antagonists at related GPCRs.

  16. GPCR Mutation information - GRIPDB | LSDB Archive [Life Science Database Archive metadata

    Lifescience Database Archive (English)

    Full Text Available switchLanguage; BLAST Search Image Search Home About Archive Update History Data List Contact us GRI...s and exposed in molecular surface. Data file File name: gripdb_mutation_info.zip File URL: ftp://ftp.biosciencedbc.jp/archive/gri...pdb/LATEST/gripdb_mutation_info.zip File size: 766 bytes Si...mple search URL http://togodb.biosciencedbc.jp/togodb/view/gripdb_mutation_info#en Data acquisition method P... Site Policy | Contact Us GPCR Mutation information - GRIPDB | LSDB Archive ...

  17. Evolutionary and molecular foundations of multiple contemporary functions of the nitroreductase superfamily.

    Science.gov (United States)

    Akiva, Eyal; Copp, Janine N; Tokuriki, Nobuhiko; Babbitt, Patricia C

    2017-11-07

    Insight regarding how diverse enzymatic functions and reactions have evolved from ancestral scaffolds is fundamental to understanding chemical and evolutionary biology, and for the exploitation of enzymes for biotechnology. We undertook an extensive computational analysis using a unique and comprehensive combination of tools that include large-scale phylogenetic reconstruction to determine the sequence, structural, and functional relationships of the functionally diverse flavin mononucleotide-dependent nitroreductase (NTR) superfamily (>24,000 sequences from all domains of life, 54 structures, and >10 enzymatic functions). Our results suggest an evolutionary model in which contemporary subgroups of the superfamily have diverged in a radial manner from a minimal flavin-binding scaffold. We identified the structural design principle for this divergence: Insertions at key positions in the minimal scaffold that, combined with the fixation of key residues, have led to functional specialization. These results will aid future efforts to delineate the emergence of functional diversity in enzyme superfamilies, provide clues for functional inference for superfamily members of unknown function, and facilitate rational redesign of the NTR scaffold. Copyright © 2017 the Author(s). Published by PNAS.

  18. GPCR Interaction - GRIPDB | LSDB Archive [Life Science Database Archive metadata

    Lifescience Database Archive (English)

    Full Text Available switchLanguage; BLAST Search Image Search Home About Archive Update History Data List Contact us GRI...ed information (disease etc.). Data file File name: gripdb_main.zip File URL: ftp://ftp.biosciencedbc.jp/archive/gripdb/LATEST/gri...godb.biosciencedbc.jp/togodb/view/gripdb_main#en Data acquisition method PDB, Refseq Data analysis method - ...Number of data entries 409 entries Data item Description GRIP ID Interaction ID Main Title Interaction title...tabase Database Description Download License Update History of This Database Site Policy | Contact Us GPCR Interaction - GRIPDB | LSDB Archive ...

  19. Internalization of G-protein-coupled receptors: Implication in receptor function, physiology and diseases.

    Science.gov (United States)

    Calebiro, Davide; Godbole, Amod

    2018-04-01

    G protein-coupled receptors (GPCRs) are the largest family of membrane receptors and mediate the effects of numerous hormones and neurotransmitters. The nearly 1000 GPCRs encoded by the human genome regulate virtually all physiological functions and are implicated in the pathogenesis of prevalent human diseases such as thyroid disorders, hypertension or Parkinson's disease. As a result, 30-50% of all currently prescribed drugs are targeting these receptors. Once activated, GPCRs induce signals at the cell surface. This is often followed by internalization, a process that results in the transfer of receptors from the plasma membrane to membranes of the endosomal compartment. Internalization was initially thought to be mainly implicated in signal desensitization, a mechanism of adaptation to prolonged receptor stimulation. However, several unexpected functions have subsequently emerged. Most notably, accumulating evidence indicates that internalization can induce prolonged receptor signaling on intracellular membranes, which is apparently required for at least some biological effects of hormones like TSH, LH and adrenaline. These findings reveal an even stronger connection between receptor internalization and signaling than previously thought. Whereas new studies are just beginning to reveal an important physiological role for GPCR signaling after internalization and ways to exploit it for therapeutic purposes, future investigations will be required to explore its involvement in human disease. Copyright © 2018 Elsevier Ltd. All rights reserved.

  20. KCTD Hetero-oligomers Confer Unique Kinetic Properties on Hippocampal GABA(B) Receptor-Induced K+ Currents

    Czech Academy of Sciences Publication Activity Database

    Fritzius, T.; Tureček, Rostislav; Seddik, R.; Kobayashi, H.; Tiao, J.; Rem, P.D.; Metz, M.; Králíková, Michaela; Bouvier, M.; Gassmann, M.; Bettler, B.

    2017-01-01

    Roč. 37, č. 5 (2017), s. 1162-1175 ISSN 0270-6474 R&D Projects: GA ČR(CZ) GA14-28334S Institutional support: RVO:68378041 Keywords : G-protein coupled receptor * GABA-B * GPCR Subject RIV: FH - Neurology OBOR OECD: Biology (theoretical, mathematical, thermal, cryobiology, biological rhythm), Evolutionary biology Impact factor: 5.988, year: 2016

  1. Primary structure and functional characterization of a Drosophila dopamine receptor with high homology to human D1/5 receptors.

    Science.gov (United States)

    Gotzes, F; Balfanz, S; Baumann, A

    1994-01-01

    Members of the superfamily of G-protein coupled receptors share significant similarities in sequence and transmembrane architecture. We have isolated a Drosophila homologue of the mammalian dopamine receptor family using a low stringency hybridization approach. The deduced amino acid sequence is approximately 70% homologous to the human D1/D5 receptors. When expressed in HEK 293 cells, the Drosophila receptor stimulates cAMP production in response to dopamine application. This effect was mimicked by SKF 38393, a specific D1 receptor agonist, but inhibited by dopaminergic antagonists such as butaclamol and flupentixol. In situ hybridization revealed that the Drosophila dopamine receptor is highly expressed in the somata of the optic lobes. This suggests that the receptor might be involved in the processing of visual information and/or visual learning in invertebrates.

  2. Heteroreceptors Modulating CGRP Release at Neurovascular Junction: Potential Therapeutic Implications on Some Vascular-Related Diseases

    Directory of Open Access Journals (Sweden)

    Abimael González-Hernández

    2016-01-01

    Full Text Available Calcitonin gene-related peptide (CGRP is a 37-amino-acid neuropeptide belonging to the calcitonin gene peptide superfamily. CGRP is a potent vasodilator with potential therapeutic usefulness for treating vascular-related disease. This peptide is primarily located on C- and Aδ-fibers, which have extensive perivascular presence and a dual sensory-efferent function. Although CGRP has two major isoforms (α-CGRP and β-CGRP, the α-CGRP is the isoform related to vascular actions. Release of CGRP from afferent perivascular nerve terminals has been shown to result in vasodilatation, an effect mediated by at least one receptor (the CGRP receptor. This receptor is an atypical G-protein coupled receptor (GPCR composed of three functional proteins: (i the calcitonin receptor-like receptor (CRLR; a seven-transmembrane protein, (ii the activity-modifying protein type 1 (RAMP1, and (iii a receptor component protein (RCP. Although under physiological conditions, CGRP seems not to play an important role in vascular tone regulation, this peptide has been strongly related as a key player in migraine and other vascular-related disorders (e.g., hypertension and preeclampsia. The present review aims at providing an overview on the role of sensory fibers and CGRP release on the modulation of vascular tone.

  3. Systematic classification of the His-Me finger superfamily.

    Science.gov (United States)

    Jablonska, Jagoda; Matelska, Dorota; Steczkiewicz, Kamil; Ginalski, Krzysztof

    2017-11-16

    The His-Me finger endonucleases, also known as HNH or ββα-metal endonucleases, form a large and diverse protein superfamily. The His-Me finger domain can be found in proteins that play an essential role in cells, including genome maintenance, intron homing, host defense and target offense. Its overall structural compactness and non-specificity make it a perfectly-tailored pathogenic module that participates on both sides of inter- and intra-organismal competition. An extremely low sequence similarity across the superfamily makes it difficult to identify and classify new His-Me fingers. Using state-of-the-art distant homology detection methods, we provide an updated and systematic classification of His-Me finger proteins. In this work, we identified over 100 000 proteins and clustered them into 38 groups, of which three groups are new and cannot be found in any existing public domain database of protein families. Based on an analysis of sequences, structures, domain architectures, and genomic contexts, we provide a careful functional annotation of the poorly characterized members of this superfamily. Our results may inspire further experimental investigations that should address the predicted activity and clarify the potential substrates, to provide more detailed insights into the fundamental biological roles of these proteins. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  4. G protein-coupled receptors: the inside story.

    Science.gov (United States)

    Jalink, Kees; Moolenaar, Wouter H

    2010-01-01

    Recent findings necessitate revision of the traditional view of G protein-coupled receptor (GPCR) signaling and expand the diversity of mechanisms by which receptor signaling influences cell behavior in general. GPCRs elicit signals at the plasma membrane and are then rapidly removed from the cell surface by endocytosis. Internalization of GPCRs has long been thought to serve as a mechanism to terminate the production of second messengers such as cAMP. However, recent studies show that internalized GPCRs can continue to either stimulate or inhibit cAMP production in a sustained manner. They do so by remaining associated with their cognate G protein subunit and adenylyl cyclase at endosomal compartments. Once internalized, the GPCRs produce cellular responses distinct from those elicited at the cell surface.

  5. Non-Dioxin-Like Polychlorinated Biphenyls Inhibit G-Protein Coupled Receptor-Mediated Ca2+ Signaling by Blocking Store-Operated Ca2+ Entry.

    Directory of Open Access Journals (Sweden)

    Se-Young Choi

    Full Text Available Polychlorinated biphenyls (PCBs are ubiquitous pollutants which accumulate in the food chain. Recently, several molecular mechanisms by which non-dioxin-like (NDL PCBs mediate neurodevelopmental and neurobehavioral toxicity have been elucidated. However, although the G-protein coupled receptor (GPCR is a significant target for neurobehavioral disturbance, our understanding of the effects of PCBs on GPCR signaling remains unclear. In this study, we investigated the effects of NDL-PCBs on GPCR-mediated Ca2+ signaling in PC12 cells. We found that ortho-substituted 2,2',6-trichlorinated biphenyl (PCB19 caused a rapid decline in the Ca2+ signaling of bradykinin, a typical Gq- and phospholipase Cβ-coupled GPCR, without any effect on its inositol 1,4,5-trisphosphate production. PCB19 reduced thapsigargin-induced sustained cytosolic Ca2+ levels, suggesting that PCB19 inhibits SOCE. The abilities of other NDL-PCBs to inhibit store-operated Ca2+ entry (SOCE were also examined and found to be of similar potencies to that of PCB19. PCB19 also showed a manner equivalent to that of known SOCE inhibitors. PCB19-mediated SOCE inhibition was confirmed by demonstrating the ability of PCB19 to inhibit the SOCE current and thapsigargin-induced Mn2+ influx. These results imply that one of the molecular mechanism by which NDL-PCBs cause neurobehavioral disturbances involves NDL-PCB-mediated inhibition of SOCE, thereby interfering with GPCR-mediated Ca2+ signaling.

  6. Ligand-independent Thrombopoietin Mutant Receptor Requires Cell Surface Localization for Endogenous Activity*

    OpenAIRE

    Marty, Caroline; Chaligné, Ronan; Lacout, Catherine; Constantinescu, Stefan N.; Vainchenker, William; Villeval, Jean-Luc

    2009-01-01

    The activating W515L mutation in the thrombopoietin receptor (MPL) has been identified in primary myelofibrosis and essential thrombocythemia. MPL belongs to a subset of the cytokine receptor superfamily that requires the JAK2 kinase for signaling. We examined whether the ligand-independent MPLW515L mutant could signal intracellularly. Addition of the endoplasmic reticulum (ER) retention KDEL sequence to the receptor C terminus efficiently locked MPLW515L within its na...

  7. Biased agonism of the calcium-sensing receptor

    DEFF Research Database (Denmark)

    Thomsen, Alex Rojas Bie; Hvidtfeldt, Maja; Bräuner-Osborne, Hans

    2012-01-01

    After the discovery of molecules modulating G protein-coupled receptors (GPCRs) that are able to selectively affect one signaling pathway over others for a specific GPCR, thereby "biasing" the signaling, it has become obvious that the original model of GPCRs existing in either an "on" or "off...... through recruitment of ß-arrestins. Next, by measuring activity of all three signaling pathways we found that barium, spermine, neomycin, and tobramycin act as biased agonist in terms of efficacy and/or potency. Finally, polyamines and aminoglycosides in general were biased in their potencies toward ERK1...

  8. Beyond Modeling: All-Atom Olfactory Receptor Model Simulations

    Directory of Open Access Journals (Sweden)

    Peter C Lai

    2012-05-01

    Full Text Available Olfactory receptors (ORs are a type of GTP-binding protein-coupled receptor (GPCR. These receptors are responsible for mediating the sense of smell through their interaction with odor ligands. OR-odorant interactions marks the first step in the process that leads to olfaction. Computational studies on model OR structures can validate experimental functional studies as well as generate focused and novel hypotheses for further bench investigation by providing a view of these interactions at the molecular level. Here we have shown the specific advantages of simulating the dynamic environment that is associated with OR-odorant interactions. We present a rigorous methodology that ranges from the creation of a computationally-derived model of an olfactory receptor to simulating the interactions between an OR and an odorant molecule. Given the ubiquitous occurrence of GPCRs in the membranes of cells, we anticipate that our OR-developed methodology will serve as a model for the computational structural biology of all GPCRs.

  9. A modeling strategy for G-protein coupled receptors

    Directory of Open Access Journals (Sweden)

    Anna Kahler

    2016-03-01

    Full Text Available Cell responses can be triggered via G-protein coupled receptors (GPCRs that interact with small molecules, peptides or proteins and transmit the signal over the membrane via structural changes to activate intracellular pathways. GPCRs are characterized by a rather low sequence similarity and exhibit structural differences even for functionally closely related GPCRs. An accurate structure prediction for GPCRs is therefore not straightforward. We propose a computational approach that relies on the generation of several independent models based on different template structures, which are subsequently refined by molecular dynamics simulations. A comparison of their conformational stability and the agreement with GPCR-typical structural features is then used to select a favorable model. This strategy was applied to predict the structure of the herpesviral chemokine receptor US28 by generating three independent models based on the known structures of the chemokine receptors CXCR1, CXCR4, and CCR5. Model refinement and evaluation suggested that the model based on CCR5 exhibits the most favorable structural properties. In particular, the GPCR-typical structural features, such as a conserved water cluster or conserved non-covalent contacts, are present to a larger extent in the model based on CCR5 compared to the other models. A final model validation based on the recently published US28 crystal structure confirms that the CCR5-based model is the most accurate and exhibits 80.8% correctly modeled residues within the transmembrane helices. The structural agreement between the selected model and the crystal structure suggests that our modeling strategy may also be more generally applicable to other GPCRs of unknown structure.

  10. Structural Insight into Specificity of Interactions between Nonconventional Three-finger Weak Toxin from Naja kaouthia (WTX) and Muscarinic Acetylcholine Receptors

    Czech Academy of Sciences Publication Activity Database

    Lyukmanova, E. N.; Shenkarev, Z. O.; Shulepko, M. A.; Paramonov, A. S.; Chugunov, A. O.; Janíčková, Helena; Dolejší, Eva; Doležal, Vladimír; Utkin, Y.N.; Tsetlin, V.I.; Arseniev, A. S.; Efremov, R. G.; Dolgikh, D. A.; Kirpichnikov, M. P.

    2015-01-01

    Roč. 290, č. 39 (2015), s. 23616-23630 ISSN 0021-9258 R&D Projects: GA ČR(CZ) GA14-05696S Institutional support: RVO:67985823 Keywords : computer modeling * G protein-coupled receptor (GPCR) * site-directed mutagenesis Subject RIV: ED - Physiology Impact factor: 4.258, year: 2015

  11. Tre1, a G protein-coupled receptor, directs transepithelial migration of Drosophila germ cells.

    Directory of Open Access Journals (Sweden)

    Prabhat S Kunwar

    2003-12-01

    Full Text Available In most organisms, germ cells are formed distant from the somatic part of the gonad and thus have to migrate along and through a variety of tissues to reach the gonad. Transepithelial migration through the posterior midgut (PMG is the first active step during Drosophila germ cell migration. Here we report the identification of a novel G protein-coupled receptor (GPCR, Tre1, that is essential for this migration step. Maternal tre1 RNA is localized to germ cells, and tre1 is required cell autonomously in germ cells. In tre1 mutant embryos, most germ cells do not exit the PMG. The few germ cells that do leave the midgut early migrate normally to the gonad, suggesting that this gene is specifically required for transepithelial migration and that mutant germ cells are still able to recognize other guidance cues. Additionally, inhibiting small Rho GTPases in germ cells affects transepithelial migration, suggesting that Tre1 signals through Rho1. We propose that Tre1 acts in a manner similar to chemokine receptors required during transepithelial migration of leukocytes, implying an evolutionarily conserved mechanism of transepithelial migration. Recently, the chemokine receptor CXCR4 was shown to direct migration in vertebrate germ cells. Thus, germ cells may more generally use GPCR signaling to navigate the embryo toward their target.

  12. Global medicinal chemistry and GPCR conference: interview with Stevan Djuric.

    Science.gov (United States)

    Djuric, Stevan

    2018-04-01

    Stevan Djuric speaks to Benjamin Walden, Commissioning Editor. Stevan Djuric is head of the global Medicinal Chemistry Leadership Team at AbbVie and is also Vice President of the Discovery Chemistry and Technology organization within their Discovery organization and chemistry outsourcing activities. He spoke at the Global-Medicinal-Chemistry and GPCR summit on the imperative to develop chemistry related technology that can reduce cycle time, cost of goods and improve probability of success. To this end, he discussed his efforts in the chemistry technology area with a focus on integrated synthesis-purification bioassay, and flow photochemistry and high temperature chemistry platforms.

  13. Orphan G protein receptor GPR55 as an emerging target in cancer therapy and management

    International Nuclear Information System (INIS)

    Leyva-Illades, Dinorah; DeMorrow, Sharon

    2013-01-01

    G protein-coupled receptors (GPCRs) modulate a vast array of cellular processes. The current review gives an overview of the general characteristics of GPCRs and their role in physiological conditions. In addition, it describes the current knowledge of the physiological and pathophysiological functions of GPR55, an orphan GPCR, and how it can be exploited as a therapeutic target to combat various cancers

  14. Mouse Hepatitis Virus Strain A59 and Blocking Antireceptor Monoclonal Antibody Bind to the N-Terminal Domain of Cellular Receptor

    Science.gov (United States)

    Dveksler, Gabriela S.; Pensiero, Michael N.; Dieffenbach, Carl W.; Cardellichio, Christine B.; Basile, Alexis A.; Elia, Patrick E.; Holmes, Kathryn V.

    1993-03-01

    Mouse hepatitis virus (MHV) strain A59 uses as cellular receptors members of the carcinoembryonic antigen family in the immunoglobulin superfamily. Recombinant receptor proteins with deletions of whole or partial immunoglobulin domains were used to identify the regions of receptor glycoprotein recognized by virus and by antireceptor monoclonal antibody CC1, which blocks infection of murine cells. Monoclonal antibody CC1 and MHV-A59 virions bound only to recombinant proteins containing the entire first domain of MHV receptor. To determine which of the proteins could serve as functional virus receptors, receptor-negative hamster cells were transfected with recombinant deletion clones and then challenged with MHV-A59 virions. Receptor activity required the entire N-terminal domain with either the second or the fourth domain and the transmembrane and cytoplasmic domains. Recombinant proteins lacking the first domain or its C-terminal portion did not serve as viral receptors. Thus, like other virus receptors in the immunoglobulin superfamily, including CD4, poliovirus receptor, and intercellular adhesion molecule 1, the N-terminal domain of MHV receptor is recognized by the virus and the blocking monoclonal antibody.

  15. The retinoid X receptor response element in the human aldehyde dehydrogenase 2 promoter is antagonized by the chicken ovalbumin upstream promoter family of orphan receptors

    NARCIS (Netherlands)

    Pinaire, J; Hasanadka, R; Fang, M; Chou, WY; Stewart, MJ; Kruijer, W; Crabb, D

    2000-01-01

    Two tandem sites in the aldehyde dehydrogenase 2 promoter (designated FP330-5' and FP330-3') that bind members of the nuclear receptor superfamily mere recently identified. Antibodies against apolipoprotein regulatory protein (ARP-1) altered DNA-protein interactions in electrophoretic mobility shift

  16. Signal transduction through the IL-4 and insulin receptor families.

    Science.gov (United States)

    Wang, L M; Keegan, A; Frankel, M; Paul, W E; Pierce, J H

    1995-07-01

    Activation of tyrosine kinase-containing receptors and intracellular tyrosine kinases by ligand stimulation is known to be crucial for mediating initial and subsequent events involved in mitogenic signal transduction. Receptors for insulin and insulin-like growth factor 1 (IGF-1) contain cytoplasmic tyrosine kinase domains that undergo autophosphorylation upon ligand stimulation. Activation of these receptors also leads to pronounced and rapid tyrosine phosphorylation of insulin receptor substrate 1 (IRS-1) in cells of connective tissue origin. A related substrate, designated 4PS, is similarly phosphorylated by insulin and IGF-1 stimulation in many hematopoietic cell types. IRS-1 and 4PS possess a number of tyrosine phosphorylation sites that are within motifs that bind specific SH2-containing molecules known to be involved in mitogenic signaling such as PI-3 kinase, SHPTP-2 (Syp) and Grb-2. Thus, they appear to act as docking substrates for a variety of signaling molecules. The majority of hematopoietic cytokines bind to receptors that do not possess intrinsic kinase activity, and these receptors have been collectively termed as members of the hematopoietin receptor superfamily. Despite their lack of tyrosine kinase domains, stimulation of these receptors has been demonstrated to activate intracellular kinases leading to tyrosine phosphorylation of multiple substrates. Recent evidence has demonstrated that activation of different members of the Janus family of tyrosine kinases is involved in mediating tyrosine phosphorylation events by specific cytokines. Stimulation of the interleukin 4 (IL-4) receptor, a member of the hematopoietin receptor superfamily, is thought to result in activation of Jak1, Jak3, and/or Fes tyrosine kinases.(ABSTRACT TRUNCATED AT 250 WORDS)

  17. Functional diversity of the superfamily of K⁺ transporters to meet various requirements.

    Science.gov (United States)

    Diskowski, Marina; Mikusevic, Vedrana; Stock, Charlott; Hänelt, Inga

    2015-09-01

    The superfamily of K+ transporters unites proteins from plants, fungi, bacteria, and archaea that translocate K+ and/or Na+ across membranes. These proteins are key components in osmotic regulation, pH homeostasis, and resistance to high salinity and dryness. The members of the superfamily are closely related to K+ channels such as KcsA but also show several striking differences that are attributed to their altered functions. This review highlights these functional differences, focusing on the bacterial superfamily members KtrB, TrkH, and KdpA. The functional variations within the family and comparison to MPM-type K+ channels are discussed in light of the recently solved structures of the Ktr and Trk systems.

  18. Conformational analysis of g protein-coupled receptor signaling by hydrogen/deuterium exchange mass spectrometry.

    Science.gov (United States)

    Li, Sheng; Lee, Su Youn; Chung, Ka Young

    2015-01-01

    Conformational change and protein-protein interactions are two major mechanisms of membrane protein signal transduction, including G protein-coupled receptors (GPCRs). Upon agonist binding, GPCRs change conformation, resulting in interaction with downstream signaling molecules such as G proteins. To understand the precise signaling mechanism, studies have investigated the structural mechanism of GPCR signaling using X-ray crystallography, nuclear magnetic resonance (NMR), or electron paramagnetic resonance. In addition to these techniques, hydrogen/deuterium exchange mass spectrometry (HDX-MS) has recently been used in GPCR studies. HDX-MS measures the rate at which peptide amide hydrogens exchange with deuterium in the solvent. Exposed or flexible regions have higher exchange rates and excluded or ordered regions have lower exchange rates. Therefore, HDX-MS is a useful tool for studying protein-protein interfaces and conformational changes after protein activation or protein-protein interactions. Although HDX-MS does not give high-resolution structures, it analyzes protein conformations that are difficult to study with X-ray crystallography or NMR. Furthermore, conformational information from HDX-MS can help in the crystallization of X-ray crystallography by suggesting highly flexible regions. Interactions between GPCRs and downstream signaling molecules are not easily analyzed by X-ray crystallography or NMR because of the large size of the GPCR-signaling molecule complexes, hydrophobicity, and flexibility of GPCRs. HDX-MS could be useful for analyzing the conformational mechanism of GPCR signaling. In this chapter, we discuss details of HDX-MS for analyzing GPCRs using the β2AR-G protein complex as a model system. © 2015 Elsevier Inc. All rights reserved.

  19. Internalization of the human CRF receptor 1 is independent of classical phosphorylation sites and of beta-arrestin 1 recruitment

    DEFF Research Database (Denmark)

    Rasmussen, Trine N; Novak, Ivana; Nielsen, Søren M

    2004-01-01

    The corticotropin releasing factor receptor 1 (CRFR1) belongs to the superfamily of G-protein coupled receptors. Though CRF is involved in the aetiology of several stress-related disorders, including depression and anxiety, details of CRFR1 regulation such as internalization remain uncharacterized...

  20. Protein 4.1, a component of the erythrocyte membrane skeleton and its related homologue proteins forming the protein 4.1/FERM superfamily.

    Directory of Open Access Journals (Sweden)

    Aleksander F Sikorski

    2007-01-01

    Full Text Available The review is focused on the domain structure and function of protein 4.1, one of the proteins belonging to the membrane skeleton. The protein 4.1 of the red blood cells (4.1R is a multifunctional protein that localizes to the membrane skeleton and stabilizes erythrocyte shape and membrane mechanical properties, such as deformability and stability, via lateral interactions with spectrin, actin, glycophorin C and protein p55. Protein 4.1 binding is modulated through the action of kinases and/or calmodulin-Ca2+. Non-erythroid cells express the 4.1R homologues: 4.1G (general type, 4.1B (brain type, and 4.1N (neuron type, and the whole group belongs to the protein 4.1 superfamily, which is characterized by the presence of a highly conserved FERM domain at the N-terminus of the molecule. Proteins 4.1R, 4.1G, 4.1N and 4.1B are encoded by different genes. Most of the 4.1 superfamily proteins also contain an actin-binding domain. To date, more than 40 members have been identified. They can be divided into five groups: protein 4.1 molecules, ERM proteins, talin-related molecules, protein tyrosine phosphatase (PTPH proteins and NBL4 proteins. We have focused our attention on the main, well known representatives of 4.1 superfamily and tried to choose the proteins which are close to 4.1R or which have distinct functions. 4.1 family proteins are not just linkers between the plasma membrane and membrane skeleton; they also play an important role in various processes. Some, such as focal adhesion kinase (FAK, non-receptor tyrosine kinase that localizes to focal adhesions in adherent cells, play the role in cell adhesion. The other members control or take part in tumor suppression, regulation of cell cycle progression, inhibition of cell proliferation, downstream signaling of the glutamate receptors, and establishment of cell polarity; some are also involved in cell proliferation, cell motility, and/or cell-to-cell communication.

  1. High-level expression, purification and characterization of a constitutively active thromboxane A2 receptor polymorphic variant.

    Directory of Open Access Journals (Sweden)

    Bing Xu

    Full Text Available G protein-coupled receptors (GPCRs exhibit some level of basal signaling even in the absence of a bound agonist. This basal or constitutive signaling can have important pathophysiological roles. In the past few years, a number of high resolution crystal structures of GPCRs have been reported, including two crystal structures of constitutively active mutants (CAM of the dim-light receptor, rhodopsin. The structural characterizations of CAMs are impeded by the lack of proper expression systems. The thromboxane A2 receptor (TP is a GPCR that mediates vasoconstriction and promotes thrombosis in response to the binding of thromboxane. Here, we report on the expression and purification of a genetic variant and CAM in TP, namely A160T, using tetracycline-inducible HEK293S-TetR and HEK293S (GnTI¯-TetR cell lines. Expression of the TP and the A160T genes in these mammalian cell lines resulted in a 4-fold increase in expression to a level of 15.8 ±0.3 pmol of receptor/mg of membrane protein. The receptors expressed in the HEK293S (GnTI(--TetR cell line showed homogeneous glycosylation. The functional yield of the receptors using a single step affinity purification was 45 µg/10⁶ cells. Temperature- dependent secondary structure changes of the purified TP and A160T receptors were characterized using circular dichroism (CD spectropolarimetry. The CD spectra shows that the loss of activity or thermal sensitivity that was previously observed for the A160T mutant, is not owing to large unfolding of the protein but rather to a more subtle effect. This is the first study to report on the successful high-level expression, purification, and biophysical characterization of a naturally occurring, diffusible ligand activated GPCR CAM.

  2. cAMP biosensors applied in molecular pharmacological studies of G protein-coupled receptors

    DEFF Research Database (Denmark)

    Mathiesen, Jesper Mosolff; Vedel, Line; Bräuner-Osborne, Hans

    2013-01-01

    end-point assays for quantifying GPCR-mediated changes in intracellular cAMP levels exist. More recently, fluorescence resonance energy transfer (FRET)-based cAMP biosensors that can quantify intracellular cAMP levels in real time have been developed. These FRET-based cAMP biosensors have been used...... primarily in single cell FRET microscopy to monitor and visualize changes in cAMP upon GPCR activation. Here, a similar cAMP biosensor with a more efficient mCerulean/mCitrine FRET pair is described for use in the 384-well plate format. After cloning and expression in HEK293 cells, the biosensor...... is characterized in the 384-well plate format and used for measuring the signaling of the G(s)-coupled ß(2)-adrenergic receptor. The procedures described may be applied for other FRET-based biosensors in terms of characterization and conversion to the 384-well plate format....

  3. N-glycosylation of the β2 adrenergic receptor regulates receptor function by modulating dimerization.

    Science.gov (United States)

    Li, Xiaona; Zhou, Mang; Huang, Wei; Yang, Huaiyu

    2017-07-01

    N-glycosylation is a common post-translational modification of G-protein-coupled receptors (GPCRs). However, it remains unknown how N-glycosylation affects GPCR signaling. β 2 adrenergic receptor (β 2 AR) has three N-glycosylation sites: Asn6, Asn15 at the N-terminus, and Asn187 at the second extracellular loop (ECL2). Here, we show that deletion of the N-glycan did not affect receptor expression and ligand binding. Deletion of the N-glycan at the N-terminus rather than Asn187 showed decreased effects on isoproterenol-promoted G-protein-dependent signaling, β-arrestin2 recruitment, and receptor internalization. Both N6Q and N15Q showed decreased receptor dimerization, while N187Q did not influence receptor dimerization. As decreased β 2 AR homodimer accompanied with reduced efficiency for receptor function, we proposed that the N-glycosylation of β 2 AR regulated receptor function by influencing receptor dimerization. To verify this hypothesis, we further paid attention to the residues at the dimerization interface. Studies of Lys60 and Glu338, two residues at the receptor dimerization interface, exhibited that the K60A/E338A showed decreased β 2 AR dimerization and its effects on receptor signaling were similar to N6Q and N15Q, which further supported the importance of receptor dimerization for receptor function. This work provides new insights into the relationship among glycosylation, dimerization, and function of GPCRs. Peptide-N-glycosidase F (PNGase F, EC 3.2.2.11); endo-β-N-acetylglucosaminidase A (Endo-A, EC 3.2.1.96). © 2017 Federation of European Biochemical Societies.

  4. Nuclear hormone receptors in parasitic helminths

    OpenAIRE

    Wu, Wenjie; LoVerde, Philip T

    2010-01-01

    Nuclear receptors (NRs) belong to a large protein superfamily that are important transcriptional modulators in metazoans. Parasitic helminths include parasitic worms from the Lophotrochozoa (Platyhelminths) and Ecdysozoa (Nematoda). NRs in parasitic helminths diverged into two different evolutionary lineages. NRs in parasitic Platyhelminths have orthologues in Deuterostomes, in arthropods or both with a feature of extensive gene loss and gene duplication within different gene groups. NRs in p...

  5. Differential transcription of the orphan receptor RORbeta in nuclear extracts derived from Neuro2A and HeLa cells.

    NARCIS (Netherlands)

    Gawlas, K.; Stunnenberg, H.G.

    2001-01-01

    An important model system for studying the process leading to productive transcription is provided by the superfamily of nuclear receptors, which are for the most part ligand-controlled transcription factors. Over the past years several 'orphan' nuclear receptors have been isolated for which no

  6. Orphan nuclear receptor TR4 and fibroblast growth factor 1 in metabolism

    NARCIS (Netherlands)

    Liu, Weilin

    2016-01-01

    Metabolic homeostasis is achieved, in part, through the coordinated activities of members of the Nuclear Receptor (NR) family, a superfamily of ligand-modulated transcription factors (TFs) that mediate responses to a wide range of lipophilic signaling molecules including lipids, steroids, retinoids,

  7. Cholesterol activates the G-protein coupled receptor Smoothened to promote Hedgehog signaling

    Science.gov (United States)

    Luchetti, Giovanni; Sircar, Ria; Kong, Jennifer H; Nachtergaele, Sigrid; Sagner, Andreas; Byrne, Eamon FX; Covey, Douglas F; Siebold, Christian; Rohatgi, Rajat

    2016-01-01

    Cholesterol is necessary for the function of many G-protein coupled receptors (GPCRs). We find that cholesterol is not just necessary but also sufficient to activate signaling by the Hedgehog (Hh) pathway, a prominent cell-cell communication system in development. Cholesterol influences Hh signaling by directly activating Smoothened (SMO), an orphan GPCR that transmits the Hh signal across the membrane in all animals. Unlike many GPCRs, which are regulated by cholesterol through their heptahelical transmembrane domains, SMO is activated by cholesterol through its extracellular cysteine-rich domain (CRD). Residues shown to mediate cholesterol binding to the CRD in a recent structural analysis also dictate SMO activation, both in response to cholesterol and to native Hh ligands. Our results show that cholesterol can initiate signaling from the cell surface by engaging the extracellular domain of a GPCR and suggest that SMO activity may be regulated by local changes in cholesterol abundance or accessibility. DOI: http://dx.doi.org/10.7554/eLife.20304.001 PMID:27705744

  8. Adipokinetic hormones and their G protein-coupled receptors emerged in Lophotrochozoa

    DEFF Research Database (Denmark)

    Li, Shizhong; Hauser, Frank; Skadborg, Signe K.

    2016-01-01

    the neuropeptide systems used by proto- or deuterostomes. An exception, however, are members of the gonadotropin-releasing hormone (GnRH) receptor superfamily, which occur in both evolutionary lineages, where GnRHs are the ligands in Deuterostomia and GnRH-like peptides, adipokinetic hormone (AKH), corazonin...

  9. Purification of family B G protein-coupled receptors using nanodiscs: Application to human glucagon-like peptide-1 receptor.

    Directory of Open Access Journals (Sweden)

    Yingying Cai

    Full Text Available Family B G protein-coupled receptors (GPCRs play vital roles in hormone-regulated homeostasis. They are drug targets for metabolic diseases, including type 2 diabetes and osteoporosis. Despite their importance, the signaling mechanisms for family B GPCRs at the molecular level remain largely unexplored due to the challenges in purification of functional receptors in sufficient amount for biophysical characterization. Here, we purified the family B GPCR human glucagon-like peptide-1 (GLP-1 receptor (GLP1R, whose agonists, e.g. exendin-4, are used for the treatment of type 2 diabetes mellitus. The receptor was expressed in HEK293S GnTl- cells using our recently developed protocol. The protocol incorporates the receptor into the native-like lipid environment of reconstituted high density lipoprotein (rHDL particles, also known as nanodiscs, immediately after the membrane solubilization step followed by chromatographic purification, minimizing detergent contact with the target receptor to reduce denaturation and prolonging stabilization of receptor in lipid bilayers without extra steps of reconstitution. This method yielded purified GLP1R in nanodiscs that could bind to GLP-1 and exendin-4 and activate Gs protein. This nanodisc purification method can potentially be a general strategy to routinely obtain purified family B GPCRs in the 10s of microgram amounts useful for spectroscopic analysis of receptor functions and activation mechanisms.

  10. Purification of family B G protein-coupled receptors using nanodiscs: Application to human glucagon-like peptide-1 receptor.

