WorldWideScience

Sample records for seven-transmembrane receptor activation

  1. Molecular pharmacological phenotyping of EBI2. An orphan seven-transmembrane receptor with constitutive activity

    DEFF Research Database (Denmark)

    Rosenkilde, Mette M; Benned-Jensen, Tau; Holst, Peter J

    2006-01-01

    Epstein-Barr virus (EBV)-induced receptor 2 (EBI2) is an orphan seven-transmembrane (7TM) receptor originally identified as the most up-regulated gene (>200-fold) in EBV-infected cells. Here we show that EBI2 signals with constitutive activity through Galpha(i) as determined by a receptor......2, showed similar expression and signaling through Galpha(i) as full-length EBI2. By using a 32P-labeled EBI2 probe we found a very high expression in lymphoid tissue (spleen and lymph node) and peripheral blood mononuclear cells and a high expression in lung tissue. Real-time PCR of EBV......-infected cells showed high expression of EBI2 during latent and lytic infection, in contrast to the EBV-encoded 7TM receptor BILF1, which was induced during lytic infection. EBI2 clustered with the orphan GPR18 by alignment analysis as well as by close proximity in the chromosomal region 13q32.3. Based...

  2. Metal ion site engineering indicates a global toggle switch model for seven-transmembrane receptor activation

    DEFF Research Database (Denmark)

    Elling, Christian E; Frimurer, Thomas M; Gerlach, Lars-Ole

    2006-01-01

    for monoamine binding in TM-III, was used as the starting point to engineer activating metal ion sites between the extracellular segments of the beta2-adrenergic receptor. Cu(II) and Zn(II) alone and in complex with aromatic chelators acted as potent (EC50 decreased to 0.5 microm) and efficacious agonists...

  3. Molecular pharmacology of promiscuous seven transmembrane receptors sensing organic nutrients

    DEFF Research Database (Denmark)

    Wellendorph, Petrine; Johansen, Lars Dan; Bräuner-Osborne, Hans

    2009-01-01

    A number of highly promiscuous seven transmembrane (7TM) receptors have been cloned and characterized within the last few years. It is noteworthy that many of these receptors are activated broadly by amino acids, proteolytic degradation products, carbohydrates, or free fatty acids and are expressed....... The promiscuous tendency in ligand recognition of these receptors is in contrast to the typical specific interaction with one physiological agonist seen for most receptors, which challenges the classic "lock-and-key" concept. We here review the molecular mechanisms of nutrient sensing of the calcium...... drug targets, to treat, for example, type II diabetes by mimicking food intake by potent agonists or positive allosteric modulators. The ligand-receptor interactions of the promiscuous receptors of organic nutrients thus remain an interesting subject of emerging functional importance....

  4. PheVI:09 (Phe6.44) as a sliding microswitch in seven-transmembrane (7TM) G protein-coupled receptor activation

    DEFF Research Database (Denmark)

    Valentin-Hansen, Louise; Holst, Birgitte; Frimurer, Thomas M

    2012-01-01

    In seven-transmembrane (7TM), G protein-coupled receptors, highly conserved residues function as microswitches, which alternate between different conformations and interaction partners in an extended allosteric interface between the transmembrane segments performing the large scale conformational...

  5. Ligand Modulation of the Epstein-Barr Virus-induced Seven-transmembrane Receptor EBI2

    DEFF Research Database (Denmark)

    Benned-Jensen, Tau; Smethurst, Christopher; Holst, Peter Johannes

    2011-01-01

    The Epstein-Barr virus-induced receptor 2 (EBI2) is a constitutively active seven-transmembrane receptor, which was recently shown to orchestrate the positioning of B cells in the follicle. To date, no ligands, endogenously or synthetic, have been identified that modulate EBI2 activity. Here we...

  6. A bioluminescence resonance energy transfer 2 (BRET2) assay for monitoring seven transmembrane receptor and insulin receptor crosstalk

    DEFF Research Database (Denmark)

    Sanni, Samra Joke; Kulahin, Nikolaj; Jorgensen, Rasmus

    2017-01-01

    The angiotensin AT1 receptor is a seven transmembrane (7TM) receptor, which mediates the regulation of blood pressure. Activation of angiotensin AT1 receptor may lead to impaired insulin signaling indicating crosstalk between angiotensin AT1 receptor and insulin receptor signaling pathways. To el...

  7. ETL, a novel seven-transmembrane receptor that is developmentally regulated in the heart. ETL is a member of the secretin family and belongs to the epidermal growth factor-seven-transmembrane subfamily.

    Science.gov (United States)

    Nechiporuk, T; Urness, L D; Keating, M T

    2001-02-09

    Using differential display of rat fetal and postnatal cardiomyocytes, we have identified a novel seven-transmembrane receptor, ETL. The cDNA-predicted amino acid sequence of ETL indicated that it encodes a 738-aa protein composed of a large extracellular domain with epidermal growth factor (EGF)-like repeats, a seven-transmembrane domain, and a short cytoplasmic tail. ETL belongs to the secretin family of G-protein-coupled peptide hormone receptors and the EGF-TM7 subfamily of receptors. The latter are characterized by a variable number of extracellular EGF and cell surface domains and conserved seven transmembrane-spanning regions. ETL mRNA expression is up-regulated in the adult rat and human heart. In situ hybridization analyses revealed expression in rat cardiomyocytes and abundant expression in vascular and bronchiolar smooth muscle cells. In COS-7 cells transfected with Myc-tagged rat ETL, rat ETL exists as a stable dimer and undergoes endoproteolytic cleavage of the extracellular domain. The proteolytic activity can be abolished by a specific mutation, T455A, in this domain. In transfected mammalian cells, ETL is associated with cell membranes and is also observed in cytoplasmic vesicles. ETL is the first seven-transmembrane receptor containing EGF-like repeats that is developmentally regulated in the heart.

  8. Promiscuous Seven Transmembrane Receptors Sensing L-α-amino Acids

    DEFF Research Database (Denmark)

    Smajilovic, Sanela; Wellendorph, Petrine; Bräuner-Osborne, Hans

    2014-01-01

    A number of nutrient sensing seven trans-membrane (7TM) receptors have been identified and characterized over the past few years. While the sensing mechanisms to carbohydrates and free fatty acids are well understood, the molecular basis of amino acid sensing has recently come to the limelight. T....... The present review describes the current status of promiscuous L-α-amino acid sensors, the calcium sensing receptor (CaSR), the GPRC6A receptor, the T1R1/T1R3 receptor and also their molecular pharmacology, expression pattern and physiological significance....

  9. Conformational constraining of inactive and active States of a seven transmembrane receptor by metal ion site engineering in the extracellular end of transmembrane segment V

    DEFF Research Database (Denmark)

    Rosenkilde, Mette M; David, Ralf; Oerlecke, Ilka

    2006-01-01

    The extracellular part of transmembrane segment V (TM-V) is expected to be involved in the activation process of 7TM receptors, but its role is far from clear. Here, we study the highly constitutively active CXC-chemokine receptor encoded by human herpesvirus 8 (ORF74-HHV8), in which a metal ion ...

  10. Agonists and inverse agonists for the herpesvirus 8-encoded constitutively active seven-transmembrane oncogene product, ORF-74

    DEFF Research Database (Denmark)

    Rosenkilde, M M; Kledal, T N; Bräuner-Osborne, Hans

    1999-01-01

    A number of CXC chemokines competed with similar, nanomolar affinity against 125I-interleukin-8 (IL-8) binding to ORF-74, a constitutively active seven-transmembrane receptor encoded by human herpesvirus 8. However, in competition against 125I-labeled growth-related oncogene (GRO)-alpha, the ORF-74...

  11. High constitutive activity of a virus-encoded seven transmembrane receptor in the absence of the conserved DRY motif (Asp-Arg-Tyr) in transmembrane helix 3

    DEFF Research Database (Denmark)

    Rosenkilde, Mette M; Kledal, Thomas N; Schwartz, Thue W

    2005-01-01

    of activated T cells. Coexpression of the ORF74-EHV2 receptor with the promiscuous G protein Gqi4myr supported the constitutive Gi activation as determined by inositol phosphate turnover and CREB activation. The constitutive activity was inhibited by nonpeptide inverse agonists with micromolar potencies...

  12. Structure, function and physiological consequences of virally encoded chemokine seven transmembrane receptors

    DEFF Research Database (Denmark)

    Rosenkilde, M M; Smit, M J; Waldhoer, M

    2008-01-01

    A number of human and animal herpes viruses encode G-protein coupled receptors with seven transmembrane (7TM) segments-most of which are clearly related to human chemokine receptors. It appears, that these receptors are used by the virus for immune evasion, cellular transformation, tissue targeting...... pathogenesis is still poorly understood. Here we focus on the current knowledge of structure, function and trafficking patterns of virally encoded chemokine receptors and further address the putative roles of these receptors in virus survival and host -cell and/or -immune system modulation. Finally, we...

  13. A bioluminescence resonance energy transfer 2 (BRET2) assay for monitoring seven transmembrane receptor and insulin receptor crosstalk.

    Science.gov (United States)

    Sanni, Samra Joke; Kulahin, Nikolaj; Jorgensen, Rasmus; Lyngsø, Christina; Gammeltoft, Steen; Hansen, Jakob Lerche

    2017-12-01

    The angiotensin AT1 receptor is a seven transmembrane (7TM) receptor, which mediates the regulation of blood pressure. Activation of angiotensin AT1 receptor may lead to impaired insulin signaling indicating crosstalk between angiotensin AT1 receptor and insulin receptor signaling pathways. To elucidate the molecular mechanisms behind this crosstalk, we applied the BRET2 technique to monitor the effect of angiotensin II on the interaction between Rluc8 tagged insulin receptor and GFP2 tagged insulin receptor substrates 1, 4, 5 (IRS1, IRS4, IRS5) and Src homology 2 domain-containing protein (Shc). We demonstrate that angiotensin II reduces the interaction between insulin receptor and IRS1 and IRS4, respectively, while the interaction with Shc is unaffected, and this effect is dependent on Gαq activation. Activation of other Gαq-coupled 7TM receptors led to a similar reduction in insulin receptor and IRS4 interactions whereas Gαs- and Gαi-coupled 7TM receptors had no effect. Furthermore, we used a panel of kinase inhibitors to show that angiotensin II engages different pathways when regulating insulin receptor interactions with IRS1 and IRS4. Angiotensin II inhibited the interaction between insulin receptor and IRS1 through activation of ERK1/2, while the interaction between insulin receptor and IRS4 was partially inhibited through protein kinase C dependent mechanisms. We conclude that the crosstalk between angiotensin AT1 receptor and insulin receptor signaling shows a high degree of specificity, and involves Gαq protein, and activation of distinct kinases. Thus, the BRET2 technique can be used as a platform for studying molecular mechanisms of crosstalk between insulin receptor and 7TM receptors.

  14. Selective elimination of high constitutive activity or chemokine binding in the human herpesvirus 8 encoded seven transmembrane oncogene ORF74

    DEFF Research Database (Denmark)

    Rosenkilde, M M; Kledal, T N; Holst, Peter Johannes

    2000-01-01

    Open reading frame 74 (ORF74) encoded by human herpesvirus 8 is a highly constitutively active seven transmembrane (7TM) receptor stimulated by angiogenic chemokines, e.g. growth-related oncogene-alpha, and inhibited by angiostatic chemokines e.g. interferon-gamma-inducible protein. Transgenic mice...... either agonist or inverse agonist modulation as well as high constitutive activity of the virally encoded oncogene ORF74 and that these mutant forms presumably can be used in transgenic animals to identify the molecular mechanism of its transforming activity....

  15. Apelin, the natural ligand of the orphan seven-transmembrane receptor APJ, inhibits human immunodeficiency virus type 1 entry

    NARCIS (Netherlands)

    Cayabyab, M.; Hinuma, S.; Farzan, M.; Choe, H.; Fukusumi, S.; Kitada, C.; Nishizawa, N.; Hosoya, M.; Nishimura, O.; Messele, T.; Pollakis, G.; Goudsmit, J.; Fujino, M.; Sodroski, J.

    2000-01-01

    In addition to the CCR5 and CXCR4 chemokine receptors, a subset of primary human immunodeficiency virus type 1 (HIV-1) isolates can also use the seven-transmembrane-domain receptor APJ as a coreceptor. A previously identified ligand of APJ, apelin, specifically inhibited the entry of primary

  16. Evolutionary and Comparative Genomics to Drive Rational Drug Design, with Particular Focus on Neuropeptide Seven-Transmembrane Receptors.

    Science.gov (United States)

    Furlong, Michael; Seong, Jae Young

    2017-01-01

    Seven transmembrane receptors (7TMRs), also known as G protein-coupled receptors, are popular targets of drug development, particularly 7TMR systems that are activated by peptide ligands. Although many pharmaceutical drugs have been discovered via conventional bulk analysis techniques the increasing availability of structural and evolutionary data are facilitating change to rational, targeted drug design. This article discusses the appeal of neuropeptide-7TMR systems as drug targets and provides an overview of concepts in the evolution of vertebrate genomes and gene families. Subsequently, methods that use evolutionary concepts and comparative analysis techniques to aid in gene discovery, gene function identification, and novel drug design are provided along with case study examples.

  17. HIV-1 Entry Cofactor: Functional cDNA Cloning of a Seven-Transmembrane, G Protein–Coupled Receptor

    OpenAIRE

    Feng, Yu; Broder, Christopher C.; Kennedy, Paul E.; Berger, Edward A.

    2011-01-01

    A cofactor for HIV-1 (human immunodeficiency virus-type 1) fusion and entry was identified with the use of a novel functional complementary DNA (cDNA) cloning strategy. This protein, designated “fusin,” is a putative G protein–coupled receptor with seven transmembrane segments. Recombinant fusin enabled CD4-expressing nonhuman cell types to support HIV-1 Env-mediated cell fusion and HIV-1 infection. Antibodies to fusin blocked cell fusion and infection with normal CD4-positive human target ce...

  18. Seven transmembrane G protein-coupled receptor repertoire of gastric ghrelin cells

    DEFF Research Database (Denmark)

    Engelstoft, Maja S; Park, Won-Mee; Sakata, Ichiro

    2013-01-01

    The molecular mechanisms regulating secretion of the orexigenic-glucoregulatory hormone ghrelin remain unclear. Based on qPCR analysis of FACS-purified gastric ghrelin cells, highly expressed and enriched 7TM receptors were comprehensively identified and functionally characterized using in vitro......, ex vivo and in vivo methods. Five Gαs-coupled receptors efficiently stimulated ghrelin secretion: as expected the β1-adrenergic, the GIP and the secretin receptors but surprisingly also the composite receptor for the sensory neuropeptide CGRP and the melanocortin 4 receptor. A number of Gαi....../o-coupled receptors inhibited ghrelin secretion including somatostatin receptors SSTR1, SSTR2 and SSTR3 and unexpectedly the highly enriched lactate receptor, GPR81. Three other metabolite receptors known to be both Gαi/o- and Gαq/11-coupled all inhibited ghrelin secretion through a pertussis toxin-sensitive Gαi...

  19. Seven Transmembrane Receptors as Shapeshifting Proteins: The Impact of Allosteric Modulation and Functional Selectivity on New Drug Discovery

    Science.gov (United States)

    Miller, Laurence J.

    2010-01-01

    It is useful to consider seven transmembrane receptors (7TMRs) as disordered proteins able to allosterically respond to a number of binding partners. Considering 7TMRs as allosteric systems, affinity and efficacy can be thought of in terms of energy flow between a modulator, conduit (the receptor protein), and a number of guests. These guests can be other molecules, receptors, membrane-bound proteins, or signaling proteins in the cytosol. These vectorial flows of energy can yield standard canonical guest allostery (allosteric modification of drug effect), effects along the plane of the cell membrane (receptor oligomerization), or effects directed into the cytosol (differential signaling as functional selectivity). This review discusses these apparently diverse pharmacological effects in terms of molecular dynamics and protein ensemble theory, which tends to unify 7TMR behavior toward cells. Special consideration will be given to functional selectivity (biased agonism and biased antagonism) in terms of mechanism of action and potential therapeutic application. The explosion of technology that has enabled observation of diverse 7TMR behavior has also shown how drugs can have multiple (pluridimensional) efficacies and how this can cause paradoxical drug classification and nomenclatures. PMID:20392808

  20. The seven-transmembrane receptor Gpr1 governs processes relevant for the antagonistic interaction of Trichoderma atroviride with its host

    Science.gov (United States)

    Omann, Markus R.; Lehner, Sylvia; Escobar Rodríguez, Carolina; Brunner, Kurt

    2012-01-01

    Mycoparasitic Trichoderma species are applied as biocontrol agents in agriculture to guard plants against fungal diseases. During mycoparasitism, Trichoderma directly interacts with phytopathogenic fungi, preceded by a specific recognition of the host and resulting in its disarming and killing. In various fungal pathogens, including mycoparasites, signalling via heterotrimeric G proteins plays a major role in regulating pathogenicity-related functions. However, the corresponding receptors involved in the recognition of host-derived signals are largely unknown. Functional characterization of Trichoderma atroviride Gpr1 revealed a prominent role of this seven-transmembrane protein of the cAMP-receptor-like family of fungal G-protein-coupled receptors in the antagonistic interaction with the host fungus and governing of mycoparasitism-related processes. Silencing of gpr1 led to an avirulent phenotype accompanied by an inability to attach to host hyphae. Furthermore, gpr1-silenced transformants were unable to respond to the presence of living host fungi with the expression of chitinase- and protease-encoding genes. Addition of exogenous cAMP was able to restore host attachment in gpr1-silenced transformants but could not restore mycoparasitic overgrowth. A search for downstream targets of the signalling pathway(s) involving Gpr1 resulted in the isolation of genes encoding e.g. a member of the cyclin-like superfamily and a small secreted cysteine-rich protein. Although silencing of gpr1 caused defects similar to those of mutants lacking the Tga3 Gα protein, no direct interaction between Gpr1 and Tga3 was observed in a split-ubiquitin two-hybrid assay. PMID:22075023

  1. A conserved aromatic lock for the tryptophan rotameric switch in TM-VI of seven-transmembrane receptors

    DEFF Research Database (Denmark)

    Holst, Birgitte; Nygaard, Rie; Hansen, Louise Valentin

    2010-01-01

    proximity. Mutational analysis was performed in the ghrelin receptor with multiple substitutions and with Ala substitutions in GPR119, GPR39, and the beta(2)-adrenergic receptor as well as the NK1 receptor. In all of these cases, it was found that mutation of the Trp-VI:13 rotameric switch itself eliminated...... could rotate into a position where it obtained an ideal aromatic-aromatic interaction with Phe-V:13. It is concluded that Phe-V:13 can serve as an aromatic lock for the proposed active conformation of the Trp-VI:13 rotameric switch, being involved in the global movement of TM-V and TM-VI in 7TM receptor...

  2. 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

    activity. We identified BILF1 receptor orthologues in 12 previously uncharacterized LCVs from nonhuman primates (NHPs) of Old and New World origin. As 7TM receptors are excellent drug targets, our unique insight into the molecular mechanism of action of the BILF1 family and into the evolution of primate......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...... EBV and 12 previously uncharacterized nonhuman primate (NHP) lymphocryptoviruses (LCVs). Phylogenetic analysis defined 3 BILF1 clades, corresponding to LCVs of New World monkeys (clade A) or Old World monkeys and great apes (clades B and C). Common functional properties were suggested by a high degree...

  3. Identification of common mechanisms by which human and mouse cytomegalovirus seven-transmembrane receptor homologues contribute to in vivo phenotypes in a mouse model

    DEFF Research Database (Denmark)

    Farrell, Helen E; Abraham, Alexander M; Cardin, Rhonda D

    2013-01-01

    The mouse cytomegalovirus chemokine receptor homologue (CKR) M33 is required for salivary gland tropism and efficient reactivation from latency, phenotypes partially rescued by the human cytomegalovirus CKR US28. Herein, we demonstrate that complementation of salivary gland tropism is mediated...

  4. Genetic dissection of the functions of the melanocortin-3 receptor, a seven-transmembrane G-protein-coupled receptor, suggests roles for central and peripheral receptors in energy homeostasis.

    Science.gov (United States)

    Begriche, Karima; Levasseur, Peter R; Zhang, Jingying; Rossi, Jari; Skorupa, Danielle; Solt, Laura A; Young, Brandon; Burris, Thomas P; Marks, Daniel L; Mynatt, Randall L; Butler, Andrew A

    2011-11-25

    The melanocortin-3 receptor (MC3R) gene is pleiotropic, influencing body composition, natriuresis, immune function, and entrainment of circadian rhythms to nutrient intake. MC3Rs are expressed in hypothalamic and limbic regions of the brain and in peripheral tissues. To investigate the roles of central MC3Rs, we inserted a "lox-stop-lox" (LoxTB) 5' of the translation initiation codon of the mouse Mc3r gene and reactivated transcription using neuron-specific Cre transgenic mice. As predicted based on earlier observations of Mc3r knock-out mice, Mc3r(TB/TB) mice displayed reduced lean mass, increased fat mass, and accelerated diet-induced obesity. Surprisingly, rescuing Mc3r expression in the nervous system using the Nestin-Cre transgene only partially rescued obesity in chow-fed conditions and had no impact on the accelerated diet-induced obesity phenotype. The ventromedial hypothalamus (VMH), a critical node in the neural networks regulating feeding-related behaviors and metabolic homeostasis, exhibits dense Mc3r expression relative to other brain regions. To target VMH MC3R expression, we used the steroidogenic factor-1 Cre transgenic mouse. Although restoring VMH MC3R signaling also had a modest impact on obesity, marked improvements in metabolic homeostasis were observed. VMH MC3R signaling was not sufficient to rescue the lean mass phenotype or the regulation of behaviors anticipating food anticipation. These results suggest that actions of MC3Rs impacting on energy homeostasis involve both central and peripheral sites of action. The impact of central MC3Rs on behavior and metabolism involves divergent pathways; VMH MC3R signaling improves metabolic homeostasis but does not significantly impact on the expression of behaviors anticipating nutrient availability.

  5. Predicting Kinase Activity in Angiotensin Receptor Phosphoproteomes Based on Sequence-Motifs and Interactions

    DEFF Research Database (Denmark)

    Bøgebo, Rikke; Horn, Heiko; Olsen, Jesper V

    2014-01-01

    Recent progress in the understanding of seven-transmembrane receptor (7TMR) signalling has promoted the development of a new generation of pathway selective ligands. The angiotensin II type I receptor (AT1aR) is one of the most studied 7TMRs with respect to selective activation of the β-arrestin ......Recent progress in the understanding of seven-transmembrane receptor (7TMR) signalling has promoted the development of a new generation of pathway selective ligands. The angiotensin II type I receptor (AT1aR) is one of the most studied 7TMRs with respect to selective activation of the β...

  6. Functionally Selective AT(1) Receptor Activation Reduces Ischemia Reperfusion Injury

    DEFF Research Database (Denmark)

    Hostrup, Anders; Christensen, Gitte Lund; Bentzen, Bo Hjort

    2012-01-01

    Angiotensin II (AngII) is a key peptide in cardiovascular homeostasis and is a ligand for the Angiotensin II type 1 and 2 seven transmembrane receptors (AT(1)R and AT(2)R). The AT(1 )receptor is a seven-transmembrane (7TM) G protein-coupled receptor (GPCR) mediating the majority of the physiologi...

  7. Structural motifs of importance for the constitutive activity of the orphan 7TM receptor EBI2: analysis of receptor activation in the absence of an agonist

    DEFF Research Database (Denmark)

    Benned-Jensen, Tau; Rosenkilde, Mette M

    2008-01-01

    of these molecular mechanisms for EBI2 activation is important from a drug development point of view, in that it may facilitate the rational design and development of small-molecule inverse agonists against EBI2 of putative importance as antiviral- or immune modulatory therapy.......The Epstein-Barr induced receptor 2 (EBI2) is a lymphocyte-expressed orphan seven transmembrane-spanning (7TM) receptor that signals constitutively through Galphai, as shown, for instance by guanosine 5'-O-(3-thio)triphosphate incorporation. Two regions of importance for the constitutive activity...

  8. Role of protease-activated receptor-2 in inflammation, and its possible implications as a putative mediator of periodontitis

    Directory of Open Access Journals (Sweden)

    M Holzhausen

    2005-03-01

    Full Text Available Proteinase-activated receptor-2 (PAR2 belongs to a novel subfamily of G-protein-coupled receptors with seven-transmembrane domains. This receptor is widely distributed throughout the body and seems to be importantly involved in inflammatory processes. PAR2 can be activated by serine proteases such as trypsin, mast cell tryptase, and bacterial proteases, such as gingipain produced by Porphyromonas gingivalis. This review describes the current stage of knowledge of the possible mechanisms that link PAR2 activation with periodontal disease, and proposes future therapeutic strategies to modulate the host response in the treatment of periodontitis.

  9. Interaction of chemokines with their receptors--from initial chemokine binding to receptor activating steps

    DEFF Research Database (Denmark)

    Thiele, Stefanie; Rosenkilde, Mette Marie

    2014-01-01

    The human chemokine system comprises 19 seven-transmembrane helix (7TM) receptors and 45 endogenous chemokines that often interact with each other in a promiscuous manner. Due to the chemokine system's primary function in leukocyte migration, it has a central role in immune homeostasis and survei......The human chemokine system comprises 19 seven-transmembrane helix (7TM) receptors and 45 endogenous chemokines that often interact with each other in a promiscuous manner. Due to the chemokine system's primary function in leukocyte migration, it has a central role in immune homeostasis...

  10. Quantitative phosphoproteomics dissection of 7TM receptor signaling using full and biased agonists

    DEFF Research Database (Denmark)

    Christensen, Gitte Lund; Kelstrup, Christian; Lyngsø, Christina

    2010-01-01

    Seven-transmembrane receptors (7TMRs) signal through the well described heterotrimeric G proteins, but can also activate G protein-independent signaling pathways of which the impact and complexity are less understood. The Angiotensin II type 1 receptor (AT1R) is a prototypical 7TMR and an importa...

  11. A constitutively active G-protein-coupled receptor causes mating self-compatibility in the mushroom Coprinus.

    Science.gov (United States)

    Olesnicky, N S; Brown, A J; Dowell, S J; Casselton, L A

    1999-01-01

    In the mushroom Coprinus cinereus, the multiallelic B mating type genes are predicted to encode a large family of seven-transmembrane domain receptors and CaaX-modified pheromones. We have shown that a single amino acid change Q229P in transmembrane domain VI of one receptor confers a self-compatible mating phenotype. Using a heterologous yeast assay, we have demonstrated that this C.cinereus pheromone receptor is a G-protein-coupled receptor and that the Q229P mutation is constitutively activating. A C.cinereus pheromone precursor was processed to an active species specifically in yeast MATa cells and activated the co-expressed wild-type receptor. Yeast cells expressing the wild-type receptor were used to test the activity of synthetic peptides, enabling us to predict the structure of the mature C.cinereus pheromone and to show that the Q229P mutation does not compromise normal receptor function. PMID:10329622

  12. The emerging role of promiscuous 7TM receptors as chemosensors for food intake

    DEFF Research Database (Denmark)

    Wellendorph, Petrine; Johansen, Lars Dan; Bräuner-Osborne, Hans

    2010-01-01

    In recent years, several highly promiscuous seven transmembrane (7TM) receptors have been cloned and characterized of which many are activated broadly by amino acids, proteolytic degradation products, carbohydrates, or free fatty acids (FFAs) and are expressed in taste tissue, the gastrointestinal...

  13. SGIP1 alters internalization and modulates signaling of activated cannabinoid receptor 1 in a biased manner

    Czech Academy of Sciences Publication Activity Database

    Hájková, Alena; Techlovská, Šárka; Dvořáková, Michaela; Chambers, Jayne Nicole; Kumpošt, Jiří; Hubálková, Pavla; Prezeau, L.; Blahoš, Jaroslav

    2016-01-01

    Roč. 107, léto (2016), s. 201-214 ISSN 0028-3908 R&D Projects: GA ČR GAP303/12/2408 Institutional support: RVO:68378050 Keywords : Seven transmembrane receptors * G-protein coupled receptors * Cannabinoid receptor 1 * Protein-protein interactions * Bias signaling * Receptor endocytosis Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 5.012, year: 2016

  14. Search for a platelet-activating factor receptor in the Trypanosoma cruzi proteome: a potential target for Chagas disease chemotherapy

    Directory of Open Access Journals (Sweden)

    Daniel Fábio Kawano

    2011-12-01

    Full Text Available Chagas disease (CD causes the highest burden of parasitic diseases in the Western Hemisphere and is therefore a priority for drug research and development. Platelet-activating factor (PAF causes the CD parasite Trypanosoma cruzi to differentiate, which suggests that the parasite may express PAF receptors. Here, we explored the T. cruzi proteome for PAF receptor-like proteins. From a total of 23,000 protein sequences, we identified 29 hypothetical proteins that are predicted to have seven transmembrane domains (TMDs, which is the main characteristic of the G protein-coupled receptors (GPCRs, including the PAF receptor. The TMDs of these sequences were independently aligned with domains from 25 animal PAF receptors and the sequences were analysed for conserved residues. The conservation score mean values for the TMDs of the hypothetical proteins ranged from 31.7-44.1%, which suggests that if the putative T. cruzi PAF receptor is among the sequences identified, the TMDs are not highly conserved. These results suggest that T. cruzi contains several GPCR-like proteins and that one of these GPCRs may be a PAF receptor. Future studies may further validate the PAF receptor as a target for CD chemotherapy.

  15. Ligand binding to G protein-coupled receptors in tethered cell membranes

    DEFF Research Database (Denmark)

    Martinez, Karen L.; Meyer, Bruno H.; Hovius, Ruud

    2003-01-01

    for the surface immobilization of membrane proteins was developed using the prototypic seven transmembrane neurokinin-1 receptor. The receptor was expressed as a biotinylated protein in mammalian cells. Membranes from cell homogenates were selectively immobilized on glass surfaces covered with streptavidin. TIRF...... measurements showed that a fluorescent agonist binds to the receptor on the sensor surface with similar affinity as to the receptor in live cells. This approach offers the possibility to investigate minute amounts of membrane protein in an active form and in its native environment without purification....

  16. Conserved waters mediate structural and functional activation of family A (rhodopsin-like) G protein-coupled receptors

    Energy Technology Data Exchange (ETDEWEB)

    Angel, T.; Chance, M; Palczewski, K

    2009-01-01

    G protein-coupled receptors with seven transmembrane {alpha}-helices (GPCRs) comprise the largest receptor superfamily and are involved in detecting a wide variety of extracellular stimuli. The availability of high-resolution crystal structures of five prototypical GPCRs, bovine and squid rhodopsin, engineered A2A-adenosine, {beta}1- and {beta}2-adrenergic receptors, permits comparative analysis of features common to these and likely all GPCRs. We provide an analysis of the distribution of water molecules in the transmembrane region of these GPCR structures and find conserved contacts with microdomains demonstrated to be involved in receptor activation. Colocalization of water molecules associating with highly conserved and functionally important residues in several of these GPCR crystal structures supports the notion that these waters are likely to be as important to proper receptor function as the conserved residues. Moreover, in the absence of large conformational changes in rhodopsin after photoactivation, we propose that ordered waters contribute to the functional plasticity needed to transmit activation signals from the retinal-binding pocket to the cytoplasmic face of rhodopsin and that fundamental features of the mechanism of activation, involving these conserved waters, are shared by many if not all family A receptors.

  17. The adhesion G protein-coupled receptor G2 (ADGRG2/GPR64) constitutively activates SRE and NFκB and is involved in cell adhesion and migration

    DEFF Research Database (Denmark)

    Cornelia Peeters, Miriam; Fokkelman, Michiel; Boogaard, Bob

    2015-01-01

    Adhesion G protein-coupled receptors (ADGRs) are believed to be activated by auto-proteolytic cleavage of their very large extracellular N-terminal domains normally acting as a negative regulator of the intrinsically constitutively active seven transmembrane domain. ADGRG2 (or GPR64) which...... intracellular signal transduction. Knockdown of ADGRG2 by siRNA in the highly motile breast cancer cell lines Hs578T and MDA-MB-231 resulted in a strong reduction in cell adhesion and subsequent cell migration which was associated with a selective reduction in RelB, an NFκB family member. It is concluded...... that the adhesion GPCR ADGRG2 is critically involved in the adhesion and migration of certain breast cancer cells through mechanisms including a non-canonical NFkB pathway and that ADGRG2 could be a target for treatment of certain types of cancer....

  18. Molecular identification of the first SIFamide receptor

    DEFF Research Database (Denmark)

    Jørgensen, Lars M; Hauser, Frank; Cazzamali, Giuseppe

    2006-01-01

    enigmatic. Here, we have identified the Drosophila gene (CG10823) coding for the SIFamide receptor. When expressed in Chinese hamster ovary cells, the receptor is only activated by Drosophila SIFamide (EC(50), 2x10(-8)M) and not by a library of 32 other insect neuropeptides and eight biogenic amines....... Database searches revealed SIFamide receptor orthologues in the genomes from the malaria mosquito Anopheles gambiae, the silkworm Bombyx mori, the red flour beetle Tribolium castaneum, and the honey bee Apis mellifera. An alignment of the five insect SIFamide or SIFamide-like receptors showed, again......, an impressive sequence conservation (67-77% amino acid sequence identities between the seven-transmembrane areas; 82-87% sequence similarities). The identification of well-conserved SIFamide receptor orthologues in all other insects with a sequenced genome, suggests that the SIFamide/receptor couple must have...

  19. Ligand-specific regulation of the extracellular surface of a G-protein-coupled receptor

    DEFF Research Database (Denmark)

    Bokoch, Michael P; Zou, Yaozhong; Rasmussen, Søren Gøgsig Faarup

    2010-01-01

    G-protein-coupled receptors (GPCRs) are seven-transmembrane proteins that mediate most cellular responses to hormones and neurotransmitters. They are the largest group of therapeutic targets for a broad spectrum of diseases. Recent crystal structures of GPCRs have revealed structural conservation...... receptor: a salt bridge linking extracellular loops 2 and 3. Small-molecule drugs that bind within the transmembrane core and exhibit different efficacies towards G-protein activation (agonist, neutral antagonist and inverse agonist) also stabilize distinct conformations of the ECS. We thereby demonstrate...

  20. Differential extracellular signal-regulated kinases 1 and 2 activation by the angiotensin type 1 receptor supports distinct phenotypes of cardiac myocytes

    DEFF Research Database (Denmark)

    Aplin, Mark; Christensen, Gitte Lund; Schneider, Mikael

    2007-01-01

    The angiotensin II (AngII) type 1 receptor (AT(1)R) is a seven-transmembrane receptor well established to activate extracellular signal-regulated kinases 1 and 2 (ERK1/2) by discrete G protein-dependent and beta-arrestin2-dependent pathways. The biological importance of this, however, remains obs...... obscure. Application of the modified analogue [Sar(1), Ile(4), Ile(8)]-AngII ([SII] AngII) allowed us to dissect the two pathways of ERK1/2 activation in native cardiac myocytes. Although cytosol-retained, the beta-arrestin2-bound pool of ERK1/2 represents an active signalling component...... that phosphorylates p90 Ribosomal S6 Kinase, a ubiquitous and versatile mediator of ERK1/2 signal transduction. Moreover, the beta-arrestin2-dependent ERK1/2 signal supports intact proliferation of cardiac myocytes. In contrast to G(q)-activated ERK1/2, and in keeping with its failure to translocate to the nucleus......, the beta-arrestin2-scaffolded pool of ERK1/2 does not phosphorylate the transcription factor Elk-1, induces no increased transcription of the immediate-early gene c-Fos, and does not entail myocyte hypertrophy. These results clearly demonstrate the biological significance of differential signalling...

  1. Uncovering the triggers for GPCR activation using solid-state NMR spectroscopy

    Science.gov (United States)

    Kimata, Naoki; Reeves, Philip J.; Smith, Steven O.

    2015-04-01

    G protein-coupled receptors (GPCRs) span cell membranes with seven transmembrane helices and respond to a diverse array of extracellular signals. Crystal structures of GPCRs have provided key insights into the architecture of these receptors and the role of conserved residues. However, the question of how ligand binding induces the conformational changes that are essential for activation remains largely unanswered. Since the extracellular sequences and structures of GPCRs are not conserved between receptor subfamilies, it is likely that the initial molecular triggers for activation vary depending on the specific type of ligand and receptor. In this article, we describe NMR studies on the rhodopsin subfamily of GPCRs and propose a mechanism for how retinal isomerization switches the receptor to the active conformation. These results suggest a general approach for determining the triggers for activation in other GPCR subfamilies using NMR spectroscopy.

  2. The Second Transmembrane Domain of the Human Type 1 Angiotensin II Receptor Participates in the Formation of the Ligand Binding Pocket and Undergoes Integral Pivoting Movement during the Process of Receptor Activation*

    Science.gov (United States)

    Domazet, Ivana; Holleran, Brian J.; Martin, Stéphane S.; Lavigne, Pierre; Leduc, Richard; Escher, Emanuel; Guillemette, Gaétan

    2009-01-01

    The octapeptide hormone angiotensin II (AngII) exerts a wide variety of cardiovascular effects through the activation of the angiotensin II type-1 (AT1) receptor, which belongs to the G protein-coupled receptor superfamily. Like other G protein-coupled receptors, the AT1 receptor possesses seven transmembrane domains that provide structural support for the formation of the ligand-binding pocket. In order to identify those residues in the second transmembrane domain (TMD2) that contribute to the formation of the binding pocket of the AT1 receptor, we used the substituted cysteine accessibility method. All of the residues within the Leu-70 to Trp-94 region were mutated one at a time to a cysteine, and, after expression in COS-7 cells, the mutant receptors were treated with the sulfhydryl-specific alkylating agent methanethiosulfonate-ethylammonium (MTSEA). MTSEA reacts selectively with water-accessible, free sulfhydryl groups of endogenous or introduced point mutation cysteines. If a cysteine is found in the binding pocket, the covalent modification will affect the binding kinetics of the ligand. MTSEA substantially decreased the binding affinity of D74C-AT1, L81C-AT1, A85C-AT1, T88C-AT1, and A89C-AT1 mutant receptors, which suggests that these residues orient themselves within the water-accessible binding pocket of the AT1 receptor. Interestingly, this pattern of acquired MTSEA sensitivity was altered for TMD2 reporter cysteines engineered in a constitutively active N111G-AT1 receptor background. Indeed, mutant D74C-N111G-AT1 became insensitive to MTSEA, whereas mutant L81C-N111G-AT1 lost some sensitivity and mutant V86C-N111G-AT1 became sensitive to MTSEA. Our results suggest that constitutive activation of the AT1 receptor causes TMD2 to pivot, bringing the top of TMD2 closer to the binding pocket and pushing the bottom of TMD2 away from the binding pocket. PMID:19276075

  3. The L-alpha-amino acid receptor GPRC6A is expressed in the islets of Langerhans but is not involved in L-arginine-induced insulin release

    DEFF Research Database (Denmark)

    Smajilovic, Sanela; Clemmensen, Christoffer; Johansen, Lars Dan

    2013-01-01

    insulin secretion; therefore, the receptor has been hypothesized to have a role in regulating glucose metabolism. In this study, we demonstrate that GPRC6A is expressed in islets of Langerhans, but activation of the receptor by L-arginine did not stimulate insulin secretion. We also investigated central......GPRC6A is a seven-transmembrane receptor activated by a wide range of L: -a-amino acids, most potently by L: -arginine and other basic amino acids. The receptor is broadly expressed, but its exact physiological role remains to be elucidated. It is well established that L: -arginine stimulates...... metabolic parameters in GPRC6A knockout mice compared with wildtype littermates and found no difference in glucose metabolism or body fat percentage when mice were administered a standard chow diet. In conclusion, our data do not support a role for GPRC6A in L-arginine-induced insulin release and glucose...

  4. Crystal structure of a multi-domain human smoothened receptor in complex with a super stabilizing ligand

    Science.gov (United States)

    Zhang, Xianjun; Zhao, Fei; Wu, Yiran; Yang, Jun; Han, Gye Won; Zhao, Suwen; Ishchenko, Andrii; Ye, Lintao; Lin, Xi; Ding, Kang; Dharmarajan, Venkatasubramanian; Griffin, Patrick R.; Gati, Cornelius; Nelson, Garrett; Hunter, Mark S.; Hanson, Michael A.; Cherezov, Vadim; Stevens, Raymond C.; Tan, Wenfu; Tao, Houchao; Xu, Fei

    2017-05-01

    The Smoothened receptor (SMO) belongs to the Class Frizzled of the G protein-coupled receptor (GPCR) superfamily, constituting a key component of the Hedgehog signalling pathway. Here we report the crystal structure of the multi-domain human SMO, bound and stabilized by a designed tool ligand TC114, using an X-ray free-electron laser source at 2.9 Å. The structure reveals a precise arrangement of three distinct domains: a seven-transmembrane helices domain (TMD), a hinge domain (HD) and an intact extracellular cysteine-rich domain (CRD). This architecture enables allosteric interactions between the domains that are important for ligand recognition and receptor activation. By combining the structural data, molecular dynamics simulation, and hydrogen-deuterium-exchange analysis, we demonstrate that transmembrane helix VI, extracellular loop 3 and the HD play a central role in transmitting the signal employing a unique GPCR activation mechanism, distinct from other multi-domain GPCRs.

  5. Mechanism for the activation of glutamate receptors

    Science.gov (United States)

    Scientists at the NIH have used a technique called cryo-electron microscopy to determine a molecular mechanism for the activation and desensitization of ionotropic glutamate receptors, a prominent class of neurotransmitter receptors in the brain and spina

  6. Urokinase-type plasminogen activator receptor

    DEFF Research Database (Denmark)

    2003-01-01

    A polypeptide presenting an epitope cross-reactive with an epitope of urokinase-type plasminogen activator receptor, and/or having uPA binding activity, is described.......A polypeptide presenting an epitope cross-reactive with an epitope of urokinase-type plasminogen activator receptor, and/or having uPA binding activity, is described....

  7. Molecular determinants of odorant receptor function in insects

    Indian Academy of Sciences (India)

    2014-07-20

    Jul 20, 2014 ... 10.1007/s12038-014-9447-7. 1. Introduction. The Odorant receptor gene family in insects encodes seven-transmembrane-domain proteins that detect a wide variety of volatile ... tations in the AgOr1 ORF were introduced using a PCR ..... insect vectors like the malaria mosquito Anopheles gambiae.

  8. CERAPP: Collaborative Estrogen Receptor Activity Prediction Project

    Data.gov (United States)

    U.S. Environmental Protection Agency — Data from a large-scale modeling project called CERAPP (Collaborative Estrogen Receptor Activity Prediction Project) demonstrating using predictive computational...

  9. Molecular cloning, genomic organization, and developmental regulation of a novel receptor from Drosophila melanogaster structurally related to members of the thyroid-stimulating hormone, follicle-stimulating hormone, luteinizing hormone/choriogonadotropin receptor family from mammals

    DEFF Research Database (Denmark)

    Hauser, F; Nothacker, H P; Grimmelikhuijzen, C J

    1997-01-01

    ); follicle-stimulating hormone (FSH); luteinizing hormone/choriogonadotropin (LH/CG)) receptor family from mammals. This homology includes a very large, extracellular N terminus (20% sequence identity with rat TSH, 19% with rat FSH, and 20% with the rat LH/CG receptor) and a seven-transmembrane region (53...

  10. The Atypical Receptor CCRL2 (C-C Chemokine Receptor-Like 2) Does Not Act As a Decoy Receptor in Endothelial Cells.

    Science.gov (United States)

    Mazzotti, Chiara; Gagliostro, Vincenzo; Bosisio, Daniela; Del Prete, Annalisa; Tiberio, Laura; Thelen, Marcus; Sozzani, Silvano

    2017-01-01

    C-C chemokine receptor-like 2 (CCRL2) is a non-signaling seven-transmembrane domain (7-TMD) receptor related to the atypical chemokine receptor (ACKR) family. ACKRs bind chemokines but do not activate G protein-dependent signaling or cell functions. ACKRs were shown to regulate immune functions in vivo by their ability to scavenge chemokines from the local environment. This study was performed to investigate whether CCRL2 shares two of the main characteristics of ACKRs, namely the ability to internalize and scavenge the ligands. Cell membrane analysis of CCRL2-transfected cells revealed a weak, constitutive, ligand-independent internalization, and recycling of CCRL2, with a kinetics that was slower than those observed with ACKR3, a prototypic ACKR, or other chemotactic signaling receptors [i.e., chemokine-like receptor 1 and C-X-C motif chemokine receptor 2]. Intracellularly, CCRL2 colocalized with early endosome antigen 1-positive and Rab5-positive vesicles and with recycling compartments mainly characterized by Rab11-positive vesicles. CCRL2-transfected cells and activated mouse blood endothelial cells, that endogenously express CCRL2, were used to investigate the scavenging ability of CCRL2. These experiments confirmed the ability of CCRL2 to bind chemerin, the only recognized ligand, but excluded the ability of CCRL2 to perform scavenging. Collectively, these results identify unique functional properties for this member of the non-signaling 7-TMD receptor family.

  11. 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

  12. Structural basis of ligand interaction with atypical chemokine receptor 3

    Science.gov (United States)

    Gustavsson, Martin; Wang, Liwen; van Gils, Noortje; Stephens, Bryan S.; Zhang, Penglie; Schall, Thomas J.; Yang, Sichun; Abagyan, Ruben; Chance, Mark R.; Kufareva, Irina; Handel, Tracy M.

    2017-01-01

    Chemokines drive cell migration through their interactions with seven-transmembrane (7TM) chemokine receptors on cell surfaces. The atypical chemokine receptor 3 (ACKR3) binds chemokines CXCL11 and CXCL12 and signals exclusively through β-arrestin-mediated pathways, without activating canonical G-protein signalling. This receptor is upregulated in numerous cancers making it a potential drug target. Here we collected over 100 distinct structural probes from radiolytic footprinting, disulfide trapping, and mutagenesis to map the structures of ACKR3:CXCL12 and ACKR3:small-molecule complexes, including dynamic regions that proved unresolvable by X-ray crystallography in homologous receptors. The data are integrated with molecular modelling to produce complete and cohesive experimentally driven models that confirm and expand on the existing knowledge of the architecture of receptor:chemokine and receptor:small-molecule complexes. Additionally, we detected and characterized ligand-induced conformational changes in the transmembrane and intracellular regions of ACKR3 that elucidate fundamental structural elements of agonism in this atypical receptor.

  13. Structural basis of ligand interaction with atypical chemokine receptor 3

    Energy Technology Data Exchange (ETDEWEB)

    Gustavsson, Martin; Wang, Liwen; van Gils, Noortje; Stephens, Bryan S.; Zhang, Penglie; Schall, Thomas J.; Yang, Sichun; Abagyan, Ruben; Chance, Mark R.; Kufareva, Irina; Handel, Tracy M.

    2017-01-18

    Chemokines drive cell migration through their interactions with seven-transmembrane (7TM) chemokine receptors on cell surfaces. The atypical chemokine receptor 3 (ACKR3) binds chemokines CXCL11 and CXCL12 and signals exclusively through β-arrestin-mediated pathways, without activating canonical G-protein signalling. This receptor is upregulated in numerous cancers making it a potential drug target. Here we collected over 100 distinct structural probes from radiolytic footprinting, disulfide trapping, and mutagenesis to map the structures of ACKR3:CXCL12 and ACKR3:small-molecule complexes, including dynamic regions that proved unresolvable by X-ray crystallography in homologous receptors. The data are integrated with molecular modelling to produce complete and cohesive experimentally driven models that confirm and expand on the existing knowledge of the architecture of receptor:chemokine and receptor:small-molecule complexes. Additionally, we detected and characterized ligand-induced conformational changes in the transmembrane and intracellular regions of ACKR3 that elucidate fundamental structural elements of agonism in this atypical receptor.

  14. Multiple melanocortin receptors are expressed in bone cells

    Science.gov (United States)

    Zhong, Qing; Sridhar, Supriya; Ruan, Ling; Ding, Ke-Hong; Xie, Ding; Insogna, Karl; Kang, Baolin; Xu, Jianrui; Bollag, Roni J.; Isales, Carlos M.

    2005-01-01

    Melanocortin receptors belong to the seven transmembrane domain, G-protein coupled family of receptors. There are five members of this receptor family labeled MC1R-MC5R. These receptors are activated by fragments derived from a larger molecule, proopiomelanocortin (POMC) and include ACTH, alpha beta and gamma-MSH and beta-endorphin. Because of in vitro and in vivo data suggesting direct effects of these POMC molecules on bone and bone turnover, we examined bone and bone derived cells for the presence of the various members of the melanocortin receptor family. We report that the five known melanocortin receptors are expressed to varying degrees in osteoblast-like and osteoclastic cells. POMC fragments increased proliferation and expression of a variety of genes in osteoblastic cells. Furthermore, POMC mRNA was detected in osteoclastic cells. These data demonstrate that POMC-derived peptide hormones acting through high affinity melanocortin receptors have specific effects on bone cells. Thus, in addition to the indirect effects of POMC-derived hormones on bone turnover through their modulation of steroid hormone secretion, POMC fragments may have direct and specific effects on bone cell subpopulations.

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

    NARCIS (Netherlands)

    Liebscher, Ines; Ackley, Brian; Araç, Demet; Ariestanti, Donna M.; Aust, Gabriela; Bae, Byoung-Il; Bista, Bigyan R.; Bridges, James P.; Duman, Joseph G.; Engel, Felix B.; Giera, Stefanie; Goffinet, André M.; Hall, Randy A.; Hamann, Jörg; Hartmann, Nicole; Lin, Hsi-Hsien; Liu, Mingyao; Luo, Rong; Mogha, Amit; Monk, Kelly R.; Peeters, Miriam C.; Prömel, Simone; Ressl, Susanne; Schiöth, Helgi B.; Sigoillot, Séverine M.; Song, Helen; Talbot, William S.; Tall, Gregory G.; White, James P.; Wolfrum, Uwe; Xu, Lei; Piao, Xianhua

    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 alpha-helix-a structural feature of all GPCRs-the class of aGPCRs is characterized by the presence of a large N-terminal extracellular

  16. G protein-coupled receptors self-assemble in dynamics simulations of model bilayers

    NARCIS (Netherlands)

    Periole, Xavier; Huber, Thomas; Marrink, Siewert-Jan; Sakmar, Thomas P.

    2007-01-01

    Many integral membrane proteins assemble to form oligomeric structures in biological membranes. In particular, seven-transmembrane helical G protein-coupled receptors (GPCRs) appear to self-assemble constitutively in membranes, but the mechanism and physiological role of this assembly are unknown.

  17. Spatially restricted expression of candidate taste receptors in the Drosophila gustatory system

    National Research Council Canada - National Science Library

    Dunipace, Leslie; Meister, Stephan; McNealy, Corum; Amrein, Hubert

    2001-01-01

    ... of seven-transmembrane receptors that are dedicated to olfactory, gustatory, or pheromonal sensory perception [1–11] As in most vertebrates, chemoreception in Drosophila can be divided into two distinct modalities—smell (olfaction), the recognition of volatile molecules, and taste (gustation), the recognition of soluble molecules [12] . In Dros...

  18. In Vivo Phosphoproteomics Analysis Reveals the Cardiac Targets of β-Adrenergic Receptor Signaling

    DEFF Research Database (Denmark)

    Lundby, Alicia; Andersen, Martin N; Steffensen, Annette B

    2013-01-01

    used quantitative in vivo phosphoproteomics to identify 670 site-specific phosphorylation changes in murine hearts in response to acute treatment with specific βAR agonists. The residues adjacent to the regulated phosphorylation sites exhibited a sequence-specific preference (R......) of the potassium channel KV7.1, increased current amplitude. Our data set represents a quantitative analysis of phosphorylated proteins regulated in vivo upon stimulation of seven-transmembrane receptors, and our findings reveal previously unknown phosphorylation sites that regulate myocardial contractility......-X-X-pS/T), and integrative analysis of sequence motifs and interaction networks suggested that the kinases AMPK (adenosine 5'-monophosphate-activated protein kinase), Akt, and mTOR (mammalian target of rapamycin) mediate βAR signaling, in addition to the well-established pathways mediated by PKA (cyclic adenosine...

  19. The significance of G protein-coupled receptor crystallography for drug discovery.

    Science.gov (United States)

    Salon, John A; Lodowski, David T; Palczewski, Krzysztof

    2011-12-01

    Crucial as molecular sensors for many vital physiological processes, seven-transmembrane domain G protein-coupled receptors (GPCRs) comprise the largest family of proteins targeted by drug discovery. Together with structures of the prototypical GPCR rhodopsin, solved structures of other liganded GPCRs promise to provide insights into the structural basis of the superfamily's biochemical functions and assist in the development of new therapeutic modalities and drugs. One of the greatest technical and theoretical challenges to elucidating and exploiting structure-function relationships in these systems is the emerging concept of GPCR conformational flexibility and its cause-effect relationship for receptor-receptor and receptor-effector interactions. Such conformational changes can be subtle and triggered by relatively small binding energy effects, leading to full or partial efficacy in the activation or inactivation of the receptor system at large. Pharmacological dogma generally dictates that these changes manifest themselves through kinetic modulation of the receptor's G protein partners. Atomic resolution information derived from increasingly available receptor structures provides an entrée to the understanding of these events and practically applying it to drug design. Supported by structure-activity relationship information arising from empirical screening, a unified structural model of GPCR activation/inactivation promises to both accelerate drug discovery in this field and improve our fundamental understanding of structure-based drug design in general. This review discusses fundamental problems that persist in drug design and GPCR structural determination.

  20. Steroid and G Protein Binding Characteristics of the Seatrout and Human Progestin Membrane Receptor Alpha Subtypes and Their Evolutionary Origins

    NARCIS (Netherlands)

    Thomas, P.; Pang, Y.; Dong, J.; Groenen, P.; Kelder, J.; Vlieg, J. de; Zhu, Y.; Tubbs, C.

    2006-01-01

    A novel progestin receptor (mPR) with seven-transmembrane domains was recently discovered in spotted seatrout and homologous genes were identified in other vertebrates. Here we show that cDNAs for the mPR alpha subtypes from spotted seatrout (st-mPRalpha) and humans (hu-mPRalpha) encode progestin

  1. Activation of Neuropeptide FF Receptors by Kisspeptin Receptor Ligands

    Science.gov (United States)

    2010-01-01

    Kisspeptin is a member of the RFamide neuropeptide family that is implicated in gonadotropin secretion. Because kisspeptin-GPR54 signaling is implicated in the neuroendocrine regulation of reproduction, GPR54 ligands represent promising therapeutic agents against endocrine secretion disorders. In the present study, the selectivity profiles of GPR54 agonist peptides were investigated for several GPCRs, including RFamide receptors. Kisspeptin-10 exhibited potent binding and activation of neuropeptide FF receptors (NPFFR1 and NPFFR2). In contrast, short peptide agonists bound with much lower affinity to NPFFRs while showing relatively high selectivity toward GPR54. The possible localization of secondary kisspeptin targets was also demonstrated by variation in the levels of GnRH release from the median eminence and the type of GPR54 agonists used. Negligible affinity of the reported NPFFR ligands to GPR54 was observed and indicates the unidirectional cross-reactivity between both ligands. PMID:24900254

  2. NMDA receptor activity in neuropsychiatric disorders

    Directory of Open Access Journals (Sweden)

    Shaheen E Lakhan

    2013-06-01

    Full Text Available N-Methyl-D-aspartate (NMDA receptors play a variety of physiologic roles and their proper signaling is essential for cellular homeostasis. Any disruption in this pathway, leading to either enhanced or decreased activity, may result in the manifestation of neuropsychiatric pathologies such as schizophrenia, mood disorders, substance induced psychosis, Huntington's disease, Alzheimer's disease, and neuropsychiatric systemic lupus erythematosus. Here, we explore the notion that the overlap in activity of at least one biochemical pathway, the NMDA receptor pathway, may be the link to understanding the overlap in psychotic symptoms between diseases. This review intends to present a broad overview of those neuropsychiatric disorders for which alternations in NMDA receptor activity is prominent thus suggesting that continued direction of pharmaceutical intervention to this pathway may present a viable option for managing symptoms.

  3. Transcriptional peroxisome proliferator-activated receptor γ ...

    African Journals Online (AJOL)

    user

    Peroxisome proliferator-activated receptor γ coactivator (PGC)-1ɑ, a well-known member of PGC-1 transcriptional coactivator's family, plays a key role in various metabolic pathways. Here, we investigated the role of PGC-1ɑ in the transformation of muscle fiber type in Schizothorax prenanti. The expression of PGC-1ɑ was ...

  4. CERAPP: Collaborative estrogen receptor activity prediction project

    DEFF Research Database (Denmark)

    Mansouri, Kamel; Abdelaziz, Ahmed; Rybacka, Aleksandra

    2016-01-01

    Background: Humans are exposed to thousands of man-made chemicals in the environment. Some chemicals mimic natural endocrine hormones and, thus, have the potential to be endocrine disruptors. Most of these chemicals have never been tested for their ability to interact with the estrogen receptor (ER......). Risk assessors need tools to prioritize chemicals for evaluation in costly in vivo tests, for instance, within the U.S. EPA Endocrine Disruptor Screening Program. oBjectives: We describe a large-scale modeling project called CERAPP (Collaborative Estrogen Receptor Activity Prediction Project...

  5. Functional analysis of the murine cytomegalovirus chemokine receptor homologue M33: ablation of constitutive signaling is associated with an attenuated phenotype in vivo

    DEFF Research Database (Denmark)

    Case, Ruth; Sharp, Emma; Benned-Jensen, Tau

    2007-01-01

    The murine cytomegalovirus (MCMV) M33 gene is conserved among all betaherpesviruses and encodes a homologue of seven-transmembrane receptors (7TMR) with the capacity for constitutive signaling. Previous studies have demonstrated that M33 is important for MCMV dissemination to or replication within...

  6. Shear stress-dependent downregulation of the adhesion-G protein-coupled receptor CD97 on circulating leukocytes upon contact with its ligand CD55

    NARCIS (Netherlands)

    Karpus, Olga N.; Veninga, Henrike; Hoek, Robert M.; Flierman, Dennis; van Buul, Jaap D.; Vandenakker, Corianne C.; VanBavel, Ed; Medof, M. Edward; van Lier, René A. W.; Reedquist, Kris A.; Hamann, Jörg

    2013-01-01

    Adhesion G protein-coupled receptors (aGPCRs) are two-subunit molecules, consisting of an adhesive extracellular α subunit that couples noncovalently to a seven-transmembrane β subunit. The cooperation between the two subunits and the effect of endogenous ligands on the functioning of aGPCRs is

  7. Functional enhancement of AT1R potency in the presence of the TPαR is revealed by a comprehensive 7TM receptor co-expression screen

    DEFF Research Database (Denmark)

    Hansen, Jonas Tind; Lyngsø, Christina; Speerschneider, Tobias

    2013-01-01

    Functional cross-talk between seven transmembrane (7TM) receptors can dramatically alter their pharmacological properties, both in vitro and in vivo. This represents an opportunity for the development of novel therapeutics that potentially target more specific biological effects while causing few...

  8. Phenobarbital and Insulin Reciprocate Activation of the Nuclear Receptor Constitutive Androstane Receptor through the Insulin Receptor.

    Science.gov (United States)

    Yasujima, Tomoya; Saito, Kosuke; Moore, Rick; Negishi, Masahiko

    2016-05-01

    Phenobarbital (PB) antagonized insulin to inactivate the insulin receptor and attenuated the insulin receptor downstream protein kinase B (AKT)-forkhead box protein O1 and extracellular signal-regulated kinase 1/2 signals in mouse primary hepatocytes and HepG2 cells. Hepatic AKT began dephosphorylation in an early stage of PB treatment, and blood glucose levels transiently increased in both wild-type and constitutive androstane receptor (CAR) knockout (KO) mice. On the other hand, blood glucose levels increased in wild-type mice, but not KO mice, in later stages of PB treatment. As a result, PB, acting as an insulin receptor antagonist, elicited CAR-independent increases and CAR-dependent decreases of blood glucose levels at these different stages of treatment, respectively. Reciprocally, insulin activation of the insulin receptor repressed CAR activation and induction of its target CYP2B6 gene in HepG2 cells. Thus, PB and insulin cross-talk through the insulin receptor to regulate glucose and drug metabolism reciprocally. Copyright © 2016 by U.S. Government work not protected by U.S. copyright.

  9. International Union of Basic and Clinical Pharmacology. [corrected]. LXXXIX. Update on the extended family of chemokine receptors and introducing a new nomenclature for atypical chemokine receptors.

    OpenAIRE

    Bachelerie Francoise; Ben-Baruch Adit; Burkhardt Amanda M.; Combadiere Christophe; Farber Joshua M.; Graham Gerard J; Horuk Richard; Sparre-Ulrich Alexander Hovard; Locati Massimo; Luster Andrew D.; Mantovani Alberto; Matsushima Kouji; Murphy Philip M.; Nibbs Robert; Nomiyama Hisayuki

    2014-01-01

    Sixteen years ago the Nomenclature Committee of the International Union of Pharmacology approved a system for naming human seven transmembrane (7TM) G protein coupled chemokine receptors the large family of leukocyte chemoattractant receptors that regulates immune system development and function in large part by mediating leukocyte trafficking. This was announced in Pharmacological Reviews in a major overview of the first decade of research in this field [Murphy PM Baggiolini M Charo IF Héber...

  10. International Union of Pharmacology. LXXXIX. Update on the Extended Family of Chemokine Receptors and Introducing a New Nomenclature for Atypical Chemokine Receptors

    OpenAIRE

    Bachelerie, Francoise; Ben-Baruch, Adit; Burkhardt, Amanda M.; Combadiere, Christophe; Farber, Joshua M.; Graham, Gerard J.; Horuk, Richard; Sparre-Ulrich, Alexander Hovard; Locati, Massimo; Luster, Andrew D.; Mantovani, Alberto; Matsushima, Kouji; Murphy, Philip M.; Nibbs, Robert; Nomiyama, Hisayuki

    2014-01-01

    Sixteen years ago, the Nomenclature Committee of the International Union of Pharmacology approved a system for naming human seven-transmembrane (7TM) G protein-coupled chemokine receptors, the large family of leukocyte chemoattractant receptors that regulates immune system development and function, in large part by mediating leukocyte trafficking. This was announced in Pharmacological Reviews in a major overview of the first decade of research in this field [Murphy PM, Baggiolini M, Charo IF,...

  11. 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.

  12. Proteinase-activated receptors (PARs) – focus on receptor-receptor-interactions and their physiological and pathophysiological impact

    Science.gov (United States)

    2013-01-01

    Proteinase-activated receptors (PARs) are a subfamily of G protein-coupled receptors (GPCRs) with four members, PAR1, PAR2, PAR3 and PAR4, playing critical functions in hemostasis, thrombosis, embryonic development, wound healing, inflammation and cancer progression. PARs are characterized by a unique activation mechanism involving receptor cleavage by different proteinases at specific sites within the extracellular amino-terminus and the exposure of amino-terminal “tethered ligand“ domains that bind to and activate the cleaved receptors. After activation, the PAR family members are able to stimulate complex intracellular signalling networks via classical G protein-mediated pathways and beta-arrestin signalling. In addition, different receptor crosstalk mechanisms critically contribute to a high diversity of PAR signal transduction and receptor-trafficking processes that result in multiple physiological effects. In this review, we summarize current information about PAR-initiated physical and functional receptor interactions and their physiological and pathological roles. We focus especially on PAR homo- and heterodimerization, transactivation of receptor tyrosine kinases (RTKs) and receptor serine/threonine kinases (RSTKs), communication with other GPCRs, toll-like receptors and NOD-like receptors, ion channel receptors, and on PAR association with cargo receptors. In addition, we discuss the suitability of these receptor interaction mechanisms as targets for modulating PAR signalling in disease. PMID:24215724

  13. One-step immunopurification and lectinochemical characterization of the Duffy atypical chemokine receptor from human erythrocytes

    OpenAIRE

    Grodecka, Magdalena; Bertrand, Olivier; Karolak, Ewa; Lisowski, Marek; Waśniowska, Kazimiera

    2012-01-01

    Duffy antigen/receptor for chemokines (DARC) is a glycosylated seven-transmembrane protein acting as a blood group antigen, a chemokine binding protein and a receptor for Plasmodium vivax malaria parasite. It is present on erythrocytes and endothelial cells of postcapillary venules. The N-terminal extracellular domain of the Duffy glycoprotein carries Fya/Fyb blood group antigens and Fy6 linear epitope recognized by monoclonal antibodies. Previously, we have shown that recombinant Duffy prote...

  14. Monoclonal Antibodies to the Human Insulin Receptor that Activate Glucose Transport but not Insulin Receptor Kinase Activity

    Science.gov (United States)

    Forsayeth, John R.; Caro, Jose F.; Sinha, Madhur K.; Maddux, Betty A.; Goldfine, Ira D.

    1987-05-01

    Three mouse monoclonal antibodies were produced that reacted with the α subunit of the human insulin receptor. All three both immunoprecipitated 125I-labeled insulin receptors from IM-9 lymphocytes and competitively inhibited 125I-labeled insulin binding to its receptor. Unlike insulin, the antibodies failed to stimulate receptor autophosphorylation in both intact IM-9 lymphocytes and purified human placental insulin receptors. Moreover, unlike insulin, the antibodies failed to stimulate receptor-mediated phosphorylation of exogenous substrates. However, like insulin, two of the three antibodies stimulated glucose transport in isolated human adipocytes. One antibody, on a molar basis, was as potent as insulin. These studies indicate, therefore, that monoclonal antibodies to the insulin receptor can mimic a major function of insulin without activating receptor kinase activity. They also raise the possibility that certain actions of insulin such as stimulation of glucose transport may not require the activation of receptor kinase activity.

  15. Peroxisome Proliferator Activated Receptors and Lipoprotein Metabolism

    Directory of Open Access Journals (Sweden)

    Sander Kersten

    2008-01-01

    Full Text Available Plasma lipoproteins are responsible for carrying triglycerides and cholesterol in the blood and ensuring their delivery to target organs. Regulation of lipoprotein metabolism takes place at numerous levels including via changes in gene transcription. An important group of transcription factors that mediates the effect of dietary fatty acids and certain drugs on plasma lipoproteins are the peroxisome proliferator activated receptors (PPARs. Three PPAR isotypes can be distinguished, all of which have a major role in regulating lipoprotein metabolism. PPARα is the molecular target for the fibrate class of drugs. Activation of PPARα in mice and humans markedly reduces hepatic triglyceride production and promotes plasma triglyceride clearance, leading to a clinically significant reduction in plasma triglyceride levels. In addition, plasma high-density lipoprotein (HDL-cholesterol levels are increased upon PPARα activation in humans. PPARγ is the molecular target for the thiazolidinedione class of drugs. Activation of PPARγ in mice and human is generally associated with a modest increase in plasma HDL-cholesterol and a decrease in plasma triglycerides. The latter effect is caused by an increase in lipoprotein lipase-dependent plasma triglyceride clearance. Analogous to PPARα, activation of PPARβ/δ leads to increased plasma HDL-cholesterol and decreased plasma triglyceride levels. In this paper, a fresh perspective on the relation between PPARs and lipoprotein metabolism is presented. The emphasis is on the physiological role of PPARs and the mechanisms underlying the effect of synthetic PPAR agonists on plasma lipoprotein levels.

  16. A library of 7TM receptor C-terminal tails. Interactions with the proposed post-endocytic sorting proteins ERM-binding phosphoprotein 50 (EBP50), N-ethylmaleimide-sensitive factor (NSF), sorting nexin 1 (SNX1), and G protein-coupled receptor-associated sorting protein (GASP)

    DEFF Research Database (Denmark)

    Heydorn, Arne; Søndergaard, Birgitte P; Ersbøll, Bjarne

    2004-01-01

    Adaptor and scaffolding proteins determine the cellular targeting, the spatial, and thereby the functional association of G protein-coupled seven-transmembrane receptors with co-receptors, transducers, and downstream effectors and the adaptors determine post-signaling events such as receptor...... sequestration through interactions, mainly with the C-terminal intracellular tails of the receptors. A library of tails from 59 representative members of the super family of seven-transmembrane receptors was probed as glutathione S-transferase fusion proteins for interactions with four different adaptor...... that the tail library provides useful information on the general importance of certain adaptor proteins, for example, in this case, ruling out EBP50 as being a broad spectrum-recycling adaptor....

  17. Model for growth hormone receptor activation based on subunit rotation within a receptor dimer

    Energy Technology Data Exchange (ETDEWEB)

    Brown, Richard J.; Adams, Julian J.; Pelekanos, Rebecca A.; Wan, Yu; McKinstry, William J.; Palethorpe, Kathryn; Seeber, Ruth M.; Monks, Thea A.; Eidne, Karin A.; Parker, Michael W.; Waters, Michael J. (UWA); (St. Vincent); (Queensland)

    2010-07-13

    Growth hormone is believed to activate the growth hormone receptor (GHR) by dimerizing two identical receptor subunits, leading to activation of JAK2 kinase associated with the cytoplasmic domain. However, we have reported previously that dimerization alone is insufficient to activate full-length GHR. By comparing the crystal structure of the liganded and unliganded human GHR extracellular domain, we show here that there is no substantial change in its conformation on ligand binding. However, the receptor can be activated by rotation without ligand by inserting a defined number of alanine residues within the transmembrane domain. Fluorescence resonance energy transfer (FRET), bioluminescence resonance energy transfer (BRET) and coimmunoprecipitation studies suggest that receptor subunits undergo specific transmembrane interactions independent of hormone binding. We propose an activation mechanism involving a relative rotation of subunits within a dimeric receptor as a result of asymmetric placement of the receptor-binding sites on the ligand.

  18. Family C 7TM receptor dimerization and activation

    DEFF Research Database (Denmark)

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

    2006-01-01

    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....

  19. Chapter 8. Activation mechanisms of chemokine receptors

    DEFF Research Database (Denmark)

    Jensen, Pia C; Rosenkilde, Mette M

    2009-01-01

    , more research has to be done in this field. Chemokine receptors are interesting tools in this matter. First, the chemokine system has a high degree of promiscuity that allows several chemokines to target one receptor in different ways, as well as a single chemokine ligand to target several receptors...

  20. Activation and allosteric modulation of a muscarinic acetylcholine receptor

    OpenAIRE

    Kruse, Andrew C.; Ring, Aaron M.; Manglik, Aashish; Hu, Jianxin; Hu, Kelly; Eitel, Katrin; Hübner, Harald; Pardon, Els; Valant, Celine; Sexton, Patrick M.; Christopoulos, Arthur; Felder, Christian C.; Gmeiner, Peter; Steyaert, Jan; Weis, William I.

    2013-01-01

    Despite recent advances in crystallography of G protein-coupled receptors (GPCRs), little is known about the mechanism of their activation process, as only the β2 adrenergic receptor (β2AR) and rhodopsin have been crystallized in fully active conformations. Here, we report the structure of an agonist-bound, active state of the human M2 muscarinic acetylcholine receptor stabilized by a G-protein mimetic camelid antibody fragment isolated by conformational selection using yeast surface display....

  1. The Role of Chemoattractant Receptors in Shaping the Tumor Microenvironment

    Directory of Open Access Journals (Sweden)

    Jiamin Zhou

    2014-01-01

    Full Text Available Chemoattractant receptors are a family of seven transmembrane G protein coupled receptors (GPCRs initially found to mediate the chemotaxis and activation of immune cells. During the past decades, the functions of these GPCRs have been discovered to not only regulate leukocyte trafficking and promote immune responses, but also play important roles in homeostasis, development, angiogenesis, and tumor progression. Accumulating evidence indicates that chemoattractant GPCRs and their ligands promote the progression of malignant tumors based on their capacity to orchestrate the infiltration of the tumor microenvironment by immune cells, endothelial cells, fibroblasts, and mesenchymal cells. This facilitates the interaction of tumor cells with host cells, tumor cells with tumor cells, and host cells with host cells to provide a basis for the expansion of established tumors and development of distant metastasis. In addition, many malignant tumors of the nonhematopoietic origin express multiple chemoattractant GPCRs that increase the invasiveness and metastasis of tumor cells. Therefore, GPCRs and their ligands constitute targets for the development of novel antitumor therapeutics.

