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Sample records for camp receptor protein

  1. Directed evolution of the Escherichia coli cAMP receptor protein at the cAMP pocket.

    Science.gov (United States)

    Gunasekara, Sanjiva M; Hicks, Matt N; Park, Jin; Brooks, Cory L; Serate, Jose; Saunders, Cameron V; Grover, Simranjeet K; Goto, Joy J; Lee, Jin-Won; Youn, Hwan

    2015-10-30

    The Escherichia coli cAMP receptor protein (CRP) requires cAMP binding to undergo a conformational change for DNA binding and transcriptional regulation. Two CRP residues, Thr(127) and Ser(128), are known to play important roles in cAMP binding through hydrogen bonding and in the cAMP-induced conformational change, but the connection between the two is not completely clear. Here, we simultaneously randomized the codons for these two residues and selected CRP mutants displaying high CRP activity in a cAMP-producing E. coli. Many different CRP mutants satisfied the screening condition for high CRP activity, including those that cannot form any hydrogen bonds with the incoming cAMP at the two positions. In vitro DNA-binding analysis confirmed that these selected CRP mutants indeed display high CRP activity in response to cAMP. These results indicate that the hydrogen bonding ability of the Thr(127) and Ser(128) residues is not critical for the cAMP-induced CRP activation. However, the hydrogen bonding ability of Thr(127) and Ser(128) was found to be important in attaining high cAMP affinity. Computational analysis revealed that most natural cAMP-sensing CRP homologs have Thr/Ser, Thr/Thr, or Thr/Asn at positions 127 and 128. All of these pairs are excellent hydrogen bonding partners and they do not elevate CRP activity in the absence of cAMP. Taken together, our analyses suggest that CRP evolved to have hydrogen bonding residues at the cAMP pocket residues 127 and 128 for performing dual functions: preserving high cAMP affinity and keeping CRP inactive in the absence of cAMP.

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

    DEFF Research Database (Denmark)

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

    2013-01-01

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

  3. cAMP receptor protein (CRP) downregulates Klebsiella pneumoniae nif promoters in Escherichia coli

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    In enteric bacteria, in response to the PTS system, the cAMP receptor protein (CRP) mediates the glucose effect, via regulating s70-dependent catabolic genes at transcriptional level. In this study, it is observed that the nitrogen fixation capacity of Klebsiella pneumoniae varies strongly when cells are grown on different carbohydrates, and this carbon effect occurs at the level of nif gene expression. Here we show that CRP can repress s54-dependent nif promoters (nifB, nifE, nifF, nifH, nifJ, nifLA and nifU), in a cAMP dependent fashion, in closed related E. coli background. Sequence analysis of these nif promoters indicates that there is no direct correlation between the fold of CRP-cAMP-mediated inhibition and the upstream cis elements at the promoters. In addition, the crp gene of K. pneumoniae has been isolated and sequenced, which is structural and functional highly homologous to that of E. coli. This suggests that CRP-cAMP-mediated inhibition on the nif promoters could be the reason for carbon effect on nitrogen fixation and thus has its physiological significance. A novel regulatory linkage between carbon metabolism and nitrogen fixation is proposed.

  4. Melanocortin 1 receptor mutations impact differentially on signalling to the cAMP and the ERK mitogen-activated protein kinase pathways.

    Science.gov (United States)

    Herraiz, Cecilia; Jiménez-Cervantes, Celia; Zanna, Paola; García-Borrón, José C

    2009-10-06

    Melanocortin 1 receptor (MC1R), a Gs protein-coupled receptor expressed in melanocytes, is a major determinant of skin pigmentation, phototype and cancer risk. MC1R activates cAMP and mitogen-activated protein kinase ERK1/ERK2 signalling. When expressed in rat pheochromocytoma cell line cells, the R151C, R160W and D294H MC1R variants associated with melanoma and impaired cAMP signalling mediated ERK activation and ERK-dependent, agonist-induced neurite outgrowth comparable with wild-type. Dose-response curves for ERK activation and cAMP production indicated higher sensitivity of the ERK response. Thus, the melanoma-associated MC1R mutations impact differently on cAMP and ERK signalling, suggesting that cAMP is not responsible for functional coupling of MC1R to the ERK cascade.

  5. Identification of the subunit of cAMP receptor protein (CRP) that functionally interacts with CytR in CRP-CytR-mediated transcriptional repression

    DEFF Research Database (Denmark)

    Meibom, K L; Kallipolitis, B H; Ebright, R H

    2000-01-01

    At promoters of the Escherichia coli CytR regulon, the cAMP receptor protein (CRP) interacts with the repressor CytR to form transcriptionally inactive CRP-CytR-promoter or (CRP)(2)-CytR-promoter complexes. Here, using "oriented heterodimer" analysis, we show that only one subunit of the CRP dimer...

  6. Computational prediction of cAMP receptor protein (CRP binding sites in cyanobacterial genomes

    Directory of Open Access Journals (Sweden)

    Su Zhengchang

    2009-01-01

    Full Text Available Abstract Background Cyclic AMP receptor protein (CRP, also known as catabolite gene activator protein (CAP, is an important transcriptional regulator widely distributed in many bacteria. The biological processes under the regulation of CRP are highly diverse among different groups of bacterial species. Elucidation of CRP regulons in cyanobacteria will further our understanding of the physiology and ecology of this important group of microorganisms. Previously, CRP has been experimentally studied in only two cyanobacterial strains: Synechocystis sp. PCC 6803 and Anabaena sp. PCC 7120; therefore, a systematic genome-scale study of the potential CRP target genes and binding sites in cyanobacterial genomes is urgently needed. Results We have predicted and analyzed the CRP binding sites and regulons in 12 sequenced cyanobacterial genomes using a highly effective cis-regulatory binding site scanning algorithm. Our results show that cyanobacterial CRP binding sites are very similar to those in E. coli; however, the regulons are very different from that of E. coli. Furthermore, CRP regulons in different cyanobacterial species/ecotypes are also highly diversified, ranging from photosynthesis, carbon fixation and nitrogen assimilation, to chemotaxis and signal transduction. In addition, our prediction indicates that crp genes in modern cyanobacteria are likely inherited from a common ancestral gene in their last common ancestor, and have adapted various cellular functions in different environments, while some cyanobacteria lost their crp genes as well as CRP binding sites during the course of evolution. Conclusion The CRP regulons in cyanobacteria are highly diversified, probably as a result of divergent evolution to adapt to various ecological niches. Cyanobacterial CRPs may function as lineage-specific regulators participating in various cellular processes, and are important in some lineages. However, they are dispensable in some other lineages. The

  7. Differential regulation of phosphorylation of the cAMP response element-binding protein after activation of EP2 and EP4 prostanoid receptors by prostaglandin E2.

    Science.gov (United States)

    Fujino, Hiromichi; Salvi, Sambhitab; Regan, John W

    2005-07-01

    The EP2 and EP4 prostanoid receptors are G-protein-coupled receptors whose activation by their endogenous ligand, prostaglandin (PG) E2, stimulates the formation of intracellular cAMP. We have previously reported that the stimulation of cAMP formation in EP4-expressing cells is significantly less than in EP2-expressing cells, despite nearly identical levels of receptor expression (J Biol Chem 277:2614-2619, 2002). In addition, a component of EP4 receptor signaling, but not of EP2 receptor signaling, was found to involve the activation of phosphatidylinositol 3-kinase (PI3K). In this study, we report that PGE2 stimulation of cells expressing either the EP2 or EP4 receptor results in the phosphorylation of the cAMP response element binding protein (CREB) at serine-133. Pretreatment of cells with N-[2-(4-bromocinnamylamino)ethyl]-5-isoquinoline (H-89), an inhibitor of protein kinase A (PKA), attenuated the PGE2-mediated phosphorylation of CREB in EP2-expressing cells, but not in EP4-expressing cells. Pretreatment of cells with wortmannin, an inhibitor of PI3K, had no effects on the PGE2-mediated phosphorylation of CREB in either EP2- or EP4-expressing cells, although it significantly increased the PGE2-mediated activation of PKA in EP4-expressing cells. However, combined pretreatment with H-89 and wortmannin blocked PGE2-mediated phosphorylation in EP2-expressing cells as well as in EP2-expressing cells. PGE2-mediated intracellular cAMP formation was not affected by pretreatment with wortmannin, or combined treatment with wortmannin and H-89, in either the EP2- or EP4-expressing cells. These findings suggest that PGE2 stimulation of EP4 receptors, but not EP2 receptors, results in the activation of a PI3K signaling pathway that inhibits the activity of PKA and that the PGE2-mediated phosphorylation of CREB by these receptors occurs through different signaling pathways

  8. Downregulation of protease-activated receptor-1 in human lung fibroblasts is specifically mediated by the prostaglandin E receptor EP2 through cAMP elevation and protein kinase A.

    Science.gov (United States)

    Sokolova, Elena; Hartig, Roland; Reiser, Georg

    2008-07-01

    Many cellular functions of lung fibroblasts are controlled by protease-activated receptors (PARs). In fibrotic diseases, PAR-1 plays a major role in controlling fibroproliferative and inflammatory responses. Therefore, in these diseases, regulation of PAR-1 expression plays an important role. Using the selective prostaglandin EP2 receptor agonist butaprost and cAMP-elevating agents, we show here that prostaglandin (PG)E(2), via the prostanoid receptor EP2 and subsequent cAMP elevation, downregulates mRNA and protein levels of PAR-1 in human lung fibroblasts. Under these conditions, the functional response of PAR-1 in fibroblasts is reduced. These effects are specific for PGE(2). Activation of other receptors coupled to cAMP elevation, such as beta-adrenergic and adenosine receptors, does not reproduce the effects of PGE(2). PGE(2)-mediated downregulation of PAR-1 depends mainly on protein kinase A activity, but does not depend on another cAMP effector, the exchange protein activated by cAMP. PGE(2)-induced reduction of PAR-1 level is not due to a decrease of PAR-1 mRNA stability, but rather to transcriptional regulation. The present results provide further insights into the therapeutic potential of PGE(2) to specifically control fibroblast function in fibrotic diseases.

  9. Enhancing E. coli tolerance towards oxidative stress via engineering its global regulator cAMP receptor protein (CRP.

    Directory of Open Access Journals (Sweden)

    Souvik Basak

    Full Text Available Oxidative damage to microbial hosts often occurs under stressful conditions during bioprocessing. Classical strain engineering approaches are usually both time-consuming and labor intensive. Here, we aim to improve E. coli performance under oxidative stress via engineering its global regulator cAMP receptor protein (CRP, which can directly or indirectly regulate redox-sensing regulators SoxR and OxyR, and other ~400 genes in E. coli. Error-prone PCR technique was employed to introduce modifications to CRP, and three mutants (OM1~OM3 were identified with improved tolerance via H(2O(2 enrichment selection. The best mutant OM3 could grow in 12 mM H(2O(2 with the growth rate of 0.6 h(-1, whereas the growth of wild type was completely inhibited at this H(2O(2 concentration. OM3 also elicited enhanced thermotolerance at 48°C as well as resistance against cumene hydroperoxide. The investigation about intracellular reactive oxygen species (ROS, which determines cell viability, indicated that the accumulation of ROS in OM3 was always lower than in WT with or without H(2O(2 treatment. Genome-wide DNA microarray analysis has shown not only CRP-regulated genes have demonstrated great transcriptional level changes (up to 8.9-fold, but also RpoS- and OxyR-regulated genes (up to 7.7-fold. qRT-PCR data and enzyme activity assay suggested that catalase (katE could be a major antioxidant enzyme in OM3 instead of alkyl hydroperoxide reductase or superoxide dismutase. To our knowledge, this is the first work on improving E. coli oxidative stress resistance by reframing its transcription machinery through its native global regulator. The positive outcome of this approach may suggest that engineering CRP can be successfully implemented as an efficient strain engineering alternative for E. coli.

  10. Engineering of global regulator cAMP receptor protein (CRP) in Escherichia coli for improved lycopene production.

    Science.gov (United States)

    Huang, Lei; Pu, Yue; Yang, Xiuliang; Zhu, Xiangcheng; Cai, Jin; Xu, Zhinan

    2015-04-10

    Transcriptional engineering has received significant attention for improving strains by modulating the behavior of transcription factors, which could be used to reprogram a series of gene transcriptions and enable multiple simultaneous modifications at the genomic level. In this study, engineering of the cAMP receptor protein (CRP) was explored with the aim of subtly balancing entire pathway networks and potentially improving lycopene production without significant genetic intervention in other pathways. Amino acid mutations were introduced to CRP by error-prone PCR, and three variants (mcrp26, mcrp159 and mcrp424) with increased lycopene productivity were screened. Combinations of three point mutations were then created via site-directed mutagenesis. The best mutant gene (mcrp26) was integrated into the genome of E. coli BW25113-BIE to replace the wild-type crp gene (MT-1), which resulted in a higher lycopene production (18.49mg/g DCW) compared to the original strain (WT). The mutant strain MT-1 was further investigated in a 10-L bench-top fermentor with a lycopene yield of 128mg/l at 20h, approximately 25% higher than WT. DNA microarray analyses showed that 396 genes (229 up-regulated and 167 down-regulated) were differentially expressed in the mutant MT-1 compared to WT. Finally, the introduction of the mutant crp gene (mcrp26) increased β-carotene production in E. coli. This is the first report of improving the phenotype for metabolite overproduction in E. coli using a CRP engineering strategy.

  11. Enhancing E. coli isobutanol tolerance through engineering its global transcription factor cAMP receptor protein (CRP).

    Science.gov (United States)

    Chong, Huiqing; Geng, Hefang; Zhang, Hongfang; Song, Hao; Huang, Lei; Jiang, Rongrong

    2014-04-01

    The limited isobutanol tolerance of Escherichia coli is a major drawback during fermentative isobutanol production. Different from classical strain engineering approaches, this work was initiated to improve E. coli isobutanol tolerance from its transcriptional level by engineering its global transcription factor cAMP receptor protein (CRP). Random mutagenesis libraries were generated by error-prone PCR of crp, and the libraries were subjected to isobutanol stress for selection. Variant IB2 (S179P, H199R) was isolated and exhibited much better growth (0.18 h(-1) ) than the control (0.05 h(-1) ) in 1.2% (v/v) isobutanol (9.6 g/L). Genome-wide DNA microarray analysis revealed that 58 and 308 genes in IB2 had differential expression (>2-fold, p < 0.05) in the absence and presence of 1% (v/v) isobutanol, respectively. When challenged with isobutanol, genes related to acid resistance (gadABCE, hdeABD), nitrate reduction (narUZYWV), flagella and fimbrial activity (lfhA, yehB, ycgR, fimCDF), and sulfate reduction and transportation (cysIJH, cysC, cysN) were the major functional groups that were up-regulated, whereas most of the down-regulated genes were enzyme (tnaA) and transporters (proVWX, manXYZ). As demonstrated by single-gene knockout experiments, gadX, nirB, rhaS, hdeB, and ybaS were found associated with strain isobutanol resistance. The intracellular reactive oxygen species (ROS) level in IB2 was only half of that of the control when facing stress, indicating that IB2 can withstand toxic isobutanol much better than the control.

  12. Enhancing E. coli tolerance towards oxidative stress via engineering its global regulator cAMP receptor protein (CRP).

    Science.gov (United States)

    Basak, Souvik; Jiang, Rongrong

    2012-01-01

    Oxidative damage to microbial hosts often occurs under stressful conditions during bioprocessing. Classical strain engineering approaches are usually both time-consuming and labor intensive. Here, we aim to improve E. coli performance under oxidative stress via engineering its global regulator cAMP receptor protein (CRP), which can directly or indirectly regulate redox-sensing regulators SoxR and OxyR, and other ~400 genes in E. coli. Error-prone PCR technique was employed to introduce modifications to CRP, and three mutants (OM1~OM3) were identified with improved tolerance via H(2)O(2) enrichment selection. The best mutant OM3 could grow in 12 mM H(2)O(2) with the growth rate of 0.6 h(-1), whereas the growth of wild type was completely inhibited at this H(2)O(2) concentration. OM3 also elicited enhanced thermotolerance at 48°C as well as resistance against cumene hydroperoxide. The investigation about intracellular reactive oxygen species (ROS), which determines cell viability, indicated that the accumulation of ROS in OM3 was always lower than in WT with or without H(2)O(2) treatment. Genome-wide DNA microarray analysis has shown not only CRP-regulated genes have demonstrated great transcriptional level changes (up to 8.9-fold), but also RpoS- and OxyR-regulated genes (up to 7.7-fold). qRT-PCR data and enzyme activity assay suggested that catalase (katE) could be a major antioxidant enzyme in OM3 instead of alkyl hydroperoxide reductase or superoxide dismutase. To our knowledge, this is the first work on improving E. coli oxidative stress resistance by reframing its transcription machinery through its native global regulator. The positive outcome of this approach may suggest that engineering CRP can be successfully implemented as an efficient strain engineering alternative for E. coli.

  13. Improving ethanol tolerance of Escherichia coli by rewiring its global regulator cAMP receptor protein (CRP.

    Directory of Open Access Journals (Sweden)

    Huiqing Chong

    Full Text Available A major challenge in bioethanol fermentation is the low tolerance of the microbial host towards the end product bioethanol. Here we report to improve the ethanol tolerance of E. coli from the transcriptional level by engineering its global transcription factor cAMP receptor protein (CRP, which is known to regulate over 400 genes in E. coli. Three ethanol tolerant CRP mutants (E1- E3 were identified from error-prone PCR libraries. The best ethanol-tolerant strain E2 (M59T had the growth rate of 0.08 h(-1 in 62 g/L ethanol, higher than that of the control at 0.06 h(-1. The M59T mutation was then integrated into the genome to create variant iE2. When exposed to 150 g/l ethanol, the survival of iE2 after 15 min was about 12%, while that of BW25113 was <0.01%. Quantitative real-time reverse transcription PCR analysis (RT-PCR on 444 CRP-regulated genes using OpenArray® technology revealed that 203 genes were differentially expressed in iE2 in the absence of ethanol, whereas 92 displayed differential expression when facing ethanol stress. These genes belong to various functional groups, including central intermediary metabolism (aceE, acnA, sdhD, sucA, iron ion transport (entH, entD, fecA, fecB, and general stress response (osmY, rpoS. Six up-regulated and twelve down-regulated common genes were found in both iE2 and E2 under ethanol stress, whereas over one hundred common genes showed differential expression in the absence of ethanol. Based on the RT-PCR results, entA, marA or bhsA was knocked out in iE2 and the resulting strains became more sensitive towards ethanol.

  14. Expression of orphan G-protein coupled receptor GPR174 in CHO cells induced morphological changes and proliferation delay via increasing intracellular cAMP

    Energy Technology Data Exchange (ETDEWEB)

    Sugita, Kazuya; Yamamura, Chiaki; Tabata, Ken-ichi [Laboratory of Pharmacoinformatics, Graduate School of Ritsumeikan University, Kusatsu, Shiga 525-8577 (Japan); Fujita, Norihisa, E-mail: nori@ph.ritsumei.ac.jp [Laboratory of Pharmacoinformatics, Graduate School of Ritsumeikan University, Kusatsu, Shiga 525-8577 (Japan); School of Pharmacy, Ristumeikan University, Kusatsu, Shiga 525-8577 (Japan)

    2013-01-04

    Highlights: Black-Right-Pointing-Pointer Expression of GPR174 in CHO cells induces morphological changes and proliferation delay. Black-Right-Pointing-Pointer These are due to increase in intracellular cAMP concentration. Black-Right-Pointing-Pointer Lysophosphatidylserine was identified to stimulate GPR174 leading to activate ACase. Black-Right-Pointing-Pointer The potencies of fatty acid moiety on LysoPS were oleoyl Greater-Than-Or-Slanted-Equal-To stearoyl > palmitoyl. Black-Right-Pointing-Pointer We propose that GPR174 is a lysophosphatidylserine receptor. -- Abstract: We established cell lines that stably express orphan GPCR GPR174 using CHO cells, and studied physiological and pharmacological features of the receptor. GPR174-expressing cells showed cell-cell adhesion with localization of actin filaments to cell membrane, and revealed significant delay of cell proliferation. Since the morphological changes of GPR174-cells were very similar to mock CHO cells treated with cholera toxin, we measured the concentration of intracellular cAMP. The results showed the concentration was significantly elevated in GPR174-cells. By measuring intracellular cAMP concentration in GPR174-cells, we screened lipids and nucleotides to identify ligands for GPR174. We found that lysophosphatidylserine (LysoPS) stimulated increase in intracellular cAMP in a dose-dependent manner. Moreover, phosphorylation of Erk was elevated by LysoPS in GPR174 cells. These LysoPS responses were inhibited by NF449, an inhibitor of G{alpha}{sub s} protein. These results suggested that GPR174 was a putative LysoPS receptor conjugating with G{alpha}{sub s}, and its expression induced morphological changes in CHO cells by constitutively activating adenylyl cycles accompanied with cell conjunctions and delay of proliferation.

  15. Functional status and relationships of melanocortin 1 receptor signaling to the cAMP and extracellular signal-regulated protein kinases 1 and 2 pathways in human melanoma cells.

    Science.gov (United States)

    Herraiz, Cecilia; Journé, Fabrice; Ghanem, Ghanem; Jiménez-Cervantes, Celia; García-Borrón, José C

    2012-12-01

    Melanocortin 1 receptor (MC1R), a major determinant of skin phototype frequently mutated in melanoma, is a Gs protein-coupled receptor that regulates pigment production in melanocytes. MC1R stimulation activates cAMP synthesis and the extracellular signal-regulated (ERK) ERK1 and ERK2. In human melanocytes, ERK activation by MC1R relies on cAMP-independent transactivation of the c-KIT receptor. Thus MC1R functional coupling to the cAMP and ERK pathways may involve different structural requirements giving raise to biased effects of skin cancer-associated mutations. We evaluated the impact of MC1R mutations on ERK activation, cAMP production and agonist binding. We found that MC1R mutations impair cAMP production much more often than ERK activation, suggesting less stringent requirements for functional coupling to the ERK pathway. We examined the crosstalk of the cAMP and ERK pathways in HBL human melanoma cells (wild-type for MC1R, NRAS and BRAF). ERK activation by constitutively active upstream effectors or pharmacological inhibition had little effect on MC1R-stimulated cAMP synthesis. High cAMP levels were compatible with normal ERK activation but, surprisingly, the adenylyl cyclase activator forskolin abolished ERK activation by MC1R, most likely by a cAMP-independent mechanism. These results indicate little crosstalk of the cAMP and ERK pathways in HBL melanoma cells. Finally, we studied cAMP accumulation in a panel of 22 human melanoma cell lines stimulated with MC1R agonists or forskolin. cAMP synthesis was often inhibited, even in cells wild-type for MC1R and NRAS. Therefore, the cAMP pathway is more frequently impaired in melanoma than could be predicted by the MC1R or NRAS genotype.

  16. G-protein-mediated interconversions of cell-surface cAMP receptors and their involvement in excitation and desensitization of guanylate cyclase in Dictyostelium discoideum

    Energy Technology Data Exchange (ETDEWEB)

    van Haastert, P.J.; de Wit, R.J.; Janssens, P.M.; Kesbeke, F.; DeGoede, J.

    1986-05-25

    In Dictyostelium discoideum cells, extracellular cAMP induces the rapid (within 2 s) activation of guanylate cyclase, which is followed by complete desensitization after about 10 s. cAMP binding to these cells is heterogeneous, showing a subclass of fast dissociating sites coupled to adenylate cyclase (A-sites) and a subclass of slowly dissociating sites coupled to guanylate cyclase (B-sites). The kinetics of the B-sites were further investigated on a seconds time scale. Statistical analysis of the association of (/sup 3/H)cAMP to the B-sites and dissociation of the complex revealed that the receptor can exist in three states which interconvert according to the following scheme. cAMP binds to the BF-state (off-rate 2.5 s) which rapidly (t1/2 = 3 s) converts to the BS-state (off-rate 15 s) and subsequently (without a detectable delay) into the BSS-state (off-rate 150 s). In membranes, both the BS- and BSS-states are converted to the BF-state by GTP and GDP, suggesting the involvement of a G-protein. Densensitized cells show a 80% reduction of the formation of the BSS-state, but no reduction of the BF- or BS-state. These data are combined into a model in which the transitions of the B-sites are mediated by a G-protein; activation of the G-protein and guanylate cyclase is associated with the transition of the BS- to the BSS-state of the receptor, whereas desensitization is associated with the inhibition of this transition.

  17. Signal Transduction in Dictyostelium fgd A Mutants with a Defective Interaction between Surface cAMP Receptors and a GTP-binding Regulatory Protein

    NARCIS (Netherlands)

    Kesbeke, Fanja; Snaar-Jagalska, B. Ewa; Haastert, Peter J.M. van

    1988-01-01

    Transmembrane signal transduction was investigated in four Dictyostelium discoideum mutants that belong to the fgd A complementation group. The results show the following. (a) Cell surface cAMP receptors are present in fgd A mutants, but cAMP does not induce any of the intracellular responses, inclu

  18. Signal transduction in Dictyostelium fgd A mutants with a defective interaction between surface cAMP receptors and a GTP-binding regulatory protein [published erratum appears in J Cell Biol 1988 Dec;107(6 Pt 1):following 2463

    OpenAIRE

    1988-01-01

    Transmembrane signal transduction was investigated in four Dictyostelium discoideum mutants that belong to the fgd A complementation group. The results show the following. (a) Cell surface cAMP receptors are present in fgd A mutants, but cAMP does not induce any of the intracellular responses, including the activation of adenylate or guanylate cyclase and chemotaxis. (b) cAMP induces down- regulation and the covalent modification (presumably phosphorylation) of the cAMP receptor. (c) The inhi...

  19. Learning and memory deficits consequent to reduction of the fragile X mental retardation protein result from metabotropic glutamate receptor-mediated inhibition of cAMP signaling in Drosophila.

    Science.gov (United States)

    Kanellopoulos, Alexandros K; Semelidou, Ourania; Kotini, Andriana G; Anezaki, Maria; Skoulakis, Efthimios M C

    2012-09-19

    Loss of the RNA-binding fragile X protein [fragile X mental retardation protein (FMRP)] results in a spectrum of cognitive deficits, the fragile X syndrome (FXS), while aging individuals with decreased protein levels present with a subset of these symptoms and tremor. The broad range of behavioral deficits likely reflects the ubiquitous distribution and multiple functions of the protein. FMRP loss is expected to affect multiple neuronal proteins and intracellular signaling pathways, whose identity and interactions are essential in understanding and ameliorating FXS symptoms. We used heterozygous mutants and targeted RNA interference-mediated abrogation in Drosophila to uncover molecular pathways affected by FMRP reduction. We present evidence that FMRP loss results in excess metabotropic glutamate receptor (mGluR) activity, attributable at least in part to elevation of the protein in affected neurons. Using high-resolution behavioral, genetic, and biochemical analyses, we present evidence that excess mGluR upon FMRP attenuation is linked to the cAMP decrement reported in patients and models, and underlies olfactory associative learning and memory deficits. Furthermore, our data indicate positive transcriptional regulation of the fly fmr1 gene by cAMP, via protein kinase A, likely through the transcription factor CREB. Because the human Fmr1 gene also contains CREB binding sites, the interaction of mGluR excess and cAMP signaling defects we present suggests novel combinatorial pharmaceutical approaches to symptom amelioration upon FMRP attenuation.

  20. Influence of cAMP receptor protein (CRP) on bacterial virulence and transcriptional regulation of allS by CRP in Klebsiella pneumoniae.

    Science.gov (United States)

    Xue, Jian; Tan, Bin; Yang, Shiya; Luo, Mei; Xia, Huiming; Zhang, Xian; Zhou, Xipeng; Yang, Xianxian; Yang, Ruifu; Li, Yingli; Qiu, Jingfu

    2016-11-15

    cAMP receptor protein (CRP) is one of the most important transcriptional regulators, which can regulate large quantities of operons in different bacteria. The gene allS was well-known as allantoin-utilizing capability and involving in bacterial virulence in Klebsiella pneumoniae (K. pneumoniae). The specific DNA recognition motif of transcription regulator CRP was found in allS promoter region. Therefore, this study is aimed to investigate the function of CRP on virulence and its transcriptional regulation mechanism to gene allS in K. pneumoniae. The wild-type (WT) K. pneumoniae NTUH-2044, crp knockout (Kp-Δcrp) and the complemented knockout (KpC-Δcrp) strains were used to determine the function of crp gene. The lacZ fusion, qRT-PCR, electrophoretic mobility shift and DNase I footprinting assays were performed to study the transcriptional regulation of CRP on allS. The result showed a decreased virulence in crp knockout strain. Complement through supplementing crp fragment in expression plasmid partially restore virulence of knockout bacteria. The CRP could bind to the allS promoter-proximal region and the binding site was further refined to be located from 60bp to 94bp upstream of the allS promoter. Based on these results, we proposed that CRP is an essential virulence regulator and knock out of crp gene will result in reduced virulence in K. pneumoniae. In the meantime, the transcription of gene allS is positively regulated by CRP via directly binding to upstream of allS promoter.

  1. Epac and PKA: a tale of two intracellular cAMP receptors

    Institute of Scientific and Technical Information of China (English)

    Xiaodong Cheng; Zhenyu Ji; Tamara Tsalkova; Fang Mei

    2008-01-01

    cAMP-mediated signaling pathways regulate a multitude of important biological processes under both physiological and pathological conditions,including diabetes,heart failure and cancer.In eukaryotic cells,the effects of cAMP are mediated by two ubiquitously expressed intracellular cAMP receptors,the classic protein kinase A (PKA)/cAMP-dependent protein kinase and the recently discovered exchange protein directly activated by cAMP(Epac)/cAMP-regulated guanine nucleotide exchange factors.Like PKA,Epac contains an evolutionally conserved cAMP binding domain that acts as a molecular switch for sensing intracellular second messenger cAMP levels to control diverse biological functions.The existence of two families of cAMP effectors provides a mechanism for a more precise and integrated control of the cAMP signaling pathways in a spatial and temporal manner.Depending upon the specific cellular environments as well as their relative abundance,distrbution and localization,Epac and PKA may act independently,converge synergistically or oppose each other in regulating a specific cellular function.

  2. Dissecting direct and indirect readout of cAMP receptor protein DNA binding using an inosine and 2,6-diaminopurine in vitro selection system

    DEFF Research Database (Denmark)

    Lindemose, Søren; Nielsen, Peter E.; Møllegaard, Niels Erik

    2008-01-01

    The DNA interaction of the Escherichia coli cyclic AMP receptor protein (CRP) represents a typical example of a dual recognition mechanism exhibiting both direct and indirect readout. We have dissected the direct and indirect components of DNA recognition by CRP employing in vitro selection of a ...

  3. Opioid receptor activation triggering downregulation of cAMP improves effectiveness of anti-cancer drugs in treatment of glioblastoma

    Science.gov (United States)

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

    2014-01-01

    Glioblastoma are the most frequent and malignant human brain tumors, having a very poor prognosis. The enhanced radio- and chemoresistance of glioblastoma and the glioblastoma stem cells might be the main reason why conventional therapies fail. The second messenger cyclic AMP (cAMP) controls cell proliferation, differentiation, and apoptosis. Downregulation of cAMP sensitizes tumor cells for anti-cancer treatment. Opioid receptor agonists triggering opioid receptors can activate inhibitory Gi proteins, which, in turn, block adenylyl cyclase activity reducing cAMP. In this study, we show that downregulation of cAMP by opioid receptor activation improves the effectiveness of anti-cancer drugs in treatment of glioblastoma. The µ-opioid receptor agonist D,L-methadone sensitizes glioblastoma as well as the untreatable glioblastoma stem cells for doxorubicin-induced apoptosis and activation of apoptosis pathways by reversing deficient caspase activation and deficient downregulation of XIAP and Bcl-xL, playing critical roles in glioblastomas’ resistance. Blocking opioid receptors using the opioid receptor antagonist naloxone or increasing intracellular cAMP by 3-isobutyl-1-methylxanthine (IBMX) strongly reduced opioid receptor agonist-induced sensitization for doxorubicin. In addition, the opioid receptor agonist D,L-methadone increased doxorubicin uptake and decreased doxorubicin efflux, whereas doxorubicin increased opioid receptor expression in glioblastomas. Furthermore, opioid receptor activation using D,L-methadone inhibited tumor growth significantly in vivo. Our findings suggest that opioid receptor activation triggering downregulation of cAMP is a promising strategy to inhibit tumor growth and to improve the effectiveness of anti-cancer drugs in treatment of glioblastoma and in killing glioblastoma stem cells. PMID:24626197

  4. The modulation of cell surface cAMP receptors from Dictyostelium disscoideum by ammonium sulfate

    NARCIS (Netherlands)

    Haastert, Peter J.M. van

    1985-01-01

    Dictyostelium discoideum cells contain a heterogeneous population of cell surface cAMP receptors with components possessing different affinities (Kd between 15 and 450 nM) and different off-rates of the cAMP-receptor complex (t½ between 0.7 and 150 s). The association of cAMP to the receptor and the

  5. [Blockade of NMDA receptor enhances corticosterone-induced downregulation of brain-derived neurotrophic factor gene expression in the rat hippocampus through cAMP response element binding protein pathway].

    Science.gov (United States)

    Feng, Hao; Lu, Li-Min; Huang, Ying; Zhu, Yi-Chun; Yao, Tai

    2005-10-25

    High concentration of corticosterone leads to morphological and functional impairments in hippocampus, ranging from a reversible atrophy of pyramidal CA3 apical dendrites to the impairment of long-term potentiation (LTP) and hippocampus-dependent learning and memory. Glutamate and N-methyl-D-aspartate (NMDA) receptor play an important role in this effect. Because of the importance of brain-derived neurotrophic factor (BDNF) in the functions of the hippocampal neurons, alteration of the expression of BDNF is thought to be involved in the corticosterone effect on the hippocampus. To determine whether change in BDNF in the hippocampus is involved in the corticosterone effect, we injected corticosterone (2 mg/kg, s.c.) to Sprague-Dawley rats and measured the mRNA, proBDNF and mature BDNF protein levels in the hippocampus. We also measured the phosphorylation level of the transcription factor cAMP response element binding protein (CREB). Furthermore, we intraperitoneally injected NMDA receptor antagonist MK801 (0.1 mg/kg) 30 min before corticosterone administration to investigate whether and how MK801 affected the regulation of BDNF gene expression by corticosterone. Our results showed that 3 h after single s.c. injection of corticsterone, the expression of BDNF mRNA, proBDNF and mature BDNF protein decreased significantly (PBDNF gene expression in the rat hippocampus by corticosterone. We also found that either applying corticosterone or co-applying corticosterone with MK801 downregulated the phosphoration level of CREB, the latter (corticosterone plus MK801) being more effective (PBDNF gene expression in the rat hippocampus through CREB pathway and that blockade of NMDA receptor enhances this effect of corticosterone in reducing BDNF expression.

  6. Ca2+ - or Phorbol Ester - Dependent Effect of ATP on a Subpopulation of cAMP Cell-Surface Receptors in Membranes from D. discoideum. A Role for Protein Kinase C

    NARCIS (Netherlands)

    Haastert, Peter J.M. van; Wit, René J.W. de; Lookeren Campagne, Michiel M. van

    1985-01-01

    D. discoideum cells contain surface receptors for the chemoattractant cAMP which are composed of fast and slowly dissociating binding sites with half-lifes of respectively about 1 s and 15 s. In membranes prepared by shearing the cells through a Nucleopore filter, ATP has no effect on cAMP-binding a

  7. -Adrenergic receptors on rat ventricular myocytes: characteristics and linkage to cAMP metabolism

    Energy Technology Data Exchange (ETDEWEB)

    Buxton, I.L.O.; Brunton, L.L.

    1986-08-01

    When incubated with purified cardiomyocytes from adult rat ventricle, the 1-antagonist (TH)prazosin binds to a single class of sites with high affinity. Competition for (TH)prazosin binding by the 2-selective antagonist yohimbine and the nonselective -antagonist phentolamine demonstrates that these receptors are of the 1-subtype. In addition, incubation of myocyte membranes with (TH)yohimbine results in no measurable specific binding. Agonist competition for (TH)prazosin binding to membranes prepared from purified myocytes demonstrates the presence of two components of binding: 28% of 1-receptors interact with norepinephrine with high affinity (K/sub D/ = 36 nM), whereas the majority of receptors (72%) have a low affinity for agonist (K/sub D/ = 2.2 M). After addition of 10 M GTP, norepinephrine competes for (TH)prazosin binding to a single class of sites with lower affinity (K/sub D/ = 2.2 M). Incubation of intact myocytes for 2 min with 1 M norepinephrine leads to significantly less cyclic AMP (cAMP) accumulation than stimulation with either norepinephrine plus prazosin or isoproterenol. Likewise, incubation of intact myocytes with 10 W M norepinephrine leads to significantly less activation of cAMP-dependent protein kinase than when myocytes are stimulated by both norepinephrine and the 1-adrenergic antagonist, prazosin or the US -adrenergic agonist, isoproterenol. They conclude that the cardiomyocyte 1 receptor is coupled to a guanine nucleotide-binding protein, that 1-receptors are functionally linked to decreased intracellular cAMP content, and that this change in cellular cAMP is expressed as described activation of cAMP-dependent protein kinase.

  8. Altered beta-adrenergic receptor-stimulated cAMP formation in cultured skin fibroblasts from Alzheimer donors.

    Science.gov (United States)

    Huang, H M; Gibson, G E

    1993-07-15

    An alteration in signal transduction systems in Alzheimer's disease would likely be of pathophysiological significance, because these steps are critical to normal brain function. Since dynamic processes are difficult to study in autopsied brain, the current studies utilized cultured skin fibroblasts. The beta-adrenergic-stimulated increase in cAMP was reduced approximately 80% in fibroblasts from Alzheimer's disease compared with age-matched controls. The deficit in Alzheimer fibroblasts in response to various adrenergic agonists paralleled their beta-adrenergic potency, and enhancement of cAMP accumulation by a non-adrenergic agonist, such as prostaglandin E1, was similar in Alzheimer and control fibroblasts. Diminished adenylate cyclase activity did not underlie these abnormalities, since direct stimulation of adenylate cyclase by forskolin elevated cAMP production equally in Alzheimer and control fibroblasts. Cholera toxin equally stimulated cAMP formation in Alzheimer and control fibroblasts. Moreover, cholera toxin partially reduced isoproterenol-induced cAMP deficit in Alzheimer fibroblasts. Pertussis toxin, on the other hand, did not alter the Alzheimer deficits. The results suggest either that the coupling of the GTP-binding protein(s) to the beta-adrenergic receptor is abnormal or that the sensitivity of receptor is altered with Alzheimer's disease. Further, any hypothesis about Alzheimer's disease must explain why a reduced beta-adrenergic-stimulated cAMP formation persists in tissue culture.

  9. The Cyclic Nucleotide Specificity of Three cAMP Receptors in Dictyostelium

    NARCIS (Netherlands)

    Johnson, Ronald L.; Haastert, Peter J.M. van; Kimmel, Alan R.; Saxe III, Charles L.; Jastorff, Bernd; Devreotes, Peter N.

    1992-01-01

    cAMP receptors mediate signal transduction pathways during development in Dictyostelium. A cAMP receptor (cAR1) has been cloned and sequenced (Klein, P., Sun, T. J., Saxe, C. L., Kimmel, A. R., Johnson, R. L., and Devreotes, P. N. (1988) Science 241, 1467-1472) and recently several other cAR genes h

  10. Exchange factors directly activated by cAMP mediate melanocortin 4 receptor-induced gene expression

    Science.gov (United States)

    Glas, Evi; Mückter, Harald; Gudermann, Thomas; Breit, Andreas

    2016-01-01

    Gs protein-coupled receptors regulate many vital body functions by activation of cAMP response elements (CRE) via cAMP-dependent kinase A (PKA)-mediated phosphorylation of the CRE binding protein (CREB). Melanocortin 4 receptors (MC4R) are prototypical Gs-coupled receptors that orchestrate the hypothalamic control of food-intake and metabolism. Remarkably, the significance of PKA for MC4R-induced CRE-dependent transcription in hypothalamic cells has not been rigorously interrogated yet. In two hypothalamic cell lines, we observed that blocking PKA activity had only weak or no effects on reporter gene expression. In contrast, inhibitors of exchange factors directly activated by cAMP-1/2 (EPAC-1/2) mitigated MC4R-induced CRE reporter activation and mRNA induction of the CREB-dependent genes c-fos and thyrotropin-releasing hormone. Furthermore, we provide first evidence that extracellular-regulated kinases-1/2 (ERK-1/2) activated by EPACs and not PKA are the elusive CREB kinases responsible for MC4R-induced CREB/CRE activation in hypothalamic cells. Overall, these data emphasize the pivotal role of EPACs rather than PKA in hypothalamic gene expression elicited by a prototypical Gs-coupled receptor. PMID:27612207

  11. Different role of cAMP dependent protein kinase and CaMKII in H3 receptor regulation of histamine synthesis and release.

    Science.gov (United States)

    Moreno-Delgado, D; Gómez-Ramírez, J; Torrent-Moreno, A; González-Sepúlveda, M; Blanco, I; Ortiz, J

    2009-12-15

    Histamine H(3) autoreceptors induce a negative feedback on histamine synthesis and release. While it is known that cAMP/cAMP dependent protein kinase (PKA) and Ca(2+)/CaMKII transduction pathways mediate H(3) effects on histamine synthesis, the pathways regulating neuronal histamine release are poorly known. Given the potential use of H(3) ligands in cognitive diseases, we have developed a technique for the determination of H(3) effects on histamine synthesis and release in brain cortical miniprisms. Potassium-induced depolarization effects were impaired by blockade of calcium entry through N and P/Q channels, as well as of CaMKII, but release was not affected by activators or inhibitors of the cAMP/PKA pathway (1-methyl-3-isobutylxanthine (IBMX), N6,2'-O-dibutyryladenosine 3',5'-cyclic monophosphate sodium salt (db-cAMP) or myristoyl PKA inhibitor peptide 14-22 (PKI(14-22)). In contrast, forskolin stimulated histamine release, although independently of PKA. Stimulation of histamine H(3) receptors with the agonist imetit markedly reduced the depolarization increase of histamine release, apparently through P/Q calcium channel inhibition. The H(3) antagonist/inverse agonist thioperamide modestly stimulated histamine release. Thioperamide effect on release was not modified by the PKA inhibitor PKI(14-22), but it was blocked by the CaMKII inhibitor KN-62. These results indicate that H(3) autoreceptors regulate neuronal histamine release (1) independently of the cAMP/PKA cascade, and (2) through modulation of calcium entry and CaMKII activation during depolarization.

  12. Dual role of cAMP and involvement of both G-proteins and ras in regulation of ERK2 in Dictyostelium discoideum.

    Science.gov (United States)

    Knetsch, M L; Epskamp, S J; Schenk, P W; Wang, Y; Segall, J E; Snaar-Jagalska, B E

    1996-07-01

    Dictyostelium discoideum expresses two Extracellular signal Regulated Kinases, ERK1 and ERK2, which are involved in growth, multicellular development and regulation of adenylyl cyclase. Binding of extracellular cAMP to cAMP receptor 1, a G-protein coupled cell surface receptor, transiently stimulates phosphorylation, activation and nuclear translocation of ERK2. Activation of ERK2 by cAMP is dependent on heterotrimeric G-proteins, since activation of ERK2 is absent in cells lacking the Galpha4 subunit. The small G-protein rasD also activates ERK2. In cells overexpressing a mutated, constitutively active rasD, ERK2 activity is elevated prior to cAMP stimulation. Intracellular cAMP and cAMP-dependent protein kinase (PKA) are essential for adaptation of the ERK2 response. This report shows that multiple signalling pathways are involved in regulation of ERK2 activity in D.discoideum.

  13. The RGS protein Crg2 regulates both pheromone and cAMP signalling in Cryptococcus neoformans.

    Science.gov (United States)

    Xue, Chaoyang; Hsueh, Yen-Ping; Chen, Lydia; Heitman, Joseph

    2008-10-01

    G proteins orchestrate critical cellular functions by transducing extracellular signals into internal signals and controlling cellular responses to environmental cues. G proteins typically function as switches that are activated by G protein-coupled receptors (GPCRs) and negatively controlled by regulator of G protein signalling (RGS) proteins. In the human fungal pathogen Cryptococcus neoformans, three G protein alpha subunits (Gpa1, Gpa2 and Gpa3) have been identified. In a previous study, we identified the RGS protein Crg2 involved in regulating the pheromone response pathway through Gpa2 and Gpa3. In this study, a role for Crg2 was established in the Gpa1-cAMP signalling pathway that governs mating and virulence. We show that Crg2 physically interacts with Gpa1 and crg2 mutations increase cAMP production. crg2 mutations also enhance mating filament hyphae production, but reduce cell-cell fusion and sporulation efficiency during mating. Although crg2 mutations and the Gpa1 dominant active allele GPA1(Q284L) enhanced melanin production under normally repressive conditions, virulence was attenuated in a murine model. We conclude that Crg2 participates in controlling both Gpa1-cAMP-virulence and pheromone-mating signalling cascades and hypothesize it may serve as a molecular interface between these two central signalling conduits.

  14. Novel mechanisms and signaling pathways of esophageal ulcer healing: the role of prostaglandin EP2 receptors, cAMP, and pCREB.

    Science.gov (United States)

    Ahluwalia, Amrita; Baatar, Dolgor; Jones, Michael K; Tarnawski, Andrzej S

    2014-09-15

    Clinical studies indicate that prostaglandins of E class (PGEs) may promote healing of tissue injury e.g., gastroduodenal and dermal ulcers. However, the precise roles of PGEs, their E-prostanoid (EP) receptors, signaling pathways including cAMP and cAMP response element-binding protein (CREB), and their relation to VEGF and angiogenesis in the tissue injury healing process remain unknown, forming the rationale for this study. Using an esophageal ulcer model in rats, we demonstrated that esophageal mucosa expresses predominantly EP2 receptors and that esophageal ulceration triggers an increase in expression of the EP2 receptor, activation of CREB (the downstream target of the cAMP signaling), and enhanced VEGF gene expression. Treatment of rats with misoprostol, a PGE1 analog capable of activating EP receptors, enhanced phosphorylation of CREB, stimulated VEGF expression and angiogenesis, and accelerated esophageal ulcer healing. In cultured human esophageal epithelial (HET-1A) cells, misoprostol increased intracellular cAMP levels (by 163-fold), induced phosphorylation of CREB, and stimulated VEGF expression. A cAMP analog (Sp-cAMP) mimicked, whereas an inhibitor of cAMP-dependent protein kinase A (Rp-cAMP) blocked, these effects of misoprostol. These results indicate that the EP2/cAMP/protein kinase A pathway mediates the stimulatory effect of PGEs on angiogenesis essential for tissue injury healing via the induction of CREB activity and VEGF expression.

  15. Inactivation of Multidrug Resistance Proteins Disrupts Both Cellular Extrusion and Intracellular Degradation of cAMP

    OpenAIRE

    Xie, Moses; Rich, Thomas C.; Scheitrum, Colleen; Conti, Marco; Richter, Wito

    2011-01-01

    In addition to xenobiotics and several other endogenous metabolites, multidrug-resistance proteins (MRPs) extrude the second-messenger cAMP from various cells. Pharmacological and/or genetic inactivation of MRPs has been shown to augment intracellular cAMP signaling, an effect assumed to be a direct consequence of the blockade of cAMP extrusion. Here we provide evidence that the augmented intracellular cAMP levels are not due exclusively to the prevention of cAMP efflux because MRP inactivati...

  16. Effect of electromagnetic field on cyclic adenosine monophosphate (cAMP) in a human mu-opioid receptor cell model.

    Science.gov (United States)

    Ross, Christina L; Teli, Thaleia; Harrison, Benjamin S

    2016-01-01

    During the cell communication process, endogenous and exogenous signaling affect normal as well as pathological developmental conditions. Exogenous influences such as extra-low-frequency electromagnetic field (EMF) have been shown to effect pain and inflammation by modulating G-protein receptors, down-regulating cyclooxygenase-2 activity, and affecting the calcium/calmodulin/nitric oxide pathway. Investigators have reported changes in opioid receptors and second messengers, such as cyclic adenosine monophosphate (cAMP), in opiate tolerance and dependence by showing how repeated exposure to morphine decreases adenylate cyclase activity causing cAMP to return to control levels in the tolerant state, and increase above control levels during withdrawal. Resonance responses to biological systems using exogenous EMF signals suggest that frequency response characteristics of the target can determine the EMF biological response. In our past research we found significant down regulation of inflammatory markers tumor necrosis factor alpha (TNF-α) and nuclear factor kappa B (NFκB) using 5 Hz EMF frequency. In this study cAMP was stimulated in Chinese Hamster Ovary (CHO) cells transfected with human mu-opioid receptors, then exposed to 5 Hz EMF, and outcomes were compared with morphine treatment. Results showed a 23% greater inhibition of cAMP-treating cells with EMF than with morphine. In order to test our results for frequency specific effects, we ran identical experiments using 13 Hz EMF, which produced results similar to controls. This study suggests the use of EMF as a complementary or alternative treatment to morphine that could both reduce pain and enhance patient quality of life without the side-effects of opiates.