    Science.gov (United States)

    Cai, Yingying; Liu, Yuting; Culhane, Kelly J; DeVree, Brian T; Yang, Yang; Sunahara, Roger K; Yan, Elsa C Y

    2017-01-01

    Family B G protein-coupled receptors (GPCRs) play vital roles in hormone-regulated homeostasis. They are drug targets for metabolic diseases, including type 2 diabetes and osteoporosis. Despite their importance, the signaling mechanisms for family B GPCRs at the molecular level remain largely unexplored due to the challenges in purification of functional receptors in sufficient amount for biophysical characterization. Here, we purified the family B GPCR human glucagon-like peptide-1 (GLP-1) receptor (GLP1R), whose agonists, e.g. exendin-4, are used for the treatment of type 2 diabetes mellitus. The receptor was expressed in HEK293S GnTl- cells using our recently developed protocol. The protocol incorporates the receptor into the native-like lipid environment of reconstituted high density lipoprotein (rHDL) particles, also known as nanodiscs, immediately after the membrane solubilization step followed by chromatographic purification, minimizing detergent contact with the target receptor to reduce denaturation and prolonging stabilization of receptor in lipid bilayers without extra steps of reconstitution. This method yielded purified GLP1R in nanodiscs that could bind to GLP-1 and exendin-4 and activate Gs protein. This nanodisc purification method can potentially be a general strategy to routinely obtain purified family B GPCRs in the 10s of microgram amounts useful for spectroscopic analysis of receptor functions and activation mechanisms.

  11. Genetic analysis of the estrogen-related receptor alpha and studies of association with obesity and type 2 diabetes

    DEFF Research Database (Denmark)

    Larsen, L H; Rose, C S; Sparsø, T

    2007-01-01

    The estrogen-related receptor alpha (ERRalpha or NR3B1) is a transcription factor from the nuclear receptor super-family, group III. The gene encoding ERRalpha (ESRRA) is located on chromosome 11q13, a region showing genetic linkage to body mass index and fat percentage. Through interaction...

  12. Metabotropic glutamate receptor 1 splice variants mGluR1a and mGluR1b combine in mGluR1a/b dimers in vivo

    Czech Academy of Sciences Publication Activity Database

    Techlovská, Šárka; Chambers, Jayne Nicole; Dvořáková, Michaela; Petralia, R.S.; Wang, Y.X.; Hájková, Alena; Franková, Daniela; Prezeau, L.; Blahoš, Jaroslav

    2014-01-01

    Roč. 86, November (2014), s. 329-326 ISSN 0028-3908 R&D Projects: GA ČR GAP303/12/2408 Institutional support: RVO:68378050 Keywords : Glutamate receptors * GPCR * alternative splicing Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 5.106, year: 2014

  13. GPCR Interaction: 157 [GRIPDB[Archive

    Lifescience Database Archive (English)

    Full Text Available ation of V1a, V2 and OT homodimers. In addition, coexpressions of different pairs of receptors (V1a/V2, V1a/...one receptor to interact with another receptor) were identical whatever the pair of receptors coexpress...ed in the cells, which suggests that the proportion of homodimers and heterodimers depends on the relative expr...ents using chimeric V1a or V2 vasopressin or oxytocin and receptors labeled on the C-terminus with luciferas...ession of each receptor. 19896898 BRET NP_000907.2 ...

  14. GPCR Interaction: 156 [GRIPDB[Archive

    Lifescience Database Archive (English)

    Full Text Available ation of V1a, V2 and OT homodimers. In addition, coexpressions of different pairs of receptors (V1a/V2, V1a/...one receptor to interact with another receptor) were identical whatever the pair of receptors coexpress...ed in the cells, which suggests that the proportion of homodimers and heterodimers depends on the relative expr...ents using chimeric V1a or V2 vasopressin or oxytocin and receptors labeled on the C-terminus with luciferas...ession of each receptor. 19896898 BRET NP_000697.1 ...

  15. GPCR Interaction: 155 [GRIPDB[Archive

    Lifescience Database Archive (English)

    Full Text Available n of V1a, V2 and OT homodimers. In addition, coexpressions of different pairs of receptors (V1a/V2, V1a/OTR ...receptor to interact with another receptor) were identical whatever the pair of receptors coexpress...ed in the cells, which suggests that the proportion of homodimers and heterodimers depends on the relative expression of each receptor. 19896898 BRET NP_000907.2 ... ... using chimeric V1a or V2 vasopressin or oxytocin and receptors labeled on the C-terminus with luciferase or

  16. Bacterial Multidrug Efflux Pumps of the Major Facilitator Superfamily as Targets for Modulation.

    Science.gov (United States)

    Kumar, Sanath; He, Guixin; Kakarla, Prathusha; Shrestha, Ugina; Ranjana, K C; Ranaweera, Indrika; Willmon, T Mark; Barr, Sharla R; Hernandez, Alberto J; Varela, Manuel F

    2016-01-01

    Causative agents of infectious disease that are multidrug resistant bacterial pathogens represent a serious public health concern due to the increasingly difficult nature of achieving efficacious clinical treatments. Of the various acquired and intrinsic antimicrobial agent resistance determinants, integral-membrane multidrug efflux pumps of the major facilitator superfamily constitute a major mechanism of bacterial resistance. The major facilitator superfamily (MFS) encompasses thousands of known related secondary active and passive solute transporters, including multidrug efflux pumps, from bacteria to humans. This review article addresses recent developments involving the targeting by various modulators of bacterial multidrug efflux pumps from the major facilitator superfamily. It is currently of tremendous interest to modulate bacterial multidrug efflux pumps in order to eventually restore the clinical efficacy of therapeutic agents against recalcitrant bacterial infections. Such MFS multidrug efflux pumps are good targets for modulation.

  17. Elaboración de un modelo 3D del receptor Humano de NK1 a partir del receptor delta Opiode

    OpenAIRE

    Vílchez Cruz, Ángela

    2015-01-01

    La publicación de la primera estructura cristalográfica de resolución atómica de un miembro de la familia de los receptores acoplados a proteínas G (GPCR) como es la Rodopsina Bovina (Rho) (1)marcó el comienzo de los estudios sobre la relación entre la actividad biológica y la estructura de estas proteínas (1,2). Si bien se han llevado a cabo numerosos estudios de difracción de rayos X de las estructuras de GPCRs desde ese momento, dada la dificultad de cristalización de es...

  18. The estrogen-related receptors and the adipocyte.

    Science.gov (United States)

    Carnesecchi, Julie; Vanacker, Jean-Marc

    2013-08-01

    The estrogen-related receptors (ERRα, β, and γ) are orphan members of the nuclear receptor superfamily. ERRα and γ are highly expressed in tissues displaying elevated energy demands and are involved in several aspects of energetic metabolism, which they regulate mostly in association with members of the PGC-1 coactivator family. These activities have mostly been documented in the liver, heart, or skeletal muscle. ERRα and γ are also highly expressed in adipocytes. Their precise roles in this cell type are less documented, although published data indicate that they contribute to cell differentiation as well as functionality. This review describes these activities.

  19. GPCRdb

    DEFF Research Database (Denmark)

    Isberg, Vignir; Mordalski, Stefan; Munk, Christian

    2016-01-01

    Recent developments in G protein-coupled receptor (GPCR) structural biology and pharmacology have greatly enhanced our knowledge of receptor structure-function relations, and have helped improve the scientific foundation for drug design studies. The GPCR database, GPCRdb, serves a dual role...... in disseminating and enabling new scientific developments by providing reference data, analysis tools and interactive diagrams. This paper highlights new features in the fifth major GPCRdb release: (i) GPCR crystal structure browsing, superposition and display of ligand interactions; (ii) direct deposition...... a smooth browsing experience and development. GPCRdb is available at http://www.gpcrdb.org/ and it's open source code at https://bitbucket.org/gpcr/protwis....

  20. Quaternary structure of the yeast pheromone receptor Ste2 in living cells.

    Science.gov (United States)

    Stoneman, Michael R; Paprocki, Joel D; Biener, Gabriel; Yokoi, Koki; Shevade, Aishwarya; Kuchin, Sergei; Raicu, Valerică

    2017-09-01

    Transmembrane proteins known as G protein-coupled receptors (GPCRs) have been shown to form functional homo- or hetero-oligomeric complexes, although agreement has been slow to emerge on whether homo-oligomerization plays functional roles. Here we introduce a platform to determine the identity and abundance of differing quaternary structures formed by GPCRs in living cells following changes in environmental conditions, such as changes in concentrations. The method capitalizes on the intrinsic capability of FRET spectrometry to extract oligomer geometrical information from distributions of FRET efficiencies (or FRET spectrograms) determined from pixel-level imaging of cells, combined with the ability of the statistical ensemble approaches to FRET to probe the proportion of different quaternary structures (such as dimers, rhombus or parallelogram shaped tetramers, etc.) from averages over entire cells. Our approach revealed that the yeast pheromone receptor Ste2 forms predominantly tetramers at average expression levels of 2 to 25 molecules per pixel (2.8·10 -6 to 3.5·10 -5 molecules/nm 2 ), and a mixture of tetramers and octamers at expression levels of 25-100 molecules per pixel (3.5·10 -5 to 1.4·10 -4 molecules/nm 2 ). Ste2 is a class D GPCR found in the yeast Saccharomyces cerevisiae of the mating type a, and binds the pheromone α-factor secreted by cells of the mating type α. Such investigations may inform development of antifungal therapies targeting oligomers of pheromone receptors. The proposed FRET imaging platform may be used to determine the quaternary structure sub-states and stoichiometry of any GPCR and, indeed, any membrane protein in living cells. This article is part of a Special Issue entitled: Interactions between membrane receptors in cellular membranes edited by Kalina Hristova. Copyright © 2016 Elsevier B.V. All rights reserved.

  1. Enhanced A3 adenosine receptor selectivity of multivalent nucleoside-dendrimer conjugates

    Directory of Open Access Journals (Sweden)

    Shainberg Asher

    2008-10-01

    Full Text Available Abstract Background An approach to use multivalent dendrimer carriers for delivery of nucleoside signaling molecules to their cell surface G protein-coupled receptors (GPCRs was recently introduced. Results A known adenosine receptor (AR agonist was conjugated to polyamidoamine (PAMAM dendrimer carriers for delivery of the intact covalent conjugate to on the cell surface. Depending on the linking moiety, multivalent conjugates of the N6-chain elongated functionalized congener ADAC (N6-[4-[[[4-[[[(2-aminoethylamino]carbonyl]methyl]anilino]carbonyl]methyl]phenyl]-adenosine achieved unanticipated high selectivity in binding to the cytoprotective human A3 AR, a class A GPCR. The key to this selectivity of > 100-fold in both radioreceptor binding (Ki app = 2.4 nM and functional assays (EC50 = 1.6 nM in inhibition of adenylate cyclase was maintaining a free amino group (secondary in an amide-linked chain. Attachment of neutral amide-linked chains or thiourea-containing chains preserved the moderate affinity and efficacy at the A1 AR subtype, but there was no selectivity for the A3 AR. Since residual amino groups on dendrimers are associated with cytotoxicity, the unreacted terminal positions of this A3 AR-selective G2.5 dendrimer were present as carboxylate groups, which had the further benefit of increasing water-solubility. The A3 AR selective G2.5 dendrimer was also visualized binding the membrane of cells expressing the A3 receptor but did not bind cells that did not express the receptor. Conclusion This is the first example showing that it is feasible to modulate and even enhance the pharmacological profile of a ligand of a GPCR based on conjugation to a nanocarrier and the precise structure of the linking group, which was designed to interact with distal extracellular regions of the 7 transmembrane-spanning receptor. This ligand tool can now be used in pharmacological models of tissue rescue from ischemia and to probe the existence of A3 AR

  2. Common gene variants in the tumor necrosis factor (TNF and TNF receptor superfamilies and NF-kB transcription factors and non-Hodgkin lymphoma risk.

    Directory of Open Access Journals (Sweden)

    Sophia S Wang

    Full Text Available A promoter polymorphism in the pro-inflammatory cytokine tumor necrosis factor (TNF (TNF G-308A is associated with increased non-Hodgkin lymphoma (NHL risk. The protein product, TNF-alpha, activates the nuclear factor kappa beta (NF-kappaB transcription factor, and is critical for inflammatory and apoptotic responses in cancer progression. We hypothesized that the TNF and NF-kappaB pathways are important for NHL and that gene variations across the pathways may alter NHL risk.We genotyped 500 tag single nucleotide polymorphisms (SNPs from 48 candidate gene regions (defined as 20 kb 5', 10 kb 3' in the TNF and TNF receptor superfamilies and the NF-kappaB and related transcription factors, in 1946 NHL cases and 1808 controls pooled from three independent population-based case-control studies. We obtained a gene region-level summary of association by computing the minimum p-value ("minP test". We used logistic regression to compute odds ratios and 95% confidence intervals for NHL and four major NHL subtypes in relation to SNP genotypes and haplotypes. For NHL, the tail strength statistic supported an overall relationship between the TNF/NF-kappaB pathway and NHL (p = 0.02. We confirmed the association between TNF/LTA on chromosome 6p21.3 with NHL and found the LTA rs2844484 SNP most significantly and specifically associated with the major subtype, diffuse large B-cell lymphoma (DLBCL (p-trend = 0.001. We also implicated for the first time, variants in NFKBIL1 on chromosome 6p21.3, associated with NHL. Other gene regions identified as statistically significantly associated with NHL included FAS, IRF4, TNFSF13B, TANK, TNFSF7 and TNFRSF13C. Accordingly, the single most significant SNPs associated with NHL were FAS rs4934436 (p-trend = 0.0024, IRF4 rs12211228 (p-trend = 0.0026, TNFSF13B rs2582869 (p-trend = 0.0055, TANK rs1921310 (p-trend = 0.0025, TNFSF7 rs16994592 (p-trend = 0.0024, and TNFRSF13C rs6002551 (p-trend = 0.0074. All associations were

  3. Action mechanisms of Liver X Receptors

    Energy Technology Data Exchange (ETDEWEB)

    Gabbi, Chiara; Warner, Margaret [Center for Nuclear Receptors and Cell Signaling, University of Houston, 3056 Cullen Blv, 77204 Houston, Texas (United States); Gustafsson, Jan-Åke, E-mail: jgustafs@central.uh.edu [Center for Nuclear Receptors and Cell Signaling, University of Houston, 3056 Cullen Blv, 77204 Houston, Texas (United States); Department of Biosciences and Nutrition, Karolinska Institutet, Novum S-141 86 (Sweden)

    2014-04-11

    Highlights: • LXRα and LXRβ are ligand-activated nuclear receptors. • They share oxysterol ligands and the same heterodimerization partner, RXR. • LXRs regulate lipid and glucose metabolism, CNS and immune functions, and water transport. - Abstract: The two Liver X Receptors, LXRα and LXRβ, are nuclear receptors belonging to the superfamily of ligand-activated transcription factors. They share more than 78% homology in amino acid sequence, a common profile of oxysterol ligands and the same heterodimerization partner, Retinoid X Receptor. LXRs play crucial roles in several metabolic pathways: lipid metabolism, in particular in preventing cellular cholesterol accumulation; glucose homeostasis; inflammation; central nervous system functions and water transport. As with all nuclear receptors, the transcriptional activity of LXR is the result of an orchestration of numerous cellular factors including ligand bioavailability, presence of corepressors and coactivators and cellular context i.e., what other pathways are activated in the cell at the time the receptor recognizes its ligand. In this mini-review we summarize the factors regulating the transcriptional activity and the mechanisms of action of these two receptors.

  4. Action mechanisms of Liver X Receptors

    International Nuclear Information System (INIS)

    Gabbi, Chiara; Warner, Margaret; Gustafsson, Jan-Åke

    2014-01-01

    Highlights: • LXRα and LXRβ are ligand-activated nuclear receptors. • They share oxysterol ligands and the same heterodimerization partner, RXR. • LXRs regulate lipid and glucose metabolism, CNS and immune functions, and water transport. - Abstract: The two Liver X Receptors, LXRα and LXRβ, are nuclear receptors belonging to the superfamily of ligand-activated transcription factors. They share more than 78% homology in amino acid sequence, a common profile of oxysterol ligands and the same heterodimerization partner, Retinoid X Receptor. LXRs play crucial roles in several metabolic pathways: lipid metabolism, in particular in preventing cellular cholesterol accumulation; glucose homeostasis; inflammation; central nervous system functions and water transport. As with all nuclear receptors, the transcriptional activity of LXR is the result of an orchestration of numerous cellular factors including ligand bioavailability, presence of corepressors and coactivators and cellular context i.e., what other pathways are activated in the cell at the time the receptor recognizes its ligand. In this mini-review we summarize the factors regulating the transcriptional activity and the mechanisms of action of these two receptors

  5. H-2RIIBP, a member of the nuclear hormone receptor superfamily that binds to both the regulatory element of major histocompatibility class I genes and the estrogen response element.

    Science.gov (United States)

    Hamada, K; Gleason, S L; Levi, B Z; Hirschfeld, S; Appella, E; Ozato, K

    1989-11-01

    Transcription of major histocompatibility complex (MHC) class I genes is regulated by the conserved MHC class I regulatory element (CRE). The CRE has two factor-binding sites, region I and region II, both of which elicit enhancer function. By screening a mouse lambda gt 11 library with the CRE as a probe, we isolated a cDNA clone that encodes a protein capable of binding to region II of the CRE. This protein, H-2RIIBP (H-2 region II binding protein), bound to the native region II sequence, but not to other MHC cis-acting sequences or to mutant region II sequences, similar to the naturally occurring region II factor in mouse cells. The deduced amino acid sequence of H-2RIIBP revealed two putative zinc fingers homologous to the DNA-binding domain of steroid/thyroid hormone receptors. Although sequence similarity in other regions was minimal, H-2RIIBP has apparent modular domains characteristic of the nuclear hormone receptors. Further analyses showed that both H-2RIIBP and the natural region II factor bind to the estrogen response element (ERE) of the vitellogenin A2 gene. The ERE is composed of a palindrome, and half of this palindrome resembles the region II binding site of the MHC CRE. These results indicate that H-2RIIBP (i) is a member of the superfamily of nuclear hormone receptors and (ii) may regulate not only MHC class I genes but also genes containing the ERE and related sequences. Sequences homologous to the H-2RIIBP gene are widely conserved in the animal kingdom. H-2RIIBP mRNA is expressed in many mouse tissues, in agreement with the distribution of the natural region II factor.

  6. Phenobarbital Meets Phosphorylation of Nuclear Receptors.

    Science.gov (United States)

    Negishi, Masahiko

    2017-05-01

    Phenobarbital was the first therapeutic drug to be characterized for its induction of hepatic drug metabolism. Essentially at the same time, cytochrome P450, an enzyme that metabolizes drugs, was discovered. After nearly 50 years of investigation, the molecular target of phenobarbital induction has now been delineated to phosphorylation at threonine 38 of the constitutive androstane receptor (NR1I3), a member of the nuclear receptor superfamily. Determining this mechanism has provided us with the molecular basis to understand drug induction of drug metabolism and disposition. Threonine 38 is conserved as a phosphorylation motif in the majority of both mouse and human nuclear receptors, providing us with an opportunity to integrate diverse functions of nuclear receptors. Here, I review the works and accomplishments of my laboratory at the National Institutes of Health National Institute of Environmental Health Sciences and the future research directions of where our study of the constitutive androstane receptor might take us. U.S. Government work not protected by U.S. copyright.

  7. Delayed internalization and lack of recycling in a beta2-adrenergic receptor fused to the G protein alpha-subunit

    Directory of Open Access Journals (Sweden)

    Floridi Aristide

    2008-10-01

    Full Text Available Abstract Background Chimeric proteins obtained by the fusion of a G protein-coupled receptor (GPCR sequence to the N-terminus of the G protein α-subunit have been extensively used to investigate several aspects of GPCR signalling. Although both the receptor and the G protein generally maintain a fully functional state in such polypeptides, original observations made using a chimera between the β2-adrenergic receptor (β2AR and Gαs indicated that the fusion to the α-subunit resulted in a marked reduction of receptor desensitization and down-regulation. To further investigate this phenomenon, we have compared the rates of internalization and recycling between wild-type and Gαs-fused β2AR. Results The rate of agonist-induced internalization, measured as the disappearance of cell surface immunofluorescence in HEK293 cells permanently expressing N-terminus tagged receptors, was reduced three-fold by receptor-G protein fusion. However, both fused and non-fused receptors translocated to the same endocytic compartment, as determined by dual-label confocal analysis of cells co-expressing both proteins and transferrin co-localization. Receptor recycling, determined as the reversion of surface immunofluorescence following the addition of antagonist to cells that were previously exposed to agonist, markedly differed between wild-type and fused receptors. While most of the internalized β2AR returned rapidly to the plasma membrane, β2AR-Gαs did not recycle, and the observed slow recovery for the fusion protein immunofluorescence was entirely accounted for by protein synthesis. Conclusion The covalent linkage between β2AR and Gαs does not appear to alter the initial endocytic translocation of the two proteins, although there is reduced efficiency. It does, however, completely disrupt the process of receptor and G protein recycling. We conclude that the physical separation between receptor and Gα is not necessary for the transit to early endosomes

  8. GPCR Interaction: 154 [GRIPDB[Archive

    Lifescience Database Archive (English)

    Full Text Available of V1a, V2 and OT homodimers. In addition, coexpressions of different pairs of receptors (V1a/V2, V1a/OTR a...eceptor to interact with another receptor) were identical whatever the pair of receptors coexpress...ed in the cells, which suggests that the proportion of homodimers and heterodimers depends on the relative expression of each receptor. 19896898 BRET NP_000045.1 ... ...using chimeric V1a or V2 vasopressin or oxytocin and receptors labeled on the C-terminus with luciferase or

  9. G protein-coupled receptor kinase-2 (GRK-2) regulates serotonin metabolism through the monoamine oxidase AMX-2 in Caenorhabditis elegans.

    Science.gov (United States)

    Wang, Jianjun; Luo, Jiansong; Aryal, Dipendra K; Wetsel, William C; Nass, Richard; Benovic, Jeffrey L

    2017-04-07

    G protein-coupled receptors (GPCRs) regulate many animal behaviors. GPCR signaling is mediated by agonist-promoted interactions of GPCRs with heterotrimeric G proteins, GPCR kinases (GRKs), and arrestins. To further elucidate the role of GRKs in regulating GPCR-mediated behaviors, we utilized the genetic model system Caenorhabditis elegans Our studies demonstrate that grk-2 loss-of-function strains are egg laying-defective and contain low levels of serotonin (5-HT) and high levels of the 5-HT metabolite 5-hydroxyindole acetic acid (5-HIAA). The egg laying defect could be rescued by the expression of wild type but not by catalytically inactive grk-2 or by the selective expression of grk-2 in hermaphrodite-specific neurons. The addition of 5-HT or inhibition of 5-HT metabolism also rescued the egg laying defect. Furthermore, we demonstrate that AMX-2 is the primary monoamine oxidase that metabolizes 5-HT in C. elegans , and we also found that grk-2 loss-of-function strains have abnormally high levels of AMX-2 compared with wild-type nematodes. Interestingly, GRK-2 was also found to interact with and promote the phosphorylation of AMX-2. Additional studies reveal that 5-HIAA functions to inhibit egg laying in a manner dependent on the 5-HT receptor SER-1 and the G protein GOA-1. These results demonstrate that GRK-2 modulates 5-HT metabolism by regulating AMX-2 function and that 5-HIAA may function in the SER-1 signaling pathway. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  10. Principles and determinants of G-protein coupling by the rhodopsin-like thyrotropin receptor.

    Directory of Open Access Journals (Sweden)

    Gunnar Kleinau

    Full Text Available In this study we wanted to gain insights into selectivity mechanisms between G-protein-coupled receptors (GPCR and different subtypes of G-proteins. The thyrotropin receptor (TSHR binds G-proteins promiscuously and activates both Gs (cAMP and Gq (IP. Our goal was to dissect selectivity patterns for both pathways in the intracellular region of this receptor. We were particularly interested in the participation of poorly investigated receptor parts.We systematically investigated the amino acids of intracellular loop (ICL 1 and helix 8 using site-directed mutagenesis alongside characterization of cAMP and IP accumulation. This approach was guided by a homology model of activated TSHR in complex with heterotrimeric Gq, using the X-ray structure of opsin with a bound G-protein peptide as a structural template.We provide evidence that ICL1 is significantly involved in G-protein activation and our model suggests potential interactions with subunits G alpha as well as G betagamma. Several amino acid substitutions impaired both IP and cAMP accumulation. Moreover, we found a few residues in ICL1 (L440, T441, H443 and helix 8 (R687 that are sensitive for Gq but not for Gs activation. Conversely, not even one residue was found that selectively affects cAMP accumulation only. Together with our previous mutagenesis data on ICL2 and ICL3 we provide here the first systematically completed map of potential interfaces between TSHR and heterotrimeric G-protein. The TSHR/Gq-heterotrimer complex is characterized by more selective interactions than the TSHR/Gs complex. In fact the receptor interface for binding Gs is a subset of that for Gq and we postulate that this may be true for other GPCRs coupling these G-proteins. Our findings support that G-protein coupling and preference is dominated by specific structural features at the intracellular region of the activated GPCR but is completed by additional complementary recognition patterns between receptor and G

  11. Principles and determinants of G-protein coupling by the rhodopsin-like thyrotropin receptor.

    Science.gov (United States)

    Kleinau, Gunnar; Jaeschke, Holger; Worth, Catherine L; Mueller, Sandra; Gonzalez, Jorge; Paschke, Ralf; Krause, Gerd

    2010-03-18

    In this study we wanted to gain insights into selectivity mechanisms between G-protein-coupled receptors (GPCR) and different subtypes of G-proteins. The thyrotropin receptor (TSHR) binds G-proteins promiscuously and activates both Gs (cAMP) and Gq (IP). Our goal was to dissect selectivity patterns for both pathways in the intracellular region of this receptor. We were particularly interested in the participation of poorly investigated receptor parts.We systematically investigated the amino acids of intracellular loop (ICL) 1 and helix 8 using site-directed mutagenesis alongside characterization of cAMP and IP accumulation. This approach was guided by a homology model of activated TSHR in complex with heterotrimeric Gq, using the X-ray structure of opsin with a bound G-protein peptide as a structural template.We provide evidence that ICL1 is significantly involved in G-protein activation and our model suggests potential interactions with subunits G alpha as well as G betagamma. Several amino acid substitutions impaired both IP and cAMP accumulation. Moreover, we found a few residues in ICL1 (L440, T441, H443) and helix 8 (R687) that are sensitive for Gq but not for Gs activation. Conversely, not even one residue was found that selectively affects cAMP accumulation only. Together with our previous mutagenesis data on ICL2 and ICL3 we provide here the first systematically completed map of potential interfaces between TSHR and heterotrimeric G-protein. The TSHR/Gq-heterotrimer complex is characterized by more selective interactions than the TSHR/Gs complex. In fact the receptor interface for binding Gs is a subset of that for Gq and we postulate that this may be true for other GPCRs coupling these G-proteins. Our findings support that G-protein coupling and preference is dominated by specific structural features at the intracellular region of the activated GPCR but is completed by additional complementary recognition patterns between receptor and G-protein subtypes.

  12. The nuclear receptor gene family in the Pacific oyster, Crassostrea gigas, contains a novel subfamily group.

    Science.gov (United States)

    Vogeler, Susanne; Galloway, Tamara S; Lyons, Brett P; Bean, Tim P

    2014-05-15

    Nuclear receptors are a superfamily of transcription factors important in key biological, developmental and reproductive processes. Several of these receptors are ligand- activated and through their ability to bind endogenous and exogenous ligands, are potentially vulnerable to xenobiotics. Molluscs are key ecological species in defining aquatic and terrestrial habitats and are sensitive to xenobiotic compounds in the environment. However, the understanding of nuclear receptor presence, function and xenobiotic disruption in the phylum Mollusca is limited. Here, forty-three nuclear receptor sequences were mined from the genome of the Pacific oyster, Crassostrea gigas. They include members of NR0-NR5 subfamilies, notably lacking any NR6 members. Phylogenetic analyses of the oyster nuclear receptors have been conducted showing the presence of a large novel subfamily group not previously reported, which is named NR1P. Homologues to all previous identified nuclear receptors in other mollusc species have also been determined including the putative heterodimer partner retinoid X receptor, estrogen receptor and estrogen related receptor. C. gigas contains a highly diverse set of nuclear receptors including a novel NR1 group, which provides important information on presence and evolution of this transcription factor superfamily in invertebrates. The Pacific oyster possesses two members of NR3, the sex steroid hormone receptor analogues, of which there are 9 in humans. This provides increasing evidence that steroid ligand specific expansion of this family is deuterostome specific. This new knowledge on divergence and emergence of nuclear receptors in C. gigas provides essential information for studying regulation of molluscan gene expression and the potential effects of xenobiotics.

  13. Regulation of β2-adrenergic receptor function by conformationally selective single-domain intrabodies

    DEFF Research Database (Denmark)

    Staus, Dean P; Wingler, Laura M; Strachan, Ryan T

    2014-01-01

    . However, a monomeric single-domain antibody (nanobody) from the Camelid family was recently found to allosterically bind and stabilize an active conformation of the β2-adrenergic receptor (β2AR). Here, we set out to study the functional interaction of 18 related nanobodies with the β2AR to investigate...... their roles as novel tools for studying GPCR biology. Our studies revealed several sequence-related nanobody families with preferences for active (agonist-occupied) or inactive (antagonist-occupied) receptors. Flow cytometry analysis indicates that all nanobodies bind to epitopes displayed...... on the intracellular receptor surface; therefore, we transiently expressed them intracellularly as "intrabodies" to test their effects on β2AR-dependent signaling. Conformational specificity was preserved after intrabody conversion as demonstrated by the ability for the intracellularly expressed nanobodies...

  14. Relationship between Apolipoprotein Superfamily and Parkinson's Disease

    Directory of Open Access Journals (Sweden)

    Lin Li

    2017-01-01

    Conclusions: The Apo superfamily has been proved to be closely involved in the initiation, progression, and prognosis of PD. Apos and their genes are of great value in predicting the susceptibility of PD and hopeful to become the target of medical intervention to prevent the onset of PD or slow down the progress. Therefore, further large-scale studies are warranted to elucidate the precise mechanisms of Apos in PD.

  15. Discovery of a novel allosteric modulator of 5-HT3 receptor

    DEFF Research Database (Denmark)

    Trattnig, Sarah M; Harpsøe, Kasper; Thygesen, Sarah B

    2012-01-01

    The ligand-gated ion channels in the Cysloop receptor superfamily mediate the effects of neurotransmitters acetylcholine, serotonin, GABA and glycine. Cysloop receptor signaling is susceptible to modulation by ligands acting through numerous allosteric sites. Here we report the discovery of a novel...... receptor guided by a homology model, PU02 is demonstrated to act through a transmembrane intersubunit site situated in the upper three helical turns of TM2 and TM3 in the (+)subunit and TM1 and TM2 in the (minus)subunit. The Ser248, Leu288, Ile290, Thr294 and Gly306 residues are identified as important...

  16. A Hydrogen-Bonded Polar Network in the Core of the Glucagon-Like Peptide-1 Receptor Is a Fulcrum for Biased Agonism: Lessons from Class B Crystal Structures

    OpenAIRE

    Wootten, Denise; Reynolds, Christopher A.; Koole, Cassandra; Smith, Kevin J.; Mobarec, Juan C.; Simms, John; Quon, Tezz; Coudrat, Thomas; Furness, Sebastian G. B.; Miller, Laurence J.; Christopoulos, Arthur; Sexton, Patrick M.

    2016-01-01

    The glucagon-like peptide 1 (GLP-1) receptor is a class B G protein-coupled receptor (GPCR) that is a key target for treatments for type II diabetes and obesity. This receptor, like other class B GPCRs, displays biased agonism, though the physiologic significance of this is yet to be elucidated. Previous work has implicated R2.60190, N3.43240, Q7.49394, and H6.52363 as key residues involved in peptide-mediated biased agonism, with R2.60190, N3.43240, and Q7.49394 predicted to form a polar int...

  17. The dopamine D2 receptor can directly recruit and activate GRK2 without G protein activation.

    Science.gov (United States)

    Pack, Thomas F; Orlen, Margo I; Ray, Caroline; Peterson, Sean M; Caron, Marc G

    2018-04-20

    The dopamine D2 receptor (D2R) is a G protein-coupled receptor (GPCR) that is critical for many central nervous system functions. The D2R carries out these functions by signaling through two transducers: G proteins and β-arrestins (βarrs). Selectively engaging either the G protein or βarr pathway may be a way to improve drugs targeting GPCRs. The current model of GPCR signal transduction posits a chain of events where G protein activation ultimately leads to βarr recruitment. GPCR kinases (GRKs), which are regulated by G proteins and whose kinase action facilitates βarr recruitment, bridge these pathways. Therefore, βarr recruitment appears to be intimately tied to G protein activation via GRKs. Here we sought to understand how GRK2 action at the D2R would be disrupted when G protein activation is eliminated and the effect of this on βarr recruitment. We used two recently developed biased D2R mutants that can preferentially interact either with G proteins or βarrs as well as a βarr-biased D2R ligand, UNC9994. With these functionally selective tools, we investigated the mechanism whereby the βarr-preferring D2R achieves βarr pathway activation in the complete absence of G protein activation. We describe how direct, G protein-independent recruitment of GRK2 drives interactions at the βarr-preferring D2R and also contributes to βarr recruitment at the WT D2R. Additionally, we found an additive interaction between the βarr-preferring D2R mutant and UNC9994. These results reveal that the D2R can directly recruit GRK2 without G protein activation and that this mechanism may have relevance to achieving βarr-biased signaling. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

  18. In silico identification, phylogeny and expression analysis of expansin superfamily in Medicago truncatula

    Directory of Open Access Journals (Sweden)

    Ying Liu

    2016-01-01

    Full Text Available Expansins are important components of plant cell walls, which are involved in the process of cell wall loosening under low extracellular pH. By using a combinational method for homology search and protein domain analysis, a total of 42 expansin genes were identified from Medicago truncatula genome in this study. They were divided into four families, based on sequence alignment and phylogenetic analysis. Gene duplication events were identified in the expansins superfamily, especially in the extension of α-expansin family. By analysis of RNA-sequencing data from National Center for Biotechnology Information, the expansin (EXP genes expressed during tissues development were characterized. Meanwhile, lots of cis-acting regulatory DNA elements in the EXP superfamily were identified, which were mainly related to plant growth and development processes. The results presented in this study are expected to facilitate further research works on this gene superfamily and provide new insights about the molecular mechanisms of expansins in M. truncatula.

  19. Self-Assembly in the Ferritin Nano-Cage Protein Superfamily

    Directory of Open Access Journals (Sweden)

    Yu Zhang

    2011-08-01

    Full Text Available Protein self-assembly, through specific, high affinity, and geometrically constraining protein-protein interactions, can control and lead to complex cellular nano-structures. Establishing an understanding of the underlying principles that govern protein self-assembly is not only essential to appreciate the fundamental biological functions of these structures, but could also provide a basis for their enhancement for nano-material applications. The ferritins are a superfamily of well studied proteins that self-assemble into hollow cage-like structures which are ubiquitously found in both prokaryotes and eukaryotes. Structural studies have revealed that many members of the ferritin family can self-assemble into nano-cages of two types. Maxi-ferritins form hollow spheres with octahedral symmetry composed of twenty-four monomers. Mini-ferritins, on the other hand, are tetrahedrally symmetric, hollow assemblies composed of twelve monomers. This review will focus on the structure of members of the ferritin superfamily, the mechanism of ferritin self-assembly and the structure-function relations of these proteins.

  20. Aromatic interactions impact ligand binding and function at serotonin 5-HT2C G protein-coupled receptors: receptor homology modelling, ligand docking, and molecular dynamics results validated by experimental studies

    Science.gov (United States)

    Córdova-Sintjago, Tania; Villa, Nancy; Fang, Lijuan; Booth, Raymond G.

    2014-02-01

    The serotonin (5-hydroxytryptamine, 5-HT) 5-HT2 G protein-coupled receptor (GPCR) family consists of types 2A, 2B, and 2C that share ∼75% transmembrane (TM) sequence identity. Agonists for 5-HT2C receptors are under development for psychoses; whereas, at 5-HT2A receptors, antipsychotic effects are associated with antagonists - in fact, 5-HT2A agonists can cause hallucinations and 5-HT2B agonists cause cardiotoxicity. It is known that 5-HT2A TM6 residues W6.48, F6.51, and F6.52 impact ligand binding and function; however, ligand interactions with these residues at the 5-HT2C receptor have not been reported. To predict and validate molecular determinants for 5-HT2C-specific activation, results from receptor homology modelling, ligand docking, and molecular dynamics simulation studies were compared with experimental results for ligand binding and function at wild type and W6.48A, F6.51A, and F6.52A point-mutated 5-HT2C receptors.