  2. Ligand-specific regulation of the extracellular surface of a G-protein-coupled receptor

    Energy Technology Data Exchange (ETDEWEB)

    Bokoch, Michael P.; Zou, Yaozhong; Rasmussen, Søren G.F.; Liu, Corey W.; Nygaard, Rie; Rosenbaum, Daniel M.; Fung, Juan José; Choi, Hee-Jung; Thian, Foon Sun; Kobilka, Tong Sun; Puglisi, Joseph D.; Weis, William I.; Pardo, Leonardo; Prosser, R. Scott; Mueller, Luciano; Kobilka, Brian K. (Stanford-MED); (Toronto); (BMS); (UAB, Spain)

    2010-01-14

    G-protein-coupled receptors (GPCRs) are seven-transmembrane proteins that mediate most cellular responses to hormones and neurotransmitters. They are the largest group of therapeutic targets for a broad spectrum of diseases. Recent crystal structures of GPCRs have revealed structural conservation extending from the orthosteric ligand-binding site in the transmembrane core to the cytoplasmic G-protein-coupling domains. In contrast, the extracellular surface (ECS) of GPCRs is remarkably diverse and is therefore an ideal target for the discovery of subtype-selective drugs. However, little is known about the functional role of the ECS in receptor activation, or about conformational coupling of this surface to the native ligand-binding pocket. Here we use NMR spectroscopy to investigate ligand-specific conformational changes around a central structural feature in the ECS of the {beta}{sub 2} adrenergic receptor: a salt bridge linking extracellular loops 2 and 3. Small-molecule drugs that bind within the transmembrane core and exhibit different efficacies towards G-protein activation (agonist, neutral antagonist and inverse agonist) also stabilize distinct conformations of the ECS. We thereby demonstrate conformational coupling between the ECS and the orthosteric binding site, showing that drugs targeting this diverse surface could function as allosteric modulators with high subtype selectivity. Moreover, these studies provide a new insight into the dynamic behaviour of GPCRs not addressable by static, inactive-state crystal structures.

  3. The WSXWS motif in cytokine receptors is a molecular switch involved in receptor activation

    DEFF Research Database (Denmark)

    Dagil, Robert; Knudsen, Maiken J.; Olsen, Johan Gotthardt

    2012-01-01

    The prolactin receptor (PRLR) is activated by binding of prolactin in a 2:1 complex, but the activation mechanism is poorly understood. PRLR has a conserved WSXWS motif generic to cytokine class I receptors. We have determined the nuclear magnetic resonance solution structure of the membrane...

  4. Interaction of CPCCOEt with a chimeric mGlu1b and calcium sensing receptor

    DEFF Research Database (Denmark)

    Bräuner-Osborne, H; Jensen, Anders A.; Krogsgaard-Larsen, P

    1999-01-01

    R) and a chimeric receptor consisting of the agonist binding amino-terminal domain (ATD) of CaR and the seven transmembrane (7TM) domain of mGlu1b (named Ca/1b). CPCCOEt inhibited responses of (S)-glutamic acid and Ca2+ at mGlu1b and Ca/1b, applied at EC50 values, with IC50 values of 10.2 microM and 13.4 micro...

  5. Characterization of a β-adrenergic-like octopamine receptor from the rice stem borer (Chilo suppressalis).

    Science.gov (United States)

    Wu, Shun-Fan; Yao, Yao; Huang, Jia; Ye, Gong-Yin

    2012-08-01

    Octopamine, the invertebrate counterpart of adrenaline and noradrenaline, plays a key role in regulation of many physiological and behavioral processes in insects. It modulates these functions through binding to specific octopamine receptors, which are typical rhodopsin-like G-protein coupled receptors. A cDNA encoding a seven-transmembrane receptor was cloned from the nerve cord of the rice stem borer, Chilo suppressalis, viz. CsOA2B2, which shares high sequence similarity to CG6989, a Drosophila β-adrenergic-like octopamine receptor (DmOctβ2R). We generated an HEK-293 cell line that stably expresses CsOA2B2 in order to examine the functional and pharmacological properties of this receptor. Activation of CsOA2B2 by octopamine increased the production of cAMP in a dose-dependent manner (EC(50)=2.33 nmol l(-1)), with a maximum response at 100 nmol l(-1). Tyramine also activated the receptor but with much less potency than octopamine. Dopamine and serotonin had marginal effects on cAMP production. Using a series of known agonists and antagonists for octopamine receptors, we observed a rather unique pharmacological profile for CsOA2B2 through measurements of cAMP. The rank order of potency of the agonists was naphazoline > clonidine. The activated effect of octopamine is abolished by co-incubation with phentolamine, mianserin or chlorpromazine. Using in vivo pharmacology, CsOA2B2 antagonists mianserin and phentolamine impaired the motor ability of individual rice stem borers. The results of the present study are important for a better functional understanding of this receptor as well as for practical applications in the development of environmentally sustainable pesticides.

  6. Cell death sensitization of leukemia cells by opioid receptor activation.

    Science.gov (United States)

    Friesen, Claudia; Roscher, Mareike; Hormann, Inis; Fichtner, Iduna; Alt, Andreas; Hilger, Ralf A; Debatin, Klaus-Michael; Miltner, Erich

    2013-05-01

    Cyclic AMP (cAMP) regulates a number of cellular processes and modulates cell death induction. cAMP levels are altered upon stimulation of specific G-protein-coupled receptors inhibiting or activating adenylyl cyclases. Opioid receptor stimulation can activate inhibitory Gi-proteins which in turn block adenylyl cyclase activity reducing cAMP. Opioids such as D,L-methadone induce cell death in leukemia cells. However, the mechanism how opioids trigger apoptosis and activate caspases in leukemia cells is not understood. In this study, we demonstrate that downregulation of cAMP induced by opioid receptor activation using the opioid D,L-methadone kills and sensitizes leukemia cells for doxorubicin treatment. Enhancing cAMP levels by blocking opioid-receptor signaling strongly reduced D,L-methadone-induced apoptosis, caspase activation and doxorubicin-sensitivity. Induction of cell death in leukemia cells by activation of opioid receptors using the opioid D,L-methadone depends on critical levels of opioid receptor expression on the cell surface. Doxorubicin increased opioid receptor expression in leukemia cells. In addition, the opioid D,L-methadone increased doxorubicin uptake and decreased doxorubicin efflux in leukemia cells, suggesting that the opioid D,L-methadone as well as doxorubicin mutually increase their cytotoxic potential. Furthermore, we found that opioid receptor activation using D,L-methadone alone or in addition to doxorubicin inhibits tumor growth significantly in vivo. These results demonstrate that opioid receptor activation via triggering the downregulation of cAMP induces apoptosis, activates caspases and sensitizes leukemia cells for doxorubicin treatment. Hence, opioid receptor activation seems to be a promising strategy to improve anticancer therapies.

  7. The Orphan Nuclear Receptor TR4 Is a Vitamin A-activated Nuclear Receptor

    Energy Technology Data Exchange (ETDEWEB)

    Zhou, X. Edward; Suino-Powell, Kelly M.; Xu, Yong; Chan, Cee-Wah; Tanabe, Osamu; Kruse, Schoen W.; Reynolds, Ross; Engel, James Douglas; Xu, H. Eric (Michigan-Med); (Van Andel)

    2015-11-30

    Testicular receptors 2 and 4 (TR2/4) constitute a subgroup of orphan nuclear receptors that play important roles in spermatogenesis, lipid and lipoprotein regulation, and the development of the central nervous system. Currently, little is known about the structural features and the ligand regulation of these receptors. Here we report the crystal structure of the ligand-free TR4 ligand binding domain, which reveals an autorepressed conformation. The ligand binding pocket of TR4 is filled by the C-terminal half of helix 10, and the cofactor binding site is occupied by the AF-2 helix, thus preventing ligand-independent activation of the receptor. However, TR4 exhibits constitutive transcriptional activity on multiple promoters, which can be further potentiated by nuclear receptor coactivators. Mutations designed to disrupt cofactor binding, dimerization, or ligand binding substantially reduce the transcriptional activity of this receptor. Importantly, both retinol and retinoic acid are able to promote TR4 to recruit coactivators and to activate a TR4-regulated reporter. These findings demonstrate that TR4 is a ligand-regulated nuclear receptor and suggest that retinoids might have a much wider regulatory role via activation of orphan receptors such as TR4.

  8. G Protein-Coupled Receptor 87: a Promising Opportunity for Cancer Drug Discovery.

    Science.gov (United States)

    Zhang, Yanhong; Scoumanne, Ariane; Chen, Xinbin

    2010-01-01

    G protein-coupled receptors (GPRs) constitute one of the largest families of membrane proteins encoded by the human genome. Upon binding to various ligands, these seven-transmembrane receptors play an essential role in many physiological processes, including neurotransmission, immunity, inflammation, regulation of mood and behavior. In view of their important functions, aberrant expression and activity of GPRs have been implicated in a wide spectrum of diseases, including tumorigenesis. GPR87, a cell surface GPR related to the LPA receptor family, is overexpressed in diverse carcinomas and plays an essential role in tumor cell survival. In our recent work, we uncovered that GPR87 expression is regulated by the tumor suppressor p53 and by DNA damage in a p53-dependent manner. Moreover, we found that a lack of GPR87 triggers an increase in p53, concomitant with a decrease in Akt, which results in the sensitization of tumor cells to DNA damage-induced apoptosis and growth suppression. Altogether, we uncovered an essential function for GPR87 in p53-dependent cell survival in response to stress signals. Due to their unique structure, localization and ligand binding ability, GPRs have been extensively used for drug development and are the most common targets of commercial drugs. Although studies are required to determine GPR87 natural ligand(s) and signaling pathways, GPR87 is undoubtedly a very promising novel target for cancer prevention and treatment.

  9. Activation of human peroxisome-activated receptor-gamma ...

    Science.gov (United States)

    Obesity in children has become an epidemic and recent research suggests a possible contribution from exposure to environmental chemicals. Several chemicals, such as phthalates, brominated flame retardants, and perfluorinated chemicals, are common in house dust on floors where children play and are suspected obesogens. Obesogens can act via a mechanism that involves activation of peroxisome proliferator-activated receptor-gamma (PPARy). A previous study found that dust collected from children’s homes binds to PPARy. Here, we investigated the ability of house dust to activate PPARy in a transiently transfected cell assay. Dust samples were collected in 2012 from carpeted and hardwood floors in children’s homes using thimbles fitted into a vacuum cleaner hose (“TEO” samples), or from homes in an adult cohort NIEHS study. Dust was extracted with 50:50 hexane:acetone, sonicated, centrifuged, and the organic layer collected. This was repeated 2X. The extracts were filtered to remove particulates, dried with purified nitrogen, and reconstituted in DMS0 at 200 ug/ul. COS-1 cells were transfected for 24 hrs with a human PPARy vector containing a luciferase reporter, and exposed for 24 hrs to negative controls water or DMSO (0.1%), positive controls Troglitazone (3 uM in water) or Rosiglitazone (100 nM in DMSO), or dust extracts serially diluted in DMEM at 50, 100, and 200 ug/ml in 0.1% DMSO. Cells were lysed and luciferase activity was measured. Data were log-tra

  10. Sugar-regulated cation channel formed by an insect gustatory receptor.

    Science.gov (United States)

    Sato, Koji; Tanaka, Kana; Touhara, Kazushige

    2011-07-12

    Insects sense the taste of foods and toxic compounds in their environment through the gustatory system. Genetic studies using fruit flies have suggested that putative seven-transmembrane gustatory receptors (Grs) expressed in gustatory sensory neurons are required for responses to specific tastants. We reconstituted sugar responses of Bombyx mori Gr-9 (BmGr-9), a silkworm Gr, in two heterologous expression systems. Xenopus oocytes or HEK293T cells expressing BmGr-9 selectively responded to D-fructose with an influx of extracellular Ca(2+) and a nonselective cation current conductance in a G protein-independent manner. Outside-out patch-clamp recording of BmGr-9-expressing cell membranes provides evidence supporting the hypothesis that BmGr-9 constitutes a ligand-gated ion channel. The fructose-activated current associated with BmGr-9 was suppressed by other hexoses, including glucose and sorbose. The activation and inhibition of insect Gr ion channels may be the molecular basis for the decoding system that discriminates subtle differences in sweet taste. Finally, Drosophila melanogaster Gr43a (DmGr43a), a BmGr-9 ortholog, also responded to D-fructose, suggesting that DmGr43a relatives appear to compose the family of fructose receptors.

  11. Activation of glucocorticoid receptors increases 5-HT2A receptor levels

    DEFF Research Database (Denmark)

    Trajkovska, Viktorija; Kirkegaard, Lisbeth; Krey, Gesa

    2009-01-01

    Major depression is associated with both dysregulation of the hypothalamic pituitary adrenal axis and serotonergic deficiency, not the least of the 5-HT2A receptor. However, how these phenomena are linked to each other, and whether a low 5-HT2A receptor level is a state or a trait marker of depre......Major depression is associated with both dysregulation of the hypothalamic pituitary adrenal axis and serotonergic deficiency, not the least of the 5-HT2A receptor. However, how these phenomena are linked to each other, and whether a low 5-HT2A receptor level is a state or a trait marker...... of depression is unknown. In mice with altered glucocorticoid receptor (GR) expression we investigated 5-HT2A receptor levels by Western blot and 3H-MDL100907 receptor binding. Serotonin fibre density was analyzed by stereological quantification of serotonin transporter immunopositive fibers. To establish...... an effect of GR activation on 5-HT2A levels, mature organotypic hippocampal cultures were exposed to corticosterone with or without GR antagonist mifepristone and mineralocorticoid receptor (MR) antagonist spironolactone. In GR under-expressing mice, hippocampal 5-HT2A receptor protein levels were decreased...

  12. Plant cysteine proteases that evoke itch activate protease-activated receptors

    Science.gov (United States)

    Reddy, V.B.; Lerner, E.A.

    2013-01-01

    Background Bromelain, ficin and papain are cysteine proteases from plants that produce itch upon injection into skin. Their mechanism of action has not been considered previously. Objectives To determine the mechanism by which these proteases function. Methods The ability of these proteases to activate protease-activated receptors was determined by ratiometric calcium imaging. Results We show here that bromelain, ficin and papain activate protease-activated receptors 2 and 4. Conclusions Bromelain, ficin and papain function as signalling molecules and activate protease-activated receptors. Activation of these receptors is the likely mechanism by which these proteases evoke itch. PMID:20491769

  13. Activation of the Innate Immune Receptors: Guardians of the Micro Galaxy : Activation and Functions of the Innate Immune Receptors.

    Science.gov (United States)

    De Nardo, Dominic

    2017-01-01

    The families of innate immune receptors are the frontline responders to danger. These superheroes of the host immune systems populate innate immune cells, surveying the extracellular environment and the intracellular endolysosomal compartments and cytosol for exogenous and endogenous danger signals. As a collective the innate immune receptors recognise a wide array of stimuli, and in response they initiate specific signalling pathways leading to activation of transcriptional or proteolytic pathways and the production of inflammatory molecules to destroy foreign pathogens and/or resolve tissue injury. In this review, I will give an overview of the innate immune system and the activation and effector functions of the families of receptors it comprises. Current key concepts will be described throughout, including innate immune memory, formation of innate immune receptor signalosomes, inflammasome formation and pyroptosis, methods of extrinsic cell communication and examples of receptor cooperation. Finally, several open questions and future directions in the field of innate immunity will be presented and discussed.

  14. Nuclear receptor corepressor-dependent repression of peroxisome-proliferator-activated receptor delta-mediated transactivation

    DEFF Research Database (Denmark)

    Krogsdam, Anne-M; Nielsen, Curt A F; Neve, Søren

    2002-01-01

    delta-RXR alpha heterodimer bound to an acyl-CoA oxidase (ACO)-type peroxisome-proliferator response element recruited a glutathione S-transferase-NCoR fusion protein in a ligand-independent manner. Contrasting with most other nuclear receptors, PPAR delta was found to interact equally well......The nuclear receptor corepressor (NCoR) was isolated as a peroxisome-proliferator-activated receptor (PPAR) delta interacting protein using the yeast two-hybrid system. NCoR interacted strongly with the ligand-binding domain of PPAR delta, whereas interactions with the ligand-binding domains...

  15. Modulation of β-catenin signaling by glucagon receptor activation.

    Directory of Open Access Journals (Sweden)

    Jiyuan Ke

    Full Text Available The glucagon receptor (GCGR is a member of the class B G protein-coupled receptor family. Activation of GCGR by glucagon leads to increased glucose production by the liver. Thus, glucagon is a key component of glucose homeostasis by counteracting the effect of insulin. In this report, we found that in addition to activation of the classic cAMP/protein kinase A (PKA pathway, activation of GCGR also induced β-catenin stabilization and activated β-catenin-mediated transcription. Activation of β-catenin signaling was PKA-dependent, consistent with previous reports on the parathyroid hormone receptor type 1 (PTH1R and glucagon-like peptide 1 (GLP-1R receptors. Since low-density-lipoprotein receptor-related protein 5 (Lrp5 is an essential co-receptor required for Wnt protein mediated β-catenin signaling, we examined the role of Lrp5 in glucagon-induced β-catenin signaling. Cotransfection with Lrp5 enhanced the glucagon-induced β-catenin stabilization and TCF promoter-mediated transcription. Inhibiting Lrp5/6 function using Dickkopf-1(DKK1 or by expression of the Lrp5 extracellular domain blocked glucagon-induced β-catenin signaling. Furthermore, we showed that Lrp5 physically interacted with GCGR by immunoprecipitation and bioluminescence resonance energy transfer assays. Together, these results reveal an unexpected crosstalk between glucagon and β-catenin signaling, and may help to explain the metabolic phenotypes of Lrp5/6 mutations.

  16. Implications of compound heterozygous insulin receptor mutations in congenital muscle fibre type disproportion myopathy for the receptor kinase activation

    DEFF Research Database (Denmark)

    Klein, H H; Müller, R; Vestergaard, H

    1999-01-01

    We studied insulin receptor kinase activation in two brothers with congenital muscle fibre type disproportion myopathy and compound heterozygous mutations of the insulin receptor gene, their parents, and their unaffected brother. In the father who has a heterozygote Arg1174-->Gln mutation, in situ...... activation of the receptor kinase in skeletal muscle was reduced about 70%. Selection of only those receptors that bound to anti-phosphotyrosine antibody showed that these receptors had normal kinase activity and that the reduction in overall kinase activity was due to the inability of about 70......% of the receptors to become insulin-dependently activated. The mother carries a point mutation at the last base pair in exon 17 which, due to abnormal alternative splicing, could lead to normally transcribed receptor or truncated receptor lacking the kinase region. Kinase activation was normal in the mother...

  17. Identification of COUP-TFII Orphan Nuclear Receptor as a Retinoic Acid-Activated Receptor

    Energy Technology Data Exchange (ETDEWEB)

    Kruse, Schoen W; Suino-Powell, Kelly; Zhou, X Edward; Kretschman, Jennifer E; Reynolds, Ross; Vonrhein, Clemens; Xu, Yong; Wang, Liliang; Tsai, Sophia Y; Tsai, Ming-Jer; Xu, H Eric [Baylor; (Van Andel); (Globel Phasing); (Grand Valley)

    2010-01-12

    The chicken ovalbumin upstream promoter-transcription factors (COUP-TFI and II) make up the most conserved subfamily of nuclear receptors that play key roles in angiogenesis, neuronal development, organogenesis, cell fate determination, and metabolic homeostasis. Although the biological functions of COUP-TFs have been studied extensively, little is known of their structural features or aspects of ligand regulation. Here we report the ligand-free 1.48 {angstrom} crystal structure of the human COUP-TFII ligand-binding domain. The structure reveals an autorepressed conformation of the receptor, where helix {alpha}10 is bent into the ligand-binding pocket and the activation function-2 helix is folded into the cofactor binding site, thus preventing the recruitment of coactivators. In contrast, in multiple cell lines, COUP-TFII exhibits constitutive transcriptional activity, which can be further potentiated by nuclear receptor coactivators. Mutations designed to disrupt cofactor binding, dimerization, and ligand binding, substantially reduce the COUP-TFII transcriptional activity. Importantly, retinoid acids are able to promote COUP-TFII to recruit coactivators and activate a COUP-TF reporter construct. Although the concentration needed is higher than the physiological levels of retinoic acids, these findings demonstrate that COUP-TFII is a ligand-regulated nuclear receptor, in which ligands activate the receptor by releasing it from the autorepressed conformation.

  18. Pyrazolodiazepine derivatives with agonist activity toward Drosophila RYamide receptor.

    Science.gov (United States)

    Yoon, Yeu Kyung; Kim, Jung Ha; Kim, Jeong-Hyun; Kwon, Jae-Young; Kim, Yong-Chul; Kim, Young-Joon; Park, Zee-Yong

    2016-10-15

    The neuropeptide Y (NPY)-like signaling is conserved broadly in many animal species, and implicated in diverse biological functions, particularly those associated with feeding and metabolism. In Drosophila, three G protein coupled receptors (GPCRs) are closely related to the vertebrate NPY receptors: RYamide receptor (RYa-R) CG5811, neuropeptide F receptor (NPFR) CG1147 and short neuropeptide F receptor (sNPF-R) CG7395. Here, we screened 442 compounds of the pyrazolodiazepine analogs library, and identified four synthetic small compounds that activate the RYa-R, but not other two receptors. Their maximum activity is about 40% of the endogenous ligand, Drosophila RYamide-1, indicating they are partial agonists. Structural comparisons of these agonists identified an active core structure, characterized by phenylalanine and lysine fused pyrazolodiazepine skeletons, which can be utilized as a lead structure for further development of more potent drugs active on mammalian NPYRs. Identification of small compound agonists selective on RYa-R of the genetically amenable insect model will facilitate future efforts to understand biological functions of RYa-R, a GPCR conserved in many species. Copyright © 2016 Elsevier Ltd. All rights reserved.

  19. Monitoring leptin activity using the chicken leptin receptor.

    Science.gov (United States)

    Hen, Gideon; Yosefi, Sera; Ronin, Ana; Einat, Paz; Rosenblum, Charles I; Denver, Robert J; Friedman-Einat, Miriam

    2008-05-01

    We report on the construction of a leptin bioassay based on the activation of chicken leptin receptor in cultured cells. A human embryonic kidney (HEK)-293 cell line, stably transfected with the full-length cDNA of chicken leptin receptor together with a STAT3-responsive reporter gene specifically responded to recombinant human and Xenopus leptins. The observed higher sensitivity of chicken leptin receptor to the former is in agreement with the degree of sequence similarity among these species (about 60 and 38% identical amino acids between humans and chickens, and between humans and Xenopus respectively). The specific activation of signal transduction through the chicken leptin receptor, shown here for the first time, suggests that the transition of Gln269 (implicated in the Gln-to-Pro Zucker fatty mutation in rats) to Glu in chickens does not impair its activity. Analysis of leptin-like activity in human serum samples of obese and lean subjects coincided well with leptin levels determined by RIA. Serum samples of pre- and post partum cows showed a tight correlation with the degree of adiposity. However, specific activation of the chicken leptin receptor in this assay was not observed with serum samples from broiler or layer chickens (representing fat and lean phenotypes respectively) or with those from turkey. Similar leptin receptor activation profiles were observed with cells transfected with human leptin receptor. Further work is needed to determine whether the lack of leptin-like activity in the chicken serum samples is due to a lack of leptin in this species or simply to a serum level of leptin that is below the detection threshold.

  20. Role of Peroxisome Proliferator-Activated Receptors in Inflammation Control

    OpenAIRE

    Jihan Youssef; Mostafa Badr

    2004-01-01

    Peroxisome proliferator-activated receptors (PPARs) were discovered over a decade ago, and were classified as orphan members of the nuclear receptor superfamily. To date, three PPAR subtypes have been discovered and characterized (PPARα, β/δ, γ). Different PPAR subtypes have been shown to play crucial roles in important diseases and conditions such as obesity, diabetes, atherosclerosis, cancer, and fertility. Among the most studied roles of PPARs is their involvement in inflammatory processes...

  1. 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.

  2. Proteinase-activated receptor-2 up-regulation by Fcγ-receptor activation in human neutrophils

    Science.gov (United States)

    St-Onge, Mireille; Lagarde, Stéphanie; Laflamme, Cynthia; Rollet-Labelle, Emmanuelle; Marois, Louis; Naccache, Paul H.; Pouliot, Marc

    2010-01-01

    We shed new light on the expression and function of the proteinase-activated receptor (PAR) family, associated with inflammation and hyperalgesia, in human granulocytes. Resting cells expressed constitutive levels of PAR-2 and PAR-3 mRNA but not PAR-1 or PAR-4. Based on flow cytometry, stimulation with opsonized bacteria (Bop) specifically up-regulated cell surface expression of PAR-2 in a concentration-dependent and time-dependent manner, independent of transcription or de novo protein synthesis. Primary granules were identified as a source of preformed PAR-2 that can readily be mobilized at the surface on fusion with the plasma membrane. Cellular response to PAR-2 activation, measured as changes in intracellular calcium concentration, was enhanced in PAR-2 up-regulated cells. Increase of cell-surface PAR-2 and of cell responsiveness were dependent specifically on the engagement of immunoglobulin (Ig)-binding receptors. Together, our results reveal that mobilization of intracellular granules, in response to Ig-receptor activation, up-regulates PAR-2 surface expression and makes neutrophils more responsive to proteinase activity. This enhanced response to PAR-2 activation indicates that molecular communication between pain and inflammation may be more important than previously believed.—St-Onge, M., Lagarde, S., Laflamme, C., Rollet-Labelle, E., Marois, L., Naccache, P. H., Pouliot, M. Proteinase-activated receptor-2 up-regulation by Fcγ-receptor activation in human neutrophils. PMID:20154268

  3. Characterization of a tyramine receptor type 2 from hemocytes of rice stem borer, Chilo suppressalis.

    Science.gov (United States)

    Wu, Shun-Fan; Xu, Gang; Ye, Gong-Yin

    2015-04-01

    Calcium acts as a second messenger in many cell types, including insect hemocytes. Intracellular calcium level has a definite role in innate and adaptive immune signaling. Biogenic amines such as octopamine (OA), tyramine (TA), dopamine (DA) and serotonin (5-HT) play various important physiological roles in insects by activating distinct G-protein-coupled receptors (GPCRs) that share a putative seven transmembrane domain structure. OA and 5-HT have been shown that can mediate insect hemocytic immune reactions to infections and invasions. Here, we showed that TA increase hemocyte spreading in the rice stem borer, Chilo suppressalis. Furthermore, we cloned a cDNA encoding a tyramine receptor type 2 from the hemocytes in the C. suppressalis, viz., CsTA2, which shares high sequence similarity to members of the invertebrate tyramine receptor family. The CsTA2 receptor was stably expressed in human embryonic kidney (HEK) 293 cells, and its ligand response has been examined. Receptor activation with TA induced a dose-dependent increase in intracellular Ca(2+) concentration ([Ca(2+)]i) in cells, with an EC50 value of 18.7±5.3 nM, whereas OA, DA, 5-HT and other potential agonists did not have this response. The mRNA is present in various tissues including nerve cord, hemocytes, fat body, midgut, Malpighian tubules, and epidermis in the larval stage. Western blot analysis and immunohistochemistry assay displayed that CsTA2 was detected and presented on hemocytes. We also showed that TA induced Ca(2+) release from the hemocytes of C. suppressalis. Copyright © 2015 Elsevier Ltd. All rights reserved.

  4. Preclinical pharmacology of bilastine, a new selective histamine H1 receptor antagonist: receptor selectivity and in vitro antihistaminic activity.

    Science.gov (United States)

    Corcóstegui, Reyes; Labeaga, Luis; Innerárity, Ana; Berisa, Agustin; Orjales, Aurelio

    2005-01-01

    This study aimed to establish the receptor selectivity and antihistaminic activity of bilastine, a new selective antihistamine receptor antagonist. In vitro experiments were conducted using a receptor binding screening panel and guinea-pig and rat tissues. Antihistaminic activity was determined using H1 receptor binding studies and in vitro H1 antagonism studies conducted in guinea-pig tissues and human cell lines. Receptor selectivity was established using a receptor binding screening panel and a receptor antagonism screening conducted in guinea-pig, rat and rabbit tissues. Inhibition of inflammatory mediators was determined through the Schultz-Dale reaction in sensitised guinea-pig ileum. Bilastine binds to histamine H1-receptors as indicated by its displacement of [3H]-pyrilamine from H1-receptors expressed in guinea-pig cerebellum and human embryonic kidney (HEK) cell lines. The studies conducted on guinea-pig smooth muscle demonstrated the capability of bilastine to antagonise H1-receptors. Bilastine is selective for histamine H1-receptors as shown in receptor-binding screening conducted to determine the binding capacity of bilastine to 30 different receptors. The specificity of its H1-receptor antagonistic activity was also demonstrated in a series of in vitro experiments conducted on guinea-pig and rat tissues. The results of these studies confirmed the lack of significant antagonism against serotonin, bradykinin, leukotriene D4, calcium, muscarinic M3-receptors, alpha1-adrenoceptors, beta2-adrenoceptors, and H2- and H3-receptors. The results of the in vitro Schultz-Dale reaction demonstrated that bilastine also has anti-inflammatory activity. These preclinical studies provide evidence that bilastine has H1- antihistamine activity, with high specificity for H1-receptors, and poor or no affinity for other receptors. Bilastine has also been shown to have anti-inflammatory properties.

  5. A facile synthesis of new 2-carboxamido-3-carboxythiophene and 4,5,6,7-tetrahydro-2-carboxamido-3-carboxythieno[2,3-c]pyridine derivatives as potential endothelin receptors ligands.

    Science.gov (United States)

    Pittalà, Valeria; Modica, Maria; Romeo, Giuseppe; Materia, Luisa; Salerno, Loredana; Siracusa, Mariangela; Cagnotto, Alfredo; Mereghetti, Ilario; Russo, Filippo

    2005-09-01

    Endothelins (ETs) are the most ubiquitous, highly potent and unusually long-lasting peptidic constrictors of human vessels known. Elevated levels of the plasma concentration of ETs were observed in several diseases such as hypertension, acute myocardial infarction, congestive heart failure, renal failure, pulmonary hypertension, and atherosclerosis. ETs exert their activities via specific seven-transmembrane, G protein-coupled receptors. To date two receptor subtypes, endothelin A (ET(A)) and endothelin B (ET(B)), have been identified and cloned. A literature survey revealed that a number of compounds that bind ET receptors with affinity and selectivity are known, nevertheless these compounds belong only to few chemical classes. The aim of this work is the identification of an "hit compound" with novel chemical structure endowed with reasonable ET affinity and selectivity. Accordingly, new variously substituted 2-carboxamido-3-carboxythiophene derivatives (29-52) were synthesized. These compounds were tested for their ability to inhibit ETs binding in radioligand binding assay using CHO cells stably expressing human ET(A) and ET(B) receptors.

  6. Abelson tyrosine kinase links PDGFbeta receptor activation to cytoskeletal regulation of NMDA receptors in CA1 hippocampal neurons

    Directory of Open Access Journals (Sweden)

    Beazely Michael A

    2008-12-01

    Full Text Available Abstract Background We have previously demonstrated that PDGF receptor activation indirectly inhibits N-methyl-D-aspartate (NMDA currents by modifying the cytoskeleton. PDGF receptor ligand is also neuroprotective in hippocampal slices and cultured neurons. PDGF receptors are tyrosine kinases that control a variety of signal transduction pathways including those mediated by PLCγ. In fibroblasts Src and another non-receptor tyrosine kinase, Abelson kinase (Abl, control PDGF receptor regulation of cytoskeletal dynamics. The mechanism whereby PDGF receptor regulates cytoskeletal dynamics in central neurons remains poorly understood. Results Intracellular applications of active Abl, but not heat-inactivated Abl, decreased NMDA-evoked currents in isolated hippocampal neurons. This mimics the effects of PDGF receptor activation in these neurons. The Abl kinase inhibitor, STI571, blocked the inhibition of NMDA currents by Abl. We demonstrate that PDGF receptors can activate Abl kinase in hippocampal neurons via mechanisms similar to those observed previously in fibroblasts. Furthermore, PDGFβ receptor activation alters the subcellular localization of Abl. Abl kinase is linked to actin cytoskeletal dynamics in many systems. We show that the inhibition of NMDA receptor currents by Abl kinase is blocked by the inclusion of the Rho kinase inhibitor, Y-27632, and that activation of Abl correlates with an increase in ROCK tyrosine phosphorylation. Conclusion This study demonstrates that PDGFβ receptors act via an interaction with Abl kinase and Rho kinase to regulated cytoskeletal regulation of NMDA receptor channels in CA1 pyramidal neurons.

  7. Neurotrophin receptors expression and JNK pathway activation in human astrocytomas

    Directory of Open Access Journals (Sweden)

    Maraziotis Theodore

    2007-10-01

    Full Text Available Abstract Background Neurotrophins are growth factors that regulate cell growth, differentiation and apoptosis in the nervous system. Their diverse actions are mediated through two different transmembrane – receptor signaling systems: Trk receptor tyrosine kinases (TrkA, TrkB, TrkC and p75NTR neurotrophin receptor. Trk receptors promote cell survival and differentiation while p75NTR induces, in most cases, the activity of JNK-p53-Bax apoptosis pathway or suppresses intracellular survival signaling cascades. Robust Trk activation blocks p75NTR -induced apoptosis by suppressing the JNK-p53-Bax pathway. The aim of this exploratory study was to investigate the expression levels of neurotrophin receptors, Trks and p75NTR, and the activation of JNK pathway in human astrocytomas and in adjacent non-neoplastic brain tissue. Methods Formalin-fixed paraffin-embedded serial sections from 33 supratentorial astrocytomas (5 diffuse fibrillary astrocytomas, WHO grade II; 6 anaplastic astrocytomas, WHO grade III; 22 glioblastomas multiforme, WHO grade IV were immunostained following microwave pretreatment. Polyclonal antibodies against TrkA, TrkB, TrkC and monoclonal antibodies against p75NTR and phosphorylated forms of JNK (pJNK and c-Jun (pc-Jun were used. The labeling index (LI, defined as the percentage of positive (labeled cells out of the total number of tumor cells counted, was determined. Results Moderate to strong, granular cytoplasmic immunoreactivity for TrkA, TrkB and TrkC receptors was detected in greater than or equal to 10% of tumor cells in the majority of tumors independently of grade; on the contrary, p75NTR receptor expression was found in a small percentage of tumor cells (~1% in some tumors. The endothelium of tumor capillaries showed conspicuous immunoreactivity for TrkB receptor. Trk immunoreactivity seemed to be localized in some neurons and astrocytes in non-neoplastic tissue. Phosphorylated forms of JNK (pJNK and c-Jun (pc-Jun were

  8. Allosteric Modulation of Protease-activated Receptor Signaling

    Science.gov (United States)

    Canto, Isabel; Soh, Unice J. K.; Trejo, JoAnn

    2014-01-01

    The protease-activated receptors (PARs) are G protein-coupled receptors (GPCRs) that are uniquely activated by proteolysis. PARs mediate hemostasis, thrombosis, inflammation, embryonic development and progression of certain malignant cancers. The family of PARs include four members: PAR1, PAR2, PAR3 and PAR4. PARs harbor a cryptic ligand sequence within the N-terminus that is exposed following proteolytic cleavage. The newly formed PAR N-terminus functions as a tethered ligand that binds intramolecularly to the receptor to trigger transmembrane signaling. This unique mechanism of activation would indicate that regardless of the activating protease, cleavage of PARs would unmask a tethered ligand sequence that would induce a similar active receptor conformation and signaling response. However, this is not the case. Recent studies demonstrate that PARs can be differentially activated by synthetic peptide agonists, proteases or through dimerization, that ultimately result in distinct cellular responses. In some cases, allosteric modulation of PARs involves compartmentalization in caveolae, plasma membrane microdomains enriched in cholesterol. Here, we discuss the mechanisms that lead to allosteric modulation of PAR signaling. PMID:22681248

  9. Epidermis-type lipoxygenase 3 regulates adipocyte differentiation and peroxisome proliferator-activated receptor gamma activity

    DEFF Research Database (Denmark)

    Hallenborg, Philip; Jørgensen, Claus; Petersen, Rasmus K

    2010-01-01

    The nuclear receptor peroxisome proliferator-activated receptor gamma (PPAR gamma) is essential for adipogenesis. Although several fatty acids and their derivatives are known to bind and activate PPAR gamma, the nature of the endogenous ligand(s) promoting the early stages of adipocyte differenti...

  10. Regulation of Proteome Maintenance Gene Expression by Activators of Peroxisome Proliferator-Activated Receptor a (PPARa)

    Science.gov (United States)

    The nuclear receptor peroxisome proliferator-activated receptor alpha (PPARa) is activated by a large number of xenobiotic and hypolipidemic compounds called peroxisome proliferator chemicals (PPC). One agonist of PPARa (WY-14,643) regulates responses in the mouse liver to chemic...

  11. Construction of a high affinity zinc binding site in the metabotropic glutamate receptor mGluR1

    DEFF Research Database (Denmark)

    Jensen, Anders A.; Sheppard, P O; Jensen, L B

    2001-01-01

    The metabotropic glutamate receptors (mGluRs) belong to family C of the G-protein-coupled receptor (GPCR) superfamily. The receptors are characterized by having unusually long amino-terminal domains (ATDs), to which agonist binding has been shown to take place. Previously, we have constructed...... of a "closed" conformation, and thus stabilizing a more or less inactive "open" form of the ATD. This study presents the first metal ion site constructed in a family C GPCR. Furthermore, it is the first time a metal ion site has been created in a region outside of the seven transmembrane regions of a GPCR...

  12. Structure and activation of the TSH receptor transmembrane domain.

    Science.gov (United States)

    Núñez Miguel, Ricardo; Sanders, Jane; Furmaniak, Jadwiga; Smith, Bernard Rees

    2017-12-01

    The thyroid-stimulating hormone receptor (TSHR) is the target autoantigen for TSHR-stimulating autoantibodies in Graves' disease. The TSHR is composed of: a leucine-rich repeat domain (LRD), a hinge region or cleavage domain (CD) and a transmembrane domain (TMD). The binding arrangements between the TSHR LRD and the thyroid-stimulating autoantibody M22 or TSH have become available from the crystal structure of the TSHR LRD-M22 complex and a comparative model of the TSHR LRD in complex with TSH, respectively. However, the mechanism by which the TMD of the TSHR and the other glycoprotein hormone receptors (GPHRs) becomes activated is unknown. We have generated comparative models of the structures of the inactive (TMD_In) and active (TMD_Ac) conformations of the TSHR, follicle-stimulating hormone receptor (FSHR) and luteinizing hormone receptor (LHR) TMDs. The structures of TMD_Ac and TMD_In were obtained using class A GPCR crystal structures for which fully active and inactive conformations were available. Most conserved motifs observed in GPCR TMDs are also observed in the amino acid sequences of GPHR TMDs. Furthermore, most GPCR TMD conserved helix distortions are observed in our models of the structures of GPHR TMDs. Analysis of these structures has allowed us to propose a mechanism for activation of GPHR TMDs. Insight into the mechanism of activation of the TSHR by both TSH and TSHR autoantibodies is likely to be useful in the development of new treatments for Graves' disease.

  13. In Vivo Imaging of Nuclear Receptor Transcriptional Activity.

    Science.gov (United States)

    Dart, D Alwyn; Bevan, Charlotte L

    2016-01-01

    Nuclear receptors drive key processes during development, reproduction, metabolism, and disease. In order to understand and analyze, as well as manipulate, their actions it is imperative that we are able to study them in whole animals and in a spatiotemporal manner. The increasing repertoire of transgenic animals, expressing reporter genes driven by a specific nuclear receptor, enables us to do this. Use of luciferase reporter genes is the method of choice of many researchers as it is well tolerated, relatively easy to use, and robust. Further, luciferase lends itself to the process as it can penetrate tissue and can be manipulated to degrade rapidly thus allowing a dynamic response. However, limited resolution, lack of quantitation, and the largely two-dimensional images acquired make it desirable to support results using ex vivo imaging and enzymatic and/or immunohistochemical analysis of dissected tissue. As well as enabling the visualization of nuclear receptor signaling in wild-type animals, crossing these mouse models with models of disease will provide invaluable information on how such signaling is dysregulated during disease progression, and how we may manipulate nuclear receptor signaling in therapy. The use of in vivo imaging therefore provides the power to determine where and when in development, aging, and disease nuclear receptors are active and how ligands or receptor modulators affect this.

  14. Protease activated receptor-2 (PAR2): possible target of phytochemicals.

    Science.gov (United States)

    Kakarala, Kavita Kumari; Jamil, Kaiser

    2015-09-01

    The use of phytochemicals either singly or in combination with other anticancer drugs comes with an advantage of less toxicity and minimal side effects. Signaling pathways play central role in cell cycle, cell growth, metabolism, etc. Thus, the identification of phytochemicals with promising antagonistic effect on the receptor/s playing key role in single transduction may have better therapeutic application. With this background, phytochemicals were screened against protease-activated receptor 2 (PAR2). PAR2 belongs to the superfamily of GPCRs and is an important target for breast cancer. Using in silico methods, this study was able to identify the phytochemicals with promising binding affinity suggesting their therapeutic potential in the treatment of breast cancer. The findings from this study acquires importance as the information on the possible agonists and antagonists of PAR2 is limited due its unique mechanism of activation.

  15. Regulation of Liver Energy Balance by the Nuclear Receptors Farnesoid X Receptor and Peroxisome Proliferator Activated Receptor α.

    Science.gov (United States)

    Kim, Kang Ho; Moore, David D

    2017-01-01

    The liver undergoes major changes in substrate utilization and metabolic output over the daily feeding and fasting cycle. These changes occur acutely in response to hormones such as insulin and glucagon, with rapid changes in signaling pathways mediated by protein phosphorylation and other post-translational modifications. They are also reflected in chronic alterations in gene expression in response to nutrient-sensitive transcription factors. Among these, the nuclear receptors farnesoid X receptor (FXR) and peroxisome proliferator activated receptor α (PPARα) provide an intriguing, coordinated response to maintain energy balance in the liver. FXR is activated in the fed state by bile acids returning to the liver, while PPARα is activated in the fasted state in response to the free fatty acids produced by adipocyte lipolysis or possibly other signals. Key Messages: Previous studies indicate that FXR and PPARα have opposing effects on each other's primary targets in key metabolic pathways including gluconeogenesis. Our more recent work shows that these 2 nuclear receptors coordinately regulate autophagy: FXR suppresses this pathway of nutrient and energy recovery, while PPARα activates it. Another recent study indicates that FXR activates the complement and coagulation pathway, while earlier studies identify this as a negative target of PPARα. Since secretion is a very energy- and nutrient-intensive process for hepatocytes, it is possible that FXR licenses it in the nutrient-rich fed state, while PPARα represses it to spare resources in the fasted state. Energy balance is a potential connection linking FXR and PPARα regulation of autophagy and secretion, 2 seemingly unrelated aspects of hepatocyte function. FXR and PPARα act coordinately to promote energy balance and homeostasis in the liver by regulating autophagy and potentially protein secretion. It is quite likely that their impact extends to additional pathways relevant to hepatic energy balance, and

  16. Modulation of transcriptional mineralocorticoid receptor activity by nitrosative stress.

    Science.gov (United States)

    Ruhs, Stefanie; Strätz, Nicole; Schlör, Kathleen; Meinel, Sandra; Mildenberger, Sigrid; Rabe, Sindy; Gekle, Michael; Grossmann, Claudia

    2012-09-01

    The mineralocorticoid receptor (MR) plays an important role in salt and water homeostasis and pathological tissue modifications, such as cardiovascular and renal fibrosis. Importantly, MR activation by aldosterone per se is not sufficient for the deleterious effects but requires the additional presence of a certain pathological milieu. Phenomenologically, this milieu could be generated by enhanced nitrosative stress. However, little is known regarding the modulation of MR transcriptional activity in a pathological milieu. The glucocorticoid receptor (GR), the closest relative of the MR, binds to the same hormone-response element but elicits protective effects on the cardiovascular system. To investigate the possible modulation of MR and GR by nitrosative stress under controlled conditions we used human embryonic kidney (HEK) cells and measured MR and GR transactivation after stimulation with the nitric oxide (NO)-donor SNAP and the peroxynitrite-donor Sin-1. In the presence of corticosteroids NO led to a general reduced corticosteroid receptor activity by repression of corticosteroid receptor-DNA interaction. The NO-induced diminished transcriptional MR activity was most pronounced during stimulation with physiological aldosterone concentrations, suggesting that NO treatment prevented its pathophysiological overactivation. In contrast, single peroxynitrite administration specifically induced the MR transactivation activity whereas genomic GR activity remained unchanged. Mechanistically, peroxynitrite permitted nuclear MR translocation whereas the cytosolic GR distribution was unaffected. Consequently, peroxynitrite represents a MR-specific aldosterone mimetic. In summary, our data indicate that the genomic function of corticosteroid receptors can be modulated by nitrosative stress which may induce the shift from physiological toward pathophysiological MR effects. Copyright © 2012 Elsevier Inc. All rights reserved.

  17. The aryl hydrocarbon receptor and glucocorticoid receptor interact to activate human metallothionein 2A

    Energy Technology Data Exchange (ETDEWEB)

    Sato, Shoko, E-mail: satosho@rs.tus.ac.jp [Laboratory of Nutrition, Graduate School of Agricultural Science, Tohoku University, Sendai 981-8555 (Japan); Shirakawa, Hitoshi, E-mail: shirakah@m.tohoku.ac.jp [Laboratory of Nutrition, Graduate School of Agricultural Science, Tohoku University, Sendai 981-8555 (Japan); Tomita, Shuhei, E-mail: tomita@med.tottori-u.ac.jp [Division of Molecular Pharmacology, Department of Pathophysiological and Therapeutic Science, Yonago 683-8503 (Japan); Tohkin, Masahiro, E-mail: tohkin@phar.nagoya-cu.ac.jp [Department of Medical Safety Science, Graduate School of Pharmaceutical Science, Nagoya City University, Nagoya 267-8603 (Japan); Gonzalez, Frank J., E-mail: gonzalef@mail.nih.gov [Laboratory of Metabolism, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892 (United States); Komai, Michio, E-mail: mkomai@m.tohoku.ac.jp [Laboratory of Nutrition, Graduate School of Agricultural Science, Tohoku University, Sendai 981-8555 (Japan)

    2013-11-15

    Although the aryl hydrocarbon receptor (AHR) and glucocorticoid receptor (GR) play essential roles in mammalian development, stress responses, and other physiological events, crosstalk between these receptors has been the subject of much debate. Metallothioneins are classic glucocorticoid-inducible genes that were reported to increase upon treatment with AHR agonists in rodent tissues and cultured human cells. In this study, the mechanism of human metallothionein 2A (MT2A) gene transcription activation by AHR was investigated. Cotreatment with 3-methylcholanthrene and dexamethasone, agonists of AHR and GR respectively, synergistically increased MT2A mRNA levels in HepG2 cells. MT2A induction was suppressed by RNA interference against AHR or GR. Coimmunoprecipitation experiments revealed a physical interaction between AHR and GR proteins. Moreover, chromatin immunoprecipitation assays indicated that AHR was recruited to the glucocorticoid response element in the MT2A promoter. Thus, we provide a novel mechanism whereby AHR modulates expression of human MT2A via the glucocorticoid response element and protein–protein interactions with GR. - Highlights: • Aryl hydrocarbon receptor forms a complex with glucocorticoid receptor in cells. • Human metallothionein gene is regulated by the AHR and GR interaction. • AHR–GR complex binds to glucocorticoid response element in metallothionein gene. • We demonstrated a novel transcriptional mechanism via AHR and GR interaction.

  18. Intramolecular Crosstalk between Catalytic Activities of Receptor Kinases

    KAUST Repository

    Kwezi, Lusisizwe

    2018-01-22

    Signal modulation is important for the growth and development of plants and this process is mediated by a number of factors including physiological growth regulators and their associated signal transduction pathways. Protein kinases play a central role in signaling, including those involving pathogen response mechanisms. We previously demonstrated an active guanylate cyclase (GC) catalytic center in the brassinosteroid insensitive receptor (AtBRI1) within an active intracellular kinase domain resulting in dual enzymatic activity. Here we propose a novel type of receptor architecture that is characterized by a functional GC catalytic center nested in the cytosolic kinase domain enabling intramolecular crosstalk. This may be through a cGMP-AtBRI1 complex forming that may induce a negative feedback mechanism leading to desensitisation of the receptor, regulated through the cGMP production pathway. We further argue that the comparatively low but highly localized cGMP generated by the GC in response to a ligand is sufficient to modulate the kinase activity. This type of receptor therefore provides a molecular switch that directly and/or indirectly affects ligand dependent phosphorylation of downstream signaling cascades and suggests that subsequent signal transduction and modulation works in conjunction with the kinase in downstream signaling.

  19. Ah receptor agonist activity in frequently consumed food items

    NARCIS (Netherlands)

    Waard, de W.J.; Aarts, J.M.M.J.G.; Peijnenburg, A.A.C.M.; Kok, de T.M.C.M.; Schooten, van F.J.; Hoogenboom, L.A.P.

    2008-01-01

    The aryl hydrocarbon receptor (AhR) receives much attention for its role in the toxicity of dioxins and dioxin-like polychlorinated biphenyls. However, many other compounds have also been reported to bind and activate AhR, of which natural food components are of special interest from a human health

  20. Nuclear Receptor Activity and Liver Cancer Lesion Progression

    Science.gov (United States)

    Nuclear receptors (NRs) are ligand-activated transcription factors that control diverse cellular processes. Chronic stimulation of some NRs is a non-genotoxic mechanism of rodent liver cancer with unclear relevance to humans. We explored this question using human CAR, PXR, PPARα,...

  1. Analysis of Chromatin Dynamics during Glucocorticoid Receptor Activation

    Science.gov (United States)

    Burd, Craig J.; Ward, James M.; Crusselle-Davis, Valerie J.; Kissling, Grace E.; Phadke, Dhiral; Shah, Ruchir R.

    2012-01-01

    Steroid hormone receptors initiate a genetic program tightly regulated by the chromatin environment of the responsive regions. Using the glucocorticoid receptor (GR) as a model factor for transcriptional initiation, we classified chromatin structure through formaldehyde-assisted isolation of regulatory elements (FAIRE). We looked at dynamic changes in FAIRE signals during GR activation specifically at regions of receptor interaction. We found a distribution of GR-responsive regions with diverse responses to activation and chromatin modulation. The majority of GR binding regions demonstrate increases in FAIRE signal in response to ligand. However, the majority GR-responsive regions shared a similar FAIRE signal in the basal chromatin state, suggesting a common chromatin structure for GR recruitment. Supporting this notion, global FAIRE sequencing (seq) data indicated an enrichment of signal surrounding the GR binding site prior to activation. Brg-1 knockdown showed response element-specific effects of ATPase-dependent chromatin remodeling. FAIRE induction was universally decreased by Brg-1 depletion, but to varying degrees in a target specific manner. Taken together, these data suggest classes of nuclear receptor response regions that react to activation through different chromatin regulatory events and identify a chromatin structure that classifies the majority of response elements tested. PMID:22451486

  2. Thrombopoietin receptor activation by myeloproliferative neoplasm associated calreticulin mutants.

    Science.gov (United States)

    Chachoua, Ilyas; Pecquet, Christian; El-Khoury, Mira; Nivarthi, Harini; Albu, Roxana-Irina; Marty, Caroline; Gryshkova, Vitalina; Defour, Jean-Philippe; Vertenoeil, Gaëlle; Ngo, Anna; Koay, Ann; Raslova, Hana; Courtoy, Pierre J; Choong, Meng Ling; Plo, Isabelle; Vainchenker, William; Kralovics, Robert; Constantinescu, Stefan N

    2016-03-10

    Mutations in the calreticulin gene (CALR) represented by deletions and insertions in exon 9 inducing a -1/+2 frameshift are associated with a significant fraction of myeloproliferative neoplasms (MPNs). The mechanisms by which CALR mutants induce MPN are unknown. Here, we show by transcriptional, proliferation, biochemical, and primary cell assays that the pathogenic CALR mutants specifically activate the thrombopoietin receptor (TpoR/MPL). No activation is detected with a battery of type I and II cytokine receptors, except granulocyte colony-stimulating factor receptor, which supported only transient and weak activation. CALR mutants induce ligand-independent activation of JAK2/STAT/phosphatydylinositol-3'-kinase (PI3-K) and mitogen-activated protein (MAP) kinase pathways via TpoR, and autonomous growth in Ba/F3 cells. In these transformed cells, no synergy is observed between JAK2 and PI3-K inhibitors in inhibiting cytokine-independent proliferation, thus showing a major difference from JAK2V617F cells where such synergy is strong. TpoR activation was dependent on its extracellular domain and its N-glycosylation, especially at N117. The glycan binding site and the novel C-terminal tail of the mutant CALR proteins were required for TpoR activation. A soluble form of TpoR was able to prevent activation of full-length TpoR provided that it was N-glycosylated. By confocal microscopy and subcellular fractionation, CALR mutants exhibit different intracellular localization from that of wild-type CALR. Finally, knocking down either MPL/TpoR or JAK2 in megakaryocytic progenitors from patients carrying CALR mutations inhibited cytokine-independent megakaryocytic colony formation. Taken together, our study provides a novel signaling paradigm, whereby a mutated chaperone constitutively activates cytokine receptor signaling. © 2016 by The American Society of Hematology.

  3. Influence of phasic and tonic dopamine release on receptor activation

    DEFF Research Database (Denmark)

    Dreyer, Jakob Kristoffer Kisbye; Herrik, Kjartan F; Berg, Rune W

    2010-01-01

    Tonic and phasic dopamine release is implicated in learning, motivation, and motor functions. However, the relationship between spike patterns in dopaminergic neurons, the extracellular concentration of dopamine, and activation of dopamine receptors remains unresolved. In the present study, we...... develop a computational model of dopamine signaling that give insight into the relationship between the dynamics of release and occupancy of D(1) and D(2) receptors. The model is derived from first principles using experimental data. It has no free parameters and offers unbiased estimation...

  4. Screening of selected pesticides for oestrogen receptor activation in vitro

    DEFF Research Database (Denmark)

    Vinggaard, Anne; Breinholt, Vibeke; Larsen, John Christian

    1999-01-01

    Twenty pesticides were tested for their ability to activate the oestrogen receptor in vitro using an,MCF7 cell proliferation assay and a Yeast Oestrogen Screen. The fungicides fenarimol, triadimefon, and triadimenol were identified as weak oestrogen receptor agonists, which at 10 mu M induces a 2.......0, 2.4, and 1.9-fold increase in proliferation of human MCF7 breast cancer cells (E3 clone). The relative proliferation efficiency (RPE) was 43-69%, indicating partial agonism at the oestrogen receptor. Several pesticides did not have any effect oil the proliferation response after 6 days of exposure......, including. chlorpyrifos, diuron, iprodion, linuron, pentachlorphenol, prochloraz, propioconazol, propyzamine, quintozen, tetrachorvinphos and tetradifon. Some pesticides resulted in a negligible proliferation response, which was nor statistically significant under the present experimental conditions...

  5. CINPA1 Is an Inhibitor of Constitutive Androstane Receptor That Does Not Activate Pregnane X Receptor

    Science.gov (United States)

    Cherian, Milu T; Lin, Wenwei; Wu, Jing

    2015-01-01

    Constitutive androstane receptor (CAR) and pregnane X receptor (PXR) are xenobiotic sensors that enhance the detoxification and elimination of xenobiotics and endobiotics by modulating the expression of genes encoding drug-metabolizing enzymes and transporters. Elevated levels of drug-metabolizing enzymes and efflux transporters, resulting from CAR activation in various cancers, promote the elimination of chemotherapeutic agents, leading to reduced therapeutic effectiveness and acquired drug resistance. CAR inhibitors, in combination with existing chemotherapeutics, could therefore be used to attenuate multidrug resistance in cancers. Interestingly, all previously reported CAR inverse-agonists are also activators of PXR, rendering them mechanistically counterproductive in tissues where both these xenobiotic receptors are present and active. We used a directed high-throughput screening approach, followed by subsequent mechanistic studies, to identify novel, potent, and specific small-molecule CAR inhibitors that do not activate PXR. We describe here one such inhibitor, CINPA1 (CAR inhibitor not PXR activator 1), capable of reducing CAR-mediated transcription with an IC50 of ∼70 nM. CINPA1 1) is a specific xenobiotic receptor inhibitor and has no cytotoxic effects up to 30 µM; 2) inhibits CAR-mediated gene expression in primary human hepatocytes, where CAR is endogenously expressed; 3) does not alter the protein levels or subcellular localization of CAR; 4) increases corepressor and reduces coactivator interaction with the CAR ligand-binding domain in mammalian two-hybrid assays; and 5) disrupts CAR binding to the promoter regions of target genes in chromatin immunoprecipitation assays. CINPA1 could be used as a novel molecular tool for understanding CAR function. PMID:25762023

  6. CINPA1 is an inhibitor of constitutive androstane receptor that does not activate pregnane X receptor.

    Science.gov (United States)

    Cherian, Milu T; Lin, Wenwei; Wu, Jing; Chen, Taosheng

    2015-05-01

    Constitutive androstane receptor (CAR) and pregnane X receptor (PXR) are xenobiotic sensors that enhance the detoxification and elimination of xenobiotics and endobiotics by modulating the expression of genes encoding drug-metabolizing enzymes and transporters. Elevated levels of drug-metabolizing enzymes and efflux transporters, resulting from CAR activation in various cancers, promote the elimination of chemotherapeutic agents, leading to reduced therapeutic effectiveness and acquired drug resistance. CAR inhibitors, in combination with existing chemotherapeutics, could therefore be used to attenuate multidrug resistance in cancers. Interestingly, all previously reported CAR inverse-agonists are also activators of PXR, rendering them mechanistically counterproductive in tissues where both these xenobiotic receptors are present and active. We used a directed high-throughput screening approach, followed by subsequent mechanistic studies, to identify novel, potent, and specific small-molecule CAR inhibitors that do not activate PXR. We describe here one such inhibitor, CINPA1 (CAR inhibitor not PXR activator 1), capable of reducing CAR-mediated transcription with an IC50 of ∼70 nM. CINPA1 1) is a specific xenobiotic receptor inhibitor and has no cytotoxic effects up to 30 µM; 2) inhibits CAR-mediated gene expression in primary human hepatocytes, where CAR is endogenously expressed; 3) does not alter the protein levels or subcellular localization of CAR; 4) increases corepressor and reduces coactivator interaction with the CAR ligand-binding domain in mammalian two-hybrid assays; and 5) disrupts CAR binding to the promoter regions of target genes in chromatin immunoprecipitation assays. CINPA1 could be used as a novel molecular tool for understanding CAR function. Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.

  7. Activation of serotonin receptors promotes microglial injury-induced motility but attenuates phagocytic activity

    NARCIS (Netherlands)

    Krabbe, Grietje; Matyash, Vitali; Pannasch, Ulrike; Mamer, Lauren; Boddeke, Hendrikus W. G. M.; Kettenmann, Helmut

    Microglia, the brain immune cell, express several neurotransmitter receptors which modulate microglial functions. In this project we studied the impact of serotonin receptor activation on distinct microglial properties as serotonin deficiency not only has been linked to a number of psychiatric

  8. Glycine Receptor α2 Subunit Activation Promotes Cortical Interneuron Migration

    Directory of Open Access Journals (Sweden)

    Ariel Avila

    2013-08-01

    Full Text Available Glycine receptors (GlyRs are detected in the developing CNS before synaptogenesis, but their function remains elusive. This study demonstrates that functional GlyRs are expressed by embryonic cortical interneurons in vivo. Furthermore, genetic disruption of these receptors leads to interneuron migration defects. We discovered that extrasynaptic activation of GlyRs containing the α2 subunit in cortical interneurons by endogenous glycine activates voltage-gated calcium channels and promotes calcium influx, which further modulates actomyosin contractility to fine-tune nuclear translocation during migration. Taken together, our data highlight the molecular events triggered by GlyR α2 activation that control cortical tangential migration during embryogenesis.

  9. Peroxisome Proliferator-Activated Receptor Alpha Target Genes

    Directory of Open Access Journals (Sweden)

    Maryam Rakhshandehroo

    2010-01-01

    Full Text Available The peroxisome proliferator-activated receptor alpha (PPARα is a ligand-activated transcription factor involved in the regulation of a variety of processes, ranging from inflammation and immunity to nutrient metabolism and energy homeostasis. PPARα serves as a molecular target for hypolipidemic fibrates drugs which bind the receptor with high affinity. Furthermore, PPARα binds and is activated by numerous fatty acids and fatty acid-derived compounds. PPARα governs biological processes by altering the expression of a large number of target genes. Accordingly, the specific role of PPARα is directly related to the biological function of its target genes. Here, we present an overview of the involvement of PPARα in lipid metabolism and other pathways through a detailed analysis of the different known or putative PPARα target genes. The emphasis is on gene regulation by PPARα in liver although many of the results likely apply to other organs and tissues as well.

  10. Peroxisome Proliferator-Activated Receptor Alpha Target Genes

    Science.gov (United States)

    Rakhshandehroo, Maryam; Knoch, Bianca; Müller, Michael; Kersten, Sander

    2010-01-01

    The peroxisome proliferator-activated receptor alpha (PPARα) is a ligand-activated transcription factor involved in the regulation of a variety of processes, ranging from inflammation and immunity to nutrient metabolism and energy homeostasis. PPARα serves as a molecular target for hypolipidemic fibrates drugs which bind the receptor with high affinity. Furthermore, PPARα binds and is activated by numerous fatty acids and fatty acid-derived compounds. PPARα governs biological processes by altering the expression of a large number of target genes. Accordingly, the specific role of PPARα is directly related to the biological function of its target genes. Here, we present an overview of the involvement of PPARα in lipid metabolism and other pathways through a detailed analysis of the different known or putative PPARα target genes. The emphasis is on gene regulation by PPARα in liver although many of the results likely apply to other organs and tissues as well. PMID:20936127

  11. Atypical chemokine receptor 4 shapes activated B cell fate.

    Science.gov (United States)

    Kara, Ervin E; Bastow, Cameron R; McKenzie, Duncan R; Gregor, Carly E; Fenix, Kevin A; Babb, Rachelle; Norton, Todd S; Zotos, Dimitra; Rodda, Lauren B; Hermes, Jana R; Bourne, Katherine; Gilchrist, Derek S; Nibbs, Robert J; Alsharifi, Mohammed; Vinuesa, Carola G; Tarlinton, David M; Brink, Robert; Hill, Geoffrey R; Cyster, Jason G; Comerford, Iain; McColl, Shaun R

    2018-01-31

    Activated B cells can initially differentiate into three functionally distinct fates-early plasmablasts (PBs), germinal center (GC) B cells, or early memory B cells-by mechanisms that remain poorly understood. Here, we identify atypical chemokine receptor 4 (ACKR4), a decoy receptor that binds and degrades CCR7 ligands CCL19/CCL21, as a regulator of early activated B cell differentiation. By restricting initial access to splenic interfollicular zones (IFZs), ACKR4 limits the early proliferation of activated B cells, reducing the numbers available for subsequent differentiation. Consequently, ACKR4 deficiency enhanced early PB and GC B cell responses in a CCL19/CCL21-dependent and B cell-intrinsic manner. Conversely, aberrant localization of ACKR4-deficient activated B cells to the IFZ was associated with their preferential commitment to the early PB linage. Our results reveal a regulatory mechanism of B cell trafficking via an atypical chemokine receptor that shapes activated B cell fate. © 2018 Kara et al.

  12. Gaddum Memorial Lecture 2014: receptors as an evolving concept: from switches to biased microprocessors

    Science.gov (United States)

    Kenakin, Terry

    2015-01-01

    This review is based on the JR Vane Medal Lecture presented at the BPS Winter Meeting in December 2014 by T. Kenakin. A recording of the lecture is included as supporting information and can also be viewed online here: https://www.youtube.com/watch?v=xrP81AQ8l-8. Pharmacological models used to describe drug agonism and antagonism have evolved over the past 20 years from a parsimonious model describing single active and inactive receptor states to models of multiconformational receptor systems modified by ligand conformational selection. These latter models describe the observed, presently underexploited, pharmacological mechanism of ligand-directed biased signalling. Biased signals can be quantified with transduction coefficients (ΔΔLog(τ/KA) values), a scale grounded in the Black/Leff operational model; this enables the optimization of biased profiles through medicinal chemistry. The past decades have also brought the availability of new technologies to measure multiple functional effects mediated by seven transmembrane receptors. These have confirmed that drugs can have many efficacies, which may be collaterally linked, that is there is no linear sequence of activities required. In addition, new functional screening assays have introduced increasing numbers of allosteric ligands into drug discovery. These molecules are permissive (they do not necessarily preclude endogenous signalling in vivo); therefore, they may allow better fine tuning of pathological physiology. The permissive quality of allosteric ligands can also change the quality of endogenous signalling efficacy (‘induced bias’) as well as the quantity of signal; in this regard, indices related to ΔΔLog(τ/KA) values (namely ΔLog(αβ) values) can be used to quantify these effects for optimization in the drug discovery process. All of these added scales of drug activity will, hopefully, allow better targeting of candidate molecules towards therapies. PMID:26075971

  13. Gaddum Memorial Lecture 2014: receptors as an evolving concept: from switches to biased microprocessors.

    Science.gov (United States)

    Kenakin, Terry

    2015-09-01

    This review is based on the JR Vane Medal Lecture presented at the BPS Winter Meeting in December 2014 by T. Kenakin. A recording of the lecture is included as supporting information and can also be viewed online here: https://www.youtube.com/watch?v=xrP81AQ8l-8. Pharmacological models used to describe drug agonism and antagonism have evolved over the past 20 years from a parsimonious model describing single active and inactive receptor states to models of multiconformational receptor systems modified by ligand conformational selection. These latter models describe the observed, presently underexploited, pharmacological mechanism of ligand-directed biased signalling. Biased signals can be quantified with transduction coefficients (ΔΔLog(τ/KA) values), a scale grounded in the Black/Leff operational model; this enables the optimization of biased profiles through medicinal chemistry. The past decades have also brought the availability of new technologies to measure multiple functional effects mediated by seven transmembrane receptors. These have confirmed that drugs can have many efficacies, which may be collaterally linked, that is there is no linear sequence of activities required. In addition, new functional screening assays have introduced increasing numbers of allosteric ligands into drug discovery. These molecules are permissive (they do not necessarily preclude endogenous signalling in vivo); therefore, they may allow better fine tuning of pathological physiology. The permissive quality of allosteric ligands can also change the quality of endogenous signalling efficacy ('induced bias') as well as the quantity of signal; in this regard, indices related to ΔΔLog(τ/KA) values (namely ΔLog(αβ) values) can be used to quantify these effects for optimization in the drug discovery process. All of these added scales of drug activity will, hopefully, allow better targeting of candidate molecules towards therapies. © 2015 The British Pharmacological Society.

  14. Hyaluronic acid induces activation of the κ-opioid receptor.

    Directory of Open Access Journals (Sweden)

    Barbara Zavan

    Full Text Available Nociceptive pain is one of the most common types of pain that originates from an injury involving nociceptors. Approximately 60% of the knee joint innervations are classified as nociceptive. The specific biological mechanism underlying the regulation of nociceptors is relevant for the treatment of symptoms affecting the knee joint. Intra-articular administration of exogenous hyaluronic acid (HA in patients with osteoarthritis (OA appears to be particularly effective in reducing pain and improving patient function.We performed an in vitro study conducted in CHO cells that expressed a panel of opioid receptors and in primary rat dorsal root ganglion (DRG neurons to determine if HA induces the activation of opioid peptide receptors (OPr using both aequorin and the fluorescent dye Fura-2/AM.Selective agonists and antagonists for each OPr expressed on CHO cells were used to test the efficacy of our in vitro model followed by stimulation with HA. The results showed that HA induces stimulatory effects on the κ receptor (KOP. These effects of HA were also confirmed in rat DRG neurons, which express endogenously the OPr.HA activates the KOP receptor in a concentration dependent manner, with a pEC(50 value of 7.57.

  15. Search for Active-State Conformation of Drug Target GPCR Using Real-Coded Genetic Algorithm

    Science.gov (United States)

    Ishino, Yoko; Harada, Takanori; Aida, Misako

    G-Protein coupled receptors (GPCRs) comprise a large superfamily of proteins and are a target for nearly 50% of drugs in clinical use today. GPCRs have a unique structural motif, seven transmembrane helices, and it is known that agonists and antagonists dock with a GPCR in its ``active'' and ``inactive'' condition, respectively. Knowing conformations of both states is eagerly anticipated for elucidation of drug action mechanism. Since GPCRs are difficult to crystallize, the 3D structures of these receptors have not yet been determined by X-ray crystallography, except the inactive-state conformation of two proteins. The conformation of them enabled the inactive form of other GPCRs to be modeled by computer-aided homology modeling. However, to date, the active form of GPCRs has not been solved. This paper describes a novel method to predict the 3D structure of an active-state GPCR aiming at molecular docking-based virtual screening using real-coded genetic algorithm (real-coded GA), receptor-ligand docking simulations, and molecular dynamics (MD) simulations. The basic idea of the method is that the MD is first used to calculate an average 3D coordinates of all atoms of a GPCR protein against heat fluctuation on the pico- or nano- second time scale, and then real-coded GA involving receptor-ligand docking simulations functions to determine the rotation angle of each helix as a movement on wider time scale. The method was validated using human leukotriene B4 receptor BLT1 as a sample GPCR. Our study demonstrated that the established evolutionary search for the active state of the leukotriene receptor provided the appropriate 3D structure of the receptor to dock with its agonists.

  16. Pyrimidinergic Receptor Activation Controls Toxoplasma gondii Infection in Macrophages.

    Directory of Open Access Journals (Sweden)

    Aline Cristina Abreu Moreira-Souza

    Full Text Available Infection by the protozoan parasite Toxoplasma gondii is highly prevalent worldwide and may have serious clinical manifestations in immunocompromised patients. T. gondii is an obligate intracellular parasite that infects almost any cell type in mammalian hosts, including immune cells. The immune cells express purinergic P2 receptors in their membrane--subdivided into P2Y and P2X subfamilies--whose activation is important for infection control. Here, we examined the effect of treatment with UTP and UDP in mouse peritoneal macrophages infected with T. gondii tachyzoites. Treatment with these nucleotides reduced parasitic load by 90%, but did not increase the levels of the inflammatory mediators NO and ROS, nor did it modulate host cell death by apoptosis or necrosis. On the other hand, UTP and UDP treatments induced early egress of tachyzoites from infected macrophages, in a Ca2+-dependent manner, as shown by scanning electron microscopy analysis, and videomicroscopy. In subsequent infections, prematurely egressed parasites had reduced infectivity, and could neither replicate nor inhibit the fusion of lysosomes to the parasitophorous vacuole. The use of selective agonists and antagonists of the receptor subtypes P2Y2 and P2Y4 and P2Y6 showed that premature parasite egress may be mediated by the activation of these receptor subtypes. Our results suggest that the activity of P2Y host cell receptors controls T. gondii infection in macrophages, highlighting the importance of pyrimidinergic signaling for innate immune system response against infection. Finally the P2Y receptors should be considered as new target for the development of drugs against T. gondii infection.

  17. Common structural basis for constitutive activity of the ghrelin receptor family

    DEFF Research Database (Denmark)

    Holst, Birgitte; Holliday, Nicholas D; Bach, Anders

    2004-01-01

    Three members of the ghrelin receptor family were characterized in parallel: the ghrelin receptor, the neurotensin receptor 2 and the orphan receptor GPR39. In transiently transfected COS-7 and human embryonic kidney 293 cells, all three receptors displayed a high degree of ligand......-independent signaling activity. The structurally homologous motilin receptor served as a constitutively silent control; upon agonist stimulation, however, it signaled with a similar efficacy to the three related receptors. The constitutive activity of the ghrelin receptor and of neurotensin receptor 2 through the G...... demonstrated that the epitope-tagged ghrelin receptor was constitutively internalized but could be trapped at the cell surface by an inverse agonist, whereas GPR39 remained at the cell surface. Mutational analysis showed that the constitutive activity of both the ghrelin receptor and GPR39 could systematically...

  18. Transcriptional activation by the thyroid hormone receptor through ligand-dependent receptor recruitment and chromatin remodelling

    DEFF Research Database (Denmark)

    Grøntved, Lars; Waterfall, Joshua J; Kim, Dong Wook

    2015-01-01

    A bimodal switch model is widely used to describe transcriptional regulation by the thyroid hormone receptor (TR). In this model, the unliganded TR forms stable, chromatin-bound complexes with transcriptional co-repressors to repress transcription. Binding of hormone dissociates co-repressors and...... process. This dynamic and ligand-dependent interaction with chromatin is likely shared by all steroid hormone receptors regardless of their capacity to repress transcription in the absence of ligand.......A bimodal switch model is widely used to describe transcriptional regulation by the thyroid hormone receptor (TR). In this model, the unliganded TR forms stable, chromatin-bound complexes with transcriptional co-repressors to repress transcription. Binding of hormone dissociates co......-repressors and facilitates recruitment of co-activators to activate transcription. Here we show that in addition to hormone-independent TR occupancy, ChIP-seq against endogenous TR in mouse liver tissue demonstrates considerable hormone-induced TR recruitment to chromatin associated with chromatin remodelling and activated...