  17. Human choriogonadotropin binds to a lutropin receptor with essentially no N-terminal extension and stimulates cAMP synthesis.

    Science.gov (United States)

    Ji, I H; Ji, T H

    1991-07-15

    The lutropin (LH) receptor, which belongs to the family of G-protein coupled receptors, consists of an extracellular hydrophilic N-terminal extension of 341 amino acids and a membrane-embedded C-terminal region of 333 amino acids. This C-terminal region comprises a short N terminus, seven transmembrane domains, three cytoplasmic loops, three exoplasmic loops, and a C terminus. Recently, it was reported that the N-terminal extension of the LH receptor alone or a naturally occurring variant LH receptor similar to the N-terminal extension is capable of binding the hormone with an affinity slightly higher than that of the native receptor. This finding raises a question as to whether the N-terminal extension represents the entire hormone binding site and, if so, how is hormone binding transduced to the activation of a G-protein? In an attempt to answer this important question, we have prepared truncated receptors containing an N-terminal extension as short as 10 amino acids. Surprisingly, the truncated receptors were not only capable of binding the hormone, albeit with low affinities, but also capable of stimulating cAMP synthesis. These results suggest a possibility that the hormone, at least in part, interacts with the membrane-embedded C-terminal region and modulates it to activate adenylate cyclase. The low hormone binding affinities of the truncated receptors taken together with high affinity hormone binding to the N-terminal extension of the LH receptor indicate the existence of two or more contact points between the receptor and the hormone.

  18. Role of exchange protein directly activated by cAMP (EPAC1) in breast cancer cell migration and apoptosis.

    Science.gov (United States)

    Kumar, Naveen; Gupta, Sonal; Dabral, Surbhi; Singh, Shailja; Sehrawat, Seema

    2017-02-16

    Despite the current progress in cancer research and therapy, breast cancer remains the leading cause of mortality among half a million women worldwide. Migration and invasion of cancer cells are associated with prevalent tumor metastasis as well as high mortality. Extensive studies have powerfully established the role of prototypic second messenger cAMP and its two ubiquitously expressed intracellular cAMP receptors namely the classic protein kinaseA/cAMP-dependent protein kinase (PKA) and the more recently discovered exchange protein directly activated by cAMP/cAMP-regulated guanine nucleotide exchange factor (EPAC/cAMP-GEF) in cell migration, cell cycle regulation, and cell death. Herein, we performed the analysis of the Cancer Genome Atlas (TCGA) dataset to evaluate the essential role of cAMP molecular network in breast cancer. We report that EPAC1, PKA, and AKAP9 along with other molecular partners are amplified in breast cancer patients, indicating the importance of this signaling network. To evaluate the functional role of few of these proteins, we used pharmacological modulators and analyzed their effect on cell migration and cell death in breast cancer cells. Hence, we report that inhibition of EPAC1 activity using pharmacological modulators leads to inhibition of cell migration and induces cell death. Additionally, we also observed that the inhibition of EPAC1 resulted in disruption of its association with the microtubule cytoskeleton and delocalization of AKAP9 from the centrosome as analyzed by in vitro imaging. Finally, this study suggests for the first time the mechanistic insights of mode of action of a primary cAMP-dependent sensor, Exchange protein activated by cAMP 1 (EPAC1), via its interaction with A-kinase anchoring protein 9 (AKAP9). This study provides a new cell signaling cAMP-EPAC1-AKAP9 direction to the development of additional biotherapeutics for breast cancer.

  19. Control of βAR- and N-methyl-D-aspartate (NMDA) Receptor-Dependent cAMP Dynamics in Hippocampal Neurons.

    Science.gov (United States)

    Chay, Andrew; Zamparo, Ilaria; Koschinski, Andreas; Zaccolo, Manuela; Blackwell, Kim T

    2016-02-01

    Norepinephrine, a neuromodulator that activates β-adrenergic receptors (βARs), facilitates learning and memory as well as the induction of synaptic plasticity in the hippocampus. Several forms of long-term potentiation (LTP) at the Schaffer collateral CA1 synapse require stimulation of both βARs and N-methyl-D-aspartate receptors (NMDARs). To understand the mechanisms mediating the interactions between βAR and NMDAR signaling pathways, we combined FRET imaging of cAMP in hippocampal neuron cultures with spatial mechanistic modeling of signaling pathways in the CA1 pyramidal neuron. Previous work implied that cAMP is synergistically produced in the presence of the βAR agonist isoproterenol and intracellular calcium. In contrast, we show that when application of isoproterenol precedes application of NMDA by several minutes, as is typical of βAR-facilitated LTP experiments, the average amplitude of the cAMP response to NMDA is attenuated compared with the response to NMDA alone. Models simulations suggest that, although the negative feedback loop formed by cAMP, cAMP-dependent protein kinase (PKA), and type 4 phosphodiesterase may be involved in attenuating the cAMP response to NMDA, it is insufficient to explain the range of experimental observations. Instead, attenuation of the cAMP response requires mechanisms upstream of adenylyl cyclase. Our model demonstrates that Gs-to-Gi switching due to PKA phosphorylation of βARs as well as Gi inhibition of type 1 adenylyl cyclase may underlie the experimental observations. This suggests that signaling by β-adrenergic receptors depends on temporal pattern of stimulation, and that switching may represent a novel mechanism for recruiting kinases involved in synaptic plasticity and memory.

  20. Control of βAR- and N-methyl-D-aspartate (NMDA Receptor-Dependent cAMP Dynamics in Hippocampal Neurons.

    Directory of Open Access Journals (Sweden)

    Andrew Chay

    2016-02-01

    Full Text Available Norepinephrine, a neuromodulator that activates β-adrenergic receptors (βARs, facilitates learning and memory as well as the induction of synaptic plasticity in the hippocampus. Several forms of long-term potentiation (LTP at the Schaffer collateral CA1 synapse require stimulation of both βARs and N-methyl-D-aspartate receptors (NMDARs. To understand the mechanisms mediating the interactions between βAR and NMDAR signaling pathways, we combined FRET imaging of cAMP in hippocampal neuron cultures with spatial mechanistic modeling of signaling pathways in the CA1 pyramidal neuron. Previous work implied that cAMP is synergistically produced in the presence of the βAR agonist isoproterenol and intracellular calcium. In contrast, we show that when application of isoproterenol precedes application of NMDA by several minutes, as is typical of βAR-facilitated LTP experiments, the average amplitude of the cAMP response to NMDA is attenuated compared with the response to NMDA alone. Models simulations suggest that, although the negative feedback loop formed by cAMP, cAMP-dependent protein kinase (PKA, and type 4 phosphodiesterase may be involved in attenuating the cAMP response to NMDA, it is insufficient to explain the range of experimental observations. Instead, attenuation of the cAMP response requires mechanisms upstream of adenylyl cyclase. Our model demonstrates that Gs-to-Gi switching due to PKA phosphorylation of βARs as well as Gi inhibition of type 1 adenylyl cyclase may underlie the experimental observations. This suggests that signaling by β-adrenergic receptors depends on temporal pattern of stimulation, and that switching may represent a novel mechanism for recruiting kinases involved in synaptic plasticity and memory.

  1. Effect of cAMP signaling on expression of glucocorticoid receptor, Bim and Bad in glucocorticoid-sensitive and resistant leukemic and multiple myeloma cells.

    Science.gov (United States)

    Dong, Hongli; Carlton, Michael E; Lerner, Adam; Epstein, Paul M

    2015-01-01

    Stimulation of cAMP signaling induces apoptosis in glucocorticoid-sensitive and resistant CEM leukemic and MM.1 multiple myeloma cell lines, and this effect is enhanced by dexamethasone in both glucocorticoid-sensitive cell types and in glucocorticoid-resistant CEM cells. Expression of the mRNA for the glucocorticoid receptor alpha (GR) promoters 1A3, 1B and 1C, expression of mRNA and protein for GR, and the BH3-only proapoptotic proteins, Bim and Bad, and the phosphorylation state of Bad were examined following stimulation of the cAMP and glucocorticoid signaling pathways. Expression levels of GR promoters were increased by cAMP and glucocorticoid signaling, but GR protein expression was little changed in CEM and decreased in MM.1 cells. Stimulation of these two signaling pathways induced Bim in CEM cells, induced Bad in MM.1 cells, and activated Bad, as indicated by its dephosphorylation on ser112, in both cell types. This study shows that leukemic and multiple myeloma cells, including those resistant to glucocorticoids, can be induced to undergo apoptosis by stimulating the cAMP signaling pathway, with enhancement by glucocorticoids, and the mechanism by which this occurs may be related to changes in Bim and Bad expression, and in all cases, to activation of Bad.

  2. cAMP signaling prevents podocyte apoptosis via activation of protein kinase A and mitochondrial fusion.

    Directory of Open Access Journals (Sweden)

    Xiaoying Li

    Full Text Available Our previous in vitro studies suggested that cyclic AMP (cAMP signaling prevents adriamycin (ADR and puromycin aminonucleoside (PAN-induced apoptosis in podocytes. As cAMP is an important second messenger and plays a key role in cell proliferation, differentiation and cytoskeleton formation via protein kinase A (PKA or exchange protein directly activated by cAMP (Epac pathways, we sought to determine the role of PKA or Epac signaling in cAMP-mediated protection of podocytes. In the ADR nephrosis model, we found that forskolin, a selective activator of adenylate cyclase, attenuated albuminuria and improved the expression of podocyte marker WT-1. When podocytes were treated with pCPT-cAMP (a selective cAMP/PKA activator, PKA activation was increased in a time-dependent manner and prevented PAN-induced podocyte loss and caspase 3 activation, as well as a reduction in mitochondrial membrane potential. We found that PAN and ADR resulted in a decrease in Mfn1 expression and mitochondrial fission in podocytes. pCPT-cAMP restored Mfn1 expression in puromycin or ADR-treated podocytes and induced Drp1 phosphorylation, as well as mitochondrial fusion. Treating podocytes with arachidonic acid resulted in mitochondrial fission, podocyte loss and cleaved caspase 3 production. Arachidonic acid abolished the protective effects of pCPT-cAMP on PAN-treated podocytes. Mdivi, a mitochondrial division inhibitor, prevented PAN-induced cleaved caspase 3 production in podocytes. We conclude that activation of cAMP alleviated murine podocyte caused by ADR. PKA signaling resulted in mitochondrial fusion in podocytes, which at least partially mediated the effects of cAMP.

  3. cAMP signaling prevents podocyte apoptosis via activation of protein kinase A and mitochondrial fusion.

    Science.gov (United States)

    Li, Xiaoying; Tao, Hua; Xie, Kewei; Ni, Zhaohui; Yan, Yucheng; Wei, Kai; Chuang, Peter Y; He, John Cijiang; Gu, Leyi

    2014-01-01

    Our previous in vitro studies suggested that cyclic AMP (cAMP) signaling prevents adriamycin (ADR) and puromycin aminonucleoside (PAN)-induced apoptosis in podocytes. As cAMP is an important second messenger and plays a key role in cell proliferation, differentiation and cytoskeleton formation via protein kinase A (PKA) or exchange protein directly activated by cAMP (Epac) pathways, we sought to determine the role of PKA or Epac signaling in cAMP-mediated protection of podocytes. In the ADR nephrosis model, we found that forskolin, a selective activator of adenylate cyclase, attenuated albuminuria and improved the expression of podocyte marker WT-1. When podocytes were treated with pCPT-cAMP (a selective cAMP/PKA activator), PKA activation was increased in a time-dependent manner and prevented PAN-induced podocyte loss and caspase 3 activation, as well as a reduction in mitochondrial membrane potential. We found that PAN and ADR resulted in a decrease in Mfn1 expression and mitochondrial fission in podocytes. pCPT-cAMP restored Mfn1 expression in puromycin or ADR-treated podocytes and induced Drp1 phosphorylation, as well as mitochondrial fusion. Treating podocytes with arachidonic acid resulted in mitochondrial fission, podocyte loss and cleaved caspase 3 production. Arachidonic acid abolished the protective effects of pCPT-cAMP on PAN-treated podocytes. Mdivi, a mitochondrial division inhibitor, prevented PAN-induced cleaved caspase 3 production in podocytes. We conclude that activation of cAMP alleviated murine podocyte caused by ADR. PKA signaling resulted in mitochondrial fusion in podocytes, which at least partially mediated the effects of cAMP.

  4. Antagonists of chemoattractants reveal separate receptors for cAMP, folic acid and pterin in Dictyostelium

    NARCIS (Netherlands)

    Haastert, Peter J.M. van; Wit, René J.W. de; Konijn, Theo M.

    1982-01-01

    Adenosine 3’,5’-monophosphate (cAMP), folic acid and pterin are chemoattractants in the cellular slime molds. The cAMP analog, 3’-amino-cAMP, inhibits a chemotactic reaction to cAMP at a concentration at which the analog is chemotactically inactive. The antagonistic effect of 3’-amino-cAMP on the ch

  5. The cyclase-associated protein CAP as regulator of cell polarity and cAMP signaling in Dictyostelium.

    Science.gov (United States)

    Noegel, Angelika A; Blau-Wasser, Rosemarie; Sultana, Hameeda; Müller, Rolf; Israel, Lars; Schleicher, Michael; Patel, Hitesh; Weijer, Cornelis J

    2004-02-01

    Cyclase-associated protein (CAP) is an evolutionarily conserved regulator of the G-actin/F-actin ratio and, in yeast, is involved in regulating the adenylyl cyclase activity. We show that cell polarization, F-actin organization, and phototaxis are altered in a Dictyostelium CAP knockout mutant. Furthermore, in complementation assays we determined the roles of the individual domains in signaling and regulation of the actin cytoskeleton. We studied in detail the adenylyl cyclase activity and found that the mutant cells have normal levels of the aggregation phase-specific adenylyl cyclase and that receptor-mediated activation is intact. However, cAMP relay that is responsible for the generation of propagating cAMP waves that control the chemotactic aggregation of starving Dictyostelium cells was altered, and the cAMP-induced cGMP production was significantly reduced. The data suggest an interaction of CAP with adenylyl cyclase in Dictyostelium and an influence on signaling pathways directly as well as through its function as a regulatory component of the cytoskeleton.

  6. PSD-95 regulates D1 dopamine receptor resensitization, but not receptor-mediated Gs-protein activation

    Institute of Scientific and Technical Information of China (English)

    Peihua Sun; Jingru Wang; Weihua Gu; Wei Cheng; Guo-zhang Jin; Eitan Friedman; Jie Zheng; Xuechu Zhen

    2009-01-01

    The present study aims to define the role of postsynaptic density (PSD)-95 in the regulation of dopamine (DA) receptor function. We found that PSD-95 physically associates with either D1 or D2 DA receptors in co-transfected HEK-293 cells. Stimulation of DA receptors altered the association between D1 receptor and PSD-95 in a time-depen-dent manner. Functional assays indicated that PSD-95 co-expression did not affect D1 receptor-stimulated cAMP pro-duction, Gs-protein activation or receptor desensitization. However, PSD-95 accelerated the recovery of internalized membrane receptors by promoting receptor recycling, thus resulting in enhanced resensitization of internalized D1 receptors. Our results provide a novel mechanism for regulating DA receptor recycling that may play an important role in postsynaptic DA functional modulation and synaptic neuroplasticity.

  7. Association of dopamine D(3) receptors with actin-binding protein 280 (ABP-280).

    Science.gov (United States)

    Li, Ming; Li, Chuanyu; Weingarten, Paul; Bunzow, James R; Grandy, David K; Zhou, Qun Yong

    2002-03-01

    Proteins that bind to G protein-coupled receptors have been identified as regulators of receptor localization and signaling. In our previous studies, a cytoskeletal protein, actin-binding protein 280 (ABP-280), was found to associate with the third cytoplasmic loop of dopamine D(2) receptors. In this study, we demonstrate that ABP-280 also interacts with dopamine D(3) receptors, but not with D(4) receptors. Similar to the dopamine D(2) receptor, the D(3)/ABP-280 association is of signaling importance. In human melanoma M2 cells lacking ABP-280, D(3) receptors were unable to inhibit forskolin-stimulated cyclic AMP (cAMP) production significantly. D(4) receptors, however, exhibited a similar degree of inhibition of forskolin-stimulated cAMP production in ABP-280-deficient M2 cells and ABP-280-replent M2 subclones (A7 cells). Further experiments revealed that the D(3)/ABP-280 interaction was critically dependent upon a 36 amino acid carboxyl domain of the D(3) receptor third loop, which is conserved in the D(2) receptor but not in the D(4) receptor. Our results demonstrate a subtype-specific regulation of dopamine D(2)-family receptor signaling by the cytoskeletal protein ABP-280.

  8. Autonomous and nonautonomous regulation of axis formation by antagonistic signaling via 7-span cAMP receptors and GSK3 in Dictyostelium.

    Science.gov (United States)

    Ginsburg, G T; Kimmel, A R

    1997-08-15

    Early during Dictyostelium development a fundamental cell-fate decision establishes the anteroposterior (prestalk/prespore) axis. Signaling via the 7-transmembrane cAMP receptor CAR4 is essential for creating and maintaining a normal pattern; car4-null alleles have decreased levels of prestalk-specific mRNAs but enhanced expression of prespore genes. car4- cells produce all of the signals required for prestalk differentiation but lack an extracellular factor necessary for prespore differentiation of wild-type cells. This secreted factor decreases the sensitivity of prespore cells to inhibition by the prestalk morphogen DIF-1. At the cell autonomous level, CAR4 is linked to intracellular circuits that activate prestalk but inhibit prespore differentiation. The autonomous action of CAR4 is antagonistic to the positive intracellular signals mediated by another cAMP receptor, CAR1 and/or CAR3. Additional data indicate that these CAR-mediated pathways converge at the serine/threonine protein kinase GSK3, suggesting that the anterior (prestalk)/posterior (prespore) axis of Dictyostelium is regulated by an ancient mechanism that is shared by the Wnt/Fz circuits for dorsoventral patterning during early Xenopus development and establishing Drosophila segment polarity.

  9. Camps 2.0: exploring the sequence and structure space of prokaryotic, eukaryotic, and viral membrane proteins.

    Science.gov (United States)

    Neumann, Sindy; Hartmann, Holger; Martin-Galiano, Antonio J; Fuchs, Angelika; Frishman, Dmitrij

    2012-03-01

    Structural bioinformatics of membrane proteins is still in its infancy, and the picture of their fold space is only beginning to emerge. Because only a handful of three-dimensional structures are available, sequence comparison and structure prediction remain the main tools for investigating sequence-structure relationships in membrane protein families. Here we present a comprehensive analysis of the structural families corresponding to α-helical membrane proteins with at least three transmembrane helices. The new version of our CAMPS database (CAMPS 2.0) covers nearly 1300 eukaryotic, prokaryotic, and viral genomes. Using an advanced classification procedure, which is based on high-order hidden Markov models and considers both sequence similarity as well as the number of transmembrane helices and loop lengths, we identified 1353 structurally homogeneous clusters roughly corresponding to membrane protein folds. Only 53 clusters are associated with experimentally determined three-dimensional structures, and for these clusters CAMPS is in reasonable agreement with structure-based classification approaches such as SCOP and CATH. We therefore estimate that ∼1300 structures would need to be determined to provide a sufficient structural coverage of polytopic membrane proteins. CAMPS 2.0 is available at http://webclu.bio.wzw.tum.de/CAMPS2.0/.

  10. G Protein-coupled Receptor Gpr4 Senses Amino Acids and Activates the cAMP-PKA Pathway in Cryptococcus neoformansD⃞

    OpenAIRE

    Xue, Chaoyang; Bahn, Yong-Sun; Cox, Gary M.; Heitman, Joseph

    2006-01-01

    The Gα protein Gpa1 governs the cAMP-PKA signaling pathway and plays a central role in virulence and differentiation in the human fungal pathogen Cryptococcus neoformans, but the signals and receptors that trigger this pathway were unknown. We identified seven putative proteins that share identity with known G protein-coupled receptors (GPCRs). One protein, Gpr4, shares limited sequence identity with the Dictyostelium discoideum cAMP receptor cAR1 and the Aspergillus nidulans GPCR protein Gpr...

  11. Control of Vibrio fischeri lux gene transcription by a cyclic AMP receptor protein-luxR protein regulatory circuit.

    OpenAIRE

    1988-01-01

    Expression of the Vibrio fischeri luminescence genes (lux genes) requires two transcriptional activators: the V. fischeri luxR gene product with autoinducer and the cyclic AMP (cAMP) receptor protein (CRP) with cAMP. It has been established that autoinducer and the luxR gene product are required for transcriptional activation of the luxICDABE operon, which contains a gene required for autoinducer synthesis and genes required for light emission. However, the role of cAMP-CRP in the induction o...

  12. The Role of Cgrp-Receptor Component Protein (Rcp in Cgrp-Mediated Signal Transduction

    Directory of Open Access Journals (Sweden)

    M. A. Prado

    2001-01-01

    Full Text Available The calcitonin gene-related peptide (CGRP-receptor component protein (RCP is a 17-kDa intracellular peripheral membrane protein required for signal transduction at CGRP receptors. To determine the role of RCP in CGRP-mediated signal transduction, RCP was depleted from NIH3T3 cells using antisense strategy. Loss of RCP protein correlated with loss of cAMP production by CGRP in the antisense cells. In contrast, loss of RCP had no effect on CGRP-mediated binding; therefore RCP is not acting as a chaperone for the CGRP receptor. Instead, RCP is a novel signal transduction molecule that couples the CGRP receptor to the cellular signal transduction machinery. RCP thus represents a prototype for a new class of signal transduction proteins that are required for regulation of G protein-coupled receptors.

  13. Hippocampal expression of synaptic structural proteins and phosphorylated cAMP response element-binding protein in a rat model of vascular dementia induced by chronic cerebral hypoperfusion

    Institute of Scientific and Technical Information of China (English)

    Hui Zhao; Zhiyong Li; Yali Wang; Qiuxia Zhang

    2012-01-01

    The present study established a rat model of vascular dementia induced by chronic cerebral hy-poperfusion through permanent ligation of bilateral common carotid arteries. At 60 days after mod-eling, escape latency and swimming path length during hidden-platform acquisition training in Morris water maze significantly increased in the model group. In addition, the number of accurate crossings over the original platform significantly decreased, hippocampal CA1 synaptophysin and growth-associated protein 43 expression significantly decreased, cAMP response element-binding protein expression remained unchanged, and phosphorylated cAMP response element-binding protein expression significantly decreased. Results suggested that abnormal expression of hippo-campal synaptic structural protein and cAMP response element-binding protein phosphorylation played a role in cognitive impairment following chronic cerebral hypoperfusion.

  14. Biological roles of cAMP: variations on a theme in the different kingdoms of life.

    Science.gov (United States)

    Gancedo, Juana M

    2013-08-01

    Cyclic AMP (cAMP) plays a key regulatory role in most types of cells; however, the pathways controlled by cAMP may present important differences between organisms and between tissues within a specific organism. Changes in cAMP levels are caused by multiple triggers, most affecting adenylyl cyclases, the enzymes that synthesize cAMP. Adenylyl cyclases form a large and diverse family including soluble forms and others with one or more transmembrane domains. Regulatory mechanisms for the soluble adenylyl cyclases involve either interaction with diverse proteins, as happens in Escherichia coli or yeasts, or with calcium or bicarbonate ions, as occurs in mammalian cells. The transmembrane cyclases can be regulated by a variety of proteins, among which the α subunit and the βγ complex from G proteins coupled to membrane receptors are prominent. cAMP levels also are controlled by the activity of phosphodiesterases, enzymes that hydrolyze cAMP. Phosphodiesterases can be regulated by cAMP, cGMP or calcium-calmodulin or by phosphorylation by different protein kinases. Regulation through cAMP depends on its binding to diverse proteins, its proximal targets, this in turn causing changes in a variety of distal targets. Specifically, binding of cAMP to regulatory subunits of cAMP-dependent protein kinases (PKAs) affects the activity of substrates of PKA, binding to exchange proteins directly activated by cAMP (Epac) regulates small GTPases, binding to transcription factors such as the cAMP receptor protein (CRP) or the virulence factor regulator (Vfr) modifies the rate of transcription of certain genes, while cAMP binding to ion channels modulates their activity directly. Further studies on cAMP signalling will have important implications, not only for advancing fundamental knowledge but also for identifying targets for the development of new therapeutic agents.

  15. Construction and characterization of the cAMP receptor protein gene deletion mutant of Salmonella typhimurium SL1344 strain%鼠伤寒沙门菌SL1344株cAMP受体蛋白基因缺失株的构建及其生物学特性

    Institute of Scientific and Technical Information of China (English)

    廖成水; 程相朝; 赵战勤; 张春杰; 李银聚; 吴庭才; 郁川; 王晓利; 胡阿勇

    2011-01-01

    The cAMP receptor protein gene(crp) deletion mutant of Salmonella typhimurium SL1344 strain was constructed by the allelic exchange introduced by the transduction of suicide plasmid.In addition,the biological characteristics of the mutant were determined.Firstly,the upstream and downstream fragments of crp gene were amplified from SL1344 strain genome.The two fragments were successively cloned into the suicide pRE112 vector to construct the recombinant suicide vector pREΔcrp harboring the 321 bp-deleted crp fragment.The recombination suicide vector was conjugated with SL1344 and the unmarked crp deleted strain without resistance was selected by two-step method and crp deletion on the genome was determined by PCR.The serotype of the mutant was 1,4,5,12:i:1,2,identical to the parent SL1344.The mutant was stable with the recombinant Δcrp gene in vitro.However,the carbohydrate fermentation or utilization assays of the mutant were differed from the parent SL1344 strain,obviously.The growth velocity of the mutant was more slowly compared with SL1344.The chicken lethal test showed that the virulence of the SL1344 Δcrp mutant strain with LD50 of 7.40×109 CFU was 32 456 times lower than the parent SL1344 strain with LD50 of 2.28×105 CFU.These results showed that the SL1344 Δcrp mutant was constructed successfully.It is likely that this Δcrp mutant could be adapted to develop attenuated Salmonella vaccine.%通过自杀性质粒介导的细菌同源重组技术,构建鼠伤寒沙门菌SL1344株的crp基因缺失疫苗候选菌株,并对其生物学特性进行初步研究。首先构建含缺失321bp crp基因的重组自杀性质粒pREΔcrp,然后利用重组自杀性质粒介导的等位基因交换技术,两步法筛选SL1344的Δcrp缺失株,用PCR鉴定结果表明该缺失株构建成功。生物学特性研究发现,缺失株ΔcrpSL1344保留了亲本菌株SL1344的血清型1,4,5,12:i:1,2,且能够稳定遗传缺失321bp的crp

  16. The Role of Cgrp-Receptor Component Protein (Rcp) in Cgrp-Mediated Signal Transduction

    OpenAIRE

    Prado, M.A.; B. Evans-Bain; Santi, S. L.; Dickerson, I M

    2001-01-01

    The calcitonin gene-related peptide (CGRP)-receptor component protein (RCP) is a 17-kDa intracellular peripheral membrane protein required for signal transduction at CGRP receptors. To determine the role of RCP in CGRP-mediated signal transduction, RCP was depleted from NIH3T3 cells using antisense strategy. Loss of RCP protein correlated with loss of cAMP production by CGRP in the antisense cells. In contrast, loss of RCP had no effect on CGRP-mediated binding; therefore RCP is not acting as...

  17. Protein intake during training sessions has no effect on performance and recovery during a strenuous training camp for elite cyclists

    OpenAIRE

    Hansen, Mette; Bangsbo, Jens; Jensen, Jørgen; Krause-Jensen, Matilde; Bibby, Bo Martin; Sollie, Ove; Hall, Ulrika Andersson; Madsen, Klavs

    2016-01-01

    BACKGROUND: Training camps for top-class endurance athletes place high physiological demands on the body. Focus on optimizing recovery between training sessions is necessary to minimize the risk of injuries and improve adaptations to the training stimuli. Carbohydrate supplementation during sessions is generally accepted as being beneficial to aid performance and recovery, whereas the effect of protein supplementation and timing is less well understood. We studied the effects of protein inges...

  18. Gene Expression Patterns Define Key Transcriptional Events InCell-Cycle Regulation By cAMP And Protein Kinase A

    Energy Technology Data Exchange (ETDEWEB)

    Zambon, Alexander C.; Zhang, Lingzhi; Minovitsky, Simon; Kanter, Joan R.; Prabhakar, Shyam; Salomonis, Nathan; Vranizan, Karen; Dubchak Inna,; Conklin, Bruce R.; Insel, Paul A.

    2005-06-01

    Although a substantial number of hormones and drugs increase cellular cAMP levels, the global impact of cAMP and its major effector mechanism, protein kinase A (PKA), on gene expression is not known. Here we show that treatment of murine wild-type S49 lymphoma cells for 24 h with 8-(4-chlorophenylthio)-cAMP (8-CPTcAMP), a PKA-selective cAMP analog, alters the expression of approx equal to 4,500 of approx. equal to 13,600 unique genes. By contrast, gene expression was unaltered in Kin- S49 cells (that lack PKA) incubated with 8-CPTcAMP. Changes in mRNA and protein expression of several cell cycle regulators accompanied cAMP-induced G1-phase cell-cycle arrest of wild-type S49 cells. Within 2h, 8-CPT-cAMP altered expression of 152 genes that contain evolutionarily conserved cAMP-response elements within 5 kb of transcriptional start sites, including the circadian clock gene Per1. Thus, cAMP through its activation of PKA produces extensive transcriptional regulation in eukaryotic cells. These transcriptional networks include a primary group of cAMP-response element-containing genes and secondary networks that include the circadian clock.

  19. Lipoic acid stimulates cAMP production via the EP2 and EP4 prostanoid receptors and inhibits IFN gamma synthesis and cellular cytotoxicity in NK cells.

    Science.gov (United States)

    Salinthone, Sonemany; Schillace, Robynn V; Marracci, Gail H; Bourdette, Dennis N; Carr, Daniel W

    2008-08-13

    The antioxidant lipoic acid (LA) treats and prevents the animal model of multiple sclerosis (MS), experimental autoimmune encephalomyelitis (EAE). In an effort to understand the therapeutic potential of LA in MS, we sought to define the cellular mechanisms that mediate the effects of LA on human natural killer (NK) cells, which are important in innate immunity as the first line of defense against invading pathogens and tumor cells. We discovered that LA stimulates cAMP production in NK cells in a dose-dependent manner. Studies using pharmacological inhibitors and receptor transfection experiments indicate that LA stimulates cAMP production via activation of the EP2 and EP4 prostanoid receptors and adenylyl cyclase. In addition, LA suppressed interleukin (IL)-12/IL-18 induced IFNgamma secretion and cytotoxicity in NK cells. These novel findings suggest that LA may inhibit NK cell function via the cAMP signaling pathway.

  20. Activation of protein kinase A and exchange protein directly activated by cAMP promotes adipocyte differentiation of human mesenchymal stem cells

    DEFF Research Database (Denmark)

    Jia, Bingbing; Madsen, Lise; Petersen, Rasmus Koefoed;

    2012-01-01

    Human mesenchymal stem cells are primary multipotent cells capable of differentiating into several cell types including adipocytes when cultured under defined in vitro conditions. In the present study we investigated the role of cAMP signaling and its downstream effectors, protein kinase A (PKA......) and exchange protein directly activated by cAMP (Epac) in adipocyte conversion of human mesenchymal stem cells derived from adipose tissue (hMADS). We show that cAMP signaling involving the simultaneous activation of both PKA- and Epac-dependent signaling is critical for this process even in the presence......(2)) may fully substitute for the cAMP-elevating agent isobutylmethylxanthine (IBMX). Moreover, selective activation of Epac-dependent signaling promoted adipocyte differentiation when the Rho-associated kinase (ROCK) was inhibited. Unlike the case for murine preadipocytes cell lines, long...

  1. Ex vivo study of 5-HT(1A) and 5-HT(7) receptor agonists and antagonists on cAMP accumulation during memory formation and amnesia.

    Science.gov (United States)

    Perez-García, G; Meneses, A

    2008-12-16

    The cyclic adenosine monophosphate (cAMP) is a second messenger and a central component of intracellular signaling pathways that regulate a wide range of biological functions, including memory. Hence, in this work, firstly the time-course of memory formation was determined in an autoshaping learning task, which had allowed the identification of testing times for increases or decreases in performance. Next, untrained, trained and overtrained groups were compared in cAMP production. Moreover, selective stimulation and antagonism of 5-HT(1A) and 5-HT(7) receptors during memory formation and cAMP production were determined. Finally, since there is scarce information about how pharmacological models of amnesia affect cAMP production, the cholinergic or glutamatergic antagonists, scopolamine and dizocilpine, were tested. The major findings of this work showed that when the time-course was determined inasmuch as training and testing sessions occurred, memory performance was graduate and progressive. Notably, for the fourth to seventh (i.e., 48-120 h following autoshaping training session) testing session performance was significantly higher from the previous ones. When animals received 5-HT(1A) and 5-HT(7) receptor agonists and antagonists or amnesic drugs significant increases or decrements in memory performance were observed at 24 and 48 h. Moreover, when ex vivo cAMP production from trained and overtrained groups were compared to untrained ones, significant differences were observed among groups and brain areas. Trained animals treated with 8-OHDPAT, AS19, 8-OHDPAT plus AS19, WAY100635, SB-269970, scopolamine or dizocilpine were compared to similar untrained groups, and eightfold-reduced cAMP production was evident, showing the importance of cAMP production in the signaling case in mammalian memory formation.

  2. Exchange Protein Directly Activated by cAMP (epac) : A Multidomain cAMP Mediator in the Regulation of Diverse Biological Functions

    NARCIS (Netherlands)

    Schmidt, Martina; Dekker, Frank J.; Maarsingh, Harm

    2013-01-01

    Since the discovery nearly 60 years ago, cAMP is envisioned as one of the most universal and versatile second messengers. The tremendous feature of cAMP to tightly control highly diverse physiologic processes, including calcium homeostasis, metabolism, secretion, muscle contraction, cell fate, and g

  3. Dynamic fluctuations provide the basis of a conformational switch mechanism in apo cyclic AMP receptor protein.

    Science.gov (United States)

    Aykaç Fas, Burcu; Tutar, Yusuf; Haliloğlu, Türkan

    2013-01-01

    Escherichia coli cyclic AMP Receptor Protein (CRP) undergoes conformational changes with cAMP binding and allosterically promotes CRP to bind specifically to the DNA. In that, the structural and dynamic properties of apo CRP prior to cAMP binding are of interest for the comprehension of the activation mechanism. Here, the dynamics of apo CRP monomer/dimer and holo CRP dimer were studied by Molecular Dynamics (MD) simulations and Gaussian Network Model (GNM). The interplay of the inter-domain hinge with the cAMP and DNA binding domains are pre-disposed in the apo state as a conformational switch in the CRP's allosteric communication mechanism. The hinge at L134-D138 displaying intra- and inter-subunit coupled fluctuations with the cAMP and DNA binding domains leads to the emergence of stronger coupled fluctuations between the two domains and describes an on state. The flexible regions at K52-E58, P154/D155 and I175 maintain the dynamic coupling of the two domains. With a shift in the inter-domain hinge position towards the N terminus, nevertheless, the latter correlations between the domains loosen and become disordered; L134-D138 dynamically interacts only with the cAMP and DNA binding domains of its own subunit, and an off state is assumed. We present a mechanistic view on how the structural dynamic units are hierarchically built for the allosteric functional mechanism; from apo CRP monomer to apo-to-holo CRP dimers.

  4. Principles and determinants of G-protein coupling by the rhodopsin-like thyrotropin receptor.

    Directory of Open Access Journals (Sweden)

    Gunnar Kleinau

    Full Text Available In this study we wanted to gain insights into selectivity mechanisms between G-protein-coupled receptors (GPCR and different subtypes of G-proteins. The thyrotropin receptor (TSHR binds G-proteins promiscuously and activates both Gs (cAMP and Gq (IP. Our goal was to dissect selectivity patterns for both pathways in the intracellular region of this receptor. We were particularly interested in the participation of poorly investigated receptor parts.We systematically investigated the amino acids of intracellular loop (ICL 1 and helix 8 using site-directed mutagenesis alongside characterization of cAMP and IP accumulation. This approach was guided by a homology model of activated TSHR in complex with heterotrimeric Gq, using the X-ray structure of opsin with a bound G-protein peptide as a structural template.We provide evidence that ICL1 is significantly involved in G-protein activation and our model suggests potential interactions with subunits G alpha as well as G betagamma. Several amino acid substitutions impaired both IP and cAMP accumulation. Moreover, we found a few residues in ICL1 (L440, T441, H443 and helix 8 (R687 that are sensitive for Gq but not for Gs activation. Conversely, not even one residue was found that selectively affects cAMP accumulation only. Together with our previous mutagenesis data on ICL2 and ICL3 we provide here the first systematically completed map of potential interfaces between TSHR and heterotrimeric G-protein. The TSHR/Gq-heterotrimer complex is characterized by more selective interactions than the TSHR/Gs complex. In fact the receptor interface for binding Gs is a subset of that for Gq and we postulate that this may be true for other GPCRs coupling these G-proteins. Our findings support that G-protein coupling and preference is dominated by specific structural features at the intracellular region of the activated GPCR but is completed by additional complementary recognition patterns between receptor and G-protein

  5. The Identification of Novel Protein-Protein Interactions in Liver that Affect Glucagon Receptor Activity.

    Directory of Open Access Journals (Sweden)

    Junfeng Han

    Full Text Available Glucagon regulates glucose homeostasis by controlling glycogenolysis and gluconeogenesis in the liver. Exaggerated and dysregulated glucagon secretion can exacerbate hyperglycemia contributing to type 2 diabetes (T2D. Thus, it is important to understand how glucagon receptor (GCGR activity and signaling is controlled in hepatocytes. To better understand this, we sought to identify proteins that interact with the GCGR to affect ligand-dependent receptor activation. A Flag-tagged human GCGR was recombinantly expressed in Chinese hamster ovary (CHO cells, and GCGR complexes were isolated by affinity purification (AP. Complexes were then analyzed by mass spectrometry (MS, and protein-GCGR interactions were validated by co-immunoprecipitation (Co-IP and Western blot. This was followed by studies in primary hepatocytes to assess the effects of each interactor on glucagon-dependent glucose production and intracellular cAMP accumulation, and then in immortalized CHO and liver cell lines to further examine cell signaling. Thirty-three unique interactors were identified from the AP-MS screening of GCGR expressing CHO cells in both glucagon liganded and unliganded states. These studies revealed a particularly robust interaction between GCGR and 5 proteins, further validated by Co-IP, Western blot and qPCR. Overexpression of selected interactors in mouse hepatocytes indicated that two interactors, LDLR and TMED2, significantly enhanced glucagon-stimulated glucose production, while YWHAB inhibited glucose production. This was mirrored with glucagon-stimulated cAMP production, with LDLR and TMED2 enhancing and YWHAB inhibiting cAMP accumulation. To further link these interactors to glucose production, key gluconeogenic genes were assessed. Both LDLR and TMED2 stimulated while YWHAB inhibited PEPCK and G6Pase gene expression. In the present study, we have probed the GCGR interactome and found three novel GCGR interactors that control glucagon

  6. Mlc is a transcriptional activator with a key role in integrating cyclic AMP receptor protein and integration host factor regulation of leukotoxin RNA synthesis in Aggregatibacter actinomycetemcomitans

    Science.gov (United States)

    Aggregatibacter actinomycetemcomitans, a periodontal pathogen, synthesizes leukotoxin (LtxA), a protein that helps the bacterium evade the host immune response. Transcription of the ltxA operon is induced during anaerobic growth. The cAMP receptor protein (CRP) indirectly increases ltxA expression...

  7. Inhibitory effect of luteolin on the odorant-induced cAMP level in HEK293 cells expressing the olfactory receptor.

    Science.gov (United States)

    Yoon, Yeo Cho; Hwang, Jin-Teak; Sung, Mi-Jeong; Wang, Shuaiyu; Munkhtugs, Davaatseren; Rhyu, Mee-Ra; Park, Jae-Ho

    2012-01-01

    Luteolin is a flavonoid in many fruits and vegetables. Although luteolin has important biological functions, including antioxidant, anti-inflammatory, antimicrobial, and neuroprotective activities, little is known about the functions of luteolin in the olfactory system. Various odorants can be detected and distinguished by using several molecular processes, including the binding of odorants to odorant receptors, activation of adenylyl cyclase (AC), changes of cyclic adenosine monophosphate (cAMP) and Ca(2+) levels in olfactory sensory neurons, as well as changes in membrane potentials and the transmission of electric signals to the brain. Because AC-cAMP signal transduction plays a pivotal role in the olfactory system, we evaluated the effects of luteolin on the AC-cAMP pathway that had been stimulated by the odorant eugenol. We demonstrated that eugenol caused an upregulation of the cAMP level and the phosphorylation of phosphokinase A (PKA, a downstream target of cAMP) in human embryonic kidney 293 (HEK293) cells expressing the murine eugenol receptor. This upregulation significantly decreased in the presence of luteolin, suggesting that luteolin inhibited the odorant-induced production of cAMP and affected the downstream phosphorylation of PKA.

  8. Calcium regulates motility and protein phosphorylation by changing cAMP and ATP concentrations in boar sperm in vitro.

    Science.gov (United States)

    Li, Xinhong; Wang, Lirui; Li, Yuhua; Zhao, Na; Zhen, Linqing; Fu, Jieli; Yang, Qiangzhen

    2016-09-01

    Considering the importance of calcium (Ca(2+)) in regulating sperm capacitation, hyperactivation and acrosome reaction, little is known about the molecular mechanism of action of this ion in this process. In the present study, assessment of the molecular mechanism from the perspective of energy metabolism occurred. Sperm motility variables were determined using computer-assisted sperm analysis (CASA) and the phosphorylation of PKA substrates, tyrosine residues and AMP-activated protein kinase (AMPK) were analyzed by Western blot. Moreover, intracellular sperm-specific glyceraldehyde 3-phosphatedehydrogenase (GAPDH) activity, 3'-5'-cyclic adenosine monophosphate (cAMP) and adenosine 5'-triphosphate (ATP) concentrations were assessed in boar sperm treated with Ca(2+). Results of the present study indicated that, under greater extracellular Ca(2+)concentrations (≥3.0mM), sperm motility and protein phosphorylation were inhibited. Interestingly, these changes were correlated with that of GAPDH activity, AMPK phosphorylation, cAMP and ATP concentrations. The negative effects of Ca(2+) on these intracellular processes were attenuated by addition of the calmodulin (CaM) inhibitor W7 and the inhibitor of calmodulin-dependent protein kinase (CaMK), KN-93. In the presence of greater extracellular Ca(2+), however, the phosphorylation pathway was suppressed by H-89. Taken together, these results suggested that Ca(2+) had a dual role in regulating boar sperm motility and protein phosphorylation due to the changes of cAMP and ATP concentrations, in response to cAMP-mediated signal transduction and the Ca(2+) signaling cascade. The present study provided some novel insights into the molecular mechanism underlying the effects of Ca(2+) on boar sperm as well as the involvement of energy metabolism in this mechanism.

  9. Novel receptors for bacterial protein toxins.

    Science.gov (United States)

    Schmidt, Gudula; Papatheodorou, Panagiotis; Aktories, Klaus

    2015-02-01

    While bacterial effectors are often directly introduced into eukaryotic target cells by various types of injection machines, toxins enter the cytosol of host cells from endosomal compartments or after retrograde transport via Golgi from the ER. A first crucial step of toxin-host interaction is receptor binding. Using optimized protocols and new methods novel toxin receptors have been identified, including metalloprotease ADAM 10 for Staphylococcus aureus α-toxin, laminin receptor Lu/BCAM for Escherichia coli cytotoxic necrotizing factor CNF1, lipolysis stimulated lipoprotein receptor (LSR) for Clostridium difficile transferase CDT and low-density lipoprotein receptor-related protein (LRP) 1 for Clostridium perfringens TpeL toxin.

  10. Protein Connectivity in Chemotaxis Receptor Complexes.

    Directory of Open Access Journals (Sweden)

    Stephan Eismann

    2015-12-01

    Full Text Available The chemotaxis sensory system allows bacteria such as Escherichia coli to swim towards nutrients and away from repellents. The underlying pathway is remarkably sensitive in detecting chemical gradients over a wide range of ambient concentrations. Interactions among receptors, which are predominantly clustered at the cell poles, are crucial to this sensitivity. Although it has been suggested that the kinase CheA and the adapter protein CheW are integral for receptor connectivity, the exact coupling mechanism remains unclear. Here, we present a statistical-mechanics approach to model the receptor linkage mechanism itself, building on nanodisc and electron cryotomography experiments. Specifically, we investigate how the sensing behavior of mixed receptor clusters is affected by variations in the expression levels of CheA and CheW at a constant receptor density in the membrane. Our model compares favorably with dose-response curves from in vivo Förster resonance energy transfer (FRET measurements, demonstrating that the receptor-methylation level has only minor effects on receptor cooperativity. Importantly, our model provides an explanation for the non-intuitive conclusion that the receptor cooperativity decreases with increasing levels of CheA, a core signaling protein associated with the receptors, whereas the receptor cooperativity increases with increasing levels of CheW, a key adapter protein. Finally, we propose an evolutionary advantage as explanation for the recently suggested CheW-only linker structures.

  11. Ontogeny of catecholamine and adenosine receptor-mediated cAMP signaling of embryonic red blood cells: role of cGMP-inhibited phosphodiesterase 3 and hemoglobin.

    Science.gov (United States)

    Baumann, R; Blass, C; Götz, R; Dragon, S

    1999-12-15

    We have previously shown that the cAMP signaling pathway controls major aspects of embryonic red blood cell (RBC) function in avian embryos (Glombitza et al, Am J Physiol 271:R973, 1996; and Dragon et al, Am J Physiol 271:R982, 1996) that are important for adaptation of the RBC gas transport properties to the progressive hypercapnia and hypoxia of later stages of avian embryonic development. Data about the ontogeny of receptor-mediated cAMP signaling are lacking. We have analyzed the response of primitive and definitive chick embryo RBC harvested from day 3 to 18 of development towards forskolin, beta-adrenergic, and A2 receptor agonists. The results show a strong response of immature definitive and primitive RBC to adenosine A2 and beta-adrenergic receptor agonists, which is drastically reduced in the last stage of development, coincident with the appearance of mature, transcriptionally inactive RBC. Modulation of cGMP-inhibited phosphodiesterase 3 (PDE3) has a controlling influence on cAMP accumulation in definitive RBC. Under physiological conditions, PDE3 is inhibited due to activation of soluble guanylyl cyclase (sGC). Inhibition of sGC with the specific inhibitor ODQ decreases receptor-mediated stimulation of cAMP production; this effect is reversed by the PDE3 inhibitor milrinone. sGC is acitivated by nitric oxide (NO), but we found no evidence for production of NO by erythrocyte NO-synthase. However, embryonic hemoglobin releases NO in an oxygen-linked manner that may activate guanylyl cyclase.

  12. Cyclic AMP-dependent protein kinase phosphorylation facilitates GABA(B) receptor-effector coupling.

    Science.gov (United States)

    Couve, A; Thomas, P; Calver, A R; Hirst, W D; Pangalos, M N; Walsh, F S; Smart, T G; Moss, S J

    2002-05-01

    GABA (gamma-aminobutyric acid)(B) receptors are heterodimeric G protein-coupled receptors that mediate slow synaptic inhibition in the central nervous system. Here we show that the functional coupling of GABA(B)R1/GABA(B)R2 receptors to inwardly rectifying K(+) channels rapidly desensitizes. This effect is alleviated after direct phosphorylation of a single serine residue (Ser892) in the cytoplasmic tail of GABA(B)R2 by cyclic AMP (cAMP)-dependent protein kinase (PKA). Basal phosphorylation of this residue is evident in rat brain membranes and in cultured neurons. Phosphorylation of Ser892 is modulated positively by pathways that elevate cAMP concentration, such as those involving forskolin and beta-adrenergic receptors. GABA(B) receptor agonists reduce receptor phosphorylation, which is consistent with PKA functioning in the control of GABA(B)-activated currents. Mechanistically, phosphorylation of Ser892 specifically enhances the membrane stability of GABA(B) receptors. We conclude that signaling pathways that activate PKA may have profound effects on GABA(B) receptor-mediated synaptic inhibition. These results also challenge the accepted view that phosphorylation is a universal negative modulator of G protein-coupled receptors.