  1. The Pseudo signal peptide of the corticotropin-releasing factor receptor type 2A prevents receptor oligomerization.

    Science.gov (United States)

    Teichmann, Anke; Rutz, Claudia; Kreuchwig, Annika; Krause, Gerd; Wiesner, Burkhard; Schülein, Ralf

    2012-08-03

    N-terminal signal peptides mediate the interaction of native proteins with the translocon complex of the endoplasmic reticulum membrane and are cleaved off during early protein biogenesis. The corticotropin-releasing factor receptor type 2a (CRF(2(a))R) possesses an N-terminal pseudo signal peptide, which represents a so far unique domain within the large protein family of G protein-coupled receptors (GPCRs). In contrast to a conventional signal peptide, the pseudo signal peptide remains uncleaved and consequently forms a hydrophobic extension at the N terminus of the receptor. The functional consequence of the presence of the pseudo signal peptide is not understood. Here, we have analyzed the significance of this domain for receptor dimerization/oligomerization in detail. To this end, we took the CRF(2(a))R and the homologous corticotropin-releasing factor receptor type 1 (CRF(1)R) possessing a conventional cleaved signal peptide and conducted signal peptide exchange experiments. Using single cell and single molecule imaging methods (fluorescence resonance energy transfer and fluorescence cross-correlation spectroscopy, respectively) as well as biochemical experiments, we obtained two novel findings; we could show that (i) the CRF(2(a))R is expressed exclusively as a monomer, and (ii) the presence of the pseudo signal peptide prevents its oligomerization. Thus, we have identified a novel functional domain within the GPCR protein family, which plays a role in receptor oligomerization and which may be useful to study the functional significance of this process in general.

  2. Peroxisome Proliferator-Activated Receptor and Vitamin D Receptor Signaling Pathways in Cancer Cells

    Directory of Open Access Journals (Sweden)

    Yasuko Kitagishi

    2013-10-01

    Full Text Available Peroxisome proliferator-activated receptors (PPARs are members of the superfamily of nuclear hormone receptors, which respond to specific ligands such as polyunsaturated fatty acids by altering gene expression. Three subtypes of this receptor have been discovered, each evolving to achieve different biological functions. Like other nuclear receptors, the transcriptional activity of PPARs is affected not only by ligand-stimulation, but also by cross-talk with other molecules. For example, both PPARs and the RXRs are ligand-activated transcription factors that coordinately regulate gene expression. In addition, PPARs and vitamin D receptor (VDR signaling pathways regulate a multitude of genes that are of importance for cellular functions including cell proliferation and cell differentiation. Interaction of the PPARs and VDR signaling pathways has been shown at the level of molecular cross-regulation of their transcription factor. A variety of ligands influencing the PPARs and VDR signaling pathways have been shown to reveal chemopreventive potential by mediating tumor suppressive activities in human cancers. Use of these compounds may represent a potential novel strategy to prevent cancers. This review summarizes the roles of the PPARs and the VDR in pathogenesis and progression of cancer.

  3. Peroxisome Proliferator-Activated Receptor and Vitamin D Receptor Signaling Pathways in Cancer Cells

    Energy Technology Data Exchange (ETDEWEB)

    Matsuda, Satoru, E-mail: smatsuda@cc.nara-wu.ac.jp; Kitagishi, Yasuko [Department of Food Science and Nutrition, Nara Women’s University, Kita-Uoya Nishimachi, Nara 630-8506 (Japan)

    2013-10-21

    Peroxisome proliferator-activated receptors (PPARs) are members of the superfamily of nuclear hormone receptors, which respond to specific ligands such as polyunsaturated fatty acids by altering gene expression. Three subtypes of this receptor have been discovered, each evolving to achieve different biological functions. Like other nuclear receptors, the transcriptional activity of PPARs is affected not only by ligand-stimulation, but also by cross-talk with other molecules. For example, both PPARs and the RXRs are ligand-activated transcription factors that coordinately regulate gene expression. In addition, PPARs and vitamin D receptor (VDR) signaling pathways regulate a multitude of genes that are of importance for cellular functions including cell proliferation and cell differentiation. Interaction of the PPARs and VDR signaling pathways has been shown at the level of molecular cross-regulation of their transcription factor. A variety of ligands influencing the PPARs and VDR signaling pathways have been shown to reveal chemopreventive potential by mediating tumor suppressive activities in human cancers. Use of these compounds may represent a potential novel strategy to prevent cancers. This review summarizes the roles of the PPARs and the VDR in pathogenesis and progression of cancer.

  4. Peroxisome Proliferator-Activated Receptor and Vitamin D Receptor Signaling Pathways in Cancer Cells

    International Nuclear Information System (INIS)

    Matsuda, Satoru; Kitagishi, Yasuko

    2013-01-01

    Peroxisome proliferator-activated receptors (PPARs) are members of the superfamily of nuclear hormone receptors, which respond to specific ligands such as polyunsaturated fatty acids by altering gene expression. Three subtypes of this receptor have been discovered, each evolving to achieve different biological functions. Like other nuclear receptors, the transcriptional activity of PPARs is affected not only by ligand-stimulation, but also by cross-talk with other molecules. For example, both PPARs and the RXRs are ligand-activated transcription factors that coordinately regulate gene expression. In addition, PPARs and vitamin D receptor (VDR) signaling pathways regulate a multitude of genes that are of importance for cellular functions including cell proliferation and cell differentiation. Interaction of the PPARs and VDR signaling pathways has been shown at the level of molecular cross-regulation of their transcription factor. A variety of ligands influencing the PPARs and VDR signaling pathways have been shown to reveal chemopreventive potential by mediating tumor suppressive activities in human cancers. Use of these compounds may represent a potential novel strategy to prevent cancers. This review summarizes the roles of the PPARs and the VDR in pathogenesis and progression of cancer

  5. Regulation of EGF receptor signaling by the MARVEL domain-containing protein CKLFSF8.

    Science.gov (United States)

    Jin, Caining; Ding, Peiguo; Wang, Ying; Ma, Dalong

    2005-11-21

    It is known that chemokine-like factor superfamily 8 (CKLFSF8), a member of the CKLF superfamily, has four putative transmembrane regions and a MARVEL domain. Its structure is similar to TM4SF11 (plasmolipin) and widely distributed in normal tissue. However, its function is not yet known. We show here that CKLFSF8 is associated with the epidermal growth factor receptor (EGFR) and that ectopic expression of CKLFSF8 in several cell lines suppresses EGF-induced cell proliferation, whereas knockdown of CKLFSF8 by siRNA promotes cell proliferation. In cells overexpressing CKLFSF8, the initial activation of EGFR was not affected, but subsequent desensitization of EGF-induced signaling occurred rapidly. This attenuation was correlated with an increased rate of receptor endocytosis. In contrast, knockdown of CKLFSF8 by siCKLFSF8 delayed EGFR endocytosis. These results identify CKLFSF8 as a novel regulator of EGF-induced signaling and indicate that the association of EGFR with four transmembrane proteins is critical for EGFR desensitization.

  6. Identification of four Drosophila allatostatins as the cognate ligands for the Drosophila orphan receptor DAR-2

    DEFF Research Database (Denmark)

    Lenz, C; Williamson, M; Hansen, G N

    2001-01-01

    The allatostatins are generally inhibitory insect neuropeptides. The Drosophila orphan receptor DAR-2 is a G-protein-coupled receptor, having 47% amino acid residue identity with another Drosophila receptor, DAR-1 (which is also called dros. GPCR, or DGR) that was previously shown...... to be the receptor for an intrinsic Drosophila A-type (cockroach-type) allatostatin. Here, we have permanently expressed DAR-2 in CHO cells and found that it is the cognate receptor for four Drosophila A-type allatostatins, the drostatins-A1 to -A4. Of all the drostatins, drostatin-A4 (Thr...... weakly in the brain. The Drosophila larval gut also contains about 20-30 endocrine cells, expressing the gene for the drostatins-A1 to -A4. We suggest, therefore, that DAR-2 mediates an allatostatin (drostatin)-induced inhibition of gut motility. This is the first report on the permanent and functional...

  7. Promoter switching allows simultaneous transcription of LANA and K14/vGPCR of Kaposi's sarcoma-associated herpesvirus

    International Nuclear Information System (INIS)

    Staudt, Michelle R.; Dittmer, Dirk P.

    2006-01-01

    Latent transcription of the latency-associated nuclear antigen (LANA/ORF73) of Kaposi's sarcoma-associated herpesvirus is driven by the LANAp-c. Complexity arises during lytic reactivation, however, as the bicistronic K14/vGPCR transcript initiates 32 bp downstream of LANAp-c in the opposite orientation. We identify an Rta/ORF50-inducible LANA promoter (LANAp-i) that is distinct from the LANAp-c. LANAp-c is unaffected by Rta/ORF50. Utilization of the second, downstream LANAp-i explains how LANA and K14/vGPCR are simultaneously transcribed during de novo infection or lytic reactivation. Transactivation of LANAp-i and K14/vGPCRp requires the C-terminal activation domain of Rta/ORF50 and is mediated by DNA-binding-dependent and -independent Rta/ORF50 mechanisms. Transcriptional profiling following viral reactivation support promoter reporter phenotypes. In sum, cis-elements within the LANAp were selected to ensure faithful expression of LANA and other genes regulated by LANAp during all stages of the KSHV lifecycle despite potential interference from K14/vGPCRp activity

  8. Peroxisome proliferator-activated receptor: effects on nutritional homeostasis, obesity and diabetes mellitus Receptores activados por los proliferadores de peroxisomas: implicaciones sobre la homeostasis nutricional, en la obesidad y en la diabetes mellitus

    OpenAIRE

    M. Viana Abranches; F. C. Esteves de Oliveira; J. Bressan

    2011-01-01

    The obesity and the metabolic disorders associated characterize the metabolic syndrome, which has increased at an alarming rate around the world. It is known that environmental and genetic factors are involved in the genesis of obesity. Peroxisome Proliferator-Activated Receptors (PPARs) stand out among these factors. They compose the nuclear receptor superfamily and there are in three isoforms (PPARα,PPARβ/δ and PPARγ), which play an important role in the regulation of...

  9. Insights into Basal Signaling Regulation, Oligomerization, and Structural Organization of the Human G-Protein Coupled Receptor 83.

    Directory of Open Access Journals (Sweden)

    Anne Müller

    Full Text Available The murine G-protein coupled receptor 83 (mGPR83 is expressed in the hypothalamus and was previously suggested to be involved in the regulation of metabolism. The neuropeptide PEN has been recently identified as a potent GPR83 ligand. Moreover, GPR83 constitutes functionally relevant hetero-oligomers with other G-protein coupled receptors (GPCR such as the ghrelin receptor (GHSR or GPR171. Previous deletion studies also revealed that the long N-terminal extracellular receptor domain (eNDo of mGPR83 may act as an intra-molecular ligand, which participates in the regulation of basal signaling activity, which is a key feature of GPCR function. Here, we investigated particular amino acids at the eNDo of human GPR83 (hGPR83 by side-directed mutagenesis to identify determinants of the internal ligand. These studies were accompanied by structure homology modeling to combine functional insights with structural information. The capacity for hetero-oligomer formation of hGPR83 with diverse family A GPCRs such as the melanocortin-4 receptor (MC4R was also investigated, with a specific emphasis on the impact of the eNDo on oligomerization and basal signaling properties. Finally, we demonstrate that hGPR83 exhibits an unusual basal signaling for different effectors, which also supports signaling promiscuity. hGPR83 interacts with a variety of hypothalamic GPCRs such as the MC4R or GHSR. These interactions are not dependent on the ectodomain and most likely occur at interfaces constituted in the transmembrane regions. Moreover, several amino acids at the transition between the eNDo and transmembrane helix 1 were identified, where mutations lead also to biased basal signaling modulation.

  10. Glucocorticoid receptor signaling in health and disease

    Science.gov (United States)

    Kadmiel, Mahita; Cidlowski, John A.

    2013-01-01

    Glucocorticoids are steroid hormones regulated in a circadian and stres-associated manner to maintain various metabolic and homeostatic functions that are necessary for life. Synthetic glucocorticoids are widely prescribed drugs for many conditions including asthma, chronic obstructive pulmonary disease (COPD), and inflammatory disorders of the eye. Research in the last few years has begun to unravel the profound complexity of glucocorticoid signaling and has contributed remarkably to improved therapeutic strategies. Glucocorticoids signal through the glucocorticoid receptor, a member of the superfamily of nuclear receptors, in both genomic and non-genomic ways in almost every tissue in the human body. In this review, we will provide an update on glucocorticoid receptor signaling and highlight the role of GR signaling in physiological and pathophysiological conditions in the major organ systems in the human body. PMID:23953592

  11. Molecular mechanism of action of pharmacoperone rescue of misrouted GPCR mutants: the GnRH receptor.

    Science.gov (United States)

    Janovick, Jo Ann; Patny, Akshay; Mosley, Ralph; Goulet, Mark T; Altman, Michael D; Rush, Thomas S; Cornea, Anda; Conn, P Michael

    2009-02-01

    The human GnRH receptor (hGnRHR), a G protein-coupled receptor, is a useful model for studying pharmacological chaperones (pharmacoperones), drugs that rescue misfolded and misrouted protein mutants and restore them to function. This technique forms the basis of a therapeutic approach of rescuing mutants associated with human disease and restoring them to function. The present study relies on computational modeling, followed by site-directed mutagenesis, assessment of ligand binding, effector activation, and confocal microscopy. Our results show that two different chemical classes of pharmacoperones act to stabilize hGnRHR mutants by bridging residues D(98) and K(121). This ligand-mediated bridge serves as a surrogate for a naturally occurring and highly conserved salt bridge (E(90)-K(121)) that stabilizes the relation between transmembranes 2 and 3, which is required for passage of the receptor through the cellular quality control system and to the plasma membrane. Our model was used to reveal important pharmacophoric features, and then identify a novel chemical ligand, which was able to rescue a D(98) mutant of the hGnRHR that could not be rescued as effectively by previously known pharmacoperones.

  12. Evolutionary history, structural features and biochemical diversity of the NlpC/P60 superfamily of enzymes.

    Science.gov (United States)

    Anantharaman, Vivek; Aravind, L

    2003-01-01

    Peptidoglycan is hydrolyzed by a diverse set of enzymes during bacterial growth, development and cell division. The N1pC/P60 proteins define a family of cell-wall peptidases that are widely represented in various bacterial lineages. Currently characterized members are known to hydrolyze D-gamma-glutamyl-meso-diaminopimelate or N-acetylmuramate-L-alanine linkages. Detailed analysis of the N1pC/P60 peptidases showed that these proteins define a large superfamily encompassing several diverse groups of proteins. In addition to the well characterized P60-like proteins, this superfamily includes the AcmB/LytN and YaeF/YiiX families of bacterial proteins, the amidase domain of bacterial and kinetoplastid glutathionylspermidine synthases (GSPSs), and several proteins from eukaryotes, phages, poxviruses, positive-strand RNA viruses, and certain archaea. The eukaryotic members include lecithin retinol acyltransferase (LRAT), nematode developmental regulator Egl-26, and candidate tumor suppressor H-rev107. These eukaryotic proteins, along with the bacterial YaeF/poxviral G6R family, show a circular permutation of the catalytic domain. We identified three conserved residues, namely a cysteine, a histidine and a polar residue, that are involved in the catalytic activities of this superfamily. Evolutionary analysis of this superfamily shows that it comprises four major families, with diverse domain architectures in each of them. Several related, but distinct, catalytic activities, such as murein degradation, acyl transfer and amide hydrolysis, have emerged in the N1pC/P60 superfamily. The three conserved catalytic residues of this superfamily are shown to be equivalent to the catalytic triad of the papain-like thiol peptidases. The predicted structural features indicate that the N1pC/P60 enzymes contain a fold similar to the papain-like peptidases, transglutaminases and arylamine acetyltransferases.

  13. Detection of Side Chain Rearrangements Mediating the Motions of Transmembrane Helices in Molecular Dynamics Simulations of G Protein-Coupled Receptors

    Directory of Open Access Journals (Sweden)

    Zied Gaieb

    Full Text Available Structure and dynamics are essential elements of protein function. Protein structure is constantly fluctuating and undergoing conformational changes, which are captured by molecular dynamics (MD simulations. We introduce a computational framework that provides a compact representation of the dynamic conformational space of biomolecular simulations. This method presents a systematic approach designed to reduce the large MD simulation spatiotemporal datasets into a manageable set in order to guide our understanding of how protein mechanics emerge from side chain organization and dynamic reorganization. We focus on the detection of side chain interactions that undergo rearrangements mediating global domain motions and vice versa. Side chain rearrangements are extracted from side chain interactions that undergo well-defined abrupt and persistent changes in distance time series using Gaussian mixture models, whereas global domain motions are detected using dynamic cross-correlation. Both side chain rearrangements and global domain motions represent the dynamic components of the protein MD simulation, and are both mapped into a network where they are connected based on their degree of coupling. This method allows for the study of allosteric communication in proteins by mapping out the protein dynamics into an intramolecular network to reduce the large simulation data into a manageable set of communities composed of coupled side chain rearrangements and global domain motions. This computational framework is suitable for the study of tightly packed proteins, such as G protein-coupled receptors, and we present an application on a seven microseconds MD trajectory of CC chemokine receptor 7 (CCR7 bound to its ligand CCL21. Keywords: Molecular dynamics, Change-point detection, Side chain reorganization, Helical domain motion, Intramolecular network, Membrane proteins, GPCR, GPCR computational modeling, GPCR allostery

  14. Differential modulation of Beta-adrenergic receptor signaling by trace amine-associated receptor 1 agonists.

    Directory of Open Access Journals (Sweden)

    Gunnar Kleinau

    Full Text Available Trace amine-associated receptors (TAAR are rhodopsin-like G-protein-coupled receptors (GPCR. TAAR are involved in modulation of neuronal, cardiac and vascular functions and they are potentially linked with neurological disorders like schizophrenia and Parkinson's disease. Subtype TAAR1, the best characterized TAAR so far, is promiscuous for a wide set of ligands and is activated by trace amines tyramine (TYR, phenylethylamine (PEA, octopamine (OA, but also by thyronamines, dopamine, and psycho-active drugs. Unfortunately, effects of trace amines on signaling of the two homologous β-adrenergic receptors 1 (ADRB1 and 2 (ADRB2 have not been clarified yet in detail. We, therefore, tested TAAR1 agonists TYR, PEA and OA regarding their effects on ADRB1/2 signaling by co-stimulation studies. Surprisingly, trace amines TYR and PEA are partial allosteric antagonists at ADRB1/2, whereas OA is a partial orthosteric ADRB2-antagonist and ADRB1-agonist. To specify molecular reasons for TAAR1 ligand promiscuity and for observed differences in signaling effects on particular aminergic receptors we compared TAAR, tyramine (TAR octopamine (OAR, ADRB1/2 and dopamine receptors at the structural level. We found especially for TAAR1 that the remarkable ligand promiscuity is likely based on high amino acid similarity in the ligand-binding region compared with further aminergic receptors. On the other hand few TAAR specific properties in the ligand-binding site might determine differences in ligand-induced effects compared to ADRB1/2. Taken together, this study points to molecular details of TAAR1-ligand promiscuity and identified specific trace amines as allosteric or orthosteric ligands of particular β-adrenergic receptor subtypes.

  15. Free fatty acid receptors and their role in regulation of energy metabolism.

    Science.gov (United States)

    Hara, Takafumi; Kimura, Ikuo; Inoue, Daisuke; Ichimura, Atsuhiko; Hirasawa, Akira

    2013-01-01

    The free fatty acid receptor (FFAR) is a G protein-coupled receptor (GPCR) activated by free fatty acids (FFAs), which play important roles not only as essential nutritional components but also as signaling molecules in numerous physiological processes. In the last decade, FFARs have been identified by the GPCR deorphanization strategy derived from the human genome database. To date, several FFARs have been identified and characterized as critical components in various physiological processes. FFARs are categorized according to the chain length of FFA ligands that activate each FFAR; FFA2 and FFA3 are activated by short chain FFAs, GPR84 is activated by medium-chain FFAs, whereas FFA1 and GPR120 are activated by medium- or long-chain FFAs. FFARs appear to act as physiological sensors for food-derived FFAs and digestion products in the gastrointestinal tract. Moreover, they are considered to be involved in the regulation of energy metabolism mediated by the secretion of insulin and incretin hormones and by the regulation of the sympathetic nerve systems, taste preferences, and inflammatory responses related to insulin resistance. Therefore, because FFARs can be considered to play important roles in physiological processes and various pathophysiological processes, FFARs have been targeted in therapeutic strategies for the treatment of metabolic disorders including type 2 diabetes and metabolic syndrome. In this review, we present a summary of recent progress regarding the understanding of their physiological roles in the regulation of energy metabolism and their potential as therapeutic targets.

  16. Histamine H3 receptor ligands in the group of (homo)piperazine derivatives.

    Science.gov (United States)

    Szczepanska, Katarzyna; Kuder, Kamil; Kiec-Kononowicz, Katarzyna

    2017-11-23

    Since its' discovery in 1983, followed by gene cloning in 1999, the histamine H3 receptor served as an outstanding target for drug discovery. The wide spectrum of possible therapeutic implications make H3R's one of the most researched areas in the vast GPCR ligands field - started from imidazole containing ligands, through various successful imidazole replacements, with recent introduction of Wakix® to pharmaceutical market. One of such replacements is piperazine moiety, a significant versatile scaffold in rational drug design for most of the GPCR ligands. Therefore, herein we review ligands built on piperazine, as well as its seven membered analogue azepine, that target H3R's and their potential therapeutical applications, in order to elucidate the current state of the art in this vast field. Due to a high level of structural divergence among compounds described herein, we decided to divide them into groups, where the key division element was the position of nitrogen basicity decreasing moieties in (homo)piperazine ring. Paying attention to a number of published structures and their overall high biological activity, one can realize that the (homo)piperazine scaffold bids a versatile template also for histamine H3 receptor ligands. With two possible substitution sites and therefore a number of possible structural combinations, piperazine derivatives stand as one of the largest group of high importance among H3R ligands. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  17. Efficient production of membrane-integrated and detergent-soluble G protein-coupled receptors in Escherichia coli.

    Science.gov (United States)

    Link, A James; Skretas, Georgios; Strauch, Eva-Maria; Chari, Nandini S; Georgiou, George

    2008-10-01

    G protein-coupled receptors (GPCRs) are notoriously difficult to express, particularly in microbial systems. Using GPCR fusions with the green fluorescent protein (GFP), we conducted studies to identify bacterial host effector genes that result in a general and significant enhancement in the amount of membrane-integrated human GPCRs that can be produced in Escherichia coli. We show that coexpression of the membrane-bound AAA+ protease FtsH greatly enhances the expression yield of four different class I GPCRs, irrespective of the presence of GFP. Using this new expression system, we produced 0.5 and 2 mg/L of detergent-solubilized and purified full-length central cannabinoid receptor (CB1) and bradykinin receptor 2 (BR2) in shake flask cultures, respectively, two proteins that had previously eluded expression in microbial systems.

  18. Structural mechanism of ligand activation in human calcium-sensing receptor

    Energy Technology Data Exchange (ETDEWEB)

    Geng, Yong; Mosyak, Lidia; Kurinov, Igor; Zuo, Hao; Sturchler, Emmanuel; Cheng, Tat Cheung; Subramanyam, Prakash; Brown, Alice P.; Brennan, Sarah C.; Mun, Hee-chang; Bush, Martin; Chen, Yan; Nguyen, Trang X.; Cao, Baohua; Chang, Donald D.; Quick, Matthias; Conigrave, Arthur D.; Colecraft, Henry M.; McDonald, Patricia; Fan, Qing R.

    2016-07-19

    Human calcium-sensing receptor (CaSR) is a G-protein-coupled receptor (GPCR) that maintains extracellular Ca2+homeostasis through the regulation of parathyroid hormone secretion. It functions as a disulfide-tethered homodimer composed of three main domains, the Venus Flytrap module, cysteine-rich domain, and seven-helix transmembrane region. Here, we present the crystal structures of the entire extracellular domain of CaSR in the resting and active conformations. We provide direct evidence that L-amino acids are agonists of the receptor. In the active structure, L-Trp occupies the orthosteric agonist-binding site at the interdomain cleft and is primarily responsible for inducing extracellular domain closure to initiate receptor activation. Our structures reveal multiple binding sites for Ca2+and PO43-ions. Both ions are crucial for structural integrity of the receptor. While Ca2+ions stabilize the active state, PO43-ions reinforce the inactive conformation. The activation mechanism of CaSR involves the formation of a novel dimer interface between subunits.

  19. External pH modulates EAG superfamily K+ channels through EAG-specific acidic residues in the voltage sensor

    Science.gov (United States)

    Kazmierczak, Marcin; Zhang, Xiaofei; Chen, Bihan; Mulkey, Daniel K.; Shi, Yingtang; Wagner, Paul G.; Pivaroff-Ward, Kendra; Sassic, Jessica K.; Bayliss, Douglas A.

    2013-01-01

    The Ether-a-go-go (EAG) superfamily of voltage-gated K+ channels consists of three functionally distinct gene families (Eag, Elk, and Erg) encoding a diverse set of low-threshold K+ currents that regulate excitability in neurons and muscle. Previous studies indicate that external acidification inhibits activation of three EAG superfamily K+ channels, Kv10.1 (Eag1), Kv11.1 (Erg1), and Kv12.1 (Elk1). We show here that Kv10.2, Kv12.2, and Kv12.3 are similarly inhibited by external protons, suggesting that high sensitivity to physiological pH changes is a general property of EAG superfamily channels. External acidification depolarizes the conductance–voltage (GV) curves of these channels, reducing low threshold activation. We explored the mechanism of this high pH sensitivity in Kv12.1, Kv10.2, and Kv11.1. We first examined the role of acidic voltage sensor residues that mediate divalent cation block of voltage activation in EAG superfamily channels because protons reduce the sensitivity of Kv12.1 to Zn2+. Low pH similarly reduces Mg2+ sensitivity of Kv10.1, and we found that the pH sensitivity of Kv11.1 was greatly attenuated at 1 mM Ca2+. Individual neutralizations of a pair of EAG-specific acidic residues that have previously been implicated in divalent block of diverse EAG superfamily channels greatly reduced the pH response in Kv12.1, Kv10.2, and Kv11.1. Our results therefore suggest a common mechanism for pH-sensitive voltage activation in EAG superfamily channels. The EAG-specific acidic residues may form the proton-binding site or alternatively are required to hold the voltage sensor in a pH-sensitive conformation. The high pH sensitivity of EAG superfamily channels suggests that they could contribute to pH-sensitive K+ currents observed in vivo. PMID:23712551

  20. External pH modulates EAG superfamily K+ channels through EAG-specific acidic residues in the voltage sensor.

    Science.gov (United States)

    Kazmierczak, Marcin; Zhang, Xiaofei; Chen, Bihan; Mulkey, Daniel K; Shi, Yingtang; Wagner, Paul G; Pivaroff-Ward, Kendra; Sassic, Jessica K; Bayliss, Douglas A; Jegla, Timothy

    2013-06-01

    The Ether-a-go-go (EAG) superfamily of voltage-gated K(+) channels consists of three functionally distinct gene families (Eag, Elk, and Erg) encoding a diverse set of low-threshold K(+) currents that regulate excitability in neurons and muscle. Previous studies indicate that external acidification inhibits activation of three EAG superfamily K(+) channels, Kv10.1 (Eag1), Kv11.1 (Erg1), and Kv12.1 (Elk1). We show here that Kv10.2, Kv12.2, and Kv12.3 are similarly inhibited by external protons, suggesting that high sensitivity to physiological pH changes is a general property of EAG superfamily channels. External acidification depolarizes the conductance-voltage (GV) curves of these channels, reducing low threshold activation. We explored the mechanism of this high pH sensitivity in Kv12.1, Kv10.2, and Kv11.1. We first examined the role of acidic voltage sensor residues that mediate divalent cation block of voltage activation in EAG superfamily channels because protons reduce the sensitivity of Kv12.1 to Zn(2+). Low pH similarly reduces Mg(2+) sensitivity of Kv10.1, and we found that the pH sensitivity of Kv11.1 was greatly attenuated at 1 mM Ca(2+). Individual neutralizations of a pair of EAG-specific acidic residues that have previously been implicated in divalent block of diverse EAG superfamily channels greatly reduced the pH response in Kv12.1, Kv10.2, and Kv11.1. Our results therefore suggest a common mechanism for pH-sensitive voltage activation in EAG superfamily channels. The EAG-specific acidic residues may form the proton-binding site or alternatively are required to hold the voltage sensor in a pH-sensitive conformation. The high pH sensitivity of EAG superfamily channels suggests that they could contribute to pH-sensitive K(+) currents observed in vivo.

  1. Phosphatidylcholine transfer protein interacts with thioesterase superfamily member 2 to attenuate insulin signaling.

    Science.gov (United States)

    Ersoy, Baran A; Tarun, Akansha; D'Aquino, Katharine; Hancer, Nancy J; Ukomadu, Chinweike; White, Morris F; Michel, Thomas; Manning, Brendan D; Cohen, David E

    2013-07-30

    Phosphatidylcholine transfer protein (PC-TP) is a phospholipid-binding protein that is enriched in liver and that interacts with thioesterase superfamily member 2 (THEM2). Mice lacking either protein exhibit improved hepatic glucose homeostasis and are resistant to diet-induced diabetes. Insulin receptor substrate 2 (IRS2) and mammalian target of rapamycin complex 1 (mTORC1) are key effectors of insulin signaling, which is attenuated in diabetes. We found that PC-TP inhibited IRS2, as evidenced by insulin-independent IRS2 activation after knockdown, genetic ablation, or chemical inhibition of PC-TP. In addition, IRS2 was activated after knockdown of THEM2, providing support for a role for the interaction of PC-TP with THEM2 in suppressing insulin signaling. Additionally, we showed that PC-TP bound to tuberous sclerosis complex 2 (TSC2) and stabilized the components of the TSC1-TSC2 complex, which functions to inhibit mTORC1. Preventing phosphatidylcholine from binding to PC-TP disrupted interactions of PC-TP with THEM2 and TSC2, and disruption of the PC-TP-THEM2 complex was associated with increased activation of both IRS2 and mTORC1. In livers of mice with genetic ablation of PC-TP or that had been treated with a PC-TP inhibitor, steady-state amounts of IRS2 were increased, whereas those of TSC2 were decreased. These findings reveal a phospholipid-dependent mechanism that suppresses insulin signaling downstream of its receptor.

  2. A single amino acid residue controls Ca2+ signaling by an octopamine receptor from Drosophila melanogaster

    OpenAIRE

    Hoff, Max; Balfanz, Sabine; Ehling, Petra; Gensch, Thomas; Baumann, Arnd

    2011-01-01

    Rhythmic activity of cells and cellular networks plays an important role in physiology. In the nervous system oscillations of electrical activity and/or second messenger concentrations are important to synchronize neuronal activity. At the molecular level, rhythmic activity can be initiated by different routes. We have recently shown that an octopamine-activated G-protein-coupled receptor (GPCR; DmOctα1Rb, CG3856) from Drosophila initiates Ca2+ oscillations. Here, we have unraveled the molecu...

  3. Novel agents acting on GABA2 receptors: potential cognitive enhancers

    International Nuclear Information System (INIS)

    Chebib, M.

    2001-01-01

    γ- Aminobutyric acid (GABA) is a low molecular weight ammo acid found throughout the central and peripheral nervous systems. It is a very flexible molecule and thus can attain a number of low-energy conformations which are recognised by a series of enzymes, receptors and transporter systems. This article will concentrate on the effects of GABA C as the major inhibitory neurotransmitter in the brain. GABA C receptors belong to the superfamily of ligand-gated ion channels that include nicotinic acetylcholine, GABA A , strychnine-sensitive glycine, and serotonin type 3 receptors. The compound outlined in this article provide us with novel leads for the design and development of compounds that may be selective for GABA receptors. Such compounds will help in the study of GABA C receptors both in vitro and in vivo, providing an insight into the role these receptors play in the brain

  4. Role of ionotropic GABA, glutamate and glycine receptors in the tonic and reflex control of cardiac vagal outflow in the rat

    Directory of Open Access Journals (Sweden)

    Goodchild Ann K

    2010-10-01

    Full Text Available Abstract Background Cardiac vagal preganglionic neurons (CVPN are responsible for the tonic, reflex and respiratory modulation of heart rate (HR. Although CVPN receive GABAergic and glutamatergic inputs, likely involved in respiratory and reflex modulation of HR respectively, little else is known regarding the functions controlled by ionotropic inputs. Activation of g-protein coupled receptors (GPCR alters these inputs, but the functional consequence is largely unknown. The present study aimed to delineate how ionotropic GABAergic, glycinergic and glutamatergic inputs contribute to the tonic and reflex control of HR and in particular determine which receptor subtypes were involved. Furthermore, we wished to establish how activation of the 5-HT1A GPCR affects tonic and reflex control of HR and what ionotropic interactions this might involve. Results Microinjection of the GABAA antagonist picrotoxin into CVPN decreased HR but did not affect baroreflex bradycardia. The glycine antagonist strychnine did not alter HR or baroreflex bradycardia. Combined microinjection of the NMDA antagonist, MK801, and AMPA antagonist, CNQX, into CVPN evoked a small bradycardia and abolished baroreflex bradycardia. MK801 attenuated whereas CNQX abolished baroreceptor bradycardia. Control intravenous injections of the 5-HT1A agonist 8-OH-DPAT evoked a small bradycardia and potentiated baroreflex bradycardia. These effects were still observed following microinjection of picrotoxin but not strychnine into CVPN. Conclusions We conclude that activation of GABAA receptors set the level of HR whereas AMPA to a greater extent than NMDA receptors elicit baroreflex changes in HR. Furthermore, activation of 5-HT1A receptors evokes bradycardia and enhances baroreflex changes in HR due to interactions with glycinergic neurons involving strychnine receptors. This study provides reference for future studies investigating how diseases alter neurochemical inputs to CVPN.

  5. Liver X receptor and peroxisome proliferator-activated receptor as integrators of lipid homeostasis and immunity.

    Science.gov (United States)

    Kidani, Yoko; Bensinger, Steven J

    2012-09-01

    Lipid metabolism has emerged as an important modulator of innate and adaptive immune cell fate and function. The lipid-activated transcription factors peroxisome proliferator-activated receptor (PPAR) α, β/δ, γ and liver X receptor (LXR) are members of the nuclear receptor superfamily that have a well-defined role in regulating lipid homeostasis and metabolic diseases. Accumulated evidence over the last decade indicates that PPAR and LXR signaling also influence multiple facets of inflammation and immunity, thereby providing important crosstalk between metabolism and immune system. Herein, we provide a brief introduction to LXR and PPAR biology and review recent discoveries highlighting the importance of PPAR and LXR signaling in the modulation of normal and pathologic states of immunity. We also examine advances in our mechanistic understanding of how nuclear receptors impact immune system function and homeostasis. Finally, we discuss whether LXRs and PPARs could be pharmacologically manipulated to provide novel therapeutic approaches for modulation of the immune system under pathologic inflammation or in the context of allergic and autoimmune disease. © 2012 John Wiley & Sons A/S.

  6. Functional importance of the Ala(116)-Pro(136) region in the calcium-sensing receptor. Constitutive activity and inverse agonism in a family C G-protein-coupled receptor

    DEFF Research Database (Denmark)

    Jensen, Anders A.; Spalding, T A; Burstein, E S

    2000-01-01

    The calcium-sensing receptor (CaR) belongs to family C of the G-protein-coupled receptor superfamily. To date 14 activating mutations in CaR showing increased sensitivity to Ca(2+) have been identified in humans with autosomal dominant hypocalcemia. Four of these activating mutations are found......, suppressed the elevated basal response of the constitutively activated Ca/1a mutants demonstrating inverse agonist activity of CPCCOEt. Taken together, our results demonstrate that the Ala(116)-Pro(136) region is of key importance for the maintenance of the inactive conformation of CaR....

  7. The p110beta isoform of phosphoinositide 3-kinase signals downstream of G protein-coupled receptors and is functionally redundant with p110gamma.

    Science.gov (United States)

    Guillermet-Guibert, Julie; Bjorklof, Katja; Salpekar, Ashreena; Gonella, Cristiano; Ramadani, Faruk; Bilancio, Antonio; Meek, Stephen; Smith, Andrew J H; Okkenhaug, Klaus; Vanhaesebroeck, Bart

    2008-06-17

    The p110 isoforms of phosphoinositide 3-kinase (PI3K) are acutely regulated by extracellular stimuli. The class IA PI3K catalytic subunits (p110alpha, p110beta, and p110delta) occur in complex with a Src homology 2 (SH2) domain-containing p85 regulatory subunit, which has been shown to link p110alpha and p110delta to Tyr kinase signaling pathways. The p84/p101 regulatory subunits of the p110gamma class IB PI3K lack SH2 domains and instead couple p110gamma to G protein-coupled receptors (GPCRs). Here, we show, using small-molecule inhibitors with selectivity for p110beta and cells derived from a p110beta-deficient mouse line, that p110beta is not a major effector of Tyr kinase signaling but couples to GPCRs. In macrophages, both p110beta and p110gamma contributed to Akt activation induced by the GPCR agonist complement 5a, but not by the Tyr kinase ligand colony-stimulating factor-1. In fibroblasts, which express p110beta but not p110gamma, p110beta mediated Akt activation by the GPCR ligands stromal cell-derived factor, sphingosine-1-phosphate, and lysophosphatidic acid but not by the Tyr kinase ligands PDGF, insulin, and insulin-like growth factor 1. Introduction of p110gamma in these cells reduced the contribution of p110beta to GPCR signaling. Taken together, these data show that p110beta and p110gamma can couple redundantly to the same GPCR agonists. p110beta, which shows a much broader tissue distribution than the leukocyte-restricted p110gamma, could thus provide a conduit for GPCR-linked PI3K signaling in the many cell types where p110gamma expression is low or absent.