  19. Neurohumoral activation in heart failure: the role of adrenergic receptors

    Directory of Open Access Journals (Sweden)

    Patricia C. Brum

    2006-09-01

    Full Text Available Heart failure (HF is a common endpoint for many forms of cardiovascular disease and a significant cause of morbidity and mortality. The development of end-stage HF often involves an initial insult to the myocardium that reduces cardiac output and leads to a compensatory increase in sympathetic nervous system activity. Acutely, the sympathetic hyperactivity through the activation of beta-adrenergic receptors increases heart rate and cardiac contractility, which compensate for decreased cardiac output. However, chronic exposure of the heart to elevated levels of catecholamines released from sympathetic nerve terminals and the adrenal gland may lead to further pathologic changes in the heart, resulting in continued elevation of sympathetic tone and a progressive deterioration in cardiac function. On a molecular level, altered beta-adrenergic receptor signaling plays a pivotal role in the genesis and progression of HF. beta-adrenergic receptor number and function are decreased, and downstream mechanisms are altered. In this review we will present an overview of the normal beta-adrenergic receptor pathway in the heart and the consequences of sustained adrenergic activation in HF. The myopathic potential of individual components of the adrenergic signaling will be discussed through the results of research performed in genetic modified animals. Finally, we will discuss the potential clinical impact of beta-adrenergic receptor gene polymorphisms for better understanding the progression of HF.A insuficiência cardíaca (IC é a via final comum da maioria das doenças cardiovasculares e uma das maiores causas de morbi-mortalidade. O desenvolvimento do estágio final da IC freqüentemente envolve um insulto inicial do miocárdio, reduzindo o débito cardíaco e levando ao aumento compensatório da atividade do sistema nervoso simpático (SNS. Existem evidências de que apesar da exposição aguda ser benéfica, exposições crônicas a elevadas concentra

  20. Protease-activated receptors – biology and role in cancer

    Directory of Open Access Journals (Sweden)

    Dominika Hempel

    2016-07-01

    Full Text Available The fact that blood coagulation disorders may accompany malignant disease is well established. However, many studies have shown that components of the haemostatic system may also elicit signaling leading to cancer developement and progression. The potential mechanism by which coagulation factors play a role in cancer invasion is not completely understood, but one hypothesis is that protease-activated receptors (PARs play a prominent role. PARs are transmembrane G-protein-coupled receptors (GPCRs that are activated by a unique proteolytic mechanism. They have important functions in haemostasis and inflammation but may also be implicated in cancer cell progression. Thrombin, tissue factor (TF and matrix metalloproteinases (MMPs are the main activators of these receptors. The mechanism of persistent activation of PARs was also described in cancer cells. Here, we discuss the physiological and pathological role of PARs with a particular focus on PARs’ contribution to cancer biology. We also present therapeutic options tailored specifically to inhibition of PAR-induced signalling in cancer patients.

  1. Activation of Penile Proadipogenic Peroxisome Proliferator-Activated Receptor with an Estrogen: Interaction with Estrogen Receptor Alpha during Postnatal Development

    Directory of Open Access Journals (Sweden)

    Mahmoud M. Mansour

    2008-01-01

    Full Text Available Exposure to the estrogen receptor alpha (ER ligand diethylstilbesterol (DES between neonatal days 2 to 12 induces penile adipogenesis and adult infertility in rats. The objective of this study was to investigate the in vivo interaction between DES-activated ER and the proadipogenic transcription factor peroxisome proliferator-activated receptor gamma (PPAR. Transcripts for PPARs , , and and 1a splice variant were detected in Sprague-Dawley normal rat penis with PPAR predominating. In addition, PPAR1b and PPAR2 were newly induced by DES. The PPAR transcripts were significantly upregulated with DES and reduced by antiestrogen ICI 182, 780. At the cellular level, PPAR protein was detected in urethral transitional epithelium and stromal, endothelial, neuronal, and smooth muscular cells. Treatment with DES activated ER and induced adipocyte differentiation in corpus cavernosum penis. Those adipocytes exhibited strong nuclear PPAR expression. These results suggest a biological overlap between PPAR and ER and highlight a mechanism for endocrine disruption.

  2. Mechanism of A2 adenosine receptor activation. I. Blockade of A2 adenosine receptors by photoaffinity labeling

    Energy Technology Data Exchange (ETDEWEB)

    Lohse, M.J.; Klotz, K.N.; Schwabe, U.

    1991-04-01

    It has previously been shown that covalent incorporation of the photoreactive adenosine derivative (R)-2-azido-N6-p-hydroxy-phenylisopropyladenosine ((R)-AHPIA) into the A1 adenosine receptor of intact fat cells leads to a persistent activation of this receptor, resulting in a reduction of cellular cAMP levels. In contrast, covalent incorporation of (R)-AHPIA into human platelet membranes, which contain only stimulatory A2 adenosine receptors, reduces adenylate cyclase stimulation via these receptors. This effect of (R)-AHPIA is specific for the A2 receptor and can be prevented by the adenosine receptor antagonist theophylline. Binding studies indicate that up to 90% of A2 receptors can be blocked by photoincorporation of (R)-AHPIA. However, the remaining 10-20% of A2 receptors are sufficient to mediate an adenylate cyclase stimulation of up to 50% of the control value. Similarly, the activation via these 10-20% of receptors occurs with a half-life that is only 2 times longer than that in control membranes. This indicates the presence of a receptor reserve, with respect to both the extent and the rate of adenylate cyclase stimulation. These observations require a modification of the models of receptor-adenylate cyclase coupling.

  3. Peroxisome Proliferator-Activated Receptors and Acute Lung Injury

    Directory of Open Access Journals (Sweden)

    Rosanna Di Paola

    2007-01-01

    Full Text Available Peroxisome proliferator-activated receptors are ligand-activated transcription factors belonging to the nuclear hormone receptor superfamily. PPARs regulate several metabolic pathways by binding to sequence-specific PPAR response elements in the promoter region of target genes, including lipid biosynthesis and glucose metabolism. Recently, PPARs and their respective ligands have been implicated as regulators of cellular inflammatory and immune responses. These molecules are thought to exert anti-inflammatory effects by negatively regulating the expression of proinflammatory genes. Several studies have demonstrated that PPAR ligands possess anti-inflammatory properties and that these properties may prove helpful in the treatment of inflammatory diseases of the lung. This review will outline the anti-inflammatory effects of PPARs and PPAR ligands and discuss their potential therapeutic effects in animal models of inflammatory lung disease.

  4. Activation of peroxisome proliferator-activated receptor gamma is crucial for antitumoral effects of 6-iodolactone

    OpenAIRE

    Nava-Villalba, Mario; Nuñez-Anita, Rosa E.; Bontempo, Alexander; Aceves, Carmen

    2015-01-01

    Background Molecular iodine (I2) exhibits antiproliferative and apoptotic effects on in vivo and in vitro cancer models. These effects are thought to be mediated by an iodinated arachidonic acid derivative, 6-iodolactone (6IL), and one of the proposed mechanisms is that 6IL activates Peroxisome Proliferator-Activated Receptors type gamma (PPARG). These receptors have been implicated in the inhibition of carcinogenic processes, in addition to their classical role in maintaining lipid and gluco...

  5. Monoclonal antibodies to the insulin receptor stimulate the intrinsic tyrosine kinase activity by cross-linking receptor molecules.

    Science.gov (United States)

    O'Brien, R M; Soos, M A; Siddle, K

    1987-12-20

    The effect of monoclonal anti-insulin receptor antibodies on the intrinsic kinase activity of solubilized receptor was investigated. Antibodies for six distinct epitopes stimulated receptor autophosphorylation and kinase activity towards exogenous substrates. This effect of antibodies was seen only within a narrow concentration range and monovalent antibody fragments were ineffective. Evidence was obtained by sucrose density-gradient centrifugation for the formation of antibody-receptor complexes which involved both inter- and intra-molecular cross-linking, although stimulation of autophosphorylation appeared to be preferentially associated with the latter. There was partial additivity between the effects of insulin and antibodies in stimulating autophosphorylation, although the sites of phosphorylation appeared identical on two-dimensional peptide maps. Antibodies for two further epitopes failed to activate receptor kinase, but inhibited its stimulation by insulin. The effects of antibodies on kinase activity paralleled their metabolic effects on adipocytes, except for one antibody which was potently insulin-like in its metabolic effects, but which antagonized insulin stimulation of kinase activity. It is concluded that antibodies activate the receptor by cross-linking subunits rather than by reacting at specific epitopes. The ability of some antibodies to activate receptor may depend on receptor environment as well as the disposition of epitopes.

  6. DHEA metabolites activate estrogen receptors alpha and beta

    OpenAIRE

    Michael Miller, Kristy K.; Al-Rayyan, Numan; Ivanova, Margarita M.; Mattingly, Kathleen A.; Ripp, Sharon L.; KLINGE, CAROLYN M.; Prough, Russell A.

    2012-01-01

    Dehydroepiandrosterone (DHEA) levels were reported to associate with increased breast cancer risk in postmenopausal women, but some carcinogen-induced rat mammary tumor studies question this claim. The purpose of this study was to determine how DHEA and its metabolites affect estrogen receptors α or β (ERα or ERβ) -regulated gene transcription and cell proliferation. In transiently transfected HEK-293 cells, androstenediol, DHEA, and DHEA-S activated ERα. In ERβ transfected HepG2 cells, andro...

  7. Methylthioadenosine reprograms macrophage activation through adenosine receptor stimulation.

    Directory of Open Access Journals (Sweden)

    Peter A Keyel

    Full Text Available Regulation of inflammation is necessary to balance sufficient pathogen clearance with excessive tissue damage. Central to regulating inflammation is the switch from a pro-inflammatory pathway to an anti-inflammatory pathway. Macrophages are well-positioned to initiate this switch, and as such are the target of multiple therapeutics. One such potential therapeutic is methylthioadenosine (MTA, which inhibits TNFα production following LPS stimulation. We found that MTA could block TNFα production by multiple TLR ligands. Further, it prevented surface expression of CD69 and CD86 and reduced NF-KB signaling. We then determined that the mechanism of this action by MTA is signaling through adenosine A2 receptors. A2 receptors and TLR receptors synergized to promote an anti-inflammatory phenotype, as MTA enhanced LPS tolerance. In contrast, IL-1β production and processing was not affected by MTA exposure. Taken together, these data demonstrate that MTA reprograms TLR activation pathways via adenosine receptors to promote resolution of inflammation.

  8. 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.

  9. Acute activation, desensitization and smoldering activation of human acetylcholine receptors.

    Science.gov (United States)

    Campling, Barbara G; Kuryatov, Alexander; Lindstrom, Jon

    2013-01-01

    The behavioral effects of nicotine and other nicotinic agonists are mediated by AChRs in the brain. The relative contribution of acute activation versus chronic desensitization of AChRs is unknown. Sustained "smoldering activation" occurs over a range of agonist concentrations at which activated and desensitized AChRs are present in equilibrium. We used a fluorescent dye sensitive to changes in membrane potential to examine the effects of acute activation and chronic desensitization by nicotinic AChR agonists on cell lines expressing human α4β2, α3β4 and α7 AChRs. We examined the effects of acute and prolonged application of nicotine and the partial agonists varenicline, cytisine and sazetidine-A on these AChRs. The range of concentrations over which nicotine causes smoldering activation of α4β2 AChRs was centered at 0.13 µM, a level found in smokers. However, nicotine produced smoldering activation of α3β4 and α7 AChRs at concentrations well above levels found in smokers. The α4β2 expressing cell line contains a mixture of two stoichiometries, namely (α4β2)2β2 and (α4β2)2α4. The (α4β2)2β2 stoichiometry is more sensitive to activation by nicotine. Sazetidine-A activates and desensitizes only this stoichiometry. Varenicline, cytisine and sazetidine-A were partial agonists on this mixture of α4β2 AChRs, but full agonists on α3β4 and α7 AChRs. It has been reported that cytisine and varenicline are most efficacious on the (α4β2)2α4 stoichiometry. In this study, we distinguish the dual effects of activation and desensitization of AChRs by these nicotinic agonists and define the range of concentrations over which smoldering activation can be sustained.

  10. Acute activation, desensitization and smoldering activation of human acetylcholine receptors.

    Directory of Open Access Journals (Sweden)

    Barbara G Campling

    Full Text Available The behavioral effects of nicotine and other nicotinic agonists are mediated by AChRs in the brain. The relative contribution of acute activation versus chronic desensitization of AChRs is unknown. Sustained "smoldering activation" occurs over a range of agonist concentrations at which activated and desensitized AChRs are present in equilibrium. We used a fluorescent dye sensitive to changes in membrane potential to examine the effects of acute activation and chronic desensitization by nicotinic AChR agonists on cell lines expressing human α4β2, α3β4 and α7 AChRs. We examined the effects of acute and prolonged application of nicotine and the partial agonists varenicline, cytisine and sazetidine-A on these AChRs. The range of concentrations over which nicotine causes smoldering activation of α4β2 AChRs was centered at 0.13 µM, a level found in smokers. However, nicotine produced smoldering activation of α3β4 and α7 AChRs at concentrations well above levels found in smokers. The α4β2 expressing cell line contains a mixture of two stoichiometries, namely (α4β22β2 and (α4β22α4. The (α4β22β2 stoichiometry is more sensitive to activation by nicotine. Sazetidine-A activates and desensitizes only this stoichiometry. Varenicline, cytisine and sazetidine-A were partial agonists on this mixture of α4β2 AChRs, but full agonists on α3β4 and α7 AChRs. It has been reported that cytisine and varenicline are most efficacious on the (α4β22α4 stoichiometry. In this study, we distinguish the dual effects of activation and desensitization of AChRs by these nicotinic agonists and define the range of concentrations over which smoldering activation can be sustained.

  11. 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...

  12. Liver X Receptors Regulate the Transcriptional Activity of the Glucocorticoid Receptor: Implications for the Carbohydrate Metabolism

    Science.gov (United States)

    Nader, Nancy; Ng, Sinnie Sin Man; Wang, Yonghong; Abel, Brent S.; Chrousos, George P.; Kino, Tomoshige

    2012-01-01

    GLUCOCORTICOIDS are steroid hormones that strongly influence intermediary carbohydrate metabolism by increasing the transcription rate of glucose-6-phosphatase (G6Pase), a key enzyme of gluconeogenesis, and suppress the immune system through the glucocorticoid receptor (GR). The liver X receptors (LXRs), on the other hand, bind to cholesterol metabolites, heterodimerize with the retinoid X receptor (RXR), and regulate the cholesterol turnover, the hepatic glucose metabolism by decreasing the expression of G6Pase, and repress a set of inflammatory genes in immune cells. Since the actions of these receptors overlap with each other, we evaluated the crosstalk between the GR- and LXR-mediated signaling systems. Transient transfection-based reporter assays and gene silencing methods using siRNAs for LXRs showed that overexpression/ligand (GW3965) activation of LXRs/RXRs repressed GR-stimulated transactivation of certain glucocorticoid response element (GRE)-driven promoters in a gene-specific fashion. Activation of LXRs by GW3965 attenuated dexamethasone-stimulated elevation of circulating glucose in rats. It also suppressed dexamethasone-induced mRNA expression of hepatic glucose-6-phosphatase (G6Pase) in rats, mice and human hepatoma HepG2 cells, whereas endogenous, unliganded LXRs were required for dexamethasone-induced mRNA expression of phosphoenolpyruvate carboxylase. In microarray transcriptomic analysis of rat liver, GW3965 differentially regulated glucocorticoid-induced transcriptional activity of about 15% of endogenous glucocorticoid-responsive genes. To examine the mechanism through which activated LXRs attenuated GR transcriptional activity, we examined LXRα/RXRα binding to GREs. Endogenous LXRα/RXRα bound GREs and inhibited GR binding to these DNA sequences both in in vitro and in vivo chromatin immunoprecipitation assays, while their recombinant proteins did so on classic or G6Pase GREs in gel mobility shift assays. We propose that administration of

  13. DMPD: Receptor tyrosine kinases and the regulation of macrophage activation. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 14726496 Receptor tyrosine kinases and the regulation of macrophage activation. Cor...(.csml) Show Receptor tyrosine kinases and the regulation of macrophage activation. PubmedID 14726496 Title ...Receptor tyrosine kinases and the regulation of macrophage activation. Authors Co

  14. Proteinase-activated receptors (PARs) as targets for antiplatelet therapy.

    Science.gov (United States)

    Cunningham, Margaret; McIntosh, Kathryn; Bushell, Trevor; Sloan, Graeme; Plevin, Robin

    2016-04-15

    Since the identification of the proteinase-activated receptor (PAR) family as mediators of serine protease activity in the 1990s, there has been tremendous progress in the elucidation of their pathophysiological roles. The development of drugs that target PARs has been the focus of many laboratories for the potential treatment of thrombosis, cancer and other inflammatory diseases. Understanding the mechanisms of PAR activation and G protein signalling pathways evoked in response to the growing list of endogenous proteases has yielded great insight into receptor regulation at the molecular level. This has led to the development of new selective modulators of PAR activity, particularly PAR1. The mixed success of targeting PARs has been best exemplified in the context of inhibiting PAR1 as a new antiplatelet therapy. The development of the competitive PAR1 antagonist, vorapaxar (Zontivity), has clearly shown the value in targeting PAR1 in acute coronary syndrome (ACS); however the severity of associated bleeding with this drug has limited its use in the clinic. Due to the efficacy of thrombin acting via PAR1, strategies to selectively inhibit specific PAR1-mediated G protein signalling pathways or to target the second thrombin platelet receptor, PAR4, are being devised. The rationale behind these alternative approaches is to bias downstream thrombin activity via PARs to allow for inhibition of pro-thrombotic pathways but maintain other pathways that may preserve haemostatic balance and improve bleeding profiles for widespread clinical use. This review summarizes the structural determinants that regulate PARs and the modulators of PAR activity developed to date. © 2016 Authors; published by Portland Press Limited.

  15. Peroxisome Proliferator–Activated Receptors and The Metabolic Syndrome

    Directory of Open Access Journals (Sweden)

    Anna Meiliana

    2009-04-01

    Full Text Available BACKGROUND: Obesity is a growing threat to global health by virtue of its association with insulin resistance, inflammation, hypertension, and dyslipidemia, collectively known as the metabolic syndrome (MetS. The nuclear receptors PPARα and PPARγ are therapeutic targets for hypertriglyceridemia and insulin resistance, respectively, and drugs that modulate these receptors are currently in clinical use. More recent work on the PPARδ has uncovered a dual benefit for both hypertriglyceridemia and insulin resistance, highlighting the broad potential of PPARs in the treatment of metabolic disease. CONTENT: We have learned much about PPARs, the metabolic fat sensors, and the molecular pathways they regulate. Through their distinct tissue distribution and specific target gene activation, the three PPARs together control diverse aspects of fatty acid metabolism, energy balance, insulin sensitivity glucose homeostasis, inflammation, hypertension and atherosclerosis. These studies have advanced our understanding of the etiology for the MetS. Mechanisms revealed by these studies highlight the importance of emerging concepts, such as the endocrine function of adipose tissue, tissue-tissue cross-talk and lipotoxicity, in the pathogenesis of type 2 diabetes mellitus and CVD. SUMMARY: The elucidation of key regulators of energy balance and insulin signaling have revolutionized our understanding of fat and sugar metabolism and their intimate link. The three ‘lipidsensing’ (PPARα, PPARγ and PPARδ exemplify this connection, regulating diverse aspects of lipid and glucose homeostasis, and serving as bonafide therapeutic targets. KEYWORDS: peroxisome proliferator, activated receptor, metabolic syndrome.

  16. Binding of Losartan to Angiotensin AT1 Receptors Increases Dopamine D1 Receptor Activation

    Science.gov (United States)

    Li, Dong; Scott, Lena; Crambert, Susanne; Zelenin, Sergey; Eklöf, Ann-Christine; Di Ciano, Luis; Ibarra, Fernando

    2012-01-01

    Signaling through both angiotensin AT1 receptors (AT1R) and dopamine D1 receptors (D1R) modulates renal sodium excretion and arterial BP. AT1R and D1R form heterodimers, but whether treatment with AT1R antagonists functionally modifies D1R via allosterism is unknown. In this study, the AT1R antagonist losartan strengthened the interaction between AT1R and D1R and increased expression of D1R on the plasma membrane in vitro. In rat proximal tubule cells that express endogenous AT1R and D1R, losartan increased cAMP generation. Losartan increased cAMP in HEK 293a cells transfected with both AT1R and D1R, but it did not increase cAMP in cells transfected with either receptor alone, suggesting that losartan induces D1R activation. Furthermore, losartan did not increase cAMP in HEK 293a cells expressing AT1R and mutant S397/S398A D1R, which disrupts the physical interaction between AT1R and D1R. In vivo, administration of a D1R antagonist significantly attenuated the antihypertensive effect of losartan in rats with renal hypertension. Taken together, these data imply that losartan might exert its antihypertensive effect both by inhibiting AT1R signaling and by enhancing D1R signaling. PMID:22193384

  17. Trace amine associated receptor 1 signaling in activated lymphocytes.

    Science.gov (United States)

    Panas, Michael W; Xie, Zhihua; Panas, Helen N; Hoener, Marius C; Vallender, Eric J; Miller, Gregory M

    2012-12-01

    Although most research to date on Trace Amine Associated Receptor 1 (TAAR1) has focused on its role in the brain, it has been recognized since its discovery in 2001 that TAAR1 mRNA is expressed in peripheral tissues as well, suggesting that this receptor may play a role in non-neurological pathways. This study reports TAAR1 expression, signaling and functionality in rhesus monkey lymphocytes. We detected a high level of TAAR1 protein in immortalized rhesus monkey B cell lines and a significant upregulation of TAAR1 protein expression in rhesus monkey lymphocytes following PHA treatment. Through screening a wide range of signaling pathways for their upregulation following TAAR1 activation by its potent agonist methamphetamine, we identified two transcription factors, CREB and NFAT, which are commonly associated with immune activation. Furthermore, we observed a TAAR1-dependent phosphorylation of PKA and PKC following treatment with methamphetamine in transfected HEK293 cells, immortalized rhesus monkey B cells and PHA-activated rhesus monkey lymphocytes. Accordingly, the high levels of TAAR1 that we observed on lymphocytes are inducible and fully functional, capable of transmitting a signal likely via PKA and PKC activation following ligand binding. More importantly, an increase in TAAR1 receptor expression is concomitant with lymphocyte immune activation, suggesting a possible role for TAAR1 in the generation or regulation of an immune response. TAAR1 is emerging as a potential therapeutic target, with regard to its ability to modulate brain monoamines. The current data raises the possibility that TAAR1-targeted drugs may also alter immune function.

  18. Phagocytic receptors activate and immune inhibitory receptor SIRPalpha inhibits phagocytosis through paxillin and cofilin

    Directory of Open Access Journals (Sweden)

    Miri eGitik

    2014-04-01

    Full Text Available The innate-immune function of phagocytosis of apoptotic cells, tissue-debris, pathogens and cancer cells is essential for homeostasis, tissue repair, fighting infection and combating malignancy. Phagocytosis is carried out in the CNS by resident microglia and in both CNS and PNS by recruited macrophages. While phagocytosis proceeds, bystander healthy cells protect themselves by sending a do not eat me message to phagocytes as CD47 on their surface ligates immune inhibitory receptor SIRPα on the surface of phagocytes and SIRPα then produces the signaling which inhibits phagocytosis. This helpful mechanism becomes harmful when tissue-debris and unhealthy cells inhibit their own phagocytosis by employing the same mechanism. However, the inhibitory signaling that SIRPα produces has not been fully revealed. We focus here on how SIRPα inhibits the phagocytosis of the tissue-debris degenerated-myelin which hinders repair in axonal injury and neurodegenerative diseases. We tested whether SIRPα inhibits phagocytosis by regulating cytoskeleton function through paxillin and cofilin since (a the cytoskeleton generates the mechanical forces that drive phagocytosis and (b both paxillin and cofilin control cytoskeleton function. Paxillin and cofilin were transiently activated in microglia as phagocytosis was activated. In contrast, paxillin and cofilin were continuously activated and phagocytosis augmented in microglia in which SIRPα expression was knocked-down by SIRPα-shRNA. Further, levels of phagocytosis, paxillin activation and cofilin activation positively correlated with one another. Taken together, these observations suggest a novel mechanism whereby paxillin and cofilin are targeted to control phagocytosis by both the activating signaling that phagocytic receptors produce by promoting the activation of paxillin and cofilin and the inhibiting signaling that immune inhibitory SIRPα produces by promoting the inactivation of paxillin and cofilin.

  19. Diabetes and obesity treatment based on dual incretin receptor activation

    DEFF Research Database (Denmark)

    Skow, M A; Bergmann, N C; Knop, F K

    2016-01-01

    The gut incretin hormones glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) are secreted after meal ingestion and work in concert to promote postprandial insulin secretion and regulate glucagon secretion. GLP-1 also slows gastric emptying and suppresses appetite......, whereas GIP seems to affect lipid metabolism. The introduction of selective GLP-1 receptor (GLP-1R) agonists for the treatment of type 2 diabetes and obesity has increased the scientific and clinical interest in incretins. Combining the body weight-lowering and glucose-lowering effects of GLP-1...... with a more potent improvement of β cell function through additional GIP action could potentially offer a more effective treatment of diabetes and obesity, with fewer adverse effects than selective GLP-1R agonists; therefore, new drugs designed to co-activate both the GIP receptor (GIPR) and the GLP-1R...

  20. Depersonalization disorder may be related to glutamate receptor activation imbalance.

    Science.gov (United States)

    Pikwer, Andreas

    2011-10-01

    Low-dose ketamine administration mimics, both clinically and on gross neuroimaging, depersonalization disorder. The perceptual effects of ketamine may be due to secondary stimulation of glutamate release and lamotrigine, possibly by inhibited glutamate release, may reduce some of ketamine's so-called dissociative effects. However, lamotrigine does not seem to be useful in the treatment of depersonalization disorder. Glutamate release in prefrontal cortex is increased by subanaesthetic doses of ketamine, resulting in increased inhibition, possibly via intercalated GABAerg cells, of projections from amygdala, affecting structures critically involved in depersonalization. I speculate that, in depersonalization disorder, the increased glutamate activity in prefrontal cortex is due to intrinsic imbalance, resulting in long-term potentiation, at the postsynaptic glutamate receptors on the GABAerg interneurons while the same receptor abnormality at the synapses on the intercalated GABAerg cells of the amygdala result in long-term depression in the case of either normal or high glutamate release. Copyright © 2011 Elsevier Ltd. All rights reserved.

  1. Identification of Modulators of the Nuclear Receptor Peroxisome Proliferator-Activated Receptor α (PPARα) in a Mouse Liver Gene Expression Compendium

    Science.gov (United States)

    The nuclear receptor family member peroxisome proliferator-activated receptor α (PPARα) is activated by therapeutic hypolipidemic drugs and environmentally-relevant chemicals to regulate genes involved in lipid transport and catabolism. Chronic activation of PPARα in rodents inc...

  2. The origin and function of the pituitary adenylate cyclase-activating polypeptide (PACAP)/glucagon superfamily.

    Science.gov (United States)

    Sherwood, N M; Krueckl, S L; McRory, J E

    2000-12-01

    The pituitary adenylate cyclase-activating polypeptide (PACAP)/ glucagon superfamily includes nine hormones in humans that are related by structure, distribution (especially the brain and gut), function (often by activation of cAMP), and receptors (a subset of seven-transmembrane receptors). The nine hormones include glucagon, glucagon-like peptide-1 (GLP-1), GLP-2, glucose-dependent insulinotropic polypeptide (GIP), GH-releasing hormone (GRF), peptide histidine-methionine (PHM), PACAP, secretin, and vasoactive intestinal polypeptide (VIP). The origin of the ancestral superfamily members is at least as old as the invertebrates; the most ancient and tightly conserved members are PACAP and glucagon. Evidence to date suggests the superfamily began with a gene or exon duplication and then continued to diverge with some gene duplications in vertebrates. The function of PACAP is considered in detail because it is newly (1989) discovered; it is tightly conserved (96% over 700 million years); and it is probably the ancestral molecule. The diverse functions of PACAP include regulation of proliferation, differentiation, and apoptosis in some cell populations. In addition, PACAP regulates metabolism and the cardiovascular, endocrine, and immune systems, although the physiological event(s) that coordinates PACAP responses remains to be identified.

  3. Structure-activity analysis of synthetic alpha-thrombin-receptor-activating peptides.

    Science.gov (United States)

    Van Obberghen-Schilling, E; Rasmussen, U B; Vouret-Craviari, V; Lentes, K U; Pavirani, A; Pouysségur, J

    1993-06-15

    alpha-Thrombin stimulates G-protein-coupled effectors leading to secretion and aggregation in human platelets, and to a mitogenic response in CCL39 hamster fibroblasts. alpha-Thrombin receptors can be activated by synthetic peptides corresponding to the receptor sequence starting with serine-42, at the proposed cleavage site. We have previously determined that the agonist domain of receptor-activating peptides resides within the five N-terminal residues [Vouret-Craviari, Van Obberghen-Schilling, Rasmussen, Pavirani, Lecocq and Pouysségur (1992) Mol. Biol. Cell. 3, 95-102], although the 7-residue peptide (SFFLRNP) corresponding to the hamster alpha-thrombin receptor was 10 times more potent than the 5-residue peptide for activation of human platelets. In the present study we have analysed the role of individual amino acids in receptor activation by using a series of modified hexa- or hepta-peptides derived from the human alpha-thrombin-receptor sequence. Cellular events examined here include phospholipase C activation, adenylyl cyclase inhibition and DNA synthesis stimulation in non-transformed CCL39 fibroblasts and a tumorigenic variant of that line (A71 cells). Modification of the peptide sequence had similar functional consequence for each of the assays described, indicating that either a unique receptor or pharmacologically indistinguishable receptor subtypes activate distinct G-protein signalling pathways. Furthermore, we found that: (1) the N-terminal serine can be replaced by small or intermediately sized amino acids (+/- hydroxyl groups) without loss of activity. However, its replacement by an aromatic side-chain or omission of the N-terminal amino group severely reduces activity. (2) An aromatic side-chain on the penultimate N-terminal residue appears to play a critical role since phenylalanine in this position can be substituted by tyrosine without complete loss of activity whereas an alanine in its place is not tolerated. (3) Deletion of the first

  4. UV ACTIVATION OF RECEPTOR TYROSINE KINASE-ACTIVITY

    NARCIS (Netherlands)

    COFFER, PJ; BURGERING, BMT; PEPPELENBOSCH, MP; BOS, JL; KRUIJER, W

    1995-01-01

    The exposure of mammalian cells to ultraviolet radiation (UV) may lead to DNA damage resulting in mutation and thus possibly cancer, while irradiation can further act as a potent tumour promoter. In addition UV induces p21ras-mediated signalling leading to activation of transcription factors such as

  5. Constitutive activity of the cannabinoid CB1 receptor regulates the function of co-expressed Mu opioid receptors.

    Science.gov (United States)

    Canals, Meritxell; Milligan, Graeme

    2008-04-25

    The human mu opioid receptor was expressed stably in Flp-In T-REx HEK293 cells. Occupancy by the agonist DAMGO (Tyr-d-Ala-Gly-N-methyl-Phe-Gly-ol) resulted in phosphorylation of the ERK1/2 MAP kinases, which was blocked by the opioid antagonist naloxone but not the cannabinoid CB1 receptor inverse agonist SR141716A. Expression of the human cannabinoid CB1 receptor in these cells from the inducible Flp-In T-REx locus did not alter expression levels of the mu opioid receptor. This allowed the cannabinoid CB1 agonist WIN55212-2 to stimulate ERK1/2 phosphorylation but resulted in a large reduction in the capacity of DAMGO to activate these kinases. Although lacking affinity for the mu opioid receptor, co-addition of SR141716A caused recovery of the effectiveness of DAMGO. In contrast co-addition of the CB1 receptor neutral antagonist O-2050 did not. Induction of the CB1 receptor also resulted in an increase of basal [(35)S]guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) binding and thereby a greatly reduced capacity of DAMGO to further stimulate [(35)S]GTPgammaS binding. CB1 inverse agonists attenuated basal [(35)S]GTPgammaS binding and restored the capacity of DAMGO to stimulate. Flp-In T-REx HEK293 cells were generated, which express the human mu opioid receptor constitutively and harbor a modified D163N cannabinoid CB1 receptor that lacks constitutive activity. Induction of expression of the modified cannabinoid CB1 receptor did not limit DAMGO-mediated ERK1/2 MAP kinase phosphorylation and did not allow SR141716A to enhance the function of DAMGO. These data indicate that it is the constitutive activity inherent in the cannabinoid CB1 receptor that reduces the capacity of co-expressed mu opioid receptor to function.

  6. Computing highly correlated positions using mutual information and graph theory for G protein-coupled receptors.

    Directory of Open Access Journals (Sweden)

    Sarosh N Fatakia

    Full Text Available G protein-coupled receptors (GPCRs are a superfamily of seven transmembrane-spanning proteins involved in a wide array of physiological functions and are the most common targets of pharmaceuticals. This study aims to identify a cohort or clique of positions that share high mutual information. Using a multiple sequence alignment of the transmembrane (TM domains, we calculated the mutual information between all inter-TM pairs of aligned positions and ranked the pairs by mutual information. A mutual information graph was constructed with vertices that corresponded to TM positions and edges between vertices were drawn if the mutual information exceeded a threshold of statistical significance. Positions with high degree (i.e. had significant mutual information with a large number of other positions were found to line a well defined inter-TM ligand binding cavity for class A as well as class C GPCRs. Although the natural ligands of class C receptors bind to their extracellular N-terminal domains, the possibility of modulating their activity through ligands that bind to their helical bundle has been reported. Such positions were not found for class B GPCRs, in agreement with the observation that there are not known ligands that bind within their TM helical bundle. All identified key positions formed a clique within the MI graph of interest. For a subset of class A receptors we also considered the alignment of a portion of the second extracellular loop, and found that the two positions adjacent to the conserved Cys that bridges the loop with the TM3 qualified as key positions. Our algorithm may be useful for localizing topologically conserved regions in other protein families.

  7. Computing highly correlated positions using mutual information and graph theory for G protein-coupled receptors.

    Science.gov (United States)

    Fatakia, Sarosh N; Costanzi, Stefano; Chow, Carson C

    2009-01-01

    G protein-coupled receptors (GPCRs) are a superfamily of seven transmembrane-spanning proteins involved in a wide array of physiological functions and are the most common targets of pharmaceuticals. This study aims to identify a cohort or clique of positions that share high mutual information. Using a multiple sequence alignment of the transmembrane (TM) domains, we calculated the mutual information between all inter-TM pairs of aligned positions and ranked the pairs by mutual information. A mutual information graph was constructed with vertices that corresponded to TM positions and edges between vertices were drawn if the mutual information exceeded a threshold of statistical significance. Positions with high degree (i.e. had significant mutual information with a large number of other positions) were found to line a well defined inter-TM ligand binding cavity for class A as well as class C GPCRs. Although the natural ligands of class C receptors bind to their extracellular N-terminal domains, the possibility of modulating their activity through ligands that bind to their helical bundle has been reported. Such positions were not found for class B GPCRs, in agreement with the observation that there are not known ligands that bind within their TM helical bundle. All identified key positions formed a clique within the MI graph of interest. For a subset of class A receptors we also considered the alignment of a portion of the second extracellular loop, and found that the two positions adjacent to the conserved Cys that bridges the loop with the TM3 qualified as key positions. Our algorithm may be useful for localizing topologically conserved regions in other protein families.

  8. Exercise reduces adipose tissue via cannabinoid receptor type 1 which is regulated by peroxisome proliferator-activated receptor-delta

    DEFF Research Database (Denmark)

    Yan, Zhen Cheng; Liu, Dao Yan; Zhang, Li Li

    2007-01-01

    Obesity is one major cardiovascular risk factor. We tested effects of endurance exercise on cannabinoid receptor type 1 (CB1) and peroxisome proliferator-activated receptor-delta (PPAR-delta)-dependent pathways in adipose tissue. Male Wistar rats were randomly assigned to standard laboratory chow...

  9. Stimulation of cannabinoid receptor 2 (CB2 suppresses microglial activation

    Directory of Open Access Journals (Sweden)

    Fernandez Francisco

    2005-12-01

    Full Text Available Abstract Background Activated microglial cells have been implicated in a number of neurodegenerative disorders, including Alzheimer's disease (AD, multiple sclerosis (MS, and HIV dementia. It is well known that inflammatory mediators such as nitric oxide (NO, cytokines, and chemokines play an important role in microglial cell-associated neuron cell damage. Our previous studies have shown that CD40 signaling is involved in pathological activation of microglial cells. Many data reveal that cannabinoids mediate suppression of inflammation in vitro and in vivo through stimulation of cannabinoid receptor 2 (CB2. Methods In this study, we investigated the effects of a cannabinoid agonist on CD40 expression and function by cultured microglial cells activated by IFN-γ using RT-PCR, Western immunoblotting, flow cytometry, and anti-CB2 small interfering RNA (siRNA analyses. Furthermore, we examined if the stimulation of CB2 could modulate the capacity of microglial cells to phagocytise Aβ1–42 peptide using a phagocytosis assay. Results We found that the selective stimulation of cannabinoid receptor CB2 by JWH-015 suppressed IFN-γ-induced CD40 expression. In addition, this CB2 agonist markedly inhibited IFN-γ-induced phosphorylation of JAK/STAT1. Further, this stimulation was also able to suppress microglial TNF-α and nitric oxide production induced either by IFN-γ or Aβ peptide challenge in the presence of CD40 ligation. Finally, we showed that CB2 activation by JWH-015 markedly attenuated CD40-mediated inhibition of microglial phagocytosis of Aβ1–42 peptide. Taken together, these results provide mechanistic insight into beneficial effects provided by cannabinoid receptor CB2 modulation in neurodegenerative diseases, particularly AD.

  10. Behavioral meaningful opioidergic stimulation activates kappa receptor gene expression

    Energy Technology Data Exchange (ETDEWEB)

    Teodorov, E. [Centro de Matemática, Computação e Cognição, Universidade Federal do ABC, São Paulo, SP (Brazil); Ferrari, M.F.R. [Departamento de Genética e Biologia Evolutiva, Instituto de Biociências, Universidade de São Paulo, São Paulo, SP (Brazil); Fior-Chadi, D.R. [Departamento de Fisiologia, Instituto de Biociências, Universidade de São Paulo, São Paulo, SP (Brazil); Camarini, R. [Departamento de Farmacologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo, SP (Brazil); Felício, L.F. [Departamento de Patologia, Faculdade de Medicina Veterinária e Zootecnia, Universidade de São Paulo, São Paulo, SP (Brazil)

    2012-06-01

    The periaqueductal gray (PAG) has been reported to be a location for opioid regulation of pain and a potential site for behavioral selection in females. Opioid-mediated behavioral and physiological responses differ according to the activity of opioid receptor subtypes. The present study investigated the effects of the peripheral injection of the kappa-opioid receptor agonist U69593 into the dorsal subcutaneous region of animals on maternal behavior and on Oprk1 gene activity in the PAG of female rats. Female Wistar rats weighing 200-250 g at the beginning of the study were randomly divided into 2 groups for maternal behavior and gene expression experiments. On day 5, pups were removed at 7:00 am and placed in another home cage that was distant from their mother. Thirty minutes after removing the pups, the dams were treated with U69593 (0.15 mg/kg, sc) or 0.9% saline (up to 1 mL/kg) and after 30 min were evaluated in the maternal behavior test. Latencies in seconds for pup retrieval, grouping, crouching, and full maternal behavior were scored. The results showed that U69593 administration inhibited maternal behavior (P < 0.05) because a lower percentage of U69593 group dams showed retrieval of first pup, retrieving all pups, grouping, crouching and displaying full maternal behavior compared to the saline group. Opioid gene expression was evaluated using real-time reverse-transcription polymerase chain reaction (RT-PCR). A single injection of U69593 increased Oprk1 PAG expression in both virgin (P < 0.05) and lactating female rats (P < 0.01), with no significant effect on Oprm1 or Oprd1 gene activity. Thus, the expression of kappa-opioid receptors in the PAG may be modulated by single opioid receptor stimulation and behavioral meaningful opioidergic transmission in the adult female might occur simultaneously to specific changes in gene expression of kappa-opioid receptor subtype. This is yet another alert for the complex role of the opioid system in female

  11. DHEA metabolites activate estrogen receptors alpha and beta

    Science.gov (United States)

    Michael Miller, Kristy K.; Al-Rayyan, Numan; Ivanova, Margarita M.; Mattingly, Kathleen A.; Ripp, Sharon L.; Klinge, Carolyn M.; Prough, Russell A.

    2012-01-01

    Dehydroepiandrosterone (DHEA) levels were reported to associate with increased breast cancer risk in postmenopausal women, but some carcinogen-induced rat mammary tumor studies question this claim. The purpose of this study was to determine how DHEA and its metabolites affect estrogen receptors α or β (ERα or ERβ) -regulated gene transcription and cell proliferation. In transiently transfected HEK-293 cells, androstenediol, DHEA, and DHEA-S activated ERα. In ERβ transfected HepG2 cells, androstenedione, DHEA, androstenediol, and 7-oxo DHEA stimulated reporter activity. ER antagonists ICI 182,780 (fulvestrant) and 4-hydroxytamoxifen, general P450 inhibitor miconazole, and aromatase inhibitor exemestane inhibited activation by DHEA or metabolites in transfected cells. ERβ-selective antagonist R,R-THC (R,R-cis-diethyl tetrahydrochrysene) inhibited DHEA and DHEA metabolite transcriptional activity in ERβ-transfected cells. Expression of endogenous estrogen-regulated genes: pS2, progesterone receptor, cathepsin D1, and nuclear respiratory factor-1 was increased by DHEA and its metabolites in an ER-subtype, gene, and cell-specific manner. DHEA metabolites, but not DHEA, competed with 17β-estradiol for ERα and ERβ binding and stimulated MCF-7 cell proliferation, demonstrating that DHEA metabolites interact directly with ERα and ERβ in vitro, modulating estrogen target genes in vivo. PMID:23123738

  12. Characterization and ligand identification of a membrane progesterone receptor in fungi: existence of a novel PAQR in Sporothrix schenckii.

    Science.gov (United States)

    Gonzalez-Velazquez, Waleska; Gonzalez-Mendez, Ricardo; Rodriguez-del Valle, Nuri

    2012-09-07

    Adaptive responses in fungi result from the interaction of membrane receptors and extracellular ligands. Many different classes of receptors have been described in eukaryotic cells. Recently a new family of receptors classified as belonging to the progesterone-adiponectin receptor (PAQR) family has been identified. These receptors have the seven transmembrane domains characteristic of G-protein coupled receptors, but their activity has not been associated directly to G proteins. They share sequence similarity to the eubacterial hemolysin III proteins. A new receptor, SsPAQR1 (Sporothrix schenckii progesterone-adiponectinQ receptor1), was identified as interacting with Sporothrix schenckii G protein alpha subunit SSG-2 in a yeast two-hybrid assay. The receptor was identified as a member of the PAQR family. The cDNA sequence revealed a predicted ORF of 1542 bp encoding a 514 amino acids protein with a calculated molecular weight of 57.8 kDa. Protein domain analysis of SsPAQR1 showed the 7 transmembrane domains (TM) characteristic of G protein coupled receptors and the presence of the distinctive motifs that characterize PAQRs. A yeast-based assay specific for PAQRs identified progesterone as the agonist. S. schenckii yeast cells exposed to progesterone (0.50 mM) showed an increase in intracellular levels of 3', 5' cyclic adenosine monophosphate (cAMP) within the first min of incubation with the hormone. Different progesterone concentrations were tested for their effect on the growth of the fungus. Cultures incubated at 35°C did not grow at concentrations of progesterone of 0.05 mM or higher. Cultures incubated at 25°C grew at all concentrations tested (0.01 mM-0.50 mM) with growth decreasing gradually with the increase in progesterone concentration. This work describes a receptor associated with a G protein alpha subunit in S. schenckii belonging to the PAQR family. Progesterone was identified as the ligand. Exposure to progesterone increased the levels of cAMP in

  13. An improved ivermectin-activated chloride channel receptor for inhibiting electrical activity in defined neuronal populations

    DEFF Research Database (Denmark)

    Lynagh, Timothy Peter; Lynch, Joseph W

    2010-01-01

    The ability to silence the electrical activity of defined neuronal populations in vivo is dramatically advancing our understanding of brain function. This technology may eventually be useful clinically for treating a variety of neuropathological disorders caused by excessive neuronal activity...... for surgically implanted stimulus delivery methods and their use of nonhuman receptors. A third silencing method, an invertebrate glutamate-gated chloride channel receptor (GluClR) activated by ivermectin, solves the stimulus delivery problem as ivermectin is a safe, well tolerated drug that reaches the brain...

  14. Dopamine Receptor Activation Increases HIV Entry into Primary Human Macrophages

    Science.gov (United States)

    Gaskill, Peter J.; Yano, Hideaki H.; Kalpana, Ganjam V.; Javitch, Jonathan A.; Berman, Joan W.

    2014-01-01

    Macrophages are the primary cell type infected with HIV in the central nervous system, and infection of these cells is a major component in the development of neuropathogenesis and HIV-associated neurocognitive disorders. Within the brains of drug abusers, macrophages are exposed to increased levels of dopamine, a neurotransmitter that mediates the addictive and reinforcing effects of drugs of abuse such as cocaine and methamphetamine. In this study we examined the effects of dopamine on HIV entry into primary human macrophages. Exposure to dopamine during infection increased the entry of R5 tropic HIV into macrophages, irrespective of the concentration of the viral inoculum. The entry pathway affected was CCR5 dependent, as antagonizing CCR5 with the small molecule inhibitor TAK779 completely blocked entry. The effect was dose-dependent and had a steep threshold, only occurring above 108 M dopamine. The dopamine-mediated increase in entry required dopamine receptor activation, as it was abrogated by the pan-dopamine receptor antagonist flupenthixol, and could be mediated through both subtypes of dopamine receptors. These findings indicate that the effects of dopamine on macrophages may have a significant impact on HIV pathogenesis. They also suggest that drug-induced increases in CNS dopamine may be a common mechanism by which drugs of abuse with distinct modes of action exacerbate neuroinflammation and contribute to HIV-associated neurocognitive disorders in infected drug abusers. PMID:25268786

  15. Synaptic activity regulates AMPA receptor trafficking through different recycling pathways

    Science.gov (United States)

    Zheng, Ning; Jeyifous, Okunola; Munro, Charlotte; Montgomery, Johanna M; Green, William N

    2015-01-01

    Changes in glutamatergic synaptic strength in brain are dependent on AMPA-type glutamate receptor (AMPAR) recycling, which is assumed to occur through a single local pathway. In this study, we present evidence that AMPAR recycling occurs through different pathways regulated by synaptic activity. Without synaptic stimulation, most AMPARs recycled in dynamin-independent endosomes containing the GTPase, Arf6. Few AMPARs recycled in dynamin-dependent endosomes labeled by transferrin receptors (TfRs). AMPAR recycling was blocked by alterations in the GTPase, TC10, which co-localized with Arf6 endosomes. TC10 mutants that reduced AMPAR recycling had no effect on increased AMPAR levels with long-term potentiation (LTP) and little effect on decreased AMPAR levels with long-term depression. However, internalized AMPAR levels in TfR-containing recycling endosomes increased after LTP, indicating increased AMPAR recycling through the dynamin-dependent pathway with synaptic plasticity. LTP-induced AMPAR endocytosis is inconsistent with local recycling as a source of increased surface receptors, suggesting AMPARs are trafficked from other sites. DOI: http://dx.doi.org/10.7554/eLife.06878.001 PMID:25970033

  16. NMDA receptor subunit expression and PAR2 receptor activation in colospinal afferent neurons (CANs) during inflammation induced visceral hypersensitivity.

    Science.gov (United States)

    Suckow, Shelby K; Caudle, Robert M

    2009-09-22

    Visceral hypersensitivity is a clinical observation made when diagnosing patients with functional bowel disorders. The cause of visceral hypersensitivity is unknown but is thought to be attributed to inflammation. Previously we demonstrated that a unique set of enteric neurons, colospinal afferent neurons (CANs), co-localize with the NR1 and NR2D subunits of the NMDA receptor as well as with the PAR2 receptor. The aim of this study was to determine if NMDA and PAR2 receptors expressed on CANs contribute to visceral hypersensitivity following inflammation. Recently, work has suggested that dorsal root ganglion (DRG) neurons expressing the transient receptor potential vanilloid-1 (TRPV1) receptor mediate inflammation induced visceral hypersensitivity. Therefore, in order to study CAN involvement in visceral hypersensitivity, DRG neurons expressing the TRPV1 receptor were lesioned with resiniferatoxin (RTX) prior to inflammation and behavioural testing. CANs do not express the TRPV1 receptor; therefore, they survive following RTX injection. RTX treatment resulted in a significant decrease in TRPV1 expressing neurons in the colon and immunohistochemical analysis revealed no change in peptide or receptor expression in CANs following RTX lesioning as compared to control data. Behavioral studies determined that both inflamed non-RTX and RTX animals showed a decrease in balloon pressure threshold as compared to controls. Immunohistochemical analysis demonstrated that the NR1 cassettes, N1 and C1, of the NMDA receptor on CANs were up-regulated following inflammation. Furthermore, inflammation resulted in the activation of the PAR2 receptors expressed on CANs. Our data show that inflammation causes an up-regulation of the NMDA receptor and the activation of the PAR2 receptor expressed on CANs. These changes are associated with a decrease in balloon pressure in response to colorectal distension in non-RTX and RTX lesioned animals. Therefore, these data suggest that CANs

  17. NMDA receptor subunit expression and PAR2 receptor activation in colospinal afferent neurons (CANs during inflammation induced visceral hypersensitivity

    Directory of Open Access Journals (Sweden)

    Caudle Robert M

    2009-09-01

    Full Text Available Abstract Background Visceral hypersensitivity is a clinical observation made when diagnosing patients with functional bowel disorders. The cause of visceral hypersensitivity is unknown but is thought to be attributed to inflammation. Previously we demonstrated that a unique set of enteric neurons, colospinal afferent neurons (CANs, co-localize with the NR1 and NR2D subunits of the NMDA receptor as well as with the PAR2 receptor. The aim of this study was to determine if NMDA and PAR2 receptors expressed on CANs contribute to visceral hypersensitivity following inflammation. Recently, work has suggested that dorsal root ganglion (DRG neurons expressing the transient receptor potential vanilloid-1 (TRPV1 receptor mediate inflammation induced visceral hypersensitivity. Therefore, in order to study CAN involvement in visceral hypersensitivity, DRG neurons expressing the TRPV1 receptor were lesioned with resiniferatoxin (RTX prior to inflammation and behavioural testing. Results CANs do not express the TRPV1 receptor; therefore, they survive following RTX injection. RTX treatment resulted in a significant decrease in TRPV1 expressing neurons in the colon and immunohistochemical analysis revealed no change in peptide or receptor expression in CANs following RTX lesioning as compared to control data. Behavioral studies determined that both inflamed non-RTX and RTX animals showed a decrease in balloon pressure threshold as compared to controls. Immunohistochemical analysis demonstrated that the NR1 cassettes, N1 and C1, of the NMDA receptor on CANs were up-regulated following inflammation. Furthermore, inflammation resulted in the activation of the PAR2 receptors expressed on CANs. Conclusion Our data show that inflammation causes an up-regulation of the NMDA receptor and the activation of the PAR2 receptor expressed on CANs. These changes are associated with a decrease in balloon pressure in response to colorectal distension in non-RTX and RTX lesioned

  18. Characterization of human endothelial cell urokinase-type plasminogen activator receptor protein and messenger RNA

    DEFF Research Database (Denmark)

    Barnathan, E S; Kuo, A; Karikó, K

    1990-01-01

    Human umbilical vein endothelial cells in culture (HUVEC) express receptors for urokinase-type plasminogen activators (u-PA). The immunochemical nature of this receptor and its relationship to u-PA receptors expressed by other cell types is unknown. Cross-linking active site-blocked u-PA to HUVEC...

  19. The phosphoproteome of toll-like receptor-activated macrophages

    DEFF Research Database (Denmark)

    Weintz, Gabriele; Olsen, Jesper Velgaard; Frühauf, Katja

    2010-01-01

    Recognition of microbial danger signals by toll-like receptors (TLR) causes re-programming of macrophages. To investigate kinase cascades triggered by the TLR4 ligand lipopolysaccharide (LPS) on systems level, we performed a global, quantitative and kinetic analysis of the phosphoproteome...... of primary macrophages using stable isotope labelling with amino acids in cell culture, phosphopeptide enrichment and high-resolution mass spectrometry. In parallel, nascent RNA was profiled to link transcription factor (TF) phosphorylation to TLR4-induced transcriptional activation. We reproducibly...

  20. Peroxisome Proliferator-Activated Receptors in Diabetic Nephropathy

    Directory of Open Access Journals (Sweden)

    Shinji Kume

    2008-01-01

    Full Text Available Diabetic nephropathy is a leading cause of end-stage renal disease, which is increasing in incidence worldwide, despite intensive treatment approaches such as glycemic and blood pressure control in patients with diabetes mellitus. New therapeutic strategies are needed to prevent the onset of diabetic nephropathy. Peroxisome proliferator-activated receptors (PPARs are ligand-activated nuclear transcription factors that play important roles in lipid and glucose homeostases. These agents might prevent the progression of diabetic nephropathy, since PPAR agonists improve dyslipidemia and insulin resistance. Furthermore, data from murine models suggest that PPAR agonists also have independent renoprotective effects by suppressing inflammation, oxidative stress, lipotoxicity, and activation of the renin-angiotensin system. This review summarizes data from clinical and experimental studies regarding the relationship between PPARs and diabetic nephropathy. The therapeutic potential of PPAR agonists in the treatment of diabetic nephropathy is also discussed.

  1. Targeting protease activated receptor-1 with P1pal-12 limits bleomycin-induced pulmonary fibrosis

    NARCIS (Netherlands)

    Lin, Cong; Duitman, Janwillem; Daalhuisen, Joost; ten Brink, Marieke; von der Thüsen, Jan; van der Poll, Tom; Borensztajn, Keren; Spek, C. Arnold

    2014-01-01

    Idiopathic pulmonary fibrosis is the most devastating fibrotic diffuse parenchymal lung disease which remains refractory to pharmacological therapies. Therefore, novel treatments are urgently required. Protease-activated receptor (PAR)-1 is a G-protein-coupled receptor that mediates critical

  2. Glucocorticoid receptor activation selectively hampers N-methyl-d-aspartate receptor dependent hippocampal synaptic plasticity in vitro.

    NARCIS (Netherlands)

    Wiegert, O.; Pu, Z.; Shor, S.; Joëls, M.; Krugers, H.

    2005-01-01

    Corticosterone and exposure to stressful experiences have been reported to decrease hippocampal synaptic plasticity, in particular when relatively mild stimulation paradigms-presumably activating predominantly N-methyl-d-aspartate receptors-are being used. Using various stimulation paradigms and

  3. The glucagon-like peptide 1 receptor agonist enhances intrinsic peroxisome proliferator-activated receptor γ activity in endothelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Onuma, Hirohisa; Inukai, Kouichi, E-mail: kinukai@ks.kyorin-u.ac.jp; Kitahara, Atsuko; Moriya, Rie; Nishida, Susumu; Tanaka, Toshiaki; Katsuta, Hidenori; Takahashi, Kazuto; Sumitani, Yoshikazu; Hosaka, Toshio; Ishida, Hitoshi

    2014-08-22

    Highlights: • PPARγ activation was involved in the GLP-1-mediated anti-inflammatory action. • Exendin-4 enhanced endogenous PPARγ transcriptional activity in HUVECs. • H89, a PKA inhibitor, abolished GLP-1-induced PPARγ enhancement. • The anti-inflammatory effects of GLP-1 may be explained by PPARγ activation. - Abstract: Recent studies have suggested glucagon-like peptide-1 (GLP-1) signaling to exert anti-inflammatory effects on endothelial cells, although the precise underlying mechanism remains to be elucidated. In the present study, we investigated whether PPARγ activation is involved in the GLP-1-mediated anti-inflammatory action on endothelial cells. When we treated HUVEC cells with 0.2 ng/ml exendin-4, a GLP-1 receptor agonist, endogenous PPARγ transcriptional activity was significantly elevated, by approximately 20%, as compared with control cells. The maximum PPARγ activity enhancing effect of exendin-4 was observed 12 h after the initiation of incubation with exendin-4. As H89, a PKA inhibitor, abolished GLP-1-induced PPARγ enhancement, the signaling downstream from GLP-1 cross-talk must have been involved in PPARγ activation. In conclusion, our results suggest that GLP-1 has the potential to induce PPARγ activity, partially explaining the anti-inflammatory effects of GLP-1 on endothelial cells. Cross-talk between GLP-1 signaling and PPARγ activation would have major impacts on treatments for patients at high risk for cardiovascular disease.

  4. "Mirror image" antagonists of thrombin-induced platelet activation based on thrombin receptor structure.

    OpenAIRE

    Hung, D T; Vu, T K; Wheaton, V I; Charo, I F; Nelken, N A; Esmon, N.; Esmon, C T; Coughlin, S R

    1992-01-01

    Platelet activation by thrombin plays a critical role in hemostasis and thrombosis. Based on structure-activity studies of a cloned platelet thrombin receptor, we designed two "mirror image" antagonists of thrombin and thrombin receptor function. First, "uncleavable" peptides mimicking the receptor domain postulated to interact with thrombin were found to be potent thrombin inhibitors. Second, proteolytically inactive mutant thrombins designed to bind but not cleave the thrombin receptor were...

  5. Role of Peroxisome Proliferator-Activated Receptors in Inflammation Control

    Directory of Open Access Journals (Sweden)

    Jihan Youssef

    2004-01-01

    Full Text Available Peroxisome proliferator-activated receptors (PPARs were discovered over a decade ago, and were classified as orphan members of the nuclear receptor superfamily. To date, three PPAR subtypes have been discovered and characterized (PPARα, β/δ, γ. Different PPAR subtypes have been shown to play crucial roles in important diseases and conditions such as obesity, diabetes, atherosclerosis, cancer, and fertility. Among the most studied roles of PPARs is their involvement in inflammatory processes. Numerous studies have revealed that agonists of PPARα and PPARγ exert anti-inflammatory effects both in vitro and in vivo. Using the carrageenan-induced paw edema model of inflammation, a recent study in our laboratories showed that these agonists hinder the initiation phase, but not the late phase of the inflammatory process. Furthermore, in the same experimental model, we recently also observed that activation of PPARδ exerted an anti-inflammatory effect. Despite the fact that exclusive dependence of these effects on PPARs has been questioned, the bulk of evidence suggests that all three PPAR subtypes, PPARα,δ,γ, play a significant role in controlling inflammatory responses. Whether these subtypes act via a common mechanism or are independent of each other remains to be elucidated. However, due to the intensity of research efforts in this area, it is anticipated that these efforts will result in the development of PPAR ligands as therapeutic agents for the treatment of inflammatory diseases.

  6. Peroxisome Proliferators-Activated Receptor (PPAR Modulators and Metabolic Disorders

    Directory of Open Access Journals (Sweden)

    Min-Chul Cho

    2008-01-01

    Full Text Available Overweight and obesity lead to an increased risk for metabolic disorders such as impaired glucose regulation/insulin resistance, dyslipidemia, and hypertension. Several molecular drug targets with potential to prevent or treat metabolic disorders have been revealed. Interestingly, the activation of peroxisome proliferator-activated receptor (PPAR, which belongs to the nuclear receptor superfamily, has many beneficial clinical effects. PPAR directly modulates gene expression by binding to a specific ligand. All PPAR subtypes (α,γ, and σ are involved in glucose metabolism, lipid metabolism, and energy balance. PPAR agonists play an important role in therapeutic aspects of metabolic disorders. However, undesired effects of the existing PPAR agonists have been reported. A great deal of recent research has focused on the discovery of new PPAR modulators with more beneficial effects and more safety without producing undesired side effects. Herein, we briefly review the roles of PPAR in metabolic disorders, the effects of PPAR modulators in metabolic disorders, and the technologies with which to discover new PPAR modulators.

  7. Antitussive activity of Withania somnifera and opioid receptors.

    Science.gov (United States)

    Nosálová, Gabriela; Sivová, Veronika; Ray, Bimalendu; Fraňová, Soňa; Ondrejka, Igor; Flešková, Dana

    2015-01-01

    Arabinogalactan is a polysaccharide isolated from the roots of the medicinal plant Withania somnifera L. It contains 65% arabinose and 18% galactose. The aim of the present study was to evaluate the antitussive activity of arabinogalactan in conscious, healthy adult guinea pigs and the role of the opioid pathway in the antitussive action. A polysaccharide extract was given orally in a dose of 50 mg/kg. Cough was induced by an aerosol of citric acid in a concentration 0.3 mol/L, generated by a jet nebulizer into a plethysmographic chamber. The intensity of cough response was defined as the number of cough efforts counted during a 3-min exposure to the aerosol. The major finding was that arabinogalactan clearly suppressed the cough reflex; the suppression was comparable with that of codeine that was taken as a reference drug. The involvement of the opioid system was tested with the use of a blood-brain barrier penetrable, naloxone hydrochloride, and non-penetrable, naloxone methiodide, to distinguish between the central and peripheral mu-opioid receptor pathways. Both opioid antagonists acted to reverse the arabinogalactan-induced cough suppression; the reversion was total over time with the latter antagonist. We failed to confirm the presence of a bronchodilating effect of the polysaccharide, which could be involved in its antitussive action. We conclude that the polysaccharide arabinogalactan from Withania somnifera has a distinct antitussive activity consisting of cough suppression and that this action involves the mu-opioid receptor pathways.

  8. Vasopeptidase-activated latent ligands of the histamine receptor-1.

    Science.gov (United States)

    Gera, Lajos; Roy, Caroline; Charest-Morin, Xavier; Marceau, François

    2013-11-01

    Whether peptidases present in vascular cells can activate prodrugs active on vascular cells has been tested with 2 potential latent ligands of the histamine H1 receptor (H1R). First, a peptide consisting of the antihistamine cetirizine (CTZ) condensed at the N-terminus of ε-aminocaproyl-bradykinin (εACA-BK) was evaluated for an antihistamine activity that could be revealed by degradation of the peptide part of the molecule. CTZ-εACA-BK had a submicromolar affinity for the BK B2 receptor (B2R; IC50 of 590 nM, [(3)H]BK binding competition), but a non-negligible affinity for the human H1 receptor (H1R; IC50 of 11 μM for [(3)H]pyrilamine binding). In the human isolated umbilical vein, a system where both endogenous B2R and H1R mediate strong contractions, CTZ-εACA-BK exerted mild antagonist effects on histamine-induced contraction that were not modified by omapatrilat or by a B2R antagonist that prevents endocytosis of the BK conjugate. Cells expressing recombinant ACE or B2R incubated with CTZ-εACA-BK did not release a competitor of [(3)H]pyrilamine binding to H1Rs. Thus, there is no evidence that CTZ-εACA-BK can release free cetirizine in biological environments. The second prodrug was a blocked agonist, L-alanyl-histamine, potentially activated by aminopeptidase N (APN). This compound did not compete for [(3)H]pyrilamine binding to H1Rs. The human umbilical vein contractility assay responded to L-alanyl-histamine (EC50 54.7 μM), but the APN inhibitor amastatin massively (17-fold) reduced its apparent potency. Amastatin did not influence the potency of histamine as a contractile agent. One of the 2 tested latent H1R ligands, L-alanyl-histamine, supported the feasibility of pro-drug activation by vascular ectopeptidases. © 2013.

  9. Inhibition of the androgen receptor activity by Coprinus comatus substances.

    Science.gov (United States)

    Dotan, Nesly; Wasser, Solomon P; Mahajna, Jamal

    2011-11-01

    Prostatic adenocarcinoma is the second leading cause of death from cancer in Western men. The common prostate cancer treatments are effective in the early stages; however, advanced prostate cancer is resilient to most of these treatments. Altered androgen receptor (AR) activity caused by point mutations or signaling mechanisms that regulate AR function has been proposed as a key mechanism in the transition to the androgen-independent stage. Our previous results demonstrated that hexane extract prepared from Coprinus comatus (C. comatus) strain 734 was able to interfere with AR activity. The current study was made to further evaluate the antiandrogenic activity of the C. comatus mushroom strain 734. Activity-guided chromatography was conducted and 2 active fractions, F-32-and F-33, were found to contain substances that were able to inhibit AR-mediated reporter activity and reduce the levels of AR and prostate-specific antigen (PSA) transcripts in LNCaP cells. Fraction F-32 also inhibited the proliferation and clonigenicity of LNCaP cells. Furthermore, F-32 was able to inhibit the binding of AR to the PSA enhancer region and to inhibit Akt-mediated AR phosphorylation at Ser 213. This study illustrated the potential of substances from the C. comatus mushroom to serve as natural antiandrogenic modulators for the treatment of prostatic disorders.

  10. Involvement of Activating NK Cell Receptors and Their Modulation in Pathogen Immunity

    Directory of Open Access Journals (Sweden)

    Francesco Marras

    2011-01-01

    Full Text Available Natural Killer (NK cells are endowed with cell-structure-sensing receptors providing inhibitory protection from self-destruction (inhibitory NK receptors, iNKRs, including killer inhibitory receptors and other molecules and rapid triggering potential leading to functional cell activation by Toll-like receptors (TLRs, cytokine receptors, and activating NK cell receptors including natural cytotoxicity receptors (NCRs, i.e., NKp46, NKp46, and NKp44. NCR and NKG2D recognize ligands on infected cells which may be endogenous or may directly bind to some structures derived from invading pathogens. In this paper, we address the known direct or indirect interactions between activating receptors and pathogens and their expression during chronic HIV and HCV infections.

  11. The first structure-activity relationship studies for designer receptors exclusively activated by designer drugs.

    Science.gov (United States)

    Chen, Xin; Choo, Hyunah; Huang, Xi-Ping; Yang, Xiaobao; Stone, Orrin; Roth, Bryan L; Jin, Jian

    2015-03-18

    Over the past decade, two independent technologies have emerged and been widely adopted by the neuroscience community for remotely controlling neuronal activity: optogenetics which utilize engineered channelrhodopsin and other opsins, and chemogenetics which utilize engineered G protein-coupled receptors (Designer Receptors Exclusively Activated by Designer Drugs (DREADDs)) and other orthologous ligand-receptor pairs. Using directed molecular evolution, two types of DREADDs derived from human muscarinic acetylcholine receptors have been developed: hM3Dq which activates neuronal firing, and hM4Di which inhibits neuronal firing. Importantly, these DREADDs were not activated by the native ligand acetylcholine (ACh), but selectively activated by clozapine N-oxide (CNO), a pharmacologically inert ligand. CNO has been used extensively in rodent models to activate DREADDs, and although CNO is not subject to significant metabolic transformation in mice, a small fraction of CNO is apparently metabolized to clozapine in humans and guinea pigs, lessening the translational potential of DREADDs. To effectively translate the DREADD technology, the next generation of DREADD agonists are needed and a thorough understanding of structure-activity relationships (SARs) of DREADDs is required for developing such ligands. We therefore conducted the first SAR studies of hM3Dq. We explored multiple regions of the scaffold represented by CNO, identified interesting SAR trends, and discovered several compounds that are very potent hM3Dq agonists but do not activate the native human M3 receptor (hM3). We also discovered that the approved drug perlapine is a novel hM3Dq agonist with >10 000-fold selectivity for hM3Dq over hM3.

  12. Thrombin stimulates insulin secretion via protease-activated receptor-3.

    Science.gov (United States)

    Hänzelmann, Sonja; Wang, Jinling; Güney, Emre; Tang, Yunzhao; Zhang, Enming; Axelsson, Annika S; Nenonen, Hannah; Salehi, Albert S; Wollheim, Claes B; Zetterberg, Eva; Berntorp, Erik; Costa, Ivan G; Castelo, Robert; Rosengren, Anders H

    2015-01-01

    The disease mechanisms underlying type 2 diabetes (T2D) remain poorly defined. Here we aimed to explore the pathophysiology of T2D by analyzing gene co-expression networks in human islets. Using partial correlation networks we identified a group of co-expressed genes ('module') including F2RL2 that was associated with glycated hemoglobin. F2Rl2 is a G-protein-coupled receptor (GPCR) that encodes protease-activated receptor-3 (PAR3). PAR3 is cleaved by thrombin, which exposes a 6-amino acid sequence that acts as a 'tethered ligand' to regulate cellular signaling. We have characterized the effect of PAR3 activation on insulin secretion by static insulin secretion measurements, capacitance measurements, studies of diabetic animal models and patient samples. We demonstrate that thrombin stimulates insulin secretion, an effect that was prevented by an antibody that blocks the thrombin cleavage site of PAR3. Treatment with a peptide corresponding to the PAR3 tethered ligand stimulated islet insulin secretion and single β-cell exocytosis by a mechanism that involves activation of phospholipase C and Ca(2+) release from intracellular stores. Moreover, we observed that the expression of tissue factor, which regulates thrombin generation, was increased in human islets from T2D donors and associated with enhanced β-cell exocytosis. Finally, we demonstrate that thrombin generation potential in patients with T2D was associated with increased fasting insulin and insulinogenic index. The findings provide a previously unrecognized link between hypercoagulability and hyperinsulinemia and suggest that reducing thrombin activity or blocking PAR3 cleavage could potentially counteract the exaggerated insulin secretion that drives insulin resistance and β-cell exhaustion in T2D.

  13. Ventromedial hypothalamic melanocortin receptor activation: regulation of activity energy expenditure and skeletal muscle thermogenesis.

    Science.gov (United States)

    Gavini, Chaitanya K; Jones, William C; Novak, Colleen M

    2016-09-15

    The ventromedial hypothalamus (VMH) and the central melanocortin system both play vital roles in regulating energy balance by modulating energy intake and utilization. Recent evidence suggests that activation of the VMH alters skeletal muscle metabolism. We show that intra-VMH melanocortin receptor activation increases energy expenditure and physical activity, switches fuel utilization to fats, and lowers work efficiency such that excess calories are dissipated by skeletal muscle as heat. We also show that intra-VMH melanocortin receptor activation increases sympathetic nervous system outflow to skeletal muscle. Intra-VMH melanocortin receptor activation also induced significant changes in the expression of mediators of energy expenditure in muscle. These results support the role of melanocortin receptors in the VMH in the modulation of skeletal muscle metabolism. The ventromedial hypothalamus (VMH) and the brain melanocortin system both play vital roles in increasing energy expenditure (EE) and physical activity, decreasing appetite and modulating sympathetic nervous system (SNS) outflow. Because of recent evidence showing that VMH activation modulates skeletal muscle metabolism, we propose the existence of an axis between the VMH and skeletal muscle, modulated by brain melanocortins, modelled on the brain control of brown adipose tissue. Activation of melanocortin receptors in the VMH of rats using a non-specific agonist melanotan II (MTII), compared to vehicle, increased oxygen consumption and EE and decreased the respiratory exchange ratio. Intra-VMH MTII enhanced activity-related EE even when activity levels were held constant. MTII treatment increased gastrocnemius muscle heat dissipation during controlled activity, as well as in the home cage. Compared to vehicle-treated rats, rats with intra-VMH melanocortin receptor activation had higher skeletal muscle norepinephrine turnover, indicating an increased SNS drive to muscle. Lastly, intra-VMH MTII induced m

  14. Activation and Regulation of Purinergic P2X Receptor Channels

    Science.gov (United States)

    Coddou, Claudio; Yan, Zonghe; Obsil, Tomas; Huidobro-Toro, J. Pablo

    2011-01-01

    Mammalian ATP-gated nonselective cation channels (P2XRs) can be composed of seven possible subunits, denoted P2X1 to P2X7. Each subunit contains a large ectodomain, two transmembrane domains, and intracellular N and C termini. Functional P2XRs are organized as homomeric and heteromeric trimers. This review focuses on the binding sites involved in the activation (orthosteric) and regulation (allosteric) of P2XRs. The ectodomains contain three ATP binding sites, presumably located between neighboring subunits and formed by highly conserved residues. The detection and coordination of three ATP phosphate residues by positively charged amino acids are likely to play a dominant role in determining agonist potency, whereas an AsnPheArg motif may contribute to binding by coordinating the adenine ring. Nonconserved ectodomain histidines provide the binding sites for trace metals, divalent cations, and protons. The transmembrane domains account not only for the formation of the channel pore but also for the binding of ivermectin (a specific P2X4R allosteric regulator) and alcohols. The N- and C- domains provide the structures that determine the kinetics of receptor desensitization and/or pore dilation and are critical for the regulation of receptor functions by intracellular messengers, kinases, reactive oxygen species and mercury. The recent publication of the crystal structure of the zebrafish P2X4.1R in a closed state provides a major advance in the understanding of this family of receptor channels. We will discuss data obtained from numerous site-directed mutagenesis experiments accumulated during the last 15 years with reference to the crystal structure, allowing a structural interpretation of the molecular basis of orthosteric and allosteric ligand actions. PMID:21737531

  15. The LAR protein tyrosine phosphatase enables PDGF beta-receptor activation through attenuation of the c-Abl kinase activity.