  13. Blocking of Exchange Proteins Directly Activated by cAMP Leads to Reduced Replication of Middle East Respiratory Syndrome Coronavirus

    Science.gov (United States)

    Tao, Xinrong; Mei, Feng; Agrawal, Anurodh; Peters, Clarence J.; Ksiazek, Thomas G.

    2014-01-01

    The outbreak of Middle East respiratory syndrome coronavirus (MERS-CoV) infections and diseases represents a potential threat for worldwide spread and requires development of effective therapeutic strategies. In this study, we revealed a novel positive function of an exchange protein directly activated by cyclic AMP 1 (cAMP-1; Epac-1) on MERS-CoV replication. Specifically, we have shown that Epac-specific inhibitor treatment or silencing Epac-1 gene expression rendered cells resistant to viral infection. We believe Epac-1 inhibitors deserve further study as potential therapeutic agents for MERS-CoV infection. PMID:24453361

  14. The Popeye Domain Containing Genes and cAMP Signaling

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    Thomas Brand

    2014-05-01

    Full Text Available 3'-5'-cyclic adenosine monophosphate (cAMP is a second messenger, which plays an important role in the heart. It is generated in response to activation of G-protein-coupled receptors (GPCRs. Initially, it was thought that protein kinase A (PKA exclusively mediates cAMP-induced cellular responses such as an increase in cardiac contractility, relaxation, and heart rate. With the identification of the exchange factor directly activated by cAMP (EPAC and hyperpolarizing cyclic nucleotide-gated (HCN channels as cAMP effector proteins it became clear that a protein network is involved in cAMP signaling. The Popeye domain containing (Popdc genes encode yet another family of cAMP-binding proteins, which are prominently expressed in the heart. Loss-of-function mutations in mice are associated with cardiac arrhythmia and impaired skeletal muscle regeneration. Interestingly, the cardiac phenotype, which is present in both, Popdc1 and Popdc2 null mutants, is characterized by a stress-induced sinus bradycardia, suggesting that Popdc proteins participate in cAMP signaling in the sinuatrial node. The identification of the two-pore channel TREK-1 and Caveolin 3 as Popdc-interacting proteins represents a first step into understanding the mechanisms of heart rate modulation triggered by Popdc proteins.

  15. Gene transfer of a β2-adrenergic receptor kinase inhibitor up-regulates the level of β2-adrenergic receptor and cAMP in the asthmatic murine lung

    Institute of Scientific and Technical Information of China (English)

    Mao Huang; Yan Wu; Xin Yao; Wuangjian Cha; Kaisheng Yin

    2005-01-01

    Objective: To investigate the effects of gene transfer of a β-adrenergic receptor(β-AR) kinase inhibitor(β ARKct)on pulmonary β2-adrenergic receptor and cAMP following β2-AR agonist treatment in asthmatic mice, and to analyze the relationship between the routes of gene delivery and the changes of β2AR and cAMP. Methods: BALB/c mice were sensitized and challenged by ovalbumin to establish the asthmatic model treated with βAR agonist ( salbutamol injected intramuscularly). The plasmid with the expression of βARKct was constructed and βARKct gene transfer was performed through intravenous injection or intratracheal instillation in asthmatic mice.The gene expression was measured with Western blot analysis, and the changes of pulmonary β-AR and cAMP evaluated by Radioimmunoassay. Results: The expression of tranfered βARKct gene was detectable in lungs and it was expressed more in the lungs of the mice receiving intratracheally plasmid than those receiving intravenously. The levels of βAR and cAMP were upregulated after using plasmid-βARKct to the asthmatic mice treated with β AR agonist. Conclusion: Our results indicated that there were down-regulation of βAR and cAMP in asthmatic mice treated with βAR agonist. Gene transfer of βARKct could inhibit the extent of the down-regulation of βAR and cAMP. The route of gene delivery could also affect the degree of up-regulation of βAR and cAMP. Gene transfer βARKct may provide a novel approach to the therapeutic strategy for asthma.

  16. cAMP prevents TNF-induced apoptosis through inhibiting DISC complex formation in rat hepatocytes.

    Science.gov (United States)

    Bhattacharjee, Rajesh; Xiang, Wenpei; Wang, Yinna; Zhang, Xiaoying; Billiar, Timothy R

    2012-06-22

    Tumor necrosis factor α (TNF) is a pleiotropic proinflammatory cytokine that plays a role in immunity and the control of cell proliferation, cell differentiation, and apoptosis. The pleiotropic nature of TNF is due to the formation of different signaling complexes upon the binding of TNF to its receptor, TNF receptor type 1 (TNFR1). TNF induces apoptosis in various mammalian cells when the cells are co-treated with a transcription inhibitor like actinomycin D (ActD). When TNFR1 is activated, it recruits an adaptor protein, TNF receptor-associated protein with death domain (TRADD), through its cytoplasmic death effector domain (DED). TRADD, in turn, recruits other signaling proteins, including TNF receptor-associated protein 2 (TRAF2) and receptor-associated protein kinase (RIPK) 1, to form a complex. Subsequently, this complex combines with FADD and procaspase-8, converts into a death-inducing signaling complex (DISC) to induce apoptosis. Cyclic AMP (cAMP) is a second messenger that regulates various cellular processes such as cell proliferation, gene expression, and apoptosis. cAMP analogues are reported to act as anti-apoptotic agents in various cell types, including hepatocytes. We found that a cAMP analogue, dibutyryl cAMP (db-cAMP), inhibits TNF+ActD-induced apoptosis in rat hepatocytes. The protein kinase A (PKA) inhibitor KT-5720 reverses this inhibitory effect of cAMP on apoptosis. Cytoprotection by cAMP involves down-regulation of various apoptotic signal regulators like TRADD and FADD and inhibition of caspase-8 and caspase-3 cleavage. We also found that cAMP exerts its affect at the proximal level of TNF signaling by inhibiting the formation of the DISC complex upon the binding of TNF to TNFR1. In conclusion, our study shows that cAMP prevents TNF+ActD-induced apoptosis in rat hepatocytes by inhibiting DISC complex formation.

  17. The Combined Inhibitory Effect of the Adenosine A1 and Cannabinoid CB1 Receptors on cAMP Accumulation in the Hippocampus Is Additive and Independent of A1 Receptor Desensitization

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    André Serpa

    2015-01-01

    Full Text Available Adenosine A1 and cannabinoid CB1 receptors are highly expressed in hippocampus where they trigger similar transduction pathways. We investigated how the combined acute activation of A1 and CB1 receptors modulates cAMP accumulation in rat hippocampal slices. The CB1 agonist WIN55212-2 (0.3–30 μM decreased forskolin-stimulated cAMP accumulation with an EC50 of 6.6 ± 2.7 μM and an Emax⁡ of 31% ± 2%, whereas for the A1 agonist, N6-cyclopentyladenosine (CPA, 10–150 nM, an EC50 of 35 ± 19 nM, and an Emax⁡ of 29% ± 5 were obtained. The combined inhibitory effect of WIN55212-2 (30 μM and CPA (100 nM on cAMP accumulation was 41% ± 6% (n=4, which did not differ (P>0.7 from the sum of the individual effects of each agonist (43% ± 8% but was different (P<0.05 from the effects of CPA or WIN55212-2 alone. Preincubation with CPA (100 nM for 95 min caused desensitization of adenosine A1 activity, which did not modify the effect of WIN55212-2 (30 μM on cAMP accumulation. In conclusion, the combined effect of CB1 and A1 receptors on cAMP formation is additive and CB1 receptor activity is not affected by short-term A1 receptor desensitization.

  18. The combined inhibitory effect of the adenosine A1 and cannabinoid CB1 receptors on cAMP accumulation in the hippocampus is additive and independent of A1 receptor desensitization.

    Science.gov (United States)

    Serpa, André; Correia, Sara; Ribeiro, Joaquim A; Sebastião, Ana M; Cascalheira, José F

    2015-01-01

    Adenosine A1 and cannabinoid CB1 receptors are highly expressed in hippocampus where they trigger similar transduction pathways. We investigated how the combined acute activation of A1 and CB1 receptors modulates cAMP accumulation in rat hippocampal slices. The CB1 agonist WIN55212-2 (0.3-30 μM) decreased forskolin-stimulated cAMP accumulation with an EC50 of 6.6±2.7 μM and an Emax of 31%±2%, whereas for the A1 agonist, N6-cyclopentyladenosine (CPA, 10-150 nM), an EC50 of 35±19 nM, and an Emax of 29%±5 were obtained. The combined inhibitory effect of WIN55212-2 (30 μM) and CPA (100 nM) on cAMP accumulation was 41%±6% (n=4), which did not differ (P>0.7) from the sum of the individual effects of each agonist (43%±8%) but was different (Peffects of CPA or WIN55212-2 alone. Preincubation with CPA (100 nM) for 95 min caused desensitization of adenosine A1 activity, which did not modify the effect of WIN55212-2 (30 μM) on cAMP accumulation. In conclusion, the combined effect of CB1 and A1 receptors on cAMP formation is additive and CB1 receptor activity is not affected by short-term A1 receptor desensitization.

  19. Modulation of dopamine D(2) receptor signaling by actin-binding protein (ABP-280).

    Science.gov (United States)

    Li, M; Bermak, J C; Wang, Z W; Zhou, Q Y

    2000-03-01

    Proteins that bind to G protein-coupled receptors have recently been identified as regulators of receptor anchoring and signaling. In this study, actin-binding protein 280 (ABP-280), a widely expressed cytoskeleton-associated protein that plays an important role in regulating cell morphology and motility, was found to associate with the third cytoplasmic loop of dopamine D(2) receptors. The specificity of this interaction was originally identified in a yeast two-hybrid screen and confirmed by protein binding. The functional significance of the D(2) receptor-ABP-280 association was evaluated in human melanoma cells lacking ABP-280. D(2) receptor agonists were less potent in inhibiting forskolin-stimulated cAMP production in these cells. Maximal inhibitory responses of D(2) receptor activation were also reduced. Further yeast two-hybrid experiments showed that ABP-280 association is critically dependent on the carboxyl domain of the D(2) receptor third cytoplasmic loop, where there is a potential serine phosphorylation site (S358). Serine 358 was replaced with aspartic acid to mimic the effects of receptor phosphorylation. This mutant (D(2)S358D) displayed compromised binding to ABP-280 and coupling to adenylate cyclase. PKC activation also generated D(2) receptor signaling attenuation, but only in ABP-containing cells, suggesting a PKC regulatory role in D(2)-ABP association. A mechanism for these results may be derived from a role of ABP-280 in the clustering of D(2) receptors, as determined by immunocytochemical analysis in ABP-deficient and replete cells. Our results suggest a new molecular mechanism of modulating D(2) receptor signaling by cytoskeletal protein interaction.

  20. Effect of Increased Cyclic AMP Concentration on Muscle Protein Synthesis and Beta-Adrenergic Receptor Expression in Chicken Skeletal Muscle Cells in Culture

    Science.gov (United States)

    Young, R. B.; Vaughn, J. R.; Bridge, K. Y.; Smith, C. K.

    1998-01-01

    Analogies of epinephrine are known to cause hypertrophy of skeletal muscle when fed to animals. These compounds presumably exert their physiological action through interaction with the P-adrenergic receptor. Since the intracellular signal generated by the Beta-adrenergic receptor is cyclic AMP (cAMP), experiments were initiated in cell culture to determine if artificial elevation of cAMP by treatment with forskolin would alter muscle protein metabolism and P-adrenergic receptor expression. Chicken skeletal muscle cells after 7 days in culture were treated with 0.2-30 micrometers forskolin for a total of three days. At the end of the treatment period, both the concentration of cAMP and the quantity of myosin heavy chain (MHC) were measured. Concentration of cAMP in forskolin-treated cells increased up to 10-fold in a dose dependent manner. In contrast, the quantity of MHC was increased approximately 50% above control cells at 0.2 micrometers forskolin, but exhibited a gradual decline at higher levels of forskolin so that the quantity of MHC in cells treated with 30 micrometers forskolin was not significantly different from controls. Curiously, the intracellular concentration of cAMP which elicited the maximum increase in the quantity of MHC was only 40% higher than cAMP concentration in control cells.

  1. Methoxychlor and its metabolite HPTE inhibit cAMP production and expression of estrogen receptors α and β in the rat granulosa cell in vitro.

    Science.gov (United States)

    Harvey, Craig N; Chen, Joseph C; Bagnell, Carol A; Uzumcu, Mehmet

    2015-01-01

    The major metabolite of the estrogenic pesticide methoxychlor (MXC) HPTE is a stronger ESR1 agonist than MXC and acts also as an ESR2 antagonist. In granulosa cells (GCs), FSH stimulates estradiol via the second messenger cAMP. HPTE inhibits estradiol biosynthesis, and this effect is greater in FSH-treated GCs than in cAMP-treated GCs. Therefore; we examined the effect of MXC/HPTE on FSH-stimulated cAMP production in cultured GCs. To test involvement of ESR-signaling, we used the ESR1 and ESR2 antagonist ICI 182,780, ESR2 selective antagonist PHTPP, and ESR2 selective agonist DPN. ESR1 and ESR2 mRNA and protein levels were quantified. Both HPTE and MXC inhibited the FSH-induced cAMP production. ICI 182,780 and PHTPP mimicked the inhibitory action of HPTE. MXC/HPTE reduced FSH-stimulated Esr2 mRNA and protein to basal levels. MXC/HPTE also inhibited FSH-stimulated Esr1. The greater inhibition on FSH-stimulated GCs is likely due to reduced cAMP level that involves ESR-signaling, through ESR2.

  2. Chaperone receptors: guiding proteins to intracellular compartments.

    Science.gov (United States)

    Kriechbaumer, Verena; von Löffelholz, Ottilie; Abell, Ben M

    2012-01-01

    Despite mitochondria and chloroplasts having their own genome, 99% of mitochondrial proteins (Rehling et al., Nat Rev Mol Cell Biol 5:519-530, 2004) and more than 95% of chloroplast proteins (Soll, Curr Opin Plant Biol 5:529-535, 2002) are encoded by nuclear DNA, synthesised in the cytosol and imported post-translationally. Protein targeting to these organelles depends on cytosolic targeting factors, which bind to the precursor, and then interact with membrane receptors to deliver the precursor into a translocase. The molecular chaperones Hsp70 and Hsp90 have been widely implicated in protein targeting to mitochondria and chloroplasts, and receptors capable of recognising these chaperones have been identified at the surface of both these organelles (Schlegel et al., Mol Biol Evol 24:2763-2774, 2007). The role of these chaperone receptors is not fully understood, but they have been shown to increase the efficiency of protein targeting (Young et al., Cell 112:41-50, 2003; Qbadou et al., EMBO J 25:1836-1847, 2006). Whether these receptors contribute to the specificity of targeting is less clear. A class of chaperone receptors bearing tetratricopeptide repeat domains is able to specifically bind the highly conserved C terminus of Hsp70 and/or Hsp90. Interestingly, at least of one these chaperone receptors can be found on each organelle (Schlegel et al., Mol Biol Evol 24:2763-2774, 2007), which suggests a universal role in protein targeting for these chaperone receptors. This review will investigate the role that chaperone receptors play in targeting efficiency and specificity, as well as examining recent in silico approaches to find novel chaperone receptors.

  3. cAMP prevents TNF-induced apoptosis through inhibiting DISC complex formation in rat hepatocytes

    Energy Technology Data Exchange (ETDEWEB)

    Bhattacharjee, Rajesh [Department of Surgery, University of Pittsburgh Medical Center, Pittsburgh, PA 15213 (United States); Xiang, Wenpei [Department of Surgery, University of Pittsburgh Medical Center, Pittsburgh, PA 15213 (United States); Family Planning Research Institute, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, People' s Republic of China (China); Wang, Yinna [Vascular Medicine Institute, University of Pittsburgh School of Medicine, 10051-5A BST 3, 3501 Fifth Avenue, Pittsburgh, PA 15261 (United States); Zhang, Xiaoying [Department of Medicine/Endocrinology Division, University of Pittsburgh Medical Center, 200 Lothrop St., Pittsburgh, PA 15213 (United States); Billiar, Timothy R., E-mail: billiartr@upmc.edu [Department of Surgery, University of Pittsburgh Medical Center, Pittsburgh, PA 15213 (United States)

    2012-06-22

    Highlights: Black-Right-Pointing-Pointer cAMP blocks cell death induced by TNF and actinomycin D in cultured hepatocytes. Black-Right-Pointing-Pointer cAMP blocks NF-{kappa}B activation induced by TNF and actinomycin D. Black-Right-Pointing-Pointer cAMP blocks DISC formation following TNF and actinomycin D exposure. Black-Right-Pointing-Pointer cAMP blocks TNF signaling at a proximal step. -- Abstract: Tumor necrosis factor {alpha} (TNF) is a pleiotropic proinflammatory cytokine that plays a role in immunity and the control of cell proliferation, cell differentiation, and apoptosis. The pleiotropic nature of TNF is due to the formation of different signaling complexes upon the binding of TNF to its receptor, TNF receptor type 1 (TNFR1). TNF induces apoptosis in various mammalian cells when the cells are co-treated with a transcription inhibitor like actinomycin D (ActD). When TNFR1 is activated, it recruits an adaptor protein, TNF receptor-associated protein with death domain (TRADD), through its cytoplasmic death effector domain (DED). TRADD, in turn, recruits other signaling proteins, including TNF receptor-associated protein 2 (TRAF2) and receptor-associated protein kinase (RIPK) 1, to form a complex. Subsequently, this complex combines with FADD and procaspase-8, converts into a death-inducing signaling complex (DISC) to induce apoptosis. Cyclic AMP (cAMP) is a second messenger that regulates various cellular processes such as cell proliferation, gene expression, and apoptosis. cAMP analogues are reported to act as anti-apoptotic agents in various cell types, including hepatocytes. We found that a cAMP analogue, dibutyryl cAMP (db-cAMP), inhibits TNF + ActD-induced apoptosis in rat hepatocytes. The protein kinase A (PKA) inhibitor KT-5720 reverses this inhibitory effect of cAMP on apoptosis. Cytoprotection by cAMP involves down-regulation of various apoptotic signal regulators like TRADD and FADD and inhibition of caspase-8 and caspase-3 cleavage. We also found

  4. Relaxin Stimulates cAMP Production in MCF-7 Cells upon Overexpression of Type V Adenylyl Cyclase

    OpenAIRE

    Nguyen, Bao T.; Dessauer, Carmen W.

    2005-01-01

    Relaxin stimulates cAMP production and activation of ERK and PI3K in THP-1 cells. Relaxin also stimulates protein kinase C zeta (PKCζ) translocation to the plasma membrane in a PI3K-dependent manner in THP-1 and MCF-7 cells. However, relaxin did not increase cAMP production in MCF-7 cells. We overexpressed different adenylyl cyclase (AC) isoforms in MCF-7 cells to examine coupling of endogenous relaxin receptors to cAMP production. Overexpression of types II and IV AC had no effect on cAMP pr...

  5. Activation of protein kinase A and exchange protein directly activated by cAMP promotes adipocyte differentiation of human mesenchymal stem cells.

    Directory of Open Access Journals (Sweden)

    Bingbing Jia

    Full Text Available Human mesenchymal stem cells are primary multipotent cells capable of differentiating into several cell types including adipocytes when cultured under defined in vitro conditions. In the present study we investigated the role of cAMP signaling and its downstream effectors, protein kinase A (PKA and exchange protein directly activated by cAMP (Epac in adipocyte conversion of human mesenchymal stem cells derived from adipose tissue (hMADS. We show that cAMP signaling involving the simultaneous activation of both PKA- and Epac-dependent signaling is critical for this process even in the presence of the strong adipogenic inducers insulin, dexamethasone, and rosiglitazone, thereby clearly distinguishing the hMADS cells from murine preadipocytes cell lines, where rosiglitazone together with dexamethasone and insulin strongly promotes adipocyte differentiation. We further show that prostaglandin I(2 (PGI(2 may fully substitute for the cAMP-elevating agent isobutylmethylxanthine (IBMX. Moreover, selective activation of Epac-dependent signaling promoted adipocyte differentiation when the Rho-associated kinase (ROCK was inhibited. Unlike the case for murine preadipocytes cell lines, long-chain fatty acids, like arachidonic acid, did not promote adipocyte differentiation of hMADS cells in the absence of a PPARγ agonist. However, prolonged treatment with the synthetic PPARδ agonist L165041 promoted adipocyte differentiation of hMADS cells in the presence of IBMX. Taken together our results emphasize the need for cAMP signaling in concert with treatment with a PPARγ or PPARδ agonist to secure efficient adipocyte differentiation of human hMADS mesenchymal stem cells.

  6. Cyclic adenosine 3'-5'-monophosphate (cAMP) exerts proliferative and anti-proliferative effects in pituitary cells of different types by activating both cAMP-dependent protein kinase A (PKA) and exchange proteins directly activated by cAMP (Epac).

    Science.gov (United States)

    Vitali, E; Peverelli, E; Giardino, E; Locatelli, M; Lasio, G B; Beck-Peccoz, P; Spada, A; Lania, A G; Mantovani, G

    2014-03-05

    In the pituitary the activation of cyclic adenosine 3'-5'-monophosphate (cAMP) dependent pathways generates proliferative signals in somatotrophs, whereas in pituitary cells of other lineages its effect remains uncertain. Moreover, the specific role of the two main cAMP effectors, protein kinase A (PKA) and exchange proteins directly activated by cAMP (Epac), has not been defined. Aim of this study was to investigate the effect of cAMP on pituitary adenomatous cells proliferation and to identify PKA and Epac differential involvement. We found that cAMP increased DNA synthesis and cyclin D1 expression in somatotropinomas, whereas it reduced both parameters in prolactinomas and nonfunctioning adenomas, these effects being replicated in corresponding cell lines. Moreover, the divergent cAMP effects were mimicked by Epac and PKA analogs, which activated Rap1 and CREB, respectively. In conclusion, we demonstrated that cAMP exerted opposite effects on different pituitary cell types proliferation, these effects being mediated by both Epac and PKA.

  7. ABP: a novel AMPA receptor binding protein.

    Science.gov (United States)

    Srivastava, S; Ziff, E B

    1999-04-30

    We review the cloning of a novel AMPA receptor binding protein (ABP) that interacts with GluR2/3 and is homologous to GRIP. ABP is enriched in the PSD with GluR2 and is localized to the PSD by EM. ABP binds GluR2 via the C-terminal VXI motif through a Class I PDZ interaction. ABP and GRIP can also homo- and heteromultimerize. Thus, ABP and GRIP may be involved in AMPA receptor regulation and localization, by linking it to other cytoskeletal or signaling molecules. We suggest that the ABP/GRIP and PSD-95 families form distinct scaffolds that anchor, respectively, AMPA and NMDA receptors. We are currently investigating proteins that bind ABP and that may regulate the AMPA receptor.

  8. Hydrogen sulfide inhibits A2A adenosine receptor agonist induced β-amyloid production in SH-SY5Y neuroblastoma cells via a cAMP dependent pathway.

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    Bhushan Vijay Nagpure

    Full Text Available Alzheimer's disease (AD is the leading cause of senile dementia in today's society. Its debilitating symptoms are manifested by disturbances in many important brain functions, which are influenced by adenosine. Hence, adenosinergic system is considered as a potential therapeutic target in AD treatment. In the present study, we found that sodium hydrosulfide (NaHS, an H2S donor, 100 µM attenuated HENECA (a selective A2A receptor agonist, 10-200 nM induced β-amyloid (1-42 (Aβ42 production in SH-SY5Y cells. NaHS also interfered with HENECA-stimulated production and post-translational modification of amyloid precursor protein (APP by inhibiting its maturation. Measurement of the C-terminal APP fragments generated from its enzymatic cleavage by β-site amyloid precursor protein cleaving enzyme 1 (BACE1 showed that NaHS did not have any significant effect on β-secretase activity. However, the direct measurements of HENECA-elevated γ-secretase activity and mRNA expressions of presenilins suggested that the suppression of Aβ42 production in NaHS pretreated cells was mediated by inhibiting γ-secretase. NaHS induced reductions were accompanied by similar decreases in intracellular cAMP levels and phosphorylation of cAMP responsive element binding protein (CREB. NaHS significantly reduced the elevated cAMP and Aβ42 production caused by forskolin (an adenylyl cyclase, AC agonist alone or forskolin in combination with IBMX (a phosphodiesterase inhibitor, but had no effect on those caused by IBMX alone. Moreover, pretreatment with NaHS significantly attenuated HENECA-elevated AC activity and mRNA expressions of various AC isoforms. These data suggest that NaHS may preferentially suppress AC activity when it was stimulated. In conclusion, H2S attenuated HENECA induced Aβ42 production in SH-SY5Y neuroblastoma cells through inhibiting γ-secretase via a cAMP dependent pathway.

  9. G Protein-Coupled Receptors in Cancer

    OpenAIRE

    Rachel Bar-Shavit; Myriam Maoz; Arun Kancharla; Jeetendra Kumar Nag; Daniel Agranovich; Sorina Grisaru-Granovsky; Beatrice Uziely

    2016-01-01

    Despite the fact that G protein-coupled receptors (GPCRs) are the largest signal-conveying receptor family and mediate many physiological processes, their role in tumor biology is underappreciated. Numerous lines of evidence now associate GPCRs and their downstream signaling targets in cancer growth and development. Indeed, GPCRs control many features of tumorigenesis, including immune cell-mediated functions, proliferation, invasion and survival at the secondary site. Technological advances ...

  10. Dynamic fluctuations provide the basis of a conformational switch mechanism in apo cyclic AMP receptor protein.

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    Burcu Aykaç Fas

    Full Text Available Escherichia coli cyclic AMP Receptor Protein (CRP undergoes conformational changes with cAMP binding and allosterically promotes CRP to bind specifically to the DNA. In that, the structural and dynamic properties of apo CRP prior to cAMP binding are of interest for the comprehension of the activation mechanism. Here, the dynamics of apo CRP monomer/dimer and holo CRP dimer were studied by Molecular Dynamics (MD simulations and Gaussian Network Model (GNM. The interplay of the inter-domain hinge with the cAMP and DNA binding domains are pre-disposed in the apo state as a conformational switch in the CRP's allosteric communication mechanism. The hinge at L134-D138 displaying intra- and inter-subunit coupled fluctuations with the cAMP and DNA binding domains leads to the emergence of stronger coupled fluctuations between the two domains and describes an on state. The flexible regions at K52-E58, P154/D155 and I175 maintain the dynamic coupling of the two domains. With a shift in the inter-domain hinge position towards the N terminus, nevertheless, the latter correlations between the domains loosen and become disordered; L134-D138 dynamically interacts only with the cAMP and DNA binding domains of its own subunit, and an off state is assumed. We present a mechanistic view on how the structural dynamic units are hierarchically built for the allosteric functional mechanism; from apo CRP monomer to apo-to-holo CRP dimers.

  11. Chronic antidepressant administration increases the expression of cAMP response element binding protein (CREB) in rat hippocampus.

    Science.gov (United States)

    Nibuya, M; Nestler, E J; Duman, R S

    1996-04-01

    The present study demonstrates that chronic, but not acute, adminstration of several different classes of antidepressants, including serotonin- and norepinephrine-selective reuptake inhibitors, increases the expression of cAMP response element binding protein (CREB) mRNA in rat hippocampus. In contrast, chronic administration of several nonantidepressant psychotropic drugs did not influence expression of CREB mRNA, demonstrating the pharmacological specificity of this effect. In situ hybridization analysis demonstrates that antidepressant administration increases expression of CREB mRNA in CA1 and CA3 pyramidal and dentate gyrus granule cell layers of the hippocampus. In addition, levels of CRE immunoreactivity and of CRE binding activity were increased by chronic antidepressant administration, which indicates that expression and function of CREB protein are increased along with its mRNA. Chronic administration of the phosphodiesterase (PDE) inhibitors rolipram or papaverine also increased expression of CREB mRNA in hippocampus, demonstrating a role for the cAMP cascade. Moreover, coadministration of rolipram with imipramine resulted in a more rapid induction of CREB than with either treatment alone. Increased expression and function of CREB suggest that specific target genes may be regulated by these treatments. We have found that levels of brain-derived neurotrophic factor (BDNF) and trkB mRNA are also increased by administration of antidepressants or PDE inhibitors. These findings indicate that upregulation of CREB is a common action of chronic antidepressant treatments that may lead to regulation of specific target genes, such as BDNF and trkB, and to the long-term effects of these treatments on brain function.

  12. Revisiting cAMP signaling in the carotid body

    Directory of Open Access Journals (Sweden)

    Ana Rita eNunes

    2014-10-01

    Full Text Available Chronic carotid body (CB activation is now recognized as being essential in the development of hypertension and promoting insulin resistance; thus, it is imperative to characterize the chemotransduction mechanisms of this organ in order to modulate its activity and improve patient outcomes. For several years, and although controversial, cyclic adenosine monophosphate (cAMP was considered an important player in initiating the activation of the CB. However, its relevance was partially displaced in the 90s by the emerging role of the mitochondria and molecules such as AMP-activated protein kinase (AMPK and O2-sensitive K+ channels. Neurotransmitters/neuromodulators binding to metabotropic receptors are essential to chemotransmission in the CB, and cAMP is central to this process. cAMP also contributes to raise intracellular Ca2+ levels, and is intimately related to the cellular energetic status (AMP/ATP ratio. Furthermore, cAMP signaling is a target of multiple current pharmacological agents used in clinical practice. This review provides an outline on 1 the classical view of the cAMP-signaling pathway in the CB that originally supported its role in the O2/CO2 sensing mechanism, 2 present recent evidence on CB cAMP neuromodulation and 3 discuss how CB activity is affected by current clinical therapies that modify cAMP-signaling, namely dopaminergic drugs, caffeine (modulation of A2A/A2B receptors and roflumilast (PDE4 inhibitors. cAMP is key to any process that involves metabotropic receptors and the intracellular pathways involved in CB disease states are likely to involve this classical second messenger. Research examining the potential modification of cAMP levels and/or interactions with molecules associated with CB hyperactivity is currently in its beginning and this review will open doors for future explorations.

  13. The second intracellular loop of the human cannabinoid CB2 receptor governs G protein coupling in coordination with the carboxyl terminal domain.

    Directory of Open Access Journals (Sweden)

    Congxia Zheng

    Full Text Available The major effects of cannabinoids and endocannabinoids are mediated via two G protein-coupled receptors, CB1 and CB2, elucidation of the mechanism and structural determinants of the CB2 receptor coupling with G proteins will have a significant impact on drug discovery. In the present study, we systematically investigated the role of the intracellular loops in the interaction of the CB2 receptor with G proteins using chimeric receptors alongside the characterization of cAMP accumulation and ERK1/2 phosphorylation. We provided evidence that ICL2 was significantly involved in G protein coupling in coordination with the C-terminal end. Moreover, a single alanine substitution of the Pro-139 in the CB2 receptor that corresponds to Leu-222 in the CB1 receptor resulted in a moderate impairment in the inhibition of cAMP accumulation, whereas mutants P139F, P139M and P139L were able to couple to the Gs protein in a CRE-driven luciferase assay. With the ERK activation experiments, we further found that P139L has the ability to activate ERK through both Gi- and Gs-mediated pathways. Our findings defined an essential role of the second intracellular loop of the CB2 receptor in coordination with the C-terminal tail in G protein coupling and receptor activation.

  14. cAMP response element binding protein is required for differentiation of respiratory epithelium during murine development.

    Directory of Open Access Journals (Sweden)

    A Daniel Bird

    Full Text Available The cAMP response element binding protein 1 (Creb1 transcription factor regulates cellular gene expression in response to elevated levels of intracellular cAMP. Creb1(-/- fetal mice are phenotypically smaller than wildtype littermates, predominantly die in utero and do not survive after birth due to respiratory failure. We have further investigated the respiratory defect of Creb1(-/- fetal mice during development. Lungs of Creb1(-/- fetal mice were pale in colour and smaller than wildtype controls in proportion to their reduced body size. Creb1(-/- lungs also did not mature morphologically beyond E16.5 with little or no expansion of airway luminal spaces, a phenotype also observed with the Creb1(-/- lung on a Crem(-/- genetic background. Creb1 was highly expressed throughout the lung at all stages examined, however activation of Creb1 was detected primarily in distal lung epithelium. Cell differentiation of E17.5 Creb1(-/- lung distal epithelium was analysed by electron microscopy and showed markedly reduced numbers of type-I and type-II alveolar epithelial cells. Furthermore, immunomarkers for specific lineages of proximal epithelium including ciliated, non-ciliated (Clara, and neuroendocrine cells showed delayed onset of expression in the Creb1(-/- lung. Finally, gene expression analyses of the E17.5 Creb1(-/- lung using whole genome microarray and qPCR collectively identified respiratory marker gene profiles and provide potential novel Creb1-regulated genes. Together, these results demonstrate a crucial role for Creb1 activity for the development and differentiation of the conducting and distal lung epithelium.

  15. [Hemophilia camps.

    Science.gov (United States)

    Juárez-Sierra, Julieta; Del Pilar Torres-Arreola, Laura; Marín-Palomares, Teresa; Dueñas-González, María Teresa; Monteros-Rincón, Martha Patricia; Osorio-Guzmán, Maricela

    2013-01-01

    We reported the experience of hemophilia camps which was accomplished with patients from hospitals of the Instituto Mexicano del Seguro Social. The aim was to prepare the families and patients regarding the disease treatment, in order to promote the self sufficiency and to know the impact of the program on the course of the disease. Surveys were applied about treatment items and personal opinions were collected. The results of the national hemophilia camp were: group of 56 patients, average 14 years, 2 % women, 51 % severe hemophilia and 43 % had hemophilic brothers. Benefits: patients increased their knowledge about earlier bleeding identification and the self-infusion method; they became aware on their responsibility in self care, timely treatment and duties at home. Hemophilia camps with patients are an option for attitude change before disease complications. Social network creation and the increase in self-sufficiency are other benefits.

  16. Regulation of transient receptor potential channels of melastatin type 8 (TRPM8): effect of cAMP, cannabinoid CB(1) receptors and endovanilloids.

    Science.gov (United States)

    De Petrocellis, Luciano; Starowicz, Katarzyna; Moriello, Aniello Schiano; Vivese, Marta; Orlando, Pierangelo; Di Marzo, Vincenzo

    2007-05-15

    The transient receptor potential channel of melastatin type 8 (TRPM8), which is gated by low (<25 degrees C) temperature and chemical compounds, is regulated by protein kinase C-mediated phosphorylation in a way opposite to that observed with the transient receptor potential channel of vanilloid type 1 (TRPV1), i.e. by being desensitized and not sensitized. As TRPV1 is sensitized also by protein kinase A (PKA)-mediated phosphorylation, we investigated the effect of two activators of the PKA pathway, 8-Br-cAMP and forskolin, on the activity of menthol and icilin at TRPM8 in HEK-293 cells stably overexpressing the channel (TRPM8-HEK-293 cells). We also studied the effect on TRPM8 of: (1) a series of compounds previously shown to activate or antagonize TRPV1, and (2) co-stimulation of transiently co-expressed cannabinoid CB(1) receptors. Both 8-Br-cAMP (100 microM) and forskolin (10 microM) right-shifted the dose-response curves for the TRPM8-mediated effect of icilin and menthol on intracellular Ca(2+). The inhibitory effects of 8-Br-cAMP and forskolin were attenuated by the selective PKA inhibitor Rp-cAMP-S. Stimulation of human CB(1) receptors transiently co-expressed in TRPM8-HEK-293 cells also inhibited TRPM8 response to icilin. Finally, some TRPV1 agonists and antagonists, but not iodinated antagonists, antagonized icilin- and much less so menthol-, induced TRPM8 activation. Importantly, the endovanilloids/endocannabinoids, anandamide and NADA, also antagonized TRPM8 at submicromolar concentrations. Although these findings need to be confirmed by experiments directly measuring TRPM8 activity in natively TRPM8-expressing cells, they support the notion that the same regulatory events have opposing actions on TRPM8 and TRPV1 receptors and identify anandamide and NADA as the first potential endogenous functional antagonists of TRPM8 channels.

  17. The CytR repressor antagonizes cyclic AMP-cyclic AMP receptor protein activation of the deoCp2 promoter of Escherichia coli K-12

    DEFF Research Database (Denmark)

    Søgaard-Andersen, L; Martinussen, J; Møllegaard, N E;

    1990-01-01

    We have investigated the regulation of the Escherichia coli deoCp2 promoter by the CytR repressor and the cyclic AMP (cAMP) receptor protein (CRP) complexed to cAMP. Promoter regions controlled by these two proteins characteristically contain tandem cAMP-CRP binding sites. Here we show that (i) Cyt......R selectively regulated cAMP-CRP-dependent initiations, although transcription started from the same site in deoCp2 in the absence or presence of cAMP-CRP; (ii) deletion of the uppermost cAMP-CRP target (CRP-2) resulted in loss of CytR regulation, but had only a minor effect on positive control by the cAMP...

  18. The role of the CGRP-receptor component protein (RCP) in adrenomedullin receptor signal transduction.

    Science.gov (United States)

    Prado, M A; Evans-Bain, B; Oliver, K R; Dickerson, I M

    2001-11-01

    G protein-coupled receptors are usually thought to act as monomer receptors that bind ligand and then interact with G proteins to initiate signal transduction. In this study we report an intracellular peripheral membrane protein named the calcitonin gene-related peptide (CGRP)-receptor component protein (RCP) required for signal transduction at the G protein-coupled receptor for adrenomedullin. Cell lines were made that expressed an antisense construct of the RCP cDNA, and in these cells diminished RCP expression correlated with loss of adrenomedullin signal transduction. In contrast, loss of RCP did not diminish receptor density or affinity, therefore RCP does not appear to act as a chaperone protein. Instead, RCP represents a novel class of protein required to couple the adrenomedullin receptor to the cellular signal transduction pathway. A candidate adrenomedullin receptor named the calcitonin receptor-like receptor (CRLR) has been described, which forms high affinity adrenomedullin receptors when co-expressed with the accessory protein receptor-activity modifying protein 2 (RAMP2). RCP co-immunoprecipitated with CRLR and RAMP2, indicating that a functional adrenomedullin receptor is composed of at least three proteins: the ligand binding protein (CRLR), an accessory protein (RAMP2), and a coupling protein for signal transduction (RCP).

  19. Genetically-encoded tools for cAMP probing and modulation in living systems.

    Directory of Open Access Journals (Sweden)

    Valeriy M Paramonov

    2015-09-01

    Full Text Available Intracellular 3'-5'-cyclic adenosine monophosphate (cAMP is one of the principal second messengers downstream of a manifold of signal transduction pathways, including the ones triggered by G protein-coupled receptors. Not surprisingly, biochemical assays for cAMP have been instrumental for basic research and drug discovery for decades, providing insights into cellular physiology and guiding pharmaceutical industry. However, despite impressive track record, the majority of conventional biochemical tools for cAMP probing share the same fundamental shortcoming - all the measurements require sample disruption for cAMP liberation. This common bottleneck, together with inherently low spatial resolution of measurements (as cAMP is typically analyzed in lysates of thousands of cells, underpin the ensuing limitations of the conventional cAMP assays: 1 genuine kinetic measurements of cAMP levels over time in a single given sample are unfeasible; 2 inability to obtain precise information on cAMP spatial distribution and transfer at subcellular levels, let alone the attempts to pinpoint dynamic interactions of cAMP and its effectors. At the same time, tremendous progress in synthetic biology over the recent years culminated in drastic refinement of our toolbox, allowing us not only to bypass the limitations of conventional assays, but to put intracellular cAMP life-span under tight control – something, that seemed scarcely attainable before. In this review article we discuss the main classes of modern genetically-encoded tools tailored for cAMP probing and modulation in living systems. We examine the capabilities and weaknesses of these different tools in the context of their operational characteristics and applicability to various experimental set-ups involving living cells, providing the guidance for rational selection of the best tools for particular needs.

  20. Caldendrin-Jacob: a protein liaison that couples NMDA receptor signalling to the nucleus.

    Directory of Open Access Journals (Sweden)

    Daniela C Dieterich

    2008-02-01

    Full Text Available NMDA (N-methyl-D-aspartate receptors and calcium can exert multiple and very divergent effects within neuronal cells, thereby impacting opposing occurrences such as synaptic plasticity and neuronal degeneration. The neuronal Ca2+ sensor Caldendrin is a postsynaptic density component with high similarity to calmodulin. Jacob, a recently identified Caldendrin binding partner, is a novel protein abundantly expressed in limbic brain and cerebral cortex. Strictly depending upon activation of NMDA-type glutamate receptors, Jacob is recruited to neuronal nuclei, resulting in a rapid stripping of synaptic contacts and in a drastically altered morphology of the dendritic tree. Jacob's nuclear trafficking from distal dendrites crucially requires the classical Importin pathway. Caldendrin binds to Jacob's nuclear localization signal in a Ca2+-dependent manner, thereby controlling Jacob's extranuclear localization by competing with the binding of Importin-alpha to Jacob's nuclear localization signal. This competition requires sustained synapto-dendritic Ca2+ levels, which presumably cannot be achieved by activation of extrasynaptic NMDA receptors, but are confined to Ca2+ microdomains such as postsynaptic spines. Extrasynaptic NMDA receptors, as opposed to their synaptic counterparts, trigger the cAMP response element-binding protein (CREB shut-off pathway, and cell death. We found that nuclear knockdown of Jacob prevents CREB shut-off after extrasynaptic NMDA receptor activation, whereas its nuclear overexpression induces CREB shut-off without NMDA receptor stimulation. Importantly, nuclear knockdown of Jacob attenuates NMDA-induced loss of synaptic contacts, and neuronal degeneration. This defines a novel mechanism of synapse-to-nucleus communication via a synaptic Ca2+-sensor protein, which links the activity of NMDA receptors to nuclear signalling events involved in modelling synapto-dendritic input and NMDA receptor-induced cellular degeneration.

  1. Coordinated induction of GST and MRP2 by cAMP in Caco-2 cells: Role of protein kinase A signaling pathway and toxicological relevance

    Energy Technology Data Exchange (ETDEWEB)

    Arana, Maite Rocío, E-mail: arana@ifise-conicet.gov.ar [Instituto de Fisiología Experimental (CONICET), Facultad de Ciencias Bioquímicas y Farmacéuticas (UNR), Suipacha 570, 2000 Rosario (Argentina); Tocchetti, Guillermo Nicolás, E-mail: gtocchetti@live.com.ar [Instituto de Fisiología Experimental (CONICET), Facultad de Ciencias Bioquímicas y Farmacéuticas (UNR), Suipacha 570, 2000 Rosario (Argentina); Domizi, Pablo, E-mail: domizi@ibr-conicet.gov.ar [Instituto de Biología Molecular y Celular de Rosario (CONICET), Facultad de Ciencias Bioquímicas y Farmacéuticas (UNR), Suipacha 570, 2000 Rosario (Argentina); Arias, Agostina, E-mail: agoarias@yahoo.com.ar [Instituto de Fisiología Experimental (CONICET), Facultad de Ciencias Bioquímicas y Farmacéuticas (UNR), Suipacha 570, 2000 Rosario (Argentina); Rigalli, Juan Pablo, E-mail: jprigalli@gmail.com [Instituto de Fisiología Experimental (CONICET), Facultad de Ciencias Bioquímicas y Farmacéuticas (UNR), Suipacha 570, 2000 Rosario (Argentina); Ruiz, María Laura, E-mail: ruiz@ifise-conicet.gov.ar [Instituto de Fisiología Experimental (CONICET), Facultad de Ciencias Bioquímicas y Farmacéuticas (UNR), Suipacha 570, 2000 Rosario (Argentina); and others

    2015-09-01

    The cAMP pathway is a universal signaling pathway regulating many cellular processes including metabolic routes, growth and differentiation. However, its effects on xenobiotic biotransformation and transport systems are poorly characterized. The effect of cAMP on expression and activity of GST and MRP2 was evaluated in Caco-2 cells, a model of intestinal epithelium. Cells incubated with the cAMP permeable analog dibutyryl cyclic AMP (db-cAMP: 1,10,100 μM) for 48 h exhibited a dose–response increase in GST class α and MRP2 protein expression. Incubation with forskolin, an activator of adenylyl cyclase, confirmed the association between intracellular cAMP and upregulation of MRP2. Consistent with increased expression of GSTα and MRP2, db-cAMP enhanced their activities, as well as cytoprotection against the common substrate 1-chloro-2,4-dinitrobenzene. Pretreatment with protein kinase A (PKA) inhibitors totally abolished upregulation of MRP2 and GSTα induced by db-cAMP. In silico analysis together with experiments consisting of treatment with db-cAMP of Caco-2 cells transfected with a reporter construct containing CRE and AP-1 sites evidenced participation of these sites in MRP2 upregulation. Further studies involving the transcription factors CREB and AP-1 (c-JUN, c-FOS and ATF2) demonstrated increased levels of total c-JUN and phosphorylation of c-JUN and ATF2 by db-cAMP, which were suppressed by a PKA inhibitor. Co-immunoprecipitation and ChIP assay studies demonstrated that db-cAMP increased c-JUN/ATF2 interaction, with further recruitment to the region of the MRP2 promoter containing CRE and AP-1 sites. We conclude that cAMP induces GSTα and MRP2 expression and activity in Caco-2 cells via the PKA pathway, thus regulating detoxification of specific xenobiotics. - Highlights: • cAMP positively modulates the expression and activity of GST and MRP2 in Caco-2 cells. • Such induction resulted in increased cytoprotection against chemical injury. • PKA

  2. Hydrogen sulfide-mediated stimulation of mitochondrial electron transport involves inhibition of the mitochondrial phosphodiesterase 2A, elevation of cAMP and activation of protein kinase A.

    Science.gov (United States)

    Módis, Katalin; Panopoulos, Panagiotis; Coletta, Ciro; Papapetropoulos, Andreas; Szabo, Csaba

    2013-11-01

    Although hydrogen sulfide (H₂S) is generally known as a mitochondrial poison, recent studies show that lower concentrations of H₂S play a physiological role in the stimulation of mitochondrial electron transport and cellular bioenergetics. This effect involves electron donation at Complex II. Other lines of recent studies demonstrated that one of the biological actions of H₂S involves inhibition of cAMP and cGMP phosphodiesterases (PDEs). Given the emerging functional role of the mitochondrial isoform of cAMP PDE (PDE2A) in the regulation of mitochondrial function the current study investigated whether cAMP-dependent mechanisms participate in the stimulatory effect of NaHS on mitochondrial function. In isolated rat liver mitochondria, partial digestion studies localized PDE2A into the mitochondrial matrix. NaHS exerted a concentration-dependent inhibitory effect on recombinant PDE2A enzyme in vitro. Moreover, NaHS induced an elevation of cAMP levels when added to isolated mitochondria and stimulated the mitochondrial electron transport. The latter effect was inhibited by Rp-cAMP, an inhibitor of the cAMP-dependent protein kinase (PKA). The current findings suggest that the direct electron donating effect of NaHS is amplified by an intramitochondrial cAMP system, which may involve the inhibition of PDE2A and subsequent, cAMP-mediated stimulation of PKA.

  3. MicroRNA-181b targets cAMP responsive element binding protein 1 in gastric adenocarcinomas.

    Science.gov (United States)

    Chen, Lin; Yang, Qian; Kong, Wei-Qing; Liu, Tao; Liu, Min; Li, Xin; Tang, Hua

    2012-07-01

    MicroRNAs are a class of small endogenous non-coding RNAs that function as post-transcriptional regulators. In our previous study, we found that miR-181b was significantly downregulated in human gastric adenocarcinoma tissue samples compared to the adjacent normal gastric tissues. In this study, we confirm the down-regulation of miR-181b in human gastric cancer cell lines versus the gastric epithelial cells. Overexpression of miR-181b suppressed the proliferation and colony formation rate of gastric cancer cells. miR-181b downregulated the expression of cAMP responsive element binding protein 1 (CREB1) by binding its 3' untranslated region. Overexpression of CREB1 counteracted the suppression of growth in gastric cancer cells caused by ectopic expression of miR-181b. These results indicate that miR-181b may function as a tumor suppressor in gastric adenocarcinoma cells through negative regulation of CREB1.