  8. The p110β isoform of phosphoinositide 3-kinase signals downstream of G protein-coupled receptors and is functionally redundant with p110γ

    Science.gov (United States)

    Guillermet-Guibert, Julie; Bjorklof, Katja; Salpekar, Ashreena; Gonella, Cristiano; Ramadani, Faruk; Bilancio, Antonio; Meek, Stephen; Smith, Andrew J. H.; Okkenhaug, Klaus; Vanhaesebroeck, Bart

    2008-01-01

    The p110 isoforms of phosphoinositide 3-kinase (PI3K) are acutely regulated by extracellular stimuli. The class IA PI3K catalytic subunits (p110α, p110β, and p110δ) occur in complex with a Src homology 2 (SH2) domain-containing p85 regulatory subunit, which has been shown to link p110α and p110δ to Tyr kinase signaling pathways. The p84/p101 regulatory subunits of the p110γ class IB PI3K lack SH2 domains and instead couple p110γ to G protein-coupled receptors (GPCRs). Here, we show, using small-molecule inhibitors with selectivity for p110β and cells derived from a p110β-deficient mouse line, that p110β is not a major effector of Tyr kinase signaling but couples to GPCRs. In macrophages, both p110β and p110γ contributed to Akt activation induced by the GPCR agonist complement 5a, but not by the Tyr kinase ligand colony-stimulating factor-1. In fibroblasts, which express p110β but not p110γ, p110β mediated Akt activation by the GPCR ligands stromal cell-derived factor, sphingosine-1-phosphate, and lysophosphatidic acid but not by the Tyr kinase ligands PDGF, insulin, and insulin-like growth factor 1. Introduction of p110γ in these cells reduced the contribution of p110β to GPCR signaling. Taken together, these data show that p110β and p110γ can couple redundantly to the same GPCR agonists. p110β, which shows a much broader tissue distribution than the leukocyte-restricted p110γ, could thus provide a conduit for GPCR-linked PI3K signaling in the many cell types where p110γ expression is low or absent. PMID:18544649

  9. Evolution of the AKH/corazonin/ACP/GnRH receptor superfamily and their ligands in the Protostomia

    DEFF Research Database (Denmark)

    Hauser, Frank; Grimmelikhuijzen, Cornelis

    2014-01-01

    In this review we trace the evolutionary connections between GnRH receptors from vertebrates and the receptors for adipokinetic hormone (AKH), AKH/corazonin-related peptide (ACP), and corazonin from arthropods. We conclude that these G protein-coupled receptors (GPCRs) are closely related and hav......QLTFSSDWSGamide), and the penis worm Priapulus caudatus (pQIFFSKGWRGamide). This is the first report, showing that AKH signaling is widespread in molluscs....

  10. EBI2, GPR18 and GPR17--three structurally related, but biologically distinct 7TM receptors

    DEFF Research Database (Denmark)

    Nørregaard, Kristine; Benned-Jensen, Tau; Rosenkilde, Mette Marie

    2011-01-01

    7TM receptors constitute one of the largest superfamilies of proteins in the human genome. They are involved in a large number of physiological and pathological processes in the human body and thus represent major and important drug targets for the pharmaceutical industry. Although the majority...... have been deorphanized, many remain orphan, and these orphan receptors constitute a large pool of potential drug targets. This review focuses on one of these orphan targets, the Epstein-Barr Virus-induced receptor 2, EBI2 (or GPR183), together with two structurally related receptors, GPR17 and GPR18...

  11. Dynamical Binding Modes Determine Agonistic and Antagonistic Ligand Effects in the Prostate-Specific G-Protein Coupled Receptor (PSGR).

    Science.gov (United States)

    Wolf, Steffen; Jovancevic, Nikolina; Gelis, Lian; Pietsch, Sebastian; Hatt, Hanns; Gerwert, Klaus

    2017-11-22

    We analysed the ligand-based activation mechanism of the prostate-specific G-protein coupled receptor (PSGR), which is an olfactory receptor that mediates cellular growth in prostate cancer cells. Furthermore, it is an olfactory receptor with a known chemically near identic antagonist/agonist pair, α- and β-ionone. Using a combined theoretical and experimental approach, we propose that this receptor is activated by a ligand-induced rearrangement of a protein-internal hydrogen bond network. Surprisingly, this rearrangement is not induced by interaction of the ligand with the network, but by dynamic van der Waals contacts of the ligand with the involved amino acid side chains, altering their conformations and intraprotein connectivity. Ligand recognition in this GPCR is therefore highly stereo selective, but seemingly lacks any ligand recognition via polar contacts. A putative olfactory receptor-based drug design scheme will have to take this unique mode of protein/ligand action into account.

  12. Exploring and Expanding the Fatty-Acid-Binding Protein Superfamily in Fasciola Species.

    Science.gov (United States)

    Morphew, Russell M; Wilkinson, Toby J; Mackintosh, Neil; Jahndel, Veronika; Paterson, Steve; McVeigh, Paul; Abbas Abidi, Syed M; Saifullah, Khalid; Raman, Muthusamy; Ravikumar, Gopalakrishnan; LaCourse, James; Maule, Aaron; Brophy, Peter M

    2016-09-02

    The liver flukes Fasciola hepatica and F. gigantica infect livestock worldwide and threaten food security with climate change and problematic control measures spreading disease. Fascioliasis is also a foodborne disease with up to 17 million humans infected. In the absence of vaccines, treatment depends on triclabendazole (TCBZ), and overuse has led to widespread resistance, compromising future TCBZ control. Reductionist biology from many laboratories has predicted new therapeutic targets. To this end, the fatty-acid-binding protein (FABP) superfamily has proposed multifunctional roles, including functions intersecting vaccine and drug therapy, such as immune modulation and anthelmintic sequestration. Research is hindered by a lack of understanding of the full FABP superfamily complement. Although discovery studies predicted FABPs as promising vaccine candidates, it is unclear if uncharacterized FABPs are more relevant for vaccine formulations. We have coupled genome, transcriptome, and EST data mining with proteomics and phylogenetics to reveal a liver fluke FABP superfamily of seven clades: previously identified clades I-III and newly identified clades IV-VII. All new clade FABPs were analyzed using bioinformatics and cloned from both liver flukes. The extended FABP data set will provide new study tools to research the role of FABPs in parasite biology and as therapy targets.

  13. TLX: An elusive receptor.

    Science.gov (United States)

    Benod, Cindy; Villagomez, Rosa; Webb, Paul

    2016-03-01

    TLX (tailless receptor) is a member of the nuclear receptor superfamily and belongs to a class of nuclear receptors for which no endogenous or synthetic ligands have yet been identified. TLX is a promising therapeutic target in neurological disorders and brain tumors. Thus, regulatory ligands for TLX need to be identified to complete the validation of TLX as a useful target and would serve as chemical probes to pursue the study of this receptor in disease models. It has recently been proved that TLX is druggable. However, to identify potent and specific TLX ligands with desirable biological activity, a deeper understanding of where ligands bind, how they alter TLX conformation and of the mechanism by which TLX mediates the transcription of its target genes is needed. While TLX is in the process of escaping from orphanhood, future ligand design needs to progress in parallel with improved understanding of (i) the binding cavity or surfaces to target with small molecules on the TLX ligand binding domain and (ii) the nature of the TLX coregulators in particular cell and disease contexts. Both of these topics are discussed in this review. Copyright © 2015 Elsevier Ltd. All rights reserved.

  14. The receptor RAGE: Bridging inflammation and cancer

    Directory of Open Access Journals (Sweden)

    Hess Jochen

    2009-05-01

    Full Text Available Abstract The receptor for advanced glycation end products (RAGE is a single transmembrane receptor of the immunoglobulin superfamily that is mainly expressed on immune cells, neurons, activated endothelial and vascular smooth muscle cells, bone forming cells, and a variety of cancer cells. RAGE is a multifunctional receptor that binds a broad repertoire of ligands and mediates responses to cell damage and stress conditions. It activates programs responsible for acute and chronic inflammation, and is implicated in a number of pathological diseases, including diabetic complications, stroke, atheriosclerosis, arthritis, and neurodegenerative disorders. The availability of Rage knockout mice has not only advanced our knowledge on signalling pathways within these pathophysiological conditions, but also on the functional importance of the receptor in processes of cancer. Here, we will summarize molecular mechanisms through which RAGE signalling contributes to the establishment of a pro-tumourigenic microenvironment. Moreover, we will review recent findings that provide genetic evidence for an important role of RAGE in bridging inflammation and cancer.

  15. Rearrangement of a polar core provides a conserved mechanism for constitutive activation of class B G protein-coupled receptors

    Science.gov (United States)

    Yin, Yanting; de Waal, Parker W.; He, Yuanzheng; Zhao, Li-Hua; Yang, Dehua; Cai, Xiaoqing; Jiang, Yi; Melcher, Karsten; Wang, Ming-Wei; Xu, H. Eric

    2017-01-01

    The glucagon receptor (GCGR) belongs to the secretin-like (class B) family of G protein-coupled receptors (GPCRs) and is activated by the peptide hormone glucagon. The structures of an activated class B GPCR have remained unsolved, preventing a mechanistic understanding of how these receptors are activated. Using a combination of structural modeling and mutagenesis studies, we present here two modes of ligand-independent activation of GCGR. First, we identified a GCGR-specific hydrophobic lock comprising Met-338 and Phe-345 within the IC3 loop and transmembrane helix 6 (TM6) and found that this lock stabilizes the TM6 helix in the inactive conformation. Disruption of this hydrophobic lock led to constitutive G protein and arrestin signaling. Second, we discovered a polar core comprising conserved residues in TM2, TM3, TM6, and TM7, and mutations that disrupt this polar core led to constitutive GCGR activity. On the basis of these results, we propose a mechanistic model of GCGR activation in which TM6 is held in an inactive conformation by the conserved polar core and the hydrophobic lock. Mutations that disrupt these inhibitory elements allow TM6 to swing outward to adopt an active TM6 conformation similar to that of the canonical β2-adrenergic receptor complexed with G protein and to that of rhodopsin complexed with arrestin. Importantly, mutations in the corresponding polar core of several other members of class B GPCRs, including PTH1R, PAC1R, VIP1R, and CRFR1, also induce constitutive G protein signaling, suggesting that the rearrangement of the polar core is a conserved mechanism for class B GPCR activation. PMID:28356352

  16. Calcium-sensing receptor: Role in health and disease

    Directory of Open Access Journals (Sweden)

    R V Thakker

    2012-01-01

    Full Text Available The calcium-sensing receptor (CaSR is a 1,078 amino acid G protein-coupled receptor (GPCR, which is predominantly expressed in the parathyroids and kidney. The CaSR allows regulation of parathyroid hormone (PTH secretion and renal tubular calcium re-absorption in response to alterations in extracellular calcium concentrations. Loss-of-function CaSR mutations have been reported in the hypercalcemic disorders of familial benign (hypocalciuric hypercalcemia (FBH or FHH, neonatal severe primary hyperparathyroidism (NSHPT, and adult primary hyperparathyroidism. However, some individuals with loss-of-function CaSR mutations remain normocalcemic. Gain-of-function CaSR mutations have been shown to result in autosomal-dominant hypocalcemia with hypercalciuria (ADHH and Bartter′s syndrome type V. CaSR auto-antibodies have been found in FHH patients who did not have loss-of-function CaSR mutations and in patients with an acquired form (i.e. autoimmune of hypoparathyroidism. Thus, abnormalities of the CaSR are associated with 4 hypercalcemic and 3 hypocalcemic disorders.

  17. Functional Diversity of Haloacid Dehalogenase Superfamily Phosphatases from Saccharomyces cerevisiae: BIOCHEMICAL, STRUCTURAL, AND EVOLUTIONARY INSIGHTS.

    Science.gov (United States)

    Kuznetsova, Ekaterina; Nocek, Boguslaw; Brown, Greg; Makarova, Kira S; Flick, Robert; Wolf, Yuri I; Khusnutdinova, Anna; Evdokimova, Elena; Jin, Ke; Tan, Kemin; Hanson, Andrew D; Hasnain, Ghulam; Zallot, Rémi; de Crécy-Lagard, Valérie; Babu, Mohan; Savchenko, Alexei; Joachimiak, Andrzej; Edwards, Aled M; Koonin, Eugene V; Yakunin, Alexander F

    2015-07-24

    The haloacid dehalogenase (HAD)-like enzymes comprise a large superfamily of phosphohydrolases present in all organisms. The Saccharomyces cerevisiae genome encodes at least 19 soluble HADs, including 10 uncharacterized proteins. Here, we biochemically characterized 13 yeast phosphatases from the HAD superfamily, which includes both specific and promiscuous enzymes active against various phosphorylated metabolites and peptides with several HADs implicated in detoxification of phosphorylated compounds and pseudouridine. The crystal structures of four yeast HADs provided insight into their active sites, whereas the structure of the YKR070W dimer in complex with substrate revealed a composite substrate-binding site. Although the S. cerevisiae and Escherichia coli HADs share low sequence similarities, the comparison of their substrate profiles revealed seven phosphatases with common preferred substrates. The cluster of secondary substrates supporting significant activity of both S. cerevisiae and E. coli HADs includes 28 common metabolites that appear to represent the pool of potential activities for the evolution of novel HAD phosphatases. Evolution of novel substrate specificities of HAD phosphatases shows no strict correlation with sequence divergence. Thus, evolution of the HAD superfamily combines the conservation of the overall substrate pool and the substrate profiles of some enzymes with remarkable biochemical and structural flexibility of other superfamily members. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  18. GPCR Interaction: 199 [GRIPDB[Archive

    Lifescience Database Archive (English)

    Full Text Available ecretin receptor (SecR), and interfaces of VPAC2R B Vasoactive intestinal polypeptide Type 2 VPAC2R C Secret...in ... SecR Experiment SecR does not interact with CTR (calcitonin receptor). 18401761 BRET, FRET NP_003373.2 ...

  19. Cloning and characterization of SCART1, a novel scavenger receptor cysteine-rich type I transmembrane molecule

    DEFF Research Database (Denmark)

    Holm, Dorte; Fink, Dorte Rosenbek; Grønlund, Jørn

    2009-01-01

    We have cloned and characterized a novel murine transmembrane molecule, mSCART1 belonging to the scavenger receptor cysteine-rich superfamily. The cDNA encodes a polypeptide chain of 989 amino acids, organized as a type I transmembrane protein that contains eight extracellular SRCR domains followed...

  20. Molecular interactions of agonist and inverse agonist ligands at serotonin 5-HT2C G protein-coupled receptors: computational ligand docking and molecular dynamics studies validated by experimental mutagenesis results

    Science.gov (United States)

    Córdova-Sintjago, Tania C.; Liu, Yue; Booth, Raymond G.

    2015-02-01

    To understand molecular determinants for ligand activation of the serotonin 5-HT2C G protein-coupled receptor (GPCR), a drug target for obesity and neuropsychiatric disorders, a 5-HT2C homology model was built according to an adrenergic β2 GPCR (β2AR) structure and validated using a 5-HT2B GPCR crystal structure. The models were equilibrated in a simulated phosphatidyl choline membrane for ligand docking and molecular dynamics studies. Ligands included (2S, 4R)-(-)-trans-4-(3'-bromo- and trifluoro-phenyl)-N,N-dimethyl-1,2,3,4-tetrahydronaphthalene-2-amine (3'-Br-PAT and 3'-CF3-PAT), a 5-HT2C agonist and inverse agonist, respectively. Distinct interactions of 3'-Br-PAT and 3'-CF3-PAT at the wild-type (WT) 5-HT2C receptor model were observed and experimental 5-HT2C receptor mutagenesis studies were undertaken to validate the modelling results. For example, the inverse agonist 3'-CF3-PAT docked deeper in the WT 5-HT2C binding pocket and altered the orientation of transmembrane helices (TM) 6 in comparison to the agonist 3'-Br-PAT, suggesting that changes in TM orientation that result from ligand binding impact function. For both PATs, mutation of 5-HT2C residues S3.36, T3.37, and F5.47 to alanine resulted in significantly decreased affinity, as predicted from modelling results. It was concluded that upon PAT binding, 5-HT2C residues T3.37 and F5.47 in TMs 3 and 5, respectively, engage in inter-helical interactions with TMs 4 and 6, respectively. The movement of TMs 5 and 6 upon agonist and inverse agonist ligand binding observed in the 5-HT2C receptor modelling studies was similar to movements reported for the activation and deactivation of the β2AR, suggesting common mechanisms among aminergic neurotransmitter GPCRs.

  1. Regulator of G protein signaling 2 (RGS2 and RGS4 form distinct G protein-dependent complexes with protease activated-receptor 1 (PAR1 in live cells.

    Directory of Open Access Journals (Sweden)

    Sungho Ghil

    Full Text Available Protease-activated receptor 1 (PAR1 is a G-protein coupled receptor (GPCR that is activated by natural proteases to regulate many physiological actions. We previously reported that PAR1 couples to Gi, Gq and G12 to activate linked signaling pathways. Regulators of G protein signaling (RGS proteins serve as GTPase activating proteins to inhibit GPCR/G protein signaling. Some RGS proteins interact directly with certain GPCRs to modulate their signals, though cellular mechanisms dictating selective RGS/GPCR coupling are poorly understood. Here, using bioluminescence resonance energy transfer (BRET, we tested whether RGS2 and RGS4 bind to PAR1 in live COS-7 cells to regulate PAR1/Gα-mediated signaling. We report that PAR1 selectively interacts with either RGS2 or RGS4 in a G protein-dependent manner. Very little BRET activity is observed between PAR1-Venus (PAR1-Ven and either RGS2-Luciferase (RGS2-Luc or RGS4-Luc in the absence of Gα. However, in the presence of specific Gα subunits, BRET activity was markedly enhanced between PAR1-RGS2 by Gαq/11, and PAR1-RGS4 by Gαo, but not by other Gα subunits. Gαq/11-YFP/RGS2-Luc BRET activity is promoted by PAR1 and is markedly enhanced by agonist (TFLLR stimulation. However, PAR1-Ven/RGS-Luc BRET activity was blocked by a PAR1 mutant (R205A that eliminates PAR1-Gq/11 coupling. The purified intracellular third loop of PAR1 binds directly to purified His-RGS2 or His-RGS4. In cells, RGS2 and RGS4 inhibited PAR1/Gα-mediated calcium and MAPK/ERK signaling, respectively, but not RhoA signaling. Our findings indicate that RGS2 and RGS4 interact directly with PAR1 in Gα-dependent manner to modulate PAR1/Gα-mediated signaling, and highlight a cellular mechanism for selective GPCR/G protein/RGS coupling.

  2. Medium-Throughput Screen of Microbially Produced Serotonin via a G-Protein-Coupled Receptor-Based Sensor.

    Science.gov (United States)

    Ehrenworth, Amy M; Claiborne, Tauris; Peralta-Yahya, Pamela

    2017-10-17

    Chemical biosensors, for which chemical detection triggers a fluorescent signal, have the potential to accelerate the screening of noncolorimetric chemicals produced by microbes, enabling the high-throughput engineering of enzymes and metabolic pathways. Here, we engineer a G-protein-coupled receptor (GPCR)-based sensor to detect serotonin produced by a producer microbe in the producer microbe's supernatant. Detecting a chemical in the producer microbe's supernatant is nontrivial because of the number of other metabolites and proteins present that could interfere with sensor performance. We validate the two-cell screening system for medium-throughput applications, opening the door to the rapid engineering of microbes for the increased production of serotonin. We focus on serotonin detection as serotonin levels limit the microbial production of hydroxystrictosidine, a modified alkaloid that could accelerate the semisynthesis of camptothecin-derived anticancer pharmaceuticals. This work shows the ease of generating GPCR-based chemical sensors and their ability to detect specific chemicals in complex aqueous solutions, such as microbial spent medium. In addition, this work sets the stage for the rapid engineering of serotonin-producing microbes.

  3. An expressed sequence tag (EST) data mining strategy succeeding in the discovery of new G-protein coupled receptors.

    Science.gov (United States)

    Wittenberger, T; Schaller, H C; Hellebrand, S

    2001-03-30

    We have developed a comprehensive expressed sequence tag database search method and used it for the identification of new members of the G-protein coupled receptor superfamily. Our approach proved to be especially useful for the detection of expressed sequence tag sequences that do not encode conserved parts of a protein, making it an ideal tool for the identification of members of divergent protein families or of protein parts without conserved domain structures in the expressed sequence tag database. At least 14 of the expressed sequence tags found with this strategy are promising candidates for new putative G-protein coupled receptors. Here, we describe the sequence and expression analysis of five new members of this receptor superfamily, namely GPR84, GPR86, GPR87, GPR90 and GPR91. We also studied the genomic structure and chromosomal localization of the respective genes applying in silico methods. A cluster of six closely related G-protein coupled receptors was found on the human chromosome 3q24-3q25. It consists of four orphan receptors (GPR86, GPR87, GPR91, and H963), the purinergic receptor P2Y1, and the uridine 5'-diphosphoglucose receptor KIAA0001. It seems likely that these receptors evolved from a common ancestor and therefore might have related ligands. In conclusion, we describe a data mining procedure that proved to be useful for the identification and first characterization of new genes and is well applicable for other gene families. Copyright 2001 Academic Press.

  4. Importance of the extracellular loops in G protein-coupled receptors for ligand recognition and receptor activation.

    Science.gov (United States)

    Peeters, M C; van Westen, G J P; Li, Q; IJzerman, A P

    2011-01-01

    G protein-coupled receptors (GPCRs) are the major drug target of medicines on the market today. Therefore, much research is and has been devoted to the elucidation of the function and three-dimensional structure of this large family of membrane proteins, which includes multiple conserved transmembrane domains connected by intra- and extracellular loops. In the last few years, the less conserved extracellular loops have garnered increasing interest, particularly after the publication of several GPCR crystal structures that clearly show the extracellular loops to be involved in ligand binding. This review will summarize the recent progress made in the clarification of the ligand binding and activation mechanism of class-A GPCRs and the role of extracellular loops in this process. Copyright © 2010 Elsevier Ltd. All rights reserved.

  5. Investigation of Membrane Receptors' Oligomers Using Fluorescence Resonance Energy Transfer and Multiphoton Microscopy in Living Cells

    Science.gov (United States)

    Mishra, Ashish K.

    Investigating quaternary structure (oligomerization) of macromolecules (such as proteins and nucleic acids) in living systems (in vivo) has been a great challenge in biophysics, due to molecular diffusion, fluctuations in several biochemical parameters such as pH, quenching of fluorescence by oxygen (when fluorescence methods are used), etc. We studied oligomerization of membrane receptors in living cells by means of Fluorescence (Forster) Resonance Energy Transfer (FRET) using fluorescent markers and two photon excitation fluorescence micro-spectroscopy. Using suitable FRET models, we determined the stoichiometry and quaternary structure of various macromolecular complexes. The proteins of interest for this work are : (1) sigma-1 receptor and (2) rhodopsin, are described as below. (1) Sigma-1 receptors are molecular chaperone proteins, which also regulate ion channels. S1R seems to be involved in substance abuse, as well as several diseases such as Alzheimer's. We studied S1R in the presence and absence of its ligands haloperidol (an antagonist) and pentazocine +/- (an agonist), and found that at low concentration they reside as a mixture of monomers and dimers and that they may form higher order oligomers at higher concentrations. (2) Rhodopsin is a prototypical G protein coupled receptor (GPCR) and is directly involved in vision. GPCRs form a large family of receptors that participate in cell signaling by responding to external stimuli such as drugs, thus being a major drug target (more than 40% drugs target GPCRs). Their oligomerization has been largely controversial. Understanding this may help to understand the functional role of GPCRs oligomerization, and may lead to the discovery of more drugs targeting GPCR oligomers. It may also contribute toward finding a cure for Retinitis Pigmentosa, which is caused by a mutation (G188R) in rhodopsin, a disease which causes blindness and has no cure so far. Comparing healthy rhodopsin's oligomeric structure with that

  6. TRAIL death receptors and cancer therapeutics

    International Nuclear Information System (INIS)

    Huang Ying; Sheikh, M. Saeed

    2007-01-01

    Tumor necrosis factor-related apoptosis inducing ligand (TRAIL) also known as Apo2L is an apoptotic molecule that belongs to the tumor necrosis factor superfamily of cytokines. It mediates its apoptotic effects via its cognate death receptors including DR4 and DR5. Agonistic monoclonal antibodies have also been developed that selectively activate TRAIL death receptors to mediate apoptosis. Multiple clinically relevant agents also upregulate the expression of TRAIL death receptors, and cooperate with TRAIL as well as DR4 and DR5-specific agonistic antibodies to exhibit tumor cell killing. TRAIL is currently in phase I clinical trials, whereas DR4 and DR5-specific agonistic antibodies have been tested in phase I and II studies. Thus, TRAIL has clearly distinguished itself from the other family members including TNF-alpha and FasL both of which could not make it to the clinic due to their toxic nature. It is therefore, evident that the future of TRAIL-based therapeutic approaches looks brighter

  7. Conformation guides molecular efficacy in docking screens of activated β-2 adrenergic G protein coupled receptor.

    Science.gov (United States)

    Weiss, Dahlia R; Ahn, SeungKirl; Sassano, Maria F; Kleist, Andrew; Zhu, Xiao; Strachan, Ryan; Roth, Bryan L; Lefkowitz, Robert J; Shoichet, Brian K

    2013-05-17

    A prospective, large library virtual screen against an activated β2-adrenergic receptor (β2AR) structure returned potent agonists to the exclusion of inverse-agonists, providing the first complement to the previous virtual screening campaigns against inverse-agonist-bound G protein coupled receptor (GPCR) structures, which predicted only inverse-agonists. In addition, two hits recapitulated the signaling profile of the co-crystal ligand with respect to the G protein and arrestin mediated signaling. This functional fidelity has important implications in drug design, as the ability to predict ligands with predefined signaling properties is highly desirable. However, the agonist-bound state provides an uncertain template for modeling the activated conformation of other GPCRs, as a dopamine D2 receptor (DRD2) activated model templated on the activated β2AR structure returned few hits of only marginal potency.

  8. Gene network inference and biochemical assessment delineates GPCR pathways and CREB targets in small intestinal neuroendocrine neoplasia.

    Directory of Open Access Journals (Sweden)

    Ignat Drozdov

    Full Text Available Small intestinal (SI neuroendocrine tumors (NET are increasing in incidence, however little is known about their biology. High throughput techniques such as inference of gene regulatory networks from microarray experiments can objectively define signaling machinery in this disease. Genome-wide co-expression analysis was used to infer gene relevance network in SI-NETs. The network was confirmed to be non-random, scale-free, and highly modular. Functional analysis of gene co-expression modules revealed processes including 'Nervous system development', 'Immune response', and 'Cell-cycle'. Importantly, gene network topology and differential expression analysis identified over-expression of the GPCR signaling regulators, the cAMP synthetase, ADCY2, and the protein kinase A, PRKAR1A. Seven CREB response element (CRE transcripts associated with proliferation and secretion: BEX1, BICD1, CHGB, CPE, GABRB3, SCG2 and SCG3 as well as ADCY2 and PRKAR1A were measured in an independent SI dataset (n = 10 NETs; n = 8 normal preparations. All were up-regulated (p<0.035 with the exception of SCG3 which was not differently expressed. Forskolin (a direct cAMP activator, 10(-5 M significantly stimulated transcription of pCREB and 3/7 CREB targets, isoproterenol (a selective ß-adrenergic receptor agonist and cAMP activator, 10(-5 M stimulated pCREB and 4/7 targets while BIM-53061 (a dopamine D(2 and Serotonin [5-HT(2] receptor agonist, 10(-6 M stimulated 100% of targets as well as pCREB; CRE transcription correlated with the levels of cAMP accumulation and PKA activity; BIM-53061 stimulated the highest levels of cAMP and PKA (2.8-fold and 2.5-fold vs. 1.8-2-fold for isoproterenol and forskolin. Gene network inference and graph topology analysis in SI NETs suggests that SI NETs express neural GPCRs that activate different CRE targets associated with proliferation and secretion. In vitro studies, in a model NET cell system, confirmed that transcriptional

  9. Proinflammatory response during Ebola virus infection of primate models: possible involvement of the tumor necrosis factor receptor superfamily.

    Science.gov (United States)

    Hensley, Lisa E; Young, Howard A; Jahrling, Peter B; Geisbert, Thomas W

    2002-03-01

    Ebola virus (EBOV) infections are characterized by dysregulation of normal host immune responses. Insight into the mechanism came from recent studies in nonhuman primates, which showed that EBOV infects cells of the mononuclear phagocyte system (MPS), resulting in apoptosis of bystander lymphocytes. In this study, we evaluated serum levels of cytokines/chemokines in EBOV-infected nonhuman primates, as possible correlates of this bystander apoptosis. Increased levels of interferon (IFN)-alpha, IFN-beta, interleukin (IL)-6, IL-18, MIP-1alpha, and MIP-1beta were observed in all EBOV-infected monkeys, indicating the occurrence of a strong proinflammatory response. To investigate the mechanism(s) involved in lymphoid apoptosis, soluble Fas (sFas) and nitrate accumulation were measured. sFas was detected in 4/9 animals, while, elevations of nitrate accumulation occurred in 3/3 animals. To further evaluate the potential role of these factors in the observed bystander apoptosis and intact animals, in vitro cultures were prepared of adherent human monocytes/macrophages (PHM), and monocytes differentiated into immature dendritic cells (DC). These cultures were infected with EBOV and analyzed for cytokine/chemokine induction and expression of apoptosis-related genes. In addition, the in vitro EBOV infection of peripheral blood mononuclear cells (PBMC) resulted in strong cytokine/chemokine induction, a marked increase in lactate dehydrogenase (LDH) activity, and an increase in the number of apoptotic lymphocytes examined by electron microscopy. Increased levels of sFAS were detected in PHM cultures, although, 90% of EBOV-infected PHM were positive for tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) by immunohistochemistry, RNA analysis, and flow cytometry. Inactivated EBOV also effected increased TRAIL expression in PHM, suggesting that the TNF receptor superfamily may be involved in apoptosis of the host lymphoid cells, and that induction may occur

  10. Transactivation of the proximal promoter of human oxytocin gene by TR4 orphan receptor

    International Nuclear Information System (INIS)

    Wang, C.-P.; Lee, Y.-F.; Chang, C.; Lee, H.-J.

    2006-01-01

    The human testicular receptor 4 (TR4) shares structural homology with members of the nuclear receptor superfamily. Some other members of this superfamily were able to regulate the transcriptional activity of the human oxytocin (OXT) promoter by binding to the first DR0 regulatory site. However, little investigation was conducted systematically in the study of the second dDR4 site of OXT proximal promoter, and the relationship between the first and the second sites of OXT promoter. Here, we demonstrated for the first time that TR4 could increase the proximal promoter activity of the human OXT gene via DR0, dDR4, and OXT (both DR0 and dDR4) elements, respectively. TR4 might induce OXT gene expression through the OXT element in a dose-dependent manner. However, there is no synergistic effect between DR0 and dDR4 elements during TR4 transactivation. Taken together, these results suggested that TR4 should be one of important regulators of OXT gene expression

  11. Evolution of Enzymatic Activities in the Enolase Superfamily: D-Mannonate Dhydratase from Novosphingobium aromaticivorans

    Energy Technology Data Exchange (ETDEWEB)

    Rakus,J.; Fedorov, A.; Fedorov, E.; Glasner, M.; Vick, J.; Babbitt, P.; Almo, S.; Gerlt, J.

    2007-01-01

    The d-mannonate dehydratase (ManD) function was assigned to a group of orthologous proteins in the mechanistically diverse enolase superfamily by screening a library of acid sugars. Structures of the wild type ManD from Novosphingobium aromaticivorans were determined at pH 7.5 in the presence of Mg2+ and also in the presence of Mg2+ and the 2-keto-3-keto-d-gluconate dehydration product; the structure of the catalytically active K271E mutant was determined at pH 5.5 in the presence of the d-mannonate substrate. As previously observed in the structures of other members of the enolase superfamily, ManD contains two domains, an N-terminal a+{beta} capping domain and a ({beta}/a)7{beta}-barrel domain. The barrel domain contains the ligands for the essential Mg2+, Asp 210, Glu 236, and Glu 262, at the ends of the third, fourth, and fifth {beta}-strands of the barrel domain, respectively. However, the barrel domain lacks both the Lys acid/base catalyst at the end of the second {beta}-strand and the His-Asp dyad acid/base catalyst at the ends of the seventh and sixth {beta}-strands, respectively, that are found in many members of the superfamily. Instead, a hydrogen-bonded dyad of Tyr 159 in a loop following the second {beta}-strand and Arg 147 at the end of the second {beta}-strand are positioned to initiate the reaction by abstraction of the 2-proton. Both Tyr 159 and His 212, at the end of the third {beta}-strand, are positioned to facilitate both syn-dehydration and ketonization of the resulting enol intermediate to yield the 2-keto-3-keto-d-gluconate product with the observed retention of configuration. The identities and locations of these acid/base catalysts as well as of cationic amino acid residues that stabilize the enolate anion intermediate define a new structural strategy for catalysis (subgroup) in the mechanistically diverse enolase superfamily. With these differences, we provide additional evidence that the ligands for the essential Mg2+ are the only

  12. Thyroid hormone and retinoic acid nuclear receptors: specific ligand-activated transcription factors

    International Nuclear Information System (INIS)

    Brtko, J.

    1998-01-01

    Transcriptional regulation by both the thyroid hormone and the vitamin A-derived 'retinoid hormones' is a critical component in controlling many aspects of higher vertebrate development and metabolism. Their functions are mediated by nuclear receptors, which comprise a large super-family of ligand-inducible transcription factors. Both the thyroid hormone and the retinoids are involved in a complex arrangement of physiological and development responses in many tissues of higher vertebrates. The functions of 3,5,3'-triiodothyronine (T 3 ), the thyromimetically active metabolite of thyroxine as well as all-trans retinoic acid, the biologically active vitamin A metabolite are mediated by nuclear receptor proteins that are members of the steroid/thyroid/retinoid hormone receptor family. The functions of all members of the receptor super family are discussed. (authors)

  13. Drug-target residence time--a case for G protein-coupled receptors.

    Science.gov (United States)

    Guo, Dong; Hillger, Julia M; IJzerman, Adriaan P; Heitman, Laura H

    2014-07-01

    A vast number of marketed drugs act on G protein-coupled receptors (GPCRs), the most successful category of drug targets to date. These drugs usually possess high target affinity and selectivity, and such combined features have been the driving force in the early phases of drug discovery. However, attrition has also been high. Many investigational new drugs eventually fail in clinical trials due to a demonstrated lack of efficacy. A retrospective assessment of successfully launched drugs revealed that their beneficial effects in patients may be attributed to their long drug-target residence times (RTs). Likewise, for some other GPCR drugs short RT could be beneficial to reduce the potential for on-target side effects. Hence, the compounds' kinetics behavior might in fact be the guiding principle to obtain a desired and durable effect in vivo. We therefore propose that drug-target RT should be taken into account as an additional parameter in the lead selection and optimization process. This should ultimately lead to an increased number of candidate drugs moving to the preclinical development phase and on to the market. This review contains examples of the kinetics behavior of GPCR ligands with improved in vivo efficacy and summarizes methods for assessing drug-target RT. © 2014 Wiley Periodicals, Inc.

  14. Rearrangement of a polar core provides a conserved mechanism for constitutive activation of class B G protein-coupled receptors.