    NARCIS (Netherlands)

    Zheng, W.; Lennartsson, J.; Hendriks, W.J.A.J.; Heldin, C.H.; Hellberg, C.

    2011-01-01

    The receptor tyrosine phosphatase (RPTP) LAR negatively regulates the activity of several receptor tyrosine kinases. To investigate if LAR affects the platelet-derived growth factor (PDGF) receptor signaling, mouse embryonic fibroblasts (MEFs) from mice where the LAR phosphatase domains were deleted

  16. β-Arrestin Recruitment and Biased Agonism at Free Fatty Acid Receptor 1*

    Science.gov (United States)

    Mancini, Arturo D.; Bertrand, Gyslaine; Vivot, Kevin; Carpentier, Éric; Tremblay, Caroline; Ghislain, Julien; Bouvier, Michel; Poitout, Vincent

    2015-01-01

    FFAR1/GPR40 is a seven-transmembrane domain receptor (7TMR) expressed in pancreatic β cells and activated by FFAs. Pharmacological activation of GPR40 is a strategy under consideration to increase insulin secretion in type 2 diabetes. GPR40 is known to signal predominantly via the heterotrimeric G proteins Gq/11. However, 7TMRs can also activate functionally distinct G protein-independent signaling via β-arrestins. Further, G protein- and β-arrestin-based signaling can be differentially modulated by different ligands, thus eliciting ligand-specific responses (“biased agonism”). Whether GPR40 engages β-arrestin-dependent mechanisms and is subject to biased agonism is unknown. Using bioluminescence resonance energy transfer-based biosensors for real-time monitoring of cell signaling in living cells, we detected a ligand-induced GPR40-β-arrestin interaction, with the synthetic GPR40 agonist TAK-875 being more effective than palmitate or oleate in recruiting β-arrestins 1 and 2. Conversely, TAK-875 acted as a partial agonist of Gq/11-dependent GPR40 signaling relative to both FFAs. Pharmacological blockade of Gq activity decreased FFA-induced insulin secretion. In contrast, knockdown or genetic ablation of β-arrestin 2 in an insulin-secreting cell line and mouse pancreatic islets, respectively, uniquely attenuated the insulinotropic activity of TAK-875, thus providing functional validation of the biosensor data. Collectively, these data reveal that in addition to coupling to Gq/11, GPR40 is functionally linked to a β-arrestin 2-mediated insulinotropic signaling axis. These observations expose previously unrecognized complexity for GPR40 signal transduction and may guide the development of biased agonists showing improved clinical profile in type 2 diabetes. PMID:26157145

  17. β-Arrestin Recruitment and Biased Agonism at Free Fatty Acid Receptor 1.

    Science.gov (United States)

    Mancini, Arturo D; Bertrand, Gyslaine; Vivot, Kevin; Carpentier, Éric; Tremblay, Caroline; Ghislain, Julien; Bouvier, Michel; Poitout, Vincent

    2015-08-21

    FFAR1/GPR40 is a seven-transmembrane domain receptor (7TMR) expressed in pancreatic β cells and activated by FFAs. Pharmacological activation of GPR40 is a strategy under consideration to increase insulin secretion in type 2 diabetes. GPR40 is known to signal predominantly via the heterotrimeric G proteins Gq/11. However, 7TMRs can also activate functionally distinct G protein-independent signaling via β-arrestins. Further, G protein- and β-arrestin-based signaling can be differentially modulated by different ligands, thus eliciting ligand-specific responses ("biased agonism"). Whether GPR40 engages β-arrestin-dependent mechanisms and is subject to biased agonism is unknown. Using bioluminescence resonance energy transfer-based biosensors for real-time monitoring of cell signaling in living cells, we detected a ligand-induced GPR40-β-arrestin interaction, with the synthetic GPR40 agonist TAK-875 being more effective than palmitate or oleate in recruiting β-arrestins 1 and 2. Conversely, TAK-875 acted as a partial agonist of Gq/11-dependent GPR40 signaling relative to both FFAs. Pharmacological blockade of Gq activity decreased FFA-induced insulin secretion. In contrast, knockdown or genetic ablation of β-arrestin 2 in an insulin-secreting cell line and mouse pancreatic islets, respectively, uniquely attenuated the insulinotropic activity of TAK-875, thus providing functional validation of the biosensor data. Collectively, these data reveal that in addition to coupling to Gq/11, GPR40 is functionally linked to a β-arrestin 2-mediated insulinotropic signaling axis. These observations expose previously unrecognized complexity for GPR40 signal transduction and may guide the development of biased agonists showing improved clinical profile in type 2 diabetes. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  18. Cyprodinil as an activator of aryl hydrocarbon receptor.

    Science.gov (United States)

    Fang, Chien-Chung; Chen, Fei-Yun; Chen, Chang-Rong; Liu, Chien-Chiang; Wong, Liang-Chi; Liu, Yi-Wen; Su, Jyan-Gwo Joseph

    2013-02-08

    Cyprodinil is a pyrimidinamine fungicide, used worldwide by agriculture. It is used to protect fruit plants and vegetables from a wide range of pathogens. Benzo[a]pyrene (BaP) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) are toxic environmental pollutants and are prototypes of aryl hydrocarbon receptor (AHR) ligands. Although the structure of cyprodinil distinctly differs from those of BaP and TCDD, our results show that cyprodinil induced nuclear translocation of the AHR, and induced the transcriptional activity of aryl hydrocarbon response element (AHRE). Cyprodinil induced the expression of cytochrome P450 (CYP) 1A1, a well-known AHR-targeted gene, in ovarian granulosa cells, HO23, and hepatoma cells, Hepa-1c1c7. Its induction did not appear in AHR signal-deficient cells, and was blocked by the AHR antagonist, CH-223191. Cyprodinil decreased AHR expression in HO23 cells, resulting in CYP1A1 expression decreasing after it peaked at 9h of treatment in HO23 cells. Dexamethasone is a synthetic agonist of glucocorticoids. Cyprodinil enhanced dexamethasone-induced gene expression, and conversely, its induction of CYP1A1 expression was decreased by dexamethasone in HO23 cells, indicating its induction of crosstalk between the AHR and glucocorticoid receptor and its role as a potential endocrine disrupter. In addition to BaP, TCDD, and an AHR agonist, β-NF, cyprodinil also phosphorylated extracellular signal-regulated kinase (ERK) in HO23 and Hepa-1c1c7 cells, indicating its deregulation of ERK activity. In summary, our results demonstrate that cyprodinil, similar to BaP, acts as an AHR activator, a potential endocrine disrupter, and an ERK disrupter. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

  19. Thrombin-Induced Podocyte Injury Is Protease-Activated Receptor Dependent.

    Science.gov (United States)

    Sharma, Ruchika; Waller, Amanda P; Agrawal, Shipra; Wolfgang, Katelyn J; Luu, Hiep; Shahzad, Khurrum; Isermann, Berend; Smoyer, William E; Nieman, Marvin T; Kerlin, Bryce A

    2017-09-01

    Nephrotic syndrome is characterized by massive proteinuria and injury of specialized glomerular epithelial cells called podocytes. Studies have shown that, whereas low-concentration thrombin may be cytoprotective, higher thrombin concentrations may contribute to podocyte injury. We and others have demonstrated that ex vivo plasma thrombin generation is enhanced during nephrosis, suggesting that thrombin may contribute to nephrotic progression. Moreover, nonspecific thrombin inhibition has been shown to decrease proteinuria in nephrotic animal models. We thus hypothesized that thrombin contributes to podocyte injury in a protease-activated receptor-specific manner during nephrosis. Here, we show that specific inhibition of thrombin with hirudin reduced proteinuria in two rat nephrosis models, and thrombin colocalized with a podocyte-specific marker in rat glomeruli. Furthermore, flow cytometry immunophenotyping revealed that rat podocytes express the protease-activated receptor family of coagulation receptors in vivo High-concentration thrombin directly injured conditionally immortalized human and rat podocytes. Using receptor-blocking antibodies and activation peptides, we determined that thrombin-mediated injury depended upon interactions between protease-activated receptor 3 and protease-activated receptor 4 in human podocytes, and between protease-activated receptor 1 and protease-activated receptor 4 in rat podocytes. Proximity ligation and coimmunoprecipitation assays confirmed thrombin-dependent interactions between human protease-activated receptor 3 and protease-activated receptor 4, and between rat protease-activated receptor 1 and protease-activated receptor 4 in cultured podocytes. Collectively, these data implicate thrombinuria as a contributor to podocyte injury during nephrosis, and suggest that thrombin and/or podocyte-expressed thrombin receptors may be novel therapeutic targets for nephrotic syndrome. Copyright © 2017 by the American Society of

  20. Peroxisome Proliferator-Activated Receptor-γ in Thyroid Autoimmunity

    Directory of Open Access Journals (Sweden)

    Silvia Martina Ferrari

    2015-01-01

    Full Text Available Peroxisome proliferator-activated receptor- (PPAR- γ expression has been shown in thyroid tissue from patients with thyroiditis or Graves’ disease and furthermore in the orbital tissue of patients with Graves’ ophthalmopathy (GO, such as in extraocular muscle cells. An increasing body of evidence shows the importance of the (C-X-C motif receptor 3 (CXCR3 and cognate chemokines (C-X-C motif ligand (CXCL9, CXCL10, and CXCL11, in the T helper 1 immune response and in inflammatory diseases such as thyroid autoimmune disorders. PPAR-γ agonists show a strong inhibitory effect on the expression and release of CXCR3 chemokines, in vitro, in various kinds of cells, such as thyrocytes, and in orbital fibroblasts, preadipocytes, and myoblasts from patients with GO. Recently, it has been demonstrated that rosiglitazone is involved in a higher risk of heart failure, stroke, and all-cause mortality in old patients. On the contrary, pioglitazone has not shown these effects until now; this favors pioglitazone for a possible use in patients with thyroid autoimmunity. However, further studies are ongoing to explore the use of new PPAR-γ agonists in the treatment of thyroid autoimmune disorders.

  1. Activation of liver X receptors with T0901317 attenuates cardiac hypertrophy in vivo

    NARCIS (Netherlands)

    Kuipers, Irma; Li, Jiang; Vreeswijk-Baudoin, Inge; Koster, Johan; van der Harst, Pim; Sillje, Herman H. W.; Kuipers, Folkert; van Veldhuisen, Dirk J.; van Gilst, Wiek H.; de Boer, Rudolf A.

    2010-01-01

    Liver X receptor (LXR) is a nuclear receptor regulating cholesterol metabolism. Liver X receptor has also been shown to exert anti-proliferative and anti-inflammatory properties. In this study, we evaluated the effect of LXR activation on cardiac hypertrophy in vitro and in vivo. Treatment with the

  2. Activation of protease-activated receptor 2 induces VEGF independently of HIF-1.

    Directory of Open Access Journals (Sweden)

    Jeppe Grøndahl Rasmussen

    Full Text Available BACKGROUND: Human adipose stem cells (hASCs can promote angiogenesis through secretion of proangiogenic factors such as vascular endothelial growth factor (VEGF. In other cell types, it has been shown that induction of VEGF is mediated by both protease activated receptor 2 (PAR2 and hypoxia inducible factor 1(HIF-1. The present study hypothesized that PAR2 stimulation through activation of kinase signaling cascades lead to induction of HIF-1 and secretion of VEGF. METHODOLOGY/PRINCIPAL FINDINGS: Immunohistochemistry revealed the expression of PAR2 receptors on the surface of hASCs. Blocking the PAR2 receptors with a specific antibody prior to trypsin treatment showed these receptors are involved in trypsin-evoked increase in VEGF secretion from hASCs. Blocking with specific kinase inhibitors suggested that that activation of MEK/ERK and PI3-kinase/Akt pathways are involved in trypsin-eveoked induction of VEGF. The effect of the trypsin treatment on the transcription of VEGF peaked at 6 hours after the treatment and was comparable to the activation observed after keeping hASCs for 24 hours at 1% oxygen. In contrast to hypoxia, trypsin alone failed to induce HIF-1 measured with ELISA, while the combination of trypsin and hypoxia had an additive effect on both VEGF transcription and secretion, results which were confirmed by Western blot. CONCLUSION: In hASCs trypsin and hypoxia induce VEGF expression through separate pathways.

  3. Truncated variants of pig somatostatin receptor subtype 5 (sst5) act as dominant-negative modulators for sst2-mediated signaling.

    Science.gov (United States)

    Durán-Prado, Mario; Gahete, Manuel D; Delgado-Niebla, Elena; Martínez-Fuentes, Antonio J; Vázquez-Martínez, Rafael; García-Navarro, Socorro; Gracia-Navarro, Francisco; Malagon, María M; Luque, Raúl M; Castaño, Justo P

    2012-12-01

    Somatostatin (SST) and its related peptide cortistatin (CORT) exert their multiple actions through binding to the SST receptor (sst) family, generally considered to comprise five G protein-coupled receptors with seven transmembrane domains (TMD), named sst1-sst5, plus a splice sst2B variant. However, we recently discovered that human and rodent sst5 gene expression also generates, through noncanonical alternative splicing, novel truncated albeit functional sst5 variants with less than seven TMD. Here, we cloned and characterized for the first time the porcine wild-type sst5 (psst5, full-length) and identified two novel truncated psst5 variants with six and three TMD, thus termed psst5TMD6 and psst5TMD3, respectively. In line with that observed in human and rodent truncated sst5 variants, psst5TMD6 and psst5TMD3 are functional (e.g., activate calcium signaling), selectively respond to SST and CORT, respectively, and exhibit specific tissue expression profiles that differ from full-length psst5 and often overlaps with psst2 expression. Moreover, fluorescence resonance energy transfer analysis shows that psst5 truncated variants physically interact with psst2, thereby altering their localization at the plasma membrane and specifically disrupting the cellular response to SST and/or CORT. These results represent the first characterization of a key porcine SST receptor, psst5, and, together with our previous results, provide strong evidence that alternative splicing-derived, truncated sst5 variants with distinct functional capacities exist in the mammalian lineage, where they can act as dominant-negative receptors, by interacting directly with long, seven TMD variants, potentially contributing to modulate normal and pathological SST and CORT signaling.

  4. Anticonvulsant activity of some vanilloid receptor agonists | Awad ...

    African Journals Online (AJOL)

    Background: Vanilloid receptors 1 (VR 1), a group of transient receptor potential channels family was cloned in 1997. They were found to be a potential target for treatment of different acute and chronic pain disorder. Recently these receptors were reported to be involved in several pathological conditions. Objectives: The ...

  5. Analysis of Transmembrane Domains 1 and 4 of the Human Angiotensin II AT1 Receptor by Cysteine-scanning Mutagenesis*

    Science.gov (United States)

    Yan, Liping; Holleran, Brian J.; Lavigne, Pierre; Escher, Emanuel; Guillemette, Gaétan; Leduc, Richard

    2010-01-01

    The octapeptide hormone angiotensin II (AngII) exerts a wide variety of cardiovascular effects through the activation of the AT1 receptor, which belongs to the G protein-coupled receptor superfamily. Like other G protein-coupled receptors, the AT1 receptor possesses seven transmembrane domains that provide structural support for the formation of the ligand-binding pocket. Here, we investigated the role of the first and fourth transmembrane domains (TMDs) in the formation of the binding pocket of the human AT1 receptor using the substituted-cysteine accessibility method. Each residue within the Phe-28(1.32)–Ile-53(1.57) fragment of TMD1 and Leu-143(4.40)–Phe-170(4.67) fragment of TMD4 was mutated, one at a time, to a cysteine. The resulting mutant receptors were expressed in COS-7 cells, which were subsequently treated with the charged sulfhydryl-specific alkylating agent methanethiosulfonate ethylammonium (MTSEA). This treatment led to a significant reduction in the binding affinity of TMD1 mutants M30C(1.34)-AT1 and T33C(1.37)-AT1 and TMD4 mutant V169C(4.66)-AT1. Although this reduction in binding of the TMD1 mutants was maintained when examined in a constitutively active receptor (N111G-AT1) background, we found that V169C(4.66)-AT1 remained unaffected when treated with MTSEA compared with untreated in this context. Moreover, the complete loss of binding observed for R167C(4.64)-AT1 was restored upon treatment with MTSEA. Our results suggest that the extracellular portion of TMD1, particularly residues Met-30(1.34) and Thr-33(1.37), as well as residues Arg-167(4.64) and Val-169(4.66) at the junction of TMD4 and the second extracellular loop, are important binding determinants within the AT1 receptor binding pocket but that these TMDs undergo very little movement, if at all, during the activation process. PMID:19940150

  6. Calcium is the switch in the moonlighting dual function of the ligand-activated receptor kinase phytosulfokine receptor 1

    KAUST Repository

    Muleya, Victor

    2014-09-23

    Background: A number of receptor kinases contain guanylate cyclase (GC) catalytic centres encapsulated in the cytosolic kinase domain. A prototypical example is the phytosulfokine receptor 1 (PSKR1) that is involved in regulating growth responses in plants. PSKR1 contains both kinase and GC activities however the underlying mechanisms regulating the dual functions have remained elusive. Findings: Here, we confirm the dual activity of the cytoplasmic domain of the PSKR1 receptor. We show that mutations within the guanylate cyclase centre modulate the GC activity while not affecting the kinase catalytic activity. Using physiologically relevant Ca2+ levels, we demonstrate that its GC activity is enhanced over two-fold by Ca2+ in a concentration-dependent manner. Conversely, increasing Ca2+ levels inhibits kinase activity up to 500-fold at 100 nM Ca2+. Conclusions: Changes in calcium at physiological levels can regulate the kinase and GC activities of PSKR1. We therefore propose a functional model of how calcium acts as a bimodal switch between kinase and GC activity in PSKR1 that could be relevant to other members of this novel class of ligand-activated receptor kinases.

  7. Hypersensitivity Induced by Activation of Spinal Cord PAR2 Receptors Is Partially Mediated by TRPV1 Receptors.

    Directory of Open Access Journals (Sweden)

    Petra Mrozkova

    Full Text Available Protease-activated receptors 2 (PAR2 and transient receptor potential vanilloid 1 (TRPV1 receptors in the peripheral nerve endings are implicated in the development of increased sensitivity to mechanical and thermal stimuli, especially during inflammatory states. Both PAR2 and TRPV1 receptors are co-expressed in nociceptive dorsal root ganglion (DRG neurons on their peripheral endings and also on presynaptic endings in the spinal cord dorsal horn. However, the modulation of nociceptive synaptic transmission in the superficial dorsal horn after activation of PAR2 and their functional coupling with TRPV1 is not clear. To investigate the role of spinal PAR2 activation on nociceptive modulation, intrathecal drug application was used in behavioural experiments and patch-clamp recordings of spontaneous, miniature and dorsal root stimulation-evoked excitatory postsynaptic currents (sEPSCs, mEPSCs, eEPSCs were performed on superficial dorsal horn neurons in acute rat spinal cord slices. Intrathecal application of PAR2 activating peptide SLIGKV-NH2 induced thermal hyperalgesia, which was prevented by pretreatment with TRPV1 antagonist SB 366791 and was reduced by protein kinases inhibitor staurosporine. Patch-clamp experiments revealed robust decrease of mEPSC frequency (62.8 ± 4.9%, increase of sEPSC frequency (127.0 ± 5.9% and eEPSC amplitude (126.9 ± 12.0% in dorsal horn neurons after acute SLIGKV-NH2 application. All these EPSC changes, induced by PAR2 activation, were prevented by SB 366791 and staurosporine pretreatment. Our results demonstrate an important role of spinal PAR2 receptors in modulation of nociceptive transmission in the spinal cord dorsal horn at least partially mediated by activation of presynaptic TRPV1 receptors. The functional coupling between the PAR2 and TRPV1 receptors on the central branches of DRG neurons may be important especially during different pathological states when it may enhance pain perception.

  8. Hypersensitivity Induced by Activation of Spinal Cord PAR2 Receptors Is Partially Mediated by TRPV1 Receptors.

    Science.gov (United States)

    Mrozkova, Petra; Spicarova, Diana; Palecek, Jiri

    2016-01-01

    Protease-activated receptors 2 (PAR2) and transient receptor potential vanilloid 1 (TRPV1) receptors in the peripheral nerve endings are implicated in the development of increased sensitivity to mechanical and thermal stimuli, especially during inflammatory states. Both PAR2 and TRPV1 receptors are co-expressed in nociceptive dorsal root ganglion (DRG) neurons on their peripheral endings and also on presynaptic endings in the spinal cord dorsal horn. However, the modulation of nociceptive synaptic transmission in the superficial dorsal horn after activation of PAR2 and their functional coupling with TRPV1 is not clear. To investigate the role of spinal PAR2 activation on nociceptive modulation, intrathecal drug application was used in behavioural experiments and patch-clamp recordings of spontaneous, miniature and dorsal root stimulation-evoked excitatory postsynaptic currents (sEPSCs, mEPSCs, eEPSCs) were performed on superficial dorsal horn neurons in acute rat spinal cord slices. Intrathecal application of PAR2 activating peptide SLIGKV-NH2 induced thermal hyperalgesia, which was prevented by pretreatment with TRPV1 antagonist SB 366791 and was reduced by protein kinases inhibitor staurosporine. Patch-clamp experiments revealed robust decrease of mEPSC frequency (62.8 ± 4.9%), increase of sEPSC frequency (127.0 ± 5.9%) and eEPSC amplitude (126.9 ± 12.0%) in dorsal horn neurons after acute SLIGKV-NH2 application. All these EPSC changes, induced by PAR2 activation, were prevented by SB 366791 and staurosporine pretreatment. Our results demonstrate an important role of spinal PAR2 receptors in modulation of nociceptive transmission in the spinal cord dorsal horn at least partially mediated by activation of presynaptic TRPV1 receptors. The functional coupling between the PAR2 and TRPV1 receptors on the central branches of DRG neurons may be important especially during different pathological states when it may enhance pain perception.

  9. High-affinity benzodiazepine receptor ligands among benzodiazepines and betacarbolines with different intrinsic activity

    Energy Technology Data Exchange (ETDEWEB)

    Yliniemelae, A.; Gynther, J. (Univ. of Kuopio (Finland)); Konschin, H.; Tylli, H. (Univ. of Helsinki (Finland)); Rouvinen, J. (Univ. of Joensuu (Finland))

    1989-01-01

    Structural and electrostatic features of diazepam, flumazenil, and methyl betacarboline-3-carboxylate (BCCM) have been investigated using the molecular superimposition method. These high-affinity benzodiazepine (BZ) receptor ligands are structurally unrelated and they have different intrinsic activity. These ligands are superimposed in such a way that common structural and electrostatic features essential for the high receptor binding affinity overlap. In addition to this binding pharmacophore, there are roughly three separate binding zones in the BZ receptor, one for each class of ligands. The intrinsic activity of BZ receptor ligands depends on the molecular structures and the way the ligand approaches the receptor.

  10. Increased peroxisome proliferator-activated receptor γ activity reduces imatinib uptake and efficacy in chronic myeloid leukemia mononuclear cells.

    Science.gov (United States)

    Wang, Jueqiong; Lu, Liu; Kok, Chung H; Saunders, Verity A; Goyne, Jarrad M; Dang, Phuong; Leclercq, Tamara M; Hughes, Timothy P; White, Deborah L

    2017-05-01

    Imatinib is actively transported by organic cation transporter-1 (OCT-1) influx transporter, and low OCT-1 activity in diagnostic chronic myeloid leukemia blood mononuclear cells is significantly associated with poor molecular response to imatinib. Herein we report that, in diagnostic chronic myeloid leukemia mononuclear cells and BCR-ABL1 + cell lines, peroxisome proliferator-activated receptor γ agonists (GW1929, rosiglitazone, pioglitazone) significantly decrease OCT-1 activity; conversely, peroxisome proliferator-activated receptor γ antagonists (GW9662, T0070907) increase OCT-1 activity. Importantly, these effects can lead to corresponding changes in sensitivity to BCR-ABL kinase inhibition. Results were confirmed in peroxisome proliferator-activated receptor γ-transduced K562 cells. Furthermore, we identified a strong negative correlation between OCT-1 activity and peroxisome proliferator-activated receptor γ transcriptional activity in diagnostic chronic myeloid leukemia patients (n=84; P BCR-ABL kinase inhibition. The inter-patient variability of peroxisome proliferator-activated receptor γ activation likely accounts for the heterogeneity observed in patient OCT-1 activity at diagnosis. Recently, the peroxisome proliferator-activated receptor γ agonist pioglitazone was reported to act synergistically with imatinib, targeting the residual chronic myeloid leukemia stem cell pool. Our findings suggest that peroxisome proliferator-activated receptor γ ligands have differential effects on circulating mononuclear cells compared to stem cells. Since the effect of peroxisome proliferator-activated receptor γ activation on imatinib uptake in mononuclear cells may counteract the clinical benefit of this activation in stem cells, caution should be applied when combining these therapies, especially in patients with high peroxisome proliferator-activated receptor γ transcriptional activity. Copyright© Ferrata Storti Foundation.

  11. Contribution of regional brain melanocortin receptor subtypes to elevated activity energy expenditure in lean, active rats

    OpenAIRE

    Shukla,Charu; Koch, Lauren G.; Britton, Steven L.; Cai, Minying; Hruby, Victor J.; Bednarek, Maria; Novak, Colleen M.

    2015-01-01

    Physical activity and non-exercise activity thermogenesis (NEAT) are crucial factors accounting for individual differences in body weight, interacting with genetic predisposition. In the brain, a number of neuroendocrine intermediates regulate food intake and energy expenditure (EE); this includes the brain melanocortin (MC) system, consisting of melanocortin peptides as well as their receptors (MCR). MC3R and MC4R have emerged as critical modulators of EE and food intake. To determine how va...

  12. The role of GH receptor tyrosine phosphorylation in Stat5 activation

    DEFF Research Database (Denmark)

    Hansen, J A; Hansen, L H; Wang, X

    1997-01-01

    . Mutated GH receptors lacking all but one of these three tyrosines are able to mediate a transcriptional response when transiently transfected into CHO cells together with a Spi 2.1 promoter/luciferase construct. Similarly, these GH receptors were found to be able to mediate activation of Stat5 DNA......-binding activity, whereas the GH receptor mutant lacking all intracellular tyrosines was not. Synthetic tyrosine phosphorylated peptides corresponding to the GH receptor sequence around the three tyrosines inhibited Stat5 DNA-binding activity while their non-phosphorylated counterparts were ineffective. Tyrosine...... phosphorylated GST-GH receptor fusion proteins specifically bound to Stat5 in extracts from COS 7 cells transfected with Stat5 cDNA. This binding could be inhibited by tyrosine phosphorylated peptides derived from the GH receptor. This study thus demonstrated that specific GH receptor tyrosine residues...

  13. Repressive effects of resveratrol on androgen receptor transcriptional activity.

    Directory of Open Access Journals (Sweden)

    Wen-feng Shi

    Full Text Available BACKGROUND: The chemopreventive effects of resveratrol (RSV on prostate cancer have been well established; the androgen receptor (AR plays pivotal roles in prostatic tumorigenesis. However, the exact underlying molecular mechanisms about the effects of RSV on AR have not been fully elucidated. A model system is needed to determine whether and how RSV represses AR transcriptional activity. METHODOLOGY: The AR cDNA was first cloned into the retroviral vector pOZ-N and then integrated into the genome of AR-negative HeLa cells to generate the AR(+ cells. The constitutively expressed AR was characterized by monitoring hormone-stimulated nuclear translocation, DNA binding, and transcriptional activation, with the AR(- cells serving as controls. AR(+ cells were treated with RSV, and both AR protein levels and AR transcriptional activity were measured simultaneously. Chromatin immunoprecipitation (ChIP assays were used to detect the effects of RSV on the recruitment of AR to its cognate element (ARE. RESULTS: AR in the AR (+ stable cell line functions in a manner similar to that of endogenously expressed AR. Using this model system we clearly demonstrated that RSV represses AR transcriptional activity independently of any effects on AR protein levels. However, neither the hormone-mediated nucleus translocation nor the AR/ARE interaction was affected by RSV treatment. CONCLUSION: We demonstrated unambiguously that RSV regulates AR target gene expression, at least in part, by repressing AR transcriptional activity. Repressive effects of RSV on AR activity result from mechanisms other than the affects of AR nuclear translocation or DNA binding.

  14. Phosphorylated Nuclear Receptor CAR Forms a Homodimer To Repress Its Constitutive Activity for Ligand Activation.

    Science.gov (United States)

    Shizu, Ryota; Osabe, Makoto; Perera, Lalith; Moore, Rick; Sueyoshi, Tatsuya; Negishi, Masahiko

    2017-05-15

    The nuclear receptor CAR (NR1I3) regulates hepatic drug and energy metabolism as well as cell fate. Its activation can be a critical factor in drug-induced toxicity and the development of diseases, including diabetes and tumors. CAR inactivates its constitutive activity by phosphorylation at threonine 38. Utilizing receptor for protein kinase 1 (RACK1) as the regulatory subunit, protein phosphatase 2A (PP2A) dephosphorylates threonine 38 to activate CAR. Here we demonstrate that CAR undergoes homodimer-monomer conversion to regulate this dephosphorylation. By coexpression of two differently tagged CAR proteins in Huh-7 cells, mouse primary hepatocytes, and mouse livers, coimmunoprecipitation and two-dimensional gel electrophoresis revealed that CAR can form a homodimer in a configuration in which the PP2A/RACK1 binding site is buried within its dimer interface. Epidermal growth factor (EGF) was found to stimulate CAR homodimerization, thus constraining CAR in its inactive form. The agonistic ligand CITCO binds directly to the CAR homodimer and dissociates phosphorylated CAR into its monomers, exposing the PP2A/RACK1 binding site for dephosphorylation. Phenobarbital, which is not a CAR ligand, binds the EGF receptor, reversing the EGF signal to monomerize CAR for its indirect activation. Thus, the homodimer-monomer conversion is the underlying molecular mechanism that regulates CAR activation, by placing phosphorylated threonine 38 as the common target for both direct and indirect activation of CAR. Copyright © 2017 American Society for Microbiology.

  15. Analysis of the epidermal growth factor receptor specific transcriptome: effect of receptor expression level and an activating mutation

    DEFF Research Database (Denmark)

    Pedersen, Mikkel W; Pedersen, Nina; Damstrup, Lars

    2005-01-01

    by interferons. Expression of this module was absent in the EGFRvIII-expressing cell line and the parental cell line. Treatment with the specific EGFR inhibitor AG1478 indicated that the regulations were primary, receptor-mediated events. Furthermore, activation of this module correlated with activation of STAT1...

  16. Pristanic acid and phytanic acid: naturally occurring ligands for the nuclear receptor peroxisome proliferator-activated receptor alpha

    NARCIS (Netherlands)

    Zomer, A. W.; van der Burg, B.; Jansen, G. A.; Wanders, R. J.; Poll-The, B. T.; van der Saag, P. T.

    2000-01-01

    Phytanic acid and pristanic acid are branched-chain fatty acids, present at micromolar concentrations in the plasma of healthy individuals. Here we show that both phytanic acid and pristanic acid activate the peroxisome proliferator-activated receptor alpha (PPARalpha) in a concentration-dependent

  17. A library of 7TM receptor C-terminal tails - Interactions with the proposed post-endocytic sorting proteins ERM-binding phosphoprotein 50 (EBP50), N-ethylmaleimide-sensitive factor (NSF), sorting nexin 1 (SNX1), and G protein-coupled receptor-associated sorting protein (GASP)

    DEFF Research Database (Denmark)

    Heydorn, A.; Sondergaard, B.P.; Ersbøll, Bjarne Kjær

    2004-01-01

    sequestration through interactions, mainly with the C-terminal intracellular tails of the receptors. A library of tails from 59 representative members of the super family of seven-transmembrane receptors was probed as glutathione S-transferase fusion proteins for interactions with four different adaptor...... only a single receptor tail, i.e. the beta(2)-adrenergic receptor, whereas N-ethylmaleimide-sensitive factor bound 11 of the tail-fusion proteins. Of the two proteins proposed to target receptors for lysosomal degradation, sorting nexin 1 (SNX1) bound 10 and the C-terminal domain of G protein......-coupled receptor-associated sorting protein bound 23 of the 59 tail proteins. Surface plasmon resonance analysis of the binding kinetics of selected hits from the glutathione S-transferase pull-down experiments, i.e. the tails of the virally encoded receptor US28 and the delta-opioid receptor, confirmed...

  18. Modulation of transcriptional mineralocorticoid receptor activity by casein kinase 2.

    Science.gov (United States)

    Ruhs, Stefanie; Strätz, Nicole; Quarch, Katja; Masch, Antonia; Schutkowski, Mike; Gekle, Michael; Grossmann, Claudia

    2017-11-10

    The pathogenesis of cardiovascular diseases is a multifunctional process in which the mineralocorticoid receptor (MR), a ligand-dependent transcription factor, is involved as proven by numerous clinical studies. The development of pathophysiological MR actions depends on the existence of additional factors e.g. inflammatory cytokines and seems to involve posttranslational MR modifications e.g. phosphorylation. Casein kinase 2 (CK2) is a ubiquitously expressed multifunctional serine/threonine kinase that can be activated under inflammatory conditions as the MR. Sequence analysis and inhibitor experiments revealed that CK2 acts as a positive modulator of MR activity by facilitating MR-DNA interaction with subsequent rapid MR degradation. Peptide microarrays and site-directed mutagenesis experiments identified the highly conserved S459 as a functionally relevant CK2 phosphorylation site of the MR. Moreover, MR-CK2 protein-protein interaction mediated by HSP90 was shown by co-immunoprecipitation. During inflammation, cytokine stimulation led to a CK2-dependent increased expression of proinflammatory genes. The additional MR activation by aldosterone during cytokine stimulation augmented CK2-dependent NFκB signaling which enhanced the expression of proinflammatory genes further. Overall, in an inflammatory environment the bidirectional CK2-MR interaction aggravate the existing pathophysiological cellular situation.

  19. Activation of transient receptor potential ankyrin 1 by eugenol.

    Science.gov (United States)

    Chung, G; Im, S T; Kim, Y H; Jung, S J; Rhyu, M-R; Oh, S B

    2014-03-07

    Eugenol is a bioactive plant extract used as an analgesic agent in dentistry. The structural similarity of eugenol to cinnamaldehyde, an active ligand for transient receptor potential ankyrin 1 (TRPA1), suggests that eugenol might produce its effect via TRPA1, in addition to TRPV1 as we reported previously. In this study, we investigated the effect of eugenol on TRPA1, by fura-2-based calcium imaging and patch clamp recording in trigeminal ganglion neurons and in a heterologous expression system. As the result, eugenol induced robust calcium responses in rat trigeminal ganglion neurons that responded to a specific TRPA1 agonist, allyl isothiocyanate (AITC), and not to capsaicin. Capsazepine, a TRPV1 antagonist failed to inhibit eugenol-induced calcium responses in AITC-responding neurons. In addition, eugenol response was observed in trigeminal ganglion neurons from TRPV1 knockout mice and human embryonic kidney 293 cell lines that express human TRPA1, which was inhibited by TRPA1-specific antagonist HC-030031. Eugenol-evoked TRPA1 single channel activity and eugenol-induced TRPA1 currents were dose-dependent with EC50 of 261.5μM. In summary, these results demonstrate that the activation of TRPA1 might account for another molecular mechanism underlying the pharmacological action of eugenol. Copyright © 2013 IBRO. Published by Elsevier Ltd. All rights reserved.

  20. Contribution of regional brain melanocortin receptor subtypes to elevated activity energy expenditure in lean, active rats.

    Science.gov (United States)

    Shukla, C; Koch, L G; Britton, S L; Cai, M; Hruby, V J; Bednarek, M; Novak, C M

    2015-12-03

    Physical activity and non-exercise activity thermogenesis (NEAT) are crucial factors accounting for individual differences in body weight, interacting with genetic predisposition. In the brain, a number of neuroendocrine intermediates regulate food intake and energy expenditure (EE); this includes the brain melanocortin (MC) system, consisting of MC peptides as well as their receptors (MCR). MC3R and MC4R have emerged as critical modulators of EE and food intake. To determine how variance in MC signaling may underlie individual differences in physical activity levels, we examined behavioral response to MC receptor agonists and antagonists in rats that show high and low levels of physical activity and NEAT, that is, high- and low-capacity runners (HCR, LCR), developed by artificial selection for differential intrinsic aerobic running capacity. Focusing on the hypothalamus, we identified brain region-specific elevations in expression of MCR 3, 4, and also MC5R, in the highly active, lean HCR relative to the less active and obesity-prone LCR. Further, the differences in activity and associated EE as a result of MCR activation or suppression using specific agonists and antagonists were similarly region-specific and directly corresponded to the differential MCR expression patterns. The agonists and antagonists investigated here did not significantly impact food intake at the doses used, suggesting that the differential pattern of receptor expression may by more meaningful to physical activity than to other aspects of energy balance regulation. Thus, MCR-mediated physical activity may be a key neural mechanism in distinguishing the lean phenotype and a target for enhancing physical activity and NEAT. Copyright © 2015 IBRO. Published by Elsevier Ltd. All rights reserved.

  1. HIV-1 activates macrophages independent of Toll-like receptors.

    Directory of Open Access Journals (Sweden)

    Joseph N Brown

    Full Text Available Macrophages provide an interface between innate and adaptive immunity and are important long-lived reservoirs for Human Immunodeficiency Virus Type-1 (HIV-1. Multiple genetic networks involved in regulating signal transduction cascades and immune responses in macrophages are coordinately modulated by HIV-1 infection.To evaluate complex interrelated processes and to assemble an integrated view of activated signaling networks, a systems biology strategy was applied to genomic and proteomic responses by primary human macrophages over the course of HIV-1 infection. Macrophage responses, including cell cycle, calcium, apoptosis, mitogen-activated protein kinases (MAPK, and cytokines/chemokines, to HIV-1 were temporally regulated, in the absence of cell proliferation. In contrast, Toll-like receptor (TLR pathways remained unaltered by HIV-1, although TLRs 3, 4, 7, and 8 were expressed and responded to ligand stimulation in macrophages. HIV-1 failed to activate phosphorylation of IRAK-1 or IRF-3, modulate intracellular protein levels of Mx1, an interferon-stimulated gene, or stimulate secretion of TNF, IL-1beta, or IL-6. Activation of pathways other than TLR was inadequate to stimulate, via cross-talk mechanisms through molecular hubs, the production of proinflammatory cytokines typical of a TLR response. HIV-1 sensitized macrophage responses to TLR ligands, and the magnitude of viral priming was related to virus replication.HIV-1 induced a primed, proinflammatory state, M1(HIV, which increased the responsiveness of macrophages to TLR ligands. HIV-1 might passively evade pattern recognition, actively inhibit or suppress recognition and signaling, or require dynamic interactions between macrophages and other cells, such as lymphocytes or endothelial cells. HIV-1 evasion of TLR recognition and simultaneous priming of macrophages may represent a strategy for viral survival, contribute to immune pathogenesis, and provide important targets for therapeutic

  2. Triclocarban mediates induction of xenobiotic metabolism through activation of the constitutive androstane receptor and the estrogen receptor alpha.

    Directory of Open Access Journals (Sweden)

    Mei-Fei Yueh

    Full Text Available Triclocarban (3,4,4'-trichlorocarbanilide, TCC is used as a broad-based antimicrobial agent that is commonly added to personal hygiene products. Because of its extensive use in the health care industry and resistance to degradation in sewage treatment processes, TCC has become a significant waste product that is found in numerous environmental compartments where humans and wildlife can be exposed. While TCC has been linked to a range of health and environmental effects, few studies have been conducted linking exposure to TCC and induction of xenobiotic metabolism through regulation by environmental sensors such as the nuclear xenobiotic receptors (XenoRs. To identify the ability of TCC to activate xenobiotic sensors, we monitored XenoR activities in response to TCC treatment using luciferase-based reporter assays. Among the XenoRs in the reporter screening assay, TCC promotes both constitutive androstane receptor (CAR and estrogen receptor alpha (ERα activities. TCC treatment to hUGT1 mice resulted in induction of the UGT1A genes in liver. This induction was dependent upon the constitutive active/androstane receptor (CAR because no induction occurred in hUGT1Car(-/- mice. Induction of the UGT1A genes by TCC corresponded with induction of Cyp2b10, another CAR target gene. TCC was demonstrated to be a phenobarbital-like activator of CAR in receptor-based assays. While it has been suggested that TCC be classified as an endocrine disruptor, it activates ERα leading to induction of Cyp1b1 in female ovaries as well as in promoter activity. Activation of ERα by TCC in receptor-based assays also promotes induction of human CYP2B6. These observations demonstrate that TCC activates nuclear xenobiotic receptors CAR and ERα both in vivo and in vitro and might have the potential to alter normal physiological homeostasis. Activation of these xenobiotic-sensing receptors amplifies gene expression profiles that might represent a mechanistic base for

  3. Triclocarban Mediates Induction of Xenobiotic Metabolism through Activation of the Constitutive Androstane Receptor and the Estrogen Receptor Alpha

    Science.gov (United States)

    Yueh, Mei-Fei; Li, Tao; Evans, Ronald M.; Hammock, Bruce; Tukey, Robert H.

    2012-01-01

    Triclocarban (3,4,4′-trichlorocarbanilide, TCC) is used as a broad-based antimicrobial agent that is commonly added to personal hygiene products. Because of its extensive use in the health care industry and resistance to degradation in sewage treatment processes, TCC has become a significant waste product that is found in numerous environmental compartments where humans and wildlife can be exposed. While TCC has been linked to a range of health and environmental effects, few studies have been conducted linking exposure to TCC and induction of xenobiotic metabolism through regulation by environmental sensors such as the nuclear xenobiotic receptors (XenoRs). To identify the ability of TCC to activate xenobiotic sensors, we monitored XenoR activities in response to TCC treatment using luciferase-based reporter assays. Among the XenoRs in the reporter screening assay, TCC promotes both constitutive androstane receptor (CAR) and estrogen receptor alpha (ERα) activities. TCC treatment to hUGT1 mice resulted in induction of the UGT1A genes in liver. This induction was dependent upon the constitutive active/androstane receptor (CAR) because no induction occurred in hUGT1Car−/− mice. Induction of the UGT1A genes by TCC corresponded with induction of Cyp2b10, another CAR target gene. TCC was demonstrated to be a phenobarbital-like activator of CAR in receptor-based assays. While it has been suggested that TCC be classified as an endocrine disruptor, it activates ERα leading to induction of Cyp1b1 in female ovaries as well as in promoter activity. Activation of ERα by TCC in receptor-based assays also promotes induction of human CYP2B6. These observations demonstrate that TCC activates nuclear xenobiotic receptors CAR and ERα both in vivo and in vitro and might have the potential to alter normal physiological homeostasis. Activation of these xenobiotic-sensing receptors amplifies gene expression profiles that might represent a mechanistic base for potential human

  4. Characterization of the intrinsic activity for a novel class of cannabinoid receptor ligands: Indole quinuclidine analogs.

    Science.gov (United States)

    Franks, Lirit N; Ford, Benjamin M; Madadi, Nikhil R; Penthala, Narsimha R; Crooks, Peter A; Prather, Paul L

    2014-08-15

    Our laboratory recently reported that a group of novel indole quinuclidine analogs bind with nanomolar affinity to cannabinoid type-1 and type-2 receptors. This study characterized the intrinsic activity of these compounds by determining whether they exhibit agonist, antagonist, or inverse agonist activity at cannabinoid type-1 and/or type-2 receptors. Cannabinoid receptors activate Gi/Go-proteins that then proceed to inhibit activity of the downstream intracellular effector adenylyl cyclase. Therefore, intrinsic activity was quantified by measuring the ability of compounds to modulate levels of intracellular cAMP in intact cells. Concerning cannabinoid type-1 receptors endogenously expressed in Neuro2A cells, a single analog exhibited agonist activity, while eight acted as neutral antagonists and two possessed inverse agonist activity. For cannabinoid type-2 receptors stably expressed in CHO cells, all but two analogs acted as agonists; these two exceptions exhibited inverse agonist activity. Confirming specificity at cannabinoid type-1 receptors, modulation of adenylyl cyclase activity by all proposed agonists and inverse agonists was blocked by co-incubation with the neutral cannabinoid type-1 antagonist O-2050. All proposed cannabinoid type-1 receptor antagonists attenuated adenylyl cyclase modulation by cannabinoid agonist CP-55,940. Specificity at cannabinoid type-2 receptors was confirmed by failure of all compounds to modulate adenylyl cyclase activity in CHO cells devoid of cannabinoid type-2 receptors. Further characterization of select analogs demonstrated concentration-dependent modulation of adenylyl cyclase activity with potencies similar to their respective affinities for cannabinoid receptors. Therefore, indole quinuclidines are a novel structural class of compounds exhibiting high affinity and a range of intrinsic activity at cannabinoid type-1 and type-2 receptors. Copyright © 2014 Elsevier B.V. All rights reserved.

  5. Characterization of the intrinsic activity for a novel class of cannabinoid receptor ligands: Indole Quinuclidine analogues

    Science.gov (United States)

    Franks, Lirit N.; Ford, Benjamin M.; Madadi, Nikhil R.; Penthala, Narsimha R.; Crooks, Peter A.; Prather, Paul L.

    2014-01-01

    Our laboratory recently reported that a group of novel indole quinuclidine analogues bind with nanomolar affinity to cannabinoid type-1 and type-2 receptors. This study characterized the intrinsic activity of these compounds by determining whether they exhibit agonist, antagonist, or inverse agonist activity at cannabinoid type-1 and/or type-2 receptors. Cannabinoid receptors activate Gi/Go-proteins that then proceed to inhibit activity of the downstream intracellular effector adenylyl cyclase. Therefore, intrinsic activity was quantified by measuring the ability of compounds to modulate levels of intracellular cAMP in intact cells. Concerning cannabinoid type-1 receptors endogenously expressed in Neuro2A cells, a single analogue exhibited agonist activity, while eight acted as neutral antagonists and two possessed inverse agonist activity. For cannabinoid type-2 receptors stably expressed in CHO cells, all but two analogues acted as agonists; these two exceptions exhibited inverse agonist activity. Confirming specificity at cannabinoid type-1 receptors, modulation of adenylyl cyclase activity by all proposed agonists and inverse agonists was blocked by co-incubation with the neutral cannabinoid type-1 antagonist O-2050. All proposed cannabinoid type-1 receptor antagonists attenuated adenylyl cyclase modulation by cannabinoid agonist CP-55,940. Specificity at cannabinoid type-2 receptors was confirmed by failure of all compounds to modulate adenylyl cyclase activity in CHO cells devoid of cannabinoid type-2 receptors. Further characterization of select analogues demonstrated concentration-dependent modulation of adenylyl cyclase activity with potencies similar to their respective affinities for cannabinoid receptors. Therefore, indole quinuclidines are a novel structural class of compounds exhibiting high affinity and a range of intrinsic activity at cannabinoid type-1 and type-2 receptors. PMID:24858620

  6. SENESCENCE-ASSOCIATED DECLINE IN HEPATIC PEROXISOMAL ENZYME ACTIVITIES CORRESPONDS WITH DIMINISHED LEVELS OF RETINOID X RECEPTOR ALPHA, BUT NOT PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR ALPHA1

    Science.gov (United States)

    AbstractAging is associated with alterations in hepatic peroxisomal metabolism and susceptibility to hepatocarcinogenecity produced by agonists of peroxisome proliferator-activated receptor alpha (PPARa). Mechanisms involved in these effects are not well understood. Howev...

  7. Functional Characterization of MC1R-TUBB3 Intergenic Splice Variants of the Human Melanocortin 1 Receptor.

    Science.gov (United States)

    Herraiz, Cecilia; Olivares, Conchi; Castejón-Griñán, Maria; Abrisqueta, Marta; Jiménez-Cervantes, Celia; García-Borrón, José Carlos

    2015-01-01

    The melanocortin 1 receptor gene (MC1R) expressed in melanocytes is a major determinant of skin pigmentation. It encodes a Gs protein-coupled receptor activated by α-melanocyte stimulating hormone (αMSH). Human MC1R has an inefficient poly(A) site allowing intergenic splicing with its downstream neighbour Tubulin-β-III (TUBB3). Intergenic splicing produces two MC1R isoforms, designated Iso1 and Iso2, bearing the complete seven transmembrane helices from MC1R fused to TUBB3-derived C-terminal extensions, in-frame for Iso1 and out-of-frame for Iso2. It has been reported that exposure to ultraviolet radiation (UVR) might promote an isoform switch from canonical MC1R (MC1R-001) to the MC1R-TUBB3 chimeras, which might lead to novel phenotypes required for tanning. We expressed the Flag epitope-tagged intergenic isoforms in heterologous HEK293T cells and human melanoma cells, for functional characterization. Iso1 was expressed with the expected size. Iso2 yielded a doublet of Mr significantly lower than predicted, and impaired intracellular stability. Although Iso1- and Iso2 bound radiolabelled agonist with the same affinity as MC1R-001, their plasma membrane expression was strongly reduced. Decreased surface expression mostly resulted from aberrant forward trafficking, rather than high rates of endocytosis. Functional coupling of both isoforms to cAMP was lower than wild-type, but ERK activation upon binding of αMSH was unimpaired, suggesting imbalanced signaling from the splice variants. Heterodimerization of differentially labelled MC1R-001 with the splicing isoforms analyzed by co-immunoprecipitation was efficient and caused decreased surface expression of binding sites. Thus, UVR-induced MC1R isoforms might contribute to fine-tune the tanning response by modulating MC1R-001 availability and functional parameters.

  8. Functional Characterization of MC1R-TUBB3 Intergenic Splice Variants of the Human Melanocortin 1 Receptor.

    Directory of Open Access Journals (Sweden)

    Cecilia Herraiz

    Full Text Available The melanocortin 1 receptor gene (MC1R expressed in melanocytes is a major determinant of skin pigmentation. It encodes a Gs protein-coupled receptor activated by α-melanocyte stimulating hormone (αMSH. Human MC1R has an inefficient poly(A site allowing intergenic splicing with its downstream neighbour Tubulin-β-III (TUBB3. Intergenic splicing produces two MC1R isoforms, designated Iso1 and Iso2, bearing the complete seven transmembrane helices from MC1R fused to TUBB3-derived C-terminal extensions, in-frame for Iso1 and out-of-frame for Iso2. It has been reported that exposure to ultraviolet radiation (UVR might promote an isoform switch from canonical MC1R (MC1R-001 to the MC1R-TUBB3 chimeras, which might lead to novel phenotypes required for tanning. We expressed the Flag epitope-tagged intergenic isoforms in heterologous HEK293T cells and human melanoma cells, for functional characterization. Iso1 was expressed with the expected size. Iso2 yielded a doublet of Mr significantly lower than predicted, and impaired intracellular stability. Although Iso1- and Iso2 bound radiolabelled agonist with the same affinity as MC1R-001, their plasma membrane expression was strongly reduced. Decreased surface expression mostly resulted from aberrant forward trafficking, rather than high rates of endocytosis. Functional coupling of both isoforms to cAMP was lower than wild-type, but ERK activation upon binding of αMSH was unimpaired, suggesting imbalanced signaling from the splice variants. Heterodimerization of differentially labelled MC1R-001 with the splicing isoforms analyzed by co-immunoprecipitation was efficient and caused decreased surface expression of binding sites. Thus, UVR-induced MC1R isoforms might contribute to fine-tune the tanning response by modulating MC1R-001 availability and functional parameters.

  9. Differential effects of exercise on brain opioid receptor binding and activation in rats.

    Science.gov (United States)

    Arida, Ricardo Mario; Gomes da Silva, Sérgio; de Almeida, Alexandre Aparecido; Cavalheiro, Esper Abrão; Zavala-Tecuapetla, Cecilia; Brand, Serge; Rocha, Luisa

    2015-01-01

    Physical exercise stimulates the release of endogenous opioid peptides supposed to be responsible for changes in mood, anxiety, and performance. Exercise alters sensitivity to these effects that modify the efficacy at the opioid receptor. Although there is evidence that relates exercise to neuropeptide expression in the brain, the effects of exercise on opioid receptor binding and signal transduction mechanisms downstream of these receptors have not been explored. Here, we characterized the binding and G protein activation of mu opioid receptor, kappa opioid receptor or delta opioid receptor in several brain regions following acute (7 days) and chronic (30 days) exercise. As regards short- (acute) or long-term effects (chronic) of exercise, overall, higher opioid receptor binding was observed in acute-exercise animals and the opposite was found in the chronic-exercise animals. The binding of [(35) S]GTPγS under basal conditions (absence of agonists) was elevated in sensorimotor cortex and hippocampus, an effect more evident after chronic exercise. Divergence of findings was observed for mu opioid receptor, kappa opioid receptor, and delta opioid receptor receptor activation in our study. Our results support existing evidence of opioid receptor binding and G protein activation occurring differentially in brain regions in response to diverse exercise stimuli. We characterized the binding and G protein activation of mu, kappa, and delta opioid receptors in several brain regions following acute (7 days) and chronic (30 days) exercise. Higher opioid receptor binding was observed in the acute exercise animal group and opposite findings in the chronic exercise group. Higher G protein activation under basal conditions was noted in rats submitted to chronic exercise, as visible in the depicted pseudo-color autoradiograms. © 2014 International Society for Neurochemistry.

  10. Analysis of the epidermal growth factor receptor specific transcriptome: effect of receptor expression level and an activating mutation.

    Science.gov (United States)

    Pedersen, Mikkel W; Pedersen, Nina; Damstrup, Lars; Villingshøj, Mette; Sønder, Søren U; Rieneck, Klaus; Bovin, Lone F; Spang-Thomsen, Mogens; Poulsen, Hans S

    2005-10-01

    Overexpression or expression of activating mutations of the epidermal growth factor receptor (EGFR) is common in cancer and correlates with neoplastic progression. The present study employed Affymetrix oligonucleotide arrays to profile genes induced by ligand-activated EGFR with the receptor either moderately expressed or overexpressed at an in-itself transforming level. These changes were compared to those induced by the naturally occurring constitutively active variant EGFRvIII. This study provides novel insight on the activities and mechanisms of EGFRvIII and EGFR mediated transformation, as genes encoding proteins with functions in promoting cell proliferation, invasion, antiapoptosis, and angiogenesis featured prominently in the EGFRvIII- and EGFR-expressing cells. Surprisingly, it was found that ligand-activated EGFR induced the expression of a large group of genes known to be inducible by interferons. Expression of this module was absent in the EGFRvIII-expressing cell line and the parental cell line. Treatment with the specific EGFR inhibitor AG1478 indicated that the regulations were primary, receptor-mediated events. Furthermore, activation of this module correlated with activation of STAT1 and STAT3. The results thus demonstrate that ligand-activated EGFR at different expression levels results in different kinetics of signaling and induction of gene expression. In addition, the constitutively active variant EGFRvIII seems to activate only a subset of signal pathways and induce a subset of genes as compared to the ligand-activated EGFR. Copyright (c) 2005 Wiley-Liss, Inc.

  11. Development of molecular imaging method for manitoring estrogen receptor activity

    Energy Technology Data Exchange (ETDEWEB)

    Jung, W. S.; Jung, J. G.; Kang, J. H.; Lee, Y. J.; Kim, K. I.; O, H. J.; Jung, J. M.; Lee, D. S.; Lee, M. C. [Seoul Nation University, Seoul (Korea, Republic of)

    2004-07-01

    Estrogen receptor is expressed in 50-60% of the breast cancer and hormone therapy is effective for only ER-positive breast cancer. Therefore, we need to know whether or not the ER is expressed in breast cancer before hormone therapy. So far, the method for monitoring ER positiveness in breast tissue is radioreceptor assay or immunohistochemistry which is invasive method due to tissue biopsy. In this study, we develop the molecular imaging method of sodium iodide symporter (NIS) gene as a reporter gene for monitoring ER activity. Because molecular imaging is evaluation method through the comparison between the image intensities obtained in vivo, molecular imaging method is noninvasive and easily quantitative. We constructed the recombinant plasmid (pERE-NIS) which NIS gene expression is controlled by estrogen response element (ERE) promoter. MCF-7, ER-expressing human breast cancer cell line, was transfected with pERE-NIS with lipofectamine (Invitrogen Co). When pERE-NIS transfected MCF-7 was treated with estradiol or tamoxifen, intracellular uptake of {sup 125}I was higher than those of non-treated. The activation of ERE by drug treatment was occurred and it was caused to expression of NIS gene. The degree of {sup 125}I uptake depend on treated drug concentration. However, in case of pERE-NIS transfected breast cancer which do not express ER, there was no response with drug treatment. Therefore, we can monitor ER functionality and the efficacy of drugs with this pERE-NIS reporter system.

  12. 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...... and pharmacological perturbations along with computational methods, and propose a kinetic model applicable to the analysis of sequential receptor activation. We envision this form of dynamic agonism as a common principle of nature to spatiotemporally encode cellular information....

  13. Receptor activity-modifying protein dependent and independent activation mechanisms in the coupling of calcitonin gene-related peptide and adrenomedullin receptors to Gs.

    Science.gov (United States)

    Woolley, Michael J; Reynolds, Christopher A; Simms, John; Walker, Christopher S; Mobarec, Juan Carlos; Garelja, Michael L; Conner, Alex C; Poyner, David R; Hay, Debbie L

    2017-10-15

    Calcitonin gene-related peptide (CGRP) or adrenomedullin (AM) receptors are heteromers of the calcitonin receptor-like receptor (CLR), a class B G protein-coupled receptor, and one of three receptor activity-modifying proteins (RAMPs). How CGRP and AM activate CLR and how this process is modulated by RAMPs is unclear. We have defined how CGRP and AM induce Gs-coupling in CLR-RAMP heteromers by measuring the effect of targeted mutagenesis in the CLR transmembrane domain on cAMP production, modeling the active state conformations of CGRP and AM receptors in complex with the Gs C-terminus and conducting molecular dynamics simulations in an explicitly hydrated lipidic bilayer. The largest effects on receptor signaling were seen with H295A5.40b, I298A5.43b, L302A5.47b, N305A5.50b, L345A6.49b and E348A6.52b, F349A6.53b and H374A7.47b (class B numbering in superscript). Many of these residues are likely to form part of a group in close proximity to the peptide binding site and link to a network of hydrophilic and hydrophobic residues, which undergo rearrangements to facilitate Gs binding. Residues closer to the extracellular loops displayed more pronounced RAMP or ligand-dependent effects. Mutation of H3747.47b to alanine increased AM potency 100-fold in the CGRP receptor. The molecular dynamics simulation showed that TM5 and TM6 pivoted around TM3. The data suggest that hydrophobic interactions are more important for CLR activation than other class B GPCRs, providing new insights into the mechanisms of activation of this class of receptor. Furthermore the data may aid in the understanding of how RAMPs modulate the signaling of other class B GPCRs. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  14. Genome-Wide Profiling of Liver X Receptor, Retinoid X Receptor, and Peroxisome Proliferator-Activated Receptor α in Mouse Liver Reveals Extensive Sharing of Binding Sites

    DEFF Research Database (Denmark)

    Boergesen, Michael; Pedersen, Thomas Åskov; Gross, Barbara

    2012-01-01

    The liver X receptors (LXRs) are nuclear receptors that form permissive heterodimers with retinoid X receptor (RXR) and are important regulators of lipid metabolism in the liver. We have recently shown that RXR agonist-induced hypertriglyceridemia and hepatic steatosis in mice are dependent on LXRs...... and correlate with an LXR-dependent hepatic induction of lipogenic genes. To further investigate the roles of RXR and LXR in the regulation of hepatic gene expression, we have mapped the ligand-regulated genome-wide binding of these factors in mouse liver. We find that the RXR agonist bexarotene primarily...... increases the genomic binding of RXR, whereas the LXR agonist T0901317 greatly increases both LXR and RXR binding. Functional annotation of putative direct LXR target genes revealed a significant association with classical LXR-regulated pathways as well as peroxisome proliferator-activated receptor (PPAR...

  15. Racial differences in B cell receptor signaling pathway activation

    Directory of Open Access Journals (Sweden)

    Longo Diane M

    2012-06-01

    Full Text Available Abstract Background Single-cell network profiling (SCNP is a multi-parametric flow cytometry-based approach that simultaneously measures basal and modulated intracellular signaling activity in multiple cell subpopulations. Previously, SCNP analysis of a broad panel of immune signaling pathways in cell subsets within PBMCs from 60 healthy donors identified a race-associated difference in B cell anti-IgD-induced PI3K pathway activity. Methods The present study extended this analysis to a broader range of signaling pathway components downstream of the B cell receptor (BCR in European Americans and African Americans using a subset of donors from the previously analyzed cohort of 60 healthy donors. Seven BCR signaling nodes (a node is defined as a paired modulator and intracellular readout were measured at multiple time points by SCNP in PBMCs from 10 healthy donors [5 African Americans (36-51 yrs, 5 European Americans (36-56 yrs, all males]. Results Analysis of BCR signaling activity in European American and African American PBMC samples revealed that, compared to the European American donors, B cells from African Americans had lower anti-IgD induced phosphorylation of multiple BCR pathway components, including the membrane proximal proteins Syk and SFK as well as proteins in the PI3K pathway (S6 and Akt, the MAPK pathways (Erk and p38, and the NF-κB pathway (NF-κB. In addition to differences in the magnitude of anti-IgD-induced pathway activation, racial differences in BCR signaling kinetic profiles were observed. Further, the frequency of IgD+ B cells differed by race and strongly correlated with BCR pathway activation. Thus, the race-related difference in BCR pathway activation appears to be attributable at least in part to a race-associated difference in IgD+ B cell frequencies. Conclusions SCNP analysis enabled the identification of statistically significant race-associated differences in BCR pathway activation within PBMC samples from

  16. Racial differences in B cell receptor signaling pathway activation.

    Science.gov (United States)

    Longo, Diane M; Louie, Brent; Mathi, Kavita; Pos, Zoltan; Wang, Ena; Hawtin, Rachael E; Marincola, Francesco M; Cesano, Alessandra

    2012-06-06

    Single-cell network profiling (SCNP) is a multi-parametric flow cytometry-based approach that simultaneously measures basal and modulated intracellular signaling activity in multiple cell subpopulations. Previously, SCNP analysis of a broad panel of immune signaling pathways in cell subsets within PBMCs from 60 healthy donors identified a race-associated difference in B cell anti-IgD-induced PI3K pathway activity. The present study extended this analysis to a broader range of signaling pathway components downstream of the B cell receptor (BCR) in European Americans and African Americans using a subset of donors from the previously analyzed cohort of 60 healthy donors. Seven BCR signaling nodes (a node is defined as a paired modulator and intracellular readout) were measured at multiple time points by SCNP in PBMCs from 10 healthy donors [5 African Americans (36-51 yrs), 5 European Americans (36-56 yrs), all males]. Analysis of BCR signaling activity in European American and African American PBMC samples revealed that, compared to the European American donors, B cells from African Americans had lower anti-IgD induced phosphorylation of multiple BCR pathway components, including the membrane proximal proteins Syk and SFK as well as proteins in the PI3K pathway (S6 and Akt), the MAPK pathways (Erk and p38), and the NF-κB pathway (NF-κB). In addition to differences in the magnitude of anti-IgD-induced pathway activation, racial differences in BCR signaling kinetic profiles were observed. Further, the frequency of IgD+ B cells differed by race and strongly correlated with BCR pathway activation. Thus, the race-related difference in BCR pathway activation appears to be attributable at least in part to a race-associated difference in IgD+ B cell frequencies. SCNP analysis enabled the identification of statistically significant race-associated differences in BCR pathway activation within PBMC samples from healthy donors. Understanding race-associated contrasts in immune

  17. Molecular Vibration-Activity Relationship in the Agonism of Adenosine Receptors

    Directory of Open Access Journals (Sweden)

    Hyun Keun Chee

    2013-12-01

    Full Text Available The molecular vibration-activity relationship in the receptor-ligand interaction of adenosine receptors was investigated by structure similarity, molecular vibration, and hierarchical clustering in a dataset of 46 ligands of adenosine receptors. The resulting dendrogram was compared with those of another kind of fingerprint or descriptor. The dendrogram result produced by corralled intensity of molecular vibrational frequency outperformed four other analyses in the current study of adenosine receptor agonism and antagonism. The tree that was produced by clustering analysis of molecular vibration patterns showed its potential for the functional classification of adenosine receptor ligands.

  18. Activation of Cannabinoid Receptor 2 Enhances Osteogenic Differentiation of Bone Marrow Derived Mesenchymal Stem Cells

    Directory of Open Access Journals (Sweden)

    Yong-Xin Sun

    2015-01-01

    Full Text Available Bone marrow derived mesenchymal stem cells (BM-MSCs are considered as the most promising cells source for bone engineering. Cannabinoid (CB receptors play important roles in bone mass turnover. The aim of this study is to test if activation of CB2 receptor by chemical agonist could enhance the osteogenic differentiation and mineralization in bone BM-MSCs. Alkaline phosphatase (ALP activity staining and real time PCR were performed to test the osteogenic differentiation. Alizarin red staining was carried out to examine the mineralization. Small interference RNA (siRNA was used to study the role of CB2 receptor in osteogenic differentiation. Results showed activation of CB2 receptor increased ALP activity, promoted expression of osteogenic genes, and enhanced deposition of calcium in extracellular matrix. Knockdown of CB2 receptor by siRNA inhibited ALP activity and mineralization. Results of immunofluorescent staining showed that phosphorylation of p38 MAP kinase is reduced by knocking down of CB2 receptor. Finally, bone marrow samples demonstrated that expression of CB2 receptor is much lower in osteoporotic patients than in healthy donors. Taken together, data from this study suggested that activation of CB2 receptor plays important role in osteogenic differentiation of BM-MSCs. Lack of CB2 receptor may be related to osteoporosis.

  19. Peroxisome proliferator-activated receptor ɣ activation induces 11β-hydroxysteroid dehydrogenase type 1 activity in human alternative macrophages

    Science.gov (United States)

    Chinetti-Gbaguidi, Giulia; Bouhlel, Mohamed Amine; Copin, Corinne; Duhem, Christian; Derudas, Bruno; Neve, Bernardette; Noel, Benoit; Eeckhoute, Jerome; Lefebvre, Philippe; Seckl, Jonathan R.; Staels, Bart

    2012-01-01

    Objectives 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) catalyses the intracellular reduction of inactive cortisone to active cortisol, the natural ligand activating the glucocorticoid receptor (GR). Peroxisome Proliferator-Activated Receptor gamma (PPARγ) is a nuclear receptor controlling inflammation, lipid metabolism and the macrophage polarization state. In this study, we investigated the impact of macrophage polarization on the expression and activity of 11β-HSD1 and the role of PPAR therein. Methods and Results 11β-HSD1 gene expression is higher in pro-inflammatory M1 and anti-inflammatory M2 macrophages than in resting macrophages (RM), whereas its activity is highest in M2 macrophages. Interestingly, PPARγ activation induces 11β-HSD1 enzyme activity in M2 macrophages, but not in RM or M1 macrophages. Consequently, human M2 macrophages displayed enhanced responsiveness to the 11β-HSD1 substrate cortisone, an effect amplified by PPAR -induction of 11β-HSD1 activity, as illustrated by an increased expression of GR target genes. Conclusions Our data identify a positive cross-talk between PPARγ and GR in human M2 macrophages via the induction of 11β-HSD1 expression and activity. PMID:22207732

  20. Urokinase plasminogen activator cleaves its cell surface receptor releasing the ligand-binding domain

    DEFF Research Database (Denmark)

    Høyer-Hansen, G; Rønne, E; Solberg, H.

    1992-01-01

    The cellular receptor for urokinase-type plasminogen activator (uPAR) is a glycolipid-anchored three-domain membrane protein playing a central role in pericellular plasminogen activation. We have found that urokinase (uPA) can cleave its receptor between domains 1 and 2 generating a cell-associat...

  1. Activation of toll-like receptors and dendritic cells by a broad range of bacterial molecules

    NARCIS (Netherlands)

    Boele, L.C.L.; Bajramovic, J.J.; Vries, A.M.M.B.C. de; Voskamp-Visser, I.A.I.; Kaman, W.E.; Kleij, D. van der

    2009-01-01

    Activation of pattern recognition receptors such as Toll-like receptors (TLRs) by pathogens leads to activation and maturation of dendritic cells (DC), which orchestrate the development of the adaptive immune response. To create an overview of the effects of a broad range of pathogenic bacteria,

  2. Antileishmanial activity of the estrogen receptor modulator raloxifene.

    Directory of Open Access Journals (Sweden)

    Juliana Q Reimão

    2014-05-01

    Full Text Available BACKGROUND: The treatment of leishmaniasis relies mostly on parenteral drugs with potentially serious adverse effects. Additionally, parasite resistance in the treatment of leishmaniasis has been demonstrated for the majority of drugs available, making the search for more effective and less toxic drugs and treatment regimens a priority for the control of leishmaniasis. The aims of this study were to evaluate the antileishmanial activity of raloxifene in vitro and in vivo and to investigate its mechanism of action against Leishmania amazonensis. METHODOLOGY/PRINCIPAL FINDINGS: Raloxifene was shown to possess antileishmanial activity in vitro against several species with EC50 values ranging from 30.2 to 38.0 µM against promastigotes and from 8.8 to 16.2 µM against intracellular amastigotes. Raloxifene's mechanism of action was investigated through transmission electron microscopy and labeling with propidium iodide, DiSBAC2(3, rhodamine 123 and monodansylcadaverine. Microscopic examinations showed that raloxifene treated parasites displayed autophagosomes and mitochondrial damage while the plasma membrane remained continuous. Nonetheless, plasma membrane potential was rapidly altered upon raloxifene treatment with initial hyperpolarization followed by depolarization. Loss of mitochondrial membrane potential was also verified. Treatment of L. amazonensis-infected BALB/c mice with raloxifene led to significant decrease in lesion size and parasite burden. CONCLUSIONS/SIGNIFICANCE: The results of this work extend the investigation of selective estrogen receptor modulators as potential candidates for leishmaniasis treatment. The antileishmanial activity of raloxifene was demonstrated in vitro and in vivo. Raloxifene produces functional disorder on the plasma membrane of L. amazonensis promastigotes and leads to functional and morphological disruption of mitochondria, which culminate in cell death.

  3. Antileishmanial Activity of the Estrogen Receptor Modulator Raloxifene

    Science.gov (United States)

    Reimão, Juliana Q.; Miguel, Danilo C.; Taniwaki, Noemi N.; Trinconi, Cristiana T.; Yokoyama-Yasunaka, Jenicer K. U.; Uliana, Silvia R. B.

    2014-01-01

    Background The treatment of leishmaniasis relies mostly on parenteral drugs with potentially serious adverse effects. Additionally, parasite resistance in the treatment of leishmaniasis has been demonstrated for the majority of drugs available, making the search for more effective and less toxic drugs and treatment regimens a priority for the control of leishmaniasis. The aims of this study were to evaluate the antileishmanial activity of raloxifene in vitro and in vivo and to investigate its mechanism of action against Leishmania amazonensis. Methodology/Principal Findings Raloxifene was shown to possess antileishmanial activity in vitro against several species with EC50 values ranging from 30.2 to 38.0 µM against promastigotes and from 8.8 to 16.2 µM against intracellular amastigotes. Raloxifene's mechanism of action was investigated through transmission electron microscopy and labeling with propidium iodide, DiSBAC2(3), rhodamine 123 and monodansylcadaverine. Microscopic examinations showed that raloxifene treated parasites displayed autophagosomes and mitochondrial damage while the plasma membrane remained continuous. Nonetheless, plasma membrane potential was rapidly altered upon raloxifene treatment with initial hyperpolarization followed by depolarization. Loss of mitochondrial membrane potential was also verified. Treatment of L. amazonensis – infected BALB/c mice with raloxifene led to significant decrease in lesion size and parasite burden. Conclusions/Significance The results of this work extend the investigation of selective estrogen receptor modulators as potential candidates for leishmaniasis treatment. The antileishmanial activity of raloxifene was demonstrated in vitro and in vivo. Raloxifene produces functional disorder on the plasma membrane of L. amazonensis promastigotes and leads to functional and morphological disruption of mitochondria, which culminate in cell death. PMID:24810565

  4. Activation of intracellular angiotensin AT2 receptors induces rapid cell death in human uterine leiomyosarcoma cells

    DEFF Research Database (Denmark)

    Zhao, Yi; Lützen, Ulf; Fritsch, Jürgen

    2015-01-01

    of apoptosis and cell death in cultured human uterine leiomyosarcoma (SK-UT-1) cells and control human uterine smooth muscle cells (HutSMC). The intracellular levels of the AT2 receptor are low in proliferating SK-UT-1 cells but the receptor is substantially up-regulated in quiescent SK-UT-1 cells with high...... densities in mitochondria. Activation of the cell membrane AT2 receptors by a concomitant treatment with angiotensin II and the AT1 receptor antagonist, losartan, induces apoptosis but does not affect the rate of cell death. We demonstrate for the first time that the high-affinity, non-peptide AT2 receptor...... agonist, Compound 21 (C21) penetrates the cell membrane of quiescent SK-UT-1 cells, activates intracellular AT2 receptors and induces rapid cell death; approximately 70% of cells died within 24 h. The cells, which escaped from the cell death, displayed activation of the mitochondrial apoptotic pathway, i...