  4. Dynamics of receptor and protein transducer homodimerisation

    Directory of Open Access Journals (Sweden)

    Kolch Walter

    2008-10-01

    Full Text Available Abstract Background Signalling pathways are complex systems in which not only simple monomeric molecules interact, but also more complex structures that include constitutive or induced protein assemblies. In particular, the hetero-and homo-dimerisation of proteins is a commonly encountered motif in signalling pathways. Several authors have suggested in recent times that dimerisation relates to a series of physical and biological outcomes used by the cell in the regulation of signal transduction. Results In this paper we investigate the role of homodimerisation in receptor-protein transducer interactions. Towards this end, mathematical modelling is used to analyse the features of such kind of interactions and to predict the behaviour of the system under different experimental conditions. A kinetic model in which the interaction between homodimers provokes a dual mechanism of activation (single and double protein transducer activation at the same time is proposed. In addition, we analyse under which conditions the use of a power-law representation for the system is useful. Furthermore, we investigate the dynamical consequences of this dual mechanism and compare the performance of the system in different simulated experimental conditions. Conclusion The analysis of our mathematical model suggests that in receptor-protein interacting systems with dual mechanism there may be a shift between double and single activation in a way that intense double protein transducer activation could initiate and dominate the signal in the short term (getting a fast intense signal, while single protein activation could control the system in the medium and long term (when input signal is weaker and decreases slowly. Our investigation suggests that homodimerisation and oligomerisation are mechanisms used to enhance and regulate the dynamic properties of the initial steps in signalling pathways.

  5. Protein kinase A and mitogen-activated protein kinase pathways mediate cAMP induction of alpha-epithelial Na+ channels (alpha-ENaC).

    Science.gov (United States)

    Mustafa, Shamimunisa B; Castro, Robert; Falck, Alison J; Petershack, Jean A; Henson, Barbara M; Mendoza, Yvonne M; Choudary, Ahsan; Seidner, Steven R

    2008-04-01

    A major mechanism for Na+ transport across epithelia occurs through epithelial Na+ channels (ENaC). ENaC is a multimeric channel consisting of three subunits (alpha, beta, and gamma). The alpha-subunit is critical for ENaC function. In specific culture conditions, the rat submandibular gland epithelial cell line (SMG-C6) demonstrates minimal Na+ transport properties and exposure to dibutyryl cAMP (DbcAMP) for up to 48 h caused an elevation of alpha-ENaC mRNA and protein expression and amiloride-sensitive short-circuit current (I(SC)). Here we examined the early signaling pathways evoked by DbcAMP which contribute to the eventual increase in Na+ transport is present. Treatment with either of the protein kinase A (PKA) inhibitors KT5720 or H-89 followed by exposure to 1 mM DbcAMP for 24 h markedly attenuated DbcAMP-induced alpha-ENaC protein formation and I(SC). Exposure of SMG-C6 cells to 1 mM DbcAMP induced a rapid, transient phosphorylation of the cAMP response element binding protein (CREB). This response was attenuated in the presence of either KT5720 or H-89. Dominant-negative CREB decreased DbcAMP-induced alpha-ENaC expression. Suppression of the extracellular signal-regulated protein kinase (ERK 1,2) with PD98059 or the p38 mitogen-activated protein kinase (MAPK) pathway with SB203580 reduced DbcAMP-induced alpha-ENaC protein levels in SMG-C6 cells. DbcAMP-induced phosphorylation of CREB was markedly attenuated by PD98059 or SB203580. DbcAMP-induced activation of the either the p38 or the ERK 1,2 MAPK pathways was abolished by either of the PKA inhibitors, H-89 or KT5720. Cross talk between these signaling pathways induced by DbcAMP via the activation of CREB appears to contribute to increased levels of alpha-ENaC observed after 24 h of treatment in SMG-C6 epithelial cells.

  6. Ligand- and cell-dependent determinants of internalization and cAMP modulation by delta opioid receptor (DOR) agonists.

    Science.gov (United States)

    Charfi, Iness; Nagi, Karim; Mnie-Filali, Ouissame; Thibault, Dominic; Balboni, Gianfranco; Schiller, Peter W; Trudeau, Louis-Eric; Pineyro, Graciela

    2014-04-01

    Signaling bias refers to G protein-coupled receptor ligand ability to preferentially activate one type of signal over another. Bias to evoke signaling as opposed to sequestration has been proposed as a predictor of opioid ligand potential for generating tolerance. Here we measured whether delta opioid receptor agonists preferentially inhibited cyclase activity over internalization in HEK cells. Efficacy (τ) and affinity (KA) values were estimated from functional data and bias was calculated from efficiency coefficients (log τ/KA). This approach better represented the data as compared to alternative methods that estimate bias exclusively from τ values. Log (τ/KA) coefficients indicated that SNC-80 and UFP-512 promoted cyclase inhibition more efficiently than DOR internalization as compared to DPDPE (bias factor for SNC-80: 50 and for UFP-512: 132). Molecular determinants of internalization were different in HEK293 cells and neurons with βarrs contributing to internalization in both cell types, while PKC and GRK2 activities were only involved in neurons. Rank orders of ligand ability to engage different internalization mechanisms in neurons were compared to rank order of E max values for cyclase assays in HEK cells. Comparison revealed a significant reversal in rank order for cyclase E max values and βarr-dependent internalization in neurons, indicating that these responses were ligand-specific. Despite this evidence, and because kinases involved in internalization were not the same across cellular backgrounds, it is not possible to assert if the magnitude and nature of bias revealed by rank orders of maximal responses is the same as the one measured in HEK cells.

  7. G Protein - Coupled Receptors [Receptores Acoplados à Proteína G

    OpenAIRE

    Lucas V. B. Hoelz; Guilherme B. L. de Freitas; Pedro Henrique M. Torres; Tácio Vinício A. Fernandes; Albuquerque, Magaly G.; Joaquim Fernando M. da Silva; Pedro G Pascutti; Ricardo B. de Alencastro

    2013-01-01

    The G protein-coupled receptors (GPCRs) constitute the largest superfamily of proteins encoded by the human genome. These receptors are membrane proteins which share a common structure of seven transmembrane helices and are involved in the cellular signal transduction through activation of heterotrimeric protein (G protein) in intracellular environment. This activation signal, mediated by the agonist binding to the extracellular domain of the receptor, is transmitted into the cell and activat...

  8. Activation of exchange protein activated by cAMP in the rat basolateral amygdala impairs reconsolidation of a memory associated with self-administered cocaine.

    Directory of Open Access Journals (Sweden)

    Xun Wan

    Full Text Available The intracellular mechanisms underlying memory reconsolidation critically involve cAMP signaling. These events were originally attributed to PKA activation by cAMP, but the identification of Exchange Protein Activated by cAMP (Epac, as a distinct mediator of cAMP signaling, suggests that cAMP-regulated processes that subserve memory reconsolidation are more complex. Here we investigated how activation of Epac with 8-pCPT-cAMP (8-CPT impacts reconsolidation of a memory that had been associated with cocaine self-administration. Rats were trained to lever press for cocaine on an FR-1 schedule, in which each cocaine delivery was paired with a tone+light cue. Lever pressing was then extinguished in the absence of cue presentations and cocaine delivery. Following the last day of extinction, rats were put in a novel context, in which the conditioned cue was presented to reactivate the cocaine-associated memory. Immediate bilateral infusions of 8-CPT into the basolateral amygdala (BLA following reactivation disrupted subsequent cue-induced reinstatement in a dose-dependent manner, and modestly reduced responding for conditioned reinforcement. When 8-CPT infusions were delayed for 3 hours after the cue reactivation session or were given after a cue extinction session, no effect on cue-induced reinstatement was observed. Co-administration of 8-CPT and the PKA activator 6-Bnz-cAMP (10 nmol/side rescued memory reconsolidation while 6-Bnz alone had no effect, suggesting an antagonizing interaction between the two cAMP signaling substrates. Taken together, these studies suggest that activation of Epac represents a parallel cAMP-dependent pathway that can inhibit reconsolidation of cocaine-cue memories and reduce the ability of the cue to produce reinstatement of cocaine-seeking behavior.

  9. The lactate receptor, G-protein-coupled receptor 81/hydroxycarboxylic acid receptor 1

    DEFF Research Database (Denmark)

    Morland, Cecilie; Lauritzen, Knut Huso; Puchades, Maja;

    2015-01-01

    We have proposed that lactate is a “volume transmitter” in the brain and underpinned this by showing that the lactate receptor, G-protein-coupled receptor 81 (GPR81, also known as HCA1 or HCAR1), which promotes lipid storage in adipocytes, is also active in the mammalian brain. This includes...... anion channels activated by depolarization. In addition to locally produced lactate, lactate produced by exercising muscle as well as exogenous HCAR1 agonists, e.g., from fruits and berries, might activate the receptor on cerebral blood vessels and brain cells....

  10. cAMP response element binding protein (CREB activates transcription via two distinct genetic elements of the human glucose-6-phosphatase gene

    Directory of Open Access Journals (Sweden)

    Stefano Luisa

    2005-01-01

    Full Text Available Abstract Background The enzyme glucose-6-phosphatase catalyzes the dephosphorylation of glucose-6-phosphatase to glucose, the final step in the gluconeogenic and glycogenolytic pathways. Expression of the glucose-6-phosphatase gene is induced by glucocorticoids and elevated levels of intracellular cAMP. The effect of cAMP in regulating glucose-6-phosphatase gene transcription was corroborated by the identification of two genetic motifs CRE1 and CRE2 in the human and murine glucose-6-phosphatase gene promoter that resemble cAMP response elements (CRE. Results The cAMP response element is a point of convergence for many extracellular and intracellular signals, including cAMP, calcium, and neurotrophins. The major CRE binding protein CREB, a member of the basic region leucine zipper (bZIP family of transcription factors, requires phosphorylation to become a biologically active transcriptional activator. Since unphosphorylated CREB is transcriptionally silent simple overexpression studies cannot be performed to test the biological role of CRE-like sequences of the glucose-6-phosphatase gene. The use of a constitutively active CREB2/CREB fusion protein allowed us to uncouple the investigation of target genes of CREB from the variety of signaling pathways that lead to an activation of CREB. Here, we show that this constitutively active CREB2/CREB fusion protein strikingly enhanced reporter gene transcription mediated by either CRE1 or CRE2 derived from the glucose-6-phosphatase gene. Likewise, reporter gene transcription was enhanced following expression of the catalytic subunit of cAMP-dependent protein kinase (PKA in the nucleus of transfected cells. In contrast, activating transcription factor 2 (ATF2, known to compete with CREB for binding to the canonical CRE sequence 5'-TGACGTCA-3', did not transactivate reporter genes containing CRE1, CRE2, or both CREs derived from the glucose-6-phosphatase gene. Conclusions Using a constitutively active CREB2

  11. The G protein-coupled receptor, class C, group 6, subtype A (GPRC6A) receptor

    DEFF Research Database (Denmark)

    Clemmensen, C; Smajilovic, S; Wellendorph, P;

    2014-01-01

    GPRC6A (G protein-coupled receptor, class C, group 6, subtype A) is a class C G protein-coupled receptor, that has been cloned from human, mouse and rat. Several groups have shown that the receptor is activated by a range of basic and small aliphatic L-α-amino acids of which L-arginine, L...

  12. The Popeye domain containing protein family – A novel class of cAMP effectors with important functions in multiple tissues

    Science.gov (United States)

    Schindler, Roland F.R.; Brand, Thomas

    2016-01-01

    Popeye domain containing (Popdc) proteins are a unique family, which combine several different properties and functions in a surprisingly complex fashion. They are expressed in multiple tissues and cell types, present in several subcellular compartments, interact with different classes of proteins, and are associated with a variety of physiological and pathophysiological processes. Moreover, Popdc proteins bind the second messenger cAMP with high affinity and it is thought that they act as a novel class of cAMP effector proteins. Here, we will review the most important findings about the Popdc family, which accumulated since its discovery about 15 years ago. We will be focussing on Popdc protein interaction and function in striated muscle tissue. However, as a full picture only emerges if all aspects are taken into account, we will also describe what is currently known about the role of Popdc proteins in epithelial cells and in various types of cancer, and discuss these findings with regard to their relevance for cardiac and skeletal muscle. PMID:26772438

  13. The Popeye domain containing protein family--A novel class of cAMP effectors with important functions in multiple tissues.

    Science.gov (United States)

    Schindler, Roland F R; Brand, Thomas

    2016-01-01

    Popeye domain containing (Popdc) proteins are a unique family, which combine several different properties and functions in a surprisingly complex fashion. They are expressed in multiple tissues and cell types, present in several subcellular compartments, interact with different classes of proteins, and are associated with a variety of physiological and pathophysiological processes. Moreover, Popdc proteins bind the second messenger cAMP with high affinity and it is thought that they act as a novel class of cAMP effector proteins. Here, we will review the most important findings about the Popdc family, which accumulated since its discovery about 15 years ago. We will be focussing on Popdc protein interaction and function in striated muscle tissue. However, as a full picture only emerges if all aspects are taken into account, we will also describe what is currently known about the role of Popdc proteins in epithelial cells and in various types of cancer, and discuss these findings with regard to their relevance for cardiac and skeletal muscle.

  14. Genetically-encoded yellow fluorescent cAMP indicator with an expanded dynamic range for dual-color imaging.

    Directory of Open Access Journals (Sweden)

    Haruki Odaka

    Full Text Available Cyclic AMP is a ubiquitous second messenger, which mediates many cellular responses mainly initiated by activation of cell surface receptors. Various Förster resonance energy transfer-based ratiometric cAMP indicators have been created for monitoring the spatial and temporal dynamics of cAMP at the single-cell level. However, single fluorescent protein-based cAMP indicators have been poorly developed, with improvement required for dynamic range and brightness. Based on our previous yellow fluorescent protein-based cAMP indicator, Flamindo, we developed an improved yellow fluorescent cAMP indicator named Flamindo2. Flamindo2 has a 2-fold expanded dynamic range and 8-fold increased brightness compared with Flamindo by optimization of linker peptides in the vicinity of the chromophore. We found that fluorescence intensity of Flamindo2 was decreased to 25% in response to cAMP. Live-cell cAMP imaging of the cytosol and nucleus in COS7 cells using Flamindo2 and nlsFlamindo2, respectively, showed that forskolin elevated cAMP levels in each compartment with different kinetics. Furthermore, dual-color imaging of cAMP and Ca2+ with Flamindo2 and a red fluorescent Ca2+ indicator, R-GECO, showed that cAMP and Ca2+ elevation were induced by noradrenaline in single HeLa cells. Our study shows that Flamindo2, which is feasible for multi-color imaging with other intracellular signaling molecules, is useful and is an alternative tool for live-cell imaging of intracellular cAMP dynamics.

  15. Genetically-Encoded Yellow Fluorescent cAMP Indicator with an Expanded Dynamic Range for Dual-Color Imaging

    Science.gov (United States)

    Odaka, Haruki; Arai, Satoshi; Inoue, Takafumi; Kitaguchi, Tetsuya

    2014-01-01

    Cyclic AMP is a ubiquitous second messenger, which mediates many cellular responses mainly initiated by activation of cell surface receptors. Various Förster resonance energy transfer-based ratiometric cAMP indicators have been created for monitoring the spatial and temporal dynamics of cAMP at the single-cell level. However, single fluorescent protein-based cAMP indicators have been poorly developed, with improvement required for dynamic range and brightness. Based on our previous yellow fluorescent protein-based cAMP indicator, Flamindo, we developed an improved yellow fluorescent cAMP indicator named Flamindo2. Flamindo2 has a 2-fold expanded dynamic range and 8-fold increased brightness compared with Flamindo by optimization of linker peptides in the vicinity of the chromophore. We found that fluorescence intensity of Flamindo2 was decreased to 25% in response to cAMP. Live-cell cAMP imaging of the cytosol and nucleus in COS7 cells using Flamindo2 and nlsFlamindo2, respectively, showed that forskolin elevated cAMP levels in each compartment with different kinetics. Furthermore, dual-color imaging of cAMP and Ca2+ with Flamindo2 and a red fluorescent Ca2+ indicator, R-GECO, showed that cAMP and Ca2+ elevation were induced by noradrenaline in single HeLa cells. Our study shows that Flamindo2, which is feasible for multi-color imaging with other intracellular signaling molecules, is useful and is an alternative tool for live-cell imaging of intracellular cAMP dynamics. PMID:24959857

  16. Structure-activity relationships of fatty acid amide ligands in activating and desensitizing G protein-coupled receptor 119.

    Science.gov (United States)

    Kumar, Pritesh; Kumar, Akhilesh; Song, Zhao-Hui

    2014-01-15

    The purpose of the current study was to apply a high throughput assay to investigate the structure-activity relationships of fatty acid amides for activating and desensitizing G protein-coupled receptor 119, a promising therapeutic target for both type 2 diabetes and obesity. A cell-based, homogenous time resolved fluorescence (HTRF) method for measuring G protein-coupled receptor 119-mediated increase of cyclic adenosine monophosphate (cAMP) levels was validated and applied in this study. Using novel fatty acid amides and detailed potency and efficacy analyses, we have demonstrated that degree of saturation in acyl chain and charged head groups of fatty acid amides have profound effects on the ability of these compounds to activate G protein-coupled receptor 119. In addition, we have demonstrated for the first time that pretreatments with G protein-coupled receptor 119 agonists desensitize the receptor and the degrees of desensitization caused by fatty acid amides correlate well with their structure-activity relationships in activating the receptor.

  17. Sodium depletion enhances renal expression of (pro)renin receptor via cyclic GMP-protein kinase G signaling pathway.

    Science.gov (United States)

    Huang, Jiqian; Siragy, Helmy M

    2012-02-01

    (Pro)renin receptor (PRR) is expressed in renal vasculature, glomeruli, and tubules. The physiological regulation of this receptor is not well established. We hypothesized that sodium depletion increases PRR expression through cGMP- protein kinase G (PKG) signaling pathway. Renal PRR expressions were evaluated in Sprague-Dawley rats on normal sodium or low-sodium diet (LS) and in cultured rat proximal tubular cells and mouse renal inner medullary collecting duct cells exposed to LS concentration. LS augmented PRR expression in renal glomeruli, proximal tubules, distal tubules, and collecting ducts. LS also increased cGMP production and PKG activity. In cells exposed to normal sodium, cGMP analog increased PKG activity and upregulated PRR expression. In cells exposed to LS, blockade of guanylyl cyclase with 1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one decreased PKG activity and downregulated PRR expression. PKG inhibition decreased phosphatase protein phosphatase 2A activity; suppressed LS-mediated phosphorylation of extracellular signal-regulated kinase, c-Jun N-terminal kinase, c-Jun, and nuclear factor-κB p65; and attenuated LS-mediated PRR upregulation. LS also enhanced DNA binding of cAMP response element binding protein 1 to cAMP response elements, nuclear factor-κB p65 to nuclear factor-κB elements, and c-Jun to activator protein 1 elements in PRR promoter in proximal tubular cells. We conclude that sodium depletion upregulates renal PRR expression via the cGMP-PKG signaling pathway by enhancing binding of cAMP response element binding protein 1, nuclear factor-κB p65, and c-Jun to PRR promotor.

  18. The angiotensin Ⅱ type 1 receptor and receptor-associated proteins

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    The mechanisms of regulation, activation and signal transduction of the angiotensin Ⅱ(Ang Ⅱ) type 1 (AT1) receptor have been studied extensively in the decade after its cloning. The AT1receptor is a major component of the renin-angiotensin system (RAS). It mediates the classical biological actions of Ang Ⅱ. Among the structures required for regulation and activation of the receptor, its carboxylterminal region plays crucial roles in receptor internalization, desensitization and phosphorylation. The mechanisms involved in heterotrimeric G-protein coupling to the receptor, activation of the downstreamsignaling pathway by G proteins and the Ang Ⅱ signal transduction pathways leading to specific cellularresponses are discussed. In addition, recent work on the identification and characterization of novel proteinsassociated with carboxyl-terminus of the AT1 receptor is presented. These novel proteins will advance ourunderstanding of how the receptor is internalized and recycled as they provide molecular mechanisms for the activation and regulation of G-protein-coupled receptors.

  19. CB1 Cannabinoid Receptors and their Associated Proteins

    Science.gov (United States)

    Howlett, Allyn C.; Blume, Lawrence C.; Dalton, George D.

    2011-01-01

    CB1 receptors are G-protein coupled receptors (GPCRs) abundant in neurons, in which they modulate neurotransmission. The CB1 receptor influence on memory and learning is well recognized, and disease states associated with CB1 receptors are observed in addiction disorders, motor dysfunction, schizophrenia, and in bipolar, depression, and anxiety disorders. Beyond the brain, CB1 receptors also function in liver and adipose tissues, vascular as well as cardiac tissue, reproductive tissues and bone. Signal transduction by CB1 receptors occurs through interaction with Gi/o proteins to inhibit adenylyl cyclase, activate mitogen-activated protein kinases (MAPK), inhibit voltage-gated Ca2+ channels, activate K+ currents (Kir), and influence Nitric Oxide (NO) signaling. CB1 receptors are observed in internal organelles as well as plasma membrane. β-Arrestins, adaptor protein AP-3, and G-protein receptor-associated sorting protein 1 (GASP1) modulate cellular trafficking. Cannabinoid Receptor Interacting Protein 1a (CRIP1a) is an accessory protein whose function has not been delineated. Factor Associated with Neutral sphingomyelinase (FAN) regulates ceramide signaling. Such diversity in cellular signaling and modulation by interacting proteins suggests that agonists and allosteric modulators could be developed to specifically regulate unique, cell type-specific responses. PMID:20166926

  20. Prophylactic Melatonin Attenuates Isoflurane-Induced Cognitive Impairment in Aged Rats through Hippocampal Melatonin Receptor 2 - cAMP Response Element Binding Signalling.

    Science.gov (United States)

    Liu, Yajie; Ni, Cheng; Li, Zhengqian; Yang, Ning; Zhou, Yang; Rong, Xiaoying; Qian, Min; Chui, Dehua; Guo, Xiangyang

    2017-03-01

    Melatonin exerts many physiological effects via melatonin receptors, among which the melatonin-2 receptor (MT2 ) plays a critical role in circadian rhythm disorders, Alzheimer's disease and other neurological disorders. A melatonin replacement strategy has been tested previously, and MT2 was a critical target during the process. cAMP response element binding (CREB) is an essential transcription factor for memory formation and could be involved in MT2 signalling. Therefore, the present study was designed to investigate the effects of prophylactic melatonin on inhaled anaesthetic isoflurane-induced cognitive impairment, and to determine whether the protective effects of melatonin are dependent on MT2 and downstream CREB signalling in the hippocampus of aged rats. The results showed that prophylactic melatonin attenuated isoflurane-induced decreases in plasma/hippocampal melatonin levels and cognitive impairment in aged rats. Furthermore, 4P-PDOT, a selective MT2 antagonist, blocked the protective effects of melatonin on isoflurane-induced decreases in both hippocampal MT2 expression and downstream CREB phosphorylation. And 4P-PDOT blocked the attenuation of melatonin on isoflurane-induced memory impairment. Collectively, the results suggest that the protective effects of prophylactic melatonin are dependent on hippocampal MT2 -CREB signalling, which could be a potential therapeutic target for anaesthetic-induced cognitive impairment.

  1. G protein-coupled receptor Gpr4 senses amino acids and activates the cAMP-PKA pathway in Cryptococcus neoformans.

    Science.gov (United States)

    Xue, Chaoyang; Bahn, Yong-Sun; Cox, Gary M; Heitman, Joseph

    2006-02-01

    The Galpha protein Gpa1 governs the cAMP-PKA signaling pathway and plays a central role in virulence and differentiation in the human fungal pathogen Cryptococcus neoformans, but the signals and receptors that trigger this pathway were unknown. We identified seven putative proteins that share identity with known G protein-coupled receptors (GPCRs). One protein, Gpr4, shares limited sequence identity with the Dictyostelium discoideum cAMP receptor cAR1 and the Aspergillus nidulans GPCR protein GprH and also shares structural similarity with the Saccharomyces cerevisiae receptor Gpr1. gpr4 mutants exhibited reduced capsule production and mating defects, similar to gpa1 mutants, and exogenous cAMP suppressed both gpr4 mutant phenotypes. Epistasis analysis provides further evidence that Gpr4 functions upstream of the Galpha subunit Gpa1. Gpr4-Gpr4 homomeric interactions were observed in the yeast two-hybrid assay, and Gpr4 was shown to physically interact with Gpa1 in the split-ubiquitin system. A Gpr4::DsRED fusion protein was localized to the plasma membrane and methionine was found to trigger receptor internalization. The analysis of intracellular cAMP levels showed that gpr4 mutants still respond to glucose but not to certain amino acids, such as methionine. Amino acids might serve as ligands for Gpr4 and could contribute to engage the cAMP-PKA pathway. Activation of the cAMP-PKA pathway by glucose and amino acids represents a nutrient coincidence detection system shared in other pathogenic fungi.

  2. Receptor-mediated stimulation of lipid signalling pathways in CHO cells elicits the rapid transient induction of the PDE1B isoform of Ca2+/calmodulin-stimulated cAMP phosphodiesterase.

    Science.gov (United States)

    Spence, S; Rena, G; Sullivan, M; Erdogan, S; Houslay, M D

    1997-01-01

    Chinese hamster ovary cells (CHO cells) do not exhibit any Ca2+/calmodulin-stimulated cAMP phosphodiesterase (PDE1) activity. Challenge of CHO cells with agonists for endogenous P2-purinoceptors, lysophosphatidic acid receptors and thrombin receptors caused a similar rapid transient induction of PDE1 activity in each instance. This was also evident on noradrenaline challenge of a cloned CHO cell line transfected so as to overexpress alpha 1B-adrenoceptors. This novel PDE1 activity appeared within about 15 min of exposure to ligands, rose to a maximum value within 30 min to 1 h and then rapidly decreased. In each case, the expression of novel PDE1 activity was blocked by the transcriptional inhibitor actinomycin D. Challenge with insulin of either native CHO cells or a CHO cell line transfected so as to overexpress the human insulin receptor failed to induce PDE1 activity. Reverse transcriptase-PCR analyses, using degenerate primers able to detect the PDE1C isoform, did not amplify any fragment from RNA preparations of CHO cells expressing PDE1 activity, although they did so from the human thyroid carcinoma FTC133 cell line. Reverse transcriptase-PCR analyses, using degenerate primers able to detect the PDE1A and PDE1B isoforms, successfully amplified a fragment of the predicted size from RNA preparations of both CHO cells expressing PDE1 activity and human Jurkat T-cells. Sequencing of the PCR products, generated using the PDE1A/B primers, yielded a novel sequence which, by analogy with sequences reported for bovine and murine PDE1B forms, suggests that the PDE1 species induced in CHO cells through protein kinase C activation and that expressed in Jurkat T-cells are PDE1B forms.

  3. Receptor component protein (RCP): a member of a multi-protein complex required for G-protein-coupled signal transduction.

    Science.gov (United States)

    Prado, M A; Evans-Bain, B; Dickerson, I M

    2002-08-01

    The calcitonin-gene-related peptide (CGRP) receptor component protein (RCP) is a 148-amino-acid intracellular protein that is required for G-protein-coupled signal transduction at receptors for the neuropeptide CGRP. RCP works in conjunction with two other proteins to constitute a functional CGRP receptor: calcitonin-receptor-like receptor (CRLR) and receptor-activity-modifying protein 1 (RAMP1). CRLR has the stereotypical seven-transmembrane topology of a G-protein-coupled receptor; it requires RAMP1 for trafficking to the cell surface and for ligand specificity, and requires RCP for coupling to the cellular signal transduction pathway. We have made cell lines that expressed an antisense construct of RCP and determined that CGRP-mediated signal transduction was reduced, while CGRP binding was unaffected. Furthermore, signalling at two other endogenous G-protein-coupled receptors was unaffected, suggesting that RCP was specific for a limited subset of receptors.

  4. G Protein-Coupled Receptors in Cancer

    Directory of Open Access Journals (Sweden)

    Rachel Bar-Shavit

    2016-08-01

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

  5. Role of Exchange Protein Activated by cAMP 1 in Regulating Rates of Microtubule Formation in Cystic Fibrosis Epithelial Cells.

    Science.gov (United States)

    Rymut, Sharon M; Ivy, Tracy; Corey, Deborah A; Cotton, Calvin U; Burgess, James D; Kelley, Thomas J

    2015-12-01

    The regulation of microtubule dynamics in cystic fibrosis (CF) epithelial cells and the consequences of reduced rates of microtubule polymerization on downstream CF cellular events, such as cholesterol accumulation, a marker of impaired intracellular transport, are explored here. It is identified that microtubules in both CF cell models and in primary CF nasal epithelial cells repolymerize at a slower rate compared with respective controls. Previous studies suggest a role for cAMP in modulating organelle transport in CF cells, implicating a role for exchange protein activated by cAMP (EPAC) 1, a regulator of microtubule elongation, as a potential mechanism. EPAC1 activity is reduced in CF cell models and in Cftr(-/-) mouse lung compared with respective non-CF controls. Stimulation of EPAC1 activity with the selective EPAC1 agonist, 8-cpt-2-O-Me-cAMP, stimulates microtubule repolymerization to wild-type rates in CF cells. EPAC1 activation also alleviates cholesterol accumulation in CF cells, suggesting a direct link between microtubule regulation and intracellular transport. To verify the relationship between transport and microtubule regulation, expression of the protein, tubulin polymerization-promoting protein, was knocked down in non-CF human tracheal (9/HTEo(-)) cells to mimic the microtubule dysregulation in CF cells. Transduced cells with short hairpin RNA targeting tubulin polymerization-promoting protein exhibit CF-like perinuclear cholesterol accumulation and other cellular manifestations of CF cells, thus supporting a role for microtubule regulation as a mechanism linking CFTR function to downstream cellular manifestation.

  6. The Vasopressin Type-2 Receptor and Prostaglandin Receptors EP2 and EP4 can Increase Aquaporin-2 Plasma Membrane Targeting Through a cAMP Independent Pathway

    DEFF Research Database (Denmark)

    Olesen, Emma Tina Bisgaard; Moeller, Hanne Bjerregaard; Assentoft, Mette;

    2016-01-01

    Apical membrane targeting of the collecting duct water channel aquaporin-2 (AQP2) is essential for body water balance. As this event is regulated by Gs coupled 7-transmembrane receptors such as the vasopressin type 2 receptor (V2R) and the prostanoid receptors EP2 and EP4, it is believed to be c...

  7. Bacillus bombysepticus α-Toxin Binding to G Protein-Coupled Receptor Kinase 2 Regulates cAMP/PKA Signaling Pathway to Induce Host Death.

    Directory of Open Access Journals (Sweden)

    Ping Lin

    2016-03-01

    Full Text Available Bacterial pathogens and their toxins target host receptors, leading to aberrant behavior or host death by changing signaling events through subversion of host intracellular cAMP level. This is an efficient and widespread mechanism of microbial pathogenesis. Previous studies describe toxins that increase cAMP in host cells, resulting in death through G protein-coupled receptor (GPCR signaling pathways by influencing adenylyl cyclase or G protein activity. G protein-coupled receptor kinase 2 (GRK2 has a central role in regulation of GPCR desensitization. However, little information is available about the pathogenic mechanisms of toxins associated with GRK2. Here, we reported a new bacterial toxin-Bacillus bombysepticus (Bb α-toxin that was lethal to host. We showed that Bb α-toxin interacted with BmGRK2. The data demonstrated that Bb α-toxin directly bound to BmGRK2 to promote death by affecting GPCR signaling pathways. This mechanism involved stimulation of Gαs, increase level of cAMP and activation of protein kinase A (PKA. Activated cAMP/PKA signal transduction altered downstream effectors that affected homeostasis and fundamental biological processes, disturbing the structural and functional integrity of cells, resulting in death. Preventing cAMP/PKA signaling transduction by inhibitions (NF449 or H-89 substantially reduced the pathogenicity of Bb α-toxin. The discovery of a toxin-induced host death specifically linked to GRK2 mediated signaling pathway suggested a new model for bacterial toxin action. Characterization of host genes whose expression and function are regulated by Bb α-toxin and GRK2 will offer a deeper understanding of the pathogenesis of infectious diseases caused by pathogens that elevate cAMP.

  8. Metabotropic Glutamate Receptors and Interacting Proteins in Epileptogenesis.

    Science.gov (United States)

    Qian, Feng; Tang, Feng-Ru

    2016-01-01

    Neurotransmitter and receptor systems are involved in different neurological and neuropsychological disorders such as Parkinson's disease, depression, Alzheimer's disease and epilepsy. Recent advances in studies of signal transduction pathways or interacting proteins of neurotransmitter receptor systems suggest that different receptor systems may share the common signal transduction pathways or interacting proteins which may be better therapeutic targets for development of drugs to effectively control brain diseases. In this paper, we reviewed metabotropic glutamate receptors (mGluRs) and their related signal transduction pathways or interacting proteins in status epilepticus and temporal lobe epilepsy, and proposed some novel therapeutical drug targets for controlling epilepsy and epileptogenesis.

  9. G protein-coupled receptor regulation: The role of protein interactions and receptor trafficking

    OpenAIRE

    Sandén, Caroline

    2009-01-01

    The superfamily of G protein-coupled receptors (GPCR) is the largest gene family in the human genome. GPCR-mediated signaling operates in every human cell, and about 50% of existing clinically useful drugs act through GPCR. Kinins are proinflammatory peptides that are rapidly produced extracellularly following pathological insults and tissue damage. These peptides act through two GPCR subtypes, B1 (B1R) and B2 (B2R), to elicit numerous inflammatory responses including vasodilatiation, increas...

  10. Protein receptor for activated C kinase 1 is involved in morphine reward in mice.

    Science.gov (United States)

    Wan, L; Su, L; Xie, Y; Liu, Y; Wang, Y; Wang, Z

    2009-07-07

    Opiate addiction is associated with upregulation of cAMP signaling in the brain. cAMP-responsive element binding protein (CREB), a nuclear transcription factor, is a downstream component of the extracellular signal-regulated protein kinase (ERK) pathway, which has been shown to regulate different physiological and psychological responses of drug addiction. RACK1, the protein receptor for activated C kinase 1, is a multifunctional scaffolding protein known to be a key regulator of various signaling cascades in the CNS. RACK1 functions specifically in integrin mediated activation of ERK cascade and targets active ERK. We examined if RACK1 is involved in the mechanism of drug addiction by regulating CREB in mouse hippocampus and prefrontal cortex. Several expressions were observed. Chronic administration of morphine made the expression of RACK1 and CREB mRNA increase in hippocampus and prefrontal cortex. The expression of RACK1 and CREB protein was strongly positive in CA1, CA3 and dentate gyrus (DG) of the hippocampus of morphine-treated mice brain, especially the pyramidal neurons in the DG of the hippocampus. Using the small interfering RNA technology, we determined that the expression of CREB mRNA was decreased in hippocampus and prefrontal cortex of morphine-treated mice. The expression of RACK1 and CREB protein was negative in CA1, CA3 and DG of hippocampus. These findings suggest that morphine reward can influence the expression of RACK1 in mouse hippocampus and prefrontal cortex through regulating CREB transcription.

  11. The urokinase receptor associated protein (uPARAP/endo180)

    DEFF Research Database (Denmark)

    Engelholm, L H; Nielsen, B S; Danø, K

    2001-01-01

    of this proteolytic system. uPARAP is a high molecular weight type-1 membrane protein, belonging to the macrophage mannose receptor protein family. On the surface of certain cells, uPARAP forms a ternary complex with the pro-form of the urokinase-type plasminogen activator (uPA) and its primary receptor (uPAR). While...

  12. Modulation of lipoprotein receptor functions by intracellular adaptor proteins.

    Science.gov (United States)

    Stolt, Peggy C; Bock, Hans H

    2006-10-01

    Members of the low density lipoprotein (LDL) receptor gene family are critically involved in a wide range of physiological processes including lipid and vitamin homeostasis, cellular migration, neurodevelopment, and synaptic plasticity, to name a few. Lipoprotein receptors exert these diverse biological functions by acting as cellular uptake receptors or by inducing intracellular signaling cascades. It was discovered that a short sequence in the intracellular region of all lipoprotein receptors, Asn-Pro-X-Tyr (NPXY) is important for mediating either endocytosis or signal transduction events, and that this motif serves as a binding site for phosphotyrosine-binding (PTB) domain containing scaffold proteins. These molecular adaptors connect the transmembrane receptors with the endocytosis machinery and regulate cellular trafficking, or function as assembly sites for dynamic multi-protein signaling complexes. Whereas the LDL receptor represents the archetype of an endocytic lipoprotein receptor, the structurally closely related apolipoprotein E receptor 2 (apoER2) and very low density lipoprotein (VLDL) receptor activate a kinase-dependent intracellular signaling cascade after binding to the neuronal signaling molecule Reelin. This review focuses on two related PTB domain containing adaptor proteins that mediate these divergent lipoprotein receptor responses, ARH (autosomal recessive hypercholesterolemia protein) and Dab1 (disabled-1), and discusses the structural and molecular basis of this different behaviour.

  13. The cAMP signaling system inhibits the repair of {gamma}-ray-induced DNA damage by promoting Epac1-mediated proteasomal degradation of XRCC1 protein in human lung cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Cho, Eun-Ah [Department of Biochemistry and Molecular Biology, Cancer Research Center, Seoul National University College of Medicine, Seoul 110-799 (Korea, Republic of); Juhnn, Yong-Sung, E-mail: juhnn@snu.ac.kr [Department of Biochemistry and Molecular Biology, Cancer Research Center, Seoul National University College of Medicine, Seoul 110-799 (Korea, Republic of)

    2012-06-01

    Highlights: Black-Right-Pointing-Pointer cAMP signaling system inhibits repair of {gamma}-ray-induced DNA damage. Black-Right-Pointing-Pointer cAMP signaling system inhibits DNA damage repair by decreasing XRCC1 expression. Black-Right-Pointing-Pointer cAMP signaling system decreases XRCC1 expression by promoting its proteasomal degradation. Black-Right-Pointing-Pointer The promotion of XRCC1 degradation by cAMP signaling system is mediated by Epac1. -- Abstract: Cyclic AMP is involved in the regulation of metabolism, gene expression, cellular growth and proliferation. Recently, the cAMP signaling system was found to modulate DNA-damaging agent-induced apoptosis by regulating the expression of Bcl-2 family proteins and inhibitors of apoptosis. Thus, we hypothesized that the cAMP signaling may modulate DNA repair activity, and we investigated the effects of the cAMP signaling system on {gamma}-ray-induced DNA damage repair in lung cancer cells. Transient expression of a constitutively active mutant of stimulatory G protein (G{alpha}sQL) or treatment with forskolin, an adenylyl cyclase activator, augmented radiation-induced DNA damage and inhibited repair of the damage in H1299 lung cancer cells. Expression of G{alpha}sQL or treatment with forskolin or isoproterenol inhibited the radiation-induced expression of the XRCC1 protein, and exogenous expression of XRCC1 abolished the DNA repair-inhibiting effect of forskolin. Forskolin treatment promoted the ubiquitin and proteasome-dependent degradation of the XRCC1 protein, resulting in a significant decrease in the half-life of the protein after {gamma}-ray irradiation. The effect of forskolin on XRCC1 expression was not inhibited by PKA inhibitor, but 8-pCPT-2 Prime -O-Me-cAMP, an Epac-selective cAMP analog, increased ubiquitination of XRCC1 protein and decreased XRCC1 expression. Knockdown of Epac1 abolished the effect of 8-pCPT-2 Prime -O-Me-cAMP and restored XRCC1 protein level following {gamma}-ray irradiation. From

  14. The CytR repressor antagonizes cyclic AMP-cyclic AMP receptor protein activation of the deoCp2 promoter of Escherichia coli K-12

    DEFF Research Database (Denmark)

    Søgaard-Andersen, Lotte; Martinussen, J; Møllegaard, N E

    1990-01-01

    We have investigated the regulation of the Escherichia coli deoCp2 promoter by the CytR repressor and the cyclic AMP (cAMP) receptor protein (CRP) complexed to cAMP. Promoter regions controlled by these two proteins characteristically contain tandem cAMP-CRP binding sites. Here we show that (i) Cyt......R selectively regulated cAMP-CRP-dependent initiations, although transcription started from the same site in deoCp2 in the absence or presence of cAMP-CRP; (ii) deletion of the uppermost cAMP-CRP target (CRP-2) resulted in loss of CytR regulation, but had only a minor effect on positive control by the c...... are required for efficient CytR repression of deoCp2. Models for the action of CytR are discussed in light of these findings....

  15. G beta gamma signaling reduces intracellular cAMP to promote meiotic progression in mouse oocytes.

    Science.gov (United States)

    Gill, Arvind; Hammes, Stephen R

    2007-02-01

    In nearly every vertebrate species, elevated intracellular cAMP maintains oocytes in prophase I of meiosis. Prior to ovulation, gonadotropins trigger various intra-ovarian processes, including the breakdown of gap junctions, the activation of EGF receptors, and the secretion of steroids. These events in turn decrease intracellular cAMP levels in select oocytes to allow meiotic progression, or maturation, to resume. Studies suggest that cAMP levels are kept elevated in resting oocytes by constitutive G protein signaling, and that the drop in intracellular cAMP that accompanies maturation may be due in part to attenuation of this inhibitory G protein-mediated signaling. Interestingly, one of these G protein regulators of meiotic arrest is the Galpha(s) protein, which stimulates adenylyl cyclase to raise intracellular cAMP in two important animal models of oocyte development: Xenopus leavis frogs and mice. In addition to G(alpha)(s), constitutive Gbetagamma activity similarly stimulates adenylyl cyclase to raise cAMP and prevent maturation in Xenopus oocytes; however, the role of Gbetagamma in regulating meiosis in mouse oocytes has not been examined. Here we show that Gbetagamma does not contribute to the maintenance of murine oocyte meiotic arrest. In fact, contrary to observations in frog oocytes, Gbetagamma signaling in mouse oocytes reduces cAMP and promotes oocyte maturation, suggesting that Gbetagamma might in fact play a positive role in promoting oocyte maturation. These observations emphasize that, while many general concepts and components of meiotic regulation are conserved from frogs to mice, specific differences exist that may lead to important insights regarding ovarian development in vertebrates.

  16. Selection for Genes Encoding Secreted Proteins and Receptors

    Science.gov (United States)

    Klein, Robert D.; Gu, Qimin; Goddard, Audrey; Rosenthal, Arnon

    1996-07-01

    Extracellular proteins play an essential role in the formation, differentiation, and maintenance of multicellular organisms. Despite that, the systematic identification of genes encoding these proteins has not been possible. We describe here a highly efficient method to isolate genes encoding secreted and membrane-bound proteins by using a single-step selection in yeast. Application of this method, termed signal peptide selection, to various tissues yielded 559 clones that appear to encode known or novel extracellular proteins. These include members of the transforming growth factor and epidermal growth factor protein families, endocrine hormones, tyrosine kinase receptors, serine/threonine kinase receptors, seven transmembrane receptors, cell adhesion molecules, extracellular matrix proteins, plasma proteins, and ion channels. The eventual identification of most, or all, extracellular signaling molecules will advance our understanding of fundamental biological processes and our ability to intervene in disease states.

  17. The use of receptor-specific antibodies to study G-protein-coupled receptors.

    Science.gov (United States)

    Gupta, Achla; Devi, Lakshmi A

    2006-07-01

    The identification of G-protein-coupled receptor (GPCR) cDNAs has facilitated a number of studies characterizing the biochemical properties of the receptor protein. Most of these studies have used antibodies directed against the epitope-tagged receptor expressed in heterologous cells, because of the lack of sensitive and selective antibodies capable of recognizing endogenous receptors in their native state. In order to facilitate studies with endogenous receptors, efforts have been made to generate receptor-type selective, sensitive antibodies that are able to recognize endogenous receptors. In this review, we discuss the strategies as well as the details of the techniques used for the generation of monoclonal and polyclonal antibodies with a focus on family A GPCRs.

  18. Compartmentalized accumulation of cAMP near complexes of multidrug resistance protein 4 (MRP4) and cystic fibrosis transmembrane conductance regulator (CFTR) contributes to drug-induced diarrhea.

    Science.gov (United States)

    Moon, Changsuk; Zhang, Weiqiang; Ren, Aixia; Arora, Kavisha; Sinha, Chandrima; Yarlagadda, Sunitha; Woodrooffe, Koryse; Schuetz, John D; Valasani, Koteswara Rao; de Jonge, Hugo R; Shanmukhappa, Shiva Kumar; Shata, Mohamed Tarek M; Buddington, Randal K; Parthasarathi, Kaushik; Naren, Anjaparavanda P

    2015-05-01

    Diarrhea is one of the most common adverse side effects observed in ∼7% of individuals consuming Food and Drug Administration (FDA)-approved drugs. The mechanism of how these drugs alter fluid secretion in the gut and induce diarrhea is not clearly understood. Several drugs are either substrates or inhibitors of multidrug resistance protein 4 (MRP4), such as the anti-colon cancer drug irinotecan and an anti-retroviral used to treat HIV infection, 3'-azido-3'-deoxythymidine (AZT). These drugs activate cystic fibrosis transmembrane conductance regulator (CFTR)-mediated fluid secretion by inhibiting MRP4-mediated cAMP efflux. Binding of drugs to MRP4 augments the formation of MRP4-CFTR-containing macromolecular complexes that is mediated via scaffolding protein PDZK1. Importantly, HIV patients on AZT treatment demonstrate augmented MRP4-CFTR complex formation in the colon, which defines a novel paradigm of drug-induced diarrhea.

  19. SOCS proteins in regulation of receptor tyrosine kinase signaling

    DEFF Research Database (Denmark)

    Kazi, Julhash U.; Kabir, Nuzhat N.; Flores Morales, Amilcar;

    2014-01-01

    . The signaling mediated by RTKs must be tightly regulated by interacting proteins including protein-tyrosine phosphatases and ubiquitin ligases. The suppressors of cytokine signaling (SOCS) family proteins are well-known negative regulators of cytokine receptors signaling consisting of eight structurally similar...

  20. Allosteric modulation of G-protein coupled receptors

    DEFF Research Database (Denmark)

    Jensen, Anders A.; Spalding, Tracy A

    2004-01-01

    The superfamily of G-protein coupled receptors (GPCRs) has more than 1000 members and is the largest family of proteins in the body. GPCRs mediate signalling of stimuli as diverse as light, ions, small molecules, peptides and proteins and are the targets for many pharmaceuticals. Most GPCR ligands...

  1. Changes of phosphorylation of cAMP response element binding protein in rat nucleus accumbens after chronic ethanol intake: naloxone reversal

    Institute of Scientific and Technical Information of China (English)

    LIJing; LIYue-Hua; YUANXiao-Ru

    2003-01-01

    AIM: To study the changes in the expression and phosphorylation of cAMP response element binding protein(CREB) in the rat nucleus accumbens after chronic ethanol intake and its withdrawal. METHODS: Ethanol wasgiven in drinking water at the concentration of 6 % (v/v), for one month. Changes in the levels of CREB andphospho-CREB (p-CREB) protein in the nucleus accumbens were measured by immunohistochemistry methods.RESULTS: Ethanol given to rats in drinking water decreased the level of p-CREB protein in the nucleus accumbens(-75 %) at the time of exposure to ethanol. The decrement of p-CREB protein in the nucleus accumbens remainedat 24 h (-35 %) and 72 h (-28 %) of ethanol withdrawal, which recovered toward control level after 7 d of ethanolwithdrawal. However, chronic ethanol, as well as ethanol withdrawal failed to produce any significant alteration inthe level of CREB protein in the nucleus accumbens. Naloxone (alone) treatment of rats had no effect on the levelsof CREB and p-CREB protein in the nucleus accumbens. However, when naloxone was administered concurrentlywith ethanol treatment, it antagonized the down-regulation of p-CREB protein in the nucleus accumbens (142 %) ofrats exposed to ethanol. CONCLUSION: A long-term intake of ethanol solution down-regulates the phosphoryla-tion of CREB in the nucleus accumbens, and those changes can be reversed by naloxone, which may be one kindof the molecular mechanisms associated with ethano1 dependence.