    Science.gov (United States)

    Yin, Yanting; de Waal, Parker W; He, Yuanzheng; Zhao, Li-Hua; Yang, Dehua; Cai, Xiaoqing; Jiang, Yi; Melcher, Karsten; Wang, Ming-Wei; Xu, H Eric

    2017-06-16

    The glucagon receptor (GCGR) belongs to the secretin-like (class B) family of G protein-coupled receptors (GPCRs) and is activated by the peptide hormone glucagon. The structures of an activated class B GPCR have remained unsolved, preventing a mechanistic understanding of how these receptors are activated. Using a combination of structural modeling and mutagenesis studies, we present here two modes of ligand-independent activation of GCGR. First, we identified a GCGR-specific hydrophobic lock comprising Met-338 and Phe-345 within the IC3 loop and transmembrane helix 6 (TM6) and found that this lock stabilizes the TM6 helix in the inactive conformation. Disruption of this hydrophobic lock led to constitutive G protein and arrestin signaling. Second, we discovered a polar core comprising conserved residues in TM2, TM3, TM6, and TM7, and mutations that disrupt this polar core led to constitutive GCGR activity. On the basis of these results, we propose a mechanistic model of GCGR activation in which TM6 is held in an inactive conformation by the conserved polar core and the hydrophobic lock. Mutations that disrupt these inhibitory elements allow TM6 to swing outward to adopt an active TM6 conformation similar to that of the canonical β 2 -adrenergic receptor complexed with G protein and to that of rhodopsin complexed with arrestin. Importantly, mutations in the corresponding polar core of several other members of class B GPCRs, including PTH1R, PAC1R, VIP1R, and CRFR1, also induce constitutive G protein signaling, suggesting that the rearrangement of the polar core is a conserved mechanism for class B GPCR activation. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  15. Beta2-adrenergic receptor homodimers: Role of transmembrane domain 1 and helix 8 in dimerization and cell surface expression.

    Science.gov (United States)

    Parmar, Vikas K; Grinde, Ellinor; Mazurkiewicz, Joseph E; Herrick-Davis, Katharine

    2017-09-01

    Even though there are hundreds of reports in the published literature supporting the hypothesis that G protein-coupled receptors (GPCR) form and function as dimers this remains a highly controversial area of research and mechanisms governing homodimer formation are poorly understood. Crystal structures revealing homodimers have been reported for many different GPCR. For adrenergic receptors, a potential dimer interface involving transmembrane domain 1 (TMD1) and helix 8 (H8) was identified in crystal structures of the beta 1 -adrenergic (β 1 -AR) and β 2 -AR. The purpose of this study was to investigate a potential role for TMD1 and H8 in dimerization and plasma membrane expression of functional β 2 -AR. Charged residues at the base of TMD1 and in the distal portion of H8 were replaced, singly and in combination, with non-polar residues or residues of opposite charge. Wild type and mutant β 2 -AR, tagged with YFP and expressed in HEK293 cells, were evaluated for plasma membrane expression and function. Homodimer formation was evaluated using bioluminescence resonance energy transfer, bimolecular fluorescence complementation, and fluorescence correlation spectroscopy. Amino acid substitutions at the base of TMD1 and in the distal portion of H8 disrupted homodimer formation and caused receptors to be retained in the endoplasmic reticulum. Mutations in the proximal region of H8 did not disrupt dimerization but did interfere with plasma membrane expression. This study provides biophysical evidence linking a potential TMD1/H8 interface with ER export and the expression of functional β 2 -AR on the plasma membrane. This article is part of a Special Issue entitled: Interactions between membrane receptors in cellular membranes edited by Kalina Hristova. Copyright © 2016 Elsevier B.V. All rights reserved.

  16. The UDP glucuronosyltransferase gene superfamily: suggested nomenclature based on evolutionary divergence

    NARCIS (Netherlands)

    Burchell, B.; Nebert, D. W.; Nelson, D. R.; Bock, K. W.; Iyanagi, T.; Jansen, P. L.; Lancet, D.; Mulder, G. J.; Chowdhury, J. R.; Siest, G.

    1991-01-01

    A nomenclature system for the UDP glucuronosyltransferase superfamily is proposed, based on divergent evolution of the genes. A total of 26 distinct cDNAs in five mammalian species have been sequenced to date. Comparison of the deduced amino acid sequences leads to the definition of two families and

  17. A global view of structure-function relationships in the tautomerase superfamily.

    Science.gov (United States)

    Davidson, Rebecca; Baas, Bert-Jan; Akiva, Eyal; Holliday, Gemma L; Polacco, Benjamin J; LeVieux, Jake A; Pullara, Collin R; Zhang, Yan Jessie; Whitman, Christian P; Babbitt, Patricia C

    2018-02-16

    The tautomerase superfamily (TSF) consists of more than 11,000 nonredundant sequences present throughout the biosphere. Characterized members have attracted much attention because of the unusual and key catalytic role of an N-terminal proline. These few characterized members catalyze a diverse range of chemical reactions, but the full scale of their chemical capabilities and biological functions remains unknown. To gain new insight into TSF structure-function relationships, we performed a global analysis of similarities across the entire superfamily and computed a sequence similarity network to guide classification into distinct subgroups. Our results indicate that TSF members are found in all domains of life, with most being present in bacteria. The eukaryotic members of the cis -3-chloroacrylic acid dehalogenase subgroup are limited to fungal species, whereas the macrophage migration inhibitory factor subgroup has wide eukaryotic representation (including mammals). Unexpectedly, we found that 346 TSF sequences lack Pro-1, of which 85% are present in the malonate semialdehyde decarboxylase subgroup. The computed network also enabled the identification of similarity paths, namely sequences that link functionally diverse subgroups and exhibit transitional structural features that may help explain reaction divergence. A structure-guided comparison of these linker proteins identified conserved transitions between them, and kinetic analysis paralleled these observations. Phylogenetic reconstruction of the linker set was consistent with these findings. Our results also suggest that contemporary TSF members may have evolved from a short 4-oxalocrotonate tautomerase-like ancestor followed by gene duplication and fusion. Our new linker-guided strategy can be used to enrich the discovery of sequence/structure/function transitions in other enzyme superfamilies. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

  18. Novel role for proteinase-activated receptor 2 (PAR2) in membrane trafficking of proteinase-activated receptor 4 (PAR4).

    Science.gov (United States)

    Cunningham, Margaret R; McIntosh, Kathryn A; Pediani, John D; Robben, Joris; Cooke, Alexandra E; Nilsson, Mary; Gould, Gwyn W; Mundell, Stuart; Milligan, Graeme; Plevin, Robin

    2012-05-11

    Proteinase-activated receptors 4 (PAR(4)) is a class A G protein-coupled receptor (GPCR) recognized through the ability of serine proteases such as thrombin and trypsin to mediate receptor activation. Due to the irreversible nature of activation, a fresh supply of receptor is required to be mobilized to the cell surface for responsiveness to agonist to be sustained. Unlike other PAR subtypes, the mechanisms regulating receptor trafficking of PAR(4) remain unknown. Here, we report novel features of the intracellular trafficking of PAR(4) to the plasma membrane. PAR(4) was poorly expressed at the plasma membrane and largely retained in the endoplasmic reticulum (ER) in a complex with the COPI protein subunit β-COP1. Analysis of the PAR(4) protein sequence identified an arginine-based (RXR) ER retention sequence located within intracellular loop-2 (R(183)AR → A(183)AA), mutation of which allowed efficient membrane delivery of PAR(4). Interestingly, co-expression with PAR(2) facilitated plasma membrane delivery of PAR(4), an effect produced through disruption of β-COP1 binding and facilitation of interaction with the chaperone protein 14-3-3ζ. Intermolecular FRET studies confirmed heterodimerization between PAR(2) and PAR(4). PAR(2) also enhanced glycosylation of PAR(4) and activation of PAR(4) signaling. Our results identify a novel regulatory role for PAR(2) in the anterograde traffic of PAR(4). PAR(2) was shown to both facilitate and abrogate protein interactions with PAR(4), impacting upon receptor localization and cell signal transduction. This work is likely to impact markedly upon the understanding of the receptor pharmacology of PAR(4) in normal physiology and disease.

  19. Basic Concepts in G-Protein-Coupled Receptor Homo- and Heterodimerization

    Directory of Open Access Journals (Sweden)

    Rafael Franco

    2007-01-01

    Full Text Available Until recently, heptahelical G-protein-coupled receptors (GPCRs were considered to be expressed as monomers on the cell surface of neuronal and non-neuronal cells. It is now becoming evident that this view must be overtly changed since these receptors can form homodimers, heterodimers, and higher-order oligomers on the plasma membrane. Here we discuss some of the basics and some new concepts of receptor homo- and heteromerization. Dimers-oligomers modify pharmacology, trafficking, and signaling of receptors. First of all, GPCR dimers must be considered as the main molecules that are targeted by neurotransmitters or by drugs. Thus, binding data must be fitted to dimer-based models. In these models, it is considered that the conformational changes transmitted within the dimer molecule lead to cooperativity. Cooperativity must be taken into account in the binding of agonists-antagonists-drugs and also in the binding of the so-called allosteric modulators. Cooperativity results from the intramolecular cross-talk in the homodimer. As an intramolecular cross-talk in the heterodimer, the binding of one neurotransmitter to one receptor often affects the binding of the second neurotransmitter to the partner receptor. Coactivation of the two receptors in a heterodimer can change completely the signaling pathway triggered by the neurotransmitter as well as the trafficking of the receptors. Heterodimer-specific drugs or dual drugs able to activate the two receptors in the heterodimer simultaneously emerge as novel and promising drugs for a variety of central nervous system (CNS therapeutic applications.

  20. Spatial and temporal expression of immunoglobulin superfamily member 1 in the rat

    NARCIS (Netherlands)

    Joustra, Sjoerd D.; Meijer, Onno C.; Heinen, Charlotte A.; Mol, Isabel M.; Laghmani, El Houari; Sengers, Rozemarijn M. A.; Carreno, Gabriela; van Trotsenburg, A. S. Paul; Biermasz, Nienke R.; Bernard, Daniel J.; Wit, Jan M.; Oostdijk, Wilma; van Pelt, Ans M. M.; Hamer, Geert; Wagenaar, Gerry T. M.

    2015-01-01

    Loss-of-function mutations in the immunoglobulin superfamily member 1 (IGSF1) gene cause an X-linked syndrome of central hypothyroidism, macroorchidism, variable prolactin and GH deficiency, delayed pubertal testosterone rise, and obesity. To understand the pathophysiology of this syndrome,

  1. The neuronal Ca(2+) -binding protein 2 (NECAB2) interacts with the adenosine A(2A) receptor and modulates the cell surface expression and function of the receptor.

    Science.gov (United States)

    Canela, Laia; Luján, Rafael; Lluís, Carme; Burgueño, Javier; Mallol, Josefa; Canela, Enric I; Franco, Rafael; Ciruela, Francisco

    2007-09-01

    Heptaspanning membrane also known as G protein-coupled receptors (GPCR) do interact with a variety of intracellular proteins whose function is regulate receptor traffic and/or signaling. Using a yeast two-hybrid screen, NECAB2, a neuronal calcium binding protein, was identified as a binding partner for the adenosine A(2A) receptor (A(2A)R) interacting with its C-terminal domain. Co-localization, co-immunoprecipitation and pull-down experiments showed a close and specific interaction between A(2A)R and NECAB2 in both transfected HEK-293 cells and also in rat striatum. Immunoelectron microscopy detection of NECAB2 and A(2A)R in the rat striatopallidal structures indicated that both proteins are co-distributed in the same glutamatergic nerve terminals. The interaction of NECAB2 with A(2A)R modulated the cell surface expression, the ligand-dependent internalization and the receptor-mediated activation of the MAPK pathway. Overall, these results show that A(2A)R interacts with NECAB2 in striatal neurones co-expressing the two proteins and that the interaction is relevant for A(2A)R function.

  2. The mechanisms behind decreased internalization of angiotensin II type 1 receptor.

    Science.gov (United States)

    Bian, Jingwei; Zhang, Suli; Yi, Ming; Yue, Mingming; Liu, Huirong

    2018-04-01

    The internalization of angiotensin II type 1 receptor (AT 1 R) plays an important role in maintaining cardiovascular homeostasis. Decreased receptor internalization is closely related to cardiovascular diseases induced by the abnormal activation of AT 1 R, such as hypertension. However, the mechanism behind reduced AT 1 R internalization is not fully understood. This review focuses on four parts of the receptor internalization process (the combination of agonists and receptors, receptor phosphorylation, endocytosis, and recycling) and summarizes the possible mechanisms by which AT 1 R internalization is reduced based on these four parts of the process. (1) The agonist has a large molecular weight or a stronger ability to hydrolyze phosphatidylinositol 4,5-bisphosphate (PtdIns (4,5) P 2 ), which can increase the consumption of PtdIns (4,5) P 2 . (2) AT 1 R phosphorylation is weakened because of an abnormal function of phosphorylated kinase or changes in phospho-barcoding and GPCR-β-arrestin complex conformation. (3) The abnormal formation of vesicles or AT 1 R heterodimers with fewer endocytic receptors results in less AT 1 R endocytosis. (4) The enhanced activity and upregulated expression of small GTP-binding protein 4 (Rab4) and 11 (Rab11), which regulate receptor recycling, and phosphatidylinositol 3-kinase increase AT 1 R recycling. In addition, lower expression of AT 1 R-associated protein (ATRAP) or higher expression of AT 1 R-associated protein 1 (ARAP1) can reduce receptor internalization. Copyright © 2018 Elsevier Inc. All rights reserved.

  3. Identification and expression of the CCAP receptor in the Chagas' disease vector, Rhodnius prolixus, and its involvement in cardiac control.

    Directory of Open Access Journals (Sweden)

    Dohee Lee

    Full Text Available Rhodnius prolixus is the vector of Chagas' disease, by virtue of transmitting the parasite Trypanosoma cruzi. There is no cure for Chagas' disease and therefore controlling R. prolixus is currently the only method of prevention. Understanding the physiology of the disease vector is an important step in developing control measures. Crustacean cardioactive peptide (CCAP is an important neuropeptide in insects because it has multiple physiological roles such as controlling heart rate and modulating ecdysis behaviour. In this study, we have cloned the cDNA sequence of the CCAP receptor (RhoprCCAPR from 5(th instar R. prolixus and found it to be a G-protein coupled receptor (GPCR. The spatial expression pattern in 5(th instars reveals that the RhoprCCAPR transcript levels are high in the central nervous system, hindgut and female reproductive systems, and lower in the salivary glands, male reproductive tissues and a pool of tissues including the dorsal vessel, trachea, and fat body. Interestingly, the RhoprCCAPR expression is increased prior to ecdysis and decreased post-ecdysis. A functional receptor expression assay confirms that the RhoprCCAPR is activated by CCAP (EC50 = 12 nM but not by adipokinetic hormone, corazonin or an extended FMRFamide. The involvement of CCAP in controlling heartbeat frequency was studied in vivo and in vitro by utilizing RNA interference. In vivo, the basal heartbeat frequency is decreased by 31% in bugs treated with dsCCAPR. Knocking down the receptor in dsCCAPR-treated bugs also resulted in loss of function of applied CCAP in vitro. This is the first report of a GPCR knock-down in R. prolixus and the first report showing that a reduction in CCAPR transcript levels leads to a reduction in cardiac output in any insect.

  4. Membrane cholesterol effect on the 5-HT2A receptor: Insights into the lipid-induced modulation of an antipsychotic drug target.

    Science.gov (United States)

    Ramírez-Anguita, Juan Manuel; Rodríguez-Espigares, Ismael; Guixà-González, Ramon; Bruno, Agostino; Torrens-Fontanals, Mariona; Varela-Rial, Alejandro; Selent, Jana

    2018-01-01

    The serotonin 5-hydroxytryptamine 2A (5-HT 2A ) receptor is a G-protein-coupled receptor (GPCR) relevant for the treatment of CNS disorders. In this regard, neuronal membrane composition in the brain plays a crucial role in the modulation of the receptor functioning. Since cholesterol is an essential component of neuronal membranes, we have studied its effect on the 5-HT 2A receptor dynamics through all-atom MD simulations. We find that the presence of cholesterol in the membrane increases receptor conformational variability in most receptor segments. Importantly, detailed structural analysis indicates that conformational variability goes along with the destabilization of hydrogen bonding networks not only within the receptor but also between receptor and lipids. In addition to increased conformational variability, we also find receptor segments with reduced variability. Our analysis suggests that this increased stabilization is the result of stabilizing effects of tightly bound cholesterol molecules to the receptor surface. Our finding contributes to a better understanding of membrane-induced alterations of receptor dynamics and points to cholesterol-induced stabilizing and destabilizing effects on the conformational variability of GPCRs. © 2017 International Union of Biochemistry and Molecular Biology, Inc.

  5. Theoretical and Computational Studies of Peptides and Receptors of the Insulin Family

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    Harish Vashisth

    2015-02-01

    Full Text Available Synergistic interactions among peptides and receptors of the insulin family are required for glucose homeostasis, normal cellular growth and development, proliferation, differentiation and other metabolic processes. The peptides of the insulin family are disulfide-linked single or dual-chain proteins, while receptors are ligand-activated transmembrane glycoproteins of the receptor tyrosine kinase (RTK superfamily. Binding of ligands to the extracellular domains of receptors is known to initiate signaling via activation of intracellular kinase domains. While the structure of insulin has been known since 1969, recent decades have seen remarkable progress on the structural biology of apo and liganded receptor fragments. Here, we review how this useful structural information (on ligands and receptors has enabled large-scale atomically-resolved simulations to elucidate the conformational dynamics of these biomolecules. Particularly, applications of molecular dynamics (MD and Monte Carlo (MC simulation methods are discussed in various contexts, including studies of isolated ligands, apo-receptors, ligand/receptor complexes and intracellular kinase domains. The review concludes with a brief overview and future outlook for modeling and computational studies in this family of proteins.

  6. Molecular characterisation of two novel maize LRR receptor-like kinases, which belong to the SERK gene family

    NARCIS (Netherlands)

    Baudino, S.; Hansen, S.; Brettschneider, R.; Hecht, V.F.G.; Dresselhaus, T.; Lörz, H.; Dumas, C.; Rogowsky, P.M.

    2001-01-01

    Genes encoding two novel members of the leucine-rich repeat receptor-like kinase (LRR-RLK) superfamily have been isolated from maize (Zea mays L.). These genes have been named ZmSERK1 and ZmSERK2 since features such as a putative leucine zipper (ZIP) and five leucine rich repeats in the

  7. mGlu5 Receptor Functional Interactions and Addiction

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    Robyn eBrown

    2012-05-01

    Full Text Available The idea of ‘receptor mosaics’ suggests that proteins can form complex and dynamic networks, with respect to time and protein make up, which has the potential to make significant contributions to the diversity and specificity of GPCR signalling, particularly in neuropharmacology, where a few key receptors have been implicated in multiple neurological and psychiatric disorders such as addiction. Metabotropic glutamate type 5 receptors (mGlu5 have been shown to heterodimerise and form complexes with other GPCRs including adenosine A2A and dopamine D2 receptors. mGlu5-containing complexes are found in the striatum, a region of the brain known to be critical for mediating the rewarding and incentive motivational properties of drugs of abuse. Indeed, initial studies indicate a role for mGlu5-containing complexes in rewarding and conditioned effects of drugs, as well as drug-seeking behaviour. This is consistent with the substantial influence that mGlu5 complexes appear to have on striatal function, regulating both GABAergic output of striatopallidal neurons and glutamatergic input from corticostriatal afferents. Given their discrete localization, mGlu5-containing complexes represent a novel way in which to minimize the off-target effects and therefore provide us with an exciting therapeutic avenue for drug discovery efforts. Indeed, the therapeutic targeting of receptor mosaics in a tissue specific or temporal manner (for example, a sub-population of receptors in a ‘pathological state’ has the potential to dramatically reduce detrimental side effects that may otherwise impair vital brain function.

  8. Evolution and Diversity of the Ras Superfamily of Small GTPases in Prokaryotes

    Science.gov (United States)

    Wuichet, Kristin; Søgaard-Andersen, Lotte

    2015-01-01

    The Ras superfamily of small GTPases are single domain nucleotide-dependent molecular switches that act as highly tuned regulators of complex signal transduction pathways. Originally identified in eukaryotes for their roles in fundamental cellular processes including proliferation, motility, polarity, nuclear transport, and vesicle transport, recent studies have revealed that single domain GTPases also control complex functions such as cell polarity, motility, predation, development and antibiotic resistance in bacteria. Here, we used a computational genomics approach to understand the abundance, diversity, and evolution of small GTPases in prokaryotes. We collected 520 small GTPase sequences present in 17% of 1,611 prokaryotic genomes analyzed that cover diverse lineages. We identified two discrete families of small GTPases in prokaryotes that show evidence of three distinct catalytic mechanisms. The MglA family includes MglA homologs, which are typically associated with the MglB GTPase activating protein, whereas members of the Rup (Ras superfamily GTPase of unknown function in prokaryotes) family are not predicted to interact with MglB homologs. System classification and genome context analyses support the involvement of small GTPases in diverse prokaryotic signal transduction pathways including two component systems, laying the foundation for future experimental characterization of these proteins. Phylogenetic analysis of prokaryotic and eukaryotic GTPases supports that the last universal common ancestor contained ancestral MglA and Rup family members. We propose that the MglA family was lost from the ancestral eukaryote and that the Ras superfamily members in extant eukaryotes are the result of vertical and horizontal gene transfer events of ancestral Rup GTPases. PMID:25480683

  9. Using sequence similarity networks for visualization of relationships across diverse protein superfamilies.

    Directory of Open Access Journals (Sweden)

    Holly J Atkinson

    Full Text Available The dramatic increase in heterogeneous types of biological data--in particular, the abundance of new protein sequences--requires fast and user-friendly methods for organizing this information in a way that enables functional inference. The most widely used strategy to link sequence or structure to function, homology-based function prediction, relies on the fundamental assumption that sequence or structural similarity implies functional similarity. New tools that extend this approach are still urgently needed to associate sequence data with biological information in ways that accommodate the real complexity of the problem, while being accessible to experimental as well as computational biologists. To address this, we have examined the application of sequence similarity networks for visualizing functional trends across protein superfamilies from the context of sequence similarity. Using three large groups of homologous proteins of varying types of structural and functional diversity--GPCRs and kinases from humans, and the crotonase superfamily of enzymes--we show that overlaying networks with orthogonal information is a powerful approach for observing functional themes and revealing outliers. In comparison to other primary methods, networks provide both a good representation of group-wise sequence similarity relationships and a strong visual and quantitative correlation with phylogenetic trees, while enabling analysis and visualization of much larger sets of sequences than trees or multiple sequence alignments can easily accommodate. We also define important limitations and caveats in the application of these networks. As a broadly accessible and effective tool for the exploration of protein superfamilies, sequence similarity networks show great potential for generating testable hypotheses about protein structure-function relationships.

  10. Using sequence similarity networks for visualization of relationships across diverse protein superfamilies.

    Science.gov (United States)

    Atkinson, Holly J; Morris, John H; Ferrin, Thomas E; Babbitt, Patricia C

    2009-01-01

    The dramatic increase in heterogeneous types of biological data--in particular, the abundance of new protein sequences--requires fast and user-friendly methods for organizing this information in a way that enables functional inference. The most widely used strategy to link sequence or structure to function, homology-based function prediction, relies on the fundamental assumption that sequence or structural similarity implies functional similarity. New tools that extend this approach are still urgently needed to associate sequence data with biological information in ways that accommodate the real complexity of the problem, while being accessible to experimental as well as computational biologists. To address this, we have examined the application of sequence similarity networks for visualizing functional trends across protein superfamilies from the context of sequence similarity. Using three large groups of homologous proteins of varying types of structural and functional diversity--GPCRs and kinases from humans, and the crotonase superfamily of enzymes--we show that overlaying networks with orthogonal information is a powerful approach for observing functional themes and revealing outliers. In comparison to other primary methods, networks provide both a good representation of group-wise sequence similarity relationships and a strong visual and quantitative correlation with phylogenetic trees, while enabling analysis and visualization of much larger sets of sequences than trees or multiple sequence alignments can easily accommodate. We also define important limitations and caveats in the application of these networks. As a broadly accessible and effective tool for the exploration of protein superfamilies, sequence similarity networks show great potential for generating testable hypotheses about protein structure-function relationships.

  11. Expression analysis of G Protein-Coupled Receptors in mouse macrophages.

    Science.gov (United States)

    Lattin, Jane E; Schroder, Kate; Su, Andrew I; Walker, John R; Zhang, Jie; Wiltshire, Tim; Saijo, Kaoru; Glass, Christopher K; Hume, David A; Kellie, Stuart; Sweet, Matthew J

    2008-04-29

    Monocytes and macrophages express an extensive repertoire of G Protein-Coupled Receptors (GPCRs) that regulate inflammation and immunity. In this study we performed a systematic micro-array analysis of GPCR expression in primary mouse macrophages to identify family members that are either enriched in macrophages compared to a panel of other cell types, or are regulated by an inflammatory stimulus, the bacterial product lipopolysaccharide (LPS). Several members of the P2RY family had striking expression patterns in macrophages; P2ry6 mRNA was essentially expressed in a macrophage-specific fashion, whilst P2ry1 and P2ry5 mRNA levels were strongly down-regulated by LPS. Expression of several other GPCRs was either restricted to macrophages (e.g. Gpr84) or to both macrophages and neural tissues (e.g. P2ry12, Gpr85). The GPCR repertoire expressed by bone marrow-derived macrophages and thioglycollate-elicited peritoneal macrophages had some commonality, but there were also several GPCRs preferentially expressed by either cell population. The constitutive or regulated expression in macrophages of several GPCRs identified in this study has not previously been described. Future studies on such GPCRs and their agonists are likely to provide important insights into macrophage biology, as well as novel inflammatory pathways that could be future targets for drug discovery.

  12. GABAB receptor cell surface export is controlled by an endoplasmic reticulum gatekeeper

    Science.gov (United States)

    Doly, Stéphane; Shirvani, Hamasseh; Gäta, Gabriel; Meye, Frank; Emerit, Michel-Boris; Enslen, Hervé; Achour, Lamia; Pardo-Lopez, Liliana; Kwon, Yang Seung; Armand, Vincent; Gardette, Robert; Giros, Bruno; Gassmann, Martin; Bettler, Bernhard; Mameli, Manuel; Darmon, Michèle; Marullo, Stefano

    2016-01-01

    Summary Endoplasmic reticulum (ER) release and cell surface export of many G protein-coupled receptors (GPCRs), are tightly regulated. For GABAB receptors of GABA, the major mammalian inhibitory neurotransmitter, the ligand-binding GB1 subunit is maintained in the ER by unknown mechanisms in the absence of hetero-dimerization with the GB2 subunit. We report that GB1 retention is regulated by a specific gatekeeper, PRAF2. This ER resident transmembrane protein binds to GB1, preventing its progression in the biosynthetic pathway. GB1 release occurs upon competitive displacement from PRAF2 by GB2. PRAF2 concentration, relative to that of GB1 and GB2, tightly controls cell surface receptor density and controls GABAB function in neurons. Experimental perturbation of PRAF2 levels in vivo caused marked hyperactivity disorders in mice. These data reveal an unanticipated major impact of specific ER gate-keepers on GPCR function and identify PRAF2 as a new molecular target with therapeutic potential for psychiatric and neurological diseases involving GABAB function. PMID:26033241

  13. Trichoderma G protein-coupled receptors: functional characterisation of a cAMP receptor-like protein from Trichoderma atroviride.

    Science.gov (United States)

    Brunner, Kurt; Omann, Markus; Pucher, Marion E; Delic, Marizela; Lehner, Sylvia M; Domnanich, Patrick; Kratochwill, Klaus; Druzhinina, Irina; Denk, Dagmar; Zeilinger, Susanne

    2008-12-01

    Galpha subunits act to regulate vegetative growth, conidiation, and the mycoparasitic response in Trichoderma atroviride. To extend our knowledge on G protein signalling, we analysed G protein-coupled receptors (GPCRs). As the genome sequence of T. atroviride is not publicly available yet, we carried out an in silico exploration of the genome database of the close relative T. reesei. Twenty genes encoding putative GPCRs distributed over eight classes and additional 35 proteins similar to the Magnaporthe grisea PTH11 receptor were identified. Subsequently, four T. atroviride GPCR-encoding genes were isolated and affiliated to the cAMP receptor-like family by phylogenetic and topological analyses. All four genes showed lowest expression on glycerol and highest mRNA levels upon carbon starvation. Transcription of gpr3 and gpr4 responded to exogenously added cAMP and the shift from liquid to solid media. gpr3 mRNA levels also responded to the presence of fungal hyphae or cellulose membranes. Further characterisation of mutants bearing a gpr1-silencing construct revealed that Gpr1 is essential for vegetative growth, conidiation and conidial germination. Four genes encoding the first GPCRs described in Trichoderma were isolated and their expression characterized. At least one of these GPCRs is important for several cellular processes, supporting the fundamental role of G protein signalling in this fungus.

  14. A highly sensitive quantitative cytosensor technique for the identification of receptor ligands in tissue extracts.

    Science.gov (United States)

    Lenkei, Z; Beaudet, A; Chartrel, N; De Mota, N; Irinopoulou, T; Braun, B; Vaudry, H; Llorens-Cortes, C

    2000-11-01

    Because G-protein-coupled receptors (GPCRs) constitute excellent putative therapeutic targets, functional characterization of orphan GPCRs through identification of their endogenous ligands has great potential for drug discovery. We propose here a novel single cell-based assay for identification of these ligands. This assay involves (a) fluorescent tagging of the GPCR, (b) expression of the tagged receptor in a heterologous expression system, (c) incubation of the transfected cells with fractions purified from tissue extracts, and (d) imaging of ligand-induced receptor internalization by confocal microscopy coupled to digital image quantification. We tested this approach in CHO cells stably expressing the NT1 neurotensin receptor fused to EGFP (enhanced green fluorescent protein), in which neurotensin promoted internalization of the NT1-EGFP receptor in a dose-dependent fashion (EC(50) = 0.98 nM). Similarly, four of 120 consecutive reversed-phase HPLC fractions of frog brain extracts promoted internalization of the NT1-EGFP receptor. The same four fractions selectively contained neurotensin, an endogenous ligand of the NT1 receptor, as detected by radioimmunoassay and inositol phosphate production. The present internalization assay provides a highly specific quantitative cytosensor technique with sensitivity in the nanomolar range that should prove useful for the identification of putative natural and synthetic ligands for GPCRs.

  15. A novel fractal approach for predicting G-protein-coupled receptors and their subfamilies with support vector machines.

    Science.gov (United States)

    Nie, Guoping; Li, Yong; Wang, Feichi; Wang, Siwen; Hu, Xuehai

    2015-01-01

    G-protein-coupled receptors (GPCRs) are seven membrane-spanning proteins and regulate many important physiological processes, such as vision, neurotransmission, immune response and so on. GPCRs-related pathways are the targets of a large number of marketed drugs. Therefore, the design of a reliable computational model for predicting GPCRs from amino acid sequence has long been a significant biomedical problem. Chaos game representation (CGR) reveals the fractal patterns hidden in protein sequences, and then fractal dimension (FD) is an important feature of these highly irregular geometries with concise mathematical expression. Here, in order to extract important features from GPCR protein sequences, CGR algorithm, fractal dimension and amino acid composition (AAC) are employed to formulate the numerical features of protein samples. Four groups of features are considered, and each group is evaluated by support vector machine (SVM) and 10-fold cross-validation test. To test the performance of the present method, a new non-redundant dataset was built based on latest GPCRDB database. Comparing the results of numerical experiments, the group of combined features with AAC and FD gets the best result, the accuracy is 99.22% and Matthew's correlation coefficient (MCC) is 0.9845 for identifying GPCRs from non-GPCRs. Moreover, if it is classified as a GPCR, it will be further put into the second level, which will classify a GPCR into one of the five main subfamilies. At this level, the group of combined features with AAC and FD also gets best accuracy 85.73%. Finally, the proposed predictor is also compared with existing methods and shows better performances.

  16. Peroxisome proliferator-activated receptor gamma signaling in human sperm physiology.

    Science.gov (United States)

    Liu, Li-Li; Xian, Hua; Cao, Jing-Chen; Zhang, Chong; Zhang, Yong-Hui; Chen, Miao-Miao; Qian, Yi; Jiang, Ming

    2015-01-01

    Peroxisome proliferator-activated receptor gamma (PPARγ) is a member of the PPARs, which are transcription factors of the steroid receptor superfamily. PPARγ acts as an important molecule for regulating energy homeostasis, modulates the hypothalamic-pituitary-gonadal (HPG) axis, and is reciprocally regulated by HPG. In the human, PPARγ protein is highly expressed in ejaculated spermatozoa, implying a possible role of PPARγ signaling in regulating sperm energy dissipation. PPARγ protein is also expressed in Sertoli cells and germ cells (spermatocytes). Its activation can be induced during capacitation and the acrosome reaction. This mini-review will focus on how PPARγ signaling may affect fertility and sperm quality and the potential reversibility of these adverse effects.

  17. Evolution of endothelin receptors in vertebrates.

    Science.gov (United States)

    Braasch, Ingo; Schartl, Manfred

    2014-12-01

    Endothelin receptors are G protein coupled receptors (GPCRs) of the β-group of rhodopsin receptors that bind to endothelin ligands, which are 21 amino acid long peptides derived from longer prepro-endothelin precursors. The most basal Ednr-like GPCR is found outside vertebrates in the cephalochordate amphioxus, but endothelin ligands are only present among vertebrates, including the lineages of jawless vertebrates (lampreys and hagfishes), cartilaginous vertebrates (sharks, rays, and chimaeras), and bony vertebrates (ray-finned fishes and lobe-finned vertebrates including tetrapods). A bona fide endothelin system is thus a vertebrate-specific innovation with important roles for regulating the cardiovascular system, renal and pulmonary processes, as well as for the development of the vertebrate-specific neural crest cell population and its derivatives. Expectedly, dysregulation of endothelin receptors and the endothelin system leads to a multitude of human diseases. Despite the importance of different types of endothelin receptors for vertebrate development and physiology, current knowledge on endothelin ligand-receptor interactions, on the expression of endothelin receptors and their ligands, and on the functional roles of the endothelin system for embryonic development and in adult vertebrates is very much biased towards amniote vertebrates. Recent analyses from a variety of vertebrate lineages, however, have shown that the endothelin system in lineages such as teleost fish and lampreys is more diverse and is divergent from the mammalian endothelin system. This diversity is mainly based on differential evolution of numerous endothelin system components among vertebrate lineages generated by two rounds of whole genome duplication (three in teleosts) during vertebrate evolution. Here we review current understanding of the evolutionary history of the endothelin receptor family in vertebrates supplemented with surveys on the endothelin receptor gene complement of

  18. Origination, expansion, evolutionary trajectory, and expression bias of AP2/ERF superfamily in Brassica napus

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    Xiaoming Song

    2016-08-01

    Full Text Available The AP2/ERF superfamily, one of the most important transcription factor families, plays crucial roles in response to biotic and abiotic stresses. So far, a comprehensive evolutionary inference of its origination and expansion has not been available. Here, we identified 515 AP2/ERF genes in B. napus, a neo-tetraploid forming ~7500 years ago, and found that 82.14% of them were duplicated in the tetraploidization. A prominent subgenome bias was revealed in gene expression, tissue-specific, and gene conversion. Moreover, a large-scale analysis across plants and alga suggested that this superfamily could have been originated from AP2 family, expanding to form other families (ERF, and RAV. This process was accompanied by duplicating and/or alternative deleting AP2 domain, intragenic domain sequence conversion, and/or by acquiring other domains, resulting in copy number variations, alternatively contributing to functional innovation. We found that significant positive selection occurred at certain critical nodes during the evolution of land plants, possibly responding to changing environment. In conclusion, the present research revealed origination, functional innovation, and evolutionary trajectory of the AP2/ERF superfamily, contributing to understanding their roles in plant stress tolerance.

  19. In-silico gene co-expression network analysis in Paracoccidioides brasiliensis with reference to haloacid dehalogenase superfamily hydrolase gene

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    Raghunath Satpathy

    2015-01-01

    Full Text Available Context: Paracoccidioides brasiliensis, a dimorphic fungus is the causative agent of paracoccidioidomycosis, a disease globally affecting millions of people. The haloacid dehalogenase (HAD superfamily hydrolases enzyme in the fungi, in particular, is known to be responsible in the pathogenesis by adhering to the tissue. Hence, identification of novel drug targets is essential. Aims: In-silico based identification of co-expressed genes along with HAD superfamily hydrolase in P. brasiliensis during the morphogenesis from mycelium to yeast to identify possible genes as drug targets. Materials and Methods: In total, four datasets were retrieved from the NCBI-gene expression omnibus (GEO database, each containing 4340 genes, followed by gene filtration expression of the data set. Further co-expression (CE study was performed individually and then a combination these genes were visualized in the Cytoscape 2. 8.3. Statistical Analysis Used: Mean and standard deviation value of the HAD superfamily hydrolase gene was obtained from the expression data and this value was subsequently used for the CE calculation purpose by selecting specific correlation power and filtering threshold. Results: The 23 genes that were thus obtained are common with respect to the HAD superfamily hydrolase gene. A significant network was selected from the Cytoscape network visualization that contains total 7 genes out of which 5 genes, which do not have significant protein hits, obtained from gene annotation of the expressed sequence tags by BLAST X. For all the protein PSI-BLAST was performed against human genome to find the homology. Conclusions: The gene co-expression network was obtained with respect to HAD superfamily dehalogenase gene in P. Brasiliensis.