  5. Structural basis for androgen receptor interdomain and coactivator interactions suggests a transition in nuclear receptor activation function dominance.

    Science.gov (United States)

    He, Bin; Gampe, Robert T; Kole, Adam J; Hnat, Andrew T; Stanley, Thomas B; An, Gang; Stewart, Eugene L; Kalman, Rebecca I; Minges, John T; Wilson, Elizabeth M

    2004-11-05

    The androgen receptor (AR) is required for male sex development and contributes to prostate cancer cell survival. In contrast to other nuclear receptors that bind the LXXLL motifs of coactivators, the AR ligand binding domain is preferentially engaged in an interdomain interaction with the AR FXXLF motif. Reported here are crystal structures of the ligand-activated AR ligand binding domain with and without bound FXXLF and LXXLL peptides. Key residues that establish motif binding specificity are identified through comparative structure-function and mutagenesis studies. A mechanism in prostate cancer is suggested by a functional AR mutation at a specificity-determining residue that recovers coactivator LXXLL motif binding. An activation function transition hypothesis is proposed in which an evolutionary decline in LXXLL motif binding parallels expansion and functional dominance of the NH(2)-terminal transactivation domain in the steroid receptor subfamily.

  6. Biological Activities of Recombinant Liver X Receptor â- Ligand ...

    African Journals Online (AJOL)

    Purpose: To investigate tetracycline-inducible expression system for producing clinically usable, highquality liver X receptor ligand-binding domain recombinant protein. Methods: In this study, we have expressed and purified the recombinant liver X receptor β-ligand binding domain proteins in E. coli using a tetracycline ...

  7. A bacterial tyrosine phosphatase inhibits plant pattern recognition receptor activation

    Science.gov (United States)

    Perception of pathogen-associated molecular patterns (PAMPs) by surface-localised pattern-recognition receptors (PRRs) is a key component of plant innate immunity. Most known plant PRRs are receptor kinases and initiation of PAMP-triggered immunity (PTI) signalling requires phosphorylation of the PR...

  8. Biological Activities of Recombinant Liver X Receptor β- Ligand ...

    African Journals Online (AJOL)

    Purpose: To investigate tetracycline-inducible expression system for producing clinically usable, high- quality liver X receptor ligand-binding domain recombinant protein. Methods: In this study, we have expressed and purified the recombinant liver X receptor β-ligand binding domain proteins in E. coli using a tetracycline ...

  9. Estrogen Receptor β Activation Rapidly Modulates Male Sexual Motivation through the Transactivation of Metabotropic Glutamate Receptor 1a

    Science.gov (United States)

    Seredynski, Aurore L.; Balthazart, Jacques; Ball, Gregory F.

    2015-01-01

    In addition to the transcriptional activity of their liganded nuclear receptors, estrogens, such as estradiol (E2), modulate cell functions, and consequently physiology and behavior, within minutes through membrane-initiated events. The membrane-associated receptors (mERs) underlying the acute effects of estrogens on behavior have mostly been documented in females where active estrogens are thought to be of ovarian origin. We determined here, by acute intracerebroventricular injections of specific agonists and antagonists, the type(s) of mERs that modulate rapid effects of brain-derived estrogens on sexual motivation in male Japanese quail. Brain aromatase blockade acutely inhibited sexual motivation. Diarylpropionitrile (DPN), an estrogen receptor β (ERβ)-specific agonist, and to a lesser extent 17α-estradiol, possibly acting through ER-X, prevented this effect. In contrast, drugs targeting ERα (PPT and MPP), GPR30 (G1 and G15), and the Gq-mER (STX) did not affect sexual motivation. The mGluR1a antagonist LY367385 significantly inhibited sexual motivation but mGluR2/3 and mGluR5 antagonists were ineffective. LY367385 also blocked the behavioral restoration induced by E2 or DPN, providing functional evidence that ERβ interacts with metabotropic glutamate receptor 1a (mGluR1a) signaling to acutely regulate male sexual motivation. Together these results show that ERβ plays a key role in sexual behavior regulation and the recently uncovered cooperation between mERs and mGluRs is functional in males where it mediates the acute effects of estrogens produced centrally in response to social stimuli. The presence of an ER–mGluR interaction in birds suggests that this mechanism emerged relatively early in vertebrate history and is well conserved. SIGNIFICANCE STATEMENT The membrane-associated receptors underlying the acute effects of estrogens on behavior have mostly been documented in females, where active estrogens are thought to be of ovarian origin. Using acute

  10. Expression of Novel Steroid/Receptors in Mammary Development: Peroxisome Proliferator Activated Receptors

    National Research Council Canada - National Science Library

    Gimble, Jeffrey

    1999-01-01

    ...) are expressed in the mammary gland and regulated during physiologic and pathologic events. The PPARs are nuclear hormone receptors which bind to fatty acids as ligands and control transcription of lipid metabolic genes...

  11. SuperBiHelix method for predicting the pleiotropic ensemble of G-protein-coupled receptor conformations.

    Science.gov (United States)

    Bray, Jenelle K; Abrol, Ravinder; Goddard, William A; Trzaskowski, Bartosz; Scott, Caitlin E

    2014-01-07

    There is overwhelming evidence that G-protein-coupled receptors (GPCRs) exhibit several distinct low-energy conformations, each of which might favor binding to different ligands and/or lead to different downstream functions. Understanding the function of such proteins requires knowledge of the ensemble of low-energy configurations that might play a role in this pleiotropic functionality. We earlier reported the BiHelix method for efficiently sampling the (12)(7) = 35 million conformations resulting from 30° rotations about the axis (η) of all seven transmembrane helices (TMHs), showing that the experimental structure is reliably selected as the best conformation from this ensemble. However, various GPCRs differ sufficiently in the tilts of the TMHs that this method need not predict the optimum conformation starting from any other template. In this paper, we introduce the SuperBiHelix method in which the tilt angles (θ, ϕ) are optimized simultaneously with rotations (η) efficiently enough that it is practical and sufficient to sample (5 × 3 × 5)(7) = 13 trillion configurations. This method can correctly identify the optimum structure of a GPCR starting with the template from a different GPCR. We have validated this method by predicting known crystal structure conformations starting from the template of a different protein structure. We find that the SuperBiHelix conformational ensemble includes the higher energy conformations associated with the active protein in addition to those associated with the more stable inactive protein. This methodology was then applied to design and experimentally confirm structures of three mutants of the CB1 cannabinoid receptor associated with different functions.

  12. Electroacupuncture inhibits NLRP3 inflammasome activation through CB2 receptors in inflammatory pain.

    Science.gov (United States)

    Gao, Fang; Xiang, Hong-Chun; Li, Hong-Ping; Jia, Min; Pan, Xiao-Li; Pan, Hui-Lin; Li, Man

    2018-01-01

    The therapeutic effect of electroacupuncture (EA) on inflammatory pain has been well recognized clinically. The inflammasome promotes the maturation of the inflammatory cytokines, and EA can stimulate cannabinoid CB2 receptors in inflamed tissues. In this study we investigated whether EA inhibits NLRP3 inflammasome activation through CB2 receptors and thus relieving inflammatory pain. Assay of Caspase-1 activity and western blotting revealed that complete Freund's adjuvant (CFA) injection activated the NLRP3 inflammasome in the skin tissue in rats, which was attenuated by EA treatment. Immunofluorescence labeling showed that NLRP3 inflammasome elicited by CFA in the skin macrophages were decreased by EA. Nociceptive behavioral tests demonstrated that in CB2 receptor knockout mice, the EA effects on NLRP3 inflammasomes were largely attenuated. In addition, in vitro studies in a macrophage cell line showed that CB2 receptor stimulation inhibited the NLRP3 inflammasome activation. Thus, our results suggest a novel signaling pathway through which CB2 receptors are involved in the analgesic effect of EA on inflammatory pain. Stimulation of CB2 receptors inhibits NLRP3 inflammasome activation in inflamed skin tissues. These results suggest that EA reduces the inflammatory pain by inhibiting the activation of NLRP3 inflammasome through CB2 receptors. Our findings provide novel information about the mechanisms through which EA and CB2 receptor activation reduce inflammatory pain. Copyright © 2017 Elsevier Inc. All rights reserved.

  13. 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.

  14. Molecular cloning and characterization of a nuclear androgen receptor activated by 11-ketotestosterone

    Directory of Open Access Journals (Sweden)

    Karlsson Johnny

    2005-08-01

    Full Text Available Abstract Although 11-ketotestosterone is a potent androgen and induces male secondary sex characteristics in many teleosts, androgen receptors with high binding affinity for 11-ketotestosterone or preferential activation by 11-ketotestosterone have not been identified. So, the mechanism by which 11-ketotestosterone exhibits such high potency remains unclear. Recently we cloned the cDNA of an 11-ketotestosterone regulated protein, spiggin, from three-spined stickleback renal tissue. As spiggin is the only identified gene product regulated by 11-ketotestosterone, the stickleback kidney is ideal for determination of the mechanism of 11-ketotestosterone gene regulation. A single androgen receptor gene with two splicing variants, belonging to the androgen receptor-β subfamily was cloned from stickleback kidney. A high affinity, saturable, single class of androgen specific binding sites, with the characteristics of an androgen receptor, was identified in renal cytosolic and nuclear fractions. Measurement of ligand binding moieties in the cytosolic and nuclear fractions as well as to the recombinant receptor revealed lower affinity for 11-ketotestosterone than for dihydrotestosterone. Treatment with different androgens did not up-regulate androgen receptor mRNA level or increase receptor abundance, suggesting that auto-regulation is not involved in differential ligand activation. However, comparison of the trans-activation potential of the stickleback androgen receptor with the human androgen receptor, in both human HepG2 cells and zebrafish ZFL cells, revealed preferential activation by 11-ketotestosterone of the stickleback receptor, but not of the human receptor. These findings demonstrate the presence of a receptor preferentially activated by 11-ketotestosterone in the three-spined stickleback, so far the only one known in any animal.

  15. Amino acid residues contributing to function of the heteromeric insect olfactory receptor complex.

    Directory of Open Access Journals (Sweden)

    Tatsuro Nakagawa

    Full Text Available Olfactory receptors (Ors convert chemical signals--the binding of odors and pheromones--to electrical signals through the depolarization of olfactory sensory neurons. Vertebrates Ors are G-protein-coupled receptors, stimulated by odors to produce intracellular second messengers that gate ion channels. Insect Ors are a heteromultimeric complex of unknown stoichiometry of two seven transmembrane domain proteins with no sequence similarity to and the opposite membrane topology of G-protein-coupled receptors. The functional insect Or comprises an odor- or pheromone-specific Or subunit and the Orco co-receptor, which is highly conserved in all insect species. The insect Or-Orco complex has been proposed to function as a novel type of ligand-gated nonselective cation channel possibly modulated by G-proteins. However, the Or-Orco proteins lack homology to any known family of ion channel and lack known functional domains. Therefore, the mechanisms by which odors activate the Or-Orco complex and how ions permeate this complex remain unknown. To begin to address the relationship between Or-Orco structure and function, we performed site-directed mutagenesis of all 83 conserved Glu, Asp, or Tyr residues in the silkmoth BmOr-1-Orco pheromone receptor complex and measured functional properties of mutant channels expressed in Xenopus oocytes. 13 of 83 mutations in BmOr-1 and BmOrco altered the reversal potential and rectification index of the BmOr-1-Orco complex. Three of the 13 amino acids (D299 and E356 in BmOr-1 and Y464 in BmOrco altered both current-voltage relationships and K(+ selectivity. We introduced the homologous Orco Y464 residue into Drosophila Orco in vivo, and observed variable effects on spontaneous and evoked action potentials in olfactory neurons that depended on the particular Or-Orco complex examined. Our results provide evidence that a subset of conserved Glu, Asp and Tyr residues in both subunits are essential for channel activity of the

  16. Comparative study on transcriptional activity of 17 parabens mediated by estrogen receptor α and β and androgen receptor.

    Science.gov (United States)

    Watanabe, Yoko; Kojima, Hiroyuki; Takeuchi, Shinji; Uramaru, Naoto; Ohta, Shigeru; Kitamura, Shigeyuki

    2013-07-01

    The structure-activity relationships of parabens which are widely used as preservatives for transcriptional activities mediated by human estrogen receptor α (hERα), hERβ and androgen receptor (hAR) were investigated. Fourteen of 17 parabens exhibited hERα and/or hERβ agonistic activity at concentrations of ≤ 1 × 10(-5)M, whereas none of the 17 parabens showed AR agonistic or antagonistic activity. Among 12 parabens with linear alkyl chains ranging in length from C₁ to C₁₂, heptylparaben (C₇) and pentylparaben (C₅) showed the most potent ERα and ERβ agonistic activity in the order of 10(-7)M and 10(-8)M, respectively, and the activities decreased in a stepwise manner as the alkyl chain was shortened to C₁ or lengthened to C₁₂. Most parabens showing estrogenic activity exhibited ERβ-agonistic activity at lower concentrations than those inducing ERα-agonistic activity. The estrogenic activity of butylparaben was markedly decreased by incubation with rat liver microsomes, and the decrease of activity was blocked by a carboxylesterase inhibitor. These results indicate that parabens are selective agonists for ERβ over ERα; their interactions with ERα/β are dependent on the size and bulkiness of the alkyl groups; and they are metabolized by carboxylesterases, leading to attenuation of their estrogenic activity. Copyright © 2013 Elsevier Ltd. All rights reserved.

  17. Phospholipase C activity affinity purifies with the Torpedo nicotinic acetylcholine receptor.

    Science.gov (United States)

    Labriola, Jonathan M; daCosta, Corrie J B; Wang, Shuzhi; Figeys, Daniel; Smith, Jeffrey C; Sturgeon, R Michel; Baenziger, John E

    2010-04-02

    Nicotinic acetylcholine receptors mediate fast synaptic transmission by fluxing ions across the membrane in response to neurotransmitter binding. We show here that during affinity purification of the nicotinic acetylcholine receptor from Torpedo, phosphatidic acid, but not other anionic or zwitterionic phospholipids, is hydrolyzed to diacylglycerol. The phospholipase C activity elutes with the acetylcholine receptor and is inhibited by a lipid phosphate phosphohydrolase inhibitor, sodium vanadate, but not a phosphatidate phosphohydrolase inhibitor, N-ethylmaleimide. Further, the hydrolysis product of phosphatidic acid, diacylglycerol, enhances the functional capabilities of the acetylcholine receptor in the presence of anionic lipids. We conclude that a phospholipase C activity, which appears to be specific for phosphatidic acid, is associated with the nicotinic acetylcholine receptor. The acetylcholine receptor may directly or indirectly influence lipid metabolism in a manner that enhances its own function.

  18. The Farnesoid X Receptor Regulates Adipocyte Differentiation and Function by Promoting Peroxisome Proliferator-activated Receptor-gamma and Interfering with the Wnt/beta-Catenin Pathways

    NARCIS (Netherlands)

    Abdelkarim, Mouaadh; Caron, Sandrine; Duhem, Christian; Prawitt, Janne; Dumont, Julie; Lucas, Anthony; Bouchaert, Emmanuel; Briand, Olivier; Brozek, John; Kuipers, Folkert; Fievet, Catherine; Cariou, Bertrand; Staels, Bart

    2010-01-01

    The bile acid receptor farnesoid X receptor (FXR) is expressed in adipose tissue, but its function remains poorly defined. Peroxisome proliferator-activated receptor-gamma (PPAR gamma) is a master regulator of adipocyte differentiation and function. The aim of this study was to analyze the role of

  19. Quantitative impedimetric NPY-receptor activation monitoring and signal pathway profiling in living cells.

    Science.gov (United States)

    te Kamp, Verena; Lindner, Ricco; Jahnke, Heinz-Georg; Krinke, Dana; Kostelnik, Katja B; Beck-Sickinger, Annette G; Robitzki, Andrea A

    2015-05-15

    Label-free and non-invasive monitoring of receptor activation and identification of the involved signal pathways in living cells is an ongoing analytic challenge and a great opportunity for biosensoric systems. In this context, we developed an impedance spectroscopy-based system for the activation monitoring of NPY-receptors in living cells. Using an optimized interdigital electrode array for sensitive detection of cellular alterations, we were able for the first time to quantitatively detect the NPY-receptor activation directly without a secondary or enhancer reaction like cAMP-stimulation by forskolin. More strikingly, we could show that the impedimetric based NPY-receptor activation monitoring is not restricted to the Y1-receptor but also possible for the Y2- and Y5-receptor. Furthermore, we could monitor the NPY-receptor activation in different cell lines that natively express NPY-receptors and proof the specificity of the observed impedimetric effect by agonist/antagonist studies in recombinant NPY-receptor expressing cell lines. To clarify the nature of the observed impedimetric effect we performed an equivalent circuit analysis as well as analyzed the role of cell morphology and receptor internalization. Finally, an antagonist based extensive molecular signal pathway analysis revealed small alterations of the actin cytoskeleton as well as the inhibition of at least L-type calcium channels as major reasons for the observed NPY-induced impedance increase. Taken together, our novel impedance spectroscopy based NPY-receptor activation monitoring system offers the opportunity to identify signal pathways as well as for novel versatile agonist/antagonist screening systems for identification of novel therapeutics in the field of obesity and cancer. Copyright © 2014 Elsevier B.V. All rights reserved.

  20. SHP-1 phosphatase activity counteracts increased T cell receptor affinity

    National Research Council Canada - National Science Library

    Hebeisen, Michael; Baitsch, Lukas; Presotto, Danilo; Baumgaertner, Petra; Romero, Pedro; Michielin, Olivier; Speiser, Daniel E; Rufer, Nathalie

    2013-01-01

    .... Preferential expression of the inhibitory receptor programmed cell death-1 (PD-1) was limited to T cells with the highest TCR affinity, correlating with full functional recovery upon PD-1 ligand 1 (PD-L1) blockade...

  1. Isolated dorsal root ganglion neurones inhibit receptor-dependent adenylyl cyclase activity in associated glial cells

    Science.gov (United States)

    Ng, KY; Yeung, BHS; Wong, YH; Wise, H

    2013-01-01

    Background and Purpose Hyper-nociceptive PGE2 EP4 receptors and prostacyclin (IP) receptors are present in adult rat dorsal root ganglion (DRG) neurones and glial cells in culture. The present study has investigated the cell-specific expression of two other Gs-protein coupled hyper-nociceptive receptor systems: β-adrenoceptors and calcitonin gene-related peptide (CGRP) receptors in isolated DRG cells and has examined the influence of neurone–glial cell interactions in regulating adenylyl cyclase (AC) activity. Experimental Approach Agonist-stimulated AC activity was determined in mixed DRG cell cultures from adult rats and compared with activity in DRG neurone-enriched cell cultures and pure DRG glial cell cultures. Key Results Pharmacological analysis showed the presence of Gs-coupled β2-adrenoceptors and CGRP receptors, but not β1-adrenoceptors, in all three DRG cell preparations. Agonist-stimulated AC activity was weakest in DRG neurone-enriched cell cultures. DRG neurones inhibited IP receptor-stimulated glial cell AC activity by a process dependent on both cell–cell contact and neurone-derived soluble factors, but this is unlikely to involve purine or glutamine receptor activation. Conclusions and Implications Gs-coupled hyper-nociceptive receptors are readily expressed on DRG glial cells in isolated cell cultures and the activity of CGRP, EP4 and IP receptors, but not β2-adrenoceptors, in glial cells is inhibited by DRG neurones. Studies using isolated DRG cells should be aware that hyper-nociceptive ligands may stimulate receptors on glial cells in addition to neurones, and that variable numbers of neurones and glial cells will influence absolute measures of AC activity and affect downstream functional responses. PMID:22924655

  2. Activation of membrane estrogen receptors attenuates opioid receptor-like1 receptor-mediated antinociception via an ERK-dependent non-genomic mechanism.

    Science.gov (United States)

    Small, K M; Nag, S; Mokha, S S

    2013-01-01

    To our knowledge, the present data are the first to demonstrate that activation of membrane estrogen receptors (mERs) abolishes opioid receptor-like 1 (ORL1) receptor-mediated analgesia via extracellular signal-regulated kinase (ERK)-dependent non-genomic mechanisms. Estrogen was shown previously to both attenuate ORL1-mediated antinociception and down-regulate the ORL1 gene expression. The present study investigated whether non-genomic mechanisms contribute to estrogen-induced attenuation of ORL1-mediated antinociception by the mERs GPR30, Gq-coupled mER, ERα, and ERβ. E2BSA [β-estradiol-6-(O-carboxymethyl)oxime: bovine serum albumin] (0.5mM), a membrane impermeant analog of estradiol, injected intrathecally immediately prior to orphanin FQ (OFQ;10 nmol), the endogenous ligand for the ORL1 receptor, abolished OFQ's antinociceptive effect in both male and ovariectomized (OVX) female rats, assessed using the heat-induced tail-flick assay. This effect was not altered by protein synthesis inhibitor, anisomycin (125 μg), given intrathecally 15 min prior to E2BSA and OFQ. Intrathecal application of selective receptor agonists permitted the relative contributions of various estrogen receptors in mediating this blockade of the antinociceptive response of OFQ. Activation of GPR30, Gq-mER, ERα, but not ERβ abolished ORL1-mediated antinociception in males and OVX females. E2BSA produced a parallel and significant increase in the phosphorylation of ERK 2 only in OVX females, and pre-treatment with MEK/ERK 1/2 inhibitor, U0126 (10 μg), blocked the mER-mediated abolition of ORL1-mediated antinociception in OVX females. Taken together, the data are consistent with the interpretations that mER activation attenuates ORL1-mediated antinociception through a non-genomic, ERK 2-dependent mechanism in females. Copyright © 2013 IBRO. Published by Elsevier Ltd. All rights reserved.

  3. The formyl peptide receptor like-1 and scavenger receptor MARCO are involved in glial cell activation in bacterial meningitis

    Directory of Open Access Journals (Sweden)

    Jansen Sandra

    2011-02-01

    Full Text Available Abstract Background Recent studies have suggested that the scavenger receptor MARCO (macrophage receptor with collagenous structure mediates activation of the immune response in bacterial infection of the central nervous system (CNS. The chemotactic G-protein-coupled receptor (GPCR formyl-peptide-receptor like-1 (FPRL1 plays an essential role in the inflammatory responses of host defence mechanisms and neurodegenerative disorders such as Alzheimer's disease (AD. Expression of the antimicrobial peptide cathelicidin CRAMP/LL-37 is up-regulated in bacterial meningitis, but the mechanisms underlying CRAMP expression are far from clear. Methods Using a rat meningitis model, we investigated the influence of MARCO and FPRL1 on rCRAMP (rat cathelin-related antimicrobial peptide expression after infection with bacterial supernatants of Streptococcus pneumoniae (SP and Neisseria meningitides (NM. Expression of FPRL1 and MARCO was analyzed by immunofluorescence and real-time RT-PCR in a rat meningitis model. Furthermore, we examined the receptor involvement by real-time RT-PCR, extracellular-signal regulated kinases 1/2 (ERK1/2 phosphorylation and cAMP level measurement in glial cells (astrocytes and microglia and transfected HEK293 cells using receptor deactivation by antagonists. Receptors were inhibited by small interference RNA and the consequences in NM- and SP-induced Camp (rCRAMP gene expression and signal transduction were determined. Results We show an NM-induced increase of MARCO expression by immunofluorescence and real-time RT-PCR in glial and meningeal cells. Receptor deactivation by antagonists and small interfering RNA (siRNA verified the importance of FPRL1 and MARCO for NM- and SP-induced Camp and interleukin-1β expression in glial cells. Furthermore, we demonstrated a functional interaction between FPRL1 and MARCO in NM-induced signalling by real-time RT-PCR, ERK1/2 phosphorylation and cAMP level measurement and show differences between

  4. Adenosine A3 receptor activation is neuroprotective against retinal neurodegeneration.

    Science.gov (United States)

    Galvao, Joana; Elvas, Filipe; Martins, Tiago; Cordeiro, M Francesca; Ambrósio, António Francisco; Santiago, Ana Raquel

    2015-11-01

    Death of retinal neural cells, namely retinal ganglion cells (RGCs), is a characteristic of several retinal neurodegenerative diseases. Although the role of adenosine A3 receptor (A3R) in neuroprotection is controversial, A3R activation has been reported to afford protection against several brain insults, with few studies in the retina. In vitro models (retinal neural and organotypic cultures) and animal models [ischemia-reperfusion (I-R) and partial optic nerve transection (pONT)] were used to study the neuroprotective properties of A3R activation against retinal neurodegeneration. The A3R selective agonist (2-Cl-IB-MECA, 1 μM) prevented apoptosis (TUNEL(+)-cells) induced by kainate and cyclothiazide (KA + CTZ) in retinal neural cultures (86.5 ± 7.4 and 37.2 ± 6.1 TUNEL(+)-cells/field, in KA + CTZ and KA + CTZ + 2-Cl-IB-MECA, respectively). In retinal organotypic cultures, 2-Cl-IB-MECA attenuated NMDA-induced cell death, assessed by TUNEL (17.3 ± 2.3 and 8.3 ± 1.2 TUNEL(+)-cells/mm(2) in NMDA and NMDA+2-Cl-IB-MECA, respectively) and PI incorporation (ratio DIV4/DIV2 3.3 ± 0.3 and 1.3 ± 0.1 in NMDA and NMDA+2-Cl-IB-MECA, respectively) assays. Intravitreal 2-Cl-IB-MECA administration afforded protection against I-R injury decreasing the number of TUNEL(+) cells by 72%, and increased RGC survival by 57%. Also, intravitreal administration of 2-Cl-IB-MECA inhibited apoptosis (from 449.4 ± 37.8 to 207.6 ± 48.9 annexin-V(+)-cells) and RGC loss (from 1.2 ± 0.6 to 8.1 ± 1.7 cells/mm) induced by pONT. This study demonstrates that 2-Cl-IB-MECA is neuroprotective to the retina, both in vitro and in vivo. Activation of A3R may have great potential in the management of retinal neurodegenerative diseases characterized by RGC death, as glaucoma and diabetic retinopathy, and ischemic diseases. Copyright © 2015 Elsevier Ltd. All rights reserved.

  5. Receptor partial agonism and method to express receptor partial activation with respect to novel Full Logistic Model of mixture toxicology.

    Science.gov (United States)

    Ezechiáš, Martin; Cajthaml, Tomáš

    2018-01-15

    Living organisms interact with various chemical compounds via receptors, which is described by the receptor theory. The affinity of the biologically active compounds toward receptors and their ability to trigger a biological or toxic signal vary substantially. In this work, we describe a new insight into understanding of the mode of action of receptor partial agonists and the receptor theory using a Full Logistic Model (FLM) of mixture toxicology. We describe the hypothesis that the effect of a partial agonist can be mathematically described via separation of agonistic and antagonistic behavior of the partial agonist where the antagonistic effect is described as an action of the compound producing zero effect. In this way, a competitive antagonist can be considered as an agonist with zero effect. This idea is also placed into a context with classical concepts, e.g., Gaddum's equation. Using the assumption that competitive antagonists are agonists with no effect, equations describing the microscopic and macroscopic equilibrium constants have been derived. Accordingly, we show that the constants could be calculated from the measured partial agonistic dose-response curve. As a consequence, we provide a simple mathematical tool for comparison of dose-response curves of drugs according to their affinities and efficacies. Copyright © 2017 Elsevier B.V. All rights reserved.

  6. A2A adenosine receptor antagonism enhances synaptic and motor effects of cocaine via CB1 cannabinoid receptor activation.

    Directory of Open Access Journals (Sweden)

    Alessandro Tozzi

    Full Text Available BACKGROUND: Cocaine increases the level of endogenous dopamine (DA in the striatum by blocking the DA transporter. Endogenous DA modulates glutamatergic inputs to striatal neurons and this modulation influences motor activity. Since D2 DA and A2A-adenosine receptors (A2A-Rs have antagonistic effects on striatal neurons, drugs targeting adenosine receptors such as caffeine-like compounds, could enhance psychomotor stimulant effects of cocaine. In this study, we analyzed the electrophysiological effects of cocaine and A2A-Rs antagonists in striatal slices and the motor effects produced by this pharmacological modulation in rodents. PRINCIPAL FINDINGS: Concomitant administration of cocaine and A2A-Rs antagonists reduced glutamatergic synaptic transmission in striatal spiny neurons while these drugs failed to produce this effect when given in isolation. This inhibitory effect was dependent on the activation of D2-like receptors and the release of endocannabinoids since it was prevented by L-sulpiride and reduced by a CB1 receptor antagonist. Combined application of cocaine and A2A-R antagonists also reduced the firing frequency of striatal cholinergic interneurons suggesting that changes in cholinergic tone might contribute to this synaptic modulation. Finally, A2A-Rs antagonists, in the presence of a sub-threshold dose of cocaine, enhanced locomotion and, in line with the electrophysiological experiments, this enhanced activity required activation of D2-like and CB1 receptors. CONCLUSIONS: The present study provides a possible synaptic mechanism explaining how caffeine-like compounds could enhance psychomotor stimulant effects of cocaine.

  7. Novel indole and azaindole (pyrrolopyridine) cannabinoid (CB) receptor agonists: design, synthesis, structure-activity relationships, physicochemical properties and biological activity

    NARCIS (Netherlands)

    Blaazer, A.R.; Lange, J.H.M.; van der Neut, M.A.W.; Mulder, A.; den Boon, F.S.; Werkman, T.R.; Kruse, C.G.; Wadman, W.J.

    2011-01-01

    The discovery, synthesis and structure-activity relationship (SAR) of a novel series of cannabinoid 1 (CB1) and cannabinoid 2 (CB2) receptor ligands are reported. Based on the aminoalkylindole class of cannabinoid receptor agonists, a biphenyl moiety was introduced as novel lipophilic indole 3-acyl

  8. Calcitonin receptor expression in medullary thyroid carcinoma.

    Science.gov (United States)

    Cappagli, Virginia; Potes, Catarina Soares; Ferreira, Luciana Bueno; Tavares, Catarina; Eloy, Catarina; Elisei, Rossella; Sobrinho-Simões, Manuel; Wookey, Peter J; Soares, Paula

    2017-01-01

    Calcitonin expression is a well-established marker for medullary thyroid carcinoma (MTC); yet the role of calcitonin receptor (CTR), its seven-transmembrane G-protein coupled receptor, remains to be established in C-cells derived thyroid tumors. The aim of this work was to investigate CTR expression in MTC and to correlate such expression with clinicopathological features in order to evaluate its possible role as a prognostic indicator of disease aggressiveness and outcome. Calcitonin receptor expression was analyzed in a series of 75 MTCs by immunohistochemistry, and by qPCR mRNA quantification in specimens from four patients. Statistical tests were used to evaluate the correlation between CTR expression and the clinicopathological and molecular characteristics of patients and tumors. Calcitonin receptor expression was detected in 62 out of 75 samples (82.7%), whereas 13 of the 75 samples (17.3%) were completely negative. CTR expression was significantly associated with expression of cytoplasmatic phosphatase and tensin homologue deleted on chromosome 10 and osteopontin, as well as with wild type RET/RAS genes and absence of tumor stroma, suggesting that CTR expression do not associate with clinicopathological signs of worse prognosis. Calcitonin receptor expression appears to be associated in MTC with more differentiated status of the neoplastic cells.

  9. Functional residues essential for the activation of the CB1 cannabinoid receptor.

    Science.gov (United States)

    Shim, Joong-Youn; Padgett, Lea

    2013-01-01

    Recently developed X-ray crystal structures of active state G-protein-coupled receptors have opened the way for detailed examination of the movement of the transmembrane (TM) helices and the specific residues involved in the receptor activation upon ligand binding. Previous modeling studies have indicated that the brain cannabinoid (CB1) receptor binds with a ligand at least in part through a hydrophobic tail on the ligand. This interaction is believed to be similar to the rotameric toggle switch proposed for the β2 adrenergic receptor (β2AR). In the present study, an active state model for the CB1 receptor, guided by the X-ray structure of the active state for β2AR, was constructed with HU210 bound as a ligand. Molecular dynamics (MD) simulations were employed to provide a smooth progression between inactive and active states of the receptor. This model was compared with our previously published CB1 receptor model to identify the functional residues that play key roles in triggering receptor conformational changes upon agonist binding. Movements in TM helices and functional residues are discussed. W279(5.43), contributing to an inward movement of the fifth TM helix (TM5) to the helical core, could serve as another rotameric switch for receptor activation. V282(5.46), interacting with the ligand's hydrophobic C3 alkyl chain, appears to play a key role in TM5 inward movement centered at L286(5.50) and subsequent coupling to V204(3.40). V204(3.40), closely interacting with the TM5 and TM6 hydrophobic patch residues in the middle of the receptor, particularly I290(5.54) and L352(6.44), appears to facilitate helical rearrangements, leading to the breakage of the ionic lock and the rotameric change of Y397(7.53), which are key features of the active state. Copyright © 2013 Elsevier Inc. All rights reserved.

  10. Plasmin Activation of Glial Cells through Protease-Activated Receptor 1

    Directory of Open Access Journals (Sweden)

    André R. Greenidge

    2013-01-01

    Full Text Available The objective of this study was to determine whether plasmin could induce morphological changes in human glial cells via PAR1. Human glioblastoma A172 cells were cultured in the presence of plasmin or the PAR1 specific activating hexapeptide, SFLLRN. Cells were monitored by flow cytometry to detect proteolytic activation of PAR1 receptor. Morphological changes were recorded by photomicroscopy and apoptosis was measured by annexinV staining. Plasmin cleaved the PAR1 receptor on glial cells at 5 minutes (P=0.02. After 30 minutes, cellular processes had begun to retract from the basal substratum and by 4 hours glial cells had become detached. Similar results were obtained by generating plasmin de novo from plasminogen. Morphological transformation was blocked by plasmin inhibitors aprotinin or epsilon-aminocaproic acid (P=0.03. Cell viability was unimpaired during early morphological changes, but by 24 hours following plasmin treatment 22% of glial cells were apoptotic. PAR1 activating peptide SFLLRN (but not inactive isomer FSLLRN promoted analogous glial cell detachment (P=0.03, proving the role for PAR1 in this process. This study has identified a plasmin/PAR1 axis of glial cell activation, linked to changes in glial cell morophology. This adds to our understanding of pathophysiological disease mechanisms of plasmin and the plasminogen system in neuroinjury.

  11. How does angiotensin AT2 receptor activation help neuronal differentiation and improve neuronal pathological situations?

    Science.gov (United States)

    Guimond, Marie-Odile; Gallo-Payet, Nicole

    2012-01-01

    The angiotensin type 2 (AT2) receptor of angiotensin II has long been thought to be limited to few tissues, with the primary effect of counteracting the angiotensin type 1 (AT1) receptor. Functional studies in neuronal cells have demonstrated AT2 receptor capability to modulate neuronal excitability, neurite elongation, and neuronal migration, suggesting that it may be an important regulator of brain functions. The observation that the AT2 receptor was expressed in brain areas implicated in learning and memory led to the hypothesis that it may also be implicated in cognitive functions. However, linking signaling pathways to physiological effects has always proven challenging since information relative to its physiological functions has mainly emerged from indirect observations, either from the blockade of the AT1 receptor or through the use of transgenic animals. From a mechanistic standpoint, the main intracellular pathways linked to AT2 receptor stimulation include modulation of phosphorylation by activation of kinases and phosphatases or the production of nitric oxide and cGMP, some of which are associated with the Gi-coupling protein. The receptor can also interact with other receptors, either G protein-coupled such as bradykinin, or growth factor receptors such as nerve growth factor or platelet-derived growth factor receptors. More recently, new advances have also led to identification of various partner proteins, thus providing new insights into this receptor’s mechanism of action. This review summarizes the recent advances regarding the signaling pathways induced by the AT2 receptor in neuronal cells, and discussed the potential therapeutic relevance of central actions of this enigmatic receptor. In particular, we highlight the possibility that selective AT2 receptor activation by non-peptide and selective agonists could represent new pharmacological tools that may help to improve impaired cognitive performance in Alzheimer’s disease and other

  12. Affinities and intrinsic activities of dopamine receptor agonists for the hD(21) and hD(4.4) receptors

    NARCIS (Netherlands)

    Lahti, RA; Mutin, A; Cochrane, EV; Tepper, PG; Dijkstra, D; Wikstrom, H; Tamminga, CA

    1996-01-01

    The affinity and intrinsic activity of dopamine receptor agonists were determined at the human dopamine hD(21) and hD(4.4) receptors. (-)-3-Hydroxy-N-n-propylpiperidine ((-)3-PPP) had an intrinsic activity of 46% and 83%, whereas (+)-N-propylnorapomorphine ((+)-NPA) had intrinsic activities of 61%

  13. A ligand channel through the G protein coupled receptor opsin.

    Directory of Open Access Journals (Sweden)

    Peter W Hildebrand

    Full Text Available The G protein coupled receptor rhodopsin contains a pocket within its seven-transmembrane helix (TM structure, which bears the inactivating 11-cis-retinal bound by a protonated Schiff-base to Lys296 in TM7. Light-induced 11-cis-/all-trans-isomerization leads to the Schiff-base deprotonated active Meta II intermediate. With Meta II decay, the Schiff-base bond is hydrolyzed, all-trans-retinal is released from the pocket, and the apoprotein opsin reloaded with new 11-cis-retinal. The crystal structure of opsin in its active Ops* conformation provides the basis for computational modeling of retinal release and uptake. The ligand-free 7TM bundle of opsin opens into the hydrophobic membrane layer through openings A (between TM1 and 7, and B (between TM5 and 6, respectively. Using skeleton search and molecular docking, we find a continuous channel through the protein that connects these two openings and comprises in its central part the retinal binding pocket. The channel traverses the receptor over a distance of ca. 70 A and is between 11.6 and 3.2 A wide. Both openings are lined with aromatic residues, while the central part is highly polar. Four constrictions within the channel are so narrow that they must stretch to allow passage of the retinal beta-ionone-ring. Constrictions are at openings A and B, respectively, and at Trp265 and Lys296 within the retinal pocket. The lysine enforces a 90 degrees elbow-like kink in the channel which limits retinal passage. With a favorable Lys side chain conformation, 11-cis-retinal can take the turn, whereas passage of the all-trans isomer would require more global conformational changes. We discuss possible scenarios for the uptake of 11-cis- and release of all-trans-retinal. If the uptake gate of 11-cis-retinal is assigned to opening B, all-trans is likely to leave through the same gate. The unidirectional passage proposed previously requires uptake of 11-cis-retinal through A and release of photolyzed all

  14. Activating prostaglandin E2 receptor subtype EP4 increases secreted mucin from airway goblet cells.

    Science.gov (United States)

    Akaba, Tomohiro; Komiya, Kosaku; Suzaki, Isao; Kozaki, Yuji; Tamaoki, Jun; Rubin, Bruce K

    2017-11-09

    Prostaglandin E2 (PGE2) is a ligand of the E-type prostanoid receptors, EP1-4. PGE2 secretion is increased in the airways of patients with asthma by secretory phospholipases A2, which also increases MUC5AC mucin in goblet cells. We hypothesized that PGE2 would also increase MUC5AC mRNA and secreted protein through specific EP receptor activation. We sought to assess the effect of specific EP receptor activation on MUC5AC secretion from ciliated-enriched cells or goblet-enriched cells induced by IL-13. We develop an enriched goblet cell epithelium by growing normal human bronchial epithelial cells at air liquid interface for 14 days in the presence of IL-13. We examined exposure to 4 specific EP receptor agonists at 24 h and 14 days in cells grown with or without IL-13 exposure, and measured MUC5AC mRNA and secreted protein, as well as airway culture morphology, and EP receptor expression. In ciliated-enriched cells grown in the absence of IL-13, the EP4 receptor agonist modestly increased both MUC5AC mRNA and secretion (p receptor agonist greatly increased both MUC5AC mRNA and protein (p receptor had no effect on secreted mucin. EP4 receptor mRNA and protein were significantly increased in goblet-enriched cells, while the other receptor mRNA were decreased. We conclude that PGE2 stimulates airway mucin production predominantly by EP4 receptor activation in association with increased EP4 receptor expression. This may contribute to mucus hypersecretion as seen in severe asthma. Copyright © 2017. Published by Elsevier Ltd.

  15. Direct evidence for GABAergic activity of Withania somnifera on mammalian ionotropic GABAA and GABAρ receptors.

    Science.gov (United States)

    Candelario, Manuel; Cuellar, Erika; Reyes-Ruiz, Jorge Mauricio; Darabedian, Narek; Feimeng, Zhou; Miledi, Ricardo; Russo-Neustadt, Amelia; Limon, Agenor

    2015-08-02

    Withania somnifera (WS) has been traditionally used in Ayurvedic medicine as a remedy for debility, stress, nervous exhaustion, insomnia, loss of memory, and to enhance cognitive function. This study provides an empirical evidence to support the traditional use of WS to aid in mental process engaging GABAergic signaling. We evaluated the effect of aqueous WS root extract (aqWS), and its two main components, withaferin A and withanolide A, on the main inhibitory receptors in the central nervous system: ionotropic GABAA receptors. The pharmacological activity of aqWS, withaferin A and withanolide A, was tested on native rat brain GABAA channels microtransplanted into Xenopus oocytes and GABAρ1 receptors heterologously expressed in oocytes. The GABAergic activity of aqWS compounds was evaluated by the two-electrode voltage-clamp method and the fingerprint of the extract was done by LC-MS. Concentration-dependent inward ion currents were elicited by aqWS in microtransplanted oocytes with an EC50 equivalent to 4.7 mg/mL and a Hill coefficient (nH) of 1.6. The GABAA receptor antagonist bicuculline blocked these currents. Our results show that aqWS activated inotropic GABAA channels but with lower efficacy compared to the endogenous agonist GABA. We also demonstrate for first time that aqWS is a potent agonist of GABAρ1 receptors. GABAρ1 receptors were 27 fold more sensitive to aqWS than GABAA receptors. Furthermore, aqWS activated GABAρ1 receptors eliciting maximum currents that were no significantly different to those produced by GABA (paired t-test; p=0.533). The differential activity on GABAA and GABA ρ1 receptors and the reported lack of significant GABA presence in WS root extract indicates that the GABAergic activity of aqWS is not mediated by GABA. WS main active components, witaferin A and withanolide A, were tested to determine if they were responsible for the activation of the GABA receptors. Neither compound activated GABAA nor GABAρ1 receptors

  16. Interaction between the p21ras GTPase activating protein and the insulin receptor

    NARCIS (Netherlands)

    Pronk, G.J.; Medema, R.H.; Burgering, B.M.T.; Clark, R.; McCormick, F.; Bos, J.L.

    1992-01-01

    We investigated the involvement of the p21ras-GTPase activating protein (GAP) in insulin-induced signal transduction. In cells overexpressing the insulin receptor, we did not observe association between GAP and the insulin receptor after insulin treatment nor the phosphorylation of GAP on tyrosine

  17. beta-agonist-induced constitutive beta(2)-adrenergic receptor activity in bovine tracheal smooth muscle

    NARCIS (Netherlands)

    de Vries, B; Meurs, H; Roffel, AF; Elzinga, CRS; Hoiting, BH; de Vries, MML; Zaagsma, J

    2000-01-01

    1 According to the two state receptor model, the beta (2)-adrenergic receptor (beta (2)-AR) isomerizes between an inactive state and a constitutively active state, which couples to the stimulatory G-protein in the absence of agonist. In bovine tracheal smooth muscle (BTSM), we investigated the

  18. Are activating mutations of the adrenocorticotropin receptor involved in adrenal cortical neoplasia?

    Science.gov (United States)

    Light, K; Jenkins, P J; Weber, A; Perrett, C; Grossman, A; Pistorello, M; Asa, S L; Clayton, R N; Clark, A J

    1995-01-01

    The objective of this study was to investigate the presence of activating mutations of the ACTH receptor gene in benign and malignant adrenocortical tumours. Genomic DNA was extracted from a variety of adrenocortical neoplasms from 16 patients and the entire ACTH receptor gene was then amplified by the polymerase chain reaction and sequenced. No mutations were detected within the coding region of the ACTH receptor gene in the 16 adrenocortical tumours studied. It is likely that mutations of the coding region of the ACTH receptor gene do not play a major role in adrenocortical tumourigenesis.

  19. Discoidin domain receptor (DDR) 1 and 2: collagen-activated tyrosine kinase receptors in the cornea.

    Science.gov (United States)

    Mohan, R R; Mohan, R R; Wilson, S E

    2001-01-01

    Discoidin domain receptor (DDR) 1 and 2 have recently been found to serve as receptors for several collagen types. These receptors have been found to modulate cell proliferation and metalloprotease expression in response to collagen stimulation. The purpose of this study was to examine expression of DDR1 and DDR2 in the cornea and to determine the effect of several collagen types on proliferation and response to pro-apoptotic cytokines by corneal fibroblasts. DDR1 and DDR2 mRNAs were detected by RT-PCR. Proteins were detected by immunocytochemistry and immunoprecipitation with Western blotting. Cell proliferation in response to acetic acid-solubilized collagen type I, II, IV, IX or X was determined by cell counting. The effect of these collagen types on Fas-stimulating antibody-induced cell death was determined by trypan blue assay. DDR1 and DDR2 mRNAs were detected in each major human cell type of the cornea. Both were also detected in ex vivo human corneal epithelium. DDR1 and DDR2 proteins were detected in all three major cell types in culture and in human corneal tissue. Collagen types I, II, IV, IX and X stimulated proliferation, but had no effect on Fas-mediated apoptosis, of corneal fibroblasts. DDR1 and DDR2 tyrosine kinase receptors are expressed in the cornea. Collagen-stimulated mitosis of corneal fibroblasts in culture is likely mediated by the DDR receptors. Collagen had no effect on Fas-mediated apoptosis of corneal fibroblasts. Copyright 2001 Academic Press.

  20. Inhibition of the activation and catalytic activity of insulin receptor kinase by zinc and other divalent metal ions.

    Science.gov (United States)

    Pang, D T; Shafer, J A

    1985-04-25

    The autophosphorylation reaction responsible for conversion of insulin receptor (from human placenta) to an active tyrosyl-protein kinase was shown to be inhibited by Zn2+ and other divalent metal ions. The order of inhibitory potency was found to be Cu2+ greater than Zn2+, Cd2+ greater than Co2+, Ni2+. Autophosphorylation of insulin receptor was almost completely blocked by 10 microM Zn2+. Zn2+, however, did not appear to affect the binding of insulin to its receptor. Histidine, a chelator of Zn2+, protected against the inhibitory effects of Zn2+. The failure of histidine to regenerate the competence of the Zn2+-inhibited receptor to undergo autophosphorylation suggested that the inhibition by Zn2+ was irreversible. In addition to inhibiting autophosphorylation, Zn2+ inhibited the tyrosyl-protein kinase activity of highly purified phosphorylated receptor. Zn2+ was also observed to inhibit phosphotyrosyl-protein phosphatase activity present in preparations of partially purified insulin receptor. These inhibitory effects of Zn2+ should be considered in the design of protocols for the isolation and handling of insulin receptor and possibly other tyrosine kinases. Additionally, the possible physiological significance of the inhibition of insulin receptor kinase by Zn2+ is discussed in light of the fact that Zn2+ is accumulated in and secreted from pancreatic islet cells together with insulin.

  1. DMPD: Proximal effects of Toll-like receptor activation in dendritic cells. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 17142025 Proximal effects of Toll-like receptor activation in dendritic cells. Watt...) (.svg) (.html) (.csml) Show Proximal effects of Toll-like receptor activation in dendritic cells. PubmedID... 17142025 Title Proximal effects of Toll-like receptor activation in dendritic ce

  2. Mechanisms of epidermal growth factor receptor signaling as characterized by patterned ligand activation and mutational analysis.

    Science.gov (United States)

    Holowka, David; Baird, Barbara

    2017-09-01

    The cell surface receptor for epidermal growth factor (EGFR), a receptor tyrosine kinase, is a key player in normal cell growth and proliferation. Mutations in this receptor often lead to oncological transformation and other pathologies. Because of its representation of the receptor tyrosine kinase family and its important role in health and disease, a broad range of studies have been carried out in many laboratories to investigate the structural basis for transmembrane receptor activation and the resulting assembly of cytosolic signaling components. This review highlights two approaches our laboratory has taken to gain more detailed information about both aspects: Surface patterned ligands to examine recruitment of the signaling machinery, and mutational analysis to examine the regulatory role of EGFR's juxtamembrane segment. 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.

  3. Structure-Activity Relationships in Human Toll-like Receptor 7-Active Imidazoquinoline Analogues

    OpenAIRE

    Shukla, Nikunj M.; Malladi, Subbalakshmi S.; Mutz, Cole A.; Balakrishna, Rajalakshmi; David, Sunil A.

    2010-01-01

    Engagement of toll-like receptors serve to link innate immune responses with adaptive immunity and can be exploited as powerful vaccine adjuvants for eliciting both primary and anamnestic immune responses. TLR7 agonists are highly immunostimulatory without inducing dominant proinflammatory cytokine responses. A structure-activity study was conducted on the TLR7-agonistic imidazoquinolines, starting with 1-(4-amino-2-((ethylamino)methyl)-1H-imidazo[4,5-c]quinolin-1-yl)-2-methylpropan-2-ol as a...

  4. Peroxisome proliferator-activated receptor-delta agonist ameliorated inflammasome activation in nonalcoholic fatty liver disease.

    Science.gov (United States)

    Lee, Hyun Jung; Yeon, Jong Eun; Ko, Eun Jung; Yoon, Eileen L; Suh, Sang Jun; Kang, Keunhee; Kim, Hae Rim; Kang, Seoung Hee; Yoo, Yang Jae; Je, Jihye; Lee, Beom Jae; Kim, Ji Hoon; Seo, Yeon Seok; Yim, Hyung Joon; Byun, Kwan Soo

    2015-12-07

    To evaluate the inflammasome activation and the effect of peroxisome proliferator-activated receptors (PPAR)-δ agonist treatment in nonalcoholic fatty liver disease (NAFLD) models. Male C57BL/6J mice were classified according to control or high fat diet (HFD) with or without PPAR-δ agonist (GW) over period of 12 wk [control, HFD, HFD + lipopolysaccharide (LPS), HFD + LPS + GW group]. HepG2 cells were exposed to palmitic acid (PA) and/or LPS in the absence or presence of GW. HFD caused glucose intolerance and hepatic steatosis. In mice fed an HFD with LPS, caspase-1 and interleukin (IL)-1β in the liver were significantly increased. Treatment with GW ameliorated the steatosis and inhibited overexpression of pro-inflammatory cytokines. In HepG2 cells, PA and LPS treatment markedly increased mRNA of several nucleotide-binding and oligomerization domain-like receptor family members (NLRP3, NLRP6, and NLRP10), caspase-1 and IL-1β. PA and LPS also exaggerated reactive oxygen species production. All of the above effects of PA and LPS were reduced by GW. GW also enhanced the phosphorylation of AMPK-α. PPAR-δ agonist reduces fatty acid-induced inflammation and steatosis by suppressing inflammasome activation. Targeting the inflammasome by the PPAR-δ agonist may have therapeutic implication for NAFLD.

  5. Iron nanomedicines induce Toll-like receptor activation, cytokine production and complement activation.

    Science.gov (United States)

    Verhoef, Johan J F; de Groot, A Marit; van Moorsel, Marc; Ritsema, Jeffrey; Beztsinna, Nataliia; Maas, Coen; Schellekens, Huub

    2017-03-01

    Approximately a dozen of intravenous iron nanomedicines gained marketing authorization in the last two decades. These products are generally considered as safe, but have been associated with an increased risk for hypersensitivity-like reactions of which the underlying mechanisms are unknown. We hypothesized that iron nanomedicines can trigger the innate immune system. We hereto investigated the physico-chemical properties of ferric gluconate, iron sucrose, ferric carboxymaltose and iron isomaltoside 1000 and comparatively studied their interaction with Toll-like receptors, the complement system and peripheral blood mononuclear cells. Two out of four formulations appeared as aggregates by Scanning Transmission Electron Microscopy analysis and were actively taken up by HEK293T- and peripheral blood mononuclear cells in a cholesterol-dependent manner. These formulations triggered in vitro activation of intracellular Toll-like receptors 3, -7 and -9 in a dose- and serum-dependent manner. In parallel experiments, we determined that these compounds activated the complement system. Finally, we found that uptake of aggregation-prone iron nanomedicines by peripheral blood mononuclear cells in whole blood induced production of the proinflammatory cytokine IL-1β, but not IL-6. Copyright © 2016 Elsevier Ltd. All rights reserved.

  6. The adipogenic acetyltransferase Tip60 targets activation function 1 of peroxisome proliferator-activated receptor gamma

    DEFF Research Database (Denmark)

    van Beekum, Olivier; Brenkman, Arjan B; Grøntved, Lars

    2008-01-01

    The transcription factor peroxisome proliferator-activated receptor gamma (PPARgamma) plays a key role in the regulation of lipid and glucose metabolism in adipocytes, by regulating their differentiation, maintenance, and function. The transcriptional activity of PPARgamma is dictated by the set...... in cells, and through use of chimeric proteins, we established that coactivation by Tip60 critically depends on the N-terminal activation function 1 of PPARgamma, a domain involved in isotype-specific gene expression and adipogenesis. Chromatin immunoprecipitation experiments showed that the endogenous Tip......60 protein is recruited to PPARgamma target genes in mature 3T3-L1 adipocytes but not in preadipocytes, indicating that Tip60 requires PPARgamma for its recruitment to PPARgamma target genes. Importantly, we show that in common with disruption of PPARgamma function, small interfering RNA...

  7. Activating effect of benzbromarone, a uricosuric drug, on peroxisome proliferator-activated receptors.

    Science.gov (United States)

    Kunishima, Chiyoko; Inoue, Ikuo; Oikawa, Toshihiro; Nakajima, Hiromu; Komoda, Tsugikazu; Katayama, Shigehiro

    2007-01-01

    Benzbromarone, a uricosuric drug, reportedly causes hepatic hypertrophy accompanied by proliferation of peroxisomes in rats. To elucidate the mechanisms underlying induction of peroxisome proliferation by benzbromarone, we examined binding affinity for peroxisome proliferator-activated receptor alpha (PPARalpha) and gamma (PPARgamma), and effects on the binding activity of PPARs with peroxisome proliferation-responsive element (PPRE) and expression of the PPARs target protein. Binding affinity of benzbromarone for PPARalpha and PPARgamma was examined by reporter gene assay. Binding activity of PPARs with PPRE was determined by electric mobility shift assay, and expression of lipoprotein lipase (LPL) and acyl-CoA synthetase (ACS) by Western blot method. Benzbromarone displayed affinity for PPARalpha and PPARgamma, and promoted binding of PPARs to PPRE. Furthermore, cultured cells with benzbromarone added showed upregulated expression of LPL and ACS. These results suggest that benzbromarone induces peroxisome proliferation in hepatocytes by binding to PPARs, and controls expression of proteins related to lipid metabolism.

  8. Activating Effect of Benzbromarone, a Uricosuric Drug, on Peroxisome Proliferator-Activated Receptors

    Directory of Open Access Journals (Sweden)

    Chiyoko Kunishima

    2007-01-01

    Full Text Available Benzbromarone, a uricosuric drug, reportedly causes hepatic hypertrophy accompanied by proliferation of peroxisomes in rats. To elucidate the mechanisms underlying induction of peroxisome proliferation by benzbromarone, we examined binding affinity for peroxisome proliferator-activated receptor α (PPARα and γ (PPARγ, and effects on the binding activity of PPARs with peroxisome proliferation-responsive element (PPRE and expression of the PPARs target protein. Binding affinity of benzbromarone for PPARα and PPARγ was examined by reporter gene assay. Binding activity of PPARs with PPRE was determined by electric mobility shift assay, and expression of lipoprotein lipase (LPL and acyl-CoA synthetase (ACS by Western blot method. Benzbromarone displayed affinity for PPARα and PPARγ, and promoted binding of PPARs to PPRE. Furthermore, cultured cells with benzbromarone added showed upregulated expression of LPL and ACS. These results suggest that benzbromarone induces peroxisome proliferation in hepatocytes by binding to PPARs, and controls expression of proteins related to lipid metabolism.

  9. Soluble ectodomain of neuroligin 1 decreases synaptic activity by activating metabotropic glutamate receptor 2

    DEFF Research Database (Denmark)

    Gjørlund, Michelle D.; Carlsen, Eva Maria Meier; Kønig, Andreas Bay

    2017-01-01

    Synaptic cell adhesion molecules represent important targets for neuronal activity-dependent proteolysis. Postsynaptic neuroligins (NLs) form trans-synaptic complexes with presynaptic neurexins (NXs). Both NXs and NLs are cleaved from the cell surface by metalloproteases in an activity......-dependent manner, releasing a soluble extracellular fragment and membrane-tethered C-terminal fragment. The cleavage of NL1 depresses synaptic transmission, but the mechanism by which this occurs is unknown. Metabotropic glutamate receptor 2 (mGluR2) are located primarily at the periphery of presynaptic terminals......, where they inhibit the formation of cyclic adenosine monophosphate (cAMP) and consequently suppress the release of glutamate and decrease synaptic transmission. In the present study, we found that the soluble ectodomain of NL1 binds to and activates mGluR2 in both neurons and heterologous cells...

  10. Influence of combined thrombin stimulation, surface activation, and receptor occupancy on organization of GPIb/IX receptors on human platelets.

    Science.gov (United States)

    White, J G; Krumwiede, M; Cocking-Johnson, D; Escolar, G

    1994-09-01

    Down-regulation and clearance of as many as 60-80% of GPIb/IX receptors from exposed surfaces on thrombin-activated platelets to channels of the open canalicular system (OCS) is considered to be a fundamental mechanism regulating platelet adhesivity in vitro and in vivo. The present study has combined thrombin stimulation in suspension, surface activation on formvar grids, receptor occupancy by von Willebrand factor (vWF) and exposure to anti-vWF antibody in an effort to demonstrate the removal of GPIb/IX receptors from activated cells. Individually the stimuli failed to cause any change in the frequency of GPIb/IX receptors. Combined, the stimuli were no more effective than when each was used alone. The only way to cause GPIb/IX to move was to add anti-vWF to thrombin-activated platelets allowed to spread on formvar grids and covered with multimers of ristocetin-activated human or bovine vWF. Translocation of GPIb/IX-vWF-anti-vWF complexes from peripheral margins into caps over cell centres, however, did not clear the peripheral zone of vWF binding capacity. Exposure of capped platelets after fixation to a second incubation with vWF demonstrated as many multimers extending from the central cap to the peripheral margins as were seen on platelets exposed a single time to vWF. Antibodies to GPIb, but not to GPIIb/IIIA, prevented the second labelling by vWF. Down-regulation or clearance of GPIb/IX, in light of this study, does not appear to be a fundamental mechanism modulating platelet adhesivity.

  11. Vitamin D receptor, Retinoid X receptor and peroxisome proliferator-activated receptor γ are overexpressed in BRCA1 mutated breast cancer and predict prognosis.

    Science.gov (United States)

    Heublein, Sabine; Mayr, Doris; Meindl, Alfons; Kircher, Alexandra; Jeschke, Udo; Ditsch, Nina

    2017-04-20

    BRCA1 mutated breast cancers are commonly diagnosed as negative for classical hormone receptors i.e. estrogen receptor, progesterone receptor and/or Her2. Due to these common targets being absent the application of anti-endocrine therapies is rather limited and a certain focus has been set on discovering alternative target molecules. We recently highlighted thyroid hormone receptors (TRs) to predict prognosis in breast cancer patients that had been diagnosed a BRCA1 germline mutation. Vitamin D Receptor (VDR), Retinoid X Receptor (RXR) and Peroxisome Proliferator-activated Receptor γ (PPARγ) are known to interact with TRs by forming functional heterodimers. Whether VDR, RXR or PPARγ are expressed in BRCA1 mutated breast cancer or may even be present in case of triple negativity is not known. Hence the current study aimed to investigate VDR, RXR and PPARγ in BRCA1 mut breast cancer and to test whether any of the three may be associated with clinico-pathological criteria including overall survival. This study analyzed VDR, RXR and PPARγ by immunohistochemistry in BRCA1 associated (n = 38) and sporadic breast cancer (n = 79). Receptors were quantified by applying an established scoring system (IR-score) and were tested for association with clinico-pathological variables. VDR, RXR and PPARγ were detected in over 90% of triple negative BRCA1 mut breast cancer and were significantly (VDR: p < 0.001, RXR: p = 0.010, PPARγ: p < 0.001) overexpressed in BRCA1 mutated as compared to sporadic cancer cases. VDR and RXR positivity predicted prolonged overall survival only in BRCA1 mutated cases while such association was not observed in sporadic breast cancer. In conclusion, this is the first study to describe VDR, RXR and PPARγ in BRCA1 mutated breast cancer. Based on the data presented here these receptors may be hypothesized to potentially evolve as interesting markers or even targets in hereditary breast cancer. However, independent studies are

  12. Epidermal Growth Factor Receptor Transactivation Is Required for Mitogen-Activated Protein Kinase Activation by Muscarinic Acetylcholine Receptors in HaCaT Keratinocytes

    Directory of Open Access Journals (Sweden)

    Wymke Ockenga

    2014-11-01

    Full Text Available Non-neuronal acetylcholine plays a substantial role in the human skin by influencing adhesion, migration, proliferation and differentiation of keratinocytes. These processes are regulated by the Mitogen-Activated Protein (MAP kinase cascade. Here we show that in HaCaT keratinocytes all five muscarinic receptor subtypes are expressed, but M1 and M3 are the subtypes involved in mitogenic signaling. Stimulation with the cholinergic agonist carbachol leads to activation of the MAP kinase extracellular signal regulated kinase, together with the protein kinase Akt. The activation is fully dependent on the transactivation of the epidermal growth factor receptor (EGFR, which even appears to be the sole pathway for the muscarinic receptors to facilitate MAP kinase activation in HaCaT cells. The transactivation pathway involves a triple-membrane-passing process, based on activation of matrix metalloproteases, and extracellular ligand release; whereas phosphatidylinositol 3-kinase, Src family kinases or protein kinase C do not appear to be involved in MAP kinase activation. Furthermore, phosphorylation, ubiquitination and endocytosis of the EGF receptor after cholinergic transactivation are different from that induced by a direct stimulation with EGF, suggesting that ligands other than EGF itself mediate the cholinergic transactivation.

  13. Utilizing the folate receptor for active targeting of cancer nanotherapeutics

    Directory of Open Access Journals (Sweden)

    Grant L. Zwicke

    2012-12-01

    Full Text Available The development of specialized nanoparticles for use in the detection and treatment of cancer is increasing. Methods are being proposed and tested that could target treatments more directly to cancer cells, which could lead to higher efficacy and reduced toxicity, possibly even eliminating the adverse effects of damage to the immune system and the loss of quick replicating cells. In this mini-review we focus on recent studies that employ folate nanoconjugates to target the folate receptor. Folate receptors are highly overexpressed on the surface of many tumor types. This expression can be exploited to target imaging molecules and therapeutic compounds directly to cancerous tissues.

  14. Trace Amine Associated Receptor 1 Signaling in Activated Lymphocytes

    OpenAIRE

    Panas, Michael W.; Xie, Zhihua; Panas, Helen N.; Hoener, Marius C.; Vallender, Eric J.; Miller, Gregory M.

    2011-01-01

    Although most research to date on Trace Amine Associated Receptor 1 (TAAR1) has focused on its role in the brain, it has been recognized since its discovery in 2001 that TAAR1 mRNA is expressed in peripheral tissues as well, suggesting that this receptor may play a role in non-neurological pathways. This study reports TAAR1 expression, signaling and functionality in rhesus monkey lymphocytes. We detected a high level of TAAR1 protein in immortalized rhesus monkey B cell lines and a significan...

  15. Plasminogen activation by receptor-bound urokinase. A kinetic study with both cell-associated and isolated receptor

    DEFF Research Database (Denmark)

    Ellis, V; Behrendt, N; Danø, K

    1991-01-01

    .67 microM, below the physiological Plg concentration of 2 microM. A concomitant 6-fold reduction in kcat resulted in an increase in the overall catalytic efficiency, kcat/Km, of 5.7-fold. This high affinity Plg activation was abolished in the presence of a Plg-binding antagonist. In contrast to intact...... cells, purified uPA receptor (isolated from phorbol 12-myristate 13-acetate-stimulated U937 cells) was observed to partially inhibit uPA-catalyzed Plg activation, although activity against low molecular weight substrates was retained. Therefore, the cellular binding of Plg appears to be of critical...

  16. Methamphetamine increases locomotion and dopamine transporter activity in dopamine d5 receptor-deficient mice.

    Directory of Open Access Journals (Sweden)

    Seiji Hayashizaki

    Full Text Available Dopamine regulates the psychomotor stimulant activities of amphetamine-like substances in the brain. The effects of dopamine are mediated through five known dopamine receptor subtypes in mammals. The functional relevance of D5 dopamine receptors in the central nervous system is not well understood. To determine the functional relevance of D5 dopamine receptors, we created D5 dopamine receptor-deficient mice and then used these mice to assess the roles of D5 dopamine receptors in the behavioral response to methamphetamine. Interestingly, D5 dopamine receptor-deficient mice displayed increased ambulation in response to methamphetamine. Furthermore, dopamine transporter threonine phosphorylation levels, which regulate amphetamine-induced dopamine release, were elevated in D5 dopamine receptor-deficient mice. The increase in methamphetamine-induced locomotor activity was eliminated by pretreatment with the dopamine transporter blocker GBR12909. Taken together, these results suggest that dopamine transporter activity and threonine phosphorylation levels are regulated by D5 dopamine receptors.

  17. Progesterone receptor structure and protease activity in primary human endometrial carcinoma.

    Science.gov (United States)

    Feil, P D; Clarke, C L; Satyaswaroop, P G

    1988-03-01

    Monoclonal antibodies were used to investigate progesterone receptor structure (isoforms) in 33 primary human endometrial tumors. The monoclonal antibodies recognized on protein blots two progesterone receptor proteins with molecular weights of 116,000 and 81,000. The Mr 116,000 protein appeared as a triplet, while a single band was found for the Mr 81,000 protein. The triplet/singlet structure was found in all progesterone receptor-positive tumors, regardless of the degree of tumor differentiation. Protease activity, which gave rise to a false-negative pattern on protein blots, was found in approximately one-half of the tumors in which it was investigated. Inclusion of a cocktail of protease inhibitors during sample preparation resulted in the maintenance of the triplet/singlet progesterone receptor structure. Mixing experiments using a progesterone receptor-rich human endometrial carcinoma (EnCa 101), which lacks protease activity, and protease-containing primary tumor homogenates indicated that the protease was leupeptin sensitive. Interestingly, while the proteolytic activity reduced or eliminated the triplet/singlet progesterone receptor structure seen on protein blot analysis, it did not affect progesterone receptor concentration measured by Scatchard analysis. Sample preparation in the presence of protease inhibitors is therefore a requisite for structural analysis of the progesterone receptor in endometrial tumors.

  18. Molecular basis for activation of G protein-coupled receptor kinases

    Energy Technology Data Exchange (ETDEWEB)

    Boguth, Cassandra A.; Singh, Puja; Huang, Chih-chin; Tesmer, John J.G. (Michigan)

    2012-03-16

    G protein-coupled receptor (GPCR) kinases (GRKs) selectively recognize and are allosterically regulated by activated GPCRs, but the molecular basis for this interaction is not understood. Herein, we report crystal structures of GRK6 in which regions known to be critical for receptor phosphorylation have coalesced to stabilize the kinase domain in a closed state and to form a likely receptor docking site. The crux of this docking site is an extended N-terminal helix that bridges the large and small lobes of the kinase domain and lies adjacent to a basic surface of the protein proposed to bind anionic phospholipids. Mutation of exposed, hydrophobic residues in the N-terminal helix selectively inhibits receptor, but not peptide phosphorylation, suggesting that these residues interact directly with GPCRs. Our structural and biochemical results thus provide an explanation for how receptor recognition, phospholipid binding, and kinase activation are intimately coupled in GRKs.

  19. The insulin and IGF1 receptor kinase domains are functional dimers in the activated state

    Science.gov (United States)

    Cabail, M. Zulema; Li, Shiqing; Lemmon, Eric; Bowen, Mark E.; Hubbard, Stevan R.; Miller, W. Todd

    2015-03-01

    The insulin receptor (IR) and insulin-like growth factor-1 receptor (IGF1R) are highly related receptor tyrosine kinases with a disulfide-linked homodimeric architecture. Ligand binding to the receptor ectodomain triggers tyrosine autophosphorylation of the cytoplasmic domains, which stimulates catalytic activity and creates recruitment sites for downstream signalling proteins. Whether the two phosphorylated tyrosine kinase domains within the receptor dimer function independently or cooperatively to phosphorylate protein substrates is not known. Here we provide crystallographic, biophysical and biochemical evidence demonstrating that the phosphorylated kinase domains of IR and IGF1R form a specific dimeric arrangement involving an exchange of the juxtamembrane region proximal to the kinase domain. In this dimer, the active position of α-helix C in the kinase N lobe is stabilized, which promotes downstream substrate phosphorylation. These studies afford a novel strategy for the design of small-molecule IR agonists as potential therapeutic agents for type 2 diabetes.

  20. Constitutive activity and ligand-dependent activation of the nuclear receptor CAR-insights from molecular dynamics simulations.

    Science.gov (United States)

    Windshügel, Björn; Poso, Antti

    2011-01-01

    The constitutive androstane receptor (CAR) possesses, unlike most other nuclear receptors, a pronounced basal activity in vitro whose structural basis is still not fully understood. Using comparative molecular dynamics simulations of CAR X-ray crystal structures, we evaluated the molecular basis for constitutive activity and ligand-dependent receptor activation. Our results suggest that a combination of van der Waals interactions and hydrogen bonds is required to maintain the activation helix in the active conformation also in absence of a ligand. Furthermore, we identified conformational rearrangements within the ligand-binding pocket upon agonist binding and an influence of CAR inducers pregnanedione and CITCO on the helical conformation of the activation helix. Based on the results a model for ligand-dependent CAR activation is suggested. Copyright © 2011 John Wiley & Sons, Ltd.

  1. Peripheral Sensitization Increases Opioid Receptor Expression and Activation by Crotalphine in Rats

    Science.gov (United States)

    Zambelli, Vanessa Olzon; Fernandes, Ana Carolina de Oliveira; Gutierrez, Vanessa Pacciari; Ferreira, Julio Cesar Batista; Parada, Carlos Amilcar; Mochly-Rosen, Daria; Cury, Yara

    2014-01-01

    Inflammation enhances the peripheral analgesic efficacy of opioid drugs, but the mechanisms involved in this phenomenon have not been fully elucidated. Crotalphine (CRP), a peptide that was first isolated from South American rattlesnake C.d. terrificus venom, induces a potent and long-lasting anti-nociceptive effect that is mediated by the activation of peripheral opioid receptors. Because the high efficacy of CRP is only observed in the presence of inflammation, we aimed to elucidate the mechanisms involved in the CRP anti-nociceptive effect induced by inflammation. Using real-time RT-PCR, western blot analysis and ELISA assays, we demonstrate that the intraplantar injection of prostaglandin E2 (PGE2) increases the mRNA and protein levels of the µ- and κ-opioid receptors in the dorsal root ganglia (DRG) and paw tissue of rats within 3 h of the injection. Using conformation state-sensitive antibodies that recognize activated opioid receptors, we show that PGE2, alone does not increase the activation of these opioid receptors but that in the presence of PGE2, the activation of specific opioid receptors by CRP and selective µ- and κ-opioid receptor agonists (positive controls) increases. Furthermore, PGE2 down-regulated the expression and activation of the δ-opioid receptor. CRP increased the level of activated mitogen-activated protein kinases in cultured DRG neurons, and this increase was dependent on the activation of protein kinase Cζ. This CRP effect was much more prominent when the cells were pretreated with PGE2. These results indicate that the expression and activation of peripheral opioid receptors by opioid-like drugs can be up- or down-regulated in the presence of an acute injury and that acute tissue injury enhances the efficacy of peripheral opioids. PMID:24594607

  2. Aromatase activity in receptor negative breast and endometrial cancer

    NARCIS (Netherlands)

    Berstein, LM; Kovalevskij, A; Larionov, A; Tsyrlina, E; Vasilyev, D; Zimarina, T; Thijssen, JHH

    Among the factors for estrogen and progesterone receptors (ER and PR) negativity of tumors of reproductive tissue special attention., is attracted by ability of the tumor produce estrogens (as intratumoral regulators of ER and PR) through reaction of aromatization. 101 samples of tumor tissue (64

  3. Environmental polycyclic aromatic hydrocarbons affect androgen receptor activation in vitro

    DEFF Research Database (Denmark)

    Vinggaard, Anne Marie; Hnida, Christina; Larsen, John Christian

    2000-01-01

    Nine structurally different polycyclic aromatic hydrocarbons (PAHs) were tested for their ability to either agonize or antagonize the human androgen receptor (hAR) in a sensitive reporter gene assay based on CHO cells transiently cotransfected with a hAR vector and an MMTV-LUC vector. Benz...