  2. Novel RNAi-mediated approach to G protein-coupled receptor deorphanization: proof of principle and characterization of a planarian 5-HT receptor.

    Directory of Open Access Journals (Sweden)

    Mostafa Zamanian

    Full Text Available G protein-coupled receptors (GPCRs represent the largest known superfamily of membrane proteins extending throughout the Metazoa. There exists ample motivation to elucidate the functional properties of GPCRs given their role in signal transduction and their prominence as drug targets. In many target organisms, these efforts are hampered by the unreliable nature of heterologous receptor expression platforms. We validate and describe an alternative loss-of-function approach for ascertaining the ligand and G protein coupling properties of GPCRs in their native cell membrane environment. Our efforts are focused on the phylum Platyhelminthes, given the heavy health burden exacted by pathogenic flatworms, as well as the role of free-living flatworms as model organisms for the study of developmental biology. RNA interference (RNAi was used in conjunction with a biochemical endpoint assay to monitor cAMP modulation in response to the translational suppression of individual receptors. As proof of principle, this approach was used to confirm the neuropeptide GYIRFamide as the cognate ligand for the planarian neuropeptide receptor GtNPR-1, while revealing its endogenous coupling to Gα(i/o. The method was then extended to deorphanize a novel Gα(s-coupled planarian serotonin receptor, DtSER-1. A bioinformatics protocol guided the selection of receptor candidates mediating 5-HT-evoked responses. These results provide functional data on a neurotransmitter central to flatworm biology, while establishing the great potential of an RNAi-based deorphanization protocol. Future work can help optimize and adapt this protocol for higher-throughput platforms as well as other phyla.

  3. [Regulation of G protein-coupled receptor kinase activity].

    Science.gov (United States)

    Haga, T; Haga, K; Kameyama, K; Nakata, H

    1994-09-01

    Recent progress on the activation of G protein-coupled receptor kinases is reviewed. beta-Adrenergic receptor kinase (beta ARK) is activated by G protein beta gamma -subunits, which interact with the carboxyl terminal portion of beta ARK. Muscarinic receptor m2-subtypes are phosphorylated by beta ARK1 in the central part of the third intracellular loop (I3). Phosphorylation of I3-GST fusion protein by beta ARK1 is synergistically stimulated by the beta gamma -subunits and mastoparan or a peptide corresponding to portions adjacent to the transmembrane segments of m2-receptors or by beta gamma -subunits and the agonist-bound I3-deleted m2 variant. These results indicate that agonist-bound receptors serve as both substrates and activators of beta ARK.

  4. Multiple Facets of cAMP Signalling and Physiological Impact: cAMP Compartmentalization in the Lung

    Directory of Open Access Journals (Sweden)

    Martina Schmidt

    2012-11-01

    Full Text Available Therapies involving elevation of the endogenous suppressor cyclic AMP (cAMP are currently used in the treatment of several chronic inflammatory disorders, including chronic obstructive pulmonary disease (COPD. Characteristics of COPD are airway obstruction, airway inflammation and airway remodelling, processes encompassed by increased airway smooth muscle mass, epithelial changes, goblet cell and submucosal gland hyperplasia. In addition to inflammatory cells, airway smooth muscle cells and (myofibroblasts, epithelial cells underpin a variety of key responses in the airways such as inflammatory cytokine release, airway remodelling, mucus hypersecretion and airway barrier function. Cigarette smoke, being next to environmental pollution the main cause of COPD, is believed to cause epithelial hyperpermeability by disrupting the barrier function. Here we will focus on the most recent progress on compartmentalized signalling by cAMP. In addition to G protein-coupled receptors, adenylyl cyclases, cAMP-specific phospho-diesterases (PDEs maintain compartmentalized cAMP signalling. Intriguingly, spatially discrete cAMP-sensing signalling complexes seem also to involve distinct members of the A-kinase anchoring (AKAP superfamily and IQ motif containing GTPase activating protein (IQGAPs. In this review, we will highlight the interaction between cAMP and the epithelial barrier to retain proper lung function and to alleviate COPD symptoms and focus on the possible molecular mechanisms involved in this process. Future studies should include the development of cAMP-sensing multiprotein complex specific disruptors and/or stabilizers to orchestrate cellular functions. Compartmentalized cAMP signalling regulates important cellular processes in the lung and may serve as a therapeutic target.

  5. Reciprocal roles of angiotensin II and Angiotensin II Receptors Blockade (ARB) in regulating Cbfa1/RANKL via cAMP signaling pathway: possible mechanism for hypertension-related osteoporosis and antagonistic effect of ARB on hypertension-related osteoporosis.

    Science.gov (United States)

    Guan, Xiao-Xu; Zhou, Yi; Li, Ji-Yao

    2011-01-01

    Hypertension is a risk factor for osteoporosis. Animal and epidemiological studies demonstrate that high blood pressure is associated with increased calcium loss, elevated parathyroid hormone, and increased calcium movement from bone. However, the mechanism responsible for hypertension-related osteoporosis remains elusive. Recent epidemiological studies indicate the benefits of Angiotensin II Receptors Blockade (ARB) on decreasing fracture risks. Since receptors for angiotensin II, the targets of ARB, are expressed in both osteoblasts and osteoclasts, we postulated that angiotensin II plays an important role in hypertension-related osteoporosis. Cbfa1 and RANKL, the important factors for maintaining bone homeostasis and key mediators in controlling osteoblast and osteoclast differentiation, are both regulated by cAMP-dependent signaling. Angiotensin II along with factors such as LDL, HDL, NO and homocysteine that are commonly altered both in hypertension and osteoporosis, can down-regulate the expression of Cbfa1 but up-regulate RANKL expression via the cAMP signaling pathway. We thus hypothesized that, by altering the ratio of Cbfa1/RANKL expression via the cAMP-dependent pathway, angiotensin II differently regulates osteoblast and osteoclast differentiation leading to enhanced bone resorption and reduced bone formation. Since ARB can antagonize the adverse effect of angiotensin II on bone by lowering cAMP levels and modifying other downstream targets, including LDL, HDL, NO and Cbfa1/RANKL, we propose the hypothesis that the antagonistic effects of ARB may also be exerted via cAMP signaling pathway.

  6. Prolactin receptor and signal transduction to milk protein genes

    Energy Technology Data Exchange (ETDEWEB)

    Djiane, J.; Daniel, N.; Bignon, C. [Unite d`Endocrinologie Moleculaire, Jouy en Josas (France)] [and others

    1994-06-01

    After cloning of the mammary gland prolactin (PRL) receptor cDNA, a functional assay was established using co-transfection of PRL receptor cDNA together with a milk protein promoter/chloramphenicol acetyl transferase (CAT) construct in Chinese hamster ovary (CHO) cells. Different mutants of the PRL receptor were tested in this CAT assay to delimit the domains in the receptor necessary for signal transduction to milk protein genes. In CHO cells stably transfected with PRL receptor cDNA, high numbers of PRL receptor are expressed. By metabolic labeling and immunoprecipitation, expressed PRL receptor was identified as a single species of 100 kDa. Using these cells, we analyzed the effects of PRL on intracellular free Ca{sup ++} concentration. PRL stimulates Ca{sup ++} entry and induces secondary Ca{sup ++} mobilization. The entry of Ca{sup ++} is a result of an increase in K{sup +} conductance that hyperpolarizes the membranes. We have also analyzed tyrosine phosphorylation induced by PRL. In CHO cells stably transfected with PRL receptor cDNA, PRL induced a very rapid and transient tyrosine phosphorylation of a 100-kDa protein which is most probably the PRL receptor. The same finding was obtained in mammary membranes after PRL injection to lactating rabbits. Whereas tyrosine kinase inhibitors genistein and lavendustin were without effect, PRL stimulation of milk protein gene promoters was partially inhibited by 2 {mu}M herbimycin in CHO cells co-transfected with PRL receptor cDNA and the {Beta} lactoglobulin CAT construct. Taken together these observations indicate that the cytoplasmic domain of the PRL receptor interacts with one or several tyrosine kinases, which may represent early postreceptor events necessary for PRL signal transduction to milk protein genes. 14 refs., 4 figs.

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

    Science.gov (United States)

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

    2014-01-01

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

  8. Serotonin signaling in Schistosoma mansoni: a serotonin-activated G protein-coupled receptor controls parasite movement.

    Directory of Open Access Journals (Sweden)

    Nicholas Patocka

    2014-01-01

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

  9. Group I Metabotropic Glutamate Receptor Interacting Proteins: Fine-Tuning Receptor Functions in Health and Disease.

    Science.gov (United States)

    Kalinowska, Magdalena; Francesconi, Anna

    2016-01-01

    Group I metabotropic glutamate receptors mediate slow excitatory neurotransmission in the central nervous system and are critical to activity-dependent synaptic plasticity, a cellular substrate of learning and memory. Dysregulated receptor signaling is implicated in neuropsychiatric conditions ranging from neurodevelopmental to neurodegenerative disorders. Importantly, group I metabotropic glutamate receptor signaling functions can be modulated by interacting proteins that mediate receptor trafficking, expression and coupling efficiency to signaling effectors. These interactions afford cell- or pathway-specific modulation to fine-tune receptor function, thus representing a potential target for pharmacological interventions in pathological conditions.

  10. Dynamic phospholipid signaling by G protein-coupled receptors

    NARCIS (Netherlands)

    Weernink, Paschal A. Oude; Han, Li; Jakobs, Karl H.; Schmidt, Martina

    2007-01-01

    G protein-coupled receptors (GPCRs) control a variety of fundamental cellular processes by regulating phospholipid signaling pathways. Essential for signaling by a large number of receptors is the hydrolysis of the membrane phosphoinositide PIP2 by phospholipase C (PLC) into the second messengers IP

  11. TLR-2 Recognizes Propionibacterium acnes CAMP Factor 1 from Highly Inflammatory Strains

    Science.gov (United States)

    Ollagnier, Guillaume; Désiré, Nathalie; Sayon, Sophie; Raingeaud, Jöel; Marcelin, Anne-Geneviève; Calvez, Vincent; Khammari, Amir; Batteux, Frédéric; Dréno, Brigitte; Dupin, Nicolas

    2016-01-01

    Background Propionibacterium acnes (P. acnes) is an anaerobic, Gram-positive bacteria encountered in inflammatory acne lesions, particularly in the pilosebaceous follicle. P. acnes triggers a strong immune response involving keratinocytes, sebocytes and monocytes, the target cells during acne development. Lipoteicoic acid and peptidoglycan induce the inflammatory reaction, but no P. acnes surface protein interacting with Toll-like receptors has been identified. P. acnes surface proteins have been extracted by lithium stripping and shown to induce CXCL8 production by keratinocytes. Methodology and principal findings Far-western blotting identified two surface proteins, of 24.5- and 27.5-kDa in size, specifically recognized by TLR2. These proteins were characterized, by LC-MS/MS, as CAMP factor 1 devoid of its signal peptide sequence, as shown by N-terminal sequencing. Purified CAMP factor 1 induces CXCL8 production by activating the CXCL8 gene promoter, triggering the synthesis of CXCL8 mRNA. Antibodies against TLR2 significantly decreased the CXCL8 response. For the 27 P. acnes strains used in this study, CAMP1-TLR2 binding intensity was modulated and appeared to be strong in type IB and II strains, which produced large amounts of CXCL8, whereas most of the type IA1 and IA2 strains presented little or no CAMP1-TLR2 binding and low levels of CXCL8 production. The nucleotide sequence of CAMP factor displays a major polymorphism, defining two distinct genetic groups corresponding to CAMP factor 1 with 14 amino-acid changes from strains phylotyped II with moderate and high levels of CAMP1-TLR2 binding activity, and CAMP factor 1 containing 0, 1 or 2 amino-acid changes from strains phylotyped IA1, IA2, or IB presenting no, weak or moderate CAMP1-TLR2 binding. Conclusions Our findings indicate that CAMP factor 1 may contribute to P. acnes virulence, by amplifying the inflammation reaction through direct interaction with TLR2. PMID:27902761

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

    DEFF Research Database (Denmark)

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

    2007-01-01

    G protein-coupled receptors (GPCRs) constitute the largest family of signaling proteins in mammals, mediating responses to hormones, neurotransmitters, and senses of sight, smell and taste. Mechanistic insight into GPCR signal transduction is limited by a paucity of high-resolution structural inf...... information. We describe the generation of a monoclonal antibody that recognizes the third intracellular loop (IL3) of the native human beta(2) adrenergic (beta(2)AR) receptor; this antibody was critical for acquiring diffraction-quality crystals....

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

    Science.gov (United States)

    Day, Peter W; Rasmussen, Søren G F; Parnot, Charles; Fung, Juan José; Masood, Asna; Kobilka, Tong Sun; Yao, Xiao-Jie; Choi, Hee-Jung; Weis, William I; Rohrer, Daniel K; Kobilka, Brian K

    2007-11-01

    G protein-coupled receptors (GPCRs) constitute the largest family of signaling proteins in mammals, mediating responses to hormones, neurotransmitters, and senses of sight, smell and taste. Mechanistic insight into GPCR signal transduction is limited by a paucity of high-resolution structural information. We describe the generation of a monoclonal antibody that recognizes the third intracellular loop (IL3) of the native human beta(2) adrenergic (beta(2)AR) receptor; this antibody was critical for acquiring diffraction-quality crystals.

  14. Effects of dopamine 1 receptor agonists on the content of cAMP in canine renal arteries%DA1受体激动剂对犬肾动脉cAMP生成量的影响

    Institute of Scientific and Technical Information of China (English)

    朱琳; 冯羡菊; 薛敬礼; 赵荣瑞

    2001-01-01

    Aim:To study the effects of DA1 receptor agonists, Fenoldopam(FODA) on the content of cAMP in canine renal arteries. Methods :Used radioimmunoassay technique to determine the content of cAMP in canine renal artery after given FODA and SCH23390. Results:FODA could increase the cAMP formation, and SCH23390 blocked the efficacy of FODA. Conclusion:The relaxing responses of renal arteries to DA receptor agonists may be related to the change of cAMP content.%目的:研究多巴胺1(DA1)受体激动剂非诺多泮(fenoldopam,FODA)对犬肾动脉cAMP含量的影响。方法:利用放射免疫分析技术,测定FODA对犬肾动脉DA1受体cAMP生成量的影响。结果:FODA可呈浓度依赖性激活肾动脉腺苷酸环化酶活性,增加cAMP生成量,选择性DA1受体阻断剂SCH23390能够显著减少FODA所引起的肾动脉cAMP生成量。结论:DA1受体激动剂对肾血管的舒张反应可能与cAMP生成量的变化有着密切关系。

  15. Real-Time G-Protein-Coupled Receptor Imaging to Understand and Quantify Receptor Dynamics

    Directory of Open Access Journals (Sweden)

    María S. Aymerich

    2011-01-01

    Full Text Available Understanding the trafficking of G-protein-coupled receptors (GPCRs and their regulation by agonists and antagonists is fundamental to develop more effective drugs. Optical methods using fluorescent-tagged receptors and spinning disk confocal microscopy are useful tools to investigate membrane receptor dynamics in living cells. The aim of this study was to develop a method to characterize receptor dynamics using this system which offers the advantage of very fast image acquisition with minimal cell perturbation. However, in short-term assays photobleaching was still a problem. Thus, we developed a procedure to perform a photobleaching-corrected image analysis. A study of short-term dynamics of the long isoform of the dopamine type 2 receptor revealed an agonist-induced increase in the mobile fraction of receptors with a rate of movement of 0.08 μm/s For long-term assays, the ratio between the relative fluorescence intensity at the cell surface versus that in the intracellular compartment indicated that receptor internalization only occurred in cells co-expressing G protein-coupled receptor kinase 2. These results indicate that the lateral movement of receptors and receptor internalization are not directly coupled. Thus, we believe that live imaging of GPCRs using spinning disk confocal image analysis constitutes a powerful tool to study of receptor dynamics.

  16. Cellular prion protein and NMDA receptor modulation: protecting against excitotoxicity

    Directory of Open Access Journals (Sweden)

    Stefanie A.G. Black

    2014-08-01

    Full Text Available Although it is well established that misfolding of the cellular prion protein (PrPC into the beta-sheet-rich, aggregated scrapie conformation (PrPSc causes a variety of transmissible spongiform encephalopathies (TSEs, the physiological roles of PrPC are still incompletely understood. There is accumulating evidence describing the roles of PrPC in neurodegeneration and neuroinflammation. Recently, we identified a functional regulation of NMDA receptors by PrPC that involves formation of a physical protein complex between these proteins. Excessive NMDA receptor activity during conditions such as ischemia mediates enhanced Ca2+ entry into cells and contributes to excitotoxic neuronal death. In addition, NMDA receptors and/or PrPC play critical roles in neuroinflammation and glial cell toxicity. Inhibition of NMDA receptor activity protects against PrPSc-induced neuronal death. Moreover, in mice lacking PrPC, infarct size is increased after focal cerebral ischemia, and absence of PrPC increases susceptibility of neurons to NMDA receptor-dependent death. Recently, PrPC was found to be a receptor for oligomeric beta-amyloid (Abeta peptides, suggesting a role for PrPC in Alzheimer’s disease. Our recent findings suggest that Abeta peptides enhance NMDA receptor current by perturbing the normal copper- and PrPC-dependent regulation of these receptors. Here, we review evidence highlighting a role for PrPC in preventing NMDA receptor-mediated excitotoxicity and inflammation. There is a need for more detailed molecular characterization of PrPC-mediated regulation of NMDA receptors, such as determining which NMDA receptor subunits mediate pathogenic effects upon loss of PrPC-mediated regulation and identifying PrPC binding site(s on the receptor. This knowledge will allow development of novel therapeutic interventions for not only TSEs, but also for Alzheimer’s disease and other neurodegenerative disorders involving dysfunction of PrPC.

  17. Cooperation between cAMP signalling and sulfonylurea in insulin secretion.

    Science.gov (United States)

    Shibasaki, T; Takahashi, T; Takahashi, H; Seino, S

    2014-09-01

    Although glucose is physiologically the most important regulator of insulin secretion, glucose-induced insulin secretion is modulated by hormonal and neural inputs to pancreatic β-cells. Most of the hormones and neurotransmitters evoke intracellular signals such as cAMP, Ca²⁺ , and phospholipid-derived molecules by activating G protein-coupled receptors (GPCRs). In particular, cAMP is a key second messenger that amplifies insulin secretion in a glucose concentration-dependent manner. The action of cAMP on insulin secretion is mediated by both protein kinase A (PKA)-dependent and Epac2A-dependent mechanisms. Many of the proteins expressed in β-cells are phosphorylated by PKA in vitro, but only a few proteins in which PKA phosphorylation directly affects insulin secretion have been identified. On the other hand, Epac2A activates the Ras-like small G protein Rap in a cAMP-dependent manner. Epac2A is also directly activated by various sulfonylureas, except for gliclazide. 8-pCPT-2'-O-Me-cAMP, an Epac-selective cAMP analogue, and glibenclamide, a sulfonylurea, synergistically activate Epac2A and Rap1, whereas adrenaline, which suppresses cAMP production in pancreatic β-cells, blocks activation of Epac2A and Rap1 by glibenclamide. Thus, cAMP signalling and sulfonylurea cooperatively activate Epac2A and Rap1. This interaction could account, at least in part, for the synergistic effects of incretin-related drugs and sulfonylureas in insulin secretion. Accordingly, clarification of the mechanism of Epac2A activation may provide therapeutic strategies to improve insulin secretion in diabetes.

  18. Anxiety and depression with neurogenesis defects in exchange protein directly activated by cAMP 2-deficient mice are ameliorated by a selective serotonin reuptake inhibitor, Prozac

    Science.gov (United States)

    Zhou, L; Ma, S L; Yeung, P K K; Wong, Y H; Tsim, K W K; So, K F; Lam, L C W; Chung, S K

    2016-01-01

    Intracellular cAMP and serotonin are important modulators of anxiety and depression. Fluoxetine, a selective serotonin reuptake inhibitor (SSRI) also known as Prozac, is widely used against depression, potentially by activating cAMP response element-binding protein (CREB) and increasing brain-derived neurotrophic factor (BDNF) through protein kinase A (PKA). However, the role of Epac1 and Epac2 (Rap guanine nucleotide exchange factors, RAPGEF3 and RAPGEF4, respectively) as potential downstream targets of SSRI/cAMP in mood regulations is not yet clear. Here, we investigated the phenotypes of Epac1 (Epac1−/−) or Epac2 (Epac2−/−) knockout mice by comparing them with their wild-type counterparts. Surprisingly, Epac2−/− mice exhibited a wide range of mood disorders, including anxiety and depression with learning and memory deficits in contextual and cued fear-conditioning tests without affecting Epac1 expression or PKA activity. Interestingly, rs17746510, one of the three single-nucleotide polymorphisms (SNPs) in RAPGEF4 associated with cognitive decline in Chinese Alzheimer's disease (AD) patients, was significantly correlated with apathy and mood disturbance, whereas no significant association was observed between RAPGEF3 SNPs and the risk of AD or neuropsychiatric inventory scores. To further determine the detailed role of Epac2 in SSRI/serotonin/cAMP-involved mood disorders, we treated Epac2−/− mice with a SSRI, Prozac. The alteration in open field behavior and impaired hippocampal cell proliferation in Epac2−/− mice were alleviated by Prozac. Taken together, Epac2 gene polymorphism is a putative risk factor for mood disorders in AD patients in part by affecting the hippocampal neurogenesis. PMID:27598965

  19. Serial femtosecond crystallography datasets from G protein-coupled receptors.

    Science.gov (United States)

    White, Thomas A; Barty, Anton; Liu, Wei; Ishchenko, Andrii; Zhang, Haitao; Gati, Cornelius; Zatsepin, Nadia A; Basu, Shibom; Oberthür, Dominik; Metz, Markus; Beyerlein, Kenneth R; Yoon, Chun Hong; Yefanov, Oleksandr M; James, Daniel; Wang, Dingjie; Messerschmidt, Marc; Koglin, Jason E; Boutet, Sébastien; Weierstall, Uwe; Cherezov, Vadim

    2016-08-01

    We describe the deposition of four datasets consisting of X-ray diffraction images acquired using serial femtosecond crystallography experiments on microcrystals of human G protein-coupled receptors, grown and delivered in lipidic cubic phase, at the Linac Coherent Light Source. The receptors are: the human serotonin receptor 2B in complex with an agonist ergotamine, the human δ-opioid receptor in complex with a bi-functional peptide ligand DIPP-NH2, the human smoothened receptor in complex with an antagonist cyclopamine, and finally the human angiotensin II type 1 receptor in complex with the selective antagonist ZD7155. All four datasets have been deposited, with minimal processing, in an HDF5-based file format, which can be used directly for crystallographic processing with CrystFEL or other software. We have provided processing scripts and supporting files for recent versions of CrystFEL, which can be used to validate the data.

  20. Using green fluorescent protein to understand the mechanisms of G-protein-coupled receptor regulation

    Directory of Open Access Journals (Sweden)

    S.S.G. Ferguson

    1998-11-01

    Full Text Available G protein-coupled receptor (GPCR activation is followed rapidly by adaptive changes that serve to diminish the responsiveness of a cell to further stimulation. This process, termed desensitization, is the consequence of receptor phosphorylation, arrestin binding, sequestration and down-regulation. GPCR phosphorylation is initiated within seconds to minutes of receptor activation and is mediated by both second messenger-dependent protein kinases and receptor-specific G protein-coupled receptor kinases (GRKs. Desensitization in response to GRK-mediated phosphorylation involves the binding of arrestin proteins that serve to sterically uncouple the receptor from its G protein. GPCR sequestration, the endocytosis of receptors to endosomes, not only contributes to the temporal desensitization of GPCRs, but plays a critical role in GPCR resensitization. GPCR down-regulation, a loss of the total cellular complement of receptors, is the consequence of both increased lysosomal degradation and decreased mRNA synthesis of GPCRs. While each of these agonist-mediated desensitization processes are initiated within a temporally dissociable time frame, recent data suggest that they are intimately related to one another. The use of green fluorescent protein from the jellyfish Aqueora victoria as an epitope tag with intrinsic fluorescence has facilitated our understanding of the relative relationship between GRK phosphorylation, arrestin binding, receptor sequestration and down-regulation.

  1. Pancreatic Beta Cell G-Protein Coupled Receptors and Second Messenger Interactions: A Systems Biology Computational Analysis.

    Science.gov (United States)

    Fridlyand, Leonid E; Philipson, Louis H

    2016-01-01

    Insulin secretory in pancreatic beta-cells responses to nutrient stimuli and hormonal modulators include multiple messengers and signaling pathways with complex interdependencies. Here we present a computational model that incorporates recent data on glucose metabolism, plasma membrane potential, G-protein-coupled-receptors (GPCR), cytoplasmic and endoplasmic reticulum calcium dynamics, cAMP and phospholipase C pathways that regulate interactions between second messengers in pancreatic beta-cells. The values of key model parameters were inferred from published experimental data. The model gives a reasonable fit to important aspects of experimentally measured metabolic and second messenger concentrations and provides a framework for analyzing the role of metabolic, hormones and neurotransmitters changes on insulin secretion. Our analysis of the dynamic data provides support for the hypothesis that activation of Ca2+-dependent adenylyl cyclases play a critical role in modulating the effects of glucagon-like peptide 1 (GLP-1), glucose-dependent insulinotropic polypeptide (GIP) and catecholamines. The regulatory properties of adenylyl cyclase isoforms determine fluctuations in cytoplasmic cAMP concentration and reveal a synergistic action of glucose, GLP-1 and GIP on insulin secretion. On the other hand, the regulatory properties of phospholipase C isoforms determine the interaction of glucose, acetylcholine and free fatty acids (FFA) (that act through the FFA receptors) on insulin secretion. We found that a combination of GPCR agonists activating different messenger pathways can stimulate insulin secretion more effectively than a combination of GPCR agonists for a single pathway. This analysis also suggests that the activators of GLP-1, GIP and FFA receptors may have a relatively low risk of hypoglycemia in fasting conditions whereas an activator of muscarinic receptors can increase this risk. This computational analysis demonstrates that study of second messenger

  2. Effects of dopamine receptor agonists on the cAMP content in arteries of the rabbit%多巴胺受体激动剂对兔动脉cAMP产生系统的影响

    Institute of Scientific and Technical Information of China (English)

    朱琳; 赵荣瑞; 张玮芳

    2000-01-01

    实验观察了选择性多巴胺(DA)DA1受体激动剂fenoldopam与DA2受体激动剂propy1-butyl-dopamine (PBDA)对兔肾动脉, 肺、肠系膜动脉和股动脉环磷酸腺苷(cAMP)产生系统的影响.结果表明: (1)除股动脉外, fenoldopam均可浓度依赖性地增加肺动脉、肾动脉和肠系膜动脉cAMP的生成量.选择性DA1受体阻断剂SCH23390可以显著阻断fenoldopam的效应, 而DA2受体阻断剂domperidone则对fenoldopam的这一效应不产生任何影响.(2) PBDA可浓度依赖性地降低股动脉的cAMP生成量, 又可浓度依赖性地激活肠系膜动脉、肺动脉和肾动脉的腺苷酸环化酶(AC)活性, 增加cAMP的生成量.(3) domperidone 可明显减小PBDA对股动脉AC活性的抑制效应, 并可使PBDA对肠系膜动脉AC活性效应增强, 但不改变PBDA对肾动脉和肺动脉的AC活性效应.(4) SCH23390不影响PBDA对股动脉AC活性的抑制效应, 可显著降低PBDA对肾动脉、肺动脉和肠系膜动脉激活AC活性的效应, cAMP的含量显著减小.以上结果提示, 在肺动脉、肾动脉和肠系膜动脉上存在DA1受体介导的cAMP产生系统, 而在股动脉仅有DA2受体介导的cAMP产生系统.在肠系膜动脉既存在DA2受体介导的cAMP产生系统, 又存在DA1受体介导的cAMP产生系统.PBDA既有通过刺激DA2受体抑制AC活性的作用, 又有刺激DA1受体而激活AC活性、增加cAMP的效应.%Effects of selective dopamine-1 (DA1) receptor agonist fenoldopam and dopamine-2 (DA2) receptor agonist propy1-butyl-dopamine (PBDA) on the cAMP generation system in renal, pulmonary, mesenteric and femoral arteries of rabbits were studied. The results are as follows. (1) Fenoldopam increased the cAMP production in a dose-dependent manner in pulmonary, renal and mesenteric arteries. This effect of fenoldopam was markedly blocked by specific DA1 receptor antagonist SCH23390, but not at all by specific DA2 receptor antagonist domperidone. (2) PBDA induced a dose

  3. The central role of cAMP in regulating Plasmodium falciparum merozoite invasion of human erythrocytes.

    Directory of Open Access Journals (Sweden)

    Amrita Dawn

    2014-12-01

    Full Text Available All pathogenesis and death associated with Plasmodium falciparum malaria is due to parasite-infected erythrocytes. Invasion of erythrocytes by P. falciparum merozoites requires specific interactions between host receptors and parasite ligands that are localized in apical organelles called micronemes. Here, we identify cAMP as a key regulator that triggers the timely secretion of microneme proteins enabling receptor-engagement and invasion. We demonstrate that exposure of merozoites to a low K+ environment, typical of blood plasma, activates a bicarbonate-sensitive cytoplasmic adenylyl cyclase to raise cytosolic cAMP levels and activate protein kinase A, which regulates microneme secretion. We also show that cAMP regulates merozoite cytosolic Ca2+ levels via induction of an Epac pathway and demonstrate that increases in both cAMP and Ca2+ are essential to trigger microneme secretion. Our identification of the different elements in cAMP-dependent signaling pathways that regulate microneme secretion during invasion provides novel targets to inhibit blood stage parasite growth and prevent malaria.

  4. Nanobody stabilization of G protein coupled receptor conformational states

    OpenAIRE

    Steyaert, Jan; K Kobilka, Brian

    2011-01-01

    Remarkable progress has been made in the field of G protein coupled receptor (GPCR) structural biology during the past four years. Several obstacles to generating diffraction quality crystals of GPCRs have been overcome by combining innovative methods ranging from protein engineering to lipid-based screens and microdiffraction technology. The initial GPCR structures represent energetically stable inactive-state conformations. However, GPCRs signal through different G protein isoforms or G pro...

  5. Lesbian camp: An unearthing.

    Science.gov (United States)

    Nielsen, Elly-Jean

    2016-01-01

    Camp-a sensibility, a style, and a form of artistic self-expression-is an elusive concept said to be in the eye of the beholder. To refute Susan Sontag's ( 1966 ) claims that camp is apolitical and not especially homosexual, a number of recent scholarly works have been geared toward revealing camp's fundamental gayness. With the odd footnote aside, lesbian camp has been collapsed into the category of gay male camp, if not eclipsed entirely. Despite the negligible efforts made to legitimize lesbian camp, there are numerous salient cultural examples one might draw on to illustrate, typify, and substantiate a lesbian camp sensibility. I lay the ground work for this scholarly exercise by outlining various definitions and critiques of camp, and by discussing its history and application to queer theory. Then, to unveil lesbian camp, three non-mutually exclusive categories are discussed: classic, erotic, and radical. By gathering various strands of inquiry, and various textual examples (e.g., photography, artistic performances, and literary tropes), this article attempts to reach a more inclusive and nuanced understanding of lesbian camp.

  6. Nonlinear pharmacokinetics of therapeutic proteins resulting from receptor mediated endocytosis.

    Science.gov (United States)

    Krippendorff, Ben-Fillippo; Kuester, Katharina; Kloft, Charlotte; Huisinga, Wilhelm

    2009-06-01

    Receptor mediated endocytosis (RME) plays a major role in the disposition of therapeutic protein drugs in the body. It is suspected to be a major source of nonlinear pharmacokinetic behavior observed in clinical pharmacokinetic data. So far, mostly empirical or semi-mechanistic approaches have been used to represent RME. A thorough understanding of the impact of the properties of the drug and of the receptor system on the resulting nonlinear disposition is still missing, as is how to best represent RME in pharmacokinetic models. In this article, we present a detailed mechanistic model of RME that explicitly takes into account receptor binding and trafficking inside the cell and that is used to derive reduced models of RME which retain a mechanistic interpretation. We find that RME can be described by an extended Michaelis-Menten model that accounts for both the distribution and the elimination aspect of RME. If the amount of drug in the receptor system is negligible a standard Michaelis-Menten model is capable of describing the elimination by RME. Notably, a receptor system can efficiently eliminate drug from the extracellular space even if the total number of receptors is small. We find that drug elimination by RME can result in substantial nonlinear pharmacokinetics. The extent of nonlinearity is higher for drug/receptor systems with higher receptor availability at the membrane, or faster internalization and degradation of extracellular drug. Our approach is exemplified for the epidermal growth factor receptor system.

  7. Loss of Kv3.1 tonotopicity and alterations in cAMP response element-binding protein signaling in central auditory neurons of hearing impaired mice.

    Science.gov (United States)

    von Hehn, Christian A A; Bhattacharjee, Arin; Kaczmarek, Leonard K

    2004-02-25

    The promoter for the kv3.1 potassium channel gene is regulated by a Ca2+-cAMP responsive element, which binds the transcription factor cAMP response element-binding protein (CREB). Kv3.1 is expressed in a tonotopic gradient within the medial nucleus of the trapezoid body (MNTB) of the auditory brainstem, where Kv3.1 levels are highest at the medial end, which corresponds to high auditory frequencies. We have compared the levels of Kv3.1, CREB, and the phosphorylated form of CREB (pCREB) in a mouse strain that maintains good hearing throughout life, CBA/J (CBA), with one that suffers early cochlear hair cell loss, C57BL/6 (BL/6). A gradient of Kv3.1 immunoreactivity in the MNTB was detected in both young (6 week) and older (8 month) CBA mice. Although no gradient of CREB was detected, pCREB-immunopositive cells were grouped together in distinct clusters along the tonotopic axis. The same pattern of Kv3.1, CREB, and pCREB localization was also found in young BL/6 mice at a time (6 weeks) when hearing is normal. In contrast, at 8 months, when hearing is impaired, the gradient of Kv3.1 was abolished. Moreover, in the older BL/6 mice there was a decrease in CREB expression along the tonotopic axis, and the pattern of pCREB labeling appeared random, with no discrete clusters of pCREB-positive cells along the tonotopic axis. Our findings are consistent with the hypothesis that ongoing activity in auditory brainstem neurons is necessary for the maintenance of Kv3.1 tonotopicity through the CREB pathway.

  8. Synaptic proteins and receptors defects in autism spectrum disorders

    Directory of Open Access Journals (Sweden)

    Jianling eChen

    2014-09-01

    Full Text Available Recent studies have found that hundreds of genetic variants, including common and rare variants, rare and de novo mutations, and common polymorphisms have contributed to the occurrence of autism spectrum disorders (ASDs. The mutations in a number of genes such as neurexin, neuroligin, postsynaptic density protein 95 (PSD-95, SH3 and multiple ankyrin repeat domains 3 (SHANK3, synapsin, gephyrin, cadherin (CDH and protocadherin (PCDH, thousand-and-one-amino acid 2 kinase (TAOK2, and contactin (CNTN, have been shown to play important roles in the development and function of synapses. In addition, synaptic receptors, such as gamma-aminobutyric acid (GABA receptors and glutamate receptors, have also been associated with ASDs. This review will primarily focus on the defects of synaptic proteins and receptors associated with ASDs and their roles in the pathogenesis of ASDs via synaptic pathways.

  9. Multiple switches in G protein-coupled receptor activation.

    Science.gov (United States)

    Ahuja, Shivani; Smith, Steven O

    2009-09-01

    The activation mechanism of G protein-coupled receptors has presented a puzzle that finally may be close to solution. These receptors have a relatively simple architecture consisting of seven transmembrane helices that contain just a handful of highly conserved amino acids, yet they respond to light and a range of chemically diverse ligands. Recent NMR structural studies on the active metarhodopsin II intermediate of the visual receptor rhodopsin, along with the recent crystal structure of the apoprotein opsin, have revealed multiple structural elements or 'switches' that must be simultaneously triggered to achieve full activation. The confluence of several required structural changes is an example of "coincidence counting", which is often used by nature to regulate biological processes. In ligand-activated G protein-coupled receptors, the presence of multiple switches may provide an explanation for the differences between full, partial and inverse agonists.

  10. [G-protein-coupled receptors targeting: the allosteric approach].

    Science.gov (United States)

    Sebag, Julien A; Pantel, Jacques

    2012-10-01

    G-protein-coupled receptors (GPCR) are a major family of drug targets. Essentially all drugs targeting these receptors on the market compete with the endogenous ligand (agonists or antagonists) for binding the receptor. Recently, non-competitive compounds binding to distinct sites from the cognate ligand were documented in various classes of these receptors. These compounds, called allosteric modulators, generally endowed of a better selectivity are able to modulate specifically the endogenous signaling of the receptor. To better understand the promising potential of this class of GPCRs targeting compounds, this review highlights the properties of allosteric modulators, the strategies used to identify them and the challenges associated with the development of these compounds.

  11. Biased and G protein-independent signaling of chemokine receptors

    Directory of Open Access Journals (Sweden)

    Anne eSteen

    2014-06-01

    Full Text Available Biased signaling or functional selectivity occurs when a 7TM receptor preferentially activates one of several available pathways. It can be divided into three distinct forms: ligand bias, receptor bias, and tissue or cell bias, where it is mediated by different ligands (on the same receptor, different receptors (with the same ligand or different tissues or cells (for the same ligand-receptor pair. Most often biased signaling is differentiated into G protein-dependent and β-arrestin-dependent signaling. Yet, it may also cover signaling differences within these groups. Moreover, it may not be absolute, i.e. full versus no activation. Here we discuss biased signaling in the chemokine system, including the structural basis for biased signaling in chemokine receptors, as well as in class A 7TM receptors in general. This includes overall helical movements and the contributions of micro-switches based on recently published 7TM crystals and molecular dynamics studies. All three forms of biased signaling are abundant in the chemokine system. This challenges our understanding of classic redundancy inevitably ascribed to this system, where multiple chemokines bind to the same receptor and where a single chemokine may bind to several receptors – in both cases with the same functional outcome. The ubiquitous biased signaling confer a hitherto unknown specificity to the chemokine system with a complex interaction pattern that is better described as promiscuous with context-defined roles and different functional outcomes in a ligand-, receptor- or cell/tissue-defined manner. As the low number of successful drug development plans implies, there are great difficulties in targeting chemokine receptors; in particular with regard to receptor antagonists as anti-inflammatory drugs. Un-defined and putative non-selective targeting of the complete cellular signaling system could be the underlying cause of lack of success. Therefore, biased ligands could be the

  12. The repertoire of trace amine G-protein-coupled receptors

    DEFF Research Database (Denmark)

    Gloriam, David E.; Bjarnadóttir, Thóra K; Yan, Yi-Lin

    2005-01-01

    Trace amines, such as tyramine, beta-phenylethylamine, tryptamine, and octopamine, are present in trace levels in nervous systems and bind a specific family of G-protein-coupled receptors (GPCR), but the function or origin of this system is not well understood. We searched the genomes of several ...... ancestor of vertebrate TA-receptors arose before the split of the ray-finned and lobe-finned fishes. The evolutionary history of the TA-receptors is more complex than for most other GPCR families and here we suggest a mechanism by which they may have arisen....

  13. Metabotropic glutamate receptors and interacting proteins: evolving drug targets.

    Science.gov (United States)

    Enz, Ralf

    2012-01-01

    The correct targeting, localization, regulation and signaling of metabotropic glutamate receptors (mGluRs) represent major mechanisms underlying the complex function of neuronal networks. These tasks are accomplished by the formation of synaptic signal complexes that integrate functionally related proteins such as neurotransmitter receptors, enzymes and scaffold proteins. By these means, proteins interacting with mGluRs are important regulators of glutamatergic neurotransmission. Most described mGluR interaction partners bind to the intracellular C-termini of the receptors. These domains are extensively spliced and phosphorylated, resulting in a high variability of binding surfaces offered to interacting proteins. Malfunction of mGluRs and associated proteins are linked to neurodegenerative and neuropsychiatric disorders including addiction, depression, epilepsy, schizophrenia, Alzheimer's, Huntington's and Parkinson's disease. MGluR associated signal complexes are dynamic structures that assemble and disassemble in response to the neuronal fate. This, in principle, allows therapeutic intervention, defining mGluRs and interacting proteins as promising drug targets. In the last years, several studies elucidated the geometry of mGluRs in contact with regulatory proteins, providing a solid fundament for the development of new therapeutic strategies. Here, I will give an overview of human disorders directly associated with mGluR malfunction, provide an up-to-date summary of mGluR interacting proteins and highlight recently described structures of mGluR domains in contact with binding partners.

  14. The effect of hypoxia on PGE2-stimulated cAMP generation in HMEC-1.

    Science.gov (United States)

    Wiktorowska-Owczarek, Anna; Owczarek, Jacek

    2015-06-01

    Prostaglandin E2 (PGE2) is generated in various cells, including endothelial cells, and is responsible for various functions, such as vascular relaxation and angiogenesis. Effects of PGE2 are mediated via receptors EP1-EP4, among which EP2 and EP4 are coupled to Gs protein which activates adenylate cyclase (AC) and cAMP synthesis. The aim of this work was to study the ability of human microvascular endothelial cells (HMEC-1) to synthesize cAMP in the presence of PGE2, and to determine the effect of hypoxia on the PGE2- stimulated cAMP level. It was decided to evaluate the effect of PGE2 on the secretion of VEGF, an inducer of angiogenesis. In summary, our findings show that PGE2 induces cAMP production, but hypoxia may impair PGE2-stimulated activity of the AC-cAMP signaling pathway. These results suggest that the cardioprotective effect of PGE2/EP4/cAMP may be attenuated during ischemia. Furthermore, this study indicates that the pro-angiogenic effect of PGE2 is not associated with VEGF secretion in HMEC-1 cells.

  15. Recreation Summer Camps 2016

    Data.gov (United States)

    Montgomery County of Maryland — List of all Camps (Register here:https://apm.activecommunities.com/montgomerycounty/Home) to include Aquatics, Basketball, Soccer, Special Interest, General Sports,...

  16. Registration Summer Camp 2016

    CERN Multimedia

    2016-01-01

    Reminder: registration for the CERN Staff Association Summer Camp is now open for children from 4 to 6 years old.   More information on the website: http://nurseryschool.web.cern.ch/. The summer camp is open to all children. The proposed cost is 480.-CHF/week, lunch included. The camp will be open weeks 27, 28, 29 and 30, from 8:30 a.m. to 5:30 p.m. For further questions, you are welcome to contact us by email at Summer.Camp@cern.ch. CERN Staff Association

  17. Reactive oxygen species decrease cAMP response element binding protein expression in cardiomyocytes via a protein kinase D1-dependent mechanism that does not require Ser133 phosphorylation.

    Science.gov (United States)

    Ozgen, Nazira; Guo, Jianfen; Gertsberg, Zoya; Danilo, Peter; Rosen, Michael R; Steinberg, Susan F

    2009-10-01

    Reactive oxygen species (ROS) exert pleiotropic effects on a wide array of signaling proteins that regulate cellular growth and apoptosis. This study shows that long-term treatment with a low concentration of H2O2 leads to the activation of signaling pathways involving extracellular signal-regulated kinase, ribosomal protein S6 kinase, and protein kinase D (PKD) that increase cAMP binding response element protein (CREB) phosphorylation at Ser(133) in cardiomyocytes. Although CREB-Ser(133) phosphorylation typically mediates cAMP-dependent increases in CREB target gene expression, the H2O2-dependent increase in CREB-Ser(133) phosphorylation is accompanied by a decrease in CREB protein abundance and no change in Cre-luciferase reporter activity. Mutagenesis studies indicate that H2O2 decreases CREB protein abundance via a mechanism that does not require CREB-Ser(133) phosphorylation. Rather, the H2O2-dependent decrease in CREB protein is prevented by the proteasome inhibitor lactacystin, by inhibitors of mitogen-activated protein kinase kinase or protein kinase C activity, or by adenoviral-mediated delivery of a small interfering RNA that decreases PKD1 expression. A PKD1-dependent mechanism that links oxidative stress to decreased CREB protein abundance is predicted to contribute to the pathogenesis of heart failure by influencing cardiac growth and apoptosis responses.

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

    DEFF Research Database (Denmark)

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

    2013-01-01

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

  19. Semiotic Selection of Mutated or Misfolded Receptor Proteins

    DEFF Research Database (Denmark)

    Giorgi, Franco; Bruni, Luis Emilio; Maggio, Roberto

    2013-01-01

    for receptor monomers to assemble along the membrane and to sustain meaningful relationships with environmental ligands. How could a cell lineage deal with these loss-of-function mutations during evolution and restrain gene redundancy accordingly? In this paper, we will be arguing that the easiest way...... for bacteria clones to accomplish this goal is by getting rid of cells expressing mutated receptor proteins. The mechanism sustaining this cell selection is also occurring in many somatic tissues and its function is currently believed to counteract in vivo protein mutagenesis. Our discussion will be mainly...

  20. Molecular pharmacology of G protein-coupled receptors.

    Science.gov (United States)

    Summers, R J

    2016-10-01

    This themed issue of the British Journal of Pharmacology stems from the eighth in the series of meetings on the Molecular Pharmacology of G protein coupled receptors (MPGPCR) held as part of a joint meeting with the Australasian Society of Clinical and Experimental Pharmacologists and Toxicologists (ASCEPT) in Melbourne Australia from 7 to 11 December 2014. Linked Articles This article is part of a themed section on Molecular Pharmacology of G Protein-Coupled Receptors. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v173.20/issuetoc.

  1. G protein-coupled receptor modulation with pepducins

    DEFF Research Database (Denmark)

    Dimond, Patricia; Carlson, Kenneth; Bouvier, Michel;

    2011-01-01

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

  2. G protein activation by G protein coupled receptors: ternary complex formation or catalyzed reaction?

    Science.gov (United States)

    Roberts, David J; Waelbroeck, Magali

    2004-09-01

    G protein coupled receptors catalyze the GDP/GTP exchange on G proteins, thereby activating them. The ternary complex model, designed to describe agonist binding in the absence of GTP, is often extended to G protein activation. This is logically unsatisfactory as the ternary complex does not accumulate when G proteins are activated by GTP. Extended models taking into account nucleotide binding exist, but fail to explain catalytic G protein activation. This review puts forward an enzymatic model of G protein activation and compares its predictions with the ternary complex model and with observed receptor phenomenon. This alternative model does not merely provide a new set of formulae but leads to a new philosophical outlook and more readily accommodates experimental observations. The ternary complex model implies that, HRG being responsible for efficient G protein activation, it should be as stable as possible. In contrast, the enzyme model suggests that although a limited stabilization of HRG facilitates GDP release, HRG should not be "too stable" as this might trap the G protein in an inactive state and actually hinder G protein activation. The two models also differ completely in the definition of the receptor "active state": the ternary complex model implies that the active state corresponds to a single active receptor conformation (HRG); in contrast, the catalytic model predicts that the active receptor state is mobile, switching smoothly through various conformations with high and low affinities for agonists (HR, HRG, HRGGDP, HRGGTP, etc.).

  3. Metalloprotease cleavage of the N terminus of the orphan G protein-coupled receptor GPR37L1 reduces its constitutive activity.

    Science.gov (United States)

    Coleman, James L J; Ngo, Tony; Schmidt, Johannes; Mrad, Nadine; Liew, Chu Kong; Jones, Nicole M; Graham, Robert M; Smith, Nicola J

    2016-04-12

    Little is known about the pharmacology or physiology of GPR37L1, a G protein (heterotrimeric guanine nucleotide-binding protein)-coupled receptor that is abundant in the cerebellum. Mice deficient in this receptor exhibit precocious cerebellar development and hypertension. We showed that GPR37L1 coupled to the G protein Gα(s) when heterologously expressed in cultured cells in the absence of any added ligand, whereas a mutant receptor that lacked the amino terminus was inactive. Conversely, inhibition of ADAMs (a disintegrin and metalloproteases) enhanced receptor activity, indicating that the presence of the amino terminus is necessary for GPR37L1 signaling. Metalloprotease-dependent processing of GPR37L1 was evident in rodent cerebellum, where we detected predominantly the cleaved, inactive form. However, comparison of the accumulation of cAMP (adenosine 3',5'-monophosphate) in response to phosphodiesterase inhibition in cerebellar slice preparations from wild-type and GPR37L1-null mice showed that some constitutive signaling remained in the wild-type mice. In reporter assays of Gα(s) or Gα(i) signaling, the synthetic, prosaposin-derived peptide prosaptide (TX14A) did not increase GPR37L1 activity. Our data indicate that GPR37L1 may be a constitutively active receptor, or perhaps its ligand is present under the conditions that we used for analysis, and that the activity of this receptor is instead controlled by signals that regulate metalloprotease activity in the tissue.