  20. The structure of the human interferon alpha/beta receptor gene.

    Science.gov (United States)

    Lutfalla, G; Gardiner, K; Proudhon, D; Vielh, E; Uzé, G

    1992-02-05

    Using the cDNA coding for the human interferon alpha/beta receptor (IFNAR), the IFNAR gene has been physically mapped relative to the other loci of the chromosome 21q22.1 region. 32,906 base pairs covering the IFNAR gene have been cloned and sequenced. Primer extension and solution hybridization-ribonuclease protection have been used to determine that the transcription of the gene is initiated in a broad region of 20 base pairs. Some aspects of the polymorphism of the gene, including noncoding sequences, have been analyzed; some are allelic differences in the coding sequence that induce amino acid variations in the resulting protein. The exon structure of the IFNAR gene and of that of the available genes for the receptors of the cytokine/growth hormone/prolactin/interferon receptor family have been compared with the predictions for the secondary structure of those receptors. From this analysis, we postulate a common origin and propose an hypothesis for the divergence from the immunoglobulin superfamily.

  1. Comparative analysis of cation/proton antiporter superfamily in plants.

    Science.gov (United States)

    Ye, Chu-Yu; Yang, Xiaohan; Xia, Xinli; Yin, Weilun

    2013-06-01

    The cation/proton antiporter superfamily is associated with the transport of monovalent cations across membranes. This superfamily was annotated in the Arabidopsis genome and some members were functionally characterized. In the present study, a systematic analysis of the cation/proton antiporter genes in diverse plant species was reported. We identified 240 cation/proton antiporters in alga, moss, and angiosperm. A phylogenetic tree was constructed showing these 240 members are separated into three families, i.e., Na(+)/H(+) exchangers, K(+) efflux antiporters, and cation/H(+) exchangers. Our analysis revealed that tandem and/or segmental duplications contribute to the expansion of cation/H(+) exchangers in the examined angiosperm species. Sliding window analysis of the nonsynonymous/synonymous substitution ratios showed some differences in the evolutionary fate of cation/proton antiporter paralogs. Furthermore, we identified over-represented motifs among these 240 proteins and found most motifs are family specific, demonstrating diverse evolution of the cation/proton antiporters among three families. In addition, we investigated the co-expressed genes of the cation/proton antiporters in Arabidopsis thaliana. The results showed some biological processes are enriched in the co-expressed genes, suggesting the cation/proton antiporters may be involved in these biological processes. Taken together, this study furthers our knowledge on cation/proton antiporters in plants. Copyright © 2013 Elsevier B.V. All rights reserved.

  2. Functional characterization of viral tumor necrosis factor receptors encoded by cyprinid herpesvirus 3 (CyHV3) genome.

    Science.gov (United States)

    Yi, Yang; Qi, Hemei; Yuan, Jimin; Wang, Rui; Weng, Shaoping; He, Jianguo; Dong, Chuanfu

    2015-08-01

    Cyprinid herpesvirus 3 (CyHV3) is a large double-stranded DNA virus of Alloherpesviridae family in the order Herpesvirales. It causes significant morbidity and mortality in common carp and its ornamental koi variety, and threatens the aquaculture industries worldwide. Mimicry of cytokines and cytokine receptors is a particular strategy for large DNA viruses in modulating the host immune response. Here, we report the identification and characterization of two novel viral homologues of tumor necrosis factor receptor (TNFR) encoded by CyHV3-ORF4 and -ORF12, respectively. CyHV3-ORF4 was identified as a homologue of HVEM and CyHV3-ORF12 as a homologue of TNFRSF1. Overexpression of ORF4 and ORF12 in zebrafish embryos results in embryonic lethality, morphological defects and increased apoptosis. Although we failed to identify any interaction between the two vTNFRs and their potential ligands in zebrafish TNF superfamily by yeast two-hybrid system, the expression of some genes in TNF superfamily or TNFR superfamily were mis-regulated in ORF4 or ORF12-overexpressing embryos, especially the death receptor zHDR and its cognate ligand DL1b. Further studies showed that the apoptosis induced by the both CyHV3 vTNFRs is mainly activated through the intrinsic apoptotic pathway and requires the crosstalk between the intrinsic and extrinsic apoptotic pathway. Additionally, using RT-qPCR and Western blot assays, the expression patterns of the both vTNFRs were also analyzed during CyHV3 productive infection. Collectively, this is the first functional study of two unique vTNFRs encoded by a herpesvirus infecting non-mammalian vertebrates, which may provide novel insights into viral immune regulation mechanism and the pathogenesis of CyHV3 infection. Copyright © 2015 Elsevier Ltd. All rights reserved.

  3. Comparative analysis of cystatin superfamily in platyhelminths.

    Directory of Open Access Journals (Sweden)

    Aijiang Guo

    Full Text Available The cystatin superfamily is comprised of cysteine proteinase inhibitors and encompasses at least 3 subfamilies: stefins, cystatins and kininogens. In this study, the platyhelminth cystatin superfamily was identified and grouped into stefin and cystatin subfamilies. The conserved domain of stefins (G, QxVxG was observed in all members of platyhelminth stefins. The three characteristics of cystatins, the cystatin-like domain (G, QxVxG, PW, a signal peptide, and one or two conserved disulfide bonds, were observed in platyhelminths, with the exception of cestodes, which lacked the conserved disulfide bond. However, it is noteworthy that cestode cystatins had two tandem repeated domains, although the second tandem repeated domain did not contain a cystatin-like domain, which has not been previously reported. Tertiary structure analysis of Taenia solium cystatin, one of the cestode cystatins, demonstrated that the N-terminus of T. solium cystatin formed a five turn α-helix, a five stranded β-pleated sheet and a hydrophobic edge, similar to the structure of chicken cystatin. Although no conserved disulfide bond was found in T. solium cystatin, the models of T. solium cystatin and chicken cystatin corresponded at the site of the first disulfide bridge of the chicken cystatin. However, the two models were not similar regarding the location of the second disulfide bridge of chicken cystatin. These results showed that T. solium cystatin and chicken cystatin had similarities and differences, suggesting that the biochemistry of T. solium cystatin could be similar to chicken cystatin in its inhibitory function and that it may have further functional roles. The same results were obtained for other cestode cystatins. Phylogenetic analysis showed that cestode cystatins constituted an independent clade and implied that cestode cystatins should be considered to have formed a new clade during evolution.

  4. The ATPase of the phi29 DNA packaging motor is a member of the hexameric AAA+ superfamily.

    Science.gov (United States)

    Schwartz, Chad; De Donatis, Gian Marco; Fang, Huaming; Guo, Peixuan

    2013-08-15

    The AAA+ superfamily of proteins is a class of motor ATPases performing a wide range of functions that typically exist as hexamers. The ATPase of phi29 DNA packaging motor has long been a subject of debate in terms of stoichiometry and mechanism of action. Here, we confirmed the stoichiometry of phi29 motor ATPase to be a hexamer and provide data suggesting that the phi29 motor ATPase is a member of the classical hexameric AAA+ superfamily. Native PAGE, EMSA, capillary electrophoresis, ATP titration, and binomial distribution assay show that the ATPase is a hexamer. Mutations in the known Walker motifs of the ATPase validated our previous assumptions that the protein exists as another member of this AAA+ superfamily. Our data also supports the finding that the phi29 DNA packaging motor uses a revolution mechanism without rotation or coiling (Schwartz et al., this issue). Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.

  5. Farnesoid X receptor: a master regulator of hepatic triglyceride and glucose homeostasis

    Science.gov (United States)

    Jiao, Yang; Lu, Yan; Li, Xiao-ying

    2015-01-01

    Non-alcoholic fatty liver disease (NAFLD) is characterized by the aberrant accumulation of triglycerides in hepatocytes in the absence of significant alcohol consumption, viral infection or other specific causes of liver disease. NAFLD has become a burgeoning health problem both worldwide and in China, but its pathogenesis remains poorly understood. Farnesoid X receptor (FXR), a member of the nuclear receptor (NR) superfamily, has been demonstrated to be the primary sensor for endogenous bile acids, and play a crucial role in hepatic triglyceride homeostasis. Deciphering the synergistic contributions of FXR to triglyceride metabolism is critical for discovering therapeutic agents in the treatment of NAFLD and hypertriglyceridemia. PMID:25500875

  6. Proteomics computational analyses suggest that baculovirus GP64 superfamily proteins are class III penetrenes

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    Garry Robert F

    2008-02-01

    Full Text Available Abstract Background Members of the Baculoviridae encode two types of proteins that mediate virus:cell membrane fusion and penetration into the host cell. Alignments of primary amino acid sequences indicate that baculovirus fusion proteins of group I nucleopolyhedroviruses (NPV form the GP64 superfamily. The structure of these viral penetrenes has not been determined. The GP64 superfamily includes the glycoprotein (GP encoded by members of the Thogotovirus genus of the Orthomyxoviridae. The entry proteins of other baculoviruses, group II NPV and granuloviruses, are class I penetrenes. Results Class III penetrenes encoded by members of the Rhabdoviridae and Herpesviridae have an internal fusion domain comprised of beta sheets, other beta sheet domains, an extended alpha helical domain, a membrane proximal stem domain and a carboxyl terminal anchor. Similar sequences and structural/functional motifs that characterize class III penetrenes are located collinearly in GP64 of group I baculoviruses and related glycoproteins encoded by thogotoviruses. Structural models based on a prototypic class III penetrene, vesicular stomatitis virus glycoprotein (VSV G, were established for Thogoto virus (THOV GP and Autographa california multiple NPV (AcMNPV GP64 demonstrating feasible cysteine linkages. Glycosylation sites in THOV GP and AcMNPV GP64 appear in similar model locations to the two glycosylation sites of VSV G. Conclusion These results suggest that proteins in the GP64 superfamily are class III penetrenes.

  7. Role in diuresis of a calcitonin receptor (GPRCAL1 expressed in a distal-proximal gradient in renal organs of the mosquito Aedes aegypti (L..

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    Hyeogsun Kwon

    Full Text Available Evolution of anthropophilic hematophagy in insects resulted in the coordination of various physiological processes for survival. In female mosquitoes, a large blood meal provides proteins for egg production and as a trade-off, rapid elimination of the excess water and solutes (Na(+, Cl(- is critical for maintaining homeostasis and removing excess weight to resume flight and avoid predation. This post-prandial excretion is achieved by the concerted action of multiple hormones. Diuresis and natriuresis elicited by the calcitonin-like diuretic hormone 31 (DH(31 are believed to be mediated by a yet uncharacterized calcitonin receptor (GPRCAL in the mosquito Malpighian tubules (MTs, the renal organs. To contribute knowledge on endocrinology of mosquito diuresis we cloned GPRCAL1 from MT cDNA. This receptor is the ortholog of the DH(31 receptor from Drosophila melanogaster that is expressed in principal cells of the fruit fly MT. Immunofluorescence similarly showed AaegGPRCAL1 is present in MT principal cells in A. aegypti, however, exhibiting an overall gradient-like pattern along the tubule novel for a GPCR in insects. Variegated, cell-specific receptor expression revealed a subpopulation of otherwise phenotypically similar principal cells. To investigate the receptor contribution to fluid elimination, RNAi was followed by urine measurement assays. In vitro, MTs from females that underwent AaegGPRcal1 knock-down exhibited up to 57% decrease in the rate of fluid secretion in response to DH(31. Live females treated with AaegGPRcal1 dsRNA exhibited 30% reduction in fluid excreted after a blood meal. The RNAi-induced phenotype demonstrates the critical contribution of this single secretin-like family B GPCR to fluid excretion in invertebrates and highlights its relevance for the blood feeding adaptation. Our results with the mosquito AaegGPRCAL1 imply that the regulatory function of calcitonin-like receptors for ion and fluid transport in renal organs

  8. Membrane and Protein Interactions of the Pleckstrin Homology Domain Superfamily

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    Marc Lenoir

    2015-10-01

    Full Text Available The human genome encodes about 285 proteins that contain at least one annotated pleckstrin homology (PH domain. As the first phosphoinositide binding module domain to be discovered, the PH domain recruits diverse protein architectures to cellular membranes. PH domains constitute one of the largest protein superfamilies, and have diverged to regulate many different signaling proteins and modules such as Dbl homology (DH and Tec homology (TH domains. The ligands of approximately 70 PH domains have been validated by binding assays and complexed structures, allowing meaningful extrapolation across the entire superfamily. Here the Membrane Optimal Docking Area (MODA program is used at a genome-wide level to identify all membrane docking PH structures and map their lipid-binding determinants. In addition to the linear sequence motifs which are employed for phosphoinositide recognition, the three dimensional structural features that allow peripheral membrane domains to approach and insert into the bilayer are pinpointed and can be predicted ab initio. The analysis shows that conserved structural surfaces distinguish which PH domains associate with membrane from those that do not. Moreover, the results indicate that lipid-binding PH domains can be classified into different functional subgroups based on the type of membrane insertion elements they project towards the bilayer.

  9. Membrane and Protein Interactions of the Pleckstrin Homology Domain Superfamily.

    Science.gov (United States)

    Lenoir, Marc; Kufareva, Irina; Abagyan, Ruben; Overduin, Michael

    2015-10-23

    The human genome encodes about 285 proteins that contain at least one annotated pleckstrin homology (PH) domain. As the first phosphoinositide binding module domain to be discovered, the PH domain recruits diverse protein architectures to cellular membranes. PH domains constitute one of the largest protein superfamilies, and have diverged to regulate many different signaling proteins and modules such as Dbl homology (DH) and Tec homology (TH) domains. The ligands of approximately 70 PH domains have been validated by binding assays and complexed structures, allowing meaningful extrapolation across the entire superfamily. Here the Membrane Optimal Docking Area (MODA) program is used at a genome-wide level to identify all membrane docking PH structures and map their lipid-binding determinants. In addition to the linear sequence motifs which are employed for phosphoinositide recognition, the three dimensional structural features that allow peripheral membrane domains to approach and insert into the bilayer are pinpointed and can be predicted ab initio. The analysis shows that conserved structural surfaces distinguish which PH domains associate with membrane from those that do not. Moreover, the results indicate that lipid-binding PH domains can be classified into different functional subgroups based on the type of membrane insertion elements they project towards the bilayer.

  10. The 2.1 Å resolution structure of cyanopindolol-bound β1-adrenoceptor identifies an intramembrane Na+ ion that stabilises the ligand-free receptor.

    Directory of Open Access Journals (Sweden)

    Jennifer L Miller-Gallacher

    Full Text Available The β1-adrenoceptor (β1AR is a G protein-coupled receptor (GPCR that is activated by the endogenous agonists adrenaline and noradrenaline. We have determined the structure of an ultra-thermostable β1AR mutant bound to the weak partial agonist cyanopindolol to 2.1 Å resolution. High-quality crystals (100 μm plates were grown in lipidic cubic phase without the assistance of a T4 lysozyme or BRIL fusion in cytoplasmic loop 3, which is commonly employed for GPCR crystallisation. An intramembrane Na+ ion was identified co-ordinated to Asp872.50, Ser1283.39 and 3 water molecules, which is part of a more extensive network of water molecules in a cavity formed between transmembrane helices 1, 2, 3, 6 and 7. Remarkably, this water network and Na+ ion is highly conserved between β1AR and the adenosine A2A receptor (rmsd of 0.3 Å, despite an overall rmsd of 2.4 Å for all Cα atoms and only 23% amino acid identity in the transmembrane regions. The affinity of agonist binding and nanobody Nb80 binding to β1AR is unaffected by Na+ ions, but the stability of the receptor is decreased by 7.5°C in the absence of Na+. Mutation of amino acid side chains that are involved in the co-ordination of either Na+ or water molecules in the network decreases the stability of β1AR by 5-10°C. The data suggest that the intramembrane Na+ and associated water network stabilise the ligand-free state of β1AR, but still permits the receptor to form the activated state which involves the collapse of the Na+ binding pocket on agonist binding.

  11. Identification and Transcriptional Modulation of the Largemouth Bass, Micropterus salmoides, Vitellogenin Receptor During Oocyte Development by Insulin and Sex Steroids1

    OpenAIRE

    Dominguez, Gustavo A.; Quattro, Joseph M.; Denslow, Nancy D.; Kroll, Kevin J.; Prucha, Melinda S.; Porak, Wesley F.; Grier, Harry J.; Sabo-Attwood, Tara L.

    2012-01-01

    Fish vitellogenin synthesized and released from the liver of oviparous animals is taken up into oocytes by the vitellogenin receptor. This is an essential process in providing nutrient yolk to developing embryos to ensure successful reproduction. Here we disclose the full length vtgr cDNA sequence for largemouth bass (LMB) that reveals greater than 90% sequence homology with other fish vtgr sequences. We classify LMB Vtgr as a member of the low density lipoprotein receptor superfamily based o...

  12. The receptor for advanced glycation end products (RAGE) and the lung.

    LENUS (Irish Health Repository)

    Buckley, Stephen T

    2010-01-01

    The receptor for advanced glycation end products (RAGE) is a member of the immunoglobulin superfamily of cell surface molecules. As a pattern-recognition receptor capable of binding a diverse range of ligands, it is typically expressed at low levels under normal physiological conditions in the majority of tissues. In contrast, the lung exhibits high basal level expression of RAGE localised primarily in alveolar type I (ATI) cells, suggesting a potentially important role for the receptor in maintaining lung homeostasis. Indeed, disruption of RAGE levels has been implicated in the pathogenesis of a variety of pulmonary disorders including cancer and fibrosis. Furthermore, its soluble isoforms, sRAGE, which act as decoy receptors, have been shown to be a useful marker of ATI cell injury. Whilst RAGE undoubtedly plays an important role in the biology of the lung, it remains unclear as to the exact nature of this contribution under both physiological and pathological conditions.

  13. Neuropeptide Y family receptors traffic via the Bardet-Biedl syndrome pathway to signal in neuronal primary cilia.

    Science.gov (United States)

    Loktev, Alexander V; Jackson, Peter K

    2013-12-12

    Human monogenic obesity syndromes, including Bardet-Biedl syndrome (BBS), implicate neuronal primary cilia in regulation of energy homeostasis. Cilia in hypothalamic neurons have been hypothesized to sense and regulate systemic energy status, but the molecular mechanism of this signaling remains unknown. Here, we report a comprehensive localization screen of 42 G-protein-coupled receptors (GPCR) revealing seven ciliary GPCRs, including the neuropeptide Y (NPY) receptors NPY2R and NPY5R. We show that mice modeling BBS disease or obese tubby mice fail to localize NPY2R to cilia in the hypothalamus and that BBS mutant mice fail to activate c-fos or decrease food intake in response to the NPY2R ligand PYY3-36. We find that cells with ciliary NPY2R show augmented PYY3-36-dependent cAMP signaling. Our data demonstrate that ciliary targeting of NPY receptors is important for controlling energy balance in mammals, revealing a physiologically defined ligand-receptor pathway signaling within neuronal cilia. Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.

  14. The Association between Gene-Environment Interactions and Diseases Involving the Human GST Superfamily with SNP Variants

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    Antoinesha L. Hollman

    2016-03-01

    Full Text Available Exposure to environmental hazards has been associated with diseases in humans. The identification of single nucleotide polymorphisms (SNPs in human populations exposed to different environmental hazards, is vital for detecting the genetic risks of some important human diseases. Several studies in this field have been conducted on glutathione S-transferases (GSTs, a phase II detoxification superfamily, to investigate its role in the occurrence of diseases. Human GSTs consist of cytosolic and microsomal superfamilies that are further divided into subfamilies. Based on scientific search engines and a review of the literature, we have found a large amount of published articles on human GST super- and subfamilies that have greatly assisted in our efforts to examine their role in health and disease. Because of its polymorphic variations in relation to environmental hazards such as air pollutants, cigarette smoke, pesticides, heavy metals, carcinogens, pharmaceutical drugs, and xenobiotics, GST is considered as a significant biomarker. This review examines the studies on gene-environment interactions related to various diseases with respect to single nucleotide polymorphisms (SNPs found in the GST superfamily. Overall, it can be concluded that interactions between GST genes and environmental factors play an important role in human diseases.

  15. G Protein-Coupled Receptor Signaling in Stem Cells and Cancer.

    Science.gov (United States)

    Lynch, Jennifer R; Wang, Jenny Yingzi

    2016-05-11

    G protein-coupled receptors (GPCRs) are a large superfamily of cell-surface signaling proteins that bind extracellular ligands and transduce signals into cells via heterotrimeric G proteins. GPCRs are highly tractable drug targets. Aberrant expression of GPCRs and G proteins has been observed in various cancers and their importance in cancer stem cells has begun to be appreciated. We have recently reported essential roles for G protein-coupled receptor 84 (GPR84) and G protein subunit Gαq in the maintenance of cancer stem cells in acute myeloid leukemia. This review will discuss how GPCRs and G proteins regulate stem cells with a focus on cancer stem cells, as well as their implications for the development of novel targeted cancer therapies.

  16. G Protein-Coupled Receptor Signaling in Stem Cells and Cancer

    Directory of Open Access Journals (Sweden)

    Jennifer R. Lynch

    2016-05-01

    Full Text Available G protein-coupled receptors (GPCRs are a large superfamily of cell-surface signaling proteins that bind extracellular ligands and transduce signals into cells via heterotrimeric G proteins. GPCRs are highly tractable drug targets. Aberrant expression of GPCRs and G proteins has been observed in various cancers and their importance in cancer stem cells has begun to be appreciated. We have recently reported essential roles for G protein-coupled receptor 84 (GPR84 and G protein subunit Gαq in the maintenance of cancer stem cells in acute myeloid leukemia. This review will discuss how GPCRs and G proteins regulate stem cells with a focus on cancer stem cells, as well as their implications for the development of novel targeted cancer therapies.

  17. Comparative genomic study of ALDH gene superfamily in Gossypium: A focus on Gossypium hirsutum under salt stress.

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    Yating Dong

    Full Text Available Aldehyde dehydrogenases (ALDHs are a superfamily of enzymes which play important role in the scavenging of active aldehydes molecules. In present work, a comprehensive whole-genomic study of ALDH gene superfamily was carried out for an allotetraploid cultivated cotton species, G. hirsutum, as well as in parallel relative to their diploid progenitors, G. arboreum and G. raimondii. Totally, 30 and 58 ALDH gene sequences belong to 10 families were identified from diploid and allotetraploid cotton species, respectively. The gene structures among the members from same families were highly conserved. Whole-genome duplication and segmental duplication might be the major driver for the expansion of ALDH gene superfamily in G. hirsutum. In addition, the expression patterns of GhALDH genes were diverse across tissues. Most GhALDH genes were induced or repressed by salt stress in upland cotton. Our observation shed lights on the molecular evolutionary properties of ALDH genes in diploid cottons and their alloallotetraploid derivatives. It may be useful to mine key genes for improvement of cotton response to salt stress.

  18. Structure and Function of the Hypertension Variant A486V of G Protein-coupled Receptor Kinase 4

    Energy Technology Data Exchange (ETDEWEB)

    Allen, Samantha J.; Parthasarathy, Gopal; Darke, Paul L.; Diehl, Ronald E.; Ford, Rachael E.; Hall, Dawn L.; Johnson, Scott A.; Reid, John C.; Rickert, Keith W.; Shipman, Jennifer M.; Soisson, Stephen M.; Zuck, Paul; Munshi, Sanjeev K.; Lumb, Kevin J. (Merck)

    2015-07-01

    G-protein-coupled receptor (GPCR) kinases (GRKs) bind to and phosphorylate GPCRs, initiating the process of GPCR desensitization and internalization. GRK4 is implicated in the regulation of blood pressure, and three GRK4 polymorphisms (R65L, A142V, and A486V) are associated with hypertension. Here, we describe the 2.6 Å structure of human GRK4α A486V crystallized in the presence of 5'-adenylyl β,γ-imidodiphosphate. The structure of GRK4α is similar to other GRKs, although slight differences exist within the RGS homology (RH) bundle subdomain, substrate-binding site, and kinase C-tail. The RH bundle subdomain and kinase C-terminal lobe form a strikingly acidic surface, whereas the kinase N-terminal lobe and RH terminal subdomain surfaces are much more basic. In this respect, GRK4α is more similar to GRK2 than GRK6. A fully ordered kinase C-tail reveals interactions linking the C-tail with important determinants of kinase activity, including the αB helix, αD helix, and the P-loop. Autophosphorylation of wild-type GRK4α is required for full kinase activity, as indicated by a lag in phosphorylation of a peptide from the dopamine D1 receptor without ATP preincubation. In contrast, this lag is not observed in GRK4α A486V. Phosphopeptide mapping by mass spectrometry indicates an increased rate of autophosphorylation of a number of residues in GRK4α A486V relative to wild-type GRK4α, including Ser-485 in the kinase C-tail.

  19. Site-specific O-glycosylation of members of the low-density lipoprotein receptor superfamily enhances ligand interactions.

    Science.gov (United States)

    Wang, Shengjun; Mao, Yang; Narimatsu, Yoshiki; Ye, Zilu; Tian, Weihua; Goth, Christoffer K; Lira-Navarrete, Erandi; Pedersen, Nis B; Benito-Vicente, Asier; Martin, Cesar; Uribe, Kepa B; Hurtado-Guerrero, Ramon; Christoffersen, Christina; Seidah, Nabil G; Nielsen, Rikke; Christensen, Erik I; Hansen, Lars; Bennett, Eric P; Vakhrushev, Sergey Y; Schjoldager, Katrine T; Clausen, Henrik

    2018-05-11

    The low-density lipoprotein receptor (LDLR) and related receptors are important for the transport of diverse biomolecules across cell membranes and barriers. Their functions are especially relevant for cholesterol homeostasis and diseases, including neurodegenerative and kidney disorders. Members of the LDLR-related protein family share LDLR class A (LA) repeats providing binding properties for lipoproteins and other biomolecules. We previously demonstrated that short linker regions between these LA repeats contain conserved O -glycan sites. Moreover, we found that O -glycan modifications at these sites are selectively controlled by the GalNAc-transferase isoform, GalNAc-T11. However, the effects of GalNAc-T11-mediated O -glycosylation on LDLR and related receptor localization and function are unknown. Here, we characterized O -glycosylation of LDLR-related proteins and identified conserved O -glycosylation sites in the LA linker regions of VLDLR, LRP1, and LRP2 (Megalin) from both cell lines and rat organs. Using a panel of gene-edited isogenic cell line models, we demonstrate that GalNAc-T11-mediated LDLR and VLDLR O -glycosylation is not required for transport and cell-surface expression and stability of these receptors but markedly enhances LDL and VLDL binding and uptake. Direct ELISA-based binding assays with truncated LDLR constructs revealed that O -glycosylation increased affinity for LDL by ∼5-fold. The molecular basis for this observation is currently unknown, but these findings open up new avenues for exploring the roles of LDLR-related proteins in disease. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

  20. Ensembler: Enabling High-Throughput Molecular Simulations at the Superfamily Scale.

    Directory of Open Access Journals (Sweden)

    Daniel L Parton

    2016-06-01

    Full Text Available The rapidly expanding body of available genomic and protein structural data provides a rich resource for understanding protein dynamics with biomolecular simulation. While computational infrastructure has grown rapidly, simulations on an omics scale are not yet widespread, primarily because software infrastructure to enable simulations at this scale has not kept pace. It should now be possible to study protein dynamics across entire (superfamilies, exploiting both available structural biology data and conformational similarities across homologous proteins. Here, we present a new tool for enabling high-throughput simulation in the genomics era. Ensembler takes any set of sequences-from a single sequence to an entire superfamily-and shepherds them through various stages of modeling and refinement to produce simulation-ready structures. This includes comparative modeling to all relevant PDB structures (which may span multiple conformational states of interest, reconstruction of missing loops, addition of missing atoms, culling of nearly identical structures, assignment of appropriate protonation states, solvation in explicit solvent, and refinement and filtering with molecular simulation to ensure stable simulation. The output of this pipeline is an ensemble of structures ready for subsequent molecular simulations using computer clusters, supercomputers, or distributed computing projects like Folding@home. Ensembler thus automates much of the time-consuming process of preparing protein models suitable for simulation, while allowing scalability up to entire superfamilies. A particular advantage of this approach can be found in the construction of kinetic models of conformational dynamics-such as Markov state models (MSMs-which benefit from a diverse array of initial configurations that span the accessible conformational states to aid sampling. We demonstrate the power of this approach by constructing models for all catalytic domains in the human

  1. TGF-β superfamily signaling in testis formation and early male germline development.

    Science.gov (United States)

    Young, Julia C; Wakitani, Shoichi; Loveland, Kate L

    2015-09-01

    The TGF-β ligand superfamily contains at least 40 members, many of which are produced and act within the mammalian testis to facilitate formation of sperm. Their progressive expression at key stages and in specific cell types determines the fertility of adult males, influencing testis development and controlling germline differentiation. BMPs are essential for the interactive instructions between multiple cell types in the early embryo that drive initial specification of gamete precursors. In the nascent foetal testis, several ligands including Nodal, TGF-βs, Activins and BMPs, serve as key masculinizing switches by regulating male germline pluripotency, somatic and germline proliferation, and testicular vascularization and architecture. In postnatal life, local production of these factors determine adult testis size by regulating Sertoli cell multiplication and differentiation, in addition to specifying germline differentiation and multiplication. Because TGF-β superfamily signaling is integral to testis formation, it affects processes that underlie testicular pathologies, including testicular cancer, and its potential to contribute to subfertility is beginning to be understood. Copyright © 2015 Elsevier Ltd. All rights reserved.

  2. Ischemic tolerance modulates TRAIL expression and its receptors and generates a neuroprotected phenotype

    OpenAIRE

    Cantarella, G; Pignataro, G; Di Benedetto, G; Anzilotti, S; Vinciguerra, A; Cuomo, O; Di Renzo, G F; Parenti, C; Annunziato, L; Bernardini, R

    2014-01-01

    TNF-related apoptosis inducing ligand (TRAIL), a member of the TNF superfamily released by microglia, appears to be involved in the induction of apoptosis following focal brain ischemia. Indeed, brain ischemia is associated with progressive enlargement of damaged areas and prominent inflammation. As ischemic preconditioning reduces inflammatory response to brain ischemia and ameliorates brain damage, the purpose of the present study was to evaluate the role of TRAIL and its receptors in strok...

  3. The metabotropic glutamate receptors: structure, activation mechanism and pharmacology.

    Science.gov (United States)

    Pin, Jean-Philippe; Acher, Francine

    2002-06-01

    The metabotropic glutamate receptors are G-protein coupled receptors (GPCR) involved in the regulation of many synapses, including most glutamatergic fast excitatory synapses. Eight subtypes have been identified that can be classified into three groups. The molecular characterization of these receptors revealed proteins much more complex than any other GPCRs. They are composed of a Venus Flytrap (VFT) module where glutamate binds, connected to a heptahelical domain responsible for G-protein coupling. Recent data including the structure of the VFT module determined with and without glutamate, indicate that these receptors function as dimers. Moreover a number of intracellular proteins can regulate their targeting and transduction mechanism. Such structural features of mGlu receptors offer multiple possibilities for synthetic compounds to modulate their activity. In addition to agonists and competitive antagonists acting at the glutamate binding site, a number of non-competitive antagonists with inverse agonist activity, and positive allosteric modulators have been discovered. These later compounds share specific properties that make them good candidates for therapeutic applications. First, their non-amino acid structure makes them pass more easily the blood brain barrier. Second, they are much more selective than any other compound identified so far, being the first subtype selective molecules. Third, for the negative modulators, their non competitive mechanism of action makes them relatively unaffected by high concentrations of glutamate that may be present in disease states (e.g. stroke, epilepsy, neuropathic pain, etc.). Fourth, like the benzodiazepines acting at the GABA(A) receptors, the positive modulators offer a new way to increase the activity of these receptors in vivo, with a low risk of inducing their desensitization. The present review article focuses on the specific structural features of these receptors and highlights the various possibilities these

  4. Representation Learning for Class C G Protein-Coupled Receptors Classification

    Directory of Open Access Journals (Sweden)

    Raúl Cruz-Barbosa

    2018-03-01

    Full Text Available G protein-coupled receptors (GPCRs are integral cell membrane proteins of relevance for pharmacology. The complete tertiary structure including both extracellular and transmembrane domains has not been determined for any member of class C GPCRs. An alternative way to work on GPCR structural models is the investigation of their functionality through the analysis of their primary structure. For this, sequence representation is a key factor for the GPCRs’ classification context, where usually, feature engineering is carried out. In this paper, we propose the use of representation learning to acquire the features that best represent the class C GPCR sequences and at the same time to obtain a model for classification automatically. Deep learning methods in conjunction with amino acid physicochemical property indices are then used for this purpose. Experimental results assessed by the classification accuracy, Matthews’ correlation coefficient and the balanced error rate show that using a hydrophobicity index and a restricted Boltzmann machine (RBM can achieve performance results (accuracy of 92.9% similar to those reported in the literature. As a second proposal, we combine two or more physicochemical property indices instead of only one as the input for a deep architecture in order to add information from the sequences. Experimental results show that using three hydrophobicity-related index combinations helps to improve the classification performance (accuracy of 94.1% of an RBM better than those reported in the literature for class C GPCRs without using feature selection methods.

  5. Investigating Internalization and Intracellular Trafficking of GPCRs

    DEFF Research Database (Denmark)

    Foster, Simon R; Bräuner-Osborne, Hans

    2017-01-01

    for signal transduction. One of the major mechanisms for GPCR regulation involves their endocytic trafficking, which serves to internalize the receptors from the plasma membrane and thereby attenuate G protein-dependent signaling. However, there is accumulating evidence to suggest that GPCRs can signal...... independently of G proteins, as well as from intracellular compartments including endosomes. It is in this context that receptor internalization and intracellular trafficking have attracted renewed interest within the GPCR field. In this chapter, we will review the current understanding and methodologies...

  6. Steroid receptor coactivator 1 deficiency increases MMTV-neu mediated tumor latency and differentiation specific gene expression, decreases metastasis, and inhibits response to PPAR ligands

    International Nuclear Information System (INIS)

    Han, Ji Seung; Crowe, David L

    2010-01-01

    The peroxisome proliferator activated receptor (PPAR) subgroup of the nuclear hormone receptor superfamily is activated by a variety of natural and synthetic ligands. PPARs can heterodimerize with retinoid X receptors, which have homology to other members of the nuclear receptor superfamily. Ligand binding to PPAR/RXRs results in recruitment of transcriptional coactivator proteins such as steroid receptor coactivator 1 (SRC-1) and CREB binding protein (CBP). Both SRC-1 and CBP are histone acetyltransferases, which by modifying nucleosomal histones, produce more open chromatin structure and increase transcriptional activity. Nuclear hormone receptors can recruit limiting amounts of coactivators from other transcription factor binding sites such as AP-1, thereby inhibiting the activity of AP-1 target genes. PPAR and RXR ligands have been used in experimental breast cancer therapy. The role of coactivator expression in mammary tumorigenesis and response to drug therapy has been the subject of recent studies. We examined the effects of loss of SRC-1 on MMTV-neu mediated mammary tumorigenesis. SRC-1 null mutation in mammary tumor prone mice increased the tumor latency period, reduced tumor proliferation index and metastasis, inhibited response to PPAR and RXR ligands, and induced genes involved in mammary gland differentiation. We also examined human breast cancer cell lines overexpressing SRC-1 or CBP. Coactivator overexpression increased cellular proliferation with resistance to PPAR and RXR ligands and remodeled chromatin of the proximal epidermal growth factor receptor promoter. These results indicate that histone acetyltransferases play key roles in mammary tumorigenesis and response to anti-proliferative therapies

  7. System and methods for predicting transmembrane domains in membrane proteins and mining the genome for recognizing G-protein coupled receptors

    Science.gov (United States)

    Trabanino, Rene J; Vaidehi, Nagarajan; Hall, Spencer E; Goddard, William A; Floriano, Wely

    2013-02-05

    The invention provides computer-implemented methods and apparatus implementing a hierarchical protocol using multiscale molecular dynamics and molecular modeling methods to predict the presence of transmembrane regions in proteins, such as G-Protein Coupled Receptors (GPCR), and protein structural models generated according to the protocol. The protocol features a coarse grain sampling method, such as hydrophobicity analysis, to provide a fast and accurate procedure for predicting transmembrane regions. Methods and apparatus of the invention are useful to screen protein or polynucleotide databases for encoded proteins with transmembrane regions, such as GPCRs.