  4. EPO-independent functional EPO receptor in breast cancer enhances estrogen receptor activity and promotes cell proliferation

    Energy Technology Data Exchange (ETDEWEB)

    Reinbothe, Susann; Larsson, Anna-Maria; Vaapil, Marica; Wigerup, Caroline [Department of Laboratory Medicine, Translational Cancer Research, Medicon Village, Lund University, SE-223 81 Lund (Sweden); CREATE Health, Sackler Faculty of Medicine, Tel-Aviv University, Tel-Aviv (Israel); Sun, Jianmin [Department of Laboratory Medicine, Translational Cancer Research, Medicon Village, Lund University, SE-223 81 Lund (Sweden); Jögi, Annika [Department of Laboratory Medicine, Translational Cancer Research, Medicon Village, Lund University, SE-223 81 Lund (Sweden); CREATE Health, Sackler Faculty of Medicine, Tel-Aviv University, Tel-Aviv (Israel); Neumann, Drorit [Department of Cell and Developmental Biology, Sackler Faculty of Medicine, Tel-Aviv University, Tel-Aviv (Israel); Rönnstrand, Lars [Department of Laboratory Medicine, Translational Cancer Research, Medicon Village, Lund University, SE-223 81 Lund (Sweden); Påhlman, Sven, E-mail: sven.pahlman@med.lu.se [Department of Laboratory Medicine, Translational Cancer Research, Medicon Village, Lund University, SE-223 81 Lund (Sweden); CREATE Health, Sackler Faculty of Medicine, Tel-Aviv University, Tel-Aviv (Israel)

    2014-02-28

    Highlights: • New anti-human EPOR antibody confirms full-length EPOR expression in breast cancer cells. • Proliferation of breast cancer cells is not affected by rhEPO treatment in vitro. • EPOR knockdown impairs proliferation of ERa positive breast cancer cells. • EPOR knockdown reduces AKT phosphorylation and ERa activity. - Abstract: The main function of Erythropoietin (EPO) and its receptor (EPOR) is the stimulation of erythropoiesis. Recombinant human EPO (rhEPO) is therefore used to treat anemia in cancer patients. However, clinical trials have indicated that rhEPO treatment might promote tumor progression and has a negative effect on patient survival. In addition, EPOR expression has been detected in several cancer forms. Using a newly produced anti-EPOR antibody that reliably detects the full-length isoform of the EPOR we show that breast cancer tissue and cells express the EPOR protein. rhEPO stimulation of cultured EPOR expressing breast cancer cells did not result in increased proliferation, overt activation of EPOR (receptor phosphorylation) or a consistent activation of canonical EPOR signaling pathway mediators such as JAK2, STAT3, STAT5, or AKT. However, EPOR knockdown experiments suggested functional EPO receptors in estrogen receptor positive (ERα{sup +}) breast cancer cells, as reduced EPOR expression resulted in decreased proliferation. This effect on proliferation was not seen in ERα negative cells. EPOR knockdown decreased ERα activity further supports a mechanism by which EPOR affects proliferation via ERα-mediated mechanisms. We show that EPOR protein is expressed in breast cancer cells, where it appears to promote proliferation by an EPO-independent mechanism in ERα expressing breast cancer cells.

  5. 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.

  6. Identifying multiple active conformations in the G protein-coupled receptor activation landscape using computational methods.

    Science.gov (United States)

    Dong, Sijia S; Goddard, William A; Abrol, Ravinder

    2017-01-01

    G protein-coupled receptors (GPCRs) are membrane proteins critical in cellular signaling, making them important targets for therapeutics. The activation of GPCRs is central to their function, requiring multiple conformations of the GPCRs in their activation landscape. To enable rational design of GPCR-targeting drugs, it is essential to obtain the ensemble of atomistic structures of GPCRs along their activation pathways. This is most challenging for structure determination experiments, making it valuable to develop reliable computational structure prediction methods. In particular, since the active-state conformations are higher in energy (less stable) than inactive-state conformations, they are difficult to stabilize. In addition, the computational methods are generally biased toward lowest energy structures by design and miss these high energy but functionally important conformations. To address this problem, we have developed a computationally efficient ActiveGEnSeMBLE method that systematically predicts multiple conformations that are likely in the GPCR activation landscape, including multiple active- and inactive-state conformations. ActiveGEnSeMBLE starts with a systematic coarse grid sampling of helix tilts/rotations (~13 trillion transmembrane domain conformations) and identifies multiple potential active-state energy wells, using the TM3-TM6 intracellular distance as a surrogate activation coordinate. These energy wells are then sampled locally using a finer grid in conformational space to find a locally minimized conformation in each energy well, which can be further relaxed using molecular dynamics (MD) simulations. This method, combining homology modeling, hierarchical complete conformational sampling, and nanosecond scale MD, provides one of the very few computational methods that predict multiple candidates for active-state conformations and is one of the most computationally affordable. © 2017 Elsevier Inc. All rights reserved.

  7. Estrogen activates rapid signaling in the brain: role of estrogen receptor alpha and estrogen receptor beta in neurons and glia.

    Science.gov (United States)

    Mhyre, A J; Dorsa, D M

    2006-01-01

    The aging process is known to coincide with a decline in circulating sex hormone levels in both men and women. Due to an increase in the average lifespan, a growing number of post-menopausal women are now receiving hormone therapy for extended periods of time. Recent findings of the Women's Health Initiative, however, have called into question the benefits of long-term hormone therapy for treating symptoms of menopause. The results of this study are still being evaluated, but it is clear that a better understanding of the molecular effects of estradiol is needed in order to develop new estrogenic compounds that activate specific mechanisms but lack adverse side effects. Traditionally, the effects of estradiol treatment have been ascribed to changes in gene expression, namely transcription at estrogen response elements. This review focuses on emerging information that estradiol can also activate a repertoire of membrane-initiated signaling pathways and that these rapid signaling events lead to functional changes at the cellular level. The various types of cells in the brain can respond differently to estradiol treatment based on the signaling properties of the cell, as well as which receptor, estrogen receptor alpha and/or estrogen receptor beta, is expressed. Taken together, these findings suggest that the estradiol-induced activation of membrane-initiated signaling pathways occurs in a cell-type specific manner and can differentially influence how the cells respond to various insults.

  8. Structural Basis for Receptor Activity-Modifying Protein-Dependent Selective Peptide Recognition by a G Protein-Coupled Receptor.

    Science.gov (United States)

    Booe, Jason M; Walker, Christopher S; Barwell, James; Kuteyi, Gabriel; Simms, John; Jamaluddin, Muhammad A; Warner, Margaret L; Bill, Roslyn M; Harris, Paul W; Brimble, Margaret A; Poyner, David R; Hay, Debbie L; Pioszak, Augen A

    2015-06-18

    Association of receptor activity-modifying proteins (RAMP1-3) with the G protein-coupled receptor (GPCR) calcitonin receptor-like receptor (CLR) enables selective recognition of the peptides calcitonin gene-related peptide (CGRP) and adrenomedullin (AM) that have diverse functions in the cardiovascular and lymphatic systems. How peptides selectively bind GPCR:RAMP complexes is unknown. We report crystal structures of CGRP analog-bound CLR:RAMP1 and AM-bound CLR:RAMP2 extracellular domain heterodimers at 2.5 and 1.8 Å resolutions, respectively. The peptides similarly occupy a shared binding site on CLR with conformations characterized by a β-turn structure near their C termini rather than the α-helical structure common to peptides that bind related GPCRs. The RAMPs augment the binding site with distinct contacts to the variable C-terminal peptide residues and elicit subtly different CLR conformations. The structures and accompanying pharmacology data reveal how a class of accessory membrane proteins modulate ligand binding of a GPCR and may inform drug development targeting CLR:RAMP complexes. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  9. Wnt/Wingless signaling through β-catenin requires the function of both LRP/Arrow and frizzled classes of receptors

    Directory of Open Access Journals (Sweden)

    Varmus Harold

    2003-05-01

    Full Text Available Abstract Background Wnt/Wingless (Wg signals are transduced by seven-transmembrane Frizzleds (Fzs and the single-transmembrane LDL-receptor-related proteins 5 or 6 (LRP5/6 or Arrow. The aminotermini of LRP and Fz were reported to associate only in the presence of Wnt, implying that Wnt ligands form a trimeric complex with two different receptors. However, it was recently reported that LRPs activate the Wnt/β-catenin pathway by binding to Axin in a Dishevelled – independent manner, while Fzs transduce Wnt signals through Dishevelled to stabilize β-catenin. Thus, it is possible that Wnt proteins form separate complexes with Fzs and LRPs, transducing Wnt signals separately, but converging downstream in the Wnt/β-catenin pathway. The question then arises whether both receptors are absolutely required to transduce Wnt signals. Results We have established a sensitive luciferase reporter assay in Drosophila S2 cells to determine the level of Wg – stimulated signaling. We demonstrate here that Wg can synergize with DFz2 and function cooperatively with LRP to activate the β-catenin/Armadillo signaling pathway. Double-strand RNA interference that disrupts the synthesis of either receptor type dramatically impairs Wg signaling activity. Importantly, the pronounced synergistic effect of adding Wg and DFz2 is dependent on Arrow and Dishevelled. The synergy requires the cysteine-rich extracellular domain of DFz2, but not its carboxyterminus. Finally, mammalian LRP6 and its activated forms, which lack most of the extracellular domain of the protein, can activate the Wg signaling pathway and cooperate with Wg and DFz2 in S2 cells. We also show that the aminoterminus of LRP/Arr is required for the synergy between Wg and DFz2. Conclusion Our study indicates that Wg signal transduction in S2 cells depends on the function of both LRPs and DFz2, and the results are consistent with the proposal that Wnt/Wg signals through the aminoterminal domains of its dual

  10. Soluble urokinase plasminogen activator receptor is associated with subclinical organ damage and cardiovascular events

    DEFF Research Database (Denmark)

    Sehestedt, T; Lyngbæk, S; Eugen-Olsen, J

    2011-01-01

    The soluble urokinase plasminogen activator receptor (suPAR) is a plasma marker of low grade inflammation and has been associated with cardiovascular risk. We wanted to investigate whether suPAR was associated with markers of subclinical organ damage.......The soluble urokinase plasminogen activator receptor (suPAR) is a plasma marker of low grade inflammation and has been associated with cardiovascular risk. We wanted to investigate whether suPAR was associated with markers of subclinical organ damage....

  11. New analogues of ACPD with selective activity for group II metabotropic glutamate receptors

    DEFF Research Database (Denmark)

    Bräuner-Osborne, Hans; Madsen, U; Mikiciuk-Olasik, E

    1997-01-01

    In this study we have determined the pharmacology of a series of 1-aminocyclopentane-1,3-dicarboxylic acid (1,3-ACPD) analogues at cloned metabotropic glutamic acid (mGlu) receptors. The new analogues comprise the four possible stereoisomers of 1-amino-1-carboxycyclopentane-3-acetic acid (1,3-hom...... showed any activity at mGlu1a or mGlu4a receptors. These homo-ACPD's show a higher degree of subtype selectivity than the parent compound (1SR,3RS)-ACPD. In addition none of the compounds demonstrated any activity at ionotropic Glu receptors....

  12. Structure-activity relationships for the antifungal activity of selective estrogen receptor antagonists related to tamoxifen.

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    Arielle Butts

    Full Text Available Cryptococcosis is one of the most important invasive fungal infections and is a significant contributor to the mortality associated with HIV/AIDS. As part of our program to repurpose molecules related to the selective estrogen receptor modulator (SERM tamoxifen as anti-cryptococcal agents, we have explored the structure-activity relationships of a set of structurally diverse SERMs and tamoxifen derivatives. Our data provide the first insights into the structural requirements for the antifungal activity of this scaffold. Three key molecular characteristics affecting anti-cryptococcal activity emerged from our studies: 1 the presence of an alkylamino group tethered to one of the aromatic rings of the triphenylethylene core; 2 an appropriately sized aliphatic substituent at the 2 position of the ethylene moiety; and 3 electronegative substituents on the aromatic rings modestly improved activity. Using a cell-based assay of calmodulin antagonism, we found that the anti-cryptococcal activity of the scaffold correlates with calmodulin inhibition. Finally, we developed a homology model of C. neoformans calmodulin and used it to rationalize the structural basis for the activity of these molecules. Taken together, these data and models provide a basis for the further optimization of this promising anti-cryptococcal scaffold.

  13. Dopaminergic activation of estrogen receptors induces fos expression within restricted regions of the neonatal female rat brain.

    Directory of Open Access Journals (Sweden)

    Kristin M Olesen

    2008-05-01

    Full Text Available Steroid receptor activation in the developing brain influences a variety of cellular processes that endure into adulthood, altering both behavior and physiology. Recent data suggests that dopamine can regulate expression of progestin receptors within restricted regions of the developing rat brain by activating estrogen receptors in a ligand-independent manner. It is unclear whether changes in neuronal activity induced by dopaminergic activation of estrogen receptors are also region specific. To investigate this question, we examined where the dopamine D1-like receptor agonist, SKF 38393, altered Fos expression via estrogen receptor activation. We report that dopamine D1-like receptor agonist treatment increased Fos protein expression within many regions of the developing female rat brain. More importantly, prior treatment with an estrogen receptor antagonist partially reduced D1-like receptor agonist-induced Fos expression only within the bed nucleus of the stria terminalis and the central amygdala. These data suggest that dopaminergic activation of estrogen receptors alters neuronal activity within restricted regions of the developing rat brain. This implies that ligand-independent activation of estrogen receptors by dopamine might organize a unique set of behaviors during brain development in contrast to the more wide spread ligand activation of estrogen receptors by estrogen.

  14. Dopaminergic Activation of Estrogen Receptors Induces Fos Expression within Restricted Regions of the Neonatal Female Rat Brain

    Science.gov (United States)

    Olesen, Kristin M.; Auger, Anthony P.

    2008-01-01

    Steroid receptor activation in the developing brain influences a variety of cellular processes that endure into adulthood, altering both behavior and physiology. Recent data suggests that dopamine can regulate expression of progestin receptors within restricted regions of the developing rat brain by activating estrogen receptors in a ligand-independent manner. It is unclear whether changes in neuronal activity induced by dopaminergic activation of estrogen receptors are also region specific. To investigate this question, we examined where the dopamine D1-like receptor agonist, SKF 38393, altered Fos expression via estrogen receptor activation. We report that dopamine D1-like receptor agonist treatment increased Fos protein expression within many regions of the developing female rat brain. More importantly, prior treatment with an estrogen receptor antagonist partially reduced D1-like receptor agonist-induced Fos expression only within the bed nucleus of the stria terminalis and the central amygdala. These data suggest that dopaminergic activation of estrogen receptors alters neuronal activity within restricted regions of the developing rat brain. This implies that ligand-independent activation of estrogen receptors by dopamine might organize a unique set of behaviors during brain development in contrast to the more wide spread ligand activation of estrogen receptors by estrogen. PMID:18478050

  15. In silico analysis of the histaprodifen induced activation pathway of the guinea-pig histamine H1-receptor

    Science.gov (United States)

    Straßer, Andrea; Wittmann, Hans-Joachim

    2010-09-01

    The binding of (partial) agonists in the binding pocket of biogenic amine receptors induces a conformational change from the inactive to the active state of the receptors. There is only little knowledge about the binding pathways of ligands into binding pocket on molecular level. So far, it was not possible with molecular dynamic simulations to observe the ligand binding and receptor activation. Furthermore, there is nearly nothing known, in which state of ligand binding, the receptor gets activated. The aim of this study was to get more detailed insight into the process of ligand binding and receptor activation. With the recently developed LigPath algorithm, we scanned the potential energy surface of the binding process of dimeric histaprodifen, a partial agonist at the histamine H1-receptor, into the guinea pig histamine H1-receptor, taking also into account the receptor activation. The calculations exhibited large conformational changes of Trp6.48 and Phe6.55 during ligand binding and receptor activation. Additionally, conformational changes were also observed for Phe6.52, Tyr6.51 and Phe6.44. Conformational changes of Trp6.48 and Phe6.52 are discussed in literature as rotamer toggle switch in context with receptor activation. Additionally, the calculations indicate that the binding of dimeric histaprodifen, accompanied by receptor activation is energetically preferred. In general, this study gives new, theoretical insights onto ligand binding and receptor activation on molecular level.

  16. Activation of sigma-1 receptor chaperone in the treatment of neuropsychiatric diseases and its clinical implication

    Directory of Open Access Journals (Sweden)

    Kenji Hashimoto

    2015-01-01

    Full Text Available Endoplasmic reticulum (ER protein sigma-1 receptor represents unique chaperone activity in the central nervous system, and it exerts a potent influence on a number of neurotransmitter systems. Several lines of evidence suggest that activation of sigma-1 receptor plays a role in the pathophysiology of neuropsychiatric diseases, as well as in the mechanisms of some therapeutic drugs and neurosteroids. Preclinical studies showed that some selective serotonin reuptake inhibitors (SSRIs; fluvoxamine, fluoxetine, excitalopram, donepezil, and ifenprodil act as sigma-1 receptor agonists. Furthermore, sigma-1 receptor agonists could improve the N-methyl-D-aspartate (NMDA antagonist phencyclidine (PCP-induced cognitive deficits in mice. A study using positron emission tomography have demonstrated that an oral administration of fluvoxamine or donepezil could bind to sigma-1 receptor in the healthy human brain, suggesting that sigma-1 receptor might be involved in the therapeutic mechanisms of these drugs. Moreover, case reports suggest that sigma-1 receptor agonists, including fluvoxamine, and ifenprodil, may be effective in the treatment of cognitive impairment in schizophrenia, delirium in elderly people, and flashbacks in post-traumatic stress disorder. In this review article, the author would like to discuss the clinical implication of sigma-1 receptor agonists, including endogenous neurosteroids, in the neuropsychiatric diseases.

  17. Activation of peroxisome proliferator-activated receptor gamma is crucial for antitumoral effects of 6-iodolactone.

    Science.gov (United States)

    Nava-Villalba, Mario; Nuñez-Anita, Rosa E; Bontempo, Alexander; Aceves, Carmen

    2015-09-17

    Molecular iodine (I2) exhibits antiproliferative and apoptotic effects on in vivo and in vitro cancer models. These effects are thought to be mediated by an iodinated arachidonic acid derivative, 6-iodolactone (6IL), and one of the proposed mechanisms is that 6IL activates Peroxisome Proliferator-Activated Receptors type gamma (PPARG). These receptors have been implicated in the inhibition of carcinogenic processes, in addition to their classical role in maintaining lipid and glucose homeostasis. The aim of this study was to determine whether PPARG participates in the 6IL antiproliferative and apoptotic effects on the mammary cancer cell line MCF-7. The 6IL/PPARG complex was inhibited by the PPARG antagonist GW9662, in both an endogenous and overexpressed (adenoviral vector infection) context, and stable PPARG-knockdown MCF-7 cells (RNA interference, confirmed with hydrolysis probes and Western blot), were used to corroborate the PPARG participation. 6IL effects on proliferation (measured by Trypan Blue exclusion) and apoptosis (phosphatidylserine identification by flow cytometer) were evaluated in conditions of chemical inhibition (GW9662) and silencing (RNA interference). A wound-healing assay was conducted on wild-type and stable PPARG-knockdown MCF-7 cells to evaluate the antimigrational effect of 6IL. Caspase-8 activity was evaluated to determine if the extrinsic pathway is involved in the effects of 6IL and I2 treatment. Antiproliferative and pro-apoptotic 6IL effects require the activation of PPARG. In addition, wound-healing assays show that 6IL is able to inhibit MCF-7 cell migration and that PPARG plays a role in this phenomenon. Finally, the data exclude the participation of the extrinsic apoptotic pathway in 6IL- and I2-induced apoptosis. These results support the previously proposed mechanism, in which the I2 effects are mediated by 6IL, and they provide further support for the use of I2 as coadjuvant in breast cancer treatment.

  18. Platelet-activating factor (PAF) receptor binding activity of the roots of Enicosanthellum pulchrum.

    Science.gov (United States)

    Nordin, Noraziah; Jalil, Juriyati; Jantan, Ibrahim; Murad, Shahnaz

    2012-03-01

    Enicosanthellum pulchrum (King) Heusden (Annonaceae) is a coniferous tree that is confined to mountain forests. The chemical constituents of this species have been studied previously; however, its biological activity has never been investigated before and is reported here for the first time. The extracts, fractions and compounds from the roots of E. pulchrum were investigated for their inhibitory effects on platelet-activating factor (PAF) receptor binding to rabbit platelets using (3)H-PAF as a ligand. The PAF receptor binding inhibitory effect using rabbit platelets was determined in vitro by measuring the difference between total amount of bound (3)H-PAF in the presence and the absence of excess unlabelled PAF. The compounds were isolated by bioassay-guided fractionation and their structures were elucidated by spectroscopic techniques. Among the extracts tested, the ethyl acetate extract was the most active with 85.6% inhibition, while hexane and methanol extracts showed 40.2 and 42.5% inhibition, respectively. Fractionation of the ethyl acetate extract using vacuum liquid chromatography (VLC) yielded six fractions AEA(I--VI). Chromatography fraction AEA(VI) yielded a new compound, 1-(2',3',4'-trimethoxyphenyl)hexan-1-ol, while fraction AEA(III) afforded three compounds, namely liriodenine, cleistopholine and dehydroanonaine. 1-(2',3',4'-Trimethoxyphenyl)hexan-1-ol, cleistopholine and dehydroanonaine showed relatively strong inhibition with IC(50) values of 26.6, 50.2 and 45.4 µM, respectively. The results suggest that these compounds could be responsible for the PAF antagonistic activity of the ethyl acetate extract of this plant.

  19. Activators of peroxisome proliferator-activated receptors protect human skin from ultraviolet-B-light-induced inflammation.

    Science.gov (United States)

    Kippenberger, S; Loitsch, S M; Grundmann-Kollmann, M; Simon, S; Dang, T A; Hardt-Weinelt, K; Kaufmann, R; Bernd, A

    2001-12-01

    Peroxisome proliferator-activated receptors (PPAR) are members of a nuclear receptor superfamily, which were initially described in the context of fatty acid degradation and adipocyte differentiation. In this study we tested the hypothesis that peroxisome proliferator-activated receptor activation also controls inflammation. In an in vitro model with human keratinocytes inflammation was mimicked by irradiation with ultraviolet B light (150 mJ per cm(2)). Activators for PPAR-alpha (WY-14,643, clofibrate) were shown to reverse ultraviolet-B-light-mediated expression of inflammatory cytokines (interleukin-6, interleukin-8). An activator preferentially for PPAR-beta (bezafibrate) did not show prominent effects on interleukin-6 and interleukin-8 expression. The anti-inflammatory action of WY-14,643 on skin cells was further demonstrated by in vivo testings in which topically applied WY-14,643 markedly increased the minimal erythema dose in ultraviolet-B-irradiated skin. Additionally, it was shown that ultraviolet B irradiation led to a decrease of all three peroxisome proliferator-activated receptor subsets at the mRNA level. Also transactivation of peroxisome proliferator response element was attenuated by ultraviolet B irradiation. The downregulation of peroxisome proliferator-activated receptors by ultraviolet B irradiation provides a possible mechanism that leads to exaggerated and prolonged inflammation. This work suggests the possibility of PPAR-alpha activators as novel nonsteroidal anti-inflammatory drugs in the topical treatment of common inflammatory skin diseases such as atopic dermatitis, psoriasis, and photodermatitis.

  20. Adenosine promotes alternative macrophage activation via A2A and A2B receptors

    Science.gov (United States)

    Csóka, Balázs; Selmeczy, Zsolt; Koscsó, Balázs; Németh, Zoltán H.; Pacher, Pál; Murray, Peter J.; Kepka-Lenhart, Diane; Morris, Sidney M.; Gause, William C.; Leibovich, S. Joseph; Haskó, György

    2012-01-01

    Adenosine has been implicated in suppressing the proinflammatory responses of classically activated macrophages induced by Th1 cytokines. Alternative macrophage activation is induced by the Th2 cytokines interleukin (IL)-4 and IL-13; however, the role of adenosine in governing alternative macrophage activation is unknown. We show here that adenosine treatment of IL-4- or IL-13-activated macrophages augments the expression of alternative macrophage markers arginase-1, tissue inhibitor of matrix metalloproteinase-1 (TIMP-1), and macrophage galactose-type C-type lectin-1. The stimulatory effect of adenosine required primarily A2B receptors because the nonselective adenosine receptor agonist 5′-N-ethylcarboxamidoadenosine (NECA) increased both arginase activity (EC50=261.8 nM) and TIMP-1 production (EC50=80.67 nM), and both pharmacologic and genetic blockade of A2B receptors prevented the effect of NECA. A2A receptors also contributed to the adenosine augmentation of IL-4-induced TIMP-1 release, as both adenosine and NECA were less efficacious in augmenting TIMP-1 release by A2A receptor-deficient than control macrophages. Of the transcription factors known to drive alternative macrophage activation, CCAAT-enhancer-binding protein β was required, while cAMP response element-binding protein and signal transducer and activator of transcription 6 were dispensable in mediating the effect of adenosine. We propose that adenosine receptor activation suppresses inflammation and promotes tissue restitution, in part, by promoting alternative macrophage activation.—Csóka, B., Selmeczy, Z., Koscsó, B., Németh, Z. H., Pacher, P., Murray, P. J., Kepka-Lenhart, D., Morris S. M., Jr., Gause, W. C., Leibovich, S. J., Haskó, G. Adenosine promotes alternative macrophage activation via A2A and A2B receptors. PMID:21926236

  1. Characterization of CB1 cannabinoid receptor immunoreactivity in postmortem human brain homogenates.

    Science.gov (United States)

    De Jesús, M López; Sallés, J; Meana, J J; Callado, L F

    2006-06-30

    The CB1 cannabinoid receptor (CB1) is the predominant type of cannabinoid receptor in the CNS, in which it displays a unique anatomical distribution and is present at higher densities than most other known seven transmembrane domain receptors. Nevertheless, as with almost all seven transmembrane domain receptors, the tertiary and quaternary structure of this receptor is still unknown. Studies of CB1 in rat cerebral tissue are scarce, and even less is known regarding the expression of CB1 in the human brain. Thus, the aim of the present work was to characterize CB1 expression in membranes from postmortem human brain using specific antisera raised against this protein. Western blot analysis of P1 and P2 fractions, and crude plasma membrane preparations from the prefrontal cortex showed that CB1 migrated as a 60 kDa monomer under reducing conditions. These data were confirmed by blotting experiments carried out with human U373MG astrocytoma cells as a positive control for CB1 expression and wild-type CHO cells as negative control. In addition, when proteins were solubilized in the absence of dithiothreitol, the anti-human CB1 antiserum detected a new band migrating at around 120 kDa corresponding in size to a putative CB1 dimer. This band was sensitive to reducing agents (50 mM dithiothreitol) and showed sodium dodecylsulphate stability, suggesting the existence of disulfide-linked CB1 dimers in the membrane preparations. Important differences in the anatomical distribution of CB1 were observed with regard to that described previously in monkey and rat; in the human brain, CB1 levels were higher in cortex and caudate than in the cerebellum.

  2. The role of transmembrane segment II in 7TM receptor activation

    DEFF Research Database (Denmark)

    Benned-Jensen, Tau; Rosenkilde, M M

    2009-01-01

    , in addition to biogenic amines and retinal a.m.o. bind to residues in this region. Consequently the major helical movements occur here during activation, as described recently in the Global Toggle Switch Model for Family A (also known as rhodopsin-like) members of the 7TM receptors. As a result, the minor......, accumulating evidence emphasize that this is not the case. In this review, we focus on TM-II with an emphasis on position II:20/2.60, and present data from structure-activity studies on a range of Family A 7TM receptors including chemokine, ghrelin and melanocortin receptors in addition to the orphan EBI2...... suggesting that TM-II has important functions for both ligand-dependent and -independent activation of 7TM receptors....

  3. Ethanol inhibits epileptiform activity and NMDA receptor-mediated synaptic transmission in rat amygdaloid slices

    Energy Technology Data Exchange (ETDEWEB)

    Gean, P.W. (Cheng-Kung Univ., Tainan (Taiwan))

    1992-02-26

    The effect of ethanol on the epileptiform activity induced by Mg{sup ++}-free solution was studied in rat amygdalar slices using intracellular recording techniques. The spontaneous and evoked epileptiform discharges consisting of an initial burst followed by afterdischarges were observed 20-30 min after switching to Mg{sup ++}-free medium. Superfusion with ethanol reversibly reduced the duration of spontaneous and evoked bursting discharges in a concentration-dependent manner. Synaptic response mediated by N-methyl-D-aspartate (NMDA) receptor activation was isolated by application of a solution containing the non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and either in Mg{sup ++}-free solution or in the presence of 50 {mu}M bicuculline. Application of ethanol reversibly suppressed the duration of NMDA receptor-mediated synaptic response. These results suggest that intoxicating concentrations of ethanol possess anticonvulsant activity through blocking the NMDA receptor-mediated synaptic excitation.

  4. Stat3 activates the receptor tyrosine kinase like orphan receptor-1 gene in chronic lymphocytic leukemia cells.

    Directory of Open Access Journals (Sweden)

    Ping Li

    Full Text Available BACKGROUND: The receptor tyrosine kinase like orphan receptor (ROR-1 gene is overexpressed in chronic lymphocytic leukemia (CLL. Because Stat3 is constitutively activated in CLL and sequence analysis revealed that the ROR1 promoter harbors gamma-interferon activation sequence-like elements typically activated by Stat3, we hypothesized that Stat3 activates ROR1. METHODOLOGY/PRINCIPAL FINDINGS: Because IL-6 induced Stat3 phosphorylation and upregulated Ror1 protein levels in MM1 cells, we used these cells as a model. We transfected MM1 cells with truncated ROR1 promoter luciferase reporter constructs and found that IL-6 induced luciferase activity of ROR1-195 and upstream constructs. Co-transfection with Stat3 siRNA reduced the IL-6-induced luciferase activity, suggesting that IL-6 induced luciferase activity by activating Stat3. EMSA and the ChIP assay confirmed that Stat3 binds ROR1, and EMSA studies identified two Stat3 binding sites. In CLL cells, EMSA and ChIP studies determined that phosphorylated Stat3 bound to the ROR1 promoter at those two ROR1 promoter sites, and ChIP analysis showed that Stat3 co-immunoprecipitated DNA of STAT3, ROR1, and several Stat3-regulated genes. Finally, like STAT3-siRNA in MM1 cells, STAT3-shRNA downregulated STAT3, ROR1, and STAT3-regulated genes and Stat3 and Ror1 protein levels in CLL cells. CONCLUSION/SIGNIFICANCE: Our data suggest that constitutively activated Stat3 binds to the ROR1 promoter and activates ROR1 in CLL cells.

  5. Soluble urokinase plasminogen activator receptor is a marker of dysmetabolism in HIV-infected patients receiving highly active antiretroviral therapy

    DEFF Research Database (Denmark)

    Andersen, Ove; Eugen-Olsen, Jesper; Kofoed, Kristian

    2008-01-01

    Circulating soluble urokinase plasminogen activator receptor (suPAR) reflects the immune and pro-inflammatory status of the HIV-infected patient. Highly active antiretroviral therapy (HAART) suppresses suPAR. Independent of the immune response to HAART, suPAR remains elevated in some HIV-infected......Circulating soluble urokinase plasminogen activator receptor (suPAR) reflects the immune and pro-inflammatory status of the HIV-infected patient. Highly active antiretroviral therapy (HAART) suppresses suPAR. Independent of the immune response to HAART, suPAR remains elevated in some HIV......-HDL-cholesterol and inversely with Rd, NOGM and limb fat (P

  6. Class A scavenger receptor promotes osteoclast differentiation via the enhanced expression of receptor activator of NF-{kappa}B (RANK)

    Energy Technology Data Exchange (ETDEWEB)

    Takemura, Kenichi [Department of Cell Pathology, Graduate School of Medical Sciences, Kumamoto University, 1-1-1 Honjo, Kumamoto 860-8556 (Japan); Department of Orthopaedic and Neuro-Musculoskeletal Surgery, Graduate School of Medical Sciences, Kumamoto University, Kumamoto (Japan); Sakashita, Naomi; Fujiwara, Yukio; Komohara, Yoshihiro; Lei, XiaoFeng; Ohnishi, Koji [Department of Cell Pathology, Graduate School of Medical Sciences, Kumamoto University, 1-1-1 Honjo, Kumamoto 860-8556 (Japan); Suzuki, Hiroshi [National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Hokkaido (Japan); Kodama, Tatsuhiko [Department of Molecular Biology and Medicine, Research Center for Advanced Science and Technology, The University of Tokyo, Tokyo (Japan); Mizuta, Hiroshi [Department of Orthopaedic and Neuro-Musculoskeletal Surgery, Graduate School of Medical Sciences, Kumamoto University, Kumamoto (Japan); Takeya, Motohiro, E-mail: takeya@kumamoto-u.ac.jp [Department of Cell Pathology, Graduate School of Medical Sciences, Kumamoto University, 1-1-1 Honjo, Kumamoto 860-8556 (Japan)

    2010-01-22

    Osteoclasts originate from bone marrow monocyte/macrophage lineage cells, and their differentiation depends on macrophage colony-stimulating factor (M-CSF) and receptor activator nuclear factor kappa B (RANK) ligand. Class A scavenger receptor (SR-A) is one of the principal functional molecules of macrophages, and its level of expression declines during osteoclast differentiation. To investigate the role of SR-A in osteoclastogenesis, we examined pathological changes in femoral bone and the expression levels of osteoclastogenesis-related molecules in SR-A{sup -/-} mice. The femoral osseous density of SR-A{sup -/-} mice was higher than that of SR-A{sup +/+} mice, and the number of multinucleated osteoclasts was significantly decreased. An in vitro differentiation assay revealed that the differentiation of multinucleated osteoclasts from bone marrow-derived progenitor cells is impaired in SR-A{sup -/-} mice. Elimination of SR-A did not alter the expression level of the M-CSF receptor, c-fms; however, the expression levels of RANK and RANK-related osteoclast-differentiation molecules such as nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 1 (NFATc1) and microphthalmia-associated transcription factor (MITF) significantly decreased. Furthermore, acetylated low-density lipoprotein (AcLDL), an SR-A ligand, significantly increased the expression level of RANK and MITF during osteoclast differentiation. These data indicate that SR-A promotes osteoclastogenesis via augmentation of the expression level of RANK and its related molecules.

  7. Toll-like receptor 3-activated macrophages confer anti-HCV activity to hepatocytes through exosomes.

    Science.gov (United States)

    Zhou, Yu; Wang, Xu; Sun, Li; Zhou, Li; Ma, Tong-Cui; Song, Li; Wu, Jian-Guo; Li, Jie-Liang; Ho, Wen-Zhe

    2016-12-01

    Exosomes are a class of cell-released small vesicles that mediate intercellular communication by delivering functional factors to recipient cells. During hepatitis C virus (HCV) infection, the interaction between liver resident macrophages and hepatocytes is a key component in liver innate immunity. In this study, we explored the role of exosomes in the delivery of innate anti-HCV factors to hepatocytes from macrophages. We showed that supernatant from TLR3-activated macrophage cultures could efficiently inhibit HCV replication in Huh7 cells. This macrophage-mediated anti-HCV activity was through exosomes because inhibiting exosomes could abrogate the action of macrophages. Further analyses demonstrated that TLR3-activated macrophages release exosomes that contain anti-HCV microRNA (miRNA)-29 family members. Inhibiting miRNA29 could restore HCV replication. These findings suggest a novel antiviral mechanism in liver innate immunity against HCV infection and provide insights to support further studies on developing exosome-based delivery system for disease treatment.-Zhou, Y., Wang, X., Sun, L., Zhou, L., Ma, T.-C., Song, L., Wu, J.-G., Li, J.-L., Ho, W.-Z. Toll-like receptor 3-activated macrophages confer anti-HCV activity to hepatocytes through exosomes. © FASEB.

  8. Structure-activity relationships in human toll-like receptor 7-active imidazoquinoline analogues.

    Science.gov (United States)

    Shukla, Nikunj M; Malladi, Subbalakshmi S; Mutz, Cole A; Balakrishna, Rajalakshmi; David, Sunil A

    2010-06-10

    Engagement of toll-like receptors serve to link innate immune responses with adaptive immunity and can be exploited as powerful vaccine adjuvants for eliciting both primary and anamnestic immune responses. TLR7 agonists are highly immunostimulatory without inducing dominant proinflammatory cytokine responses. A structure-activity study was conducted on the TLR7-agonistic imidazoquinolines, starting with 1-(4-amino-2-((ethylamino)methyl)-1H-imidazo[4,5-c]quinolin-1-yl)-2-methylpropan-2-ol as a lead. Modifications of the secondary amine of the C2 ethylaminomethylene side chain are poorly tolerated. The 4-amino group must be retained for activity. Replacement of the imidazole ring of the scaffold with triazole or cyclic urea led to complete loss of activity. A systematic exploration of N(1)-benzyl-C2-alkyl substituents showed a very distinct relationship between alkyl length and TLR7-agonistic potency with the optimal compound bearing a C2-n-butyl group. Transposition of the N(1) and C2 substituents led to the identification of an extremely active TLR7-agonistic compound with an EC(50) value of 8.6 nM. The relative potencies in human TLR7-based primary reporter gene assays were paralleled by interferon-alpha induction activities in whole human blood models.

  9. Tonically Active α5GABAA Receptors Reduce Motoneuron Excitability and Decrease the Monosynaptic Reflex

    Directory of Open Access Journals (Sweden)

    Martha Canto-Bustos

    2017-09-01

    Full Text Available Motoneurons, the final common path of the Central Nervous System (CNS, are under a complex control of its excitability in order to precisely translate the interneuronal pattern of activity into skeletal muscle contraction and relaxation. To fulfill this relevant function, motoneurons are provided with a vast repertoire of receptors and channels, including the extrasynaptic GABAA receptors which have been poorly investigated. Here, we confirmed that extrasynaptic α5 subunit-containing GABAA receptors localize with choline acetyltransferase (ChAT positive cells, suggesting that these receptors are expressed in turtle motoneurons as previously reported in rodents. In these cells, α5GABAA receptors are activated by ambient GABA, producing a tonic shunt that reduces motoneurons’ membrane resistance and affects their action potential firing properties. In addition, α5GABAA receptors shunted the synaptic excitatory inputs depressing the monosynaptic reflex (MSR induced by activation of primary afferents. Therefore, our results suggest that α5GABAA receptors may play a relevant physiological role in motor control.

  10. Molecular mechanism of 7TM receptor activation--a global toggle switch model

    DEFF Research Database (Denmark)

    Schwartz, Thue W; Frimurer, Thomas M; Holst, Birgitte

    2006-01-01

    The multitude of chemically highly different agonists for 7TM receptors apparently do not share a common binding mode or active site but nevertheless act through induction of a common molecular activation mechanism. A global toggle switch model is proposed for this activation mechanism to reconcile...

  11. Gastrin-Releasing Peptide Receptor Mediates Activation of the Epidermal Growth Factor Receptor in Lung Cancer Cells

    Directory of Open Access Journals (Sweden)

    Sufi Mary Thomas

    2005-04-01

    Full Text Available Gastrin-releasing peptide receptor (GRPR and the epidermal growth factor receptor (EGFR are expressed in several cancers including non-small cell lung carcinoma (NSCLC. Here we demonstrate the activation of EGFR by the GRPR ligand, gastrin-releasing peptide (GRP, in NSCLC cells. GRP induced rapid activation of p44/42 MAPK in lung cancer cells through EGFR. GRP-mediated activation of MAPK in NSCLC cells was abrogated by pretreatment with the anti-EGFR-neutralizing antibody, C225. Pretreatment of NSCLC cells with neutralizing antibodies to the EGFR ligands, TGF-α or HB-EGF, also decreased GRP-mediated MAPK activation. On matrix metalloproteinase (MMP inhibition, GRP failed to activate MAPK in NSCLC cells. EGF and GRP both stimulated NSCLC proliferation, and inhibition of either EGFR or GRPR resulted in cell death. Combining a GRPR antagonist with the EGFR tyrosine kinase inhibitor, gefitinib, resulted in additive cytotoxic effects. Additive effects were seen at gefitinib concentrations from 1 to 18μM, encompassing the ID50 values of both gefitinib-sensitive and gefitinib-resistant NSCLC cell lines. Because a major effect of GRPR appears to be promoting the release of EGFR ligand, this study suggests that a greater inhibition of cell proliferation may occur by abrogating EGFR ligand release in consort with inhibition of EGFR.

  12. GABAA receptor modulation by piperine and a non-TRPV1 activating derivative☆

    Science.gov (United States)

    Khom, Sophia; Strommer, Barbara; Schöffmann, Angela; Hintersteiner, Juliane; Baburin, Igor; Erker, Thomas; Schwarz, Thomas; Schwarzer, Christoph; Zaugg, Janine; Hamburger, Matthias; Hering, Steffen

    2013-01-01

    The action of piperine (the pungent component of pepper) and its derivative SCT-66 ((2E,4E)-5-(1,3-benzodioxol-5-yl))-N,N-diisobutyl-2,4-pentadienamide) on different gamma-aminobutyric acid (GABA) type A (GABAA) receptors, transient-receptor-potential-vanilloid-1 (TRPV1) receptors and behavioural effects were investigated. GABAA receptor subtypes and TRPV1 receptors were expressed in Xenopus laevis oocytes. Modulation of GABA-induced chloride currents (IGABA) by piperine and SCT-66 and activation of TRPV1 was studied using the two-microelectrode-voltage-clamp technique and fast perfusion. Their effects on explorative behaviour, thermoregulation and seizure threshold were analysed in mice. Piperine acted with similar potency on all GABAA receptor subtypes (EC50 range: 42.8 ± 7.6 μM (α2β2)–59.6 ± 12.3 μM (α3β2)). IGABA modulation by piperine did not require the presence of a γ2S-subunit, suggesting a binding site involving only α and β subunits. IGABA activation was slightly more efficacious on receptors formed from β2/3 subunits (maximal IGABA stimulation through α1β3 receptors: 332 ± 64% and α1β2: 271 ± 36% vs. α1β1: 171 ± 22%, p piperine, with different subunit-dependence. Both compounds induced anxiolytic, anticonvulsant effects and reduced locomotor activity; however, SCT-66 induced stronger anxiolysis without decreasing body temperature and without the proconvulsive effects of TRPV1 activation and thus may serve as a scaffold for the development of novel GABAA receptor modulators. PMID:23623790

  13. P2X7 receptor and NLRP3 inflammasome activation in head and neck cancer.

    Science.gov (United States)

    Bae, Ju Young; Lee, Sang-Woo; Shin, Yong-Hwan; Lee, Jong-Ho; Jahng, Jeong Won; Park, Kyungpyo

    2017-07-25

    In this study, we investigated purinergic receptor P2X7 and NACHT, LRR and PYD domains-containing protein 3 (NLRP3) inflammasome expressions, and their role in head and neck cancer. We found upregulation of purinergic receptor P2X7 and all NLRP3 inflammasome components in biopsied head and neck squamous cell carcinoma tissues. Similarly, the expression of purinergic receptor P2X7, apoptosis-associated speck-like protein containing CARD, and pro-form caspase 1 in A253 cells derived from epidermoid carcinoma were highly upregulated in comparison to normal Human Salivary Gland cell line. Active caspase-1 and its final product, active interleukin-1β, both increased in primed A253 cells stimulated with purinergic receptor P2X7 agonists, while this elevated NLRP3 inflammasome activity was suppressed by purinergic receptor P2X7 antagonists. However, we observed none of these effects in Human Salivary Gland cells. Inhibition of both NLRP3 inflammasome and purinergic receptor P2X7 led to the significant cell death of primed A253 cells, but had no effect on the viability of primed HSG cells or the primary cultured human fibroblast cells. Furthermore, inhibition of either purinergic receptor P2X7 or NLRP3 inflammasome decreased invasiveness of A253, and this effect became more evident when both purinergic receptor P2X7 and NLRP3 inflammasome were simultaneously blocked. Therefore, it is concluded that the purinergic receptor P2X7 and the activation of NLRP3 inflammasome play important roles in the survival and invasiveness of head and neck squamous cell carcinoma in humans.

  14. Soluble Urokinase Plasminogen Activator Receptor and Outcomes in Patients with Diabetes on Hemodialysis.

    Science.gov (United States)

    Drechsler, Christiane; Hayek, Salim S; Wei, Changli; Sever, Sanja; Genser, Bernd; Krane, Vera; Meinitzer, Andreas; März, Winfried; Wanner, Christoph; Reiser, Jochen

    2017-08-07

    Soluble urokinase plasminogen activator receptor is a novel biomarker strongly predictive of cardiovascular outcomes implicated in the pathogenesis of kidney disease. Soluble urokinase plasminogen activator receptor levels, however, correlate with declining kidney function. It is unclear whether soluble urokinase plasminogen activator receptor levels remain associated with outcomes in patients with ESRD. We measured plasma soluble urokinase plasminogen activator receptor levels in 1175 patients (mean age =66±8 years old, 54% men) with type 2 diabetes mellitus on hemodialysis participating in the German Diabetes and Dialysis Study followed for a median of 4 years for outcomes including all-cause death, cardiovascular events, and infection-related mortality. Survival analysis was performed using stepwise Cox proportional hazards models adjusted for potential confounders. Also, adjustments were made for inflammatory markers (C-reactive protein and leukocyte count) and the oxidative stress marker asymmetric dimethyl arginine to investigate potential mediators of the relationship between soluble urokinase plasminogen activator receptor and outcomes. Median soluble urokinase plasminogen activator receptor levels were 10,521 pg/ml (interquartile range, 9105-12,543 pg/ml). When stratified by tertiles, patients with soluble urokinase plasminogen activator receptor >11,633 pg/ml (third tertile) had adjusted 1.6-fold higher mortality (hazard ratio, 1.60; 95% confidence interval, 1.27 to 2.03) compared with those with low soluble urokinase plasminogen activator receptor soluble urokinase plasminogen activator receptor to a risk factor model modestly improved risk discrimination for all-cause death (ΔC statistic, 0.02; 95% confidence interval, 0.00 to 0.03) and cardiovascular events (ΔC statistic, 0.02; 95% confidence interval, 0.00 to 0.05). The association of soluble urokinase plasminogen activator receptor levels with outcomes persists in patients on hemodialysis

  15. Activation of peroxisome proliferator-activated receptor-{alpha} enhances fatty acid oxidation in human adipocytes

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Joo-Young; Hashizaki, Hikari; Goto, Tsuyoshi; Sakamoto, Tomoya; Takahashi, Nobuyuki [Laboratory of Molecular Function of Food, Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011 (Japan); Kawada, Teruo, E-mail: fat@kais.kyoto-u.ac.jp [Laboratory of Molecular Function of Food, Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011 (Japan)

    2011-04-22

    Highlights: {yields} PPAR{alpha} activation increased mRNA expression levels of adipocyte differentiation marker genes and GPDH activity in human adipocytes. {yields} PPAR{alpha} activation also increased insulin-dependent glucose uptake in human adipocytes. {yields} PPAR{alpha} activation did not affect lipid accumulation in human adipocytes. {yields} PPAR{alpha} activation increased fatty acid oxidation through induction of fatty acid oxidation-related genes in human adipocytes. -- Abstract: Peroxisome proliferator-activated receptor-{alpha} (PPAR{alpha}) is a key regulator for maintaining whole-body energy balance. However, the physiological functions of PPAR{alpha} in adipocytes have been unclarified. We examined the functions of PPAR{alpha} using human multipotent adipose tissue-derived stem cells as a human adipocyte model. Activation of PPAR{alpha} by GW7647, a potent PPAR{alpha} agonist, increased the mRNA expression levels of adipocyte differentiation marker genes such as PPAR{gamma}, adipocyte-specific fatty acid-binding protein, and lipoprotein lipase and increased both GPDH activity and insulin-dependent glucose uptake level. The findings indicate that PPAR{alpha} activation stimulates adipocyte differentiation. However, lipid accumulation was not changed, which is usually observed when PPAR{gamma} is activated. On the other hand, PPAR{alpha} activation by GW7647 treatment induced the mRNA expression of fatty acid oxidation-related genes such as CPT-1B and AOX in a PPAR{alpha}-dependent manner. Moreover, PPAR{alpha} activation increased the production of CO{sub 2} and acid soluble metabolites, which are products of fatty acid oxidation, and increased oxygen consumption rate in human adipocytes. The data indicate that activation of PPAR{alpha} stimulates both adipocyte differentiation and fatty acid oxidation in human adipocytes, suggesting that PPAR{alpha} agonists could improve insulin resistance without lipid accumulation in adipocytes. The expected

  16. Protease activated receptors (PARS) mediation in gyroxin biological activity; Mediacao dos receptores ativados por proteases (PARs) em atividades biologicas da giroxina

    Energy Technology Data Exchange (ETDEWEB)

    Silva, Jose Alberto Alves da

    2009-07-01

    Gyroxin is a serine protease enzyme from the South American rattlesnake (Crotalus durissus terrificus) venom; it is only partially characterized and has multiple activities. Gyroxin induces blood coagulation, blood pressure decrease and a neurotoxic behavior named barrel rotation. The mechanisms involved in this neurotoxic activity are not known. Whereas gyroxin is a member of enzymes with high potential to become a new drug with clinical applications such as thrombin, batroxobin, ancrod, tripsyn and kalicrein, it is important to find out how gyroxin works. The analysis on agarose gel electrophoresis and circular dichroism confirmed the molecules' integrity and purity. The gyroxin intravenous administration in mice proved its neurotoxicity (barrel rotation). In vivo studies employing intravital microscopy proved that gyroxin induces vasodilation with the participation of protease activated receptors (PARs), nitric oxide and Na+K+ATPase. The leukocytes' adherence and rolling counting indicated that gyroxin has no pro inflammatory activity. Gyroxin induced platelet aggregation, which was blocked by inhibitors of PAR1 and PAR4 receptors (SCH 79797 and tcY-NH{sub 2}, respectively). Finally, it was proved that the gyroxin temporarily alter the permeability of the blood brain barrier (BBB). Our study has shown that both the protease-activated receptors and nitric oxide are mediators involved in the biological activities of gyroxin. (author)

  17. Activation of islet 5-HT4 receptor regulates glycemic control through promoting insulin secretion.

    Science.gov (United States)

    Chen, Hui; Hong, Feng; Chen, Ye; Li, Ji; Yao, Yuan-Sheng; Zhang, Yue; Zheng, Li-Fei; Zhu, Jin-Xia

    2016-10-15

    Mosapride, a gastrointestinal prokinetic drug, is an agonist of 5-hydroxytryptamine (5-HT) receptor 4 that also reduces blood glucose. Whether 5-HT4 receptor is distributed in pancreatic islets and whether mosapride can directly stimulate insulin secretion is unclear. In the present study, the protein expression and cellular location of 5-HT4 receptor in pancreas was detected through western blotting and immunofluorescence. The acute effects of 5-HT4 receptor agonists, mosapride and prucalopride, on insulin secretion were investigated in vivo and in vitro in normal and alloxan-induced diabetes rats. The results indicated that 5-HT4 receptor immunoreactivity was co-existed in the islets insulin-immunoreactive cells of rat, mouse, pig and human. However the immunoreactive cells of insulin and 5-HT4 receptor and the protein expression of 5-HT4 receptor were significantly decreased in the pancreas of alloxan-induced diabetes rats. In normal rats, mosapride and prucalopride decreased blood glucose and increased insulin secretion during glucose tolerance test, in association with an increase in glucose-stimulated insulin secretion, which was abolished by the 5-HT4 receptor antagonist GR113808. In diabetes rats, mosapride and prucalopride failed to improve blood glucose and insulin levels in the group of 180mg/kg alloxan, but increased glucose-stimulated insulin secretion in the group of 120mg/kg alloxan in vitro. We conclude that 5-HT4 receptor is distributed in the islet β cell. Activation of 5-HT4 receptor is able to stimulate insulin secretion directly, thereby reduce blood glucose. The study provides important experimental evidences for the 5-HT4 receptor regulating insulin secretion and acting as a potential drug target in diabetes treatment. Copyright © 2016 Elsevier B.V. All rights reserved.

  18. Activation and modulation of recombinantly expressed serotonin receptor type 3A by terpenes and pungent substances.

    Science.gov (United States)

    Ziemba, Paul M; Schreiner, Benjamin S P; Flegel, Caroline; Herbrechter, Robin; Stark, Timo D; Hofmann, Thomas; Hatt, Hanns; Werner, Markus; Gisselmann, Günter

    2015-11-27

    Serotonin receptor type 3 (5-HT3 receptor) is a ligand-gated ion channel that is expressed in the central nervous system (CNS) as well as in the peripheral nervous system (PNS). The receptor plays an important role in regulating peristalsis of the gastrointestinal tract and in functions such as emesis, cognition and anxiety. Therefore, a variety of pharmacologically active substances target the 5-HT3 receptor to treat chemotherapy-induced nausea and vomiting. The 5-HT3 receptors are activated, antagonized, or modulated by a wide range of chemically different substances, such as 2-methyl-serotonin, phenylbiguanide, setrones, or cannabinoids. Whereas the action of all of these substances is well described, less is known about the effect of terpenoids or fragrances on 5-HT3A receptors. In this study, we screened a large number of natural odorous and pungent substances for their pharmacological action on recombinantly expressed human 5-HT3A receptors. The receptors were functionally expressed in Xenopus oocytes and characterized by electrophysiological recordings using the two-electrode voltage-clamp technique. A screening of two odorous mixes containing a total of 200 substances revealed that the monoterpenes, thymol and carvacrol, act as both weak partial agonists and positive modulators on the 5-HT3A receptor. In contrast, the most effective blockers were the terpenes, citronellol and geraniol, as well as the pungent substances gingerol, capsaicin and polygodial. In our study, we identified new modulators of 5-HT3A receptors out of the classes of monoterpenes and vanilloid substances that frequently occur in various plants. Copyright © 2015. Published by Elsevier Inc.

  19. G-protein-Coupled Receptors and Their (Bio Chemical Significance Win 2012 Nobel Prize in Chemistry

    Directory of Open Access Journals (Sweden)

    Hsi-Hsien Lin

    2013-06-01

    Full Text Available G-protein-coupled receptors (GPCRs are seven transmembrane cell surface proteins specialized in cellular communication. These receptors represent a major gateway through which cells convert external cues into intracellular signals and respond with appropriate actions. While the effects of hormones, neurotransmitters, and drugs on cells, tissues, organs, and even whole organisms are well described, the molecular identity of the direct targets and the diverse signaling mechanisms of these biological ligands have been slow and hard to define. The Nobel Prize in Chemistry for the year 2012 acknowledges the importance of GPCRs in these processes, especially for the contribution of Profs Robert J. Lefkowitz and Brian K. Kobilka to the studies of GPCRs. In this brief review, the seminal works accomplished by the two GPCR pioneers are summarized and the (bio chemical significance of GPCRs in health and disease is discussed.

  20. The expression and activation of protease-activated receptor-2 correlate with skin color.

    Science.gov (United States)

    Babiarz-Magee, Laura; Chen, Nannan; Seiberg, Miri; Lin, Connie B

    2004-06-01

    Skin color results from the production and distribution of melanin in the epidermis. The protease-activated receptor-2 (PAR-2), expressed on keratinocytes but not on melanocytes, is involved in melanosome uptake via phagocytosis, and modulation of PAR-2 activation affects skin color. The pattern of melanosome distribution within the epidermis is skin color-dependent. In vitro, this distribution pattern is regulated by the ethnic origin of the keratinocytes, not the melanocytes. Therefore, we hypothesized that PAR-2 may play a role in the modulation of pigmentation in a skin type-dependent manner. We examined the expression of PAR-2 and its activator, trypsin, in human skins with different pigmentary levels. Here we show that PAR-2 and trypsin are expressed in higher levels, and are differentially localized in highly pigmented, relative to lightly pigmented skins. Moreover, highly pigmented skins exhibit an increase in PAR-2-specific protease cleavage ability. Microsphere phagocytosis was more efficient in keratinocytes from highly pigmented skins, and PAR-2 induced phagocytosis resulted in more efficient microsphere ingestion and more compacted microsphere organization in dark skin-derived keratinocytes. These results demonstrate that PAR-2 expression and activity correlate with skin color, suggesting the involvement of PAR-2 in ethnic skin color phenotypes.

  1. Structural plasticity of GABAergic axons is regulated by network activity and GABAA receptor activation

    Directory of Open Access Journals (Sweden)

    Anne eSchuemann

    2013-06-01

    Full Text Available Coordinated changes at excitatory and inhibitory synapses are essential for normal brain development and function. It is well established that excitatory neurons undergo structural changes, but our knowledge about inhibitory structural plasticity is rather scarce. Here we present a quantitative analysis of the dynamics of GABAergic boutons in the dendritic region of the hippocampal CA1 area using time-lapse two-photon imaging in organotypic hippocampal cultures from GAD65-GFP mice. We show that ~20% of inhibitory boutons are not stable. They are appearing, disappearing and reappearing at specific locations along the inhibitory axon and reflect immature or incomplete synapses. Furthermore, we observed that persistent boutons show large volume fluctuations over several hours, suggesting that presynaptic content of inhibitory synapses is not constant. Our data show that inhibitory boutons are highly dynamic structures and suggest that inhibitory axons are continuously probing potential locations for inhibitory synapse formation by redistributing presynaptic material along the axon.In addition, we found that neuronal activity affects the exploratory dynamics of inhibitory axons. Blocking network activity rapidly reduces the number of transient boutons, whereas enhancing activity reduces the number of persistent inhibitory boutons, possibly reflecting enhanced competition between boutons along the axon. The latter effect requires signaling through GABAA receptors. We propose that activity-dependent regulation of bouton dynamics contributes to inhibitory synaptic plasticity.

  2. Peroxisome Proliferator-Activated Receptor and Retinoic X Receptor in Alcoholic Liver Disease

    Directory of Open Access Journals (Sweden)

    Tommaso Mello

    2009-01-01

    Full Text Available A growing number of new studies demonstrate that nuclear receptors are involved in the development of alcoholic liver disease (ALD. Ethanol metabolism and RXR/PPAR functions are tightly interconnected in the liver. Several ethanol metabolizing enzymes are potently regulated by RXR and PPAR after alcohol consumption. The increased ethanol metabolism, in turn, leads to alteration of the redox balance of the cells and impairment of RXR/PPAR functions by direct and indirect effects of acetaldehyde, resulting in deranged lipid metabolism, oxidative stress, and release of proinflammatory cytokines. The use of animal models played a crucial role in understanding the molecular mechanisms of ALD. In this paper we summarize the reciprocal interactions between ethanol metabolism and RXR/PPAR functions. In conclusion, RXR and PPAR play a central role in the onset and perpetuation of the mechanisms underling all steps of the clinical progression in ALD.

  3. PKCζ regulates Notch receptor routing and activity in a Notch signaling-dependent manner.

    Science.gov (United States)

    Sjöqvist, Marika; Antfolk, Daniel; Ferraris, Saima; Rraklli, Vilma; Haga, Cecilia; Antila, Christian; Mutvei, Anders; Imanishi, Susumu Y; Holmberg, Johan; Jin, Shaobo; Eriksson, John E; Lendahl, Urban; Sahlgren, Cecilia

    2014-04-01

    Activation of Notch signaling requires intracellular routing of the receptor, but the mechanisms controlling the distinct steps in the routing process is poorly understood. We identify PKCζ as a key regulator of Notch receptor intracellular routing. When PKCζ was inhibited in the developing chick central nervous system and in cultured myoblasts, Notch-stimulated cells were allowed to undergo differentiation. PKCζ phosphorylates membrane-tethered forms of Notch and regulates two distinct routing steps, depending on the Notch activation state. When Notch is activated, PKCζ promotes re-localization of Notch from late endosomes to the nucleus and enhances production of the Notch intracellular domain, which leads to increased Notch activity. In the non-activated state, PKCζ instead facilitates Notch receptor internalization, accompanied with increased ubiquitylation and interaction with the endosomal sorting protein Hrs. Collectively, these data identify PKCζ as a key regulator of Notch trafficking and demonstrate that distinct steps in intracellular routing are differentially modulated depending on Notch signaling status.

  4. T cell receptor dwell times control the kinase activity of Zap70.

    Science.gov (United States)

    Klammt, Christian; Novotná, Lucie; Li, Dongyang T; Wolf, Miriam; Blount, Amy; Zhang, Kai; Fitchett, Jonathan R; Lillemeier, Björn F

    2015-09-01

    Kinase recruitment to membrane receptors is essential for signal transduction. However, the underlying regulatory mechanisms are poorly understood. We investigated how conformational changes control T cell receptor (TCR) association and activity of the kinase Zap70. Structural analysis showed that TCR binding or phosphorylation of Zap70 triggers a transition from a closed, autoinhibited conformation to an open conformation. Using Zap70 mutants with defined conformations, we found that TCR dwell times controlled Zap70 activity. The closed conformation minimized TCR dwell times and thereby prevented activation by membrane-associated kinases. Parallel recruitment of coreceptor-associated Lck kinase to the TCR ensured Zap70 phosphorylation and stabilized Zap70 TCR binding. Our study suggests that the dynamics of cytosolic enzyme recruitment to the plasma membrane regulate the activity and function of receptors lacking intrinsic catalytic activity.

  5. receptores

    Directory of Open Access Journals (Sweden)

    Salete Regina Daronco Benetti

    2006-01-01

    Full Text Available Se trata de un estudio etnográfico, que tuvo lo objetivo de interpretar el sistema de conocimiento y del significado atribuidos a la sangre referente a la transfusión sanguínea por los donadores y receptores de un banco de sangre. Para la colecta de las informaciones se observaron los participantes y la entrevista etnográfica se realizó el análisis de dominio, taxonómicos y temáticos. Los dominios culturales fueron: la sangre es vida: fuente de vida y alimento valioso; creencias religiosas: fuentes simbólicas de apoyos; donación sanguínea: un gesto colaborador que exige cuidarse, gratifica y trae felicidad; donación sanguínea: fuente simbólica de inseguridad; estar enfermo es una condición para realizar transfusión sanguínea; transfusión sanguínea: esperanza de vida; Creencias populares: transfusión sanguínea como riesgo para la salud; donadores de sangre: personas benditas; donar y recibir sangre: como significado de felicidad. Temática: “líquido precioso que origina, sostiene, modifica la vida, provoca miedo e inseguridad”.

  6. 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.

  7. Structure and activation of the TSH receptor transmembrane domain

    OpenAIRE

    N??ez Miguel, Ricardo; Sanders, Jane; Furmaniak, Jadwiga; Smith, Bernard Rees

    2016-01-01

    Purpose The thyroid-stimulating hormone receptor (TSHR) is the target autoantigen for TSHR-stimulating autoantibodies in Graves? disease. The TSHR is composed of: a leucine-rich repeat domain (LRD), a hinge region or cleavage domain (CD) and a transmembrane domain (TMD). The binding arrangements between the TSHR LRD and the thyroid-stimulating autoantibody M22 or TSH have become available from the crystal structure of the TSHR LRD?M22 complex and a comparative model of the TSHR LRD in complex...

  8. Molecular architecture of mouse activating NKR-P1 receptors

    Czech Academy of Sciences Publication Activity Database

    Kolenko, Petr; Rozbeský, Daniel; Vaněk, Ondřej; Kopecký, V. Jr.; Hofbauerová, Kateřina; Novák, Petr; Pompach, Petr; Hašek, Jindřich; Skálová, Tereza; Bezouška, Karel; Dohnálek, Jan

    2011-01-01

    Roč. 175, č. 3 (2011), s. 434-441 ISSN 1047-8477 R&D Projects: GA MŠk 1M0505; GA ČR GA303/09/0477; GA ČR GD305/09/H008; GA ČR GA305/07/1073; GA ČR GAP207/10/1040; GA AV ČR KJB101120805 Institutional research plan: CEZ:AV0Z40500505; CEZ:AV0Z50200510 Keywords : NK cell * C-type lectin-like receptor * X-ray structure Subject RIV: CE - Biochemistry Impact factor: 3.406, year: 2011

  9. Neonatal Diabetes Caused by Activating Mutations in the Sulphonylurea Receptor

    Directory of Open Access Journals (Sweden)

    Peter Proks

    2013-06-01

    Full Text Available Adenosine triphosphate (ATP-sensitive potassium (KATP channels in pancreatic β-cells play a crucial role in insulin secretion and glucose homeostasis. These channels are composed of two subunits: a pore-forming subunit (Kir6.2 and a regulatory subunit (sulphonylurea receptor-1. Recent studies identified large number of gain of function mutations in the regulatory subunit of the channel which cause neonatal diabetes. Majority of mutations cause neonatal diabetes alone, however some lead to a severe form of neonatal diabetes with associated neurological complications. This review focuses on the functional effects of these mutations as well as the implications for treatment.

  10. Activation of muscarinic receptors in porcine airway smooth muscle elicits a transient increase in phospholipase D activity.

    Science.gov (United States)

    Mamoon, A M; Smith, J; Baker, R C; Farley, J M

    1999-01-01

    Phospholipase D (PLD) is a phosphodiesterase that catalyses hydrolysis of phosphatidylcholine to produce phosphatidic acid and choline. In the presence of ethanol, PLD also catalyses the formation of phosphatidylethanol, which is a unique characteristic of this enzyme. Muscarinic receptor-induced changes in the activity of PLD were investigated in porcine tracheal smooth muscle by measuring the formation of [3H]phosphatidic acid ([3H]PA) and [3H]phosphatidylethanol ([3H]PEth) after labeling the muscle strips with [3H]palmitic acid. The cholinergic receptor agonist acetylcholine (Ach) significantly but transiently increased formation of both [3H]PA and [3H]PEth in a concentration-dependent manner (>105-400% vs. controls in the presence of 10(-6) to 10(-4) M Ach) when pretreated with 100 mM ethanol. The Ach receptor-mediated increase in PLD activity was inhibited by atropine (10(-6) M), indicating that activation of PLD occurred via muscarinic receptors. Activation of protein kinase C (PKC) by phorbol-12-myristate-13-acetate (PMA) increased PLD activity that was effectively blocked by the PKC inhibitors calphostin C (10(-8) to 10(-6) M) and GFX (10(-8) to 10(-6) M). Ach-induced increases in PLD activity were also significantly, but incompletely, inhibited by both GFX and calphostin C. From the present data, we conclude that in tracheal smooth muscle, muscarinic acetylcholine receptor-induced PLD activation is transient in nature and coupled to these receptors via PKC. However, PKC activation is not solely responsible for Ach-induced activation of PLD in porcine tracheal smooth muscle.

  11. Activation of GLP-1 Receptor Promotes Bone Marrow Stromal Cell Osteogenic Differentiation through β-Catenin

    Directory of Open Access Journals (Sweden)

    Jingru Meng

    2016-04-01

    Full Text Available Glucagon-like peptide 1 (GLP-1 plays an important role in regulating bone remodeling, and GLP-1 receptor agonist shows a positive relationship with osteoblast activity. However, GLP-1 receptor is not found in osteoblast, and the mechanism of GLP-1 receptor agonist on regulating bone remodeling is unclear. Here, we show that the GLP-1 receptor agonist exendin-4 (Ex-4 promoted bone formation and increased bone mass and quality in a rat unloading-induced bone loss model. These functions were accompanied by an increase in osteoblast number and serum bone formation markers, while the adipocyte number was decreased. Furthermore, GLP-1 receptor was detected in bone marrow stromal cells (BMSCs, but not in osteoblast. Activation of GLP-1 receptor by Ex-4 promoted the osteogenic differentiation and inhibited BMSC adipogenic differentiation through regulating PKA/β-catenin and PKA/PI3K/AKT/GSK3β signaling. These findings reveal that GLP-1 receptor regulates BMSC osteogenic differentiation and provide a molecular basis for therapeutic potential of GLP-1 against osteoporosis.

  12. Dimerization by a cytokine receptor is necessary for constitutive activation of JAK2V617F.

    Science.gov (United States)

    Lu, Xiaohui; Huang, Lily Jun-Shen; Lodish, Harvey F

    2008-02-29

    The majority of the BCR-ABL-negative myeloproliferative disorders express the mutant JAK2, JAK2V617F. Previously we showed that constitutive activation of this oncogenic JAK2 mutant in Ba/F3 or 32D cells requires coexpression of a cognate homodimeric cytokine receptor, such as the EpoR. However, overexpression of JAK2V617F in Ba/F3 cells renders them cytokine-independent for growth in the absence of an exogenous cytokine receptor. Here, we demonstrated that JAK2V617F domains required for receptor association are essential for cytokine-independent growth by overexpressed JAK2V617F, suggesting JAK2V617F is binding to an unknown endogenous cytokine receptor(s) for its activation. We further showed that disruption of EpoR dimerization by coexpressing a truncated EpoR disrupted JAK2V617F-mediated transformation, indicating that EpoR dimerization plays an essential role in the activation of JAK2V617F. Interestingly, coexpression of JAK2V617F with EpoR mutants that retain JAK2 binding but are defective in mediating Epo-dependent JAK2 activation due to mutations in a conserved juxtamembrane motif does lead to cytokine-independent activation of JAK2V617F. Overall, these findings confirm that JAK2V617F requires binding to a dimerized cytokine receptor for its activation, and that the key EpoR juxtamembrane regulatory motif essential for Epo-dependent JAK2 activation is not essential for the activation of JAK2V617F. The structure of the activated JAK2V617F is thus likely to be different from that of the activated wild-type JAK2, raising the possibility of developing a specifically targeted therapy for myeloproliferative disorders.

  13. Biased agonism as a mechanism for differential signaling by chemokine receptors.

    Science.gov (United States)

    Rajagopal, Sudarshan; Bassoni, Daniel L; Campbell, James J; Gerard, Norma P; Gerard, Craig; Wehrman, Tom S

    2013-12-06

    Chemokines display considerable promiscuity with multiple ligands and receptors shared in common, a phenomenon that is thought to underlie their biochemical "redundancy." Their receptors are part of a larger seven-transmembrane receptor superfamily, commonly referred to as G protein-coupled receptors, which have been demonstrated to be able to signal with different efficacies to their multiple downstream signaling pathways, a phenomenon referred to as biased agonism. Biased agonism has been primarily reported as a phenomenon of synthetic ligands, and the biologic prevalence and importance of such signaling are unclear. Here, to assess the presence of biased agonism that may underlie differential signaling by chemokines targeting the same receptor, we performed a detailed pharmacologic analysis of a set of chemokine receptors with multiple endogenous ligands using assays for G protein signaling, β-arrestin recruitment, and receptor internalization. We found that chemokines targeting the same receptor can display marked differences in their efficacies for G protein- or β-arrestin-mediated signaling or receptor internalization. This ligand bias correlates with changes in leukocyte migration, consistent with different mechanisms underlying the signaling downstream of these receptors induced by their ligands. These findings demonstrate that biased agonism is a common and likely evolutionarily conserved biological mechanism for generating qualitatively distinct patterns of signaling via the same receptor in response to different endogenous ligands.

  14. Histamine induces microglia activation and dopaminergic neuronal toxicity via H1 receptor activation.

    Science.gov (United States)

    Rocha, Sandra M; Saraiva, Tatiana; Cristóvão, Ana C; Ferreira, Raquel; Santos, Tiago; Esteves, Marta; Saraiva, Cláudia; Je, Goun; Cortes, Luísa; Valero, Jorge; Alves, Gilberto; Klibanov, Alexander; Kim, Yoon-Seong; Bernardino, Liliana

    2016-06-04

    Histamine is an amine widely known as a peripheral inflammatory mediator and as a neurotransmitter in the central nervous system. Recently, it has been suggested that histamine acts as an innate modulator of microglial activity. Herein, we aimed to disclose the role of histamine in microglial phagocytic activity and reactive oxygen species (ROS) production and to explore the consequences of histamine-induced neuroinflammation in dopaminergic (DA) neuronal survival. The effect of histamine on phagocytosis was assessed both in vitro by using a murine N9 microglial cell line and primary microglial cell cultures and in vivo. Cells were exposed to IgG-opsonized latex beads or phosphatidylserine (PS) liposomes to evaluate Fcγ or PS receptor-mediated microglial phagocytosis, respectively. ROS production and protein levels of NADPH oxidases and Rac1 were assessed as a measure of oxidative stress. DA neuronal survival was evaluated in vivo by counting the number of tyrosine hydroxylase-positive neurons in the substantia nigra (SN) of mice. We found that histamine triggers microglial phagocytosis via histamine receptor 1 (H1R) activation and ROS production via H1R and H4R activation. By using apocynin, a broad NADPH oxidase (Nox) inhibitor, and Nox1 knockout mice, we found that the Nox1 signaling pathway is involved in both phagocytosis and ROS production induced by histamine in vitro. Interestingly, both apocynin and annexin V (used as inhibitor of PS-induced phagocytosis) fully abolished the DA neurotoxicity induced by the injection of histamine in the SN of adult mice in vivo. Blockade of H1R protected against histamine-induced Nox1 expression and death of DA neurons in vivo. Overall, our results highlight the relevance of histamine in the modulation of microglial activity that ultimately may interfere with neuronal survival in the context of Parkinson's disease (PD) and, eventually, other neurodegenerative diseases which are accompanied by microglia

  15. Cow's milk increases the activities of human nuclear receptors peroxisome proliferator-activated receptors alpha and delta and retinoid X receptor alpha involved in the regulation of energy homeostasis, obesity, and inflammation.