  4. Protease-Activated Receptors and other G-Protein-Coupled Receptors: the Melanoma Connection

    OpenAIRE

    Rosero, Rebecca A.; Villares, Gabriel J.; Bar-Eli, Menashe

    2016-01-01

    The vast array of G-protein-coupled receptors (GPCRs) play crucial roles in both physiological and pathological processes, including vision, coagulation, inflammation, autophagy, and cell proliferation. GPCRs also affect processes that augment cell proliferation and metastases in many cancers including melanoma. Melanoma is the deadliest form of skin cancer, yet limited therapeutic modalities are available to patients with metastatic melanoma. Studies have found that both chemokine receptors ...

  5. Adenylyl cyclase-associated protein 1 is a receptor for human resistin and mediates inflammatory actions of human monocytes.

    Science.gov (United States)

    Lee, Sahmin; Lee, Hyun-Chae; Kwon, Yoo-Wook; Lee, Sang Eun; Cho, Youngjin; Kim, Joonoh; Lee, Soobeom; Kim, Ju-Young; Lee, Jaewon; Yang, Han-Mo; Mook-Jung, Inhee; Nam, Ky-Youb; Chung, Junho; Lazar, Mitchell A; Kim, Hyo-Soo

    2014-03-04

    Human resistin is a cytokine that induces low-grade inflammation by stimulating monocytes. Resistin-mediated chronic inflammation can lead to obesity, atherosclerosis, and other cardiometabolic diseases. Nevertheless, the receptor for human resistin has not been clarified. Here, we identified adenylyl cyclase-associated protein 1 (CAP1) as a functional receptor for human resistin and clarified its intracellular signaling pathway to modulate inflammatory action of monocytes. We found that human resistin directly binds to CAP1 in monocytes and upregulates cyclic AMP (cAMP) concentration, protein kinase A (PKA) activity, and NF-κB-related transcription of inflammatory cytokines. Overexpression of CAP1 in monocytes enhanced the resistin-induced increased activity of the cAMP-dependent signaling. Moreover, CAP1-overexpressed monocytes aggravated adipose tissue inflammation in transgenic mice that express human resistin from their monocytes. In contrast, suppression of CAP1 expression abrogated the resistin-mediated inflammatory activity both in vitro and in vivo. Therefore, CAP1 is the bona fide receptor for resistin leading to inflammation in humans.

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

    Energy Technology Data Exchange (ETDEWEB)

    Kruse, Andrew C.; Manglik, Aashish; Kobilka, Brian K., E-mail: kobilka@stanford.edu [Stanford University, 279 Campus Drive, Stanford, CA 94305 (United States); Weis, William I., E-mail: kobilka@stanford.edu [Stanford University, 279 Campus Drive, Stanford, CA 94305 (United States); Stanford University, Fairchild Building, Stanford, CA 94305 (United States)

    2013-11-01

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

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

    Directory of Open Access Journals (Sweden)

    Kai Yang

    2014-02-01

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

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

    DEFF Research Database (Denmark)

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

    2013-01-01

    G protein-coupled receptors (GPCRs) constitute the largest family of membrane proteins in the human genome. Their signaling is regulated by scaffold proteins containing PDZ domains, but although these interactions are important for GPCR function, they are still poorly understood. We here present...... with colocalization of the full-length proteins in cells and with previous studies, we suggest that the range of relevant interactions might extend to interactions with K i = 450 µM in the in vitro assays. Within this range, we identify novel PSD-95 interactions with the chemokine receptor CXCR2, the neuropeptide Y...

  9. Membrane cholesterol access into a G-protein-coupled receptor

    Science.gov (United States)

    Guixà-González, Ramon; Albasanz, José L.; Rodriguez-Espigares, Ismael; Pastor, Manuel; Sanz, Ferran; Martí-Solano, Maria; Manna, Moutusi; Martinez-Seara, Hector; Hildebrand, Peter W.; Martín, Mairena; Selent, Jana

    2017-02-01

    Cholesterol is a key component of cell membranes with a proven modulatory role on the function and ligand-binding properties of G-protein-coupled receptors (GPCRs). Crystal structures of prototypical GPCRs such as the adenosine A2A receptor (A2AR) have confirmed that cholesterol finds stable binding sites at the receptor surface suggesting an allosteric role of this lipid. Here we combine experimental and computational approaches to show that cholesterol can spontaneously enter the A2AR-binding pocket from the membrane milieu using the same portal gate previously suggested for opsin ligands. We confirm the presence of cholesterol inside the receptor by chemical modification of the A2AR interior in a biotinylation assay. Overall, we show that cholesterol's impact on A2AR-binding affinity goes beyond pure allosteric modulation and unveils a new interaction mode between cholesterol and the A2AR that could potentially apply to other GPCRs.

  10. Membrane cholesterol access into a G-protein-coupled receptor

    Science.gov (United States)

    Guixà-González, Ramon; Albasanz, José L.; Rodriguez-Espigares, Ismael; Pastor, Manuel; Sanz, Ferran; Martí-Solano, Maria; Manna, Moutusi; Martinez-Seara, Hector; Hildebrand, Peter W.; Martín, Mairena; Selent, Jana

    2017-01-01

    Cholesterol is a key component of cell membranes with a proven modulatory role on the function and ligand-binding properties of G-protein-coupled receptors (GPCRs). Crystal structures of prototypical GPCRs such as the adenosine A2A receptor (A2AR) have confirmed that cholesterol finds stable binding sites at the receptor surface suggesting an allosteric role of this lipid. Here we combine experimental and computational approaches to show that cholesterol can spontaneously enter the A2AR-binding pocket from the membrane milieu using the same portal gate previously suggested for opsin ligands. We confirm the presence of cholesterol inside the receptor by chemical modification of the A2AR interior in a biotinylation assay. Overall, we show that cholesterol's impact on A2AR-binding affinity goes beyond pure allosteric modulation and unveils a new interaction mode between cholesterol and the A2AR that could potentially apply to other GPCRs. PMID:28220900

  11. Scrum Code Camps

    DEFF Research Database (Denmark)

    Pries-Heje, Jan; Pries-Heje, Lene; Dahlgaard, Bente

    2013-01-01

    is required. In this paper we present the design of such a new approach, the Scrum Code Camp, which can be used to assess agile team capability in a transparent and consistent way. A design science research approach is used to analyze properties of two instances of the Scrum Code Camp where seven agile teams...

  12. Orienteering in Camping.

    Science.gov (United States)

    Larson, Elston F.

    One of the recent developments in camping is "orienteering", a program using a map and compass. Orienteering can be dovetailed into an overall camping program and used to "point up" the entire program, or it can be confined to a single simple game. The arrangement depends on the situation. The minimum age of the participants should be about 9 or…

  13. Camp's "Disneyland" Effect.

    Science.gov (United States)

    Renville, Gary

    1999-01-01

    Describes the positive mental, physical, and social growth impacts that the camping experience had on the author, and urges camp program evaluation to plan and implement such changes. Sidebar lists steps of effective evaluation: program goals and objectives, goals of evaluation, implementation of evaluation, data analysis, and findings and…

  14. Friends' Discovery Camp

    Science.gov (United States)

    Seymour, Seth

    2008-01-01

    This article features Friends' Discovery Camp, a program that allows children with and without autism spectrum disorder to learn and play together. In Friends' Discovery Camp, campers take part in sensory-rich experiences, ranging from hands-on activities and performing arts to science experiments and stories teaching social skills. Now in its 7th…

  15. G-protein-coupled receptors for free fatty acids

    DEFF Research Database (Denmark)

    Milligan, Graeme; Ulven, Trond; Murdoch, Hannah;

    2014-01-01

    It is becoming evident that nutrients and metabolic intermediates derived from such nutrients regulate cellular function by activating a number of cell-surface G-protein coupled receptors (GPCRs). Until now, members of the GPCR family have largely been considered as the molecular targets that com...

  16. Pathogen receptor discovery with a microfluidic human membrane protein array

    Science.gov (United States)

    Glick, Yair; Ben-Ari, Ya’ara; Drayman, Nir; Pellach, Michal; Neveu, Gregory; Boonyaratanakornkit, Jim; Avrahami, Dorit; Einav, Shirit; Oppenheim, Ariella

    2016-01-01

    The discovery of how a pathogen invades a cell requires one to determine which host cell receptors are exploited. This determination is a challenging problem because the receptor is invariably a membrane protein, which represents an Achilles heel in proteomics. We have developed a universal platform for high-throughput expression and interaction studies of membrane proteins by creating a microfluidic-based comprehensive human membrane protein array (MPA). The MPA is, to our knowledge, the first of its kind and offers a powerful alternative to conventional proteomics by enabling the simultaneous study of 2,100 membrane proteins. We characterized direct interactions of a whole nonenveloped virus (simian virus 40), as well as those of the hepatitis delta enveloped virus large form antigen, with candidate host receptors expressed on the MPA. Selected newly discovered membrane protein–pathogen interactions were validated by conventional methods, demonstrating that the MPA is an important tool for cellular receptor discovery and for understanding pathogen tropism. PMID:27044079

  17. Interaction of G protein coupled receptors and cholesterol.

    Science.gov (United States)

    Gimpl, Gerald

    2016-09-01

    G protein coupled receptors (GPCRs) form the largest receptor superfamily in eukaryotic cells. Owing to their seven transmembrane helices, large parts of these proteins are embedded in the cholesterol-rich plasma membrane bilayer. Thus, GPCRs are always in proximity to cholesterol. Some of them are functionally dependent on the specific presence of cholesterol. Over the last years, enormous progress on receptor structures has been achieved. While lipophilic ligands other than cholesterol have been shown to bind either inside the helix bundle or at the receptor-lipid interface, the binding site of cholesterol was either a single transmembrane helix or a groove between two or more transmembrane helices. A clear preference for one of the two membrane leaflets has not been observed. Not surprisingly, many hydrophobic residues (primarily leucine and isoleucine) were found to be involved in cholesterol binding. In most cases, the rough β-face of cholesterol contacted the transmembrane helix bundle rather than the surrounding lipid matrix. The polar hydroxy group of cholesterol was localized near the water-membrane interface with potential hydrogen bonding to residues in receptor loop regions. Although a canonical motif, designated as CCM site, was detected as a specific cholesterol binding site in case of the β2AR, this site was not found to be occupied by cholesterol in other GPCRs possessing the same motif. Cholesterol-receptor interactions can increase the compactness of the receptor structure and are able to enhance the conformational stability towards active or inactive receptor states. Overall, all current data suggest a high plasticity of cholesterol interaction sites in GPCRs.

  18. G-protein-coupled receptors and localized signaling in the primary cilium during ventral neural tube patterning.

    Science.gov (United States)

    Hwang, Sun-Hee; Mukhopadhyay, Saikat

    2015-01-01

    The primary cilium is critical in sonic hedgehog (Shh)-dependent ventral patterning of the vertebrate neural tube. Most mutants that cause disruption of the cilium result in decreased Shh signaling in the neural tube. In contrast, mutations in the intraflagellar complex A (IFT-A) and the tubby family protein, Tulp3, result in increased Shh signaling in the neural tube. Proteomic analysis of Tulp3-binding proteins first pointed to the role of the IFT-A complex in trafficking Tulp3 into the cilia. Tulp3 directs trafficking of rhodopsin family G-protein-coupled receptors (GPCRs) to the cilia, suggesting the role of a GPCR in mediating the paradoxical effects of the Tulp3/IFT-A complex in causing increased Shh signaling. Gpr161 has recently been identified as a Tulp3/IFT-A-regulated GPCR that localizes to the primary cilium. A null knock-out mouse model of Gpr161 phenocopies Tulp3 and IFT-A mutants, and causes increased Shh signaling throughout the neural tube. In the absence of Shh, the bifunctional Gli transcription factors are proteolytically processed into repressor forms in a protein kinase A (PKA) -dependent and cilium-dependent manner. Gpr161 activity results in increased cAMP levels in a Gαs -coupled manner, and determines processing of Gli3. Shh signaling also results in removal of Gpr161 from the cilia, suggesting that Gpr161 functions in a positive feedback loop in the Shh pathway. As PKA-null and Gαs mutant embryos also exhibit increased Shh signaling in the neural tube, Gpr161 is a strong candidate for a GPCR that regulates ciliary cAMP levels, and activates PKA in close proximity to the cilia.

  19. The G protein-coupled bile acid receptor, TGR5, stimulates gallbladder filling.

    Science.gov (United States)

    Li, Tingting; Holmstrom, Sam R; Kir, Serkan; Umetani, Michihisa; Schmidt, Daniel R; Kliewer, Steven A; Mangelsdorf, David J

    2011-06-01

    TGR5 is a G protein-coupled bile acid receptor present in brown adipose tissue and intestine, where its agonism increases energy expenditure and lowers blood glucose. Thus, it is an attractive drug target for treating human metabolic disease. However, TGR5 is also highly expressed in gallbladder, where its functions are less well characterized. Here, we demonstrate that TGR5 stimulates the filling of the gallbladder with bile. Gallbladder volume was increased in wild-type but not Tgr5(-/-) mice by administration of either the naturally occurring TGR5 agonist, lithocholic acid, or the synthetic TGR5 agonist, INT-777. These effects were independent of fibroblast growth factor 15, an enteric hormone previously shown to stimulate gallbladder filling. Ex vivo analyses using gallbladder tissue showed that TGR5 activation increased cAMP concentrations and caused smooth muscle relaxation in a TGR5-dependent manner. These data reveal a novel, gallbladder-intrinsic mechanism for regulating gallbladder contractility. They further suggest that TGR5 agonists should be assessed for effects on human gallbladder as they are developed for treating metabolic disease.

  20. Direct interactions between calcitonin-like receptor (CLR) and CGRP-receptor component protein (RCP) regulate CGRP receptor signaling.

    Science.gov (United States)

    Egea, Sophie C; Dickerson, Ian M

    2012-04-01

    Calcitonin gene-related peptide (CGRP) is a neuropeptide with multiple neuroendocrine roles, including vasodilation, migraine, and pain. The receptor for CGRP is a G protein-coupled receptor (GPCR) that requires three proteins for function. CGRP binds to a heterodimer composed of the GPCR calcitonin-like receptor (CLR) and receptor activity-modifying protein (RAMP1), a single transmembrane protein required for pharmacological specificity and trafficking of the CLR/RAMP1 complex to the cell surface. In addition, the CLR/RAMP1 complex requires a third protein named CGRP-receptor component protein (RCP) for signaling. Previous studies have demonstrated that depletion of RCP from cells inhibits CLR signaling, and in vivo studies have demonstrated that expression of RCP correlates with CLR signaling and CGRP efficacy. It is not known whether RCP interacts directly with CLR to exert its effect. The current studies identified a direct interaction between RCP and an intracellular domain of CLR using yeast two-hybrid analysis and coimmunoprecipitation. When this interacting domain of CLR was expressed as a soluble fusion protein, it coimmunoprecipitated with RCP and inhibited signaling from endogenous CLR. Expression of this dominant-negative domain of CLR did not significantly inhibit trafficking of CLR to the cell surface, and thus RCP may not have a chaperone function for CLR. Instead, RCP may regulate CLR signaling in the cell membrane, and direct interaction between RCP and CLR is required for CLR activation. To date, RCP has been found to interact only with CLR and represents a novel neuroendocrine regulatory step in GPCR signaling.

  1. Cell-Surface Receptors Transactivation Mediated by G Protein-Coupled Receptors

    Directory of Open Access Journals (Sweden)

    Fabio Cattaneo

    2014-10-01

    Full Text Available G protein-coupled receptors (GPCRs are seven transmembrane-spanning proteins belonging to a large family of cell-surface receptors involved in many intracellular signaling cascades. Despite GPCRs lack intrinsic tyrosine kinase activity, tyrosine phosphorylation of a tyrosine kinase receptor (RTK occurs in response to binding of specific agonists of several such receptors, triggering intracellular mitogenic cascades. This suggests that the notion that GPCRs are associated with the regulation of post-mitotic cell functions is no longer believable. Crosstalk between GPCR and RTK may occur by different molecular mechanism such as the activation of metalloproteases, which can induce the metalloprotease-dependent release of RTK ligands, or in a ligand-independent manner involving membrane associated non-receptor tyrosine kinases, such as c-Src. Reactive oxygen species (ROS are also implicated as signaling intermediates in RTKs transactivation. Intracellular concentration of ROS increases transiently in cells stimulated with GPCR agonists and their deliberated and regulated generation is mainly catalyzed by enzymes that belong to nicotinamide adenine dinucleotide phosphate (NADPH oxidase family. Oxidation and/or reduction of cysteine sulfhydryl groups of phosphatases tightly controls the activity of RTKs and ROS-mediated inhibition of cellular phosphatases results in an equilibrium shift from the non-phosphorylated to the phosphorylated state of RTKs. Many GPCR agonists activate phospholipase C, which catalyze the hydrolysis of phosphatidylinositol 4,5-bis-phosphate to produce inositol 1,4,5-triphosphate and diacylglicerol. The consequent mobilization of Ca2+ from endoplasmic reticulum leads to the activation of protein kinase C (PKC isoforms. PKCα mediates feedback inhibition of RTK transactivation during GPCR stimulation. Recent data have expanded the coverage of transactivation to include Serine/Threonine kinase receptors and Toll-like receptors

  2. Lipoic acid stimulates cAMP production via the EP2 and EP4 prostanoid receptors and inhibits IFN gamma synthesis and cellular cytotoxicity in NK cells

    OpenAIRE

    Salinthone, Sonemany; Schillace, Robynn V.; Marracci, Gail H.; Bourdette, Dennis N.; Carr, Daniel W.

    2008-01-01

    The antioxidant lipoic acid (LA) treats and prevents the animal model of multiple sclerosis (MS), experimental autoimmune encephalomyelitis (EAE). In an effort to understand the therapeutic potential of LA in MS, we sought to define the cellular mechanisms that mediate the effects of LA on human natural killer (NK) cells, which are important in innate immunity as the first line of defense against invading pathogens and tumor cells. We discovered that LA stimulates cAMP production in NK cells ...

  3. Ca2+ participates in α1B-adrenoceptor-mediated cAMP response in HEK293 cells

    Institute of Scientific and Technical Information of China (English)

    Yao SONG; Yun-fang LI; Er-dan DONG; Qi-de HAN; You-yi ZHANG

    2005-01-01

    Aim: To investigate the α1B-adrenoceptor (α1B-AR)-mediated cAMP response and underlying mechanisms in HEK293 cells. Methods: Full-length cDNA encoding α1B-AR was transfected into HEK293 cells using the calcium phosphate precipitation method, and α1B-AR expression and cAMP accumulation were determined by using the saturation radioligand binding assay and ion-exchange chromatography, respectively. Results: Under agonist stimulation, α1B-AR mediated cAMP synthesis in HEK293 cells, and blockade by PLC-PKC or tyrosine kinase did not reduce cAMP accumulation induced by NE. Pretreatment with pertussis toxin(PTX) had little effect on basal cAMP accumulation as well as norepinephrine(NE)-stimulated cAMP accumulation. In addition, pretreatment with cholera toxin(CTX) neither mimicked nor blocked the effect induced by NE. The extracellular Ca2+ chelator egtazic acid (EGTA), nonselective Ca2+ channel blocker CdC12 and calmodulin (CaM) inhibitor W-7 significantly reduced NE-induced cAMP accumulation from 1.59%±0.47% to 1.00%±0.31%, 0.78%±0.23%, and 0.90%±0.40%,respectively. Conclusion: By coupling with a PTX-insensitive G protein, α1B-AR promotes Ca2+ influx via receptor-dependent Ca2+ channels, then Ca2+ is linked to CaM to form a Ca2+-CaM complex, which stimulates adenylyl cyclase (AC),thereby increasing the cAMP production in HEK293 cell lines.

  4. Detection of novelty, but not memory of spatial habituation, is associated with an increase in phosphorylated cAMP response element-binding protein levels in the hippocampus.

    Science.gov (United States)

    Winograd, Milena; Viola, Haydée

    2004-01-01

    There is a growing body of evidence showing that the formation of associative memories is associated with an increase in phosphorylated cAMP response element-binding protein (pCREB) levels. We recently reported increased pCREB levels in the rat hippocampus after an exploration to a novel environment. In the present work, we studied whether this increment in CREB activation is associated with the formation of memory of habituation to a novel environment or with the detection of novelty. Rats were submitted to consecutive open field sessions at 3-h intervals. Measurement of the hippocampal pCREB level, carried out 1 h after each training session, showed that (1) it did not increase when rats explored a familiar environment; (2) it did not increase after a reexposure that improves the memory of habituation; (3) it increased after a brief novel exploration unable to form memory of habituation; and (4) it increased in amnesic rats for spatial habituation. Taken as a whole, our results suggest that the elevated pCREB level after a single open field exploration is not associated with the memory formation of habituation. It is indeed associated with the detection of a novel environment.

  5. Characterization of G-protein coupled receptor kinase interaction with the neurokinin-1 receptor using bioluminescence resonance energy transfer

    DEFF Research Database (Denmark)

    Jorgensen, Rasmus; Holliday, Nicholas D; Hansen, Jakob L

    2007-01-01

    To analyze the interaction between the neurokinin-1 (NK-1) receptor and G-protein coupled receptor kinases (GRKs), we performed bioluminescence resonance energy transfer(2) (BRET(2)) measurements between the family A NK-1 receptor and GRK2 and GRK5 as well as their respective kinase-inactive muta......To analyze the interaction between the neurokinin-1 (NK-1) receptor and G-protein coupled receptor kinases (GRKs), we performed bioluminescence resonance energy transfer(2) (BRET(2)) measurements between the family A NK-1 receptor and GRK2 and GRK5 as well as their respective kinase...

  6. A receptor for infectious and cellular prion protein

    Directory of Open Access Journals (Sweden)

    V.R. Martins

    1999-07-01

    Full Text Available Prions are an unconventional form of infectious agents composed only of protein and involved in transmissible spongiform encephalopathies in humans and animals. The infectious particle is composed by PrPsc which is an isoform of a normal cellular glycosyl-phosphatidylinositol (GPI anchored protein, PrPc, of unknown function. The two proteins differ only in conformation, PrPc is composed of 40% a helix while PrPsc has 60% ß-sheet and 20% a helix structure. The infection mechanism is trigged by interaction of PrPsc with cellular prion protein causing conversion of the latter's conformation. Therefore, the infection spreads because new PrPsc molecules are generated exponentially from the normal PrPc. The accumulation of insoluble PrPsc is probably one of the events that lead to neuronal death. Conflicting data in the literature showed that PrPc internalization is mediated either by clathrin-coated pits or by caveolae-like membranous domains. However, both pathways seem to require a third protein (a receptor or a prion-binding protein either to make the connection between the GPI-anchored molecule to clathrin or to convert PrPc into PrPsc. We have recently characterized a 66-kDa membrane receptor which binds PrPc in vitro and in vivo and mediates the neurotoxicity of a human prion peptide. Therefore, the receptor should have a role in the pathogenesis of prion-related diseases and in the normal cellular process. Further work is necessary to clarify the events triggered by the association of PrPc/PrPsc with the receptor.

  7. Computational methods for studying G protein-coupled receptors (GPCRs).

    Science.gov (United States)

    Kaczor, Agnieszka A; Rutkowska, Ewelina; Bartuzi, Damian; Targowska-Duda, Katarzyna M; Matosiuk, Dariusz; Selent, Jana

    2016-01-01

    The functioning of GPCRs is classically described by the ternary complex model as the interplay of three basic components: a receptor, an agonist, and a G protein. According to this model, receptor activation results from an interaction with an agonist, which translates into the activation of a particular G protein in the intracellular compartment that, in turn, is able to initiate particular signaling cascades. Extensive studies on GPCRs have led to new findings which open unexplored and exciting possibilities for drug design and safer and more effective treatments with GPCR targeting drugs. These include discovery of novel signaling mechanisms such as ligand promiscuity resulting in multitarget ligands and signaling cross-talks, allosteric modulation, biased agonism, and formation of receptor homo- and heterodimers and oligomers which can be efficiently studied with computational methods. Computer-aided drug design techniques can reduce the cost of drug development by up to 50%. In particular structure- and ligand-based virtual screening techniques are a valuable tool for identifying new leads and have been shown to be especially efficient for GPCRs in comparison to water-soluble proteins. Modern computer-aided approaches can be helpful for the discovery of compounds with designed affinity profiles. Furthermore, homology modeling facilitated by a growing number of available templates as well as molecular docking supported by sophisticated techniques of molecular dynamics and quantitative structure-activity relationship models are an excellent source of information about drug-receptor interactions at the molecular level.

  8. Shc adaptor proteins are key transducers of mitogenic signaling mediated by the G protein-coupled thrombin receptor

    DEFF Research Database (Denmark)

    Chen, Y; Grall, D; Salcini, A E

    1996-01-01

    The serine protease thrombin activates G protein signaling systems that lead to Ras activation and, in certain cells, proliferation. Whereas the steps leading to Ras activation by G protein-coupled receptors are not well defined, the mechanisms of Ras activation by receptor tyrosine kinases have...... kinase activation, gene induction and cell growth. From these data, we conclude that Shc represents a crucial point of convergence between signaling pathways activated by receptor tyrosine kinases and G protein-coupled receptors....

  9. In vitro translation of androgen receptor cRNA results in an activated androgen receptor protein

    NARCIS (Netherlands)

    G.G.J.M. Kuiper (George); P.E. de Ruiter (Petra); J. Trapman (Jan); G.W. Jenster (Guido); A.O. Brinkmann (Albert)

    1993-01-01

    textabstractTranslation of androgen receptor (AR) cRNA in a reticulocyte lysate and subsequent analysis of the translation products by SDS/PAGE showed a protein with an apparent molecular mass of 108 kDa. Scatchard-plot analysis revealed a single binding component with

  10. Interaction of structure-specific and promiscuous G-protein-coupled receptors mediates small-molecule signaling in Caenorhabditis elegans.

    Science.gov (United States)

    Park, Donha; O'Doherty, Inish; Somvanshi, Rishi K; Bethke, Axel; Schroeder, Frank C; Kumar, Ujendra; Riddle, Donald L

    2012-06-19

    A chemically diverse family of small-molecule signals, the ascarosides, control developmental diapause (dauer), olfactory learning, and social behaviors of the nematode model organism, Caenorhabditis elegans. The ascarosides act upstream of conserved signaling pathways, including the insulin, TGF-β, serotonin, and guanylyl cyclase pathways; however, the sensory processes underlying ascaroside function are poorly understood. Because ascarosides often are multifunctional and show strongly synergistic effects, characterization of their receptors will be essential for understanding ascaroside biology and may provide insight into molecular mechanisms that produce synergistic outcomes in small-molecule sensing. Based on DAF-8 immunoprecipitation, we here identify two G-protein-coupled receptors, DAF-37 and DAF-38, which cooperatively mediate ascaroside perception. daf-37 mutants are defective in all responses to ascr#2, one of the most potent dauer-inducing ascarosides, although this mutant responds normally to other ascarosides. In contrast, daf-38 mutants are partially defective in responses to several different ascarosides. Through cell-specific overexpression, we show that DAF-37 regulates dauer when expressed in ASI neurons and adult behavior when expressed in ASK neurons. Using a photoaffinity-labeled ascr#2 probe and amplified luminescence assays (AlphaScreen), we demonstrate that ascr#2 binds to DAF-37. Photobleaching fluorescent energy transfer assays revealed that DAF-37 and DAF-38 form heterodimers, and we show that heterodimerization strongly increases cAMP inhibition in response to ascr#2. These results suggest that that the ascarosides' intricate signaling properties result in part from the interaction of highly structure-specific G-protein-coupled receptors such as DAF-37 with more promiscuous G-protein-coupled receptors such as DAF-38.

  11. 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

    2013-01-01

    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. PMID:23633472

  12. Regulation of melanogenesis: the role of cAMP and MITF

    Directory of Open Access Journals (Sweden)

    Michał Otręba

    2012-01-01

    Full Text Available The article presents the melanogenesis pathway and the role of cyclic adenosine monophosphate (cAMP and microphthalmia transcription factor (MITF in regulation of this process. Products of melanogenesis are eu- and/or pheomelanins synthesized in a multistage process of tyrosine oxidation and polymerization. The conversions require the presence of tyrosinase (TYR, key enzyme, tyrosine hydroxylase isoform I (THI and tyrosinase related proteins (TRP1 and TRP2. Many types of signal molecules and transcription factors participate in regulation of melanin synthesis, but the most important are cAMP and MITF. cAMP is the second messenger in the intracellular signal cascade, which is synthesized from adenosine triphosphate (ATP by adenylyl cyclase, activated among others by the melanocortin receptor and the αS subunit of G protein. The signal molecule cAMP regulates MITF, TYR, THI, GTP-cyclohydroxylase I (GTP-CHI transcription and phenylalanine hydroxylase (PAH phosphorylation at Ser16 by protein kinase A (PKA. Mutations of genes encoding proteins belonging to the cAMP signal cascade may lead to McCune-Albright and Carney syndromes. MITF is one of the most important nuclear transcription factors regulating melanogenesis. Currently 10 isoforms of human MITF are known, but in melanocytes only MITF-M, MITF-Mdel, MITF-A and MITF-H occur. MITF transcription factor regulates melanogenesis by activation of tyrosinase, TRP1 and TRP2 transcription. It also affects expression of other factors regulating melanosome maturation, biogenesis and transport. Moreover, it regulates melanocyte proliferation and protection against apoptosis. Mutations of the MITF gene may lead to hereditary diseases: Waardenburg type II and Tietz syndromes.

  13. The Cyclase-associated Protein CAP as Regulator of Cell Polarity and cAMP Signaling in Dictyostelium

    OpenAIRE

    Noegel, Angelika A; Blau-Wasser, Rosemarie; Sultana, Hameeda; Müller, Rolf; Israel, Lars; Schleicher, Michael; Patel, Hitesh; Weijer, Cornelis J

    2004-01-01

    Cyclase-associated protein (CAP) is an evolutionarily conserved regulator of the G-actin/F-actin ratio and, in yeast, is involved in regulating the adenylyl cyclase activity. We show that cell polarization, F-actin organization, and phototaxis are altered in a Dictyostelium CAP knockout mutant. Furthermore, in complementation assays we determined the roles of the individual domains in signaling and regulation of the actin cytoskeleton. We studied in detail the adenylyl cyclase activity and fo...

  14. Interaction of Hepatitis C virus proteins with pattern recognition receptors

    Directory of Open Access Journals (Sweden)

    Imran Muhammad

    2012-06-01

    Full Text Available Abstract Hepatitis C virus (HCV is an important human pathogen that causes acute and chronic hepatitis, cirrhosis and hepatocellular carcinoma worldwide. This positive stranded RNA virus is extremely efficient in establishing persistent infection by escaping immune detection or hindering the host immune responses. Recent studies have discovered two important signaling pathways that activate the host innate immunity against viral infection. One of these pathways utilizes members of Toll-like receptor (TLR family and the other uses the RNA helicase retinoic acid inducible gene I (RIG-I as the receptors for intracellular viral double stranded RNA (dsRNA, and activation of transcription factors. In this review article, we summarize the interaction of HCV proteins with various host receptors/sensors through one of these two pathways or both, and how they exploit these interactions to escape from host defense mechanisms. For this purpose, we searched data from Pubmed and Google Scholar. We found that three HCV proteins; Core (C, non structural 3/4 A (NS3/4A and non structural 5A (NS5A have direct interactions with these two pathways. Core protein only in the monomeric form stimulates TLR2 pathway assisting the virus to evade from the innate immune system. NS3/4A disrupts TLR3 and RIG-1 signaling pathways by cleaving Toll/IL-1 receptor domain-containing adapter inducing IFN-beta (TRIF and Cardif, the two important adapter proteins of these signaling cascades respectively, thus halting the defense against HCV. NS5A downmodulates the expressions of NKG2D on natural killer cells (NK cells via TLR4 pathway and impairs the functional ability of these cells. TLRs and RIG-1 pathways have a central role in innate immunity and despite their opposing natures to HCV proteins, when exploited together, HCV as an ever developing virus against host immunity is able to accumulate these mechanisms for near unbeatable survival.

  15. Protein Kinase C and Toll-Like Receptor Signaling

    Directory of Open Access Journals (Sweden)

    Daniel J. Loegering

    2011-01-01

    Full Text Available Protein kinase C (PKC is a family of kinases that are implicated in a plethora of diseases, including cancer and cardiovascular disease. PKC isoforms can have different, and sometimes opposing, effects in these disease states. Toll-like receptors (TLRs are a family of pattern recognition receptors that bind pathogens and stimulate the secretion of cytokines. It has long been known that PKC inhibitors reduce LPS-stimulated cytokine secretion by macrophages, linking PKC activation to TLR signaling. Recent studies have shown that PKC-α, -δ, -ε, and -ζ are directly involved in multiple steps in TLR pathways. They associate with the TLR or proximal components of the receptor complex. These isoforms are also involved in the downstream activation of MAPK, RhoA, TAK1, and NF-κB. Thus, PKC activation is intimately involved in TLR signaling and the innate immune response.

  16. The multiligand α2-macroglobulin receptor/low density lipoprotein receptor-related protein

    DEFF Research Database (Denmark)

    Gliemann, Jørgen; Nykjær, Anders; Petersen, Claus Munck;

    1994-01-01

    The fusion of separate lines of research has greatly helped in elucidating the function of the giant members of the low density lipoprotein (LDL) receptor (LDLR) supergene family. The cDNA encoding a large protein structurally closely related to LDLR, and hence named LDLR-related protein (LRP...... ab~y. ~af finity chromatography using immobilized a2M in the receptor-active conformation (a2M*). The first fusion occurred with the demonstration of identity between a2MR and LRP.'.' The receptor was thought to play the role of a double agent: as symbolized by its double name. The next fusion arose...... from the observation that affinity-purified a2MR/LRP contains a 40-kDa5.8 or 39-kDa6.' protein, designated a2MRAP, in addition to the a2MFULRP a- and P-chains. cDNA cloning" disclosed the 323-residue protein as both the human homologue of mouse heparin binding protein 44 (see reference 11) and...

  17. Protein intake during training sessions has no effect on performance and recovery during a strenuous training camp for elite cyclists

    DEFF Research Database (Denmark)

    Hansen, Mette; Bangsbo, Jens; Jensen, Jørgen;

    2016-01-01

    collected in the morning after overnight fasting during the week and analyzed for biochemical markers of muscle damage, stress, and immune function. RESULTS: In both groups, 5-min all-out performance was reduced after the first training session and at day 6 compared to before the first training session......, with no difference between groups. Peak power in the sprint test did not change significantly between tests or between groups. In addition, changes in markers for muscle damage, stress, and immune function were not significantly influenced by treatment. CONCLUSIONS: Intake of protein combined with carbohydrate...

  18. Human μ—opioid receptor overexpressed in Sf9 insect cells functionally coupled to endogenous Gi/0 proteins

    Institute of Scientific and Technical Information of China (English)

    WEIQIANG; QINGXIANGSHEN; 等

    2000-01-01

    Human μ-opioid receptor(HμOR) with a tag of six consecutive histidines at its carboxyl terminus has been expressed in recombinant baculovirus infected Sf9 insect cells.The maximal binding capacity for the [3H] diprenorphine and [3H] ohmefentanyl (Ohm) were 9.1±0.7 and 6.52±0.23 nmol/g protein,respectively.The [3H] diprenorphine or [3H] Ohm binding to the receptor expressed in Sf9 cells was strongly inhibited by μ-selective agonists [D-Ala2,N-methyl-Phe4,glyol5] enkephalin(DAGO),Ohm,and morphine,but neither by δ nor by κ selective agonist.Na+ (100mM) and GTP(50μM) could reduce HμOR agonists etorphine and Ohm affinity binding to the overexpressed HμOR.μ-selective agonists DAGO and Ohm effectively stimulated [35S]GTP γS binding (EC50=2.7nM and 6.9 nM)and inhibited forskolin-stimulated cAMP accumulation(IC50=0.9 nM and 0.3 nM).The agonist-dependent effects could be blocked by opioid antagonist naloxone or by pretreatment of cells with pertussis toxin (PTX).These results demonstrated that HμOR overexpressed in Sf9 insect cells functionally coupled to endogenous Ci/o proteins.

  19. A stimulus-specific role for CREB-binding protein (CBP) in T cell receptor-activated tumor necrosis factor gene expression

    Science.gov (United States)

    Falvo, James V.; Brinkman, Brigitta M. N.; Tsytsykova, Alla V.; Tsai, Eunice Y.; Yao, Tso-Pang; Kung, Andrew L.; Goldfeld, Anne E.

    2000-04-01

    The cAMP response element binding protein (CREB)-binding protein (CBP)/p300 family of coactivator proteins regulates gene transcription through the integration of multiple signal transduction pathways. Here, we show that induction of tumor necrosis factor (TNF-) gene expression in T cells stimulated by engagement of the T cell receptor (TCR) or by virus infection requires CBP/p300. Strikingly, in mice lacking one copy of the CBP gene, TNF- gene induction by TCR activation is inhibited, whereas virus induction of the TNF- gene is not affected. Consistent with these findings, the transcriptional activity of CBP is strongly potentiated by TCR activation but not by virus infection of T cells. Thus, CBP gene dosage and transcriptional activity are critical in TCR-dependent TNF-α gene expression, demonstrating a stimulus-specific requirement for CBP in the regulation of a specific gene.

  20. Hitler's Death Camps.

    Science.gov (United States)

    Wieser, Paul

    1995-01-01

    Presents a high school lesson on Hitler's death camps and the widespread policy of brutality and oppression against European Jews. Includes student objectives, instructional procedures, and a chart listing the value of used clothing taken from the Jews. (CFR)

  1. CDC Disease Detective Camp

    Centers for Disease Control (CDC) Podcasts

    2010-08-02

    The CDC Disease Detective Camp gives rising high school juniors and seniors exposure to key aspects of the CDC, including basic epidemiology, infectious and chronic disease tracking, public health law, and outbreak investigations. The camp also helps students explore careers in public health.  Created: 8/2/2010 by Centers for Disease Control and Prevention (CDC).   Date Released: 8/2/2010.

  2. Treponema pallidum receptor binding proteins interact with fibronectin

    Energy Technology Data Exchange (ETDEWEB)

    Peterson, K.M.; Baseman, J.B.; Alderete, J.F.

    1983-06-01

    Analysis of plasma proteins avidly bound to T. pallidum surfaces revealed the ability of T. pallidum to acquire numerous host macromolecules. No acquisition was evident by the avirulent spirochete, T. phagedenis biotype Reiter. Western blotting technology using hyperimmune antifibronectin serum as a probe revealed the ability of virulent treponemes to avidly bind fibronectin from a complex medium such as plasma. The specificity of the tiplike adherence of motile T. pallidum to fibronectin-coated glass surfaces and to fibronectin on HEp-2 cells was reinforced by the observation that pretreatment of coverslips or cell monolayers with monospecific antiserum against fibronectin substantially reduced T. pallidum attachment. The stoichiometric binding of T. pallidum to fibronectin-coated coverslips and the inability of unlabeled or /sup 35/S-radiolabeled treponemes to interact with glass surfaces treated with other plasma proteins further established the specific nature of the interaction between virulent T. pallidum and fibronectin. The avid association between three outer envelope proteins of T. pallidum and fibronectin was also demonstrated. These treponemal surface proteins have been previously identified as putative receptor-binding proteins responsible for T. pallidum parasitism of host cells. The data suggest that surface fibronectin mediates tip-oriented attachment of T. pallidum to host cells via a receptor-ligand mechanism of recognition.

  3. Stabilization of G protein-coupled receptors by point mutations

    Directory of Open Access Journals (Sweden)

    Franziska eHeydenreich

    2015-04-01

    Full Text Available G protein-coupled receptors (GPCRs are flexible integral membrane proteins involved in transmembrane signaling. Their involvement in many physiological processes makes them interesting targets for drug development. Determination of the structure of these receptors will help to design more specific drugs, however, their structural characterization has so far been hampered by the low expression and their inherent instability in detergents which made protein engineering indispensable for structural and biophysical characterization.Several approaches to stabilize the receptors in a particular conformation have led to breakthroughs in GPCR structure determination. These include truncations of the flexible regions, stabilization by antibodies and nanobodies, fusion partners, high affinity and covalently bound ligands as well as conformational stabilization by mutagenesis. In this review we focus on stabilization of GPCRs by insertion of point mutations, which lead to increased conformational and thermal stability as well as improved expression levels. We summarize existing mutagenesis strategies with different coverage of GPCR sequence space and depth of information, design and transferability of mutations and the molecular basis for stabilization. We also discuss whether mutations alter the structure and pharmacological properties of GPCRs.

  4. G protein coupled receptors as targets for next generation pesticides.

    Science.gov (United States)

    Audsley, Neil; Down, Rachel E

    2015-12-01

    There is an on-going need for the discovery and development of new pesticides due to the loss of existing products through the continuing development of resistance, the desire for products with more favourable environmental and toxicological profiles and the need to implement the principles of integrated pest management. Insect G protein coupled receptors (GPCRs) have important roles in modulating biology, physiology and behaviour, including reproduction, osmoregulation, growth and development. Modifying normal receptor function by blocking or over stimulating its actions may either result in the death of a pest or disrupt its normal fitness or reproductive capacity to reduce pest populations. Hence GPCRs offer potential targets for the development of next generation pesticides providing opportunities to discover new chemistries for invertebrate pest control. Such receptors are important targets for pharmaceutical drugs, but are under-exploited by the agro-chemical industry. The octopamine receptor agonists are the only pesticides with a recognized mode of action, as described in the classification scheme developed by the Insecticide Resistance Action Committee, that act via a GPCR. The availability of sequenced insect genomes has facilitated the characterization of insect GPCRs, but the development and utilization of screening assays to identify lead compounds has been slow. Various studies using knock-down technologies or applying the native ligands and/or neuropeptide analogues to pest insects in vivo, have however demonstrated that modifying normal receptor function can have an insecticidal effect. This review presents examples of potential insect neuropeptide receptors that are potential targets for lead compound development, using case studies from three representative pest species, Tribolium castaneum, Acyrthosiphon pisum, and Drosophila suzukii. Functional analysis studies on T. castaneum suggest that GPCRs involved in growth and development (eclosion

  5. Procedure for Calculation of Potency and Efficacy for Ligands Acting on G(s)- and G (i)-Coupled Receptors

    DEFF Research Database (Denmark)

    Meier, Eddi; Schousboe, Arne; Belhage, Bo

    2012-01-01

    Structure activity relationship (SAR) analyses of pharmacological data of compounds constitute an important part of the discovery process in the design of new drug candidates with improved pharmacological properties. In particular G-Protein Coupled Receptors (GPCRs) associated with the cAMP second...... messenger systems G(s) and G(i) have constituted one of the most widely used basis for pharmacological in vitro assays for assessing functional receptor effects. Such assays are based on Radio Immuno Assay (RIA) analysis to measure the cellular cAMP concentration as readout of receptor activation....... It appears, however, to be a common practice to omit the use of cAMP standard curves to transform the measured signals (cpm or cps) into cAMP concentrations on which estimations of potencies (EC(50) values) and efficacies (E(MAX) values) in G(s) and G(i) coupled receptor stimulation are based. Such practice...

  6. Bidirectional regulation of the cAMP response element binding protein encodes spatial map alignment in prism-adapting barn owls.

    Science.gov (United States)

    Nichols, Grant S; DeBello, William M

    2008-10-01

    The barn owl midbrain contains mutually aligned maps of auditory and visual space. Throughout life, map alignment is maintained through the actions of an instructive signal that encodes the magnitude of auditory-visual mismatch. The intracellular signaling pathways activated by this signal are unknown. Here we tested the hypothesis that CREB (cAMP response element-binding protein) provides a cell-specific readout of instructive information. Owls were fitted with prismatic or control spectacles and provided rich auditory-visual experience: hunting live mice. CREB activation was analyzed within 30 min of hunting using phosphorylation state-specific CREB (pCREB) and CREB antibodies, confocal imaging, and immunofluorescence measurements at individual cell nuclei. In control owls or prism-adapted owls, which experience small instructive signals, the frequency distributions of pCREB/CREB values obtained for cell nuclei within the external nucleus of the inferior colliculus (ICX) were unimodal. In contrast, in owls adapting to prisms or readapting to normal conditions, the distributions were bimodal: certain cells had received a signal that positively regulated CREB and, by extension, transcription of CREB-dependent genes, whereas others received a signal that negatively regulated it. These changes were restricted to the subregion of the inferior colliculus that received optically displaced input, the rostral ICX, and were not evident in the caudal ICX or central nucleus. Finally, the topographic pattern of CREB regulation was patchy, not continuous, as expected from the actions of a topographically precise signal encoding discrete events. These results support a model in which the magnitude of CREB activation within individual cells provides a readout of the instructive signal that guides plasticity and learning.

  7. Aldose Reductase Regulates Microglia/Macrophages Polarization Through the cAMP Response Element-Binding Protein After Spinal Cord Injury in Mice.

    Science.gov (United States)

    Zhang, Qian; Bian, Ganlan; Chen, Peng; Liu, Ling; Yu, Caiyong; Liu, Fangfang; Xue, Qian; Chung, Sookja K; Song, Bing; Ju, Gong; Wang, Jian

    2016-01-01

    Inflammatory reactions are the most critical pathological processes occurring after spinal cord injury (SCI). Activated microglia/macrophages have either detrimental or beneficial effects on neural regeneration based on their functional polarized M1/M2 subsets. However, the mechanism of microglia/macrophage polarization to M1/M2 at the injured spinal cord environment remains unknown. In this study, wild-type (WT) or aldose reductase (AR)-knockout (KO) mice were subjected to SCI by a spinal crush injury model. The expression pattern of AR, behavior tests for locomotor activity, and lesion size were assessed at between 4 h and 28 days after SCI. We found that the expression of AR is upregulated in microglia/macrophages after SCI in WT mice. In AR KO mice, SCI led to smaller injury lesion areas compared to WT. AR deficiency-induced microglia/macrophages induce the M2 rather than the M1 response and promote locomotion recovery after SCI in mice. In the in vitro experiments, microglia cell lines (N9 or BV2) were treated with the AR inhibitor (ARI) fidarestat. AR inhibition caused 4-hydroxynonenal (HNE) accumulation, which induced the phosphorylation of the cAMP response element-binding protein (CREB) to promote Arg1 expression. KG501, the specific inhibitor of phosphorylated CREB, could cancel the upregulation of Arg1 by ARI or HNE stimulation. Our results suggest that AR works as a switch which can regulate microglia by polarizing cells to either the M1 or the M2 phenotype under M1 stimulation based on its states of activity. We suggest that inhibiting AR may be a promising therapeutic method for SCI in the future.

  8. Novel cAMP targets in cell proliferation

    NARCIS (Netherlands)

    Kuiperij, Hinke Bertha

    2004-01-01

    cAMP is a second messenger that plays a role in a wide variety of biological processes, one of which is the regulation of cell proliferation. Adenylate cyclases generate cAMP in the cell upon activation, followed by binding to and activation of its direct targets, PKA and Epac. PKA is a protein kina

  9. Mechanisms of regulation and function of G-protein-coupled receptor kinases

    Institute of Scientific and Technical Information of China (English)

    Wen Yang; Shi-Hai Xia

    2006-01-01

    G-protein-coupled receptor kinases (GRKs) interact with the agonist-activated form of G-protein-coupled receptor (GPCR) to affect receptor phosphorylation and to initiate profound impairment of receptor signaling,or desensitization. GPCR forms the largest family of cell surface receptors, and defects in GRK function have the potential consequence to affect GPCR-stimulated biological responses in many pathological situations.

  10. Cultured lymphocytes from alcoholic subjects have altered cAMP signal transduction.

    OpenAIRE

    Nagy, L E; Diamond, I; Gordon, A.

    1988-01-01

    Previous work has shown that freshly isolated lymphocytes from alcoholic subjects show significantly reduced basal and adenosine receptor-stimulated cAMP levels. This decrease could be due to ethanol-induced cellular adaptation or to a genetic difference in the regulation of cAMP signal transduction. Therefore, we cultured human lymphocytes in defined medium without ethanol for 7-8 days and then examined differences in receptor-dependent cAMP accumulation between lymphocytes from alcoholic an...