  8. Activation of the kinin B1 receptor attenuates melanoma tumor growth and metastasis.

    Directory of Open Access Journals (Sweden)

    Patricia Dillenburg-Pilla

    Full Text Available Melanoma is a very aggressive tumor that does not respond well to standard therapeutic approaches, such as radio- and chemotherapies. Furthermore, acquiring the ability to metastasize in melanoma and many other tumor types is directly related to incurable disease. The B1 kinin receptor participates in a variety of cancer-related pathophysiological events, such as inflammation and angiogenesis. Therefore, we investigated whether this G protein-coupled receptor plays a role in tumor progression. We used a murine melanoma cell line that expresses the kinin B1 receptor and does not express the kinin B2 receptor to investigate the precise contribution of activation of the B1 receptor in tumor progression and correlated events using various in vitro and in vivo approaches. Activation of the kinin B1 receptor in the absence of B2 receptor inhibits cell migration in vitro and decreases tumor formation in vivo. Moreover, tumors formed from cells stimulated with B1-specific agonist showed several features of decreased aggressiveness, such as smaller size and infiltration of inflammatory cells within the tumor area, higher levels of pro-inflammatory cytokines implicated in the host anti-tumor immune response, lower number of cells undergoing mitosis, a poorer vascular network, no signs of invasion of surrounding tissues or metastasis and increased animal survival. Our findings reveal that activation of the kinin B1 receptor has a host protective role during murine melanoma tumor progression, suggesting that the B1 receptor could be a new anti-tumor GPCR and provide new opportunities for therapeutic targeting.

  9. A highly conserved glycine within linker I and the extreme C terminus of G protein alpha subunits interact cooperatively in switching G protein-coupled receptor-to-effector specificity

    DEFF Research Database (Denmark)

    Kostenis, Evi; Martini, Lene; Ellis, James

    2004-01-01

    Numerous studies have attested to the importance of the extreme C terminus of G protein alpha subunits in determining their selectivity of receptor recognition. We have previously reported that a highly conserved glycine residue within linker I is important for constraining the fidelity of receptor...... recognition by Galpha(q) proteins. Herein, we explored whether both modules (linker I and extreme C terminus) interact cooperatively in switching G protein-coupled receptor (GPCR)-to-effector specificity and created as models mutant Galpha(q) proteins in which glycine was replaced with various amino acids...... and the C-terminal five Galpha(q) residues with the corresponding Galpha(i) or Galpha(s) sequence. Coupling properties of the mutated Galpha(q) proteins were determined after coexpression with a panel of 13 G(i)-and G(s) -selective receptors and compared with those of Galpha proteins modified in only one...

  10. Poxvirus-encoded TNF decoy receptors inhibit the biological activity of transmembrane TNF.

    Science.gov (United States)

    Pontejo, Sergio M; Alejo, Ali; Alcami, Antonio

    2015-10-01

    Poxviruses encode up to four different soluble TNF receptors, named cytokine response modifier B (CrmB), CrmC, CrmD and CrmE. These proteins mimic the extracellular domain of the cellular TNF receptors to bind and inhibit the activity of TNF and, in some cases, other TNF superfamily ligands. Most of these ligands are released after the enzymic cleavage of a membrane precursor. However, transmembrane TNF (tmTNF) is not only a precursor of soluble TNF but also exerts specific pro-inflammatory and immunological activities. Here, we report that viral TNF receptors bound and inhibited tmTNF and describe some interesting differences in their activity against the soluble cytokine. Thus, CrmE, which does not inhibit mouse soluble TNF, could block murine tmTNF-induced cytotoxicity. We propose that this anti-tmTNF effect should be taken into consideration when assessing the role of viral TNF decoy receptors in the pathogenesis of poxvirus.

  11. An atlas of Caenorhabditis elegans chemoreceptor expression.

    Directory of Open Access Journals (Sweden)

    Berta Vidal

    2018-01-01

    Full Text Available One goal of modern day neuroscience is the establishment of molecular maps that assign unique features to individual neuron types. Such maps provide important starting points for neuron classification, for functional analysis, and for developmental studies aimed at defining the molecular mechanisms of neuron identity acquisition and neuron identity diversification. In this resource paper, we describe a nervous system-wide map of the potential expression sites of 244 members of the largest gene family in the C. elegans genome, rhodopsin-like (class A G-protein-coupled receptor (GPCR chemoreceptors, using classic gfp reporter gene technology. We cover representatives of all sequence families of chemoreceptor GPCRs, some of which were previously entirely uncharacterized. Most reporters are expressed in a very restricted number of cells, often just in single cells. We assign GPCR reporter expression to all but two of the 37 sensory neuron classes of the sex-shared, core nervous system. Some sensory neurons express a very small number of receptors, while others, particularly nociceptive neurons, coexpress several dozen GPCR reporter genes. GPCR reporters are also expressed in a wide range of inter- and motorneurons, as well as non-neuronal cells, suggesting that GPCRs may constitute receptors not just for environmental signals, but also for internal cues. We observe only one notable, frequent association of coexpression patterns, namely in one nociceptive amphid (ASH and two nociceptive phasmid sensory neurons (PHA, PHB. We identified GPCRs with sexually dimorphic expression and several GPCR reporters that are expressed in a left/right asymmetric manner. We identified a substantial degree of GPCR expression plasticity; particularly in the context of the environmentally-induced dauer diapause stage when one third of all tested GPCRs alter the cellular specificity of their expression within and outside the nervous system. Intriguingly, in a number of

  12. Xenobiotics and the Glucocorticoid Receptor

    International Nuclear Information System (INIS)

    Gulliver, Linda S M

    2017-01-01

    Glucocorticoid Receptor (GR) is present in virtually every human cell type. Representing a nuclear receptor superfamily, GR has several different isoforms essentially acting as ligand-dependent transcription factors, regulating glucocorticoid-responsive gene expression in both a positive and a negative manner. Although the natural ligand of the Glucocorticoid Receptor, glucocorticoids (GC) represent only some of the multiple ligands for GR. Xenobiotics, ubiquitous in the environment, bind to GR and are also capable of activating or repressing GR gene expression, thereby modulating GR cell and tissue-specific downstream effects in a multitude of ways that include responses to inflammatory, allergic, metabolic, neoplastic and autoimmune processes. Many xenobiotics, if inadequately metabolized by xenobiotic metabolizing enzymes and not wholly eliminated, could have deleterious toxic effects with potentially lethal consequences. This review examines GR, the genomic and non-genomic actions of natural and synthetic GC and the body's handling of xenobiotic compounds, before reviewing what is presently known about GR's interactions with many of the more commonly encountered and some of the less well known GR-associated xenobiotics. GR promiscuity and crosstalk with other signaling pathways is discussed, alongside novel roles for GR that include mood disorder and addiction. A knowledge of GR interactions with xenobiotics is increasingly relevant when considering aging populations and the related prevalence of neoplastic disease, together with growing concerns around human exposure to mixtures of chemicals in the environment. Furthermore, escalating rates of obesity, Type 2 diabetes; autoimmune, allergy, addiction and mood disorder-related pathologies, require novel targeted interventions and GR appears a promising pharmacological candidate. - Highlights: • Biological impact of xenobiotics acting through Glucocorticoid Receptor. • Promiscuity of Glucocorticoid

  13. Xenobiotics and the Glucocorticoid Receptor

    Energy Technology Data Exchange (ETDEWEB)

    Gulliver, Linda S M, E-mail: linda.gulliver@otago.ac.nz

    2017-03-15

    Glucocorticoid Receptor (GR) is present in virtually every human cell type. Representing a nuclear receptor superfamily, GR has several different isoforms essentially acting as ligand-dependent transcription factors, regulating glucocorticoid-responsive gene expression in both a positive and a negative manner. Although the natural ligand of the Glucocorticoid Receptor, glucocorticoids (GC) represent only some of the multiple ligands for GR. Xenobiotics, ubiquitous in the environment, bind to GR and are also capable of activating or repressing GR gene expression, thereby modulating GR cell and tissue-specific downstream effects in a multitude of ways that include responses to inflammatory, allergic, metabolic, neoplastic and autoimmune processes. Many xenobiotics, if inadequately metabolized by xenobiotic metabolizing enzymes and not wholly eliminated, could have deleterious toxic effects with potentially lethal consequences. This review examines GR, the genomic and non-genomic actions of natural and synthetic GC and the body's handling of xenobiotic compounds, before reviewing what is presently known about GR's interactions with many of the more commonly encountered and some of the less well known GR-associated xenobiotics. GR promiscuity and crosstalk with other signaling pathways is discussed, alongside novel roles for GR that include mood disorder and addiction. A knowledge of GR interactions with xenobiotics is increasingly relevant when considering aging populations and the related prevalence of neoplastic disease, together with growing concerns around human exposure to mixtures of chemicals in the environment. Furthermore, escalating rates of obesity, Type 2 diabetes; autoimmune, allergy, addiction and mood disorder-related pathologies, require novel targeted interventions and GR appears a promising pharmacological candidate. - Highlights: • Biological impact of xenobiotics acting through Glucocorticoid Receptor. • Promiscuity of Glucocorticoid

  14. In brown adipocytes, adrenergically induced β1-/β3-(Gs)-, α2-(Gi)- and α1-(Gq)-signalling to Erk1/2 activation is not mediated via EGF receptor transactivation

    International Nuclear Information System (INIS)

    Wang, Yanling; Fälting, Johanna M.; Mattsson, Charlotte L.; Holmström, Therése E.; Nedergaard, Jan

    2013-01-01

    Brown adipose tissue is unusual in that the neurotransmitter norepinephrine influences cell destiny in ways generally associated with effects of classical growth factors: regulation of cell proliferation, of apoptosis, and progression of differentiation. The norepinephrine effects are mediated through G-protein-coupled receptors; further mediation of such stimulation to e.g. Erk1/2 activation is in cell biology in general accepted to occur through transactivation of the EGF receptor (by external or internal pathways). We have examined here the significance of such transactivation in brown adipocytes. Stimulation of mature brown adipocytes with cirazoline (α 1 -adrenoceptor coupled via G q ), clonidine (α 2 via G i ) or CL316243 (β 3 via G s ) or via β 1 -receptors significantly activated Erk1/2. Pretreatment with the EGF receptor kinase inhibitor AG1478 had, remarkably, no significant effect on Erk1/2 activation induced by any of these adrenergic agonists (although it fully abolished EGF-induced Erk1/2 activation), demonstrating absence of EGF receptor-mediated transactivation. Results with brown preadipocytes (cells in more proliferative states) were not qualitatively different. Joint stimulation of all adrenoceptors with norepinephrine did not result in synergism on Erk1/2 activation. AG1478 action on EGF-stimulated Erk1/2 phosphorylation showed a sharp concentration–response relationship (IC 50 0.3 µM); a minor apparent effect of AG1478 on norepinephrine-stimulated Erk1/2 phosphorylation showed nonspecific kinetics, implying caution in interpretation of partial effects of AG1478 as reported in other systems. Transactivation of the EGF receptor is clearly not a universal prerequisite for coupling of G-protein coupled receptors to Erk1/2 signalling cascades. - Highlights: • In brown adipocytes, norepinephrine regulates proliferation, apoptosis, differentiation. • EGF receptor transactivation is supposed to mediate GPCR-induced Erk1/2 activation. •

  15. GPCR Interaction: 184 [GRIPDB[Archive

    Lifescience Database Archive (English)

    Full Text Available tion of C5aR dimers.Upon C5a stimulation, mutant receptors did not internalize when individually express...ed. C5a stimulation of cells that co-expressed wild type C5aR together with either un...terfaces of C5aR A Complement component C5a C5aR A Complement component C5a C5aR Experiment Neither an intact NH2 term...inus nor the presence of COOH-terminal phosphorylation sites appeared to be required for the forma...liganded or phosphorylation-deficient mutant resulted in co-internalization of mutant receptors with C5aR. 18772131 NH2-terminally modified C5aR mutant NP_001727.1 ...

  16. The heterotrimeric G protein Gβ1 interacts with the catalytic subunit of protein phosphatase 1 and modulates G protein-coupled receptor signaling in platelets.

    Science.gov (United States)

    Pradhan, Subhashree; Khatlani, Tanvir; Nairn, Angus C; Vijayan, K Vinod

    2017-08-11

    Thrombosis is caused by the activation of platelets at the site of ruptured atherosclerotic plaques. This activation involves engagement of G protein-coupled receptors (GPCR) on platelets that promote their aggregation. Although it is known that protein kinases and phosphatases modulate GPCR signaling, how serine/threonine phosphatases integrate with G protein signaling pathways is less understood. Because the subcellular localization and substrate specificity of the catalytic subunit of protein phosphatase 1 (PP1c) is dictated by PP1c-interacting proteins, here we sought to identify new PP1c interactors. GPCRs signal via the canonical heterotrimeric Gα and Gβγ subunits. Using a yeast two-hybrid screen, we discovered an interaction between PP1cα and the heterotrimeric G protein Gβ 1 subunit. Co-immunoprecipitation studies with epitope-tagged PP1c and Gβ 1 revealed that Gβ 1 interacts with the PP1c α, β, and γ1 isoforms. Purified PP1c bound to recombinant Gβ 1 -GST protein, and PP1c co-immunoprecipitated with Gβ 1 in unstimulated platelets. Thrombin stimulation of platelets induced the dissociation of the PP1c-Gβ 1 complex, which correlated with an association of PP1c with phospholipase C β3 (PLCβ3), along with a concomitant dephosphorylation of the inhibitory Ser 1105 residue in PLCβ3. siRNA-mediated depletion of GNB1 (encoding Gβ 1 ) in murine megakaryocytes reduced protease-activated receptor 4, activating peptide-induced soluble fibrinogen binding. Thrombin-induced aggregation was decreased in PP1cα -/- murine platelets and in human platelets treated with a small-molecule inhibitor of Gβγ. Finally, disruption of PP1c-Gβ 1 complexes with myristoylated Gβ 1 peptides containing the PP1c binding site moderately decreased thrombin-induced human platelet aggregation. These findings suggest that Gβ 1 protein enlists PP1c to modulate GPCR signaling in platelets.

  17. Stability for Function Trade-Offs in the Enolase Superfamily 'Catalytic Module'

    Energy Technology Data Exchange (ETDEWEB)

    Nagatani, R.A.; Gonzalez, A.; Shoichet, B.K.; Brinen, L.S.; Babbitt, P.C.; /UC, San Francisco /SLAC, SSRL

    2007-07-12

    Enzyme catalysis reflects a dynamic interplay between charged and polar active site residues that facilitate function, stabilize transition states, and maintain overall protein stability. Previous studies show that substituting neutral for charged residues in the active site often significantly stabilizes a protein, suggesting a stability trade-off for functionality. In the enolase superfamily, a set of conserved active site residues (the ''catalytic module'') has repeatedly been used in nature in the evolution of many different enzymes for the performance of unique overall reactions involving a chemically diverse set of substrates. This catalytic module provides a robust solution for catalysis that delivers the common underlying partial reaction that supports all of the different overall chemical reactions of the superfamily. As this module has been so broadly conserved in the evolution of new functions, we sought to investigate the extent to which it follows the stability-function trade-off. Alanine substitutions were made for individual residues, groups of residues, and the entire catalytic module of o-succinylbenzoate synthase (OSBS), a member of the enolase superfamily from Escherichia coli. Of six individual residue substitutions, four (K131A, D161A, E190A, and D213A) substantially increased protein stability (by 0.46-4.23 kcal/mol), broadly consistent with prediction of a stability-activity trade-off. The residue most conserved across the superfamily, E190, is by far the most destabilizing. When the individual substitutions were combined into groups (as they are structurally and functionally organized), nonadditive stability effects emerged, supporting previous observations that residues within the module interact as two functional groups within a larger catalytic system. Thus, whereas the multiple-mutant enzymes D161A/E190A/D213A and K131A/K133A/D161A/E190A/D213A/K235A (termed 3KDED) are stabilized relative to the wild-type enzyme (by 1

  18. SVM-Fold: a tool for discriminative multi-class protein fold and superfamily recognition.

    Science.gov (United States)

    Melvin, Iain; Ie, Eugene; Kuang, Rui; Weston, Jason; Stafford, William Noble; Leslie, Christina

    2007-05-22

    Predicting a protein's structural class from its amino acid sequence is a fundamental problem in computational biology. Much recent work has focused on developing new representations for protein sequences, called string kernels, for use with support vector machine (SVM) classifiers. However, while some of these approaches exhibit state-of-the-art performance at the binary protein classification problem, i.e. discriminating between a particular protein class and all other classes, few of these studies have addressed the real problem of multi-class superfamily or fold recognition. Moreover, there are only limited software tools and systems for SVM-based protein classification available to the bioinformatics community. We present a new multi-class SVM-based protein fold and superfamily recognition system and web server called SVM-Fold, which can be found at http://svm-fold.c2b2.columbia.edu. Our system uses an efficient implementation of a state-of-the-art string kernel for sequence profiles, called the profile kernel, where the underlying feature representation is a histogram of inexact matching k-mer frequencies. We also employ a novel machine learning approach to solve the difficult multi-class problem of classifying a sequence of amino acids into one of many known protein structural classes. Binary one-vs-the-rest SVM classifiers that are trained to recognize individual structural classes yield prediction scores that are not comparable, so that standard "one-vs-all" classification fails to perform well. Moreover, SVMs for classes at different levels of the protein structural hierarchy may make useful predictions, but one-vs-all does not try to combine these multiple predictions. To deal with these problems, our method learns relative weights between one-vs-the-rest classifiers and encodes information about the protein structural hierarchy for multi-class prediction. In large-scale benchmark results based on the SCOP database, our code weighting approach

  19. A comprehensive analysis of the Omp85/TpsB protein superfamily structural diversity, taxonomic occurrence, and evolution

    Science.gov (United States)

    Heinz, Eva; Lithgow, Trevor

    2014-01-01

    Members of the Omp85/TpsB protein superfamily are ubiquitously distributed in Gram-negative bacteria, and function in protein translocation (e.g., FhaC) or the assembly of outer membrane proteins (e.g., BamA). Several recent findings are suggestive of a further level of variation in the superfamily, including the identification of the novel membrane protein assembly factor TamA and protein translocase PlpD. To investigate the diversity and the causal evolutionary events, we undertook a comprehensive comparative sequence analysis of the Omp85/TpsB proteins. A total of 10 protein subfamilies were apparent, distinguished in their domain structure and sequence signatures. In addition to the proteins FhaC, BamA, and TamA, for which structural and functional information is available, are families of proteins with so far undescribed domain architectures linked to the Omp85 β-barrel domain. This study brings a classification structure to a dynamic protein superfamily of high interest given its essential function for Gram-negative bacteria as well as its diverse domain architecture, and we discuss several scenarios of putative functions of these so far undescribed proteins. PMID:25101071

  20. Development of a human vasopressin V1a-receptor antagonist from an evolutionary-related insect neuropeptide

    Science.gov (United States)

    di Giglio, Maria Giulia; Muttenthaler, Markus; Harpsøe, Kasper; Liutkeviciute, Zita; Keov, Peter; Eder, Thomas; Rattei, Thomas; Arrowsmith, Sarah; Wray, Susan; Marek, Ales; Elbert, Tomas; Alewood, Paul F.; Gloriam, David E.; Gruber, Christian W.

    2017-02-01

    Characterisation of G protein-coupled receptors (GPCR) relies on the availability of a toolbox of ligands that selectively modulate different functional states of the receptors. To uncover such molecules, we explored a unique strategy for ligand discovery that takes advantage of the evolutionary conservation of the 600-million-year-old oxytocin/vasopressin signalling system. We isolated the insect oxytocin/vasopressin orthologue inotocin from the black garden ant (Lasius niger), identified and cloned its cognate receptor and determined its pharmacological properties on the insect and human oxytocin/vasopressin receptors. Subsequently, we identified a functional dichotomy: inotocin activated the insect inotocin and the human vasopressin V1b receptors, but inhibited the human V1aR. Replacement of Arg8 of inotocin by D-Arg8 led to a potent, stable and competitive V1aR-antagonist ([D-Arg8]-inotocin) with a 3,000-fold binding selectivity for the human V1aR over the other three subtypes, OTR, V1bR and V2R. The Arg8/D-Arg8 ligand-pair was further investigated to gain novel insights into the oxytocin/vasopressin peptide-receptor interaction, which led to the identification of key residues of the receptors that are important for ligand functionality and selectivity. These observations could play an important role for development of oxytocin/vasopressin receptor modulators that would enable clear distinction of the physiological and pathological responses of the individual receptor subtypes.

  1. Vitamin D receptor B1 and exon 1d: functional and evolutionary analysis.

    Science.gov (United States)

    Gardiner, Edith M; Esteban, Luis M; Fong, Colette; Allison, Susan J; Flanagan, Judith L; Kouzmenko, Alexander P; Eisman, John A

    2004-05-01

    The vitamin D receptor (VDR) shares a conserved structural and functional organization with other nuclear receptor (NR) superfamily members. For many NRs, N-terminal variant isoforms that display distinct cell-, stage- and promoter-specific actions have been identified. The novel VDR isoform VDRB1, with a 50 amino acid N-terminal extension, is produced from low abundance transcripts that contain exon 1d of the human VDR locus. There is evidence for the conservation of this exon in other mammalian and avian species. The transactivation differences between VDRB1 and the original VDR, clarified here, provide insights into mechanisms that may contribute to functional differences and potentially distinct physiological roles for these two VDR isoforms.

  2. Trace Amine-Associated Receptor 1 – Family Archetype or Iconoclast?

    Science.gov (United States)

    Grandy, David K.

    2009-01-01

    Interest has recently been rekindled in receptors that are activated by low molecular weight, non-catecholic, biogenic amines that are typically found as trace constituents of various vertebrate and invertebrate tissues and fluids. The timing of this resurgent focus on receptors activated by the ‘trace amines’ (TAs) β-phenylethylamine (PEA), tyramine (TYR), octopamine (OCT), synephrine (SYN), and tryptamine (TRYP) is the direct result of two publications that appeared in 2001 describing the cloning of a novel G protein-coupled receptor (GPCR) referred to by their discoverers as TA1 (Borowsky et al., 2001) and TAR1 (Bunzow et al., 2001). When heterologously expressed in Xenopus laevis oocytes and various eukaryotic cell lines recombinant rodent and human TA receptors dose-dependently couple to the stimulation of cAMP production. Structure-activity profiling based on this functional response has revealed that in addition to the TAs, other biologically active compounds containing a 2 carbon aliphatic side chain linking an amino group to at least one benzene ring are potent and efficacious TA receptor agonists with amphetamine, methamphetamine, 3-iodothyronamine, thyronamine, and dopamine among the most notable. Almost 100 years after the search for TA receptors began numerous TA1/TAR1-related sequences, now called Trace Amine-Associated Receptors (TAARs), have been identified in the genome of every species of vertebrate examined to date. Consequently, even though heterologously expressed TAAR1 fits the pharmacological criteria established for a bona fide TA receptor a major challenge for those working in the field is to discern the in vivo pharmacology and physiology of each purported member of this extended family of GPCRs. Only then will it be possible to establish whether TAAR1 is the family archetype or an iconoclast. PMID:17888514

  3. Crystallographic analysis of murine constitutive androstane receptor ligand-binding domain complexed with 5α-androst-16-en-3α-ol

    International Nuclear Information System (INIS)

    Vincent, Jeremy; Shan, Li; Fan, Ming; Brunzelle, Joseph S.; Forman, Barry M.; Fernandez, Elias J.

    2004-01-01

    The purification and structure determination of the murine constitutive androstane receptor bound to its inverse agonist/antagonist androstenol is described. The constitutive androstane receptor (CAR) is a member of the nuclear receptor superfamily. In contrast to classical nuclear receptors, which possess small-molecule ligand-inducible activity, CAR exhibits constitutive transcriptional activity in the apparent absence of ligand. CAR is among the most important transcription factors; it coordinately regulates the expression of microsomal cytochrome P450 genes and other drug-metabolizing enzymes. The murine CAR ligand-binding domain (LBD) was coexpressed with the steroid receptor coactivator protein (SRC-1) receptor-interacting domain (RID) in Escherichia coli. The mCAR LBD subunit was purified away from SRC-1 by affinity, anion-exchange and size-exclusion chromatography, crystallized with androstenol and the structure of the complex determined by molecular replacement

  4. The apelin receptor influences biomechanical and morphological properties of endothelial cells.

    Science.gov (United States)

    Strohbach, Anne; Pennewitz, Malte; Glaubitz, Michael; Palankar, Raghavendra; Groß, Stefan; Lorenz, Florian; Materzok, Ilka; Rong, Alena; Busch, Mathias C; Felix, Stephan B; Delcea, Mihaela; Busch, Raila

    2018-08-01

    The adaption of endothelial cells to local flow conditions is a multifunctional process which leads to distinct alterations in cell shape, the subcellular distribution of structural proteins, and cellular function. G-protein-coupled receptors (GPCRs) have been identified to be fundamentally involved in such processes. Recently, we and others have shown that the expression of the endothelial GPCR apelin receptor (APJ) is regulated by fluid flow and that activation of APJ participates in signaling pathways which are related to processes of mechanotransduction. The present study aims to illuminate these findings by further visualization of APJ function. We show that APJ is located to the cellular junctions and might thus be associated with platelet endothelial cell adhesion molecule-1 (PECAM-1) in human umbilical vein endothelial cells (HUVEC). Furthermore, siRNA-mediated silencing of APJ expression influences the shear-induced adaption of HUVEC in terms of cytoskeletal remodeling, cellular elasticity, cellular motility, attachment, and distribution of adhesion complexes. Taken together, our results demonstrate that APJ is crucial for complemented endothelial adaption to local flow conditions. © 2018 Wiley Periodicals, Inc.

  5. Induction of selective blood-tumor barrier permeability and macromolecular transport by a biostable kinin B1 receptor agonist in a glioma rat model.

    Science.gov (United States)

    Côté, Jérôme; Bovenzi, Veronica; Savard, Martin; Dubuc, Céléna; Fortier, Audrey; Neugebauer, Witold; Tremblay, Luc; Müller-Esterl, Werner; Tsanaclis, Ana-Maria; Lepage, Martin; Fortin, David; Gobeil, Fernand

    2012-01-01

    Treatment of malignant glioma with chemotherapy is limited mostly because of delivery impediment related to the blood-brain tumor barrier (BTB). B1 receptors (B1R), inducible prototypical G-protein coupled receptors (GPCR) can regulate permeability of vessels including possibly that of brain tumors. Here, we determine the extent of BTB permeability induced by the natural and synthetic peptide B1R agonists, LysdesArg(9)BK (LDBK) and SarLys[dPhe(8)]desArg(9)BK (NG29), in syngeneic F98 glioma-implanted Fischer rats. Ten days after tumor inoculation, we detected the presence of B1R on tumor cells and associated vasculature. NG29 infusion increased brain distribution volume and uptake profiles of paramagnetic probes (Magnevist and Gadomer) at tumoral sites (T(1)-weighted imaging). These effects were blocked by B1R antagonist and non-selective cyclooxygenase inhibitors, but not by B2R antagonist and non-selective nitric oxide synthase inhibitors. Consistent with MRI data, systemic co-administration of NG29 improved brain tumor delivery of Carboplatin chemotherapy (ICP-Mass spectrometry). We also detected elevated B1R expression in clinical samples of high-grade glioma. Our results documented a novel GPCR-signaling mechanism for promoting transient BTB disruption, involving activation of B1R and ensuing production of COX metabolites. They also underlined the potential value of synthetic biostable B1R agonists as selective BTB modulators for local delivery of different sized-therapeutics at (peri)tumoral sites.

  6. Agonist Binding to Chemosensory Receptors: A Systematic Bioinformatics Analysis

    Directory of Open Access Journals (Sweden)

    Fabrizio Fierro

    2017-09-01

    Full Text Available Human G-protein coupled receptors (hGPCRs constitute a large and highly pharmaceutically relevant membrane receptor superfamily. About half of the hGPCRs' family members are chemosensory receptors, involved in bitter taste and olfaction, along with a variety of other physiological processes. Hence these receptors constitute promising targets for pharmaceutical intervention. Molecular modeling has been so far the most important tool to get insights on agonist binding and receptor activation. Here we investigate both aspects by bioinformatics-based predictions across all bitter taste and odorant receptors for which site-directed mutagenesis data are available. First, we observe that state-of-the-art homology modeling combined with previously used docking procedures turned out to reproduce only a limited fraction of ligand/receptor interactions inferred by experiments. This is most probably caused by the low sequence identity with available structural templates, which limits the accuracy of the protein model and in particular of the side-chains' orientations. Methods which transcend the limited sampling of the conformational space of docking may improve the predictions. As an example corroborating this, we review here multi-scale simulations from our lab and show that, for the three complexes studied so far, they significantly enhance the predictive power of the computational approach. Second, our bioinformatics analysis provides support to previous claims that several residues, including those at positions 1.50, 2.50, and 7.52, are involved in receptor activation.

  7. GPCR Interaction: 188 [GRIPDB[Archive

    Lifescience Database Archive (English)

    Full Text Available ivation are reduced by activation of CXCR4 in advance. In addition, calcium mobilization assays show these cross-desensitization...ion is not responsible for desensitization between these receptors. 18533278 Calcium mobilization assay NP_000903.2 ...

  8. GPCR Interaction: 187 [GRIPDB[Archive

    Lifescience Database Archive (English)

    Full Text Available tivation are reduced by activation of CXCR4 in advance. In addition, calcium mobilization assays show these cross-desensitization...tion is not responsible for desensitization between these receptors. 18533278 Calcium mobilization assay NP_001008540.1 ...

  9. Chemogenetic Modulation of G Protein-Coupled Receptor Signalling in Visual Attention Research

    DEFF Research Database (Denmark)

    Jørgensen, Søren H; Fitzpatrick, Ciarán Martin; Gether, Ulrik

    2017-01-01

    Exclusively Activated by Designer Drugs (DREADDs). The DREADD technology is an emerging and transformative method that allows selective manipulation of G protein-coupled receptor (GPCR) signalling, and its broad-ranging usefulness in attention research is now beginning to emerge. We first describe......Attention is a fundamental cognitive process involved in nearly all aspects of life. Abnormal attentional control is a symptom of many neurological disorders, most notably recognized in ADHD (attention deficit hyperactivity disorder). Although attentional performance and its malfunction has been...... the different DREADDs available and explain how unprecedented specificity of neuronal signalling can be achieved using DREADDs. We next discuss various studies performed in animal models of visual attention, where different brain regions and neuronal populations have been probed by DREADDs. We highlight...

  10. Nucleoside conjugates of quantum dots for characterization of G protein-coupled receptors: strategies for immobilizing A2A adenosine receptor agonists

    Directory of Open Access Journals (Sweden)

    Gao Zhan-Guo

    2010-05-01

    Full Text Available Abstract Background Quantum dots (QDs are crystalline nanoparticles that are compatible with biological systems to provide a chemically and photochemically stable fluorescent label. New ligand probes with fluorescent reporter groups are needed for detection and characterization of G protein-coupled receptors (GPCRs. Results Synthetic strategies for coupling the A2A adenosine receptor (AR agonist CGS21680 (2-[4-(2-carboxyethylphenylethylamino]-5'-N-ethylcarboxamidoadenosine to functionalized QDs were explored. Conjugates tethered through amide-linked chains and poly(ethyleneglycol (PEG displayed low solubility and lacked receptor affinity. The anchor to the dendron was either through two thiol groups of (R-thioctic acid or through amide formation to a commercial carboxy-derivatized QD. The most effective approach was to use polyamidoamine (PAMAM D5 dendrons as multivalent spacer groups, grafted on the QD surface through a thioctic acid moiety. In radioligand binding assays, dendron nucleoside conjugate 11 displayed a moderate affinity at the human A2AAR (Kiapp 1.02 ± 0.15 μM. The QD conjugate of increased water solubility 13, resulting from the anchoring of this dendron derivative, interacted with the receptor with Kiapp of 118 ± 54 nM. The fluorescence emission of 13 occurred at 565 nm, and the presence of the pendant nucleoside did not appreciably quench the fluorescence. Conclusions This is a feasibility study to demonstrate a means of conjugating to a QD a small molecular pharmacophore of a GPCR that is relatively hydrophobic. Further enhancement of affinity by altering the pharmacophore or the linking structures will be needed to make useful affinity probes.

  11. Binding Mode Prediction of 5-Hydroxytryptamine 2C Receptor Ligands by Homology Modeling and Molecular Docking Analysis

    Energy Technology Data Exchange (ETDEWEB)

    Ahmed, Asif; Nagarajan, Shanthi; Doddareddy, Munikumar Reddy; Cho, Yong Seo; Pae, Ae Nim [Korea Institute of Science and Technology, Seoul (Korea, Republic of)

    2011-06-15

    Serotonin or 5-hydroxytryptamine subtype 2C (5-HT{sub 2C}) receptor belongs to class A amine subfamily of Gprotein- coupled receptor (GPCR) super family and its ligands has therapeutic promise as anti-depressant and -obesity agents. So far, bovine rhodopsin from class A opsin subfamily was the mostly used X-ray crystal template to model this receptor. Here, we explained homology model using beta 2 adrenergic receptor (β2AR), the model was energetically minimized and validated by flexible ligand docking with known agonists and antagonists. In the active site Asp134, Ser138 of transmembrane 3 (TM3), Arg195 of extracellular loop 2 (ECL2) and Tyr358 of TM7 were found as important residues to interact with agonists. In addition to these, V208 of ECL2 and N351 of TM7 was found to interact with antagonists. Several conserved residues including Trp324, Phe327 and Phe328 were also found to contribute hydrophobic interaction. The predicted ligand binding mode is in good agreement with published mutagenesis and homology model data. This new template derived homology model can be useful for further virtual screening based lead identification.

  12. Evolution of Enzymatic Activities in the Enolase Superfamily: L-Rhamnonate Dehydratase

    Energy Technology Data Exchange (ETDEWEB)

    Rakus,J.; Fedorov, A.; Fedorov, E.; Glaner, M.; Hubbard, B.; Delli, J.; Babbitt, P.; Almo, S.; Gerlt, J.

    2008-01-01

    The l-rhamnonate dehydratase (RhamD) function was assigned to a previously uncharacterized family in the mechanistically diverse enolase superfamily that is encoded by the genome of Escherichia coli K-12. We screened a library of acid sugars to discover that the enzyme displays a promiscuous substrate specificity: l-rhamnonate (6-deoxy-l-mannonate) has the 'best' kinetic constants, with l-mannonate, l-lyxonate, and d-gulonate dehydrated less efficiently. Crystal structures of the RhamDs from both E. coli K-12 and Salmonella typhimurium LT2 (95% sequence identity) were obtained in the presence of Mg2+; the structure of the RhamD from S. typhimurium was also obtained in the presence of 3-deoxy-l-rhamnonate (obtained by reduction of the product with NaBH4). Like other members of the enolase superfamily, RhamD contains an N-terminal a + {beta} capping domain and a C-terminal ({beta}/a)7{beta}-barrel (modified TIM-barrel) catalytic domain with the active site located at the interface between the two domains. In contrast to other members, the specificity-determining '20s loop' in the capping domain is extended in length and the '50s loop' is truncated. The ligands for the Mg2+ are Asp 226, Glu 252 and Glu 280 located at the ends of the third, fourth and fifth {beta}-strands, respectively. The active site of RhamD contains a His 329-Asp 302 dyad at the ends of the seventh and sixth {beta}-strands, respectively, with His 329 positioned to function as the general base responsible for abstraction of the C2 proton of l-rhamnonate to form a Mg2+-stabilized enediolate intermediate. However, the active site does not contain other acid/base catalysts that have been implicated in the reactions catalyzed by other members of the MR subgroup of the enolase superfamily. Based on the structure of the liganded complex, His 329 also is expected to function as the general acid that both facilitates departure of the 3-OH group in a syn-dehydration reaction and

  13. Molecular Insights into the Transmembrane Domain of the Thyrotropin Receptor.

    Directory of Open Access Journals (Sweden)

    Vanessa Chantreau

    Full Text Available The thyrotropin receptor (TSHR is a G protein-coupled receptor (GPCR that is member of the leucine-rich repeat subfamily (LGR. In the absence of crystal structure, the success of rational design of ligands targeting the receptor internal cavity depends on the quality of the TSHR models built. In this subfamily, transmembrane helices (TM 2 and 5 are characterized by the absence of proline compared to most receptors, raising the question of the structural conformation of these helices. To gain insight into the structural properties of these helices, we carried out bioinformatics and experimental studies. Evolutionary analysis of the LGR family revealed a deletion in TM5 but provided no information on TM2. Wild type residues at positions 2.58, 2.59 or 2.60 in TM2 and/or at position 5.50 in TM5 were substituted to proline. Depending on the position of the proline substitution, different effects were observed on membrane expression, glycosylation, constitutive cAMP activity and responses to thyrotropin. Only proline substitution at position 2.59 maintained complex glycosylation and high membrane expression, supporting occurrence of a bulged TM2. The TSHR transmembrane domain was modeled by homology with the orexin 2 receptor, using a protocol that forced the deletion of one residue in the TM5 bulge of the template. The stability of the model was assessed by molecular dynamics simulations. TM5 straightened during the equilibration phase and was stable for the remainder of the simulations. Our data support a structural model of the TSHR transmembrane domain with a bulged TM2 and a straight TM5 that is specific of glycoprotein hormone receptors.