    Science.gov (United States)

    Suhara, W; Koide, H; Okuzawa, T; Hayashi, D; Hashimoto, T; Kojo, H

    2009-09-01

    The nuclear peroxisome proliferator-activated receptors (PPAR) have been shown to play crucial roles in regulating energy homeostasis including lipid and carbohydrate metabolism, inflammatory responses, and cell proliferation, differentiation, and survival. Because PPAR agonists have the potential to prevent or ameliorate diseases such as hyperlipidemia, diabetes, atherosclerosis, and obesity, we have explored new natural agonists for PPAR. For this purpose, cow's milk was tested for agonistic activity toward human PPAR subtypes using a reporter gene assay. Milk increased human PPARalpha activity in a dose-dependent manner with a 3.2-fold increase at 0.5% (vol/vol). It also enhanced human PPARdelta activity in a dose-dependent manner with an 11.5-fold increase at 0.5%. However, it only slightly affected human PPARgamma activity. Ice cream, butter, and yogurt also increased the activities of PPARalpha and PPARdelta, whereas vegetable cream affected activity of PPARdelta but not PPARalpha. Skim milk enhanced the activity of PPAR to a lesser degree than regular milk. Milk and fresh cream increased the activity of human retinoid X receptor (RXR)alpha as well as PPARalpha and PPARdelta, whereas neither affected vitamin D3 receptor, estrogen receptors alpha and beta, or thyroid receptors alpha and beta. Both milk and fresh cream were shown by quantitative real-time PCR to increase the quantity of mRNA for uncoupling protein 2 (UCP2), an energy expenditure gene, in a dose-dependent manner. The increase in UCP2 mRNA was found to be reduced by treatment with PPARdelta-short interfering (si)RNA. This study unambiguously clarified at the cellular level that cow's milk increased the activities of human PPARalpha, PPARdelta, and RXRalpha. The possible role in enhancing the activities of PPARalpha, PPARdelta, and RXRalpha, and the health benefits of cow's milk were discussed.

  16. Peroxisome proliferator-activated receptor activators protect sebocytes from apoptosis: a new treatment modality for acne?

    Science.gov (United States)

    Schuster, M; Zouboulis, C C; Ochsendorf, F; Müller, J; Thaçi, D; Bernd, A; Kaufmann, R; Kippenberger, S

    2011-01-01

    The main function of the human sebaceous gland is sebum excretion. Increased sebum levels combined with follicular hyperkeratinization are a prerequisite of acne vulgaris. As peroxisome proliferator-activated receptors (PPARs) are known to control lipid metabolism in several human tissues they have been considered to be involved in the pathogenesis of acne vulgaris. To investigate the effect of activators of PPAR-α (WY14643), PPAR-γ (rosiglitazone) and PPAR-δ (L-165.041) on basal and staurosporine-induced apoptosis in the human sebocyte cell line SZ95 in vitro. After defining the basal effects of PPAR activators on membrane integrity (lactate dehydrogenase release) and DNA synthesis (5-bromodeoxyuridine incorporation), apoptosis was determined by the release of histone-associated DNA fragments. The underlying signalling events were detected by Western blotting and the use of specific inhibitors against p44/42 and protein kinase B (PKB)/Akt. PPAR activators of all three subsets offer antiapoptotic effects, with L-165.041 being the most potent. This compound induced the activation of PKB/Akt and p44/42, two kinases involved in antiapoptosis and proliferation, respectively. An inhibition of these kinases by specific inhibitors reversed the suppression of histone-associated DNA fragments by L-165.041, indicating that these signalling pathways participate in the observed antiapoptotic effect. The present data suggest that activators of PPAR, in particular of the δ subset, might have beneficial effects on acne vulgaris by inhibiting the release of lipids in the context of sebocyte apoptosis. © 2010 The Authors. BJD © 2010 British Association of Dermatologists 2010.

  17. Decay of an active GPCR: Conformational dynamics govern agonist rebinding and persistence of an active, yet empty, receptor state.

    Science.gov (United States)

    Schafer, Christopher T; Fay, Jonathan F; Janz, Jay M; Farrens, David L

    2016-10-18

    Here, we describe two insights into the role of receptor conformational dynamics during agonist release (all-trans retinal, ATR) from the visual G protein-coupled receptor (GPCR) rhodopsin. First, we show that, after light activation, ATR can continually release and rebind to any receptor remaining in an active-like conformation. As with other GPCRs, we observe that this equilibrium can be shifted by either promoting the active-like population or increasing the agonist concentration. Second, we find that during decay of the signaling state an active-like, yet empty, receptor conformation can transiently persist after retinal release, before the receptor ultimately collapses into an inactive conformation. The latter conclusion is based on time-resolved, site-directed fluorescence labeling experiments that show a small, but reproducible, lag between the retinal leaving the protein and return of transmembrane helix 6 (TM6) to the inactive conformation, as determined from tryptophan-induced quenching studies. Accelerating Schiff base hydrolysis and subsequent ATR dissociation, either by addition of hydroxylamine or introduction of mutations, further increased the time lag between ATR release and TM6 movement. These observations show that rhodopsin can bind its agonist in equilibrium like a traditional GPCR, provide evidence that an active GPCR conformation can persist even after agonist release, and raise the possibility of targeting this key photoreceptor protein by traditional pharmaceutical-based treatments.

  18. Structural basis for specific ligation of the peroxisome proliferator-activated receptor ?

    OpenAIRE

    Wu, Chyuan-Chuan; Baiga, Thomas J.; Downes, Michael; La Clair, James J.; Atkins, Annette R.; Richard, Stephane B.; Fan, Weiwei; Stockley-Noel, Theresa A.; Bowman, Marianne E; Noel, Joseph P.; Evans, Ronald M.

    2017-01-01

    Clinical treatments for metabolic diseases rely on agents with high selectivity to specific targets often within a class of structurally and functionally related proteins. In this paper, we uncover physical and chemical features governing selective small-molecule binding to peroxisome proliferator-activated receptor (PPAR) ? concomitant with distinct conformational changes in the receptor, key to therapeutic modulation of lipid catabolism, transport, and storage. These studies reveal the subt...

  19. Structure-activity relationships of constrained phenylethylamine ligands for the serotonin 5-ht2 receptors

    DEFF Research Database (Denmark)

    Isberg, Vignir; Paine, James; Leth-Petersen, Sebastian

    2013-01-01

    Serotonergic ligands have proven effective drugs in the treatment of migraine, pain, obesity, and a wide range of psychiatric and neurological disorders. There is a clinical need for more highly 5-HT2 receptor subtype-selective ligands and the most attention has been given to the phenethylamine......-activity relationships of constrained phenethylamines and contributes towards the development of 5-HT2 receptor subtype-selective ligands....

  20. Placental Implications of Peroxisome Proliferator-Activated Receptors in Gestation and Parturition

    OpenAIRE

    Borel, Valerie; Gallot, Denis; Marceau, Geoffroy; Sapin, Vincent; Blanchon, Loïc

    2008-01-01

    The placenta is a transitory structure indispensable for the proper development of the embryo and fetus during mammalian gestation. Like other members of the nuclear receptor family, the peroxisome proliferator-activated receptors (PPARs) are known to be involved in the physiological and pathological events occurring during the placentation. This placental involvement has been recently reviewed focusing on the early stages of placental development (implantation and invasion, etc.), mouse PP...

  1. Protease-activated receptor signalling by coagulation proteases in endothelial cells.

    Science.gov (United States)

    Rezaie, Alireza R

    2014-11-01

    Endothelial cells express several types of integral membrane protein receptors, which upon interaction and activation by their specific ligands, initiate a signalling network that links extracellular cues in circulation to various biological processes within a plethora of cells in the vascular system. A small family of G-protein coupled receptors, termed protease-activated receptors (PAR1-4), can be specifically activated by coagulation proteases, thereby modulating a diverse array of cellular activities under various pathophysiological conditions. Thrombin and all vitamin K-dependent coagulation proteases, with the exception of factor IXa for which no PAR signalling has been attributed, can selectively activate cell surface PARs on the vasculature. Thrombin can activate PAR1, PAR3 and PAR4, but not PAR2 which can be specifically activated by factors VIIa and Xa. The mechanistic details of the specificity of PAR signalling by coagulation proteases are the subject of extensive investigation by many research groups worldwide. However, analysis of PAR signalling data in the literature has proved to be challenging since a single coagulation protease can elicit different signalling responses through activation of the same PAR receptor in endothelial cells. This article is focused on briefly reviewing the literature with respect to determinants of the specificity of PAR signalling by coagulation proteases with special emphasis on the mechanism of PAR1 signalling by thrombin and activated protein C in endothelial cells.

  2. Activation of liver X receptor-alpha reduces activation of the renal and cardiac renin-angiotensin-aldosterone system

    NARCIS (Netherlands)

    Kuipers, Irma; van der Harst, Pim; Kuipers, Folkert; van Genne, Linda; Goris, Maaike; Lehtonen, Jukka Y.; van Veldhuisen, Dirk J.; van Gilst, Wiek H.; de Boer, Rudolf A.

    Liver X receptor (LXR)-alpha is a pivotal player in reverse cholesterol metabolism. Recently, LXR-alpha was implicated as an immediate regulator of renin expression in a cAMP-responsive manner. To determine whether long-term LXR-alpha activation affects activation of the renal and cardiac

  3. Modulation of mesenteric collecting lymphatic contractions by σ1-receptor activation and nitric oxide production.

    Science.gov (United States)

    Trujillo, Andrea N; Katnik, Christopher; Cuevas, Javier; Cha, Byeong Jake; Taylor-Clark, Thomas E; Breslin, Jerome W

    2017-10-01

    Recently, it has been reported that a σ-receptor antagonist could reduce inflammation-induced edema. Lymphatic vessels play an essential role in removing excess interstitial fluid. We tested the hypothesis that activation of σ-receptors would reduce or weaken collecting lymphatic contractions. We used isolated, cannulated rat mesenteric collecting lymphatic vessels to study contractions in response to the σ-receptor agonist afobazole in the absence and presence of different σ-receptor antagonists. We used RT-PCR and Western blot analysis to investigate whether these vessels express the σ 1 -receptor and immunofluorescence confocal microscopy to examine localization of the σ 1 -receptor in the collecting lymphatic wall. Using N -nitro-l-arginine methyl ester (l-NAME) pretreatment before afobazole in isolated lymphatics, we tested the role of nitric oxide (NO) signaling. Finally, we used 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate fluorescence as an indicator to test whether afobazole increases NO release in cultured lymphatic endothelial cells. Our results show that afobazole (50-150 µM) elevated end-systolic diameter and generally reduced pump efficiency and that this response could be partially blocked by the σ 1 -receptor antagonists BD 1047 and BD 1063 but not by the σ 2 -receptor antagonist SM-21. σ 1 -Receptor mRNA and protein were detected in lysates from isolated rat mesenteric collecting lymphatics. Confocal images with anti-σ 1 -receptor antibody labeling suggested localization in the lymphatic endothelium. Blockade of NO synthases with l-NAME inhibited the effects of afobazole. Finally, afobazole elicited increases in NO production from cultured lymphatic endothelial cells. Our findings suggest that the σ 1 -receptor limits collecting lymphatic pumping through a NO-dependent mechanism. NEW & NOTEWORTHY Relatively little is known about the mechanisms that govern contractions of lymphatic vessels. σ 1 -Receptor activation has been

  4. Natural compounds regulate energy metabolism by the modulating the activity of lipid-sensing nuclear receptors.

    Science.gov (United States)

    Goto, Tsuyoshi; Kim, Young-Il; Takahashi, Nobuyuki; Kawada, Teruo

    2013-01-01

    Obesity causes excess fat accumulation in various tissues, most notoriously in the adipose tissue, along with other insulin-responsive organs such as skeletal muscle and the liver, which predisposes an individual to the development of metabolic abnormalities. The molecular mechanisms underlying obesity-induced metabolic abnormalities have not been completely elucidated; however, in recent years, the search for therapies to prevent the development of obesity and obesity-associated metabolic disorders has increased. It is known that several nuclear receptors, when activated by specific ligands, regulate carbohydrate and lipid metabolism at the transcriptional level. The expression of lipid metabolism-related enzymes is directly regulated by the activity of various nuclear receptors via their interaction with specific response elements in promoters of those genes. Many natural compounds act as ligands of nuclear receptors and regulate carbohydrate and lipid metabolism by regulating the activities of these nuclear receptors. In this review, we describe our current knowledge of obesity, the role of lipid-sensing nuclear receptors in energy metabolism, and several examples of food factors that act as agonists or antagonists of nuclear receptors, which may be useful for the management of obesity and the accompanying energy metabolism abnormalities. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. Fatty acylated caveolin-2 is a substrate of insulin receptor tyrosine kinase for insulin receptor substrate-1-directed signaling activation.

    Science.gov (United States)

    Kwon, Hayeong; Lee, Jaewoong; Jeong, Kyuho; Jang, Donghwan; Pak, Yunbae

    2015-05-01

    Here, we demonstrate that insulin receptor (IR) tyrosine kinase catalyzes Tyr-19 and Tyr-27 phosphorylation of caveolin-2 (cav-2), leading to stimulation of signaling proteins downstream of IR, and that the catalysis is dependent on fatty acylation status of cav-2, promoting its interaction with IR. Cav-2 is myristoylated at Gly-2 and palmitoylated at Cys-109, Cys-122, and Cys-145. The fatty acylation deficient mutants are unable to localize in the plasma membrane and not phosphorylated by IR tyrosine kinase. IR interacts with the C-terminal domain of cav-2 containing the cysteines for palmitoylation. IR mutants, Y999F and K1057A, but not W1220S, fail interaction with cav-2. Insulin receptor substrate-1 (IRS-1) is recruited to interact with the IR-catalyzed phospho-tyrosine cav-2, which facilitates IRS-1 association with and activation by IR to initiate IRS-1-mediated downstream signaling. Cav-2 fatty acylation and tyrosine phosphorylation are necessary for the IRS-1-dependent PI3K-Akt and ERK activations responsible for glucose uptake and cell survival and proliferation. In conclusion, fatty acylated cav-2 is a new substrate of IR tyrosine kinase, and the fatty acylation and phosphorylation of cav-2 present novel mechanisms by which insulin signaling is activated. Copyright © 2014 Elsevier B.V. All rights reserved.

  6. Topological and functional characterization of an insect gustatory receptor.

    Directory of Open Access Journals (Sweden)

    Hui-Jie Zhang

    Full Text Available Insect gustatory receptors are predicted to have a seven-transmembrane structure and are distantly related to insect olfactory receptors, which have an inverted topology compared with G-protein coupled receptors, including mammalian olfactory receptors. In contrast, the topology of insect gustatory receptors remains unknown. Except for a few examples from Drosophila, the specificity of individual insect gustatory receptors is also unknown. In this study, the total number of identified gustatory receptors in Bombyx mori was expanded from 65 to 69. BmGr8, a silkmoth gustatory receptor from the sugar receptor subfamily, was expressed in insect cells. Membrane topology studies on BmGr8 indicate that, like insect olfactory receptors, it has an inverted topology relative to G protein-coupled receptors. An orphan GR from the bitter receptor family, BmGr53, yielded similar results. We infer, from the finding that two distantly related BmGrs have an intracellular N-terminus and an odd number of transmembrane spans, that this is likely to be a general topology for all insect gustatory receptors. We also show that BmGr8 functions independently in Sf9 cells and responds in a concentration-dependent manner to the polyalcohols myo-inositol and epi-inositol but not to a range of mono- and di-saccharides. BmGr8 is the first chemoreceptor shown to respond specifically to inositol, an important or essential nutrient for some Lepidoptera. The selectivity of BmGr8 responses is consistent with the known responses of one of the gustatory receptor neurons in the lateral styloconic sensilla of B. mori, which responds to myo-inositol and epi-inositol but not to allo-inositol.

  7. Topological and functional characterization of an insect gustatory receptor.

    Science.gov (United States)

    Zhang, Hui-Jie; Anderson, Alisha R; Trowell, Stephen C; Luo, A-Rong; Xiang, Zhong-Huai; Xia, Qing-You

    2011-01-01

    Insect gustatory receptors are predicted to have a seven-transmembrane structure and are distantly related to insect olfactory receptors, which have an inverted topology compared with G-protein coupled receptors, including mammalian olfactory receptors. In contrast, the topology of insect gustatory receptors remains unknown. Except for a few examples from Drosophila, the specificity of individual insect gustatory receptors is also unknown. In this study, the total number of identified gustatory receptors in Bombyx mori was expanded from 65 to 69. BmGr8, a silkmoth gustatory receptor from the sugar receptor subfamily, was expressed in insect cells. Membrane topology studies on BmGr8 indicate that, like insect olfactory receptors, it has an inverted topology relative to G protein-coupled receptors. An orphan GR from the bitter receptor family, BmGr53, yielded similar results. We infer, from the finding that two distantly related BmGrs have an intracellular N-terminus and an odd number of transmembrane spans, that this is likely to be a general topology for all insect gustatory receptors. We also show that BmGr8 functions independently in Sf9 cells and responds in a concentration-dependent manner to the polyalcohols myo-inositol and epi-inositol but not to a range of mono- and di-saccharides. BmGr8 is the first chemoreceptor shown to respond specifically to inositol, an important or essential nutrient for some Lepidoptera. The selectivity of BmGr8 responses is consistent with the known responses of one of the gustatory receptor neurons in the lateral styloconic sensilla of B. mori, which responds to myo-inositol and epi-inositol but not to allo-inositol.

  8. 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.

  9. A peptide antagonist of the ErbB1 receptor inhibits receptor activation, tumor cell growth and migration in vitro and xenograft tumor growth in vivo

    DEFF Research Database (Denmark)

    Xu, Ruodan; Povlsen, Gro Klitgaard; Soroka, Vladislav

    2010-01-01

    The epidermal growth factor family of receptor tyrosine kinases (ErbBs) plays essential roles in tumorigenesis and cancer disease progression, and therefore has become an attractive target for structure-based drug design. ErbB receptors are activated by ligand-induced homo- and heterodimerization...... lung cancer cell line A549. The Inherbin3 peptide may be a useful tool for investigating the mechanisms of ErbB receptor homo- and heterodimerization. Moreover, the here described biological effects of Inherbin3 suggest that peptide-based targeting of ErbB receptor dimerization is a promising anti-cancer...

  10. Pharmacologic implications of peroxisome proliferator activated receptors (PPAR) Implicaciones farmacológicas de los receptores activados por los proliferadores de peroxisomas (PPAR)

    OpenAIRE

    Luis Carlos Mejía Rivera; Sergio Parra

    2001-01-01

    PPAR are a group of proteins, members of the receptors located within the nucleus. These receptors modulate DNA transcriptional activity by binding to specific response elements on target genes. To date, three main types of PPAR have been identified designed α, δand γthese receptors are involved in the regulation of diferent metabolic processes, being the group of receptors more intensely studied. PPARαare greatly involved in both catabolism of fatty acids and transport of extracellular li...

  11. Morphine withdrawal enhances constitutive μ-opioid receptor activity in the ventral tegmental area

    NARCIS (Netherlands)

    Meye, F.J.; van Zessen, R.; Smidt, M.P.; Adan, R.A.H.; Ramakers, G.M.J.

    2012-01-01

    μ-opioid receptors (MORs) in the ventral tegmental area (VTA) are pivotally involved in addictive behavior. While MORs are typically activated by opioids, they can also become constitutively active in the absence of any agonist. In the current study, we present evidence that MOR constitutive

  12. Prediction of in vitro and in vivo oestrogen receptor activity using hierarchical clustering

    Science.gov (United States)

    In this study, hierarchical clustering classification models were developed to predict in vitro and in vivo oestrogen receptor (ER) activity. Classification models were developed for binding, agonist, and antagonist in vitro ER activity and for mouse in vivo uterotrophic ER bindi...

  13. Eph receptors are involved in the activity-dependent synaptic wiring in the mouse cerebellar cortex.

    Directory of Open Access Journals (Sweden)

    Roberta Cesa

    2011-04-01

    Full Text Available Eph receptor tyrosine kinases are involved in many cellular processes. In the developing brain, they act as migratory and cell adhesive cues while in the adult brain they regulate dendritic spine plasticity. Here we show a new role for Eph receptor signalling in the cerebellar cortex. Cerebellar Purkinje cells are innervated by two different excitatory inputs. The climbing fibres contact the proximal dendritic domain of Purkinje cells, where synapse and spine density is low; the parallel fibres contact the distal dendritic domain, where synapse and spine density is high. Interestingly, Purkinje cells have the intrinsic ability to generate a high number of spines over their entire dendritic arborisations, which can be innervated by the parallel fibres. However, the climbing fibre input continuously exerts an activity-dependent repression on parallel fibre synapses, thus confining them to the distal Purkinje cell dendritic domain. Such repression persists after Eph receptor activation, but is overridden by Eph receptor inhibition with EphA4/Fc in neonatal cultured cerebellar slices as well as mature acute cerebellar slices, following in vivo infusion of the EphA4/Fc inhibitor and in EphB receptor-deficient mice. When electrical activity is blocked in vivo by tetrodotoxin leading to a high spine density in Purkinje cell proximal dendrites, stimulation of Eph receptor activation recapitulates the spine repressive effects of climbing fibres. These results suggest that Eph receptor signalling mediates the repression of spine proliferation induced by climbing fibre activity in Purkinje cell proximal dendrites. Such repression is necessary to maintain the correct architecture of the cerebellar cortex.

  14. Modulating activity of M1 receptor to the reaction of ileal smooth muscle

    Directory of Open Access Journals (Sweden)

    Izabela Glaza

    2011-08-01

    Full Text Available Background:The subject of the study was determination of the effect of drugs on ileal smooth muscle contraction induced by activation of M1 type muscarinic receptors. Drugs that have an effect on muscarinic receptors are divided to agonists, with close ties to the receptor and high internal activity and antagonists, with no internal activity. Conducted experiments tested interactions between a broad-spectrum agonist of muscarinic receptors, carbachol and a selective muscarinic receptor antagonist of M1 type, pirenzepine.Material/Methods:Testing was conducted on tissues isolated from rat’s intestine. Male Wistar rats with weight between 220 g and 360 g were anesthetized by intraperitoneal injection of urethane (120 mg/kg. Concentration-effect curves were determined with the use of cumulated concentration method, in accordance with the van Rossum method (1963 in Kenakin modification (2006.Results:The purpose of the study was determination of concentration-effect curves for carbachol. This curve was compared with the curve of receptor occupation depending on concentration of this drug. Based on concentration-effect curves, the average value of EC50 was calculated for carbachol, amounting to 2.44×10–6 [M/l].Conclusions:The results confirmed that atropine is effective in stopping contractions caused by carbachol, meeting the conditions of competitive antagonists. Atropine caused the shift of curves for carbachol to the right. Pirenzepine, selectively blocking muscarinic receptors of M1 type gave similar results. It was proved that in the preparation of gastric fundus smooth muscle, M1 type receptors occur not only presynaptically, but also postsynaptically.

  15. Platelet-activating factor (PAF) receptor as a promising target for cancer cell repopulation after radiotherapy

    Science.gov (United States)

    da Silva-Jr, I A; Chammas, R; Lepique, A P; Jancar, S

    2017-01-01

    A major drawback of radiotherapy is the accelerated growth of the surviving tumor cells. Radiotherapy generates a variety of lipids that bind to the receptor for platelet-activating factor, expressed by cells in the tumor microenvironment. In the present study, using the TC-1 tumor cell line, we found that irradiation induced a twofold increase in receptor expression and generated agonists of receptor. Irradiated cells induced a 20-fold increase in live TC-1 proliferation in vitro. Furthermore, subcutaneous co-injection of irradiated TC-1 cells with TC-1 expressing luciferase (TC-1 fluc+) markedly increased TC-1 fluc+ proliferation in a receptor-dependent way. Moreover we used a human carcinoma cell line not expressing the PAF receptor (KBM) and the same cell transfected with the receptor gene (KBP). Following co-injection of live KBP cells with irradiated KBM in RAG mice, the tumor growth was significantly increased compared with tumor formed following co-injection of live KBM with irradiated KBM. This tumor cell repopulation correlated with increased infiltration of tumor-promoting macrophages (CD206+). We propose that receptor represents a possible target for improving the efficacy of radiotherapy through inhibition of tumor repopulation. PMID:28134937

  16. Non-Neuronal Functions of the M2 Muscarinic Acetylcholine Receptor

    Directory of Open Access Journals (Sweden)

    Ritva Tikkanen

    2013-04-01

    Full Text Available Acetylcholine is an important neurotransmitter whose effects are mediated by two classes of receptors. The nicotinic acetylcholine receptors are ion channels, whereas the muscarinic receptors belong to the large family of G protein coupled seven transmembrane helix receptors. Beyond its function in neuronal systems, it has become evident that acetylcholine also plays an important role in non-neuronal cells such as epithelial and immune cells. Furthermore, many cell types in the periphery are capable of synthesizing acetylcholine and express at least some of the receptors. In this review, we summarize the non-neuronal functions of the muscarinic acetylcholine receptors, especially those of the M2 muscarinic receptor in epithelial cells. We will review the mechanisms of signaling by the M2 receptor but also the cellular trafficking and ARF6 mediated endocytosis of this receptor, which play an important role in the regulation of signaling events. In addition, we provide an overview of the M2 receptor in human pathological conditions such as autoimmune diseases and cancer.

  17. Investigation of in vitro Opioid Receptor Binding Activities of Some Turkish Salvia species

    Directory of Open Access Journals (Sweden)

    Özge Gündüz Çınar

    2011-01-01

    Full Text Available Kappa Opioid Peptide Receptor (KOPr activation produces analgesic, psychotomimetic, diuretic and antipruritic effects. KOPr ligands are investigated for their potential roles in the treatment of addiction, depression, feeding behavior, psychosis and schizophrenia. In this study the methanolic extracts of a number of Salvia species which are native to Turkey (S. tomentosa, S. tchihatcheffii , S. rosifolia, S. dichroantha and S. sclarea were tested for their potential binding to opioid receptors in rat brain membranes and Chinese Hamster Ovary Cells expressing human KOPr (CHO-KOPh. [ 3H]Diprenorphine, an unselective opioid antagonist, was utilized in the radioligand receptor binding assays. All extracts (0.11 mg/ml inhibited the [ 3H]Diprenorphine binding with ranging KOPr binding affinities. More than 50% inhibition of diprenorphine binding was shown only with Salvia dichroantha and Salvia sclarea both in rat brain membranes and CHO-KOPh membranes.Among them Salvia sclarea deserves further investigation for its active component(s and its pharmacological characterization. This study clearly demonstrates the potential opioid receptor binding activities of several Turkish Salvia species. This work constitutes the first study on in vitro opioid receptor binding activities of Salvia species from the Turkish flora.

  18. Inhibition of receptor-bound urokinase by plasminogen-activator inhibitors

    DEFF Research Database (Denmark)

    Ellis, Vincent John; Wun, T C; Behrendt, N

    1990-01-01

    Urokinase-type plasminogen activator (uPA) binds to a specific receptor on various cell types, the bound molecule retaining its enzymatic activity against plasminogen. We have now investigated whether receptor-bound uPA also retains the ability to react with and be inhibited by plasminogen...... activator inhibitors (PAI-1 and PAI-2). uPA bound to its receptor on human U937 monocyte-like cells was inhibited by PAI-1 (in its active form in the presence of vitronectin fragments) with an association rate constant of 4.5 x 10(6) M-1 s-1, which was 40% lower than that obtained for uPA in solution (7.9 x...... 10(6) M-1 s-1). The inhibition of uPA by PAI-2 was decreased to a similar extent by receptor binding, falling from 5.3 x 10(5) to 3.3 x 10(5) M-1 s-1. Stimulation of U937 cells with phorbol 12-myristate 13-acetate was accompanied by a further reduction in receptor-bound uPA inhibition by PAI-1...

  19. Cardiovascular response, feeding behavior and locomotor activity in mice lacking the NPY Y1 receptor.

    Science.gov (United States)

    Pedrazzini, T; Seydoux, J; Künstner, P; Aubert, J F; Grouzmann, E; Beermann, F; Brunner, H R

    1998-06-01

    Neuropeptide Y (NPY) is a 36-amino-acid neurotransmitter which is widely distributed throughout the central and peripheral nervous system. NPY involvement has been suggested in various physiological responses including cardiovascular homeostasis and the hypothalamic control of food intake. At least six subtypes of NPY receptors have been described. Because of the lack of selective antagonists, the specific role of each receptor subtype has been difficult to establish. Here we describe mice deficient for the expression of the Y1 receptor subtype. Homozygous mutant mice demonstrate a complete absence of blood pressure response to NPY, whereas they retain normal response to other vasoconstrictors. Daily food intake, as well as NPY-stimulated feeding, are only slightly diminished, whereas fast-induced refeeding is markedly reduced. Adult mice lacking the NPY Y1 receptor are characterized by increased body fat with no change in protein content. The higher energetic efficiency of mutant mice might result, in part, from the lower metabolic rate measured during the active period, associated with reduced locomotor activity. These results demonstrate the importance of NPY Y1 receptors in NPY-mediated cardiovascular response and in the regulation of body weight through central control of energy expenditure. In addition, these data are also indicative of a role for the Y1 receptor in the control of food intake.

  20. Urotensin II and urotensin II-related peptide activate somatostatin receptor subtypes 2 and 5.

    Science.gov (United States)

    Malagon, Maria M; Molina, Marcelo; Gahete, Manuel D; Duran-Prado, Mario; Martinez-Fuentes, Antonio J; Alcain, Francisco J; Tonon, Marie-Christine; Leprince, Jérôme; Vaudry, Hubert; Castaño, Justo P; Vazquez-Martinez, Rafael

    2008-05-01

    The UII and urotensin II-related peptide (URP) genes belong to the same superfamily as the somatostatin gene. It has been previously shown that somatostatin activates the UII-receptor (UTR). In contrast, the possible interaction between UII and URP and somatostatin receptors has remained scarcely analyzed. Herein, we have investigated the effects of UII and URP on cell proliferation and free cytosolic Ca2+ concentration ([Ca2+]i) in CHO-K1 cells stably expressing the porcine somatostatin receptor subtypes sst2 and sst5. Results show that both UII and URP induce stimulation of cell proliferation mediated by sst2 receptors and UII provokes inhibition of cell proliferation mediated by sst5 receptors. UII and URP also provoked an increase of [Ca2+]i in both sst2- and sst5-transfected cells. Together, our present data demonstrate that UII and URP directly activate sst2 and sst5 and thus mimic the effect of somatostatin on its cognate receptors.

  1. DREADDs (designer receptors exclusively activated by designer drugs): chemogenetic tools with therapeutic utility.

    Science.gov (United States)

    Urban, Daniel J; Roth, Bryan L

    2015-01-01

    In the past decade, emerging synthetic biology technologies such as chemogenetics have dramatically transformed how pharmacologists and systems biologists deconstruct the involvement of G protein-coupled receptors (GPCRs) in a myriad of physiological and translational settings. Here we highlight a specific chemogenetic application that extends the utility of the concept of RASSLs (receptors activated solely by synthetic ligands): We have dubbed it DREADDs (designer receptors exclusively activated by designer drugs). As we show in this review, DREADDs are now used ubiquitously to modulate GPCR activity noninvasively in vivo. Results from these studies have directly implicated GPCR signaling in a large number of therapeutically relevant contexts. We also highlight recent applications of DREADD technology that have illuminated GPCR signaling processes that control pathways relevant to the treatment of eating disorders, obesity, and obesity-associated metabolic abnormalities. Additionally, we provide an overview of the potential utility of chemogenetic technologies for transformative therapeutics.

  2. TRA-418, a novel compound having both thromboxane A(2) receptor antagonistic and prostaglandin I(2) receptor agonistic activities: its antiplatelet effects in human and animal platelets.

    Science.gov (United States)

    Yamada, N; Miyamoto, M; Isogaya, M; Suzuki, M; Ikezawa, S; Ohno, M; Otake, A; Umemura, K

    2003-08-01

    TRA-418 is a novel compound that has been found in our screening for compounds having both thromboxane A2 (TP) receptor antagonistic and prostaglandin I2 (IP) receptor agonistic activities. In the binding assays, TRA-418 showed a 10-fold higher affinity to TP-receptors than IP-receptors. TRA-418 inhibited platelet aggregation induced by the TP-receptor agonist, U-46619 and by arachidonic acid at concentrations lower than those required for inhibition of ADP-induced aggregations. Furthermore, TRA-418 inhibited not only platelet aggregation induced by ADP alone, but also that induced by ADP in the presence of the TP-receptor antagonist, SQ-29548. When the IC50 values of TRA-418 for platelet aggregation were estimated in platelet preparations from monkeys, dogs, cats, and rats using ADP and arachidonic acid as the platelet stimulating agents, it was found that the values estimated in monkey platelets were quite similar to those estimated in human platelets. In ex vivo platelet aggregation in monkeys, TRA-418 exhibited significant inhibitory effects on arachidonic acid-induced aggregation in platelet preparations from monkeys treated at 3 micro g kg min-1 or higher doses, where neither a significant decrease in blood pressure nor a significant increase in heart rate was observed. These results are consistent with the fact that TRA-418 has a relatively potent TP-receptor antagonistic activity together with a relatively weak IP-receptor agonistic activity.

  3. Importance of constitutive activity and arrestin-independent mechanisms for intracellular trafficking of the ghrelin receptor

    DEFF Research Database (Denmark)

    Holliday, Nicholas D; Holst, Birgitte; Rodionova, Elena A

    2007-01-01

    The ghrelin receptor (GhrelinR) and its related orphan GPR39 each display constitutive signaling, but only GhrelinRs undergo basal internalization. Here we investigate these differences by considering the roles of the C tail receptor domains for constitutive internalization and activity....... Furthermore the interaction between phosphorylated receptors and beta-arrestin adaptor proteins has been examined. Replacement of the FLAG-tagged GhrelinR C tail with the equivalent GPR39 domain (GhR-39 chimera) preserved G(q) signaling. However in contrast to the GhrelinR, GhR-39 receptors exhibited no basal...... and substantially decreased agonist-induced internalization in transiently transfected HEK293 cells. Internalized GhrelinR and GhR-39 were predominantly localized to recycling compartments, identified with transferrin and the monomeric G proteins Rab5 and Rab11. Both the inverse agonist [d-Arg(1), d-Phe(5), d-Trp(7...

  4. An atypical residue in the pore of Varroa destructor GABA-activated RDL receptors affects picrotoxin block and thymol modulation

    OpenAIRE

    Price, Kerry L.; Lummis, Sarah C.R.

    2014-01-01

    GABA-activated RDL receptors are the insect equivalent of mammalian GABAA receptors, and play a vital role in neurotransmission and insecticide action. Here we clone the pore lining M2 region of the Varroa mite RDL receptor and show that it has 4 atypical residues when compared to M2 regions of most other insects, including bees, which are the major host of Varroa mites. We create mutant Drosophila RDL receptors containing these substitutions and characterise their effects on function. Using ...

  5. Activation of µ-opioid receptors and block of KIR3 potassium channels and NMDA receptor conductance by l- and d-methadone in rat locus coeruleus

    Science.gov (United States)

    Matsui, Aya; Williams, John T

    2010-01-01

    BACKGROUND AND PURPOSE Methadone activates opioid receptors to increase a potassium conductance mediated by G-protein-coupled, inwardly rectifying, potassium (KIR3) channels. Methadone also blocks KIR3 channels and N-methyl-D-aspartic acid (NMDA) receptors. However, the concentration dependence and stereospecificity of receptor activation and channel blockade by methadone on single neurons has not been characterized. EXPERIMENTAL APPROACH Intracellular and whole-cell recording were made from locus coeruleus neurons in brain slices and the activation of µ-opioid receptors and blockade of KIR3 and NMDA channels with l- and d-methadone was examined. KEY RESULTS The potency of l-methadone, measured by the amplitude of hyperpolarization was 16.5-fold higher than with d-methadone. A maximum hyperpolarization was caused by both enantiomers (∼30 mV); however, the maximum outward current measured with whole-cell voltage-clamp recording was smaller than the current induced by [Met]5enkephalin. The KIR3 conductance induced by activation of α2-adrenoceptors was decreased with high concentrations of l- and d-methadone (10–30 µM). In addition, methadone blocked the resting inward rectifying conductance (KIR). Both l- and d-methadone blocked the NMDA receptor-dependent current. The block of NMDA receptor-dependent current was voltage-dependent suggesting that methadone acted as a channel blocker. CONCLUSIONS AND IMPLICATIONS Methadone activated µ-opioid receptors at low concentrations in a stereospecific manner. KIR3 and NMDA receptor channel block was not stereospecific and required substantially higher concentrations. The separation in the concentration range suggests that the activation of µ-opioid receptors rather than the channel blocking properties mediate both the therapeutic and toxic actions of methadone. PMID:20659105

  6. Ultraslow Water-Mediated Transmembrane Interactions Regulate the Activation of A2A Adenosine Receptor

    Science.gov (United States)

    Lee, Yoonji; Kim, Songmi; Choi, Sun; Hyeon, Changbong

    2016-09-01

    Water molecules inside G-protein coupled receptor have recently been spotlighted in a series of crystal structures. To decipher the dynamics and functional roles of internal waters in GPCR activity, we studied A$_{\\text{2A}}$ adenosine receptor using $\\mu$sec-molecular dynamics simulations. Our study finds that the amount of water flux across the transmembrane (TM) domain varies depending on the receptor state, and that the water molecules of the TM channel in the active state flow three times slower than those in the inactive state. Depending on the location in solvent-protein interface as well as the receptor state, the average residence time of water in each residue varies from $\\sim\\mathcal{O}(10^2)$ psec to $\\sim\\mathcal{O}(10^2)$ nsec. Especially, water molecules, exhibiting ultraslow relaxation ($\\sim\\mathcal{O}(10^2)$ nsec) in the active state, are found around the microswitch residues that are considered activity hotspots for GPCR function. A continuous allosteric network spanning the TM domain, arising from water-mediated contacts, is unique in the active state, underscoring the importance of slow waters in the GPCR activation.

  7. Therapeutic actions of an insulin receptor activator and a novel peroxisome proliferator-activated receptor gamma agonist in the spontaneously hypertensive obese rat model of metabolic syndrome X.

    Science.gov (United States)

    Velliquette, Rodney A; Friedman, Jacob E; Shao, J; Zhang, Bei B; Ernsberger, Paul

    2005-07-01

    Insulin resistance clusters with hyperlipidemia, impaired glucose tolerance, and hypertension as metabolic syndrome X. We tested a low molecular weight insulin receptor activator, demethylasterriquinone B-1 (DMAQ-B1), and a novel indole peroxisome proliferator-activated receptor gamma agonist, 2-(2-(4-phenoxy-2-propylphenoxy)ethyl)indole-5-acetic acid (PPEIA), in spontaneously hypertensive obese rats (SHROB), a genetic model of syndrome X. Agents were given orally for 19 days. SHROB showed fasting normoglycemia but impaired glucose tolerance after an oral load, as shown by increased glucose area under the curve (AUC) [20,700 mg x min/ml versus 8100 in lean spontaneously hypertensive rats (SHR)]. Insulin resistance was indicated by 20-fold excess fasting insulin and increased insulin AUC (6300 ng x min/ml versus 990 in SHR). DMAQ-B1 did not affect glucose tolerance (glucose AUC = 21,300) but reduced fasting insulin 2-fold and insulin AUC (insulin AUC = 4300). PPEIA normalized glucose tolerance (glucose AUC = 9100) and reduced insulin AUC (to 3180) without affecting fasting insulin. PPEIA also increased food intake, fat mass, and body weight gain (81 +/- 12 versus 45 +/- 8 g in untreated controls), whereas DMAQ-B1 had no effect on body weight but reduced subscapular fat mass. PPEIA but not DMAQ-B1 reduced blood pressure. In skeletal muscle, insulin-stimulated phosphorylation of the insulin receptor and insulin receptor substrate protein 1-associated phosphatidylinositol 3-kinase activity were decreased by 40 to 55% in SHROB relative to lean SHR. PPEIA, but not DMAQ-B1, enhanced both insulin actions. SHROB also showed severe hypertriglyceridemia (355 +/- 42 mg/dl versus 65 +/- 3 in SHR) attenuated by both agents (DMAQ-B1, 228 +/- 18; PPEIA, 79 +/- 3). Both these novel antidiabetic agents attenuate insulin resistance and hypertriglyceridemia associated with metabolic syndrome but via distinct mechanisms.

  8. Receptor-mediated regional sympathetic nerve activation by leptin.

    OpenAIRE

    Haynes, W G; Morgan, D. A.; Walsh, S.A.; Mark, A. L.; Sivitz, W I

    1997-01-01

    Leptin is a peptide hormone produced by adipose tissue which acts centrally to decrease appetite and increase energy expenditure. Although leptin increases norepinephrine turnover in thermogenic tissues, the effects of leptin on directly measured sympathetic nerve activity to thermogenic and other tissues are not known. We examined the effects of intravenous leptin and vehicle on sympathetic nerve activity to brown adipose tissue, kidney, hindlimb, and adrenal gland in anesthetized Sprague-Da...

  9. Investigation of Prolactin Receptor Activation and Blockade Using Time-Resolved Fluorescence Resonance Energy Transfer

    Directory of Open Access Journals (Sweden)

    Estelle eTallet

    2011-09-01

    Full Text Available The prolactin receptor (PRLR is emerging as a therapeutic target in oncology. Knowledge-based drug design led to the development of a pure PRLR antagonist (Del1-9-G129R-hPRL that was recently shown to prevent PRL-induced mouse prostate tumorogenesis. In humans, the first gain-of-function mutation of the PRLR (PRLRI146L was recently identified in breast tumor patients. At the molecular level, the actual mechanism of action of these two novel players in the PRL system remains elusive. In this study, we addressed whether constitutive PRLR activation (PRLRI146L or PRLR blockade (antagonist involved alteration of receptor oligomerization and/or of inter-chain distances compared to unstimulated and PRL-stimulated PRLR. Using a combination of various biochemical and spectroscopic approaches (co-IP, blue-native electrophoresis, BRET1, we demonstrated that preformed PRLR homodimers are altered neither by PRL- or I146L-induced receptor triggering, nor by antagonist-mediated blockade. These findings were confirmed using a novel time-resolved fluorescence resonance energy transfer (TR-FRET technology that allows monitoring distance changes between cell-surface tagged receptors. This technology revealed that PRLR blockade or activation did not involve detectable distance changes between extracellular domains of receptor chains within the dimer. This study merges with our previous structural investigations suggesting that the mechanism of PRLR activation solely involves intermolecular contact adaptations leading to subtle intramolecular rearrangements.

  10. RGS4 regulates partial agonism of the M2 muscarinic receptor-activated K+ currents.

    Science.gov (United States)

    Chen, I-Shan; Furutani, Kazuharu; Inanobe, Atsushi; Kurachi, Yoshihisa

    2014-03-15

    Partial agonists are used clinically to avoid overstimulation of receptor-mediated signalling, as they produce a submaximal response even at 100% receptor occupancy. The submaximal efficacy of partial agonists is due to conformational change of the agonist-receptor complex, which reduces effector activation. In addition to signalling activators, several regulators help control intracellular signal transductions. However, it remains unclear whether these signalling regulators contribute to partial agonism. Here we show that regulator of G-protein signalling (RGS) 4 is a determinant for partial agonism of the M2 muscarinic receptor (M2R). In rat atrial myocytes, pilocarpine evoked smaller G-protein-gated K(+) inwardly rectifying (KG) currents than those evoked by ACh. In a Xenopus oocyte expression system, pilocarpine acted as a partial agonist in the presence of RGS4 as it did in atrial myocytes, while it acted like a full agonist in the absence of RGS4. Functional couplings within the agonist-receptor complex/G-protein/RGS4 system controlled the efficacy of pilocarpine relative to ACh. The pilocarpine-M2R complex suppressed G-protein-mediated activation of KG currents via RGS4. Our results demonstrate that partial agonism of M2R is regulated by the RGS4-mediated inhibition of G-protein signalling. This finding helps us to understand the molecular components and mechanism underlying the partial agonism of M2R-mediated physiological responses.

  11. Glucocorticoid receptor mediated suppression of natural killer cell activity: identification of associated deacetylase and corepressor molecules.

    Science.gov (United States)

    Bush, Kristin A; Krukowski, Karen; Eddy, Justin L; Janusek, Linda Witek; Mathews, Herbert L

    2012-01-01

    Physical and psychological stressors reduce natural killer cell function. This reduction in cellular function results from stress-induced release of glucocorticoids. Glucocorticoids act upon natural killer cells to deacetylate and transrepress immune response genes through epigenetic processes. However, other than the glucocorticoid receptor, the proteins that participate in this process are not well described in natural killer cells. The purpose of this study was to identify the proteins associated with the glucocorticoid receptor that are likely epigenetic participants in this process. Treatment of natural killer cells with the synthetic glucocorticoid, dexamethasone, produced a significant time dependent reduction in natural killer cell activity as early as 8h post treatment. This reduction in natural killer cell activity was preceded by nuclear localization of the glucocorticoid receptor with histone deacetylase 1 and the corepressor, SMRT. Other class I histone deacetylases were not associated with the glucocorticoid receptor nor was the corepressor NCoR. These results demonstrate histone deacetylase 1 and SMRT to associate with the ligand activated glucocorticoid receptor within the nuclei of natural killer cells and to be the likely participants in the histone deacetylation and transrepression that accompanies glucocorticoid mediated reductions in natural killer cell function. Copyright © 2012 Elsevier Inc. All rights reserved.

  12. Phasic dopamine release drives rapid activation of striatal D2-receptors

    Science.gov (United States)

    Marcott, Pamela F; Mamaligas, Aphroditi A; Ford, Christopher P

    2014-01-01

    Summary Striatal dopamine transmission underlies numerous goal-directed behaviors. Medium spiny neurons (MSNs) are a major target of dopamine in the striatum. However, as dopamine does not directly evoke a synaptic event in MSNs, the time course of dopamine signaling in these cells remains unclear. To examine how dopamine release activates D2-receptors on MSNs, G-protein activated inwardly rectifying potassium (GIRK2; Kir 3.2) channels were virally overexpressed in the striatum and the resulting outward currents were used as a sensor of D2-receptor activation. Electrical and optogenetic stimulation of dopamine terminals evoked robust D2-receptor inhibitory post-synaptic currents (IPSCs) in GIRK2-expressing MSNs that occurred in under a second. Evoked D2-IPSCs could be driven by repetitive stimulation and were not occluded by background dopamine tone. Together, the results indicate that D2-receptors on MSNs exhibit functional low affinity and suggest that striatal D2-receptors can encode both tonic and phasic dopamine signals. PMID:25242218

  13. Receptor-mediated enhancement of beta adrenergic drug activity by ascorbate in vitro and in vivo.

    Directory of Open Access Journals (Sweden)

    Patrick F Dillon

    Full Text Available RATIONALE: Previous in vitro research demonstrated that ascorbate enhances potency and duration of activity of agonists binding to alpha 1 adrenergic and histamine receptors. OBJECTIVES: Extending this work to beta 2 adrenergic systems in vitro and in vivo. METHODS: Ultraviolet spectroscopy was used to study ascorbate binding to adrenergic receptor preparations and peptides. Force transduction studies on acetylcholine-contracted trachealis preparations from pigs and guinea pigs measured the effect of ascorbate on relaxation due to submaximal doses of beta adrenergic agonists. The effect of inhaled albuterol with and without ascorbate was tested on horses with heaves and sheep with carbachol-induced bronchoconstriction. MEASUREMENTS: Binding constants for ascorbate binding to beta adrenergic receptor were derived from concentration-dependent spectral shifts. Dose- dependence curves were obtained for the relaxation of pre-contracted trachealis preparations due to beta agonists in the presence and absence of varied ascorbate. Tachyphylaxis and fade were also measured. Dose response curves were determined for the effect of albuterol plus-and-minus ascorbate on airway resistance in horses and sheep. MAIN RESULTS: Ascorbate binds to the beta 2 adrenergic receptor at physiological concentrations. The receptor recycles dehydroascorbate. Physiological and supra-physiological concentrations of ascorbate enhance submaximal epinephrine and isoproterenol relaxation of trachealis, producing a 3-10-fold increase in sensitivity, preventing tachyphylaxis, and reversing fade. In vivo, ascorbate improves albuterol's effect on heaves and produces a 10-fold enhancement of albuterol activity in "asthmatic" sheep. CONCLUSIONS: Ascorbate enhances beta-adrenergic activity via a novel receptor-mediated mechanism; increases potency and duration of beta adrenergic agonists effective in asthma and COPD; prevents tachyphylaxis; and reverses fade. These novel effects are

  14. Emergence of constitutively active estrogen receptor-α mutations in pretreated advanced estrogen receptor positive breast cancer

    Science.gov (United States)

    Meric-Bernstam, Funda; Gonzalez-Angulo, Ana Maria; Ferrer-Lozano, Jaime; Perez-Fidalgo, Jose A.; Cristofanilli, Massimo; Gómez, Henry; Arteaga, Carlos L.; Giltnane, Jennifer; Balko, Justin M.; Cronin, Maureen T; Jarosz, Mirna; Sun, James; Hawryluk, Matthew; Lipson, Doron; Otto, Geoff; Ross, Jeffrey S; Dvir, Addie; Soussan-Gutman, Lior; Wolf, Ido; Rubinek, Tamar; Gilmore, Lauren; Schnitt, Stuart; Come, Steven E.; Pusztai, Lajos; Stephens, Philip; Brown, Myles; Miller, Vincent A.

    2014-01-01

    Purpose We undertook this study to determine the prevalence of estrogen receptor (ER) α (ESR1) mutations throughout the natural history of hormone dependent breast cancer and to delineate the functional roles of the most commonly detected alterations. Experimental Design We studied a total of 249 tumor specimens from 208 patients. The specimens include 134 ER positive (ER+/HER2–) and, as controls, 115 ER negative (ER−) tumors. The ER+ samples consist of 58 primary breast cancers and 76 metastatic samples. All tumors were sequenced to high unique coverage using next generation sequencing targeting the coding sequence of the estrogen receptor and an additional 182 cancer-related genes. Results Recurring somatic mutations in codons 537 and 538 within the ligand-binding domain of ER were detected in ER+ metastatic disease. Overall, the frequency of these mutations was 12% (9/76, 95% CI 6%-21%) in metastatic tumors and in a subgroup of patients who received an average of 7 lines of treatment the frequency was 20% (5/25, 95% CI 7%-41%). These mutations were not detected in primary or treatment naïve ER+ cancer or in any stage of ER− disease. Functional studies in cell line models demonstrate that these mutations render estrogen receptor constitutive activity and confer partial resistance to currently available endocrine treatments. Conclusions In this study we show evidence for the temporal selection of functional ESR1 mutations as potential drivers of endocrine resistance during the progression of ER positive breast cancer. PMID:24398047

  15. Synthetic studies of neoclerodane diterpenes from Salvia divinorum: preparation and opioid receptor activity of salvinicin analogues.

    Science.gov (United States)

    Simpson, Denise S; Katavic, Peter L; Lozama, Anthony; Harding, Wayne W; Parrish, Damon; Deschamps, Jeffrey R; Dersch, Christina M; Partilla, John S; Rothman, Richard B; Navarro, Hernan; Prisinzano, Thomas E

    2007-07-26

    Further modification of salvinorin A (1a), the major active component of Salvia divinorum, has resulted in the synthesis of novel neoclerodane diterpenes with opioid receptor affinity and activity. We report in this study that oxadiazole 11a and salvidivin A (12a), a photooxygenation product of 1a, have been identified as the first neoclerodane diterpenes with kappa antagonist activity. This indicates that additional structural modifications of 1a may lead to analogues with higher potency and utility as drug abuse medications.

  16. GABAA receptors in visual and auditory cortex and neural activity changes during basic visual stimulation

    Science.gov (United States)

    Qin, Pengmin; Duncan, Niall W.; Wiebking, Christine; Gravel, Paul; Lyttelton, Oliver; Hayes, Dave J.; Verhaeghe, Jeroen; Kostikov, Alexey; Schirrmacher, Ralf; Reader, Andrew J.; Northoff, Georg

    2012-01-01

    Recent imaging studies have demonstrated that levels of resting γ-aminobutyric acid (GABA) in the visual cortex predict the degree of stimulus-induced activity in the same region. These studies have used the presentation of discrete visual stimulus; the change from closed eyes to open also represents a simple visual stimulus, however, and has been shown to induce changes in local brain activity and in functional connectivity between regions. We thus aimed to investigate the role of the GABA system, specifically GABAA receptors, in the changes in brain activity between the eyes closed (EC) and eyes open (EO) state in order to provide detail at the receptor level to complement previous studies of GABA concentrations. We conducted an fMRI study involving two different modes of the change from EC to EO: an EO and EC block design, allowing the modeling of the haemodynamic response, followed by longer periods of EC and EO to allow the measuring of functional connectivity. The same subjects also underwent [18F]Flumazenil PET to measure GABAA receptor binding potentials. It was demonstrated that the local-to-global ratio of GABAA receptor binding potential in the visual cortex predicted the degree of changes in neural activity from EC to EO. This same relationship was also shown in the auditory cortex. Furthermore, the local-to-global ratio of GABAA receptor binding potential in the visual cortex also predicted the change in functional connectivity between the visual and auditory cortex from EC to EO. These findings contribute to our understanding of the role of GABAA receptors in stimulus-induced neural activity in local regions and in inter-regional functional connectivity. PMID:23293594

  17. GABAA receptors in visual and auditory cortex and neural activity changes during basic visual stimulation

    Directory of Open Access Journals (Sweden)

    Pengmin eQin

    2012-12-01

    Full Text Available Recent imaging studies have demonstrated that levels of resting GABA in the visual cortex predict the degree of stimulus-induced activity in the same region. These studies have used the presentation of discrete visual stimulus; the change from closed eyes to open also represents a simple visual stimulus, however, and has been shown to induce changes in local brain activity and in functional connectivity between regions. We thus aimed to investigate the role of the GABA system, specifically GABAA receptors, in the changes in brain activity between the eyes closed (EC and eyes open (EO state in order to provide detail at the receptor level to complement previous studies of GABA concentrations. We conducted an fMRI study involving two different modes of the change from EC to EO: An EO and EC block design, allowing the modelling of the haemodynamic response, followed by longer periods of EC and EO to allow the measuring of functional connectivity. The same subjects also underwent [18F]Flumazenil PET measure GABAA receptor binding potentials. It was demonstrated that the local-to-global ratio of GABAA receptor binding potential in the visual cortex predicted the degree of changes in neural activity from EC to EO. This same relationship was also shown in the auditory cortex. Furthermore, the local-to-global ratio of GABAA receptor binding potential in the visual cortex also predicts the change of functional connectivity between visual and auditory cortex from EC to EO. These findings contribute to our understanding of the role of GABAA receptors in stimulus-induced neural activity in local regions and in inter-regional functional connectivity.

  18. Denatonium induces secretion of glucagon-like peptide-1 through activation of bitter taste receptor pathways

    Science.gov (United States)

    Kim, Ki-Suk; Egan, Josephine M.

    2016-01-01

    Aims/hypothesis This study was designed to ascertain whether human enteroendocrine cells express bitter taste receptors, and whether activation of these receptors with bitter-tasting ligands induces secretion of glucagon-like peptide-1 (GLP-1) and peptide YY (PYY). Methods We used human enteroendocrine NCI-H716 cells, isolated duodenal segments from mice, and whole mice as our experimental systems for investigating stimuli and mechanisms underlying GLP-1- and PYY-stimulated release. We measured hormone levels by ELISA and determined bitter taste receptor expression by real-time quantitative PCR. We adopted a pharmacological approach using inhibitors and enhancers of downstream signalling pathways known to be involved in bitter taste transduction in taste bud cells to investigate these pathways in NCI-H716 cells. Results Using a pharmacological approach, we identified signalling pathways triggered by the denatonium benzoate (DB)-activated bitter receptors. This involved activation of α-gustducin (Gαgust)—the specific G-protein subunit that is also present in taste bud cells—reduction of intracellular cAMP levels and enhancement of phospholipase C (PLC) activity, which ultimately led to increased intracellular calcium concentrations and hormone release. Gavage of DB, followed by gavage of glucose, to db/db mice stimulated GLP-1 and subsequent insulin secretion, leading to lower blood glucose levels. Conclusions/interpretation Our study demonstrates that activation of gut-expressed bitter taste receptors stimulates GLP-1 secretion in a PLC-dependent manner. In diabetic mice, DB (a ligand of bitter taste receptor cells), when given via gavage, lowers blood glucose levels in diabetic mice after oral glucose administration, through increased secretion of GLP-1. PMID:25016595

  19. Activation of NMDA receptors in lumbar spinothalamic cells is required for ejaculation.

    Science.gov (United States)

    Staudt, Michael D; de Oliveira, Cleusa V R; Lehman, Michael N; McKenna, Kevin E; Coolen, Lique M

    2011-04-01

    The sexual reflex ejaculation is controlled by a spinal ejaculation generator located in the lumbosacral spinal cord. A population of spinothalamic (LSt) neurons forms a key component of this generator, as manipulations of LSt cells either block or trigger ejaculation. However, it is currently unknown which afferent signals contribute to the activation of LSt cells and ejaculation. The current study tested the hypothesis that glutamate, via activation of N-Methyl-D-aspartic acid (NMDA) receptors in LSt cells, is a key regulator of ejaculation. Expression of phosphorylated NMDA receptor subunit 1 (NR1) was investigated following mating, or following ejaculation induced by electrical stimulation of the dorsal penile nerve (DPN) in anesthetized, spinalized male rats. Next, the effects of intraspinal delivery of NMDA receptor antagonist AP-5 on DPN stimulation-induced ejaculation were examined. Moreover, the ability of intraspinal delivery of NMDA to trigger ejaculation was examined. Finally, the site of action of NMDA was determined by studying effects of NMDA in male rats with LSt cell-specific lesions. Expression of NR1 and phosphorylated NR1 in LSt cells was analyzed. Electromyographic recordings of the bulbocavernosus muscle (BCM) were recorded in anesthetized, spinalized rats following stimulation of the DPN and delivery of AP-5 or NMDA. Results indicate that the NR1 receptors are activated in LSt cells following ejaculation in mating animals or induced by DPN stimulation in anesthetized, spinalized animals. Moreover, NR1 activation in LSt cells is an essential trigger for rhythmic BCM bursting, as DPN stimulation-induced reflexes were absent following administration of NMDA receptor antagonist in the L3-L4 spinal area, and were triggered by NMDA. NMDA effects were dependent on intact LSt cells and were absent in LSt-lesioned males. These results demonstrate that glutamate, via activation of NMDA receptors in LSt cells, is a key afferent signal for ejaculation.

  20. Activation of mu opioid receptors sensitizes transient receptor potential vanilloid type 1 (TRPV1 via β-arrestin-2-mediated cross-talk.

    Directory of Open Access Journals (Sweden)

    Matthew P Rowan

    Full Text Available The transient receptor potential family V1 channel (TRPV1 is activated by multiple stimuli, including capsaicin, acid, endovanilloids, and heat (>42C. Post-translational modifications to TRPV1 result in dynamic changes to the sensitivity of receptor activation. We have previously demonstrated that β-arrestin2 actively participates in a scaffolding mechanism to inhibit TRPV1 phosphorylation, thereby reducing TRPV1 sensitivity. In this study, we evaluated the effect of β-arrestin2 sequestration by G-protein coupled receptors (GPCRs on thermal and chemical activation of TRPV1. Here we report that activation of mu opioid receptor by either morphine or DAMGO results in β-arrestin2 recruitment to mu opioid receptor in sensory neurons, while activation by herkinorin does not. Furthermore, treatment of sensory neurons with morphine or DAMGO stimulates β-arrestin2 dissociation from TRPV1 and increased sensitivity of the receptor. Conversely, herkinorin treatment has no effect on TRPV1 sensitivity. Additional behavioral studies indicate that GPCR-driven β-arrestin2 sequestration plays an important peripheral role in the development of thermal sensitivity. Taken together, the reported data identify a novel cross-talk mechanism between GPCRs and TRPV1 that may contribute to multiple clinical conditions.

  1. Mineralocorticoid receptor inhibits CREB signaling by calcineurin activation.

    Science.gov (United States)

    Grossmann, Claudia; Wuttke, Martin; Ruhs, Stefanie; Seiferth, Anja; Mildenberger, Sigrid; Rabe, Sindy; Schwerdt, Gerald; Gekle, Michael

    2010-06-01

    We investigated the interaction of MR with cAMP-response element binding protein (CREB) and provide a mechanistic explanation and insights into the cellular relevance. MR --> CREB crosstalk was assessed in vascular smooth muscle cells and heterologous expression systems. Experiments were designed in a way that only one variable changed at a time and the respective vehicles served as controls. MR, but not GR, activation (aldosterone or hydrocortisone, IC(50), approximately 0.3 nM) inhibits CREB transcriptional activity induced by stimulation of beta1/2-adrenoceptors and adenylyl cyclase or addition of membrane-permeable cAMP up to 70% within 2 h after addition. The MR DNA-binding domain is not required for this inhibition. cAMP formation is virtually unchanged, whereas MR exerts a robust inhibition of CREB(S133) phosphorylation via calcineurin/PP2B activation without changes in PP2B-Aalpha or beta expression. In parallel, the PP2B-sensitive NFaT-pathway is activated. The inhibitory crosstalk attenuates CREB-induced glucose-6-phosphate dehydrogenase expression. Overall, transcriptional relevant MR --> CREB crosstalk occurs at the level of CREB phosphorylation by enhanced calcineurin activity, enables GRE-independent genomic signaling of MR, and is of potential pathophysiological relevance.

  2. Activation of the nuclear receptor PPARγ by metabolites isolated from sage (Salvia officinalis L.).

    Science.gov (United States)

    Christensen, K B; Jørgensen, M; Kotowska, D; Petersen, R K; Kristiansen, K; Christensen, L P

    2010-10-28

    Salvia officinalis has been used as a traditional remedy against diabetes in many countries and its glucose-lowering effects have been demonstrated in animal studies. The active compounds and their possible mode of action are still unknown although it has been suggested that diterpenes may be responsible for the anti-diabetic effect of Salvia officinalis. To investigate whether the reported anti-diabetic effects of Salvia officinalis are related to activation of the nuclear receptor peroxisome proliferator-activated receptor (PPAR)γ and to identify the bioactive constituents. From a dichloromethane extract of Salvia officinalis able to activate PPARγ several major metabolites were isolated by chromatographic techniques. To assess bioactivity of the isolated metabolites a PPARγ transactivation assay was used. Eight diterpenes were isolated and identified including a new abietane diterpene being the epirosmanol ester of 12-O-methyl carnosic acid and 20-hydroxyferruginol, which was isolated from Salvia officinalis for the first time, as well as viridiflorol, oleanolic acid, and α-linolenic acid. 12-O-methyl carnosic acid and α-linolenic acid were able to significantly activate PPARγ whereas the remaining metabolites were either unable to activate PPARγ or yielded insignificant activation. Selected metabolites from Salvia officinalis were able to activate PPARγ and hence, the anti-diabetic activity of this plant could in part be mediated through this nuclear receptor. Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.

  3. Prefrontal cortical network activity: Opposite effects of psychedelic hallucinogens and D1/D5 dopamine receptor activation.

    Science.gov (United States)

    Lambe, E K; Aghajanian, G K

    2007-03-30

    The fine-tuning of network activity provides a modulating influence on how information is processed and interpreted in the brain. Here, we use brain slices of rat prefrontal cortex to study how recurrent network activity is affected by neuromodulators known to alter normal cortical function. We previously determined that glutamate spillover and stimulation of extrasynaptic N-methyl-d-aspartic acid (NMDA) receptors are required to support hallucinogen-induced cortical network activity. Since microdialysis studies suggest that psychedelic hallucinogens and dopamine D1/D5 receptor agonists have opposite effects on extracellular glutamate in prefrontal cortex, we hypothesized that these two families of psychoactive drugs would have opposite effects on cortical network activity. We found that network activity can be enhanced by 2,5-dimethoxy-4-iodoamphetamine (DOI) (a psychedelic hallucinogen that is a partial agonist of 5-HT(2A/2C) receptors) and suppressed by the selective D1/D5 agonist SKF 38393. This suppression could be mimicked by direct activation of adenylyl cyclase with forskolin or by addition of a cAMP analog. These findings are consistent with previous work showing that activation of adenylyl cyclase can upregulate neuronal glutamate transporters, thereby decreasing synaptic spillover of glutamate. Consistent with this hypothesis, a low concentration of the glutamate transporter inhibitor threo-beta-benzoylaspartic acid (TBOA) restored electrically-evoked recurrent activity in the presence of a selective D1/D5 agonist, whereas recurrent activity in the presence of a low level of the GABA(A) antagonist bicuculline was not resistant to suppression by the D1/D5 agonist. The tempering of network UP states by D1/D5 receptor activation may have implications for the proposed use of D1/D5 agonists in the treatment of schizophrenia.

  4. Nifedipine inhibits advanced glycation end products (AGEs) and their receptor (RAGE) interaction-mediated proximal tubular cell injury via peroxisome proliferator-activated receptor-gamma activation

    Energy Technology Data Exchange (ETDEWEB)

    Matsui, Takanori [Department of Pathophysiology and Therapeutics of Diabetic Vascular Complications, Kurume University School of Medicine, Kurume (Japan); Yamagishi, Sho-ichi, E-mail: shoichi@med.kurume-u.ac.jp [Department of Pathophysiology and Therapeutics of Diabetic Vascular Complications, Kurume University School of Medicine, Kurume (Japan); Takeuchi, Masayoshi [Department of Pathophysiological Science, Faculty of Pharmaceutical Science, Hokuriku University, Kanazawa (Japan); Ueda, Seiji; Fukami, Kei; Okuda, Seiya [Department of Medicine, Kurume University School of Medicine, Kurume (Japan)

    2010-07-23

    Research highlights: {yields} Nifedipine inhibited the AGE-induced up-regulation of RAGE mRNA levels in tubular cells, which was prevented by GW9662, an inhibitor of peroxisome proliferator-activated receptor-{gamma}. {yields} GW9662 treatment alone increased RAGE mRNA levels in tubular cells. {yields} Nifedipine inhibited the AGE-induced reactive oxygen species generation, NF-{kappa}B activation and increases in intercellular adhesion molecule-1 and transforming growth factor-{beta} gene expression in tubular cells, all of which were blocked by GW9662. -- Abstract: There is a growing body of evidence that advanced glycation end products (AGEs) and their receptor (RAGE) interaction evokes oxidative stress generation and subsequently elicits inflammatory and fibrogenic reactions, thereby contributing to the development and progression of diabetic nephropathy. We have previously found that nifedipine, a calcium-channel blocker (CCB), inhibits the AGE-induced mesangial cell damage in vitro. However, effects of nifedipine on proximal tubular cell injury remain unknown. We examined here whether and how nifedipine blocked the AGE-induced tubular cell damage. Nifedipine, but not amlodipine, a control CCB, inhibited the AGE-induced up-regulation of RAGE mRNA levels in tubular cells, which was prevented by the simultaneous treatment of GW9662, an inhibitor of peroxisome proliferator-activated receptor-{gamma} (PPAR{gamma}). GW9662 treatment alone was found to increase RAGE mRNA levels in tubular cells. Further, nifedipine inhibited the AGE-induced reactive oxygen species generation, NF-{kappa}B activation and increases in intercellular adhesion molecule-1 and transforming growth factor-beta gene expression in tubular cells, all of which were blocked by GW9662. Our present study provides a unique beneficial aspect of nifedipine on diabetic nephropathy; it could work as an anti-oxidative and anti-inflammatory agent against AGEs in tubular cells by suppressing RAGE expression

  5. Peroxisome Proliferator-Activated Receptor Ligands and Their Role in Chronic Myeloid Leukemia: Therapeutic Strategies.

    Science.gov (United States)

    Yousefi, Bahman; Samadi, Nasser; Baradaran, Behzad; Shafiei-Irannejad, Vahid; Zarghami, Nosratollah

    2016-07-01

    Imatinib therapy remains the gold standard for treatment of chronic myeloid leukemia; however, the acquired resistance to this therapeutic agent in patients has urged the scientists to devise modalities for overcoming this chemoresistance. For this purpose, initially therapeutic agents with higher tyrosine kinase activity were introduced, which had the potential for inhibiting even mutant forms of Bcr-Abl. Furthermore, coupling imatinib with peroxisome proliferator-activated receptor ligands also showed beneficial effects in chronic myeloid leukemia cell proliferation. These combination protocols inhibited cell growth and induced apoptosis as well as differentiation in chronic myeloid leukemia cell lines. In addition, peroxisome proliferator-activated receptors ligands increased imatinib uptake by upregulating the expression of human organic cation transporter 1. Taken together, peroxisome proliferator-activated receptors ligands are currently being considered as novel promising therapeutic candidates for chronic myeloid leukemia treatment, because they can synergistically enhance the efficacy of imatinib. In this article, we reviewed the potential of peroxisome proliferator-activated receptors ligands for use in chronic myeloid leukemia treatment. The mechanism of action of these therapeutics modalities are also presented in detail. © 2016 John Wiley & Sons A/S.

  6. Direct activation of Transient Receptor Potential Vanilloid 1(TRPV1 by Diacylglycerol (DAG

    Directory of Open Access Journals (Sweden)

    Oh Seog

    2008-10-01

    Full Text Available Abstract The capsaicin receptor, known as transient receptor potential channel vanilloid subtype 1 (TRPV1, is activated by a wide range of noxious stimulants and putative ligands such as capsaicin, heat, pH, anandamide, and phosphorylation by protein kinase C (PKC. However, the identity of endogenous activators for TRPV1 under physiological condition is still debated. Here, we report that diacylglycerol (DAG directly activates TRPV1 channel in a membrane-delimited manner in rat dorsal root ganglion (DRG neurons. 1-oleoyl-2-acetyl-sn-glycerol (OAG, a membrane-permeable DAG analog, elicited intracellular Ca2+ transients, cationic currents and cobalt uptake that were blocked by TRPV1-selective antagonists, but not by inhibitors of PKC and DAG lipase in rat DRG neurons or HEK 293 cells heterologously expressing TRPV1. OAG induced responses were about one fifth of capsaicin induced signals, suggesting that OAG displays partial agonism. We also found that endogenously produced DAG can activate rat TRPV1 channels. Mutagenesis of rat TRPV1 revealed that DAG-binding site is at Y511, the same site for capsaicin binding, and PtdIns(4,5P2binding site may not be critical for the activation of rat TRPV1 by DAG in heterologous system. We propose that DAG serves as an endogenous ligand for rat TRPV1, acting as an integrator of Gq/11-coupled receptors and receptor tyrosine kinases that are linked to phospholipase C.