  11. Human μ-opioid receptor overexpressed in Sf9 insect cells functionally coupled to endogenous Gi/o proteins

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Human μ-opioid receptor (HμOR) with a tag of six consecutive histidines at its carboxyl terminus had been expressed in recombinant baculovirus infected Sf9 insect cells.The maximal binding capacity for the [3H] diprenorphine and [3H]ohmefentanyl (Ohm) were 9.1 ± 0.7 and 6.52 ±0.23 nmol/g protein, respectively. The [3H] diprenorphine or [3H] Ohm binding to the receptor expressed in Sf9 cells was strongly inhibited by μ-selective agonists [D-Ala2, N-methyl-Phe4, glyol5]enkephalin (DAGO), Ohm, and morphine, but neither by δ nor by κ selective agonist. Na+ (100 mM) and GTP (50μM) could reduce HμOR agonists etorphine and Ohm affinity binding to the overexpressed HμOR.μ-selective agonists DAGO and Ohm effectively stimulated [35S]GTPγS binding (EC50 = 2.7nM and 6.9 nM) and inhibited forskolin- stimulated cAMP accumulation (IC50 = 0.9 nM and 0.3 nM). The agonist-dependent effects could be blocked by opioid antagonist naloxone or by pretreatment of cells with pertussis toxin (PTX). These results demonstrated that HμOR overexpressed in Sf9 insect cells functionally coupled to endogenous Gi/o proteins.Key words: Human μ-opioid receptor (Hμ OR), Sf9 insect cells, pertussis toxin (PTX), endogenous Gi/o proteins.

  12. Collagenase-3 binds to a specific receptor and requires the low density lipoprotein receptor-related protein for internalization

    Science.gov (United States)

    Barmina, O. Y.; Walling, H. W.; Fiacco, G. J.; Freije, J. M.; Lopez-Otin, C.; Jeffrey, J. J.; Partridge, N. C.

    1999-01-01

    We have previously identified a specific receptor for collagenase-3 that mediates the binding, internalization, and degradation of this ligand in UMR 106-01 rat osteoblastic osteosarcoma cells. In the present study, we show that collagenase-3 binding is calcium-dependent and occurs in a variety of cell types, including osteoblastic and fibroblastic cells. We also present evidence supporting a two-step mechanism of collagenase-3 binding and internalization involving both a specific collagenase-3 receptor and the low density lipoprotein receptor-related protein. Ligand blot analysis shows that (125)I-collagenase-3 binds specifically to two proteins ( approximately 170 kDa and approximately 600 kDa) present in UMR 106-01 cells. Western blotting identified the 600-kDa protein as the low density lipoprotein receptor-related protein. Our data suggest that the 170-kDa protein is a specific collagenase-3 receptor. Low density lipoprotein receptor-related protein-null mouse embryo fibroblasts bind but fail to internalize collagenase-3, whereas UMR 106-01 and wild-type mouse embryo fibroblasts bind and internalize collagenase-3. Internalization, but not binding, is inhibited by the 39-kDa receptor-associated protein. We conclude that the internalization of collagenase-3 requires the participation of the low density lipoprotein receptor-related protein and propose a model in which the cell surface interaction of this ligand requires a sequential contribution from two receptors, with the collagenase-3 receptor acting as a high affinity primary binding site and the low density lipoprotein receptor-related protein mediating internalization.

  13. Effects of ohmefentanyl stereoisomers on phosphorylation of cAMP- response element binding protein in cultured rat hippocampal neurons%羟甲芬太尼立体异构体对培养的海马神经元cAMP-反应元件结合蛋白磷酸化的影响

    Institute of Scientific and Technical Information of China (English)

    高灿; 谌立伟; 陶亦敏; 陈洁; 徐学军; 池志强

    2003-01-01

    AIM: To define the effects and signal pathways of ohmefentanyl stereoisomers [(-)-cis-(3R,4S,2'R) OMF (F9202), (+)-cis-(3R, 4S, 2′S) OMF (F9204), and (-)-cis-(3S, 4S, 2′R) OMF (F9203)] on the phosphorylation of cAMP-re sponse element binding protein (CREB) in cultured rat hippocampal neurons. METHODS: The effects of the three OMF stereoisomers and morphine (Mor) on cAMP accumulation and CREB phosphorylation were monitored by radioimmunoassay and Western blot analysis, respectively. RESULTS: The three OMF stereoisomers and Mor could all partially inhibit forskolin-stimulated (25 μmol/L, 15 min) cAMP accumulation in a dose-dependent manner and this effect could be reversed by naloxone. F9202, F9204, and Mor could significantly increase CREB phospho rylation from 2.88 to 3.59 folds over control levels after 30-min exposure. This effect was reversed by naloxone,but F9203 failed to increase CREB phosphorylation. KN-62 and staurosporine significantly blocked the opioidsinduced CREB phosphorylation, while H-89 and PD 98059 had no effect on the actions. CONCLUSION: Mor,F9202, and F9204, which could induce psychological dependence affected via the μ-opioid receptor, stimulated intracellular signal pathways involving Ca2+/calmodulin-dependent protein kinases (CCDPK) and protein kinase C (PKC) pathways, which in turn initiated CREB phosphorylation. F9203, which could not induce dependence, had no effect on CREB phosphorylation in hippocampal neurons. The increased CREB phosphorylation in hippocampal neurons may play a role in opioids dependence.

  14. Production of a bioengineered G-protein coupled receptor of human formyl peptide receptor 3.

    Directory of Open Access Journals (Sweden)

    Xiaoqiang Wang

    Full Text Available G-protein coupled receptors (GPCRs participate in a wide range of vital regulations of our physiological actions. They are also of pharmaceutical importance and have become many therapeutic targets for a number of disorders and diseases. Purified GPCR-based approaches including structural study and novel biophysical and biochemical function analyses are increasingly being used in GPCR-directed drug discovery. Before these approaches become routine, however, several hurdles need to be overcome; they include overexpression, solubilization, and purification of large quantities of functional and stable receptors on a regular basis. Here we report milligram production of a human formyl peptide receptor 3 (FPR3. FPR3 comprises a functionally distinct GPCR subfamily that is involved in leukocyte chemotaxis and activation. The bioengineered FPR3 was overexpressed in stable tetracycline-inducible mammalian cell lines (HEK293S. After a systematic detergent screening, fos-choline-14 (FC-14 was selected for subsequent solubilization and purification processes. A two-step purification method, immunoaffinity using anti-rho-tag monoclonal antibody 1D4 and gel filtration, was used to purify the receptors to near homogeneity. Immunofluorescence analysis showed that expressed FPR3 was predominantly displayed on cellular membrane. Secondary structural analysis using circular dichroism showed that the purified FPR3 receptor was correctly folded with >50% α-helix, which is similar to other known GPCR secondary structures. Our method can readily produce milligram quantities of human FPR3, which would facilitate in developing human FPR as therapeutic drug targets.

  15. A kinase-anchoring proteins and adenylyl cyclase in cardiovascular physiology and pathology.

    Science.gov (United States)

    Efendiev, Riad; Dessauer, Carmen W

    2011-10-01

    3'-5'-Cyclic adenosine monophosphate (cAMP), generated by adenylyl cyclase (AC), serves as a second messenger in signaling pathways regulating many aspects of cardiac physiology, including contraction rate and action potential duration, and in the pathophysiology of hypertrophy and heart failure. A kinase-anchoring proteins localize the effect of cAMP in space and time by organizing receptors, AC, protein kinase A, and other components of the cAMP cascade into multiprotein complexes. In this review, we discuss how the interaction of A kinase-anchoring proteins with distinct AC isoforms affects cardiovascular physiology.

  16. Role of G protein-coupled receptors in inflammation

    Institute of Scientific and Technical Information of China (English)

    Lei SUN; Richard DYE

    2012-01-01

    G protein-coupled receptors (GPCRs) play important roles in inflammation.Inflammatory cells such as polymorphonuclear leuko-cytes (PMN),monocytes and macrophages express a large number of GPCRs for classic chemoattractants and chemokines.These receptors are critical to the migration of phagocytes and their accumulation at sites of inflammation,where these cells can exacer-bate inflammation but also contribute to its resolution.Besides chemoattractant GPCRs,protease activated receptors (PARs) such as PAR1 are involved in the regulation of vascular endothelial permeability.Prostaglandin receptors play different roles in inflam-matory cell activation,and can mediate both proinflammatory and anti-inflammatory functions.Many GPCRs present in inflammatory cells also mediate transcription factor activation,resulting in the synthesis and secretion of inflammatory factors and,in some cases,molecules that suppress inflammation.An understanding of the signaling paradigms of GPCRs in inflammatory cells is likely to facilitate translational research and development of improved anti-inflammatory therapies.

  17. Competing G protein-coupled receptor kinases balance G protein and β-arrestin signaling.

    Science.gov (United States)

    Heitzler, Domitille; Durand, Guillaume; Gallay, Nathalie; Rizk, Aurélien; Ahn, Seungkirl; Kim, Jihee; Violin, Jonathan D; Dupuy, Laurence; Gauthier, Christophe; Piketty, Vincent; Crépieux, Pascale; Poupon, Anne; Clément, Frédérique; Fages, François; Lefkowitz, Robert J; Reiter, Eric

    2012-06-26

    Seven-transmembrane receptors (7TMRs) are involved in nearly all aspects of chemical communications and represent major drug targets. 7TMRs transmit their signals not only via heterotrimeric G proteins but also through β-arrestins, whose recruitment to the activated receptor is regulated by G protein-coupled receptor kinases (GRKs). In this paper, we combined experimental approaches with computational modeling to decipher the molecular mechanisms as well as the hidden dynamics governing extracellular signal-regulated kinase (ERK) activation by the angiotensin II type 1A receptor (AT(1A)R) in human embryonic kidney (HEK)293 cells. We built an abstracted ordinary differential equations (ODE)-based model that captured the available knowledge and experimental data. We inferred the unknown parameters by simultaneously fitting experimental data generated in both control and perturbed conditions. We demonstrate that, in addition to its well-established function in the desensitization of G-protein activation, GRK2 exerts a strong negative effect on β-arrestin-dependent signaling through its competition with GRK5 and 6 for receptor phosphorylation. Importantly, we experimentally confirmed the validity of this novel GRK2-dependent mechanism in both primary vascular smooth muscle cells naturally expressing the AT(1A)R, and HEK293 cells expressing other 7TMRs.

  18. Protein receptor-independent plasma membrane remodeling by HAMLET

    DEFF Research Database (Denmark)

    Nadeem, Aftab; Sanborn, Jeremy; Gettel, Douglas L.;

    2015-01-01

    A central tenet of signal transduction in eukaryotic cells is that extra-cellular ligands activate specific cell surface receptors, which orchestrate downstream responses. This "protein-centric" view is increasingly challenged by evidence for the involvement of specialized membrane domains...... in signal transduction. Here, we propose that membrane perturbation may serve as an alternative mechanism to activate a conserved cell-death program in cancer cells. This view emerges from the extraordinary manner in which HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells) kills a wide range...... of tumor cells in vitro and demonstrates therapeutic efficacy and selectivity in cancer models and clinical studies. We identify a "receptor independent" transformation of vesicular motifs in model membranes, which is paralleled by gross remodeling of tumor cell membranes. Furthermore, we find that HAMLET...

  19. Analysis of odorant receptor protein function in the yellow fever mosquito, aedes aegypti

    Science.gov (United States)

    Odorant receptors (ORs) in insects are ligand-gated ion channels comprised of two subunits: a variable receptor and an obligatory co-receptor (Orco). This protein receptor complex of unknown stoichiometry interacts with an odor molecule leading to changes in permeability of the sensory dendrite, th...

  20. Novel cAMP signalling paradigms: therapeutic implications for airway disease

    OpenAIRE

    Billington, Charlotte K; Hall, Ian P

    2012-01-01

    Since its discovery over 50 years ago, cAMP has been the archetypal second messenger introducing students to the concept of cell signalling at the simplest level. As explored in this review, however, there are many more facets to cAMP signalling than the path from Gs-coupled receptor to adenylyl cyclase (AC) to cAMP to PKA to biological effect. After a brief description of this canonical cAMP signalling pathway, a snapshot is provided of the novel paradigms of cAMP signalling. As in the airwa...

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

    DEFF Research Database (Denmark)

    Araç, Demet; Aust, Gabriela; Calebiro, Davide;

    2012-01-01

    G protein-coupled receptors (GPCRs) comprise an expanded superfamily of receptors in the human genome. Adhesion class G protein-coupled receptors (adhesion-GPCRs) form the second largest class of GPCRs. Despite the abundance, size, molecular structure, and functions in facilitating cell and matrix...

  2. Protein intake during training sessions has no effect on performance and recovery during a strenuous training camp for elite cyclists

    DEFF Research Database (Denmark)

    Hansen, Mette; Bangsbo, Jens; Jensen, Jørgen

    2016-01-01

    BACKGROUND: Training camps for top-class endurance athletes place high physiological demands on the body. Focus on optimizing recovery between training sessions is necessary to minimize the risk of injuries and improve adaptations to the training stimuli. Carbohydrate supplementation during sessi...

  3. The MprF protein is required for lysinylation of phospholipids in listerial membranes and confers resistance to cationic antimicrobial peptides (CAMPs) on Listeria monocytogenes

    NARCIS (Netherlands)

    Thedieck, Kathrin; Hain, Torsten; Mohamed, Walid; Tindall, Brian J; Nimtz, Manfred; Chakraborty, Trinad; Wehland, Jürgen; Jänsch, Lothar

    2006-01-01

    Pathogenic bacteria have to cope with defence mechanisms mediated by adaptive and innate immunity of the host cells. Cationic antimicrobial peptides (CAMPs) represent one of the most effective components of the host innate immune response. Here we establish the function of Lmo1695, a member of the V

  4. Regulation of synaptic strength at mixed synapses: effects of dopamine receptor blockade and protein kinase C activation.

    Science.gov (United States)

    Silva, A; Kumar, S; Pereda, A; Faber, D S

    1995-11-01

    Previous studies of the mixed excitatory synapses between eighth nerve afferents and the lateral dendrite of the goldfish Mauthner (M-) cell have shown that synaptic strength is enhanced for an hour or longer following either repeated brief tetanizations or local extracellular applications of dopamine. Both the initial electrotonic coupling potential, mediated via current flow through gap junctions, and the subsequent chemically mediated excitatory postsynaptic potentials (EPSPs) are potentiated. Different second messenger pathways are implicated in the postsynaptic induction of these potentiations, with a Ca2+ influx presumably triggering the activity dependent long-term potentiations (LTP) and dopamine acting via a cAMP dependent pathway. Experiments performed to determine whether the LTP involves a stimulus-induced release of dopamine or requires a background level of dopamine receptor activation suggest neither is the case, as tetanization in the presence of a D1 receptor antagonist, which blocks the dopamine effects, produced an LTP comparable to that in the absence of the blocker. The effects of Ca2+ are presumably not due to protein kinase C (PKC) activation, since phorbol esters had no effect on the mixed excitatory synaptic responses, although they did enhance the frequency of spontaneously occurring inhibitory PSPs.

  5. G Protein-Coupled Receptor Signaling Analysis Using Homogenous Time-Resolved Förster Resonance Energy Transfer (HTRF® Technology

    Directory of Open Access Journals (Sweden)

    Lenea Nørskov-Lauritsen

    2014-02-01

    Full Text Available Studying multidimensional signaling of G protein-coupled receptors (GPCRs in search of new and better treatments requires flexible, reliable and sensitive assays in high throughput screening (HTS formats. Today, more than half of the detection techniques used in HTS are based on fluorescence, because of the high sensitivity and rich signal, but quenching, optical interferences and light scattering are serious drawbacks. In the 1990s the HTRF® (Cisbio Bioassays, Codolet, France technology based on Förster resonance energy transfer (FRET in a time-resolved homogeneous format was developed. This improved technology diminished the traditional drawbacks. The optimized protocol described here based on HTRF® technology was used to study the activation and signaling pathways of the calcium-sensing receptor, CaSR, a GPCR responsible for maintaining calcium homeostasis. Stimulation of the CaSR by agonists activated several pathways, which were detected by measuring accumulation of the second messengers D-myo-inositol 1-phosphate (IP1 and cyclic adenosine 3',5'-monophosphate (cAMP, and by measuring the phosphorylation of extracellular signal-regulated kinase 1 and 2 (ERK1/2. Here we show how an optimized HTRF® platform with numerous advantages compared to previous assays provides a substantial and robust mode of investigating GPCR signaling. It is furthermore discussed how these assays can be optimized and miniaturized to meet HTS requirements and for screening compound libraries.

  6. G protein-coupled receptor signaling analysis using homogenous time-resolved Förster resonance energy transfer (HTRF®) technology.

    Science.gov (United States)

    Nørskov-Lauritsen, Lenea; Thomsen, Alex Rojas Bie; Bräuner-Osborne, Hans

    2014-02-13

    Studying multidimensional signaling of G protein-coupled receptors (GPCRs) in search of new and better treatments requires flexible, reliable and sensitive assays in high throughput screening (HTS) formats. Today, more than half of the detection techniques used in HTS are based on fluorescence, because of the high sensitivity and rich signal, but quenching, optical interferences and light scattering are serious drawbacks. In the 1990s the HTRF® (Cisbio Bioassays, Codolet, France) technology based on Förster resonance energy transfer (FRET) in a time-resolved homogeneous format was developed. This improved technology diminished the traditional drawbacks. The optimized protocol described here based on HTRF® technology was used to study the activation and signaling pathways of the calcium-sensing receptor, CaSR, a GPCR responsible for maintaining calcium homeostasis. Stimulation of the CaSR by agonists activated several pathways, which were detected by measuring accumulation of the second messengers D-myo-inositol 1-phosphate (IP1) and cyclic adenosine 3',5'-monophosphate (cAMP), and by measuring the phosphorylation of extracellular signal-regulated kinase 1 and 2 (ERK1/2). Here we show how an optimized HTRF® platform with numerous advantages compared to previous assays provides a substantial and robust mode of investigating GPCR signaling. It is furthermore discussed how these assays can be optimized and miniaturized to meet HTS requirements and for screening compound libraries.

  7. Molecular characterization of a novel human hybrid-type receptor that binds the alpha2-macroglobulin receptor-associated protein

    DEFF Research Database (Denmark)

    Jacobsen, Linda; Madsen, P; Moestrup, S K;

    1996-01-01

    the corresponding cDNA. The gene, designated SORL1, maps to chromosome 11q 23/24 and encodes a 2214-residue type 1 receptor containing a furin cleavage site immediately preceding the N terminus determined in the purified protein. The receptor, designated sorLA-1, has a short cytoplasmic tail containing a tyrosine......-based internalization signal and a large external part containing (from the N-terminal): 1) a segment homologous to domains in the yeast vacuolar protein sorting 10 protein, Vps10p, that binds carboxypeptidase Y, 2) five tandemly arranged YWTD repeats and a cluster of 11 class A repeats characteristic of the low...... density lipoprotein receptor gene family receptors, and 3) six tandemly arranged fibronectin type III repeats also found in certain neural adhesion proteins. sorLA-1 may therefore be classified as a hybrid receptor. Northern blotting revealed specific mRNA transcripts in brain, spinal cord, and testis...

  8. Rehabilitating camp cities : community driven planning for urbanised refugee camps

    OpenAIRE

    Misselwitz, Philipp

    2009-01-01

    Focussing on Palestine refugee camps in the Near East, this dissertation aims to shed light on the potential relevance of urban planning to refugee camp environments worldwide. In particular, there is a focus on the role architects and urban planners can play in facilitating participatory planning processes as well as providing guidance and expertise in the development of a spatial vision for Camp Cities. Part I - The Urbanisation of Refugee Camps as a Global Challenge The first part o...

  9. G Protein-Coupled Receptor Signaling in Stem Cells and Cancer

    OpenAIRE

    Lynch, Jennifer R.; Jenny Yingzi Wang

    2016-01-01

    G protein-coupled receptors (GPCRs) are a large superfamily of cell-surface signaling proteins that bind extracellular ligands and transduce signals into cells via heterotrimeric G proteins. GPCRs are highly tractable drug targets. Aberrant expression of GPCRs and G proteins has been observed in various cancers and their importance in cancer stem cells has begun to be appreciated. We have recently reported essential roles for G protein-coupled receptor 84 (GPR84) and G protein subunit Gαq in ...

  10. Molecular basis for amino acid sensing by family C G-protein-coupled receptors

    DEFF Research Database (Denmark)

    Wellendorph, Petrine; Bräuner-Osborne, Hans

    2009-01-01

    -alpha;-amino acid receptor G-protein-coupled receptor family C, group 6, subtype A (GPRC6A) and seven orphan receptors. Aside from the orphan receptors, the family C GPCRs are dimeric receptors characterized by a large extracellular Venus flytrap domain which bind the endogenous agonists. Except from the GABA(B1......-2) and T1R2-3 receptor, all receptors are either activated or positively modulated by amino acids. In this review, we outline mutational, biophysical and structural studies which have elucidated the interaction of the amino acids with the Venus flytrap domains, molecular mechanisms of receptor selectivity...

  11. The camp model for entrepreneurship teaching

    DEFF Research Database (Denmark)

    Bager, Torben

    2011-01-01

    Artiklen omhandler brugen af camps i entrepreneurship undervising - illustreret med danske camp eksempler Udgivelsesdato: online 31.03.2010......Artiklen omhandler brugen af camps i entrepreneurship undervising - illustreret med danske camp eksempler Udgivelsesdato: online 31.03.2010...

  12. Cannabinoid CB1 receptor-interacting proteins: novel targets for central nervous system drug discovery?

    Science.gov (United States)

    Smith, Tricia H; Sim-Selley, Laura J; Selley, Dana E

    2010-06-01

    The main pharmacological effects of marijuana, as well as synthetic and endogenous cannabinoids, are mediated through G-protein-coupled receptors (GPCRs), including CB(1) and CB(2) receptors. The CB(1) receptor is the major cannabinoid receptor in the central nervous system and has gained increasing interest as a target for drug discovery for treatment of nausea, cachexia, obesity, pain, spasticity, neurodegenerative diseases and mood and substance abuse disorders. Evidence has accumulated to suggest that CB(1) receptors, like other GPCRs, interact with and are regulated by several other proteins beyond the established role of heterotrimeric G-proteins. These proteins, which include the GPCR kinases, beta-arrestins, GPCR-associated sorting proteins, factor associated with neutral sphingomyelinase, other GPCRs (heterodimerization) and the novel cannabinoid receptor-interacting proteins: CRIP(1a/b), are thought to play important roles in the regulation of intracellular trafficking, desensitization, down-regulation, signal transduction and constitutive activity of CB(1) receptors. This review examines CB(1) receptor-interacting proteins, including heterotrimeric G-proteins, but with particular emphasis on non-G-protein entities, that might comprise the CB(1) receptosomal complex. The evidence for direct interaction with CB(1) receptors and potential functional roles of these interacting proteins is discussed, as are future directions and challenges in this field with an emphasis on the possibility of eventually targeting these proteins for drug discovery.

  13. Mapping functional group free energy patterns at protein occluded sites: nuclear receptors and G-protein coupled receptors.

    Science.gov (United States)

    Lakkaraju, Sirish Kaushik; Yu, Wenbo; Raman, E Prabhu; Hershfeld, Alena V; Fang, Lei; Deshpande, Deepak A; MacKerell, Alexander D

    2015-03-23

    Occluded ligand-binding pockets (LBP) such as those found in nuclear receptors (NR) and G-protein coupled receptors (GPCR) represent a significant opportunity and challenge for computer-aided drug design. To determine free energies maps of functional groups of these LBPs, a Grand-Canonical Monte Carlo/Molecular Dynamics (GCMC/MD) strategy is combined with the Site Identification by Ligand Competitive Saturation (SILCS) methodology. SILCS-GCMC/MD is shown to map functional group affinity patterns that recapitulate locations of functional groups across diverse classes of ligands in the LBPs of the androgen (AR) and peroxisome proliferator-activated-γ (PPARγ) NRs and the metabotropic glutamate (mGluR) and β2-adreneric (β2AR) GPCRs. Inclusion of protein flexibility identifies regions of the binding pockets not accessible in crystal conformations and allows for better quantitative estimates of relative ligand binding affinities in all the proteins tested. Differences in functional group requirements of the active and inactive states of the β2AR LBP were used in virtual screening to identify high efficacy agonists targeting β2AR in Airway Smooth Muscle (ASM) cells. Seven of the 15 selected ligands were found to effect ASM relaxation representing a 46% hit rate. Hence, the method will be of use for the rational design of ligands in the context of chemical biology and the development of therapeutic agents.

  14. Biotechnological Fluorescent Ligands of the Bradykinin B1 Receptor: Protein Ligands for a Peptide Receptor.

    Directory of Open Access Journals (Sweden)

    Xavier Charest-Morin

    Full Text Available The bradykinin (BK B1 receptor (B1R is a peculiar G protein coupled receptor that is strongly regulated to the point of being inducible in immunopathology. Limited clinical evidence suggests that its expression in peripheral blood mononuclear cells is a biomarker of active inflammatory states. In an effort to develop a novel imaging/diagnostic tool, we report the rational design and testing of a fusion protein that is a ligand of the human B1R but not likely to label peptidases. This ligand is composed of a fluorescent protein (FP (enhanced green FP [EGFP] or mCherry prolonged at its N-terminus by a spacer peptide and a classical peptide agonist or antagonist (des-Arg9-BK, [Leu8]des-Arg9-BK, respectively. The design of the spacer-ligand joint peptide was validated by a competition assay for [3H]Lys-des-Arg9-BK binding to the human B1R applied to 4 synthetic peptides of 18 or 19 residues. The labeling of B1R-expressing cells with EGFP or mCherry fused with 7 of such peptides was performed in parallel (microscopy. Both assays indicated that the best design was FP-(Asn-Glyn-Lys-des-Arg9-BK; n = 15 was superior to n = 5, suggesting benefits from minimizing steric hindrance between the FP and the receptor. Cell labeling concerned mostly plasma membranes and was inhibited by a B1R antagonist. EGFP-(Asn-Gly15-Lys-des-Arg9-BK competed for the binding of [3H]Lys-des-Arg9-BK to human recombinant B1R, being only 10-fold less potent than the unlabeled form of Lys-des-Arg9-BK to do so. The fusion protein did not label HEK 293a cells expressing recombinant human BK B2 receptors or angiotensin converting enzyme. This study identifies a modular C-terminal sequence that can be adapted to protein cargoes, conferring high affinity for the BK B1R, with possible applications in diagnostic cytofluorometry, histology and drug delivery (e.g., in oncology.

  15. Cyclic AMP enhances TGFβ responses of breast cancer cells by upregulating TGFβ receptor I expression.

    Directory of Open Access Journals (Sweden)

    Ilka Oerlecke

    Full Text Available Cellular functions are regulated by complex networks of many different signaling pathways. The TGFβ and cAMP pathways are of particular importance in tumor progression. We analyzed the cross-talk between these pathways in breast cancer cells in 2D and 3D cultures. We found that cAMP potentiated TGFβ-dependent gene expression by enhancing Smad3 phosphorylation. Higher levels of total Smad3, as observed in 3D-cultured cells, blocked this effect. Two Smad3 regulating proteins, YAP (Yes-associated protein and TβRI (TGFβ receptor 1, were responsive to cAMP. While YAP had little effect on TGFβ-dependent expression and Smad3 phosphorylation, a constitutively active form of TβRI mimicked the cAMP effect on TGFβ signaling. In 3D-cultured cells, which show much higher levels of TβRI and cAMP, TβRI was unresponsive to cAMP. Upregulation of TβRI expression by cAMP was dependent on transcription. A proximal TβRI promoter fragment was moderately, but significantly activated by cAMP suggesting that cAMP increases TβRI expression at least partially by activating TβRI transcription. Neither the cAMP-responsive element binding protein (CREB nor the TβRI-regulating transcription factor Six1 was required for the cAMP effect. An inhibitor of histone deacetylases alone or together with cAMP increased TβRI expression by a similar extent as cAMP alone suggesting that cAMP may exert its effect by interfering with histone acetylation. Along with an additive stimulatory effect of cAMP and TGFβ on p21 expression an additive inhibitory effect of these agents on proliferation was observed. Finally, we show that mesenchymal stem cells that interact with breast cancer cells can simultaneously activate the cAMP and TGFβ pathways. In summary, these data suggest that combined effects of cAMP and TGFβ, as e.g. induced by mesenchymal stem cells, involve the upregulation of TβRI expression on the transcriptional level, likely due to changes in histone acetylation

  16. Role of CGRP-Receptor Component Protein (RCP) in CLR/RAMP Function

    OpenAIRE

    Dickerson, Ian M.

    2013-01-01

    The receptor for calcitonin gene-related peptide (CGRP) and adrenomedullin (AM) requires an intracellular peripheral membrane protein named CGRP-receptor component protein (RCP) for signaling. RCP is required for CGRP and AM receptor signaling, and it has recently been discovered that RCP enables signaling by binding directly to the receptor. RCP is present in most immortalized cell lines, but in vivo RCP expression is limited to specific subsets of cells, usually co-localizing with CGRP-cont...

  17. Gc protein-derived macrophage-activating factor (GcMAF) stimulates cAMP formation in human mononuclear cells and inhibits angiogenesis in chick embryo chorionallantoic membrane assay

    OpenAIRE

    2010-01-01

    Abstract: The effects of Gc protein-derived macrophage-activating factor (GcMAF) have been studied in cancer and other conditions where angiogenesis is deregulated. In this study, we demonstrate for the first time that the mitogenic response of human peripheral blood mononuclear cells (PBMCs) to GcMAF was associated with 3'-5'-cyclic adenosine monophosphate (cAMP) formation. The effect was dose dependent, and maximal stimulation was achieved using 0.1 ng/ml. Heparin inhibited the stimulatory ...

  18. Base Camp Design Simulation Training

    Science.gov (United States)

    2011-07-01

    The Army needs officers and noncommissioned officers with requisite base camp competencies. The Army’s Field Manual (FM) 3-34.400 defines a Base Camp...reason, we designed a 600-man base camp on VBS2TM from an AutoCAD diagram found on the Theater Construction Management System (version 3.2). Known

  19. Adenosine activates ATP-sensitive potassium channels in arterial myocytes via A2 receptors and cAMP-dependent protein kinase.

    Science.gov (United States)

    Kleppisch, T; Nelson, M T

    1995-01-01

    The mechanism by which the endogenous vasodilator adenosine causes ATP-sensitive potassium (KATP) channels in arterial smooth muscle to open was investigated by the whole-cell patch-clamp technique. Adenosine induced voltage-independent, potassium-selective currents, which were inhibited by glibenclamide, a blocker of KATP currents. Glibenclamide-sensitive currents were also activated by the selective adenosine A2-receptor agonist 2-p-(2-carboxethyl)-phenethylamino-5'-N- ethylcarboxamidoadenosine hydrochloride (CGS-21680), whereas 2-chloro-N6-cyclopentyladenosine (CCPA), a selective adenosine A1-receptor agonist, failed to induce potassium currents. Glibenclamide-sensitive currents induced by adenosine and CGS-21680 were largely reduced by blockers of the cAMP-dependent protein kinase (Rp-cAMP[S], H-89, protein kinase A inhibitor peptide). Therefore, we conclude that adenosine can activate KATP currents in arterial smooth muscle through the following pathway: (i) Adenosine stimulates A2 receptors, which activates adenylyl cyclase; (ii) the resulting increase intracellular cAMP stimulates protein kinase A, which, probably through a phosphorylation step, opens KATP channels. PMID:8618917

  20. Evolution of a G protein-coupled receptor response by mutations in regulatory network interactions

    DEFF Research Database (Denmark)

    Di Roberto, Raphaël B; Chang, Belinda; Trusina, Ala;

    2016-01-01

    All cellular functions depend on the concerted action of multiple proteins organized in complex networks. To understand how selection acts on protein networks, we used the yeast mating receptor Ste2, a pheromone-activated G protein-coupled receptor, as a model system. In Saccharomyces cerevisiae...

  1. Ancestral reconstruction of the ligand-binding pocket of Family C G protein-coupled receptors

    OpenAIRE

    Kuang, Donghui; Yao, Yi; MacLean, David; Wang, Minghua; Hampson, David R.; Chang, Belinda S. W.

    2006-01-01

    The metabotropic glutamate receptors (mGluRs) within the Family C subclass of G protein-coupled receptors are crucial modulators of synaptic transmission. However, their closest relatives include a diverse group of sensory receptors whose biological functions are not associated with neurotransmission, raising the question of the evolutionary origin of amino acid-binding Family C receptors. A common feature of most, if not all, functional Family C receptors is the presence of an amino acid-bin...

  2. A modeling strategy for G-protein coupled receptors

    Directory of Open Access Journals (Sweden)

    Anna Kahler

    2016-03-01

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

  3. The tandem endocytic receptors megalin and cubilin are important proteins in renal pathology.

    Science.gov (United States)

    Verroust, Pierre J; Birn, Henrik; Nielsen, Rikke; Kozyraki, Renata; Christensen, Erik Ilsø

    2002-09-01

    The molecular mechanisms controlling proximal tubule reabsorption of proteins have been much elucidated in recent years. Megalin and cubilin constitute two important endocytic receptor proteins involved in this process. Although structurally very different the two receptor proteins interact to mediate the reabsorption of a large number of filtered proteins, including carrier proteins important for transport and cellular uptake of several vitamins, lipids and other nutrients. Dysfunction of either protein results in tubular proteinuria and is associated with specific changes in vitamin metabolism due to the defective proximal tubular reabsorption of carrier proteins. Additional focus on the two receptors is attracted by the possible pathogenic role of excessive tubular protein uptake during conditions of increased filtration of proteins, and by recent findings implicating members of the low density lipoprotein-receptor family, which includes megalin, in the transduction of signals by association with cytoplasmic proteins.

  4. Phenotype, virulence and immunogenicity of Edwardsiella ictaluri cyclic adenosine 3',5'-monophosphate receptor protein (Crp) mutants in catfish host.

    Science.gov (United States)

    Santander, Javier; Mitra, Arindam; Curtiss, Roy

    2011-12-01

    Edwardsiella ictaluri is an Enterobacteriaceae that causes lethal enteric septicemia in catfish. Being a mucosal facultative intracellular pathogen, this bacterium is an excellent candidate to develop immersion-oral live attenuated vaccines for the catfish aquaculture industry. Deletion of the cyclic 3',5'-adenosine monophosphate (cAMP) receptor protein (crp) gene in several Enterobacteriaceae has been utilized in live attenuated vaccines for mammals and birds. Here we characterize the crp gene and report the effect of a crp deletion in E. ictaluri. The E. ictaluri crp gene and encoded protein are similar to other Enterobacteriaceae family members, complementing Salmonella enterica Δcrp mutants in a cAMP-dependent fashion. The E. ictaluri Δcrp-10 in-frame deletion mutant demonstrated growth defects, loss of maltose utilization, and lack of flagella synthesis. We found that the E. ictaluri Δcrp-10 mutant was attenuated, colonized lymphoid tissues, and conferred immune protection against E. ictaluri infection to zebrafish (Danio rerio) and catfish (Ictalurus punctatus). Evaluation of the IgM titers indicated that bath immunization with the E. ictaluri Δcrp-10 mutant triggered systemic and skin immune responses in catfish. We propose that deletion of the crp gene in E. ictaluri is an effective strategy to develop immersion live attenuated antibiotic-sensitive vaccines for the catfish aquaculture industry.

  5. Neuronal low-density lipoprotein receptor-related protein 1 binds and endocytoses prion fibrils via receptor cluster 4

    DEFF Research Database (Denmark)

    Jen, Angela; Parkyn, Celia J; Mootoosamy, Roy C;

    2010-01-01

    For infectious prion protein (designated PrP(Sc)) to act as a template to convert normal cellular protein (PrP(C)) to its distinctive pathogenic conformation, the two forms of prion protein (PrP) must interact closely. The neuronal receptor that rapidly endocytoses PrP(C) is the low-density lipop......For infectious prion protein (designated PrP(Sc)) to act as a template to convert normal cellular protein (PrP(C)) to its distinctive pathogenic conformation, the two forms of prion protein (PrP) must interact closely. The neuronal receptor that rapidly endocytoses PrP(C) is the low......-density lipoprotein receptor-related protein 1 (LRP1). We show here that on sensory neurons LRP1 is also the receptor that binds and rapidly endocytoses smaller oligomeric forms of infectious prion fibrils, and recombinant PrP fibrils. Although LRP1 binds two molecules of most ligands independently to its receptor...... clusters 2 and 4, PrP(C) and PrP(Sc) fibrils bind only to receptor cluster 4. PrP(Sc) fibrils out-compete PrP(C) for internalization. When endocytosed, PrP(Sc) fibrils are routed to lysosomes, rather than recycled to the cell surface with PrP(C). Thus, although LRP1 binds both forms of PrP, it traffics...

  6. The alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein binds and internalizes Pseudomonas exotoxin A.

    Science.gov (United States)

    Kounnas, M Z; Morris, R E; Thompson, M R; FitzGerald, D J; Strickland, D K; Saelinger, C B

    1992-06-25

    The alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein (alpha 2 MR/LRP) is a large cell-surface glycoprotein consisting of a 515-kDa and an 85-kDa polypeptide; this receptor is thought to be responsible for the binding and endocytosis of activated alpha 2-macroglobulin and apoE-enriched beta-very low density lipoprotein. A similar high molecular weight glycoprotein has been identified as a potential receptor for Pseudomonas exotoxin A (PE). We demonstrate that the alpha 2 MR/LRP and the PE-binding glycoprotein have a similar mobility upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis and are immunologically indistinguishable. Furthermore, affinity-purified alpha 2 MR/LRP binds specifically to PE but not to a mutant toxin defective in its ability to bind cells. The 39-kDa receptor-associated protein, which blocks binding of ligands to alpha 2 MR/LRP, also prevents binding and subsequent toxicity of PE for mouse fibroblasts. The concentration of receptor-associated protein that was required to reduce binding and toxicity to 50% was approximately 14 nM, a value virtually identical to the KD measured for the interaction of receptor-associated protein with the purified receptor. Overall, the studies strongly suggest that the alpha 2 MR/LRP is responsible for internalizing PE.

  7. Involvement of G proteins and cAMP in the production of chitinolytic enzymes by Trichoderma harzianum Envolvimento de proteínas G e cAMP na produção de enzimas quitinolíticas por Trichoderma harzianum

    Directory of Open Access Journals (Sweden)

    Alexandre A.P. Firmino

    2002-06-01

    Full Text Available The effect of G protein modulators and cyclic AMP (cAMP on N-acetylglucosaminidase (NAGase production was investigated during 84 h of growth of a Trichoderma harzianum strain in chitin-containing medium. Caffeine (5 mM, N6--2'-O-dibutyryladenosine 3'5'-cyclic monophosphate sodium salt (dBcAMP (1 mM and 3-isobutyl-1-methylxanthine (IBMX (2 mM decreased extracellular NAGase activity by 80%, 77% and 37%, respectively. AlCl3/KF (100 µM/10 mM and 200 µM/ 20 mM decreased the activity by 85% and 95%, respectively. Cholera (10 µ/mL and pertussis (20 µ/mL toxins also affected NAGase activity, causing a decrease of approximately 75%. Upon all treatments, protein bands of approximately 73 kDa, 68 kDa and 45 kDa had their signals diminished whilst a 50 kDa band was enhanced only by treatment with cholera and pertussis toxins. N-terminal sequencing analysis identified the 73 kDa and 68 kDa proteins as being T. harzianum NAGase in two different truncated forms whereas the 45 kDa band comprised a T. harzianum endochitinase. The 50 kDa protein showed sequence similarity to Coriolus vesicolor cellobiohydrolase. The above results suggest that a signaling pathway comprising G-proteins, adenylate cyclase and cAMP may be involved in the synthesis of T. harzianum chitinases.O efeito de cAMP e de moduladores de proteínas G sobre a produção de N-acetilglicosaminidase (NAGase foi investigado durante o crescimento de Trichoderma harzianum em meio contendo quitina. Cafeína (5 mM, dBcAMP (1mM e IBMX (2 mM provocaram diminuições na atividade extracelular de NAGase em 80%, 77% e 37%, respectivamente. Por outro lado, a presença de AlCl3/KF nas concentrações de 100 µM/10 mM e 200 µM/ 20 mM causou decréscimo na atividade em 85% e 95%, respectivamente. A toxina do cólera (10 µ/mL e a toxina pertussis (20 µ/mL também afetaram a atividade de NAGase, causando um decréscimo de aproximadamente 75%. Análises eletroforéticas mostraram que todos os tratamentos

  8. S-Nitrosothiols modulate G protein-coupled receptor signaling in a reversible and highly receptor-specific manner

    Directory of Open Access Journals (Sweden)

    Mönkkönen Kati S

    2005-04-01

    Full Text Available Abstract Background Recent studies indicate that the G protein-coupled receptor (GPCR signaling machinery can serve as a direct target of reactive oxygen species, including nitric oxide (NO and S-nitrosothiols (RSNOs. To gain a broader view into the way that receptor-dependent G protein activation – an early step in signal transduction – might be affected by RSNOs, we have studied several receptors coupling to the Gi family of G proteins in their native cellular environment using the powerful functional approach of [35S]GTPγS autoradiography with brain cryostat sections in combination with classical G protein activation assays. Results We demonstrate that RSNOs, like S-nitrosoglutathione (GSNO and S-nitrosocysteine (CysNO, can modulate GPCR signaling via reversible, thiol-sensitive mechanisms probably involving S-nitrosylation. RSNOs are capable of very targeted regulation, as they potentiate the signaling of some receptors (exemplified by the M2/M4 muscarinic cholinergic receptors, inhibit others (P2Y12 purinergic, LPA1lysophosphatidic acid, and cannabinoid CB1 receptors, but may only marginally affect signaling of others, such as adenosine A1, μ-opioid, and opiate related receptors. Amplification of M2/M4 muscarinic responses is explained by an accelerated rate of guanine nucleotide exchange, as well as an increased number of high-affinity [35S]GTPγS binding sites available for the agonist-activated receptor. GSNO amplified human M4 receptor signaling also under heterologous expression in CHO cells, but the effect diminished with increasing constitutive receptor activity. RSNOs markedly inhibited P2Y12 receptor signaling in native tissues (rat brain and human platelets, but failed to affect human P2Y12 receptor signaling under heterologous expression in CHO cells, indicating that the native cellular signaling partners, rather than the P2Y12 receptor protein, act as a molecular target for this action. Conclusion These in vitro studies

  9. Tachykinin-Related Peptides Share a G Protein-Coupled Receptor with Ion Transport Peptide-Like in the Silkworm Bombyx mori.

    Directory of Open Access Journals (Sweden)

    Chiaki Nagai-Okatani

    Full Text Available Recently, we identified an orphan Bombyx mori neuropeptide G protein-coupled receptor (BNGR-A24 as an ion transport peptide-like (ITPL receptor. BNGR-A24 belongs to the same clade as BNGR-A32 and -A33, which were recently identified as natalisin receptors. Since these three BNGRs share high similarities with known receptors for tachykinin-related peptides (TRPs, we examined whether these BNGRs can function as physiological receptors for five endogenous B. mori TRPs (TK-1-5. In a heterologous expression system, BNGR-A24 acted as a receptor for all five TRPs. In contrast, BNGR-A32 responded only to TK-5, and BNGR-A33 did not respond to any of the TRPs. These findings are consistent with recent studies on the ligand preferences for B. mori natalisins. Furthermore, we evaluated whether the binding of ITPL and TRPs to BNGR-A24 is competitive by using a Ca2+ imaging assay. Concomitant addition of a TRP receptor antagonist, spantide I, reduced the responses of BNGR-A24 not only to TK-4 but also to ITPL. The results of a binding assay using fluorescent-labeled BNGR-A24 and ligands demonstrated that the binding of ITPL to BNGR-A24 was inhibited by TK-4 as well as by spantide I, and vice versa. In addition, the ITPL-induced increase in cGMP levels of BNGR-A24-expressing BmN cells was suppressed by the addition of excess TK-4 or spantide I. The intracellular levels of cAMP and cGMP, as second messenger candidates of the TRP signaling, were not altered by the five TRPs, suggesting that these peptides act via different signaling pathways from cAMP and cGMP signaling at least in BmN cells. Taken together, the present findings suggest that ITPL and TRPs are endogenous orthosteric ligands of BNGR-A24 that may activate discrete signaling pathways. This receptor, which shares orthosteric ligands, may constitute an important model for studying ligand-biased signaling.

  10. Dopamine receptor-interacting protein 78 acts as a molecular chaperone for CCR5 chemokine receptor signaling complex organization.

    Directory of Open Access Journals (Sweden)

    Yi-Qun Kuang

    Full Text Available Chemokine receptors are members of the G protein-coupled receptor (GPCR family. CCR5 and CXCR4 act as co-receptors for human immunodeficiency virus (HIV and several efforts have been made to develop ligands to inhibit HIV infection by blocking those receptors. Removal of chemokine receptors from the cell surface using polymorphisms or other means confers some levels of immunity against HIV infection. Up to now, very limited success has been obtained using ligand therapies so we explored potential avenues to regulate chemokine receptor expression at the plasma membrane. We identified a molecular chaperone, DRiP78, that interacts with both CXCR4 and CCR5, but not the heterodimer formed by these receptors. We further characterized the effects of DRiP78 on CCR5 function. We show that the molecular chaperone inhibits CCR5 localization to the plasma membrane. We identified the interaction region on the receptor, the F(x6LL motif, and show that upon mutation of this motif the chaperone cannot interact with the receptor. We also show that DRiP78 is involved in the assembly of CCR5 chemokine signaling complex as a homodimer, as well as with the Gαi protein. Finally, modulation of DRiP78 levels will affect receptor functions, such as cell migration in cells that endogenously express CCR5. Our results demonstrate that modulation of the functions of a chaperone can affect signal transduction at the cell surface.

  11. Dopamine receptor-interacting protein 78 acts as a molecular chaperone for CCR5 chemokine receptor signaling complex organization.

    Science.gov (United States)

    Kuang, Yi-Qun; Charette, Nicholle; Frazer, Jennifer; Holland, Patrick J; Attwood, Kathleen M; Dellaire, Graham; Dupré, Denis J

    2012-01-01

    Chemokine receptors are members of the G protein-coupled receptor (GPCR) family. CCR5 and CXCR4 act as co-receptors for human immunodeficiency virus (HIV) and several efforts have been made to develop ligands to inhibit HIV infection by blocking those receptors. Removal of chemokine receptors from the cell surface using polymorphisms or other means confers some levels of immunity against HIV infection. Up to now, very limited success has been obtained using ligand therapies so we explored potential avenues to regulate chemokine receptor expression at the plasma membrane. We identified a molecular chaperone, DRiP78, that interacts with both CXCR4 and CCR5, but not the heterodimer formed by these receptors. We further characterized the effects of DRiP78 on CCR5 function. We show that the molecular chaperone inhibits CCR5 localization to the plasma membrane. We identified the interaction region on the receptor, the F(x)6LL motif, and show that upon mutation of this motif the chaperone cannot interact with the receptor. We also show that DRiP78 is involved in the assembly of CCR5 chemokine signaling complex as a homodimer, as well as with the Gαi protein. Finally, modulation of DRiP78 levels will affect receptor functions, such as cell migration in cells that endogenously express CCR5. Our results demonstrate that modulation of the functions of a chaperone can affect signal transduction at the cell surface.

  12. The octopamine receptor OAMB mediates ovulation via Ca2+/calmodulin-dependent protein kinase II in the Drosophila oviduct epithelium.

    Directory of Open Access Journals (Sweden)

    Hyun-Gwan Lee

    Full Text Available Ovulation is an essential physiological process in sexual reproduction; however, the underlying cellular mechanisms are poorly understood. We have previously shown that OAMB, a Drosophila G-protein-coupled receptor for octopamine (the insect counterpart of mammalian norepinephrine, is required for ovulation induced upon mating. OAMB is expressed in the nervous and reproductive systems and has two isoforms (OAMB-AS and OAMB-K3 with distinct capacities to increase intracellular Ca2+ or intracellular Ca2+ and cAMP in vitro. Here, we investigated tissue specificity and intracellular signals required for OAMB's function in ovulation. Restricted OAMB expression in the adult oviduct epithelium, but not the nervous system, reinstated ovulation in oamb mutant females, in which either OAMB isoform was sufficient for the rescue. Consistently, strong immunoreactivities for both isoforms were observed in the wild-type oviduct epithelium. To delineate the cellular mechanism by which OAMB regulates ovulation, we explored protein kinases functionally interacting with OAMB by employing a new GAL4 driver with restricted expression in the oviduct epithelium. Conditional inhibition of Ca2+/Calmodulin-dependent protein kinase II (CaMKII, but not protein kinase A or C, in the oviduct epithelium inhibited ovulation. Moreover, constitutively active CaMKII, but not protein kinase A, expressed only in the adult oviduct epithelium fully rescued the oamb female's phenotype, demonstrating CaMKII as a major downstream molecule conveying the OAMB's ovulation signal. This is consistent with the ability of both OAMB isoforms, whose common intracellular signal in vitro is Ca2+, to reinstate ovulation in oamb females. These observations reveal the critical roles of the oviduct epithelium and its cellular components OAMB and CaMKII in ovulation. It is conceivable that the OAMB-mediated cellular activities stimulated upon mating are crucial for secretory activities suitable for egg

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

    DEFF Research Database (Denmark)

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

    2005-01-01

    G protein-coupled receptors (GPCRs) have been one of the most productive classes of drug targets for several decades, and new technologies for GPCR-based discovery promise to keep this field active for years to come. While molecular screens for GPCR receptor agonist- and antagonist-based drugs...... as valuable discovery tools for several years. The application of high content cell-based screening to GPCR discovery has opened up additional possibilities, such as direct tracking of GPCRs, G proteins and other signaling pathway components using intracellular translocation assays. These assays provide...... the capability to probe GPCR function at the cellular level with better resolution than has previously been possible, and offer practical strategies for more definitive selectivity evaluation and counter-screening in the early stages of drug discovery. The potential of cell-based translocation assays for GPCR...

  14. Extracellular signal-regulated kinases control expression of G protein-coupled receptor kinase 2 (GRK2)

    DEFF Research Database (Denmark)

    Theilade, Juliane; Lerche Hansen, Jakob; Haunsø, Stig;

    2002-01-01

    G protein-coupled receptor kinase 2 (GRK2) phosphorylates G protein-coupled receptors resulting in uncoupling from G proteins. Receptors modulate GRK2 expression, however the mechanistic basis for this effect is largely unknown. Here we report a novel mechanism by which receptors use...

  15. The Emerging Mutational Landscape of G-proteins and G-protein Coupled Receptors in Cancer

    OpenAIRE

    O’Hayre, Morgan; Vázquez-Prado, José; Kufareva, Irina; Stawiski, Eric W.; Handel, Tracy M.; Seshagiri, Somasekar; Gutkind, J. Silvio

    2013-01-01

    Aberrant expression and activity of G proteins and G protein coupled receptors (GPCRs) are frequently associated with tumorigenesis. Deep sequencing studies show that 4.2% of tumors carry activating mutations in GNAS (encoding Gαs), and that oncogenic activating mutants in genes encoding Gαq family members (GNAQ or GNA11) are present in ~66% and ~6% of melanomas arising in the eye and skin, respectively. Furthermore, nearly 20% of human tumors harbor mutations in GPCRs. Many human cancer-asso...

  16. Regulation of Toll-like receptor 4-mediated immune responses through Pasteurella multocida toxin-induced G protein signalling

    Directory of Open Access Journals (Sweden)

    Hildebrand Dagmar

    2012-08-01

    Full Text Available Abstract Background Lipopolysaccharide (LPS-triggered Toll-like receptor (TLR 4-signalling belongs to the key innate defence mechanisms upon infection with Gram-negative bacteria and triggers the subsequent activation of adaptive immunity. There is an active crosstalk between TLR4-mediated and other signalling cascades to secure an effective immune response, but also to prevent excessive inflammation. Many pathogens induce signalling cascades via secreted factors that interfere with TLR signalling to modify and presumably escape the host response. In this context heterotrimeric G proteins and their coupled receptors have been recognized as major cellular targets. Toxigenic strains of Gram-negative Pasteurella multocida produce a toxin (PMT that constitutively activates the heterotrimeric G proteins Gαq, Gα13 and Gαi independently of G protein-coupled receptors through deamidation. PMT is known to induce signalling events involved in cell proliferation, cell survival and cytoskeleton rearrangement. Results Here we show that the activation of heterotrimeric G proteins through PMT suppresses LPS-stimulated IL-12p40 production and eventually impairs the T cell-activating ability of LPS-treated monocytes. This inhibition of TLR4-induced IL-12p40 expression is mediated by Gαi-triggered signalling as well as by Gβγ-dependent activation of PI3kinase and JNK. Taken together we propose the following model: LPS stimulates TLR4-mediated activation of the NFĸB-pathway and thereby the production of TNF-α, IL-6 and IL-12p40. PMT inhibits the production of IL-12p40 by Gαi-mediated inhibition of adenylate cyclase and cAMP accumulation and by Gβγ-mediated activation of PI3kinase and JNK activation. Conclusions On the basis of the experiments with PMT this study gives an example of a pathogen-induced interaction between G protein-mediated and TLR4-triggered signalling and illustrates how a bacterial toxin is able to interfere with the host’s immune

  17. Abnormalities in osteoclastogenesis and decreased tumorigenesis in mice deficient for ovarian cancer G protein-coupled receptor 1.

    Directory of Open Access Journals (Sweden)

    Hui Li

    Full Text Available Ovarian cancer G protein-coupled receptor 1 (OGR1 has been shown to be a proton sensing receptor in vitro. We have shown that OGR1 functions as a tumor metastasis suppressor gene when it is over-expressed in human prostate cancer cells in vivo. To examine the physiological functions of OGR1, we generated conditional OGR1 deficient mice by homologous recombination. OGR1 deficient mice were viable and upon gross-inspection appeared normal. Consistent with in vitro studies showing that OGR1 is involved in osteoclastogenesis, reduced osteoclasts were detected in OGR1 deficient mice. A pH-dependent osteoclasts survival effect was also observed. However, overall abnormality in the bones of these animals was not observed. In addition, melanoma cell tumorigenesis was significantly inhibited in OGR1 deficient mice. OGR1 deficient mice in the mixed background produced significantly less peritoneal macrophages when stimulated with thioglycolate. These macrophages also showed altered extracellular signal-regulated kinases (ERK activation and nitric oxide (NO production in response to lipopolysaccharide. OGR1-dependent pH responses assessed by cAMP production and cell survival in macrophages or brown fat cells were not observed, presumably due to the presence of other proton sensing receptors in these cells. Our results indicate that OGR1's role in osteoclastogenesis is not strong enough to affect overall bone development and its role in tumorigenesis warrants further investigation. The mice generated can be potentially used for several disease models, including cancers or osteoclast-related diseases.

  18. Lactate Receptor Sites Link Neurotransmission, Neurovascular Coupling, and Brain Energy Metabolism

    DEFF Research Database (Denmark)

    Lauritzen, Knut H; Morland, Cecilie; Puchades, Maja;

    2013-01-01

    The G-protein-coupled lactate receptor, GPR81 (HCA1), is known to promote lipid storage in adipocytes by downregulating cAMP levels. Here, we show that GPR81 is also present in the mammalian brain, including regions of the cerebral neocortex and hippocampus, where it can be activated by physiolog......The G-protein-coupled lactate receptor, GPR81 (HCA1), is known to promote lipid storage in adipocytes by downregulating cAMP levels. Here, we show that GPR81 is also present in the mammalian brain, including regions of the cerebral neocortex and hippocampus, where it can be activated...

  19. Interleukin-1 receptor accessory protein interacts with the type II interleukin-1 receptor.

    Science.gov (United States)

    Malinowsky, D; Lundkvist, J; Layé, S; Bartfai, T

    1998-06-16

    Stably transfected HEK-293 cells express on their surface the murine type II IL-1 receptor (mIL-1RII) as demonstrated by FACS analysis using the mAb 4E2, however binding of [125I]-hrIL-1beta to these cells is nearly absent. Saturable high affinity binding of [125I]-hrIL-1beta is observed when the murine IL-1 receptor accessory protein (mIL-1RAcP) is coexpressed with mIL-1RII. Binding of [125I]-hrIL-1beta to mIL-1RII-mIL-1RAcP complex can be inhibited either with antibodies to mIL-1RII (mAb 4E2), or by antibodies to mIL-1RAcP (mAb 4C5). The number of high affinity binding sites in cells stably transfected with the cDNA for mIL-1RII is dependent on the dose of cDNA for mIL-1RAcP used to transfect the cells. The high affinity complex between mIL-1RII and mIL-1RAcP is not preformed by interaction between the intracellular domains of these two transmembrane proteins, rather it appears to require the extracellular portions of mIL-1RII and mIL-1RAcP and the presence of a ligand. We suggest that in addition to its earlier described decoy receptor role, IL-1RII may modulate the responsiveness of cells to IL-1 by binding the IL-1RAcP in unproductive/non-signalling complexes and thus reducing the number of signalling IL-1RI-IL-1RAcP-agonist complexes when IL-1 is bound.

  20. Uncovering Molecular Bases Underlying Bone Morphogenetic Protein Receptor Inhibitor Selectivity.

    Directory of Open Access Journals (Sweden)

    Abdelaziz Alsamarah

    Full Text Available Abnormal alteration of bone morphogenetic protein (BMP signaling is implicated in many types of diseases including cancer and heterotopic ossifications. Hence, small molecules targeting BMP type I receptors (BMPRI to interrupt BMP signaling are believed to be an effective approach to treat these diseases. However, lack of understanding of the molecular determinants responsible for the binding selectivity of current BMP inhibitors has been a big hindrance to the development of BMP inhibitors for clinical use. To address this issue, we carried out in silico experiments to test whether computational methods can reproduce and explain the high selectivity of a small molecule BMP inhibitor DMH1 on BMPRI kinase ALK2 vs. the closely related TGF-β type I receptor kinase ALK5 and vascular endothelial growth factor receptor type 2 (VEGFR2 tyrosine kinase. We found that, while the rigid docking method used here gave nearly identical binding affinity scores among the three kinases; free energy perturbation coupled with Hamiltonian replica-exchange molecular dynamics (FEP/H-REMD simulations reproduced the absolute binding free energies in excellent agreement with experimental data. Furthermore, the binding poses identified by FEP/H-REMD led to a quantitative analysis of physical/chemical determinants governing DMH1 selectivity. The current work illustrates that small changes in the binding site residue type (e.g. pre-hinge region in ALK2 vs. ALK5 or side chain orientation (e.g. Tyr219 in caALK2 vs. wtALK2, as well as a subtle structural modification on the ligand (e.g. DMH1 vs. LDN193189 will cause distinct binding profiles and selectivity among BMP inhibitors. Therefore, the current computational approach represents a new way of investigating BMP inhibitors. Our results provide critical information for designing exclusively selective BMP inhibitors for the development of effective pharmacotherapy for diseases caused by aberrant BMP signaling.

  1. Snapin interacts with G-protein coupled receptor PKR2.

    Science.gov (United States)

    Song, Jian; Li, Jie; Liu, Hua-die; Liu, Wei; Feng, Yong; Zhou, Xiao-Tao; Li, Jia-Da

    2016-01-15

    Mutations in Prokineticin receptor 2 (PKR2), a G-protein-coupled receptor, have been identified in patients with Kallmann syndrome and/or idiopathic hypogonadotropic hypogonadism, characterized by delayed puberty and infertility. In this study, we performed yeast two-hybrid screening by using PKR2 C-terminus (amino acids 333-384) as a bait, and identified Snapin as a novel interaction partner for PKR2. The interaction of Snapin and PKR2 was confirmed in GST pull-down and co-immunoprecipitation studies. We further demonstrated that two α-helix domains in Snapin are required for the interaction. And the interactive motifs of PKR2 were mapped to YFK (343-345) and HWR (351-353), which shared a similar sequence of two aromatic amino acids followed by a basic amino acid. Disruption of Snapin-PKR2 interaction did not affect PKR2 signaling, but increased the ligand-induced degradation, implying a role of Snapin in the trafficking of PKR2.

  2. Fucosylation and protein glycosylation create functional receptors for cholera toxin

    Science.gov (United States)

    Wands, Amberlyn M; Fujita, Akiko; McCombs, Janet E; Cervin, Jakob; Dedic, Benjamin; Rodriguez, Andrea C; Nischan, Nicole; Bond, Michelle R; Mettlen, Marcel; Trudgian, David C; Lemoff, Andrew; Quiding-Järbrink, Marianne; Gustavsson, Bengt; Steentoft, Catharina; Clausen, Henrik; Mirzaei, Hamid; Teneberg, Susann; Yrlid, Ulf; Kohler, Jennifer J

    2015-01-01

    Cholera toxin (CT) enters and intoxicates host cells after binding cell surface receptors using its B subunit (CTB). The ganglioside (glycolipid) GM1 is thought to be the sole CT receptor; however, the mechanism by which CTB binding to GM1 mediates internalization of CT remains enigmatic. Here we report that CTB binds cell surface glycoproteins. Relative contributions of gangliosides and glycoproteins to CTB binding depend on cell type, and CTB binds primarily to glycoproteins in colonic epithelial cell lines. Using a metabolically incorporated photocrosslinking sugar, we identified one CTB-binding glycoprotein and demonstrated that the glycan portion of the molecule, not the protein, provides the CTB interaction motif. We further show that fucosylated structures promote CTB entry into a colonic epithelial cell line and subsequent host cell intoxication. CTB-binding fucosylated glycoproteins are present in normal human intestinal epithelia and could play a role in cholera. DOI: http://dx.doi.org/10.7554/eLife.09545.001 PMID:26512888

  3. Protein receptor-independent plasma membrane remodeling by HAMLET

    DEFF Research Database (Denmark)

    Nadeem, Aftab; Sanborn, Jeremy; Gettel, Douglas L.

    2015-01-01

    in signal transduction. Here, we propose that membrane perturbation may serve as an alternative mechanism to activate a conserved cell-death program in cancer cells. This view emerges from the extraordinary manner in which HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells) kills a wide range...... of tumor cells in vitro and demonstrates therapeutic efficacy and selectivity in cancer models and clinical studies. We identify a "receptor independent" transformation of vesicular motifs in model membranes, which is paralleled by gross remodeling of tumor cell membranes. Furthermore, we find that HAMLET...... accumulates within these de novo membrane conformations and define membrane blebs as cellular compartments for direct interactions of HAMLET with essential target proteins such as the Ras family of GTPases. Finally, we demonstrate lower sensitivity of healthy cell membranes to HAMLET challenge. These features...

  4. Molecular dynamics techniques for modeling G protein-coupled receptors.

    Science.gov (United States)

    McRobb, Fiona M; Negri, Ana; Beuming, Thijs; Sherman, Woody

    2016-10-01

    G protein-coupled receptors (GPCRs) constitute a major class of drug targets and modulating their signaling can produce a wide range of pharmacological outcomes. With the growing number of high-resolution GPCR crystal structures, we have the unprecedented opportunity to leverage structure-based drug design techniques. Here, we discuss a number of advanced molecular dynamics (MD) techniques that have been applied to GPCRs, including long time scale simulations, enhanced sampling techniques, water network analyses, and free energy approaches to determine relative binding free energies. On the basis of the many success stories, including those highlighted here, we expect that MD techniques will be increasingly applied to aid in structure-based drug design and lead optimization for GPCRs.

  5. Engineering therapeutic antibodies targeting G-protein-coupled receptors.

    Science.gov (United States)

    Jo, Migyeong; Jung, Sang Taek

    2016-02-05

    G-protein-coupled receptors (GPCRs) are one of the most attractive therapeutic target classes because of their critical roles in intracellular signaling and their clinical relevance to a variety of diseases, including cancer, infection and inflammation. However, high conformational variability, the small exposed area of extracellular epitopes and difficulty in the preparation of GPCR antigens have delayed both the isolation of therapeutic anti-GPCR antibodies as well as studies on the structure, function and biochemical mechanisms of GPCRs. To overcome the challenges in generating highly specific anti-GPCR antibodies with enhanced efficacy and safety, various forms of antigens have been successfully designed and employed for screening with newly emerged systems based on laboratory animal immunization and high-throughput-directed evolution.

  6. Running Boot Camp

    CERN Document Server

    Toporek, Chuck

    2008-01-01

    When Steve Jobs jumped on stage at Macworld San Francisco 2006 and announced the new Intel-based Macs, the question wasn't if, but when someone would figure out a hack to get Windows XP running on these new "Mactels." Enter Boot Camp, a new system utility that helps you partition and install Windows XP on your Intel Mac. Boot Camp does all the heavy lifting for you. You won't need to open the Terminal and hack on system files or wave a chicken bone over your iMac to get XP running. This free program makes it easy for anyone to turn their Mac into a dual-boot Windows/OS X machine. Running Bo

  7. Involvement of the G-protein-coupled receptor 4 in RANKL expression by osteoblasts in an acidic environment

    Energy Technology Data Exchange (ETDEWEB)

    Okito, Asuka [Department of Cellular Physiological Chemistry, Tokyo Medical and Dental University, Tokyo (Japan); Department of Orthodontic Science, Tokyo Medical and Dental University, Tokyo (Japan); Nakahama, Ken-ichi, E-mail: nakacell@tmd.ac.jp [Department of Cellular Physiological Chemistry, Tokyo Medical and Dental University, Tokyo (Japan); Akiyama, Masako [Department of Cellular Physiological Chemistry, Tokyo Medical and Dental University, Tokyo (Japan); Ono, Takashi [Department of Orthodontic Science, Tokyo Medical and Dental University, Tokyo (Japan); Morita, Ikuo [Department of Cellular Physiological Chemistry, Tokyo Medical and Dental University, Tokyo (Japan)

    2015-03-06

    Osteoclast activity is enhanced in acidic environments following systemic or local inflammation. However, the regulatory mechanism of receptor activator of NF-κB ligand (RANKL) expression in osteoblasts under acidic conditions is not fully understood. In the present paper, we detected the mRNA expression of the G-protein-coupled receptor (GPR) proton sensors GPR4 and GPR65 (T-cell death-associated gene 8, TDAG8), in osteoblasts. RANKL expression and the cyclic AMP (cAMP) level in osteoblasts were up-regulated under acidic culture conditions. Acidosis-induced up-regulation of RANKL was abolished by the protein kinase A inhibitor H89. To clarify the role of GPR4 in RANKL expression, GPR4 gain and loss of function experiments were performed. Gene knockdown and forced expression of GPR4 caused reduction and induction of RANKL expression, respectively. These results suggested that, at least in part, RANKL expression by osteoblasts in an acidic environment was mediated by cAMP/PKA signaling resulting from GPR4 activation. A comprehensive microarray analysis of gene expression of osteoblasts revealed that, under acidic conditions, the phenotype of osteoblasts was that of an osteoclast supporting cell rather than that of a mineralizing cell. These findings will contribute to a molecular understanding of bone disruption in an acidic environment. - Highlights: • RANKL expression was increased in osteoblasts under acidosis via cAMP/PKA pathway. • GRP4 knockdown resulted in decrease of RANKL expression. • GRP4 overexpression resulted in increase of RANKL expression. • Osteoblast mineralization was reduced under acidic condition.

  8. Development of a novel molecular sensor for imaging estrogen receptor-coactivator protein-protein interactions.

    Directory of Open Access Journals (Sweden)

    Madryn C Lake

    Full Text Available Anti-estrogens, in particular tissue selective anti-estrogens, have been the bedrock of adjuvant therapy for patients with estrogen receptor alpha (ERα positive breast cancer. Though current therapies have greatly enhanced patient prognosis, there continues to be an impetus for the development of improved anti-estrogens. ERα is a nuclear receptor transcription factor which activates gene expression through the recruitment of transcriptional coactivator proteins. The SRC family of coactivators, which includes AIB1, has been shown to be of particular importance for ERα mediated transcription. ERα-AIB1 interactions are indicative of gene expression and are inhibited by anti-estrogen treatment. We have exploited the interaction between ERα and AIB1 as a novel method for imaging ERα activity using a split luciferase molecular sensor. By producing a range of ERα ligand binding domain (ER-LBD and AIB1 nuclear receptor interacting domain (AIB-RID N- and C-terminal firefly luciferase fragment fusion proteins, constructs which exhibited more than a 10-fold increase in luciferase activity with E2 stimulation were identified. The specificity of the E2-stimulated luciferase activity to ERα-AIB1 interaction was validated through Y537S and L539/540A ER-LBD fusion protein mutants. The primed nature of the split luciferase assay allowed changes in ERα activity, with respect to the protein-protein interactions preceding transcription, to be assessed soon after drug treatment. The novel assay split luciferase detailed in this report enabled modulation of ERα activity to be sensitively imaged in vitro and in living subjects and potentially holds much promise for imaging the efficacy of novel ERα specific therapies.

  9. Endocytosis and membrane receptor internalization: implication of F-BAR protein Carom.

    Science.gov (United States)

    Xu, Yanjie; Xia, Jixiang; Liu, Suxuan; Stein, Sam; Ramon, Cueto; Xi, Hang; Wang, Luqiao; Xiong, Xinyu; Zhang, Lixiao; He, Dingwen; Yang, William; Zhao, Xianxian; Cheng, Xiaoshu; Yang, Xiaofeng; Wang, Hong

    2017-03-01

    Endocytosis is a cellular process mostly responsible for membrane receptor internalization. Cell membrane receptors bind to their ligands and form a complex which can be internalized. We previously proposed that F-BAR protein initiates membrane curvature and mediates endocytosis via its binding partners. However, F-BAR protein partners involved in membrane receptor endocytosis and the regulatory mechanism remain unknown. In this study, we established database mining strategies to explore mechanisms underlying receptor-related endocytosis. We identified 34 endocytic membrane receptors and 10 regulating proteins in clathrin-dependent endocytosis (CDE), a major process of membrane receptor internalization. We found that F-BAR protein FCHSD2 (Carom) may facilitate endocytosis via 9 endocytic partners. Carom is highly expressed, along with highly expressed endocytic membrane receptors and partners, in endothelial cells and macrophages. We established 3 models of Carom-receptor complexes and their intracellular trafficking based on protein interaction and subcellular localization. We conclude that Carom may mediate receptor endocytosis and transport endocytic receptors to the cytoplasm for receptor signaling and lysosome/proteasome degradation, or to the nucleus for RNA processing, gene transcription and DNA repair.

  10. Deletion of G-protein-coupled receptor 55 promotes obesity by reducing physical activity

    Science.gov (United States)

    Cannabinoid receptor 1 (CB1) is the best-characterized cannabinoid receptor, and CB1 antagonists are used in clinical trials to treat obesity. Because of the wide range of CB1 functions, the side effects of CB1 antagonists pose serious concerns. G-protein-coupled receptor 55 (GPR55) is an atypical c...

  11. A Summer Camp

    Institute of Scientific and Technical Information of China (English)

    2012-01-01

    正This summer,I had some special days.I joined Dongzhou International Educational Exchange Summer Camp. First,I will tell you about our foreign teachers,they are Shrina and Rebecca. They are friendly and beautiful.They are students at Oxford University. We talked about many things:famous people,subjects in England,different jobs, our deal days,western star signs,what can we say in a restaurant and so on.

  12. Interprofessional Flight Camp.

    Science.gov (United States)

    Alfes, Celeste M; Rowe, Amanda S

    2016-01-01

    The Dorothy Ebersbach Academic Center for Flight Nursing in Cleveland, OH, holds an annual flight camp designed for master's degree nursing students in the acute care nurse practitioner program, subspecializing in flight nursing at the Frances Payne Bolton School of Nursing at Case Western Reserve University. The weeklong interprofessional training is also open to any health care provider working in an acute care setting and focuses on critical care updates, trauma, and emergency care within the critical care transport environment. This year, 29 graduate nursing students enrolled in a master's degree program from Puerto Rico attended. Although the emergency department in Puerto Rico sees and cares for trauma patients, there is no formal trauma training program. Furthermore, the country only has 1 rotor wing air medical transport service located at the Puerto Rico Medical Center in San Juan. Flight faculty and graduate teaching assistants spent approximately 9 months planning for their participation in our 13th annual flight camp. Students from Puerto Rico were extremely pleased with the learning experiences at camp and expressed particular interest in having more training time within the helicopter flight simulator.

  13. Intracellular adenosine 3',5'-phosphate formation is essential for down-regulation of surface adenosine 3',5'-phosphate receptors in Dictyostelium

    NARCIS (Netherlands)

    Haastert, Peter J.M. van

    1994-01-01

    Dictyostelium discoideum cells contain cell surface cyclic AMP (cAMP) receptors that bind cAMP as a first messenger and intracellular cAMP receptors that bind cAMP as a second messenger. Prolonged incubation of Dictyostelium cells with cAMP induces a sequential process of phosphorylation, sequestrat

  14. Cannabinoid receptor-interacting protein 1a modulates CB1 receptor signaling and regulation.

    Science.gov (United States)

    Smith, Tricia H; Blume, Lawrence C; Straiker, Alex; Cox, Jordan O; David, Bethany G; McVoy, Julie R Secor; Sayers, Katherine W; Poklis, Justin L; Abdullah, Rehab A; Egertová, Michaela; Chen, Ching-Kang; Mackie, Ken; Elphick, Maurice R; Howlett, Allyn C; Selley, Dana E

    2015-04-01

    Cannabinoid CB1 receptors (CB1Rs) mediate the presynaptic effects of endocannabinoids in the central nervous system (CNS) and most behavioral effects of exogenous cannabinoids. Cannabinoid receptor-interacting protein 1a (CRIP1a) binds to the CB1R C-terminus and can attenuate constitutive CB1R-mediated inhibition of Ca(2+) channel activity. We now demonstrate cellular colocalization of CRIP1a at neuronal elements in the CNS and show that CRIP1a inhibits both constitutive and agonist-stimulated CB1R-mediated guanine nucleotide-binding regulatory protein (G-protein) activity. Stable overexpression of CRIP1a in human embryonic kidney (HEK)-293 cells stably expressing CB1Rs (CB1-HEK), or in N18TG2 cells endogenously expressing CB1Rs, decreased CB1R-mediated G-protein activation (measured by agonist-stimulated [(35)S]GTPγS (guanylyl-5'-[O-thio]-triphosphate) binding) in both cell lines and attenuated inverse agonism by rimonabant in CB1-HEK cells. Conversely, small-interfering RNA-mediated knockdown of CRIP1a in N18TG2 cells enhanced CB1R-mediated G-protein activation. These effects were not attributable to differences in CB1R expression or endocannabinoid tone because CB1R levels did not differ between cell lines varying in CRIP1a expression, and endocannabinoid levels were undetectable (CB1-HEK) or unchanged (N18TG2) by CRIP1a overexpression. In CB1-HEK cells, 4-hour pretreatment with cannabinoid agonists downregulated CB1Rs and desensitized agonist-stimulated [(35)S]GTPγS binding. CRIP1a overexpression attenuated CB1R downregulation without altering CB1R desensitization. Finally, in cultured autaptic hippocampal neurons, CRIP1a overexpression attenuated both depolarization-induced suppression of excitation and inhibition of excitatory synaptic activity induced by exogenous application of cannabinoid but not by adenosine A1 agonists. These results confirm that CRIP1a inhibits constitutive CB1R activity and demonstrate that CRIP1a can also inhibit agonist

  15. Protein phosphatase 1 suppresses androgen receptor ubiquitylation and degradation.

    Science.gov (United States)

    Liu, Xiaming; Han, Weiwei; Gulla, Sarah; Simon, Nicholas I; Gao, Yanfei; Cai, Changmeng; Yang, Hongmei; Zhang, Xiaoping; Liu, Jihong; Balk, Steven P; Chen, Shaoyong

    2016-01-12

    The phosphoprotein phosphatases are emerging as important androgen receptor (AR) regulators in prostate cancer (PCa). We reported previously that the protein phosphatase 1 catalytic subunit (PP1α) can enhance AR activity by dephosphorylating a site in the AR hinge region (Ser650) and thereby decrease AR nuclear export. In this study we show that PP1α increases the expression of wildtype as well as an S650A mutant AR, indicating that it is acting through one or more additional mechanisms. We next show that PP1α binds primarily to the AR ligand binding domain and decreases its ubiquitylation and degradation. Moreover, we find that the PP1α inhibitor tautomycin increases phosphorylation of AR ubiquitin ligases including SKP2 and MDM2 at sites that enhance their activity, providing a mechanism by which PP1α may suppress AR degradation. Significantly, the tautomycin mediated decrease in AR expression was most pronounced at low androgen levels or in the presence of the AR antagonist enzalutamide. Consistent with this finding, the sensitivity of LNCaP and C4-2 PCa cells to tautomycin, as assessed by PSA synthesis and proliferation, was enhanced at low androgen levels or by treatment with enzalutamide. Together these results indicate that PP1α may contribute to stabilizing AR protein after androgen deprivation therapies, and that targeting PP1α or the AR-PP1α interaction may be effective in castration-resistant prostate cancer (CRPC).

  16. Protein receptor-independent plasma membrane remodeling by HAMLET: a tumoricidal protein-lipid complex.

    Science.gov (United States)

    Nadeem, Aftab; Sanborn, Jeremy; Gettel, Douglas L; James, Ho C S; Rydström, Anna; Ngassam, Viviane N; Klausen, Thomas Kjær; Pedersen, Stine Falsig; Lam, Matti; Parikh, Atul N; Svanborg, Catharina

    2015-11-12

    A central tenet of signal transduction in eukaryotic cells is that extra-cellular ligands activate specific cell surface receptors, which orchestrate downstream responses. This ''protein-centric" view is increasingly challenged by evidence for the involvement of specialized membrane domains in signal transduction. Here, we propose that membrane perturbation may serve as an alternative mechanism to activate a conserved cell-death program in cancer cells. This view emerges from the extraordinary manner in which HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells) kills a wide range of tumor cells in vitro and demonstrates therapeutic efficacy and selectivity in cancer models and clinical studies. We identify a ''receptor independent" transformation of vesicular motifs in model membranes, which is paralleled by gross remodeling of tumor cell membranes. Furthermore, we find that HAMLET accumulates within these de novo membrane conformations and define membrane blebs as cellular compartments for direct interactions of HAMLET with essential target proteins such as the Ras family of GTPases. Finally, we demonstrate lower sensitivity of healthy cell membranes to HAMLET challenge. These features suggest that HAMLET-induced curvature-dependent membrane conformations serve as surrogate receptors for initiating signal transduction cascades, ultimately leading to cell death.

  17. Computational Modeling for the Activation Cycle of G-proteins by G-protein-coupled Receptors

    Directory of Open Access Journals (Sweden)

    Yifei Bao

    2010-10-01

    Full Text Available In this paper, we survey five different computational modeling methods. For comparison, we use the activation cycle of G-proteins that regulate cellular signaling events downstream of G-protein-coupled receptors (GPCRs as a driving example. Starting from an existing Ordinary Differential Equations (ODEs model, we implement the G-protein cycle in the stochastic Pi-calculus using SPiM, as Petri-nets using Cell Illustrator, in the Kappa Language using Cellucidate, and in Bio-PEPA using the Bio-PEPA eclipse plug in. We also provide a high-level notation to abstract away from communication primitives that may be unfamiliar to the average biologist, and we show how to translate high-level programs into stochastic Pi-calculus processes and chemical reactions.

  18. Protein kinase A increases type-2 inositol 1,4,5-trisphosphate receptor activity by phosphorylation of serine 937.

    Science.gov (United States)

    Betzenhauser, Matthew J; Fike, Jenna L; Wagner, Larry E; Yule, David I

    2009-09-11

    Protein kinase A (PKA) phosphorylation of inositol 1,4,5-trisphosphate receptors (InsP(3)Rs) represents a mechanism for shaping intracellular Ca(2+) signals following a concomitant elevation in cAMP. Activation of PKA results in enhanced Ca(2+) release in cells that express predominantly InsP(3)R2. PKA is known to phosphorylate InsP(3)R2, but the molecular determinants of this effect are not known. We have expressed mouse InsP(3)R2 in DT40-3KO cells that are devoid of endogenous InsP(3)R and examined the effects of PKA phosphorylation on this isoform in unambiguous isolation. Activation of PKA increased Ca(2+) signals and augmented the single channel open probability of InsP(3)R2. A PKA phosphorylation site unique to the InsP(3)R2 was identified at Ser(937). The enhancing effects of PKA activation on this isoform required the phosphorylation of Ser(937), since replacing this residue with alanine eliminated the positive effects of PKA activation. These results provide a mechanism responsible for the enhanced Ca(2+) signaling following PKA activation in cells that express predominantly InsP(3)R2.

  19. The Relationship Among HOXA10, Estrogen Receptor α, Progesterone Receptor, and Progesterone Receptor B Proteins in Rectosigmoid Endometriosis

    Science.gov (United States)

    Pereira, Ricardo Mendes Alves; da Rocha, André Monteiro; Cogliati, Bruno; Baracat, Edmund Chada; Taylor, Hugh S.; da Motta, Eduardo Leme Alves; Serafini, Paulo Cesar

    2015-01-01

    Background: Very few studies have evaluated the expression of homeobox A10 (HOXA10) and steroid (estrogen and progesterone) receptors exclusively in deep endometriosis. Conclusions drawn from studies evaluating peritoneal and ovarian endometriosis are usually generalized to explain the pathogenesis of the disease as a whole. We aimed to evaluate the expression of HOXA10, estrogen receptor α (ER-α), progesterone receptor (PR), and PR-B in rectosigmoid endometriosis (RE), a typical model of deep disease. Methods: We used RE samples from 18 consecutive patients to construct tissue microarray blocks. Nine patients each were operated during the proliferative and secretory phases of the menstrual cycle. We quantified the expressions of proteins by immunohistochemistry using the modified Allred score. Result: The HOXA10 was expressed in the stroma of nodules during the secretory phase in 5 of the 18 patients. Expression of ER-α (in 16 of 18 patients), PR (in 17 of 18 patients), and PR-B (17 of 18 patients) was moderate to strong in the glands and stroma of nodules during both phases. Expression of both PR (P = .023) and PR-B (P = .024) was significantly greater during the secretory phase. Conclusion: The HOXA10 is expressed in RE, where it likely imparts the de novo identity of endometriotic lesions. The ER-α, PR, and PR-B are strongly expressed in RE, which differs from previous studies investigating peritoneal and ovarian lesions. This suggests different routes of pathogenesis for each of the 3 types of endometriosis. PMID:25217304

  20. G Protein-Coupled Receptors: Extranuclear Mediators for the Non-Genomic Actions of Steroids

    OpenAIRE

    Chen Wang; Yi Liu; Ji-Min Cao

    2014-01-01

    Steroids hormones possess two distinct actions, a delayed genomic effect and a rapid non-genomic effect. Rapid steroid-triggered signaling is mediated by specific receptors localized most often to the plasma membrane. The nature of these receptors is of great interest and accumulated data suggest that G protein-coupled receptors (GPCRs) are appealing candidates. Increasing evidence regarding the interaction between steroids and specific membrane proteins, as well as the involvement of G prot...

  1. Activation of a GTP-binding protein and a GTP-binding-protein-coupled receptor kinase (beta-adrenergic-receptor kinase-1) by a muscarinic receptor m2 mutant lacking phosphorylation sites.

    Science.gov (United States)

    Kameyama, K; Haga, K; Haga, T; Moro, O; Sadée, W

    1994-12-01

    A mutant of the human muscarinic acetylcholine receptor m2 subtype (m2 receptor), lacking a large part of the third intracellular loop, was expressed and purified using the baculovirus/insect cell culture system. The mutant was not phosphorylated by beta-adrenergic-receptor kinase, as expected from the previous assignment of phosphorylation sites to the central part of the third intracellular loop. However, the m2 receptor mutant was capable of stimulating beta-adrenergic-receptor-kinase-1-mediated phosphorylation of a glutathione S-transferase fusion protein containing the m2 phosphorylation sites in an agonist-dependent manner. Both mutant and wild-type m2 receptors reconstituted with the guanine-nucleotide-binding regulatory proteins (G protein), G(o) and G(i)2, displayed guanine-nucleotide-sensitive high-affinity agonist binding, as assessed by displacement of [3H]quinuclidinyl-benzilate binding with carbamoylcholine, and both stimulated guanosine 5'-3-O-[35S]thiotriphosphate ([35S]GTP[S]) binding in the presence of carbamoylcholine and GDP. The Ki values of carbamoylcholine effects on [3H]quinuclidinyl-benzilate binding were indistinguishable for the mutant and wild-type m2 receptors. Moreover, the phosphorylation of the wild-type m2 receptor by beta-adrenergic-receptor kinase-1 did not affect m2 interaction with G proteins as assessed by the binding of [3H]quinuclidinyl benzilate or [35S]GTP[S]. These results indicate that (a) the m2 receptor serves both as an activator and as a substrate of beta-adrenergic-receptor kinase, and (b) a large part of the third intracellular loop of the m2 receptor does not contribute to interaction with G proteins and its phosphorylation by beta-adrenergic-receptor kinase does not uncouple the receptor and G proteins in reconstituted lipid vesicles.

  2. Bicarbonate-responsive “soluble” adenylyl cyclase defines a nuclear cAMP microdomain

    Science.gov (United States)

    Zippin, Jonathan H.; Farrell, Jeanne; Huron, David; Kamenetsky, Margarita; Hess, Kenneth C.; Fischman, Donald A.; Levin, Lonny R.; Buck, Jochen

    2004-01-01

    Bicarbonate-responsive “soluble” adenylyl cyclase resides, in part, inside the mammalian cell nucleus where it stimulates the activity of nuclear protein kinase A to phosphorylate the cAMP response element binding protein (CREB). The existence of this complete and functional, nuclear-localized cAMP pathway establishes that cAMP signals in intracellular microdomains and identifies an alternate pathway leading to CREB activation. PMID:14769862

  3. Bicarbonate-responsive “soluble” adenylyl cyclase defines a nuclear cAMP microdomain

    OpenAIRE

    2004-01-01

    Bicarbonate-responsive “soluble” adenylyl cyclase resides, in part, inside the mammalian cell nucleus where it stimulates the activity of nuclear protein kinase A to phosphorylate the cAMP response element binding protein (CREB). The existence of this complete and functional, nuclear-localized cAMP pathway establishes that cAMP signals in intracellular microdomains and identifies an alternate pathway leading to CREB activation.

  4. Severe malaria is associated with parasite binding to endothelial protein C receptor

    DEFF Research Database (Denmark)

    Turner, Louise; Lavstsen, Thomas; Berger, Sanne S;

    2013-01-01

    . falciparum erythrocyte membrane protein 1 (PfEMP1) family and receptors on the endothelial lining. Severe childhood malaria is associated with expression of specific PfEMP1 subtypes containing domain cassettes (DCs) 8 and 13 (ref. 3), but the endothelial receptor for parasites expressing these proteins...... was unknown. Here we identify endothelial protein C receptor (EPCR), which mediates the cytoprotective effects of activated protein C, as the endothelial receptor for DC8 and DC13 PfEMP1. We show that EPCR binding is mediated through the amino-terminal cysteine-rich interdomain region (CIDRα1) of DC8...... and group A PfEMP1 subfamilies, and that CIDRα1 interferes with protein C binding to EPCR. This PfEMP1 adhesive property links P. falciparum cytoadhesion to a host receptor involved in anticoagulation and endothelial cytoprotective pathways, and has implications for understanding malaria pathology...

  5. Effect of neoadjuvant chemotherapy on low-density lipoprotein (LDL) receptor and LDL receptor-related protein 1 (LRP-1) receptor in locally advanced breast cancer

    Energy Technology Data Exchange (ETDEWEB)

    Pires, L.A. [Laboratório de Metabolismo de Lípides, Instituto do Coração, Faculdade de Medicina, Hospital das Clínicas, Universidade de São Paulo, São Paulo, SP (Brazil); Departamento de Ginecologia, Faculdade de Medicina, Hospital das Clínicas, Universidade de São Paulo, São Paulo, SP (Brazil); Hegg, R. [Departamento de Ginecologia, Faculdade de Medicina, Hospital das Clínicas, Universidade de São Paulo, São Paulo, SP (Brazil); Freitas, F.R.; Tavares, E.R.; Almeida, C.P. [Laboratório de Metabolismo de Lípides, Instituto do Coração, Faculdade de Medicina, Hospital das Clínicas, Universidade de São Paulo, São Paulo, SP (Brazil); Baracat, E.C. [Departamento de Ginecologia, Faculdade de Medicina, Hospital das Clínicas, Universidade de São Paulo, São Paulo, SP (Brazil); Maranhão, R.C. [Laboratório de Metabolismo de Lípides, Instituto do Coração, Faculdade de Medicina, Hospital das Clínicas, Universidade de São Paulo, São Paulo, SP (Brazil); Faculdade de Ciências Farmacêuticas, Universidade de São Paulo, São Paulo, SP (Brazil)

    2012-05-04

    Low-density lipoprotein (LDL) receptors are overexpressed in most neoplastic cell lines and provide a mechanism for the internalization and concentration of drug-laden nanoemulsions that bind to these receptors. The aim of the present study was to determine whether the administration of standard chemotherapeutic schemes can alter the expression of LDL and LDL receptor-related protein 1 (LRP-1) receptors in breast carcinoma. Fragments of tumoral and normal breast tissue from 16 consecutive volunteer women with breast cancer in stage II or III were obtained from biopsies before the beginning of neoadjuvant chemotherapy and after chemotherapy, from fragments excised during mastectomy. Tissues were analyzed by immunohistochemistry for both receptors. Because complete response to treatment was achieved in 4 patients, only the tumors from 12 were analyzed. Before chemotherapy, there was overexpression of LDL receptor in the tumoral tissue compared to normal breast tissue in 8 of these patients. LRP-1 receptor overexpression was observed in tumors of 4 patients. After chemotherapy, expression of both receptors decreased in the tumors of 6 patients, increased in 4 and was unchanged in 2. Nonetheless, even when chemotherapy reduced receptors expression, the expression was still above normal. The fact that chemotherapy does not impair LDL receptors expression supports the use of drug carrier systems that target neoplastic cells by the LDL receptor endocytic pathway in patients on conventional chemotherapy.

  6. Identification of a preassembled TRH receptor-G(q/11) protein complex in HEK293 cells.

    Science.gov (United States)

    Drastichova, Zdenka; Novotny, Jiri

    2012-01-01

    Protein-protein interactions define specificity in signal transduction and these interactions are central to transmembrane signaling by G-protein-coupled receptors (GPCRs). It is not quite clear, however, whether GPCRs and the regulatory trimeric G-proteins behave as freely and independently diffusible molecules in the plasma membrane or whether they form some preassociated complexes. Here we used clear-native polyacrylamide gel electrophoresis (CN-PAGE) to investigate the presumed coupling between thyrotropin-releasing hormone (TRH) receptor and its cognate G(q/11) protein in HEK293 cells expressing high levels of these proteins. Under different solubilization conditions, the TRH receptor (TRH-R) was identified to form a putative pentameric complex composed of TRH-R homodimer and G(q/11) protein. The presumed association of TRH-R with G(q/11)α or Gβ proteins in plasma membranes was verified by RNAi experiments. After 10- or 30-min hormone treatment, TRH-R signaling complexes gradually dissociated with a concomitant release of receptor homodimers. These observations support the model in which GPCRs can be coupled to trimeric G-proteins in preassembled signaling complexes, which might be dynamically regulated upon receptor activation. The precoupling of receptors with their cognate G-proteins can contribute to faster G-protein activation and subsequent signal transfer into the cell interior.

  7. Antibody-protein A conjugated quantum dots for multiplexed imaging of surface receptors in living cells.

    Science.gov (United States)

    Jin, Takashi; Tiwari, Dhermendra K; Tanaka, Shin-Ichi; Inouye, Yasushi; Yoshizawa, Keiko; Watanabe, Tomonobu M

    2010-11-01

    To use quantum dots (QDs) as fluorescent probes for receptor imaging, QD surface should be modified with biomolecules such as antibodies, peptides, carbohydrates, and small-molecule ligands for receptors. Among these QDs, antibody conjugated QDs are the most promising fluorescent probes. There are many kinds of coupling reactions that can be used for preparing antibody conjugated QDs. Most of the antibody coupling reactions, however, are non-selective and time-consuming. In this paper, we report a facile method for preparing antibody conjugated QDs for surface receptor imaging. We used ProteinA as an adaptor protein for binding of antibody to QDs. By using ProteinA conjugated QDs, various types of antibodies are easily attached to the surface of the QDs via non-covalent binding between the F(c) (fragment crystallization) region of antibody and ProteinA. To show the utility of ProteinA conjugated QDs, HER2 (anti-human epidermal growth factor receptor 2) in KPL-4 human breast cancer cells were stained by using anti-HER2 antibody conjugated ProteinA-QDs. In addition, multiplexed imaging of HER2 and CXCR4 (chemokine receptor) in the KPL-4 cells was performed. The result showed that CXCR4 receptors coexist with HER2 receptors in the membrane surface of KPL-4 cells. ProteinA mediated antibody conjugation to QDs is very useful to prepare fluorescent probes for multiplexed imaging of surface receptors in living cells.

  8. Identifying neuropeptide and protein hormone receptors in Drosophila melanogaster by exploiting genomic data

    DEFF Research Database (Denmark)

    Hauser, Frank; Williamson, Michael; Cazzamali, Giuseppe

    2006-01-01

    Most neuropeptide and protein hormone receptors belong to the large superfamily of G-protein-coupled receptors (GPCRs). These cell membrane proteins steer many important processes such as development, reproduction, homeostasis and behaviour when activated by their corresponding ligands. The first...... insect genome, that of the fruitfly Drosophila melanogaster, was sequenced in 2000, and about 200 GPCRs have been annnotated in this model insect. About 50 of these receptors were predicted to have neuropeptides or protein hormones as their ligands. Since 2000, the cDNAs of most of these candidate...

  9. N-terminal T4 lysozyme fusion facilitates crystallization of a G protein coupled receptor.

    Directory of Open Access Journals (Sweden)

    Yaozhong Zou

    Full Text Available A highly crystallizable T4 lysozyme (T4L was fused to the N-terminus of the β(2 adrenergic receptor (β(2AR, a G-protein coupled receptor (GPCR for catecholamines. We demonstrate that the N-terminal fused T4L is sufficiently rigid relative to the receptor to facilitate crystallogenesis without thermostabilizing mutations or the use of a stabilizing antibody, G protein, or protein fused to the 3rd intracellular loop. This approach adds to the protein engineering strategies that enable crystallographic studies of GPCRs alone or in complex with a signaling partner.

  10. Registration Day-Camp 2016

    CERN Multimedia

    Nursery School

    2016-01-01

    Reminder Registration for the CERN Staff Association Day-camp are open for children from 4 to 6 years old More information on the website: http://nurseryschool.web.cern.ch/. The day-camp is open to all children. An inscription per week is proposed, cost 480.-CHF/week, lunch included The camp will be open weeks 27, 28, 29 and 30, from 8:30 am to 5:30 pm. For further questions, thanks you for contacting us by email at Summer.Camp@cern.ch.

  11. Expression profiling of cumulus cells reveals functional changes during ovulation and central roles of prostaglandin EP2 receptor in cAMP signaling.

    Science.gov (United States)

    Tamba, Shigero; Yodoi, Rieko; Morimoto, Kazushi; Inazumi, Tomoaki; Sukeno, Mamiko; Segi-Nishida, Eri; Okuno, Yasushi; Tsujimoto, Gozoh; Narumiya, Shuh; Sugimoto, Yukihiko

    2010-06-01

    To understand the role of prostaglandin (PG) receptor EP2 (Ptger2) signaling in ovulation and fertilization, we investigated time-dependent expression profiles in wild-type (WT) and Ptger2(-/-) cumuli before and after ovulation by using microarrays. We prepared cumulus cells from mice just before and 3, 9 and 14 h after human chorionic gonadotropin injection. Key genes including cAMP-related and epidermal growth factor (EGF) genes, as well as extracellular matrix- (ECM-) related and chemokine genes were up-regulated in WT cumuli at 3 h and 14 h, respectively. Ptger2 deficiency differently affected the expression of many of the key genes at 3 h and 14 h. These results indicate that the gene expression profile of cumulus cells greatly differs before and after ovulation, and in each situation, PGE(2)-EP2 signaling plays a critical role in cAMP-regulated gene expression in the cumulus cells under physiological conditions.

  12. Peroxisome proliferator-activated receptor delta (PPARdelta )-mediated regulation of preadipocyte proliferation and gene expression is dependent on cAMP signaling

    DEFF Research