  14. Signaling-sensitive amino acids surround the allosteric ligand binding site of the thyrotropin receptor.

    Science.gov (United States)

    Kleinau, Gunnar; Haas, Ann-Karin; Neumann, Susanne; Worth, Catherine L; Hoyer, Inna; Furkert, Jens; Rutz, Claudia; Gershengorn, Marvin C; Schülein, Ralf; Krause, Gerd

    2010-07-01

    The thyrotropin receptor [thyroid-stimulating hormone receptor (TSHR)], a G-protein-coupled receptor (GPCR), is endogenously activated by thyrotropin, which binds to the extracellular region of the receptor. We previously identified a low-molecular-weight (LMW) agonist of the TSHR and predicted its allosteric binding pocket within the receptor's transmembrane domain. Because binding of the LMW agonist probably disrupts interactions or leads to formation of new interactions among amino acid residues surrounding the pocket, we tested whether mutation of residues at these positions would lead to constitutive signaling activity. Guided by molecular modeling, we performed site-directed mutagenesis of 24 amino acids in this spatial region, followed by functional characterization of the mutant receptors in terms of expression and signaling, measured as cAMP accumulation. We found that mutations V421I, Y466A, T501A, L587V, M637C, M637W, S641A, Y643F, L645V, and Y667A located in several helices exhibit constitutive activity. Of note is mutation M637W at position 6.48 in transmembrane helix 6, which has a significant effect on the interaction of the receptor with the LMW agonist. In summary, we found that a high proportion of residues in several helices surrounding the allosteric binding site of LMW ligands in the TSHR when mutated lead to constitutively active receptors. Our findings of signaling-sensitive residues in this region of the transmembrane bundle may be of general importance as this domain appears to be evolutionarily retained among GPCRs.

  15. GPCR Interaction: 207 [GRIPDB[Archive

    Lifescience Database Archive (English)

    Full Text Available LP2) and secretin (SecR), and interfaces of GLP2 B Glucagon-like peptide Type 2 GLP2 C Secretin ... SecR Experi...ment SecR does not interact with CTR (calcitonin receptor). 18401761 BRET, FRET NP_004237.1 ...

  16. Short interspersed elements (SINEs) of the Geomyoidea superfamily rodents.

    Science.gov (United States)

    Gogolevsky, Konstantin P; Kramerov, Dmitri A

    2006-05-24

    A new short interspersed element (SINE) was isolated from the genome of desert kangaroo rat (Dipodomys deserti) using single-primer PCR. This SINE consists of two monomers: the left monomer (IDL) resembles rodent ID element and other tRNAAla(CGC)-derived SINEs, whereas the right one (Geo) shows no similarity with known SINE sequences. PCR and hybridization analyses demonstrated that IDL-Geo SINE is restricted to the rodent superfamily Geomyoidea (families Geomyidea and Heteromyidea). Isolation and analysis of IDL-Geo from California pocket mouse (Chaetodipus californicus) and Botta's pocket gopher (Thomomys bottae) revealed some species-specific features of this SINE family. The structure and evolution of known dimeric SINEs are discussed.

  17. In silico Exploration of the Conformational Universe of GPCRs.

    Science.gov (United States)

    Rodríguez-Espigares, Ismael; Kaczor, Agnieszka A; Selent, Jana

    2016-07-01

    The structural plasticity of G protein coupled receptors (GPCRs) leads to a conformational universe going from inactive to active receptor states with several intermediate states. Many of them have not been captured yet and their role for GPCR activation is not well understood. The study of this conformational space and the transition dynamics between different receptor populations is a major challenge in molecular biophysics. The rational design of effector molecules that target such receptor populations allows fine-tuning receptor signalling with higher specificity to produce drugs with safer therapeutic profiles. In this minireview, we outline highly conserved receptor regions which are considered determinant for the establishment of distinct receptor states. We then discuss in-silico approaches such as dimensionality reduction methods and Markov State Models to explore the GPCR conformational universe and exploit the obtained conformations through structure-based drug design. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  18. β-arrestins negatively control human adrenomedullin type 1-receptor internalization

    International Nuclear Information System (INIS)

    Kuwasako, Kenji; Kitamura, Kazuo; Nagata, Sayaka; Sekiguchi, Toshio; Danfeng, Jiang; Murakami, Manabu; Hattori, Yuichi; Kato, Johji

    2017-01-01

    Adrenomedullin (AM) is a potent hypotensive peptide that exerts a powerful variety of protective effects against multiorgan damage through the AM type 1 receptor (AM 1 receptor), which consists of the calcitonin receptor-like receptor (CLR) and receptor activity-modifying protein 2 (RAMP2). Two β-arrestin (β-arr) isoforms, β-arr-1 and β-arr-2, play a central role in the agonist-induced internalization of many receptors for receptor resensitization. Notably, β-arr-biased agonists are now being tested in phase II clinical trials, targeting acute pain and acute heart failure. Here, we examined the effects of β-arr-1 and β-arr-2 on human AM 1 receptor internalization. We constructed a V5-tagged chimera in which the cytoplasmic C-terminal tail (C-tail) of CLR was replaced with that of the β 2 -adrenergic receptor (β 2 -AR), and it was transiently transfected into HEK-293 cells that stably expressed RAMP2. The cell-surface expression and internalization of the wild-type or chimeric receptor were quantified by flow cytometric analysis. The [ 125 I]AM binding and the AM-induced cAMP production of these receptors were also determined. Surprisingly, the coexpression of β-arr-1 or -2 resulted in significant decreases in AM 1 receptor internalization without affecting AM binding and signaling prior to receptor internalization. Dominant-negative (DN) β-arr-1 or -2 also significantly decreased AM-induced AM 1 receptor internalization. In contrast, the AM-induced internalization of the chimeric AM 1 receptor was markedly augmented by the cotransfection of β-arr-1 or -2 and significantly reduced by the coexpression of DN-β-arr-1 or -2. These results were consistent with those seen for β 2 -AR. Thus, both β-arrs negatively control AM 1 receptor internalization, which depends on the C-tail of CLR. - Highlights: • We found that β-arrestins 1 and 2 negatively control agonist-induced GPCR internalization. • β-arrestins 1 and 2 significantly inhibits the AM

  19. FTZ-Factor1 and Fushi tarazu interact via conserved nuclear receptor and coactivator motifs

    Science.gov (United States)

    Schwartz, Carol J.E.; Sampson, Heidi M.; Hlousek, Daniela; Percival-Smith, Anthony; Copeland, John W.R.; Simmonds, Andrew J.; Krause, Henry M.

    2001-01-01

    To activate transcription, most nuclear receptor proteins require coactivators that bind to their ligand-binding domains (LBDs). The Drosophila FTZ-Factor1 (FTZ-F1) protein is a conserved member of the nuclear receptor superfamily, but was previously thought to lack an AF2 motif, a motif that is required for ligand and coactivator binding. Here we show that FTZ-F1 does have an AF2 motif and that it is required to bind a coactivator, the homeodomain-containing protein Fushi tarazu (FTZ). We also show that FTZ contains an AF2-interacting nuclear receptor box, the first to be found in a homeodomain protein. Both interaction motifs are shown to be necessary for physical interactions in vitro and for functional interactions in developing embryos. These unexpected findings have important implications for the conserved homologs of the two proteins. PMID:11157757

  20. Aldehyde Dehydrogenase Gene Superfamily in Populus: Organization and Expression Divergence between Paralogous Gene Pairs.

    Science.gov (United States)

    Tian, Feng-Xia; Zang, Jian-Lei; Wang, Tan; Xie, Yu-Li; Zhang, Jin; Hu, Jian-Jun

    2015-01-01

    Aldehyde dehydrogenases (ALDHs) constitute a superfamily of NAD(P)+-dependent enzymes that catalyze the irreversible oxidation of a wide range of reactive aldehydes to their corresponding nontoxic carboxylic acids. ALDHs have been studied in many organisms from bacteria to mammals; however, no systematic analyses incorporating genome organization, gene structure, expression profiles, and cis-acting elements have been conducted in the model tree species Populus trichocarpa thus far. In this study, a comprehensive analysis of the Populus ALDH gene superfamily was performed. A total of 26 Populus ALDH genes were found to be distributed across 12 chromosomes. Genomic organization analysis indicated that purifying selection may have played a pivotal role in the retention and maintenance of PtALDH gene families. The exon-intron organizations of PtALDHs were highly conserved within the same family, suggesting that the members of the same family also may have conserved functionalities. Microarray data and qRT-PCR analysis indicated that most PtALDHs had distinct tissue-specific expression patterns. The specificity of cis-acting elements in the promoter regions of the PtALDHs and the divergence of expression patterns between nine paralogous PtALDH gene pairs suggested that gene duplications may have freed the duplicate genes from the functional constraints. The expression levels of some ALDHs were up- or down-regulated by various abiotic stresses, implying that the products of these genes may be involved in the adaptation of Populus to abiotic stresses. Overall, the data obtained from our investigation contribute to a better understanding of the complexity of the Populus ALDH gene superfamily and provide insights into the function and evolution of ALDH gene families in vascular plants.

  1. Aldehyde Dehydrogenase Gene Superfamily in Populus: Organization and Expression Divergence between Paralogous Gene Pairs.

    Directory of Open Access Journals (Sweden)

    Feng-Xia Tian

    Full Text Available Aldehyde dehydrogenases (ALDHs constitute a superfamily of NAD(P+-dependent enzymes that catalyze the irreversible oxidation of a wide range of reactive aldehydes to their corresponding nontoxic carboxylic acids. ALDHs have been studied in many organisms from bacteria to mammals; however, no systematic analyses incorporating genome organization, gene structure, expression profiles, and cis-acting elements have been conducted in the model tree species Populus trichocarpa thus far. In this study, a comprehensive analysis of the Populus ALDH gene superfamily was performed. A total of 26 Populus ALDH genes were found to be distributed across 12 chromosomes. Genomic organization analysis indicated that purifying selection may have played a pivotal role in the retention and maintenance of PtALDH gene families. The exon-intron organizations of PtALDHs were highly conserved within the same family, suggesting that the members of the same family also may have conserved functionalities. Microarray data and qRT-PCR analysis indicated that most PtALDHs had distinct tissue-specific expression patterns. The specificity of cis-acting elements in the promoter regions of the PtALDHs and the divergence of expression patterns between nine paralogous PtALDH gene pairs suggested that gene duplications may have freed the duplicate genes from the functional constraints. The expression levels of some ALDHs were up- or down-regulated by various abiotic stresses, implying that the products of these genes may be involved in the adaptation of Populus to abiotic stresses. Overall, the data obtained from our investigation contribute to a better understanding of the complexity of the Populus ALDH gene superfamily and provide insights into the function and evolution of ALDH gene families in vascular plants.

  2. Protease-Activated Receptor 4 (PAR4): A Promising Target for Antiplatelet Therapy.

    Science.gov (United States)

    Rwibasira Rudinga, Gamariel; Khan, Ghulam Jilany; Kong, Yi

    2018-02-14

    Cardiovascular diseases (CVDs) are currently among the leading causes of death worldwide. Platelet aggregation is a key cellular component of arterial thrombi and major cause of CVDs. Protease-activated receptors (PARs), including PAR1, PAR2, PAR3 and PAR4, fall within a subfamily of seven-transmembrane G-protein-coupled receptors (GPCR). Human platelets express PAR1 and PAR4, which contribute to the signaling transduction processes. In association with CVDs, PAR4 not only contributes to platelet activation but also is a modulator of cellular responses that serve as hallmarks of inflammation. Although several antiplatelet drugs are available on the market, they have many side effects that limit their use. Emerging evidence shows that PAR4 targeting is a safer strategy for preventing thrombosis and consequently may improve the overall cardiac safety profile. Our present review summarizes the PAR4 structural characteristics, activation mechanism, role in the pathophysiology of diseases and understanding the association of PAR4 targeting for improved cardiac protection. Conclusively, this review highlights the importance of PAR4 antagonists and its potential utility in different CVDs.

  3. Development of full sweet, umami, and bitter taste responsiveness requires Regulator of G protein Signaling-21 (RGS21).

    Science.gov (United States)

    Schroer, Adam B; Gross, Joshua D; Kaski, Shane W; Wix, Kim; Siderovski, David P; Vandenbeuch, Aurelie; Setola, Vincent

    2018-04-26

    The mammalian tastes of sweet, umami, and bitter are initiated by activation of G protein-coupled receptors (GPCRs) of the T1R and T2R families on taste receptor cells. GPCRs signal via nucleotide exchange and hydrolysis, the latter hastened by GTPase-accelerating proteins (GAPs) that include the Regulators of G protein Signaling (RGS) protein family. We previously reported that RGS21, uniquely expressed in Type II taste receptor cells, decreases the potency of bitter-stimulated T2R signaling in cultured cells, consistent with its in vitro GAP activity. However, the role of RGS21 in organismal responses to GPCR-mediated tastants was not established. Here, we characterized mice lacking the Rgs21 fifth exon. Eliminating Rgs21 expression had no effect on body mass accumulation (a measure of alimentation), fungiform papillae number and morphology, circumvallate papillae morphology, and taste bud number. Two-bottle preference tests, however, revealed that Rgs21-null mice have blunted aversion to quinine and denatonium, and blunted preference for monosodium glutamate, the sweeteners sucrose and SC45647, and (surprisingly) NaCl. Observed reductions in GPCR-mediated tastant responses upon Rgs21 loss are opposite to original expectations, given that loss of RGS21 -- a GPCR signaling negative regulator -- should lead to increased responsiveness to tastant-mediated GPCR signaling (all else being equal). Yet, reduced organismal tastant responses are consistent with observations of reduced chorda tympani nerve recordings in Rgs21-null mice. Reduced tastant-mediated responses and behaviors exhibited by adult mice lacking Rgs21 expression since birth have thus revealed an underappreciated requirement for a GPCR GAP to establish the full character of tastant signaling.

  4. Do TRPC channels support working memory? Comparing modulations of TRPC channels and working memory through G-protein coupled receptors and neuromodulators.

    Science.gov (United States)

    Reboreda, Antonio; Theissen, Frederik M; Valero-Aracama, Maria J; Arboit, Alberto; Corbu, Mihaela A; Yoshida, Motoharu

    2018-03-01

    Working memory is a crucial ability we use in daily life. However, the cellular mechanisms supporting working memory still remain largely unclear. A key component of working memory is persistent neural firing which is believed to serve short-term (hundreds of milliseconds up to tens of seconds) maintenance of necessary information. In this review, we will focus on the role of transient receptor potential canonical (TRPC) channels as a mechanism underlying persistent firing. Many years of in vitro work have been suggesting a crucial role of TRPC channels in working memory and temporal association tasks. If TRPC channels are indeed a central mechanism for working memory, manipulations which impair or facilitate working memory should have a similar effect on TRPC channel modulation. However, modulations of working memory and TRPC channels were never systematically compared, and it remains unanswered whether TRPC channels indeed contribute to working memory in vivo or not. In this article, we review the effects of G-protein coupled receptors (GPCR) and neuromodulators, including acetylcholine, noradrenalin, serotonin and dopamine, on working memory and TRPC channels. Based on comparisons, we argue that GPCR and downstream signaling pathways that activate TRPC, generally support working memory, while those that suppress TRPC channels impair it. However, depending on the channel types, areas, and systems tested, this is not the case in all studies. Further work to clarify involvement of specific TRPC channels in working memory tasks and how they are affected by neuromodulators is still necessary in the future. Copyright © 2018 Elsevier B.V. All rights reserved.

  5. ATAR, a novel tumor necrosis factor receptor family member, signals through TRAF2 and TRAF5.

    Science.gov (United States)

    Hsu, H; Solovyev, I; Colombero, A; Elliott, R; Kelley, M; Boyle, W J

    1997-05-23

    Members of tumor necrosis factor receptor (TNFR) family signal largely through interactions with death domain proteins and TRAF proteins. Here we report the identification of a novel TNFR family member ATAR. Human and mouse ATAR contain 283 and 276 amino acids, respectively, making them the shortest known members of the TNFR superfamily. The receptor is expressed mainly in spleen, thymus, bone marrow, lung, and small intestine. The intracellular domains of human and mouse ATAR share only 25% identity, yet both interact with TRAF5 and TRAF2. This TRAF interaction domain resides at the C-terminal 20 amino acids. Like most other TRAF-interacting receptors, overexpression of ATAR activates the transcription factor NF-kappaB. Co-expression of ATAR with TRAF5, but not TRAF2, results in synergistic activation of NF-kappaB, suggesting potentially different roles for TRAF2 and TRAF5 in post-receptor signaling.

  6. RTA, a candidate G protein-coupled receptor: Cloning, sequencing, and tissue distribution

    International Nuclear Information System (INIS)

    Ross, P.C.; Figler, R.A.; Corjay, M.H.; Barber, C.M.; Adam, N.; Harcus, D.R.; Lynch, K.R.

    1990-01-01

    Genomic and cDNA clones, encoding a protein that is a member of the guanine nucleotide-binding regulatory protein (G protein)-coupled receptor superfamily, were isolated by screening rat genomic and thoracic aorta cDNA libraries with an oligonucleotide encoding a highly conserved region of the M 1 muscarinic acetylcholine receptor. Sequence analyses of these clones showed that they encode a 343-amino acid protein (named RTA). The RTA gene is single copy, as demonstrated by restriction mapping and Southern blotting of genomic clones and rat genomic DNA. RTA RNA sequences are relatively abundant throughout the gut, vas deferens, uterus, and aorta but are only barely detectable (on Northern blots) in liver, kidney, lung, and salivary gland. In the rat brain, RTA sequences are markedly abundant in the cerebellum. TRA is most closely related to the mas oncogene (34% identity), which has been suggested to be a forebrain angiotensin receptor. They conclude that RTA is not an angiotensin receptor; to date, they have been unable to identify its ligand

  7. High throughput mutagenesis for identification of residues regulating human prostacyclin (hIP) receptor expression and function.

    Science.gov (United States)

    Bill, Anke; Rosethorne, Elizabeth M; Kent, Toby C; Fawcett, Lindsay; Burchell, Lynn; van Diepen, Michiel T; Marelli, Anthony; Batalov, Sergey; Miraglia, Loren; Orth, Anthony P; Renaud, Nicole A; Charlton, Steven J; Gosling, Martin; Gaither, L Alex; Groot-Kormelink, Paul J

    2014-01-01

    The human prostacyclin receptor (hIP receptor) is a seven-transmembrane G protein-coupled receptor (GPCR) that plays a critical role in vascular smooth muscle relaxation and platelet aggregation. hIP receptor dysfunction has been implicated in numerous cardiovascular abnormalities, including myocardial infarction, hypertension, thrombosis and atherosclerosis. Genomic sequencing has discovered several genetic variations in the PTGIR gene coding for hIP receptor, however, its structure-function relationship has not been sufficiently explored. Here we set out to investigate the applicability of high throughput random mutagenesis to study the structure-function relationship of hIP receptor. While chemical mutagenesis was not suitable to generate a mutagenesis library with sufficient coverage, our data demonstrate error-prone PCR (epPCR) mediated mutagenesis as a valuable method for the unbiased screening of residues regulating hIP receptor function and expression. Here we describe the generation and functional characterization of an epPCR derived mutagenesis library compromising >4000 mutants of the hIP receptor. We introduce next generation sequencing as a useful tool to validate the quality of mutagenesis libraries by providing information about the coverage, mutation rate and mutational bias. We identified 18 mutants of the hIP receptor that were expressed at the cell surface, but demonstrated impaired receptor function. A total of 38 non-synonymous mutations were identified within the coding region of the hIP receptor, mapping to 36 distinct residues, including several mutations previously reported to affect the signaling of the hIP receptor. Thus, our data demonstrates epPCR mediated random mutagenesis as a valuable and practical method to study the structure-function relationship of GPCRs.

  8. High throughput mutagenesis for identification of residues regulating human prostacyclin (hIP receptor expression and function.

    Directory of Open Access Journals (Sweden)

    Anke Bill

    Full Text Available The human prostacyclin receptor (hIP receptor is a seven-transmembrane G protein-coupled receptor (GPCR that plays a critical role in vascular smooth muscle relaxation and platelet aggregation. hIP receptor dysfunction has been implicated in numerous cardiovascular abnormalities, including myocardial infarction, hypertension, thrombosis and atherosclerosis. Genomic sequencing has discovered several genetic variations in the PTGIR gene coding for hIP receptor, however, its structure-function relationship has not been sufficiently explored. Here we set out to investigate the applicability of high throughput random mutagenesis to study the structure-function relationship of hIP receptor. While chemical mutagenesis was not suitable to generate a mutagenesis library with sufficient coverage, our data demonstrate error-prone PCR (epPCR mediated mutagenesis as a valuable method for the unbiased screening of residues regulating hIP receptor function and expression. Here we describe the generation and functional characterization of an epPCR derived mutagenesis library compromising >4000 mutants of the hIP receptor. We introduce next generation sequencing as a useful tool to validate the quality of mutagenesis libraries by providing information about the coverage, mutation rate and mutational bias. We identified 18 mutants of the hIP receptor that were expressed at the cell surface, but demonstrated impaired receptor function. A total of 38 non-synonymous mutations were identified within the coding region of the hIP receptor, mapping to 36 distinct residues, including several mutations previously reported to affect the signaling of the hIP receptor. Thus, our data demonstrates epPCR mediated random mutagenesis as a valuable and practical method to study the structure-function relationship of GPCRs.

  9. Crystal structure of the human OX2 orexin receptor bound to the insomnia drug suvorexant

    Science.gov (United States)

    Yin, Jie; Mobarec, Juan Carlos; Kolb, Peter; Rosenbaum, Daniel M.

    2015-03-01

    The orexin (also known as hypocretin) G protein-coupled receptors (GPCRs) respond to orexin neuropeptides in the central nervous system to regulate sleep and other behavioural functions in humans. Defects in orexin signalling are responsible for the human diseases of narcolepsy and cataplexy; inhibition of orexin receptors is an effective therapy for insomnia. The human OX2 receptor (OX2R) belongs to the β branch of the rhodopsin family of GPCRs, and can bind to diverse compounds including the native agonist peptides orexin-A and orexin-B and the potent therapeutic inhibitor suvorexant. Here, using lipid-mediated crystallization and protein engineering with a novel fusion chimaera, we solved the structure of the human OX2R bound to suvorexant at 2.5 Å resolution. The structure reveals how suvorexant adopts a π-stacked horseshoe-like conformation and binds to the receptor deep in the orthosteric pocket, stabilizing a network of extracellular salt bridges and blocking transmembrane helix motions necessary for activation. Computational docking suggests how other classes of synthetic antagonists may interact with the receptor at a similar position in an analogous π-stacked fashion. Elucidation of the molecular architecture of the human OX2R expands our understanding of peptidergic GPCR ligand recognition and will aid further efforts to modulate orexin signalling for therapeutic ends.

  10. Pharmacogenomics of GPCR Drug Targets

    DEFF Research Database (Denmark)

    Hauser, Alexander Sebastian; Chavali, Sreenivas; Masuho, Ikuo

    2018-01-01

    Natural genetic variation in the human genome is a cause of individual differences in responses to medications and is an underappreciated burden on public health. Although 108 G-protein-coupled receptors (GPCRs) are the targets of 475 (∼34%) Food and Drug Administration (FDA)-approved drugs and a...

  11. Molecular characterization of a rat α2B-adrenergic receptor

    International Nuclear Information System (INIS)

    Zeng, D.; Harrison, J.K.; D'Angelo, D.D.; Barber, C.M.; Tucker, A.L.; Lu, Z.; Lynch, K.R.

    1990-01-01

    α 2 -Adrenergic receptors comprise a heterogeneous population based on pharmacologic and molecular evidence. The authors have isolated a cDNA clone (pRNGα 2 ) encoding a rat α 2 -adrenergic receptor. A rat kidney cDNA library was screened with an oligonucleotide complementary to a highly conserved region found in all biogenic amine receptors described to date. The deduced amino acid sequence displays many features of guanyl nucleotide-binding protein-coupled receptors except it does not have a consensus N-linked glycosylation site near the amino terminus. Membranes prepared from COS cells transfected with pRNGα 2 DNA display high affinity an saturable binding to [ 3 H]rauwolscine. Competition curve data analysis shows that RNGα 2 protein binds to a variety of adrenergic drugs with the following rank order of potency: yohimbine ≥ chlorpromazine > prazosin ≥ clonidine > norepinephrine ≥ oxymetazoline. RNGα 2 RNA accumulates in both rat kidney and neonatal rat lung. When a cysteine residue (Cys-169) that is conserved among all members of the seven-transmembrane-region superfamily is changed to phenylalanine, the RNGα 2 protein fails to bind [ 3 H]rauwolscine after expression in COS cells. They conclude that pRNGα 2 likely represents a cDNA for a rat α 2B -adrenergic receptor

  12. OX1 and OX2 orexin/hypocretin receptor pharmacogenetics

    Directory of Open Access Journals (Sweden)

    Miles Douglas Thompson

    2014-05-01

    Full Text Available Orexin/hypocretin peptide mutations are rare in humans. Even though human narcolepsy is associated with orexin deficiency, this is only extremely rarely due to mutations in the gene coding prepro-orexin, the precursor for both orexin peptides. In contrast, coding and non-coding variants of the OX1 and OX2 orexin receptors have been identified in many human populations; sometimes, these have been associated with disease phenotype, although most confer a relatively low risk. In most cases, these studies have been based on a candidate gene hypothesis that predicts involvement of orexins in the relevant pathophysiological processes. In the current review, the known human OX1/HCRTR1 and OX2/HCRTR2 genetic variants/polymorphisms as well as studies concerning their involvement in disorders such as narcolepsy, excessive daytime sleepiness, cluster headache, polydipsia-hyponatremia in schizophrenia, and affective disorders are presented. In most cases, the functional cellular or pharmacological correlates of orexin variants have not been investigated  with the exception of the possible impact of an amino acid 10 Pro/Ser variant of OX2 on orexin potency  leaving conclusions on the nature of the receptor variant effects speculative. Nevertheless, we present perspectives that could shape the basis for further studies. The pharmacology and other properties of the orexin receptor variants are discussed in the context of GPCR signaling. Since orexinergic therapeutics are emerging, the impact of receptor variants on the affinity or potency of ligands deserves consideration. This perspective (pharmacogenetics is also discussed in the review.

  13. Radical SAM, A Novel Protein Superfamily Linking Unresolved Steps in Familiar Biosynthetic Pathways with Radical Mechanisms: Functional Characterization Using New Analysis and Information Visualization Methods

    Energy Technology Data Exchange (ETDEWEB)

    Sofia, Heidi J.; Chen, Guang; Hetzler, Elizabeth G.; Reyes Spindola, Jorge F.; Miller, Nancy E.

    2001-03-01

    A large protein superfamily with over 500 members has been discovered and analyzed using powerful new bioinformatics and information visualization methods. Evidence exists that these proteins generate a 5?-deoxyadenosyl radical by reductive cleavage of S-adenosylmethionine (SAM) through an unusual Fe-S center. Radical SAM superfamily proteins function in DNA precursor, vitamin, cofactor, antibiotic, and herbicide biosynthesis in a collection of basic and familiar pathways. One of the members is interferon-inducible and is considered a candidate drug target for osteoporosis. The identification of this superfamily suggests that radical-based catalysis is important in a number of previously well-studied but unresolved biochemical pathways.

  14. Elabela-apelin receptor signaling pathway is functional in mammalian systems.

    Science.gov (United States)

    Wang, Zhi; Yu, Daozhan; Wang, Mengqiao; Wang, Qilong; Kouznetsova, Jennifer; Yang, Rongze; Qian, Kun; Wu, Wenjun; Shuldiner, Alan; Sztalryd, Carole; Zou, Minghui; Zheng, Wei; Gong, Da-Wei

    2015-02-02

    Elabela (ELA) or Toddler is a recently discovered hormone which is required for normal development of heart and vasculature through activation of apelin receptor (APJ), a G protein-coupled receptor (GPCR), in zebrafish. The present study explores whether the ELA-APJ signaling pathway is functional in the mammalian system. Using reverse-transcription PCR, we found that ELA is restrictedly expressed in human pluripotent stem cells and adult kidney whereas APJ is more widely expressed. We next studied ELA-APJ signaling pathway in reconstituted mammalian cell systems. Addition of ELA to HEK293 cells over-expressing GFP-AJP fusion protein resulted in rapid internalization of the fusion receptor. In Chinese hamster ovarian (CHO) cells over-expressing human APJ, ELA suppresses cAMP production with EC50 of 11.1 nM, stimulates ERK1/2 phosphorylation with EC50 of 14.3 nM and weakly induces intracellular calcium mobilization. Finally, we tested ELA biological function in human umbilical vascular endothelial cells and showed that ELA induces angiogenesis and relaxes mouse aortic blood vessel in a dose-dependent manner through a mechanism different from apelin. Collectively, we demonstrate that the ELA-AJP signaling pathways are functional in mammalian systems, indicating that ELA likely serves as a hormone regulating the circulation system in adulthood as well as in embryonic development.

  15. The specific monomer/dimer equilibrium of the corticotropin-releasing factor receptor type 1 is established in the endoplasmic reticulum.

    Science.gov (United States)

    Teichmann, Anke; Gibert, Arthur; Lampe, André; Grzesik, Paul; Rutz, Claudia; Furkert, Jens; Schmoranzer, Jan; Krause, Gerd; Wiesner, Burkhard; Schülein, Ralf

    2014-08-29

    G protein-coupled receptors (GPCRs) represent the most important drug targets. Although the smallest functional unit of a GPCR is a monomer, it became clear in the past decades that the vast majority of the receptors form dimers. Only very recently, however, data were presented that some receptors may in fact be expressed as a mixture of monomers and dimers and that the interaction of the receptor protomers is dynamic. To date, equilibrium measurements were restricted to the plasma membrane due to experimental limitations. We have addressed the question as to where this equilibrium is established for the corticotropin-releasing factor receptor type 1. By developing a novel approach to analyze single molecule fluorescence cross-correlation spectroscopy data for intracellular membrane compartments, we show that the corticotropin-releasing factor receptor type 1 has a specific monomer/dimer equilibrium that is already established in the endoplasmic reticulum (ER). It remains constant at the plasma membrane even following receptor activation. Moreover, we demonstrate for seven additional GPCRs that they are expressed in specific but substantially different monomer/dimer ratios. Although it is well known that proteins may dimerize in the ER in principle, our data show that the ER is also able to establish the specific monomer/dimer ratios of GPCRs, which sheds new light on the functions of this compartment. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  16. Meeting report - TGF-β superfamily: signaling in development and disease.

    Science.gov (United States)

    Zhang, Ying E; Newfeld, Stuart J

    2013-11-01

    The latest advances on the transforming growth factor β (TGF-β) and bone morphogenetic protein (BMP) signaling pathways were reported at the July 2013 FASEB Summer Research Conference 'The TGF-β Superfamily: Development and Disease'. The meeting was held in Steamboat Springs, Colorado, USA at 6700 feet above sea level in the Rocky Mountains. This was the seventh biannual meeting in the series. In attendance were investigators from a broad range of disciplines with a common interest in the mechanics of TGF-β and BMP signaling pathways, their normal developmental and homeostatic functions, and the diseases associated with pathway misregulation.

  17. Structure of TTHA1623, a novel metallo-β-lactamase superfamily protein from Thermus thermophilus HB8

    International Nuclear Information System (INIS)

    Yamamura, Akihiro; Okada, Akitoshi; Kameda, Yasuhiro; Ohtsuka, Jun; Nakagawa, Noriko; Ebihara, Akio; Nagata, Koji; Tanokura, Masaru

    2009-01-01

    The crystal structures of TTHA1623 from T. thermophilus HB8 in an iron-bound and a zinc-bound form have been determined to 2.8 and 2.2 Å resolution, respectively. TTHA1623 is a metallo-β-lactamase superfamily protein from the extremely thermophilic bacterium Thermus thermophilus HB8. Homologues of TTHA1623 exist in a wide range of bacteria and archaea and one eukaryote, Giardia lamblia, but their function remains unknown. To analyze the structural properties of TTHA1623, the crystal structures of its iron-bound and zinc-bound forms have been determined to 2.8 and 2.2 Å resolution, respectively. TTHA1623 possesses an αββα-fold similar to that of other metallo-β-lactamase superfamily proteins with glyoxalase II-type metal coordination. However, TTHA1623 exhibits a putative substrate-binding pocket with a unique shape

  18. RFamide Peptides: Structure, Function, Mechanisms and Pharmaceutical Potential

    Science.gov (United States)

    Findeisen, Maria; Rathmann, Daniel; Beck-Sickinger, Annette G.

    2011-01-01

    Different neuropeptides, all containing a common carboxy-terminal RFamide sequence, have been characterized as ligands of the RFamide peptide receptor family. Currently, five subgroups have been characterized with respect to their N-terminal sequence and hence cover a wide pattern of biological functions, like important neuroendocrine, behavioral, sensory and automatic functions. The RFamide peptide receptor family represents a multiligand/multireceptor system, as many ligands are recognized by several GPCR subtypes within one family. Multireceptor systems are often susceptible to cross-reactions, as their numerous ligands are frequently closely related. In this review we focus on recent results in the field of structure-activity studies as well as mutational exploration of crucial positions within this GPCR system. The review summarizes the reported peptide analogs and recently developed small molecule ligands (agonists and antagonists) to highlight the current understanding of the pharmacophoric elements, required for affinity and activity at the receptor family. Furthermore, we address the biological functions of the ligands and give an overview on their involvement in physiological processes. We provide insights in the knowledge for the design of highly selective ligands for single receptor subtypes to minimize cross-talk and to eliminate effects from interactions within the GPCR system. This will support the drug development of members of the RFamide family.

  19. RFamide Peptides: Structure, Function, Mechanisms and Pharmaceutical Potential

    Directory of Open Access Journals (Sweden)

    Maria Findeisen

    2011-09-01

    Full Text Available Different neuropeptides, all containing a common carboxy-terminal RFamide sequence, have been characterized as ligands of the RFamide peptide receptor family. Currently, five subgroups have been characterized with respect to their N-terminal sequence and hence cover a wide pattern of biological functions, like important neuroendocrine, behavioral, sensory and automatic functions. The RFamide peptide receptor family represents a multiligand/multireceptor system, as many ligands are recognized by several GPCR subtypes within one family. Multireceptor systems are often susceptible to cross-reactions, as their numerous ligands are frequently closely related. In this review we focus on recent results in the field of structure-activity studies as well as mutational exploration of crucial positions within this GPCR system. The review summarizes the reported peptide analogs and recently developed small molecule ligands (agonists and antagonists to highlight the current understanding of the pharmacophoric elements, required for affinity and activity at the receptor family. Furthermore, we address the biological functions of the ligands and give an overview on their involvement in physiological processes. We provide insights in the knowledge for the design of highly selective ligands for single receptor subtypes to minimize cross-talk and to eliminate effects from interactions within the GPCR system. This will support the drug development of members of the RFamide family.

  20. Transcription control and neuronal differentiation by agents that activate the LXR nuclear receptor family.

    Science.gov (United States)

    Schmidt, A; Vogel, R; Holloway, M K; Rutledge, S J; Friedman, O; Yang, Z; Rodan, G A; Friedman, E

    1999-09-10

    LXR and PPAR receptors belong to the nuclear receptor superfamily of transcriptional activating factors. Using ligand-dependent transcription assays, we found that 5-tetradecyloxy-2-furancarboxylic acid (TOFA) transactivates chimeric receptors composed of the glucocorticoid receptor DNA binding domain and the ligand binding regions of PPARalpha, PPARbeta (NUC-1) and LXRbeta (NER) receptors. In the same assays, ligands for PPARs (oleic acid, WY-14643 and L-631,033) and LXRs (hydroxycholesterols) maintain their respective receptor selectivity. TOFA and hydroxycholesterols also stimulate transcription from a minimal fibrinogen promoter that is under the control of AP-1 or NF-kappaB transcription factor binding sites. In addition to their effects on transcription, these LXRbeta activators induce neuronal differentiation in rat pheochromocytoma cells. TOFA and the natural LXR agonist, 22 (R)-hydroxycholesterol, stimulate neurite outgrowth in 55 and 28% of cells, respectively. No neurite outgrowth was induced by the related 22(S)-hydroxycholesterol, which does not activate the LXR family. These results suggest that the hydroxycholesterol signaling pathway has a complex effect on transcription that mediates the activity of TOFA and hydroxycholesterol on neuronal differentiation in pheochromocytoma cells.