  7. Extracellular polysaccharides produced by Ganoderma formosanum stimulate macrophage activation via multiple pattern-recognition receptors

    Science.gov (United States)

    2012-01-01

    Background The fungus of Ganoderma is a traditional medicine in Asia with a variety of pharmacological functions including anti-cancer activities. We have purified an extracellular heteropolysaccharide fraction, PS-F2, from the submerged mycelia culture of G. formosanum and shown that PS-F2 exhibits immunostimulatory activities. In this study, we investigated the molecular mechanisms of immunostimulation by PS-F2. Results PS-F2-stimulated TNF-α production in macrophages was significantly reduced in the presence of blocking antibodies for Dectin-1 and complement receptor 3 (CR3), laminarin, or piceatannol (a spleen tyrosine kinase inhibitor), suggesting that PS-F2 recognition by macrophages is mediated by Dectin-1 and CR3 receptors. In addition, the stimulatory effect of PS-F2 was attenuated in the bone marrow-derived macrophages from C3H/HeJ mice which lack functional Toll-like receptor 4 (TLR4). PS-F2 stimulation triggered the phosphorylation of mitogen-activated protein kinases JNK, p38, and ERK, as well as the nuclear translocation of NF-κB, which all played essential roles in activating TNF-α expression. Conclusions Our results indicate that the extracellular polysaccharides produced by G. formosanum stimulate macrophages via the engagement of multiple pattern-recognition receptors including Dectin-1, CR3 and TLR4, resulting in the activation of Syk, JNK, p38, ERK, and NK-κB and the production of TNF-α. PMID:22883599

  8. Identification of chemical modulators of the constitutive activated receptor (CAR) in a gene expression compendium

    Science.gov (United States)

    Oshida, Keiyu; Vasani, Naresh; Jones, Carlton; Moore, Tanya; Hester, Susan; Nesnow, Stephen; Auerbach, Scott; Geter, David R.; Aleksunes, Lauren M.; Thomas, Russell S.; Applegate, Dawn; Klaassen, Curtis D.; Corton, J. Christopher

    2015-01-01

    The nuclear receptor family member constitutive activated receptor (CAR) is activated by structurally diverse drugs and environmentally-relevant chemicals leading to transcriptional regulation of genes involved in xenobiotic metabolism and transport. Chronic activation of CAR increases liver cancer incidence in rodents, whereas suppression of CAR can lead to steatosis and insulin insensitivity. Here, analytical methods were developed to screen for chemical treatments in a gene expression compendium that lead to alteration of CAR activity. A gene expression biomarker signature of 83 CAR-dependent genes was identified using microarray profiles from the livers of wild-type and CAR-null mice after exposure to three structurally-diverse CAR activators (CITCO, phenobarbital, TCPOBOP). A rank-based algorithm (Running Fisher’s algorithm (p-value ≤ 10-4)) was used to evaluate the similarity between the CAR biomarker signature and a test set of 28 and 32 comparisons positive or negative, respectively, for CAR activation; the test resulted in a balanced accuracy of 97%. The biomarker signature was used to identify chemicals that activate or suppress CAR in an annotated mouse liver/primary hepatocyte gene expression database of ~1850 comparisons. CAR was activated by 1) activators of the aryl hydrocarbon receptor (AhR) in wild-type but not AhR-null mice, 2) pregnane X receptor (PXR) activators in wild-type and to lesser extents in PXR-null mice, and 3) activators of PPARα in wild-type and PPARα-null mice. CAR was consistently activated by five conazole fungicides and four perfluorinated compounds. Comparison of effects in wild-type and CAR-null mice showed that the fungicide propiconazole increased liver weight and hepatocyte proliferation in a CAR-dependent manner, whereas the perfluorinated compound perfluorooctanoic acid (PFOA) increased these endpoints in a CAR-independent manner. A number of compounds suppressed CAR coincident with increases in markers of

  9. Internalization mechanisms of the epidermal growth factor receptor after activation with different ligands.

    Directory of Open Access Journals (Sweden)

    Lasse Henriksen

    Full Text Available The epidermal growth factor receptor (EGFR regulates normal growth and differentiation, but dysregulation of the receptor or one of the EGFR ligands is involved in the pathogenesis of many cancers. There are eight ligands for EGFR, however most of the research into trafficking of the receptor after ligand activation focuses on the effect of epidermal growth factor (EGF and transforming growth factor-α (TGF-α. For a long time it was believed that clathrin-mediated endocytosis was the major pathway for internalization of the receptor, but recent work suggests that different pathways exist. Here we show that clathrin ablation completely inhibits internalization of EGF- and TGF-α-stimulated receptor, however the inhibition of receptor internalization in cells treated with heparin-binding EGF-like growth factor (HB-EGF or betacellulin (BTC was only partial. In contrast, clathrin knockdown fully inhibits EGFR degradation after all ligands tested. Furthermore, inhibition of dynamin function blocked EGFR internalization after stimulation with all ligands. Knocking out a number of clathrin-independent dynamin-dependent pathways of internalization had no effect on the ligand-induced endocytosis of the EGFR. We suggest that EGF and TGF-α lead to EGFR endocytosis mainly via the clathrin-mediated pathway. Furthermore, we suggest that HB-EGF and BTC also lead to EGFR endocytosis via a clathrin-mediated pathway, but can additionally use an unidentified internalization pathway or better recruit the small amount of clathrin remaining after clathrin knockdown.

  10. Structural linkage between ligand discrimination and receptor activation by type I interferons.

    Science.gov (United States)

    Thomas, Christoph; Moraga, Ignacio; Levin, Doron; Krutzik, Peter O; Podoplelova, Yulia; Trejo, Angelica; Lee, Choongho; Yarden, Ganit; Vleck, Susan E; Glenn, Jeffrey S; Nolan, Garry P; Piehler, Jacob; Schreiber, Gideon; Garcia, K Christopher

    2011-08-19

    Type I Interferons (IFNs) are important cytokines for innate immunity against viruses and cancer. Sixteen human type I IFN variants signal through the same cell-surface receptors, IFNAR1 and IFNAR2, yet they can evoke markedly different physiological effects. The crystal structures of two human type I IFN ternary signaling complexes containing IFNα2 and IFNω reveal recognition modes and heterotrimeric architectures that are unique among the cytokine receptor superfamily but conserved between different type I IFNs. Receptor-ligand cross-reactivity is enabled by conserved receptor-ligand "anchor points" interspersed among ligand-specific interactions that "tune" the relative IFN-binding affinities, in an apparent extracellular "ligand proofreading" mechanism that modulates biological activity. Functional differences between IFNs are linked to their respective receptor recognition chemistries, in concert with a ligand-induced conformational change in IFNAR1, that collectively control signal initiation and complex stability, ultimately regulating differential STAT phosphorylation profiles, receptor internalization rates, and downstream gene expression patterns. Copyright © 2011 Elsevier Inc. All rights reserved.

  11. Opioid Receptor Activity and Analgesic Potency of DPDPE Peptide Analogues Containing a Xylene Bridge.

    Science.gov (United States)

    Stefanucci, Azzurra; Novellino, Ettore; Mirzaie, Sako; Macedonio, Giorgia; Pieretti, Stefano; Minosi, Paola; Szűcs, Edina; Erdei, Anna I; Zádor, Ferenc; Benyhe, Sándor; Mollica, Adriano

    2017-04-13

    d-Pen 2 ,d-Pen 5 enkephalin (DPDPE) is one of the most selective synthetic peptide agonists targeting the δ-opioid receptor. Three cyclic analogues of DPDPE containing a xylene bridge in place of disulfide bond have been synthesized and fully characterized as opioid receptors agonists. The in vitro activity was investigated showing a good affinity of 7a - c for μ- and δ-receptors. In vivo biological assays revealed that 7b is the most potent analogue with the ability to maintain high level of analgesia from 15 to 60 min following intracerebroventricular (i.c.v.) administration, whereas DPDPE was slightly active until 45 min. Compound 7b induced long lasting analgesia also after subcutaneous administration, whereas DPDPE was inactive.

  12. GIRK channel activation via adenosine or muscarinic receptors has similar effects on rat atrial electrophysiology

    DEFF Research Database (Denmark)

    Wang, Xiaodong; Liang, Bo; Skibsbye, Lasse

    2013-01-01

    G protein-coupled inwardly rectifying K+ channels (GIRK) are important in the regulation of heart rate and atrial electrophysiology. GIRK channels are activated by G protein-coupled receptors, including muscarinic M2 receptors and adenosine A1 receptors. The aim of this study was to characterize...... and compare the electrophysiological effects of acetylcholine (ACh) and adenosine on GIRK channels in rat atria. Action potential duration at 90% repolarization (APD90), effective refractory period (ERP), and resting membrane potential (RMP) were investigated in isolated rat atria by intracellular recordings....... Both the adenosine analog N6-cyclopentyladenosine (CPA) and ACh profoundly shortened APD90 and ERP and hyperpolarized the RMP. No additive or synergistic effect of CPA and ACh coapplication was observed. To antagonize GIRK channel activation, the specific inhibitor rTertiapin Q (TTQ) was applied...

  13. Anti-tumor Activity of Toll-Like Receptor 7 Agonists

    Directory of Open Access Journals (Sweden)

    Huju Chi

    2017-05-01

    Full Text Available Toll-like receptors (TLRs are a class of pattern recognition receptors that play a bridging role in innate immunity and adaptive immunity. The activated TLRs not only induce inflammatory responses, but also elicit the development of antigen specific immunity. TLR7, a member of TLR family, is an intracellular receptor expressed on the membrane of endosomes. TLR7 can be triggered not only by ssRNA during viral infections, but also by immune modifiers that share a similar structure to nucleosides. Its powerful immune stimulatory action can be potentially used in the anti-tumor therapy. This article reviewed the anti-tumor activity and mechanism of TLR7 agonists that are frequently applied in preclinical and clinical investigations, and mainly focused on small synthetic molecules, including imiquimod, resiquimod, gardiquimod, and 852A, etc.

  14. The influence of selective vitamin D receptor activator paricalcitol on cardiovascular system and cardiorenal protection

    Directory of Open Access Journals (Sweden)

    Duplancic D

    2013-02-01

    Full Text Available Darko Duplancic,1 Marijan Cesarik,2 Nikola Kolja Poljak,3 Maja Radman,4 Vedran Kovacic,5 Josipa Radic,5 Veljko Rogosic61Department of Cardiology, School of Medicine in Split, University Hospital Split, Split, Croatia; 2Department for Neurology, County General Hospital Pozega, Pozega, Croatia; 3Department of Otorhinolaryngology, 4Department of Endocrinology, 5Department of Nephrology, 6Department of Ophthalmology, School of Medicine in Split, University Hospital Split, Split, CroatiaAbstract: The ubiquitous distribution of vitamin D receptors in the human body is responsible for the pleiotropic effects of vitamin D-receptor activation. We discuss the possible beneficial effects of a selective activator of vitamin D receptor, paricalcitol, on the cardiovascular system in chronic heart failure patients and chronic kidney patients, in light of new trials. Paricalcitol should provide additional clinical benefits over the standard treatment for chronic kidney and heart failure, especially in cases of cardiorenal syndrome.Keywords: vitamin D deficiency, cardiovascular system, albuminuria, inflammation

  15. Rapid regulation of endoplasmic reticulum dynamics in dendritic spines by NMDA receptor activation.

    Science.gov (United States)

    Ng, Ai Na; Doherty, Andrew J; Lombroso, Paul J; Emptage, Nigel J; Collingridge, Graham L

    2014-08-19

    Endoplasmic reticulum (ER) is motile within dendritic spines, but the mechanisms underlying its regulation are poorly understood. To address this issue, we have simultaneously imaged morphology and ER content of dendritic spines in cultured dissociated mouse hippocampal neurons. Over a 10 min period, spines were highly dynamic, with spines both increasing and decreasing in volume. ER was present in approximately 50% of spines and was also highly dynamic, with a net increase over this period of time. Inhibition of the endogenous activation of NMDA receptors resulted in a reduction in ER growth. Conversely, augmentation of the synaptic activation of NMDA receptors, by elimination of striatal-enriched protein tyrosine phosphatase (STEP), resulted in enhanced ER growth. Therefore, NMDA receptors rapidly regulate spine ER dynamics.

  16. Haemolysis induced by α-toxin from Staphylococcus aureus requires P2X receptor activation

    DEFF Research Database (Denmark)

    Skals, Marianne Gerberg; Leipziger, Jens Georg; Prætorius, Helle

    2011-01-01

    -forming bacterial toxins. In this context, it is essential to know whether this is specific to HlyA-induced cell damage or if other bacterial pore-forming toxins involve purinergic signals to orchestrate haemolysis. Here, we investigate if the haemolysis produced by α-toxin from Staphylococcus aureus involves P2...... receptor activation. We observed that α-toxin-induced haemolysis is completely blocked by the unselective P2 receptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid. Moreover, several selective blockers of P2X1 and P2X7 ionotropic receptors abolished haemolysis in murine and equine...... erythrocytes. Inhibitors of pannexin channels partially reduced the α-toxin induced lysis. Thus, we conclude that α-toxin, similar to HlyA from E. coli produces cell damage by specific activation of a purinergic signalling cascade. These data indicate that pore-forming toxins in general require purinergic...

  17. Structure-dependent binding and activation of perfluorinated compounds on human peroxisome proliferator-activated receptor γ

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Lianying [State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, P.O. Box 2871, 18 Shuangqing Road, Beijing 100085 (China); College of Life Science, Dezhou University, Dezhou 253023 (China); Ren, Xiao-Min; Wan, Bin [State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, P.O. Box 2871, 18 Shuangqing Road, Beijing 100085 (China); Guo, Liang-Hong, E-mail: LHGuo@rcees.ac.cn [State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, P.O. Box 2871, 18 Shuangqing Road, Beijing 100085 (China)

    2014-09-15

    Perfluorinated compounds (PFCs) have been shown to disrupt lipid metabolism and even induce cancer in rodents through activation of peroxisome proliferator-activated receptors (PPARs). Lines of evidence showed that PPARα was activated by PFCs. However, the information on the binding interactions between PPARγ and PFCs and subsequent alteration of PPARγ activity is still limited and sometimes inconsistent. In the present study, in vitro binding of 16 PFCs to human PPARγ ligand binding domain (hPPARγ-LBD) and their activity on the receptor in cells were investigated. The results showed that the binding affinity was strongly dependent on their carbon number and functional group. For the eleven perfluorinated carboxylic acids (PFCAs), the binding affinity increased with their carbon number from 4 to 11, and then decreased slightly. The binding affinity of the three perfluorinated sulfonic acids (PFSAs) was stronger than their PFCA counterparts. No binding was detected for the two fluorotelomer alcohols (FTOHs). Circular dichroim spectroscopy showed that PFC binding induced distinctive structural change of the receptor. In dual luciferase reporter assays using transiently transfected Hep G2 cells, PFCs acted as hPPARγ agonists, and their potency correlated with their binding affinity with hPPARγ-LBD. Molecular docking showed that PFCs with different chain length bind with the receptor in different geometry, which may contribute to their differences in binding affinity and transcriptional activity. - Highlights: • Binding affinity between PFCs and PPARγ was evaluated for the first time. • The binding strength was dependent on fluorinated carbon chain and functional group. • PFC binding induced distinctive structural change of the receptor. • PFCs could act as hPPARγ agonists in Hep G2 cells.

  18. Structural rearrangement of the intracellular domains during AMPA receptor activation

    DEFF Research Database (Denmark)

    Zachariassen, Linda Grønborg; Katchan, Ljudmila; Jensen, Anna Guldvang

    2016-01-01

    subunit constructs that had normal activity and displayed intrareceptor FRET. We used fluorescence lifetime imaging microscopy (FLIM) in live HEK293 cells to determine distinct steady-state FRET efficiencies in the presence of different ligands, suggesting a dynamic picture of the resting state. Patch...

  19. Monoclonal Antibodies to the Thyrotropin Receptor

    Directory of Open Access Journals (Sweden)

    Takao Ando

    2005-01-01

    Full Text Available The thyrotropin receptor (TSHR is a seven transmembrane G-protein linked glycoprotein expressed on the thyroid cell surface and which, under the regulation of TSH, controls the production and secretion of thyroid hormone from the thyroid gland. This membrane protein is also a major target antigen in the autoimmune thyroid diseases. In Graves' disease, autoantibodies to the TSHR (TSHR-Abs stimulate the TSHR to produce thyroid hormone excessively. In autoimmune thyroid failure, some patients exhibit TSHR-Abs which block TSH action on the receptor. There have been many attempts to generate human stimulating TSHR-mAbs, but to date, only one pathologically relevant human stimulating TSHR-mAb has been isolated. Most mAbs to the TSHR have been derived from rodents immunized with TSHR antigen from bacteria or insect cells. These antigens lacked the native conformation of the TSHR and the resulting mAbs were exclusively blocking or neutral TSHR-mAbs. However, mAbs raised against intact native TSHR antigen have included stimulating mAbs. One such stimulating mAb has demonstrated a number of differences in its regulation of TSHR post-translational processing. These differences are likely to be reflective of TSHR-Abs seen in Graves' disease.

  20. Palliation of bone cancer pain by antagonists of platelet-activating factor receptors.

    Directory of Open Access Journals (Sweden)

    Katsuya Morita

    Full Text Available Bone cancer pain is the most severe among cancer pain and is often resistant to current analgesics. Thus, the development of novel analgesics effective at treating bone cancer pain are desired. Platelet-activating factor (PAF receptor antagonists were recently demonstrated to have effective pain relieving effects on neuropathic pain in several animal models. The present study examined the pain relieving effect of PAF receptor antagonists on bone cancer pain using the femur bone cancer (FBC model in mice. Animals were injected with osteolytic NCTC2472 cells into the tibia, and subsequently the effects of PAF receptor antagonists on pain behaviors were evaluated. Chemical structurally different type of antagonists, TCV-309, BN 50739 and WEB 2086 ameliorated the allodynia and improved pain behaviors such as guarding behavior and limb-use abnormalities in FBC model mice. The pain relieving effects of these antagonists were achieved with low doses and were long lasting. Blockade of spinal PAF receptors by intrathecal injection of TCV-309 and WEB 2086 or knockdown of the expression of spinal PAF receptor protein by intrathecal transfer of PAF receptor siRNA also produced a pain relieving effect. The amount of an inducible PAF synthesis enzyme, lysophosphatidylcholine acyltransferase 2 (LPCAT2 protein significantly increased in the spinal cord after transplantation of NCTC 2472 tumor cells into mouse tibia. The combination of morphine with PAF receptor antagonists develops marked enhancement of the analgesic effect against bone cancer pain without affecting morphine-induced constipation. Repeated administration of TCV-309 suppressed the appearance of pain behaviors and prolonged survival of FBC mice. The present results suggest that PAF receptor antagonists in combination with, or without, opioids may represent a new strategy for the treatment of persistent bone cancer pain and improve the quality of life of patients.

  1. Cu-Zn superoxide dismutase activates muscarinic acetylcholine M1 receptor pathway in neuroblastoma cells.

    Science.gov (United States)

    Damiano, Simona; Petrozziello, Tiziana; Ucci, Valentina; Amente, Stefano; Santillo, Mariarosaria; Mondola, Paolo

    2013-01-01

    Muscarinic receptors (mAChRs) control several neuronal functions and are widely expressed in the central nervous system (CNS): M1 subtype represents the predominant mAChR in the CNS. Previously, we showed that antioxidant enzyme Cu-Zn superoxide dismutase (SOD1) is secreted by many cellular lines and specifically interacts with cell surface membrane of human neuroblastoma SK-N-BE cells thus activating phospholipase C (PLC) transduction pathway and increasing intracellular calcium concentration ([Ca(2+)](i)). In addition, we demonstrated that a small amount of SOD1 is contained in large core dense vesicles and that it is secreted in response to depolarization induced by elevated extracellular K(+) concentration. In the present study, we investigated the involvement of muscarinic M1 receptors in SOD1-induced activation of PLC transduction pathway. We showed that, in SK-N-BE cells, SOD1 was able to activate muscarinic M1 receptor producing a phosphorylation of ERK 1/2 and Akt in dose- and time-dependent manner. Interestingly, in the presence of the M1 antagonist pirenzepine, ERK 1/2 and Akt phosphorylation induced by SOD1 was remarkably prevented. This effect was mimicked by knocking-down M1 receptor using two sequences of RNA silencing (siRNA). At functional level, siRNAs against M1 receptor were able to prevent the increase in [Ca(2+)](i) induced by SOD1. The same inhibitory effect on [Ca(2+)](i) changes was produced by the M1 antagonist pirenzepine. Collectively, the results of this study demonstrated that SOD1 could activate a transductional pathway through the involvement of M1 muscarinic receptor. Copyright © 2012 Elsevier Inc. All rights reserved.

  2. Rapid activation of inwardly rectifying potassium channels by immobile G-protein-coupled receptors.

    Science.gov (United States)

    Lober, Robert M; Pereira, Miguel A; Lambert, Nevin A

    2006-11-29

    G-protein-coupled receptors (GPCRs) mediate slow synaptic transmission and many other effects of small molecule and peptide neurotransmitters. In the standard model of GPCR signaling, receptors and G-proteins diffuse laterally within the plane of the plasma membrane and encounter each other by random collision. This model predicts that signaling will be most efficient if both GPCRs and G-proteins are free to diffuse, thus maximizing collision frequency. However, neuronal GPCRs are often recruited to and enriched at specific synaptic locations, suggesting receptor mobility is restricted in these cells. Here, we test the hypothesis that restricting GPCR mobility impairs signaling in neurons by limiting the frequency of collisions between receptors and G-proteins. Mu-opioid receptors (MORs) were immobilized on the surface of cerebellar granule neurons by avidin-mediated cross-linking, and inwardly rectifying potassium (GIRK) channels were used as rapid indicators of G-protein activation. Mobile and immobile MORs activated GIRK channels with the same onset kinetics and agonist sensitivity in these neurons. In a heterologous expression system, GFP (green fluorescent protein)-tagged G alpha(oA) subunits remained mobile after cross-linking, but their mobility was reduced in the presence of immobile MORs, suggesting that these receptors and subunits were transiently precoupled. In addition, channel activation could be reconstituted with immobile GPCRs, G-protein heterotrimers, and GIRK channels. These results show that collision frequency is not rate-limiting for G-protein activation in CNS neurons, and are consistent with the idea that signaling components are compartmentalized or preassembled.

  3. The silencing mediator of retinoic acid and thyroid hormone receptor (SMRT) corepressor is required for full estrogen receptor alpha transcriptional activity.

    Science.gov (United States)

    Peterson, Theresa J; Karmakar, Sudipan; Pace, Margaret C; Gao, Tong; Smith, Carolyn L

    2007-09-01

    Multiple factors influence estrogen receptor alpha (ERalpha) transcriptional activity. Current models suggest that the silencing mediator of retinoic acid and thyroid hormone receptor (SMRT) corepressor functions within a histone deactylase-containing protein complex that binds to antiestrogen-bound ERalpha and contributes to negative regulation of gene expression. In this report, we demonstrate that SMRT is required for full agonist-dependent ERalpha activation. Chromatin immunoprecipitation assays demonstrate that SMRT, like ERalpha and the SRC-3 coactivator, is recruited to an estrogen-responsive promoter in estrogen-treated MCF-7 cells. Depletion of SMRT, but not histone deacetylases 1 or 3, negatively impacts estradiol-stimulated ERalpha transcriptional activity, while exogenous expression of SMRT's receptor interaction domains blocks ERalpha activity, indicating a functional interaction between this corepressor and agonist-bound ERalpha. Stimulation of estradiol-induced ERalpha activity by SMRT overexpression occurred in HeLa and MCF-7 cells, but not HepG2 cells, indicating that these positive effects are cell type specific. Similarly, the ability of SMRT depletion to promote the agonist activity of tamoxifen was observed for HeLa but not MCF-7 cells. Furthermore, impairment of agonist-stimulated activity by SMRT depletion is specific to ERalpha and not observed for receptors for vitamin D, androgen, or thyroid hormone. Nuclear receptor corepressor (N-CoR) depletion increased the transcriptional activity of all four tested receptors. SMRT is required for full expression of the ERalpha target genes cyclin D1, BCL-2, and progesterone receptor but not pS2, and its depletion significantly attenuated estrogen-dependent proliferation of MCF-7 cells. Taken together, these data indicate that SMRT, in conjunction with gene-specific and cell-dependent factors, is required for positively regulating agonist-dependent ERalpha transcriptional activity.

  4. Activation of muscarinic acetylcholine receptors elicits pigment granule dispersion in retinal pigment epithelium isolated from bluegill

    Directory of Open Access Journals (Sweden)

    Crittenden Elizabeth L

    2004-07-01

    Full Text Available Abstract Background In fish, melanin pigment granules in the retinal pigment epithelium disperse into apical projections as part of the suite of responses the eye makes to bright light conditions. This pigment granule dispersion serves to reduce photobleaching and occurs in response to neurochemicals secreted by the retina. Previous work has shown that acetylcholine may be involved in inducing light-adaptive pigment dispersion. Acetylcholine receptors are of two main types, nicotinic and muscarinic. Muscarinic receptors are in the G-protein coupled receptor superfamily, and five different muscarinic receptors have been molecularly cloned in human. These receptors are coupled to adenylyl cyclase, calcium mobilization and ion channel activation. To determine the receptor pathway involved in eliciting pigment granule migration, we isolated retinal pigment epithelium from bluegill and subjected it to a battery of cholinergic agents. Results The general cholinergic agonist carbachol induces pigment granule dispersion in isolated retinal pigment epithelium. Carbachol-induced pigment granule dispersion is blocked by the muscarinic antagonist atropine, by the M1 antagonist pirenzepine, and by the M3 antagonist 4-DAMP. Pigment granule dispersion was also induced by the M1 agonist 4-[N-(4-chlorophenyl carbamoyloxy]-4-pent-2-ammonium iodide. In contrast the M2 antagonist AF-DX 116 and the M4 antagonist tropicamide failed to block carbachol-induced dispersion, and the M2 agonist arecaidine but-2-ynyl ester tosylate failed to elicit dispersion. Conclusions Our results suggest that carbachol-mediated pigment granule dispersion occurs through the activation of Modd muscarinic receptors, which in other systems couple to phosphoinositide hydrolysis and elevation of intracellular calcium. This conclusion must be corroborated by molecular studies, but suggests Ca2+-dependent pathways may be involved in light-adaptive pigment dispersion.

  5. Retinoids induce integrin-independent lymphocyte adhesion through RAR-α nuclear receptor activity

    Energy Technology Data Exchange (ETDEWEB)

    Whelan, Jarrett T.; Wang, Lei; Chen, Jianming; Metts, Meagan E.; Nasser, Taj A.; McGoldrick, Liam J. [Department of Biochemistry and Molecular Biology, The Brody School of Medicine at East Carolina University, Greenville, NC 27834 (United States); Bridges, Lance C., E-mail: bridgesl@ecu.edu [Department of Biochemistry and Molecular Biology, The Brody School of Medicine at East Carolina University, Greenville, NC 27834 (United States); East Carolina Diabetes and Obesity Institute, The Brody School of Medicine at East Carolina University, Greenville, NC 27834 (United States)

    2014-11-28

    Highlights: • Transcription and translation are required for retinoid-induced lymphocyte adhesion. • RAR activation is sufficient to induced lymphocyte cell adhesion. • Vitamin D derivatives inhibit RAR-prompted lymphocyte adhesion. • Adhesion occurs through a novel binding site within ADAM disintegrin domains. • RARα is a key nuclear receptor for retinoid-dependent lymphocyte cell adhesion. - Abstract: Oxidative metabolites of vitamin A, in particular all-trans-retinoic acid (atRA), have emerged as key factors in immunity by specifying the localization of immune cells to the gut. Although it is appreciated that isomers of retinoic acid activate the retinoic acid receptor (RAR) and retinoid X receptor (RXR) family of nuclear receptors to elicit cellular changes, the molecular details of retinoic acid action remain poorly defined in immune processes. Here we employ a battery of agonists and antagonists to delineate the specific nuclear receptors utilized by retinoids to evoke lymphocyte cell adhesion to ADAM (adisintegrin and metalloprotease) protein family members. We report that RAR agonism is sufficient to promote immune cell adhesion in both immortal and primary immune cells. Interestingly, adhesion occurs independent of integrin function, and mutant studies demonstrate that atRA-induced adhesion to ADAM members required a distinct binding interface(s) as compared to integrin recognition. Anti-inflammatory corticosteroids as well as 1,25-(OH){sub 2}D{sub 3}, a vitamin D metabolite that prompts immune cell trafficking to the skin, potently inhibited the observed adhesion. Finally, our data establish that induced adhesion was specifically attributable to the RAR-α receptor isotype. The current study provides novel molecular resolution as to which nuclear receptors transduce retinoid exposure into immune cell adhesion.

  6. Activation of muscarinic acetylcholine receptors elicits pigment granule dispersion in retinal pigment epithelium isolated from bluegill

    Science.gov (United States)

    González, Alfredo; Crittenden, Elizabeth L; García, Dana M

    2004-01-01

    Background In fish, melanin pigment granules in the retinal pigment epithelium disperse into apical projections as part of the suite of responses the eye makes to bright light conditions. This pigment granule dispersion serves to reduce photobleaching and occurs in response to neurochemicals secreted by the retina. Previous work has shown that acetylcholine may be involved in inducing light-adaptive pigment dispersion. Acetylcholine receptors are of two main types, nicotinic and muscarinic. Muscarinic receptors are in the G-protein coupled receptor superfamily, and five different muscarinic receptors have been molecularly cloned in human. These receptors are coupled to adenylyl cyclase, calcium mobilization and ion channel activation. To determine the receptor pathway involved in eliciting pigment granule migration, we isolated retinal pigment epithelium from bluegill and subjected it to a battery of cholinergic agents. Results The general cholinergic agonist carbachol induces pigment granule dispersion in isolated retinal pigment epithelium. Carbachol-induced pigment granule dispersion is blocked by the muscarinic antagonist atropine, by the M1 antagonist pirenzepine, and by the M3 antagonist 4-DAMP. Pigment granule dispersion was also induced by the M1 agonist 4-[N-(4-chlorophenyl) carbamoyloxy]-4-pent-2-ammonium iodide. In contrast the M2 antagonist AF-DX 116 and the M4 antagonist tropicamide failed to block carbachol-induced dispersion, and the M2 agonist arecaidine but-2-ynyl ester tosylate failed to elicit dispersion. Conclusions Our results suggest that carbachol-mediated pigment granule dispersion occurs through the activation of Modd muscarinic receptors, which in other systems couple to phosphoinositide hydrolysis and elevation of intracellular calcium. This conclusion must be corroborated by molecular studies, but suggests Ca2+-dependent pathways may be involved in light-adaptive pigment dispersion. PMID:15251036

  7. Agonist activity of LSD and lisuride at cloned 5HT2A and 5HT2C receptors.

    Science.gov (United States)

    Egan, C T; Herrick-Davis, K; Miller, K; Glennon, R A; Teitler, M

    1998-04-01

    Evidence from studies with phenylisopropylamine hallucinogens indicates that the 5HT2A receptor is the likely target for the initiation of events leading to hallucinogenic activity associated with LSD and related drugs. Recently, lisuride (a purported non-hallucinogenic congener of LSD) was reported to be a potent antagonist at the 5HT2C receptor and an agonist at the 5HT2A receptor. LSD exhibited agonist activity at both receptors. These data were interpreted as indicating that the 5HT2C receptor might be the initiating site of action for hallucinogens. To test this hypothesis, recombinant cells expressing 5HT2A and 5HT2C receptors were used to determine the actions of LSD and lisuride. LSD and lisuride were potent partial agonists at 5HT2A receptors with EC50 values of 7.2 nM and 17 nM, respectively. Also, LSD and lisuride were partial agonists at 5HT2C receptors with EC50 values of 27 nM and 94 nM, respectively. We conclude that lisuride and LSD have similar actions at 5HT2A and 5HT2C receptors in recombinant cells. As agonist activity at brain 5HT2A receptors has been associated with hallucinogenic activity, these results indicate that lisuride may possess hallucinogenic activity, although the psychopharmacological effects of lisuride appear to be different from the hallucinogenic effects of LSD.

  8. Activation of overexpressed receptors for insulin and epidermal growth factor interferes in mitogenic signaling without affecting the activation of p21ras

    NARCIS (Netherlands)

    Osterop, A.P.R.M.; Medema, R.H.; Ouwens, D.M.; Zon, G.C.M. van der; Möller, W.; Maassen, J.A.

    1994-01-01

    Activated receptors with a tyrosine kinase activity induce a variety of responses like changes in the differentiation and mitogenic status of cells. These responses are mediated in part by p21ras. Some of these activated receptors induce in certain cell types a pronounced, but transient, increase in

  9. Effects of histamine H1 receptor signaling on glucocorticoid receptor activity. Role of canonical and non-canonical pathways

    NARCIS (Netherlands)

    Zappia, C.D.; Granja-Galeano, G.; Fernández, N.; Shayo, C.; Davio, C.; Fitzsimons, C.P.; Monczor, F.

    2015-01-01

    Histamine H1 receptor (H1R) antagonists and glucocorticoid receptor (GR) agonists are used to treat inflammatory conditions such as allergic rhinitis, atopic dermatitis and asthma. Consistent with the high morbidity levels of such inflammatory conditions, these receptors are the targets of a vast

  10. Activation of 5-HT2A receptor disrupts rat maternal behavior.

    Science.gov (United States)

    Gao, Jun; Wu, Ruiyong; Davis, Collin; Li, Ming

    2018-01-01

    Serotonin 5-HT2A receptor is widely distributed in the central nervous system and plays an important role in sensorimotor function, emotion regulation, motivation, executive control, learning and memory. We investigated its role in rat maternal behavior, a naturalistic behavior encompassing many psychological functions that the 5-HT2A receptor is involved in. We first showed that activation of 5-HT2A receptor by TCB-2 (a highly selective 5-HT2A agonist, 1, 2.5 or 5.0 mg/kg) disrupted maternal behavior dose-dependently, and this effect was reduced by pretreatment with a 5-HT2A receptor antagonist MDL 100907, but exacerbated by pretreatment with a 5-HT2C receptor antagonist SB242084 and a 5-HT2C receptor agonist MK212, indicating that the maternal disruptive effect of 5-HT2A activation is receptor-specific and can be modulated by 5-HT2C receptor bidirectionally. We then microinjected TCB-2 into two brain regions important for the normal expression of maternal behavior: the medial prefrontal cortex (mPFC) and the medial preoptic area (mPOA) and found that only acute intra-mPFC infusion of TCB-2 suppressed pup retrieval, whereas intra-mPOA had no effect. Finally, using c-Fos immunohistochemistry, we identified that the ventral bed nucleus of stria terminalis (vBNST), the central amygdala (CeA), and the dorsal raphe (DR) were additionally involved in the maternal-disruptive effect of TCB-2. These findings suggest that the 5-HT2A receptor in the mPFC and other maternally related regions is required for the normal expression of maternal behavior through its intrinsic action or interactions with other receptors (e.g. 5-HT2C). Functional disruption of this neuroreceptor system might contribute to postpartum mental disorders (e.g. depression and psychosis) that impair the quality of maternal care. Copyright © 2017 Elsevier Ltd. All rights reserved.

  11. Solubilization of phencyclidine receptors from rat cerebral cortex in an active ligand binding site

    Energy Technology Data Exchange (ETDEWEB)

    McVittie, L.D.; Sibley, D.R.

    1989-01-01

    A phencyclidine (PCP) receptor binding site has been solubilized in an active ligand-binding state from rat cerebral cortical membranes with sodium deoxycholate. Optimal receptor solubilization occurs at a detergent/protein ratio of 0.5 (w/w); for 5 mg protein/ml solubilized with 0.25% sodium deoxycholate, about 60% of the protein and 25% of the receptor is solubilized. Specific binding of either (/sup 3/H)-N-(1-(2-thienyl)cyclohexyl)piperidine ((/sup 3/H)TCP) or (/sup 3/H)MK-801 is measurable by filtration through Sephadex G-50 columns or glass fiber filters; more than 60% of the binding activity is stable after 48 h at 4/degrees/C. In the presence of detergent, (/sup 3/H)TCP binding exhibits a K/sub d/ of 250 nM, a B/sub max/ of 0.56 pmol/mg protein, and a pharmacological profile consistent with that of the membrane-bound PCP receptor, although most drugs bind with affinities 2 to 8 fold lower than in membranes. Upon reduction of detergent concentration, binding parameters approximate those for the membrane-bound receptor (/sup 3/H)TCP binding: K/sub d/ = 48 nM, M/sub max/ = 1.13 pmol/mg protein.

  12. The role of chemokines and chemokine receptors in eosinophil activation during inflammatory allergic reactions

    Directory of Open Access Journals (Sweden)

    Oliveira S.H.P.

    2003-01-01

    Full Text Available Chemokines are important chemotactic cytokines that play a fundamental role in the trafficking of leukocytes to sites of inflammation. They are also potent cell-activating factors, inducing cytokine and histamine release and free radical production, a fact that makes them particularly important in the pathogenesis of allergic inflammation. The action of chemokines is regulated at the level of agonist production and processing as well as at the level of receptor expression and coupling. Therefore, an analysis of the ligands must necessarily consider receptors. Eosinophils are target cells involved in the allergic inflammatory response since they are able to release a wide variety of mediators including CC and CXC chemokines and express their receptors. These mediators could damage the airway epithelial cells and might be important to stimulate other cells inducing an amplification of the allergic response. This review focuses on recently emerging data pertaining to the importance of chemokines and chemokine receptors in promoting eosinophil activation and migration during the allergic inflammatory process. The analysis of the function of eosinophils and their chemokine receptors during allergic inflammation might be a good approach to understanding the determinants of asthma severity and to developing novel therapies.

  13. Dreamlike effects of LSD on waking imagery in humans depend on serotonin 2A receptor activation.

    Science.gov (United States)

    Kraehenmann, Rainer; Pokorny, Dan; Vollenweider, Leonie; Preller, Katrin H; Pokorny, Thomas; Seifritz, Erich; Vollenweider, Franz X

    2017-07-01

    Accumulating evidence indicates that the mixed serotonin and dopamine receptor agonist lysergic acid diethylamide (LSD) induces an altered state of consciousness that resembles dreaming. This study aimed to test the hypotheses that LSD produces dreamlike waking imagery and that this imagery depends on 5-HT2A receptor activation and is related to subjective drug effects. Twenty-five healthy subjects performed an audiorecorded guided mental imagery task 7 h after drug administration during three drug conditions: placebo, LSD (100 mcg orally) and LSD together with the 5-HT2A receptor antagonist ketanserin (40 mg orally). Cognitive bizarreness of guided mental imagery reports was quantified as a standardised formal measure of dream mentation. State of consciousness was evaluated using the Altered State of Consciousness (5D-ASC) questionnaire. LSD, compared with placebo, significantly increased cognitive bizarreness (p LSD-induced increase in cognitive bizarreness was positively correlated with the LSD-induced loss of self-boundaries and cognitive control (p LSD-induced increases in cognitive bizarreness and changes in state of consciousness were fully blocked by ketanserin. LSD produced mental imagery similar to dreaming, primarily via activation of the 5-HT2A receptor and in relation to loss of self-boundaries and cognitive control. Future psychopharmacological studies should assess the differential contribution of the D2/D1 and 5-HT1A receptors to cognitive bizarreness.

  14. Peroxisome proliferator-activated receptor gamma coactivator-1 alpha acts as a tumor suppressor in hepatocellular carcinoma.

    Science.gov (United States)

    Liu, Rui; Zhang, Haiyang; Zhang, Yan; Li, Shuang; Wang, Xinyi; Wang, Xia; Wang, Cheng; Liu, Bin; Zen, Ke; Zhang, Chen-Yu; Zhang, Chunni; Ba, Yi

    2017-04-01

    Peroxisome proliferator-activated receptor gamma coactivator-1 alpha plays a crucial role in regulating the biosynthesis of mitochondria, which is closely linked to the energy metabolism in various tumors. This study investigated the regulatory role of peroxisome proliferator-activated receptor gamma coactivator-1 alpha in the pathogenesis of hepatocellular carcinoma. In this study, the changes of peroxisome proliferator-activated receptor gamma coactivator-1 alpha messenger RNA levels between normal human liver and hepatocellular carcinoma tissue were examined by quantitative reverse transcription polymerase chain reaction. Knockdown of peroxisome proliferator-activated receptor gamma coactivator-1 alpha was conducted by RNA interference in the human liver cell line L02, while overexpression of peroxisome proliferator-activated receptor gamma coactivator-1 alpha was conducted by adenovirus encoding peroxisome proliferator-activated receptor gamma coactivator-1 alpha complementary DNA in the human hepatocarcinoma cell line HepG2. Cellular morphological changes were observed via optical and electron microscopy. Cellular apoptosis was determined by Hoechst 33258 staining. In addition, the expression levels of 21,400 genes in tissues and cells were detected by microarray. It was shown that peroxisome proliferator-activated receptor gamma coactivator-1 alpha expression was significantly downregulated in hepatocellular carcinoma compared with normal liver tissues. After knockdown of peroxisome proliferator-activated receptor gamma coactivator-1 alpha expression in L02 cells, cells reverted to immature and dedifferentiated morphology exhibiting cancerous tendency. Apoptosis occurred in the HepG2 cells after transfection by adenovirus encoding peroxisome proliferator-activated receptor gamma coactivator-1 alpha. Microarray analysis showed consistent results. The results suggest that peroxisome proliferator-activated receptor gamma coactivator-1 alpha acts as a tumor

  15. Activation of dorsal horn cannabinoid CB2 receptor suppresses the expression of P2Y12 and P2Y13 receptors in neuropathic pain rats.

    Science.gov (United States)

    Niu, Juan; Huang, Dujuan; Zhou, Rui; Yue, MingXia; Xu, Tao; Yang, Junna; He, Li; Tian, Hong; Liu, XiaoHong; Zeng, Junwei

    2017-09-12

    More evidence suggests that dorsal spinal cord microglia is an important site contributing to CB2 receptor-mediated analgesia. The upregulation of P2Y12 and P2Y13 purinoceptors in spinal dorsal horn microglia is involved in the development of pain behavior caused by peripheral nerve injury. However, it is not known whether the expression of P2Y12 and P2Y13 receptors at spinal dorsal horn will be influenced after CB2 receptor activation in neuropathic pain rats. Chronic constriction injury (CCI) and intrathecal ADPbetaS injection were performed in rats to induce neuropathic pain. The paw withdrawal latency (PWL) was used to evaluate thermal hyperalgesia in neuropathic rats. The expression of P2Y12 and P2Y13 receptors, p-p38MAPK, and NF-kappaBp65 was detected with RT-PCR and western blotting analysis. Treatment with AM1241 produces a pronounced inhibition of CCI-induced thermal hyperalgesia and significantly inhibited the increased expression of P2Y12 and P2Y13 receptors at the mRNA and protein levels, which open up the possibility that P2Y12 and P2Y13 receptor expression are downregulated by CB2 receptor agonist AM1241 in CCI rats. Western blot analysis demonstrated that AM1241 reduced the elevated expression of p-p38MAPK and NF-κBp65 in the dorsal spinal cord induced by CCI. After administration with either SB203580 (p38MAPK inhibitor) or PDTC (NF-kappaB inhibitor), the levels of P2Y13 receptor expression in the dorsal spinal cord were lower than those in the CCI group. However, in CCI rats, the increased expression of P2Y12 receptor was prevented by intrathecal administration of PDTC but not by SB203580. In addition, minocycline significantly decreased the increased expression of P2Y12 and P2Y13 receptors. The similar results can be observed in ADPbetaS-treated rats. Intrathecal injection of ADPbataS causes thermal hyperalgesia and increased expression of P2Y12 and P2Y13 receptors in the dorsal spinal cord of naive rats. Moreover, intrathecal injection of AM1241

  16. Norepinephrine directly activates adult hippocampal precursors via β3 adrenergic receptors

    Science.gov (United States)

    Jhaveri, Dhanisha J.; Mackay, Eirinn W.; Hamlin, Adam S.; Marathe, Swananda V.; Nandam, L. Sanjay; Vaidya, Vidita A.; Bartlett, Perry F.

    2010-01-01

    Adult hippocampal neurogenesis is a critical form of cellular plasticity that is greatly influenced by neural activity. Among the neurotransmitters that are widely implicated in regulating this process are serotonin and norepinephrine, levels of which are modulated by stress, depression and clinical antidepressants. However, studies to date have failed to address a direct role for either neurotransmitter in regulating hippocampal precursor activity. Here we show that norepinephrine but not serotonin directly activates self-renewing and multipotent neural precursors, including stem cells, from the hippocampus of adult mice. Mechanistically, we provide evidence that β3 adrenergic receptors, which are preferentially expressed on a Hes5-expressing precursor population in the subgranular zone (SGZ), mediate this norepinephrine-dependent activation. Moreover, intrahippocampal injection of a selective β3 adrenergic receptor agonist in vivo increases the number of proliferating cells in the SGZ. Similarly, systemic injection of the β adrenergic receptor agonist isoproterenol not only results in enhancement of proliferation in the SGZ but also leads to an increase in the percentage of nestin/glial fibrillary acidic protein double-positive neural precursors in vivo. Finally, using a novel ex vivo ‘slice-sphere’ assay that maintains an intact neurogenic niche, we demonstrate that antidepressants that selectively block the reuptake of norepinephrine, but not serotonin, robustly increase hippocampal precursor activity via β adrenergic receptors. These findings suggest that the activation of neurogenic precursors and stem cells via β3 adrenergic receptors could be a potent mechanism to increase neuronal production, providing a putative target for the development of novel antidepressants. PMID:20164362

  17. Cannabinoid CB1 receptor activation, pharmacological blockade, or genetic ablation affects the function of the muscarinic auto- and heteroreceptor.

    Science.gov (United States)

    Schulte, Kirsten; Steingrüber, Nina; Jergas, Bernd; Redmer, Agnes; Kurz, Christina Maria; Buchalla, Rainer; Lutz, Beat; Zimmer, Andreas; Schlicker, Eberhard

    2012-04-01

    Different types of presynaptic inhibitory Gα(i/o) protein-coupled receptors usually do not act independently of each other but rather pre-activation of receptor X impairs the effect mediated via receptor Y. It is, however, unknown whether this interaction extends to the cannabinoid CB(1) receptor on cholinergic neurones and hence we studied whether its activation, pharmacological blockade, or genetic inactivation affects the function of other presynaptic inhibitory receptors. The electrically evoked acetylcholine or noradrenaline release was determined in superfused rodent tissues preincubated with (3)H-choline or (3)H-noradrenaline. The muscarinic M(2) receptor, Gα(i), and Gα(o) proteins were determined in hippocampal synaptosomes by Western blotting. Hippocampal anandamide and 2-arachidonoyl glycerol levels were determined by LC-MS/MS. The inhibitory effect of the muscarinic receptor agonist oxotremorine on acetylcholine release in hippocampal slices was increased by genetic CB(1) receptor ablation (mouse) and the CB(1) antagonist rimonabant (rat but not mouse) and decreased by a cannabinoid receptor agonist (mouse). In mouse tissues, CB(1) receptor ablation also increased the effect of a δ opioid receptor agonist on acetylcholine release in the hippocampus and the effect of oxotremorine on noradrenaline release in the vas deferens. CB(1) receptor ablation, to a very slight extent, increased Gα(o) protein levels without affecting either Gα(i) and M(2) receptor protein or the levels of anandamide and 2-arachidonoyl glycerol in the hippocampus. In conclusion, the CB(1) receptor shows an inhibitory interaction with the muscarinic and δ opioid receptor on cholinergic neurones in the rodent hippocampus and with the muscarinic receptor on noradrenergic neurones in the mouse vas deferens.

  18. Quantitation of the receptor for urokinase plasminogen activator by enzyme-linked immunosorbent assay

    DEFF Research Database (Denmark)

    Rønne, E; Behrendt, N; Ploug, M

    1994-01-01

    Binding of the urokinase plasminogen activator (uPA) to a specific cell surface receptor (uPAR) plays a crucial role in proteolysis during tissue remodelling and cancer invasion. An immunosorbent assay for the quantitation of uPAR has now been developed. This assay is based on two monoclonal anti...

  19. Soluble Urokinase Plasminogen Activator Receptor for Risk Prediction in Patients Admitted with Acute Chest Pain

    DEFF Research Database (Denmark)

    Lyngbæk, Stig; Andersson, Charlotte; Marott, Jacob L

    2013-01-01

    Plasma concentrations of soluble urokinase plasminogen activator receptor (suPAR) predict mortality in several clinical settings, but the long-term prognostic importance of suPAR in chest pain patients admitted on suspicion of non-ST-segment elevation acute coronary syndrome (NSTEACS) is uncertain....

  20. Adaptive and innate immune reactions regulating mast cell activation: from receptor-mediated signaling to responses

    DEFF Research Database (Denmark)

    Tkaczyk, Christine; Jensen, Bettina M; Iwaki, Shoko

    2006-01-01

    In this article, we have described studies that have demonstrated that mast cells can be activated as a consequence of adaptive and innate immune reactions and that these responses can be modified by ligands for other receptors expressed on the surface of mast cells. These various stimuli...

  1. Protease activated receptor-1 regulates macrophage-mediated cellular senescence : A risk for idiopathic pulmonary fibrosis

    NARCIS (Netherlands)

    Lin, Cong; Rezaee, Farhad; Waasdorp, Maaike; Shi, Kun; van der Poll, Tom; Borensztajn, Keren; Spek, C. Arnold

    2015-01-01

    Idiopathic pulmonary fibrosis (IPF) is a destructive disease in part resulting from premature or mature cellular aging. Protease-activated receptor-1 (PAR-1) recently emerged as a critical component in the context of fibrotic lung diseases. Therefore, we aimed to study the role of macrophages in

  2. Activation of the Ah receptor by extracts of dietary herbal supplements, vegetables, and fruits

    NARCIS (Netherlands)

    Jeuken, A.; Keser, B.J.G.; Khan, E.; Brouwer, A.; Koeman, J.H.; Denison, M.S.

    2003-01-01

    The aryl hydrocarbon receptor (AhR) is a ligand-dependent transcription factor that can be activated by a structurally diverse range of synthetic and natural chemicals, and it mediates the toxic and biological effects of environmental contaminants such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD).

  3. Activation of the Ah receptor by extracts of dietary herbal supplements, vegetables and fruits

    NARCIS (Netherlands)

    Jeuken, A.; Keser, B.J.G.; Khan, E.; Brouwer, A.; Koeman, J.; Denison, M.S.

    2003-01-01

    The aryl hydrocarbon receptor (AhR) is a ligand-dependent transcription factor that can be activated by a structurally diverse range of synthetic and natural chemicals, and it mediates the toxic and biological effects of environmental contaminants such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD).

  4. B cell activation involves nanoscale receptor reorganizations and inside-out signaling by Syk.

    Science.gov (United States)

    Kläsener, Kathrin; Maity, Palash C; Hobeika, Elias; Yang, Jianying; Reth, Michael

    2014-06-24

    Binding of antigen to the B cell antigen receptor (BCR) initiates a multitude of events resulting in B cell activation. How the BCR becomes signaling-competent upon antigen binding is still a matter of controversy. Using a high-resolution proximity ligation assay (PLA) to monitor the conformation of the BCR and its interactions with co-receptors at a 10-20 nm resolution, we provide direct evidence for the opening of BCR dimers during B cell activation. We also show that upon binding Syk opens the receptor by an inside-out signaling mechanism that amplifies BCR signaling. Furthermore, we found that on resting B cells, the coreceptor CD19 is in close proximity with the IgD-BCR and on activated B cells with the IgM-BCR, indicating nanoscale reorganization of receptor clusters during B cell activation.DOI: http://dx.doi.org/10.7554/eLife.02069.001. Copyright © 2014, Kläsener et al.

  5. Circulating intact and cleaved forms of the urokinase-type plasminogen activator receptor

    DEFF Research Database (Denmark)

    Røndbjerg, Tine Thurison; Christensen, Ib J; Lund, Ida K

    2015-01-01

    BACKGROUND: High levels of circulating forms of the urokinase-type plasminogen activator receptor (uPAR) are significantly associated to poor prognosis in cancer patients. Our aim was to determine biological variations and reference intervals of the uPAR forms in blood, and in addition, to test t...

  6. Deletion of protease-activated receptor 2 prolongs survival of scrapie-inoculated mice

    Czech Academy of Sciences Publication Activity Database

    Matěj, R.; Olejár, Tomáš; Janoušková, O.; Holada, K.

    2012-01-01

    Roč. 93, č. 9 (2012), s. 2057-2061 ISSN 0022-1317 Institutional support: RVO:67985823 Keywords : protease-activated receptor (PAR2) * scrapie * neurodegenerative disorders Subject RIV: FN - Epidemiology, Contagious Diseases ; Clinical Immunology Impact factor: 3.127, year: 2012

  7. RECEPTOR POTENTIAL AND LIGHT-INDUCED MITOCHONDRIAL ACTIVATION IN BLOWFLY PHOTORECEPTOR MUTANTS

    NARCIS (Netherlands)

    MOJET, MH; TINBERGEN, J; STAVENGA, DG

    1991-01-01

    1. Simultaneous measurements of the receptor potential and the light-induced mitochondrial activation were performed in white-eyed blowflies Calliphora vicina, mutant chalky, and Lucilia cuprina, mutants w(F) and w'nss. The intensity dependence and the temporal dynamics were investigated. 2. The

  8. Whole Body Activity Retentions in the Peptide Receptor Radionuclide Therapy with Lu-177

    NARCIS (Netherlands)

    Liu, B.

    2013-01-01

    The patients with the neuroendocrine tumours (liver, spleen, etc.) often need treatment by the Peptide Receptor Radionuclide Therapy with 177Lu. The amount of 177Lu activity in the body of patients has to be known accurately for assessment of the dosimetry and for evaluation of the effectiveness of

  9. Pharmacological Targeting of Protease-Activated Receptor 2 Affords Protection from Bleomycin-Induced Pulmonary Fibrosis

    NARCIS (Netherlands)

    Lin, Cong; von der Thüsen, Jan; Daalhuisen, Joost; ten Brink, Marieke; Crestani, Bruno; van der Poll, Tom; Borensztajn, Keren; Spek, C. Arnold

    2015-01-01

    Idiopathic pulmonary fibrosis is the most devastating diffuse fibrosing lung disease that remains refractory to therapy. Despite increasing evidence that protease-activated receptor 2 (PAR-2) contributes to fibrosis, its importance in pulmonary fibrosis is under debate. We addressed whether PAR-2

  10. Pharmacological targeting of protease-activated receptor 2 affords protection from bleomycin-induced pulmonary fibrosis

    NARCIS (Netherlands)

    C. Lin (Cong); J. von der Thusen (Jan); J. Daalhuisen (Joost); M. Ten Brink (Marieke); B. Crestani (Bruno); T. van der Poll (Tom); K. Borensztajn (Keren); C. Arnold Spek (C.)

    2015-01-01

    textabstractIdiopathic pulmonary fibrosis is the most devastating diffuse fibrosing lung disease that remains refractory to therapy. Despite increasing evidence that protease-activated receptor 2 (PAR-2) contributes to fibrosis, its importance in pulmonary fibrosis is under debate. We addressed

  11. Protease-activated receptor (PAR)-2 is required for PAR-1 signalling in pulmonary fibrosis

    NARCIS (Netherlands)

    Lin, Cong; von der Thüsen, Jan; Daalhuisen, Joost; ten Brink, Marieke; Crestani, Bruno; van der Poll, Tom; Borensztajn, Keren; Spek, C. Arnold

    2015-01-01

    Idiopathic pulmonary fibrosis is the most devastating diffuse fibrosing lung disease of unknown aetiology. Compelling evidence suggests that both protease-activated receptor (PAR)-1 and PAR-2 participate in the development of pulmonary fibrosis. Previous studies have shown that bleomycin-induced

  12. Blood coagulation-(in)dependent protease activated receptor signaling in pancreatic cancer

    NARCIS (Netherlands)

    Shi, K.

    2015-01-01

    There is no doubt that pancreatic cancer is one of the most lethal diseases in general and current treatment options are limited. The studies described in this thesis pinpoint Protease activated receptors (PARs) as key players in pancreatic cancer progression. Inhibition of PAR-1 may be clinical

  13. Domains of the growth hormone receptor required for association and activation of JAK2 tyrosine kinase

    DEFF Research Database (Denmark)

    VanderKuur, J A; Wang, X; Zhang, L

    1994-01-01

    Growth hormone (GH) has recently been shown to activate the GH receptor (GHR)-associated tyrosine kinase JAK2. In the present study, regions of the GHR required for JAK2 association with GHR were identified. GH-dependent JAK2 association with GHR was detected in Chinese hamster ovary (CHO) cells...

  14. Discovery of indole alkaloids with cannabinoid CB1 receptor antagonistic activity.

    Science.gov (United States)

    Kitajima, Mariko; Iwai, Masumi; Kikura-Hanajiri, Ruri; Goda, Yukihiro; Iida, Mitsuru; Yabushita, Hisatoshi; Takayama, Hiromitsu

    2011-04-01

    Three indole alkaloids, voacamine (1), 3,6-oxidovoacangine (2), and a new alkaloid, 5-hydroxy-3,6-oxidovoacangine (3), isolated from Voacanga africana were found to exhibit potent cannabinoid CB1 receptor antagonistic activity. This is the first example of CB1 antagonists derived from natural alkaloids. Copyright © 2011 Elsevier Ltd. All rights reserved.

  15. Soluble Urokinase-Type Plasminogen Activator Receptor Levels in Patients With Schizophrenia

    DEFF Research Database (Denmark)

    Nielsen, Jimmi; Røge, Rasmus; Pristed, Sofie Gry

    2015-01-01

    BACKGROUND: The etiology of schizophrenia remains largely unknown but alterations in the immune system may be involved. In addition to the psychiatric symptoms, schizophrenia is also associated with up to 20 years reduction in life span. Soluble urokinase-type plasminogen activator receptor (su...

  16. Soluble urokinase plasminogen activator receptor (suPAR) in acute care

    DEFF Research Database (Denmark)

    Rasmussen, Line Jee Hartmann; Ladelund, Steen; Haupt, Thomas Huneck

    2016-01-01

    OBJECTIVE: Soluble urokinase plasminogen activator receptor (suPAR) is an inflammatory biomarker associated with presence and progression of disease and with increased risk of mortality. We aimed to evaluate the unspecific biomarker suPAR as a prognostic marker in patients admitted to acute care...

  17. Activation of peroxisome proliferator?activated receptor gamma induces anti-inflammatory properties in the chicken free avian respiratory macrophages

    OpenAIRE

    Mutua, Mbuvi P.; Steinaa, Lucilla; Shadrack, Muya M.; Muita, Gicheru M.

    2015-01-01

    Background Activation of peroxisome proliferator activated receptor gamma (PPAR ?) in the alveolar macrophages (AM) by selective synthetic PPAR ? ligands, improves the ability of the cells to resolve inflammation. In birds, respiratory macrophages are known as free avian respiratory macrophages (FARM) and show distinct functional differences from AM. The effects of treating FARM with PPAR ? ligands are unclear. Methods FARM were harvested by lavage of chicken respiratory tract and their morph...

  18. Stretch-Induced Mitogen-Activated Protein Kinase Activation in Lung Fibroblasts Is Independent of Receptor Tyrosine Kinases

    OpenAIRE

    Boudreault, Francis; Tschumperlin, Daniel J.

    2009-01-01

    Lung growth and remodeling are modulated by mechanical stress, with fibroblasts thought to play a leading role. Little mechanistic information is available about how lung fibroblasts respond to mechanical stress. We exposed cultured lung fibroblasts to tonic stretch and measured changes in phosphorylation status of mitogen-activated protein kinases (MAPKs), selected receptor tyrosine kinases (RTKs), and phospholipase Cγ1 (PLCγ1) and activation of the small G-protein Ras. Human lung fibroblast...

  19. Mechanisms of peroxisome proliferator activated receptor γ regulation by non-steroidal anti-inflammatory drugs.

    Science.gov (United States)

    Puhl, Ana C; Milton, Flora A; Cvoro, Aleksandra; Sieglaff, Douglas H; Campos, Jéssica C L; Bernardes, Amanda; Filgueira, Carly S; Lindemann, Jan Lammel; Deng, Tuo; Neves, Francisco A R; Polikarpov, Igor; Webb, Paul

    2015-01-01

    Non-steroidal anti-inflammatory drugs (NSAIDs) display anti-inflammatory, antipyretic and analgesic properties by inhibiting cyclooxygenases and blocking prostaglandin production. Previous studies, however, suggested that some NSAIDs also modulate peroxisome proliferator activated receptors (PPARs), raising the possibility that such off target effects contribute to the spectrum of clinically relevant NSAID actions. In this study, we set out to understand how peroxisome proliferator activated receptor-γ (PPARγ/PPARG) interacts with NSAIDs using X-ray crystallography and to relate ligand binding modes to effects on receptor activity. We find that several NSAIDs (sulindac sulfide, diclofenac, indomethacin and ibuprofen) bind PPARγ and modulate PPARγ activity at pharmacologically relevant concentrations. Diclofenac acts as a partial agonist and binds to the PPARγ ligand binding pocket (LBP) in typical partial agonist mode, near the β-sheets and helix 3. By contrast, two copies of indomethacin and sulindac sulfide bind the LBP and, in aggregate, these ligands engage in LBP contacts that resemble agonists. Accordingly, both compounds, and ibuprofen, act as strong partial agonists. Assessment of NSAID activities in PPARγ-dependent 3T3-L1 cells reveals that NSAIDs display adipogenic activities and exclusively regulate PPARγ-dependent target genes in a manner that is consistent with their observed binding modes. Further, PPARγ knockdown eliminates indomethacin activities at selected endogenous genes, confirming receptor-dependence of observed effects. We propose that it is important to consider how individual NSAIDs interact with PPARγ to understand their activities, and that it will be interesting to determine whether high dose NSAID therapies result in PPAR activation.

  20. Neuron to astrocyte communication via cannabinoid receptors is necessary for sustained epileptiform activity in rat hippocampus.

    Directory of Open Access Journals (Sweden)

    Guyllaume Coiret

    Full Text Available Astrocytes are integral functional components of synapses, regulating transmission and plasticity. They have also been implicated in the pathogenesis of epilepsy, although their precise roles have not been comprehensively characterized. Astrocytes integrate activity from neighboring synapses by responding to neuronally released neurotransmitters such as glutamate and ATP. Strong activation of astrocytes mediated by these neurotransmitters can promote seizure-like activity by initiating a positive feedback loop that induces excessive neuronal discharge. Recent work has demonstrated that astrocytes express cannabinoid 1 (CB1 receptors, which are sensitive to endocannabinoids released by nearby pyramidal cells. In this study, we tested whether this mechanism also contributes to epileptiform activity. In a model of 4-aminopyridine induced epileptic-like activity in hippocampal slice cultures, we show that pharmacological blockade of astrocyte CB1 receptors did not modify the initiation, but significantly reduced the maintenance of epileptiform discharge. When communication in astrocytic networks was disrupted by chelating astrocytic calcium, this CB1 receptor-mediated modulation of epileptiform activity was no longer observed. Thus, endocannabinoid signaling from neurons to astrocytes represents an additional significant factor in the maintenance of epileptiform activity in the hippocampus.

  1. Multiple autophosphorylation sites of the epidermal growth factor receptor are essential for receptor kinase activity and internalization. Contrasting significance of tyrosine 992 in the native and truncated receptors

    DEFF Research Database (Denmark)

    Sorkin, A; Helin, K; Waters, C M

    1992-01-01

    The role of epidermal growth factor (EGF) receptor autophosphorylation sites in the regulation of receptor functions has been studied using cells transfected with mutant EGF receptors. Simultaneous point mutation of 4 tyrosines (Y1068, Y1086, Y1148, Y1173) to phenylalanine, as well as removal of ...

  2. Activation of dopamine receptors in the nucleus accumbens promotes sucrose-reinforced cued approach behavior

    Directory of Open Access Journals (Sweden)

    Saleem M. Nicola

    2016-07-01

    Full Text Available Dopamine receptor activation in the nucleus accumbens (NAc promotes vigorous environmentally-cued food-seeking in hungry rats. Rats fed ad libitum, however, respond to fewer food-predictive cues, particularly when the value of food reward is low. Here, we investigated whether this difference could be due to differences in the degree of dopamine receptor activation in the NAc. First, we observed that although rats given ad libitum access to chow in their home cages approached a food receptacle in response to reward-predictive cues, the number of such approaches declined as animals accumulated food rewards. Intriguingly, cued approach to food occurred in clusters, with several cued responses followed by successive non-responses. This pattern suggested that behavior was dictated by transitions between two states, responsive and non-responsive. Injection of D1 or D2 dopamine receptor agonists into the NAc dose-dependently increased cue responding by promoting transitions to the responsive state and by preventing transitions to the non-responsive state. In contrast, antagonists of either D1 or D2 receptors promoted long bouts of non-responding by inducing transitions to the non-responsive state and by preventing transitions to the responsive state. Moreover, locomotor behavior during the inter-trial interval was correlated with the responsive state, and was also increased by dopamine receptor agonists. These results suggest that activation of NAc dopamine receptors plays an important role in regulating the probability of approach to food under conditions of normative satiety.

  3. Insulin receptor activation and down-regulation by cationic lipid transfection reagents

    Directory of Open Access Journals (Sweden)

    Renström Ing-Marie

    2004-01-01

    Full Text Available Abstract Background Transfection agents comprised of cationic lipid preparations are widely used to transfect cell lines in culture with specific recombinant complementary DNA molecules. We have found that cells in culture are often resistant to stimulation with insulin subsequent to treatment with transfection agents such as LipofectAMINE 2000™ and FuGENE-6™. This is seen with a variety of different readouts, including insulin receptor signalling, glucose uptake into muscle cells, phosphorylation of protein kinase B and reporter gene activity in a variety of different cell types Results We now show that this is due in part to the fact that cationic lipid agents activate the insulin receptor fully during typical transfection experiments, which is then down-regulated. In attempts to circumvent this problem, we investigated the effects of increasing concentrations of LipofectAMINE 2000™ on insulin receptor phosphorylation in Chinese hamster ovary cells expressing the human insulin receptor. In addition, the efficiency of transfection that is supported by the same concentrations of transfection reagent was studied by using a green fluorescent protein construct. Our data indicate that considerably lower concentrations of LipofectAMINE 2000™ can be used than are recommended by the manufacturers. This is without sacrificing transfection efficiency markedly and avoids the problem of reducing insulin receptor expression in the cells. Conclusion Widely-used cationic lipid transfection reagents cause a state of insulin unresponsiveness in cells in culture due to fully activating and subsequently reducing the expression of the receptor in cells. This phenomenon can be avoided by reducing the concentration of reagent used in the transfection process.

  4. 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...... recent advances in understanding the biological functions, signaling mechanisms, and disease associations of the aGPCRs....

  5. 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.

  6. Discoidin domain receptor 1 is activated independently of beta(1) integrin

    DEFF Research Database (Denmark)

    Vogel, W; Brakebusch, C; Fässler, R

    2000-01-01

    with kinase activity or membrane-anchored proteins serving as coreceptors. In particular, the role of the collagen-binding integrins alpha(1)beta(1) or alpha(2)beta(1) in the DDR activation process is undefined. Here, we provide three lines of evidence suggesting that DDR1 signaling is distinct from integrin...... for alpha(2)beta(1) integrin or in cells with a targeted deletion of the beta(1) integrin gene. Finally, we show that overexpression of dominant negative DDR1 in the myoblast cell line C2C12 blocks cellular differentiation and the formation of myofibers....... activation. First we demonstrate that the enzymatic activity of DDR1 is essential for receptor tyrosine phosphorylation. Collagen-induced DDR receptor autophosphorylation can be blocked either by a dominant negative mutant or by a preparation of recombinant extracellular domain. Second, we show DDR1 signals...

  7. Activated factor X signaling via protease-activated receptor 2 suppresses pro-inflammatory cytokine production from LPS-stimulated myeloid cells.

    LENUS (Irish Health Repository)

    Gleeson, Eimear M

    2013-07-19

    Vitamin K-dependent proteases generated in response to vascular injury and infection enable fibrin clot formation, but also trigger distinct immuno-regulatory signaling pathways on myeloid cells. Factor Xa, a protease crucial for blood coagulation, also induces protease-activated receptor-dependent cell signaling. Factor Xa can bind both monocytes and macrophages, but whether factor Xa-dependent signaling stimulates or suppresses myeloid cell cytokine production in response to Toll-like receptor activation is not known. In this study, exposure to factor Xa significantly impaired pro-inflammatory cytokine production from lipopolysaccharide-treated peripheral blood mononuclear cells, THP-1 monocytic cells and murine macrophages. Furthermore, factor Xa inhibited nuclear factor-kappa B activation in THP-1 reporter cells, requiring phosphatidylinositide 3-kinase activity for its anti-inflammatory effect. Active-site blockade, γ-carboxyglutamic acid domain truncation and a peptide mimic of the factor Xa inter-epidermal growth factor-like region prevented factor Xa inhibition of lipopolysaccharide-induced tumour necrosis factor-α release. In addition, factor Xa anti-inflammatory activity was markedly attenuated by the presence of an antagonist of protease-activated receptor 2, but not protease-activated receptor 1. The key role of protease-activated receptor 2 in eliciting factor Xa-dependent anti-inflammatory signaling on macrophages was further underscored by the inability of factor Xa to mediate inhibition of tumour necrosis factor-α and interleukin-6 release from murine bone marrow-derived protease-activated receptor 2-deficient macrophages. We also show for the first time that, in addition to protease-activated receptor 2, factor Xa requires a receptor-associated protein-sensitive low-density lipoprotein receptor to inhibit lipopolysaccharide-induced cytokine production. Collectively, this study supports a novel function for factor Xa as an endogenous, receptor

  8. Evidence for constitutively-active adenosine receptors at mammalian motor nerve endings

    OpenAIRE

    Searl, Timothy J; Silinsky, Eugene M

    2012-01-01

    A study was made to determine if constitutively active adenosine receptors are present at mouse motor nerve endings. In preparations blocked by low Ca2+ / high Mg2+ solution, 8-cyclopentyl-1,3,dipropylxanthine (CPX, 10–100 nM), which has been reported to be both an A1 adenosine receptor antagonist and inverse agonist, produced a dose-dependent increase in the number of acetylcholine quanta released by a nerve impulse. Adenosine deaminase, which degrades ambient adenosine into its inactive con...

  9. The human receptor for urokinase plasminogen activator. NH2-terminal amino acid sequence and glycosylation variants

    DEFF Research Database (Denmark)

    Behrendt, N; Rønne, E; Ploug, M

    1990-01-01

    The receptor for human urokinase-type plasminogen activator (u-PA) was purified from phorbol 12-myristate 13-acetate-stimulated U937 cells by temperature-induced phase separation of detergent extracts, followed by affinity chromatography with immobilized diisopropyl fluorophosphate-treated u....... The protein has a high content of cysteine residues. The NH2-terminal sequence is not closely related to any known sequence. The identification of the purified and sequenced protein with the human u-PA receptor is based on the following findings: 1) the ability of the purified protein to bind u-PA and its...

  10. Altered circadian food anticipatory activity rhythms in PACAP receptor 1 (PAC1) deficient mice

    DEFF Research Database (Denmark)

    Hannibal, Jens; Georg, Birgitte; Fahrenkrug, Jan

    2016-01-01

    Light signals from intrinsically photosensitive retinal ganglion cells (ipRGCs) entrain the circadian clock and regulate negative masking. Two neurotransmitters, glutamate and Pituitary Adenylate Cyclase Activating Polypeptide (PACAP), found in the ipRGCs transmit light signals to the brain via...... glutamate receptors and the specific PACAP type 1 (PAC1) receptor. Light entrainment occurs during the twilight zones and has little effect on clock phase during daytime. When nocturnal animals have access to food only for a few hours during the resting phase at daytime, they adapt behavior...

  11. Peroxisome Proliferator-Activated Receptor (PPAR): Balance for Survival in Parasitic Infections

    OpenAIRE

    Chan, Marion M.; Evans, Kyle W.; Moore, Andrea R.; Dunne Fong

    2010-01-01

    Parasitic infections induce a magnitude of host responses. At the opposite ends of the spectrum are those that ensure the host's needs to eliminate the invaders and to minimize damage to its own tissues. This review analyzes how parasites would manipulate immunity by activating the immunosuppressive nuclear factor, peroxisome proliferator-activated receptors (PPARs) with type 2 cytokines and free fatty acids from arachidonic acid metabolism. PPARs limit the action of type 1 immunity, in w...

  12. Protease-activated receptor 2 suppresses lymphangiogenesis and subsequent lymph node metastasis in a murine pancreatic cancer model

    NARCIS (Netherlands)

    Shi, Kun; Queiroz, Karla C. S.; Roelofs, Joris J. T. H.; van Noesel, Carel J. M.; Richel, Dirk J.; Spek, C. Arnold

    2014-01-01

    Protease-activated receptor-2 (PAR-2) is a G protein-coupled receptor that functions as a cell-surface sensor for coagulation factors and other proteases associated with the tumour microenvironment. Pancreatic cancer cells express high levels of PAR-2 and activation of PAR-2 may induce their

  13. The orphan nuclear receptor Rev-Erbalpha is a peroxisome proliferator-activated receptor (PPAR) gamma target gene and promotes PPARgamma-induced adipocyte differentiation

    DEFF Research Database (Denmark)

    Fontaine, Coralie; Dubois, Guillaume; Duguay, Yannick

    2003-01-01

    Rev-Erbalpha (NR1D1) is an orphan nuclear receptor encoded on the opposite strand of the thyroid receptor alpha gene. Rev-Erbalpha mRNA is induced during adipocyte differentiation of 3T3-L1 cells, and its expression is abundant in rat adipose tissue. Peroxisome proliferator-activated receptor gamma...... (PPARgamma) (NR1C3) is a nuclear receptor controlling adipocyte differentiation and insulin sensitivity. Here we show that Rev-Erbalpha expression is induced by PPARgamma activation with rosiglitazone in rat epididymal and perirenal adipose tissues in vivo as well as in 3T3-L1 adipocytes in vitro...... of functional PPARgamma response element. Finally, ectopic expression of Rev-Erbalpha in 3T3-L1 preadipocytes potentiated adipocyte differentiation induced by the PPARgamma ligand rosiglitazone. These results identify Rev-Erbalpha as a target gene of PPARgamma in adipose tissue and demonstrate a role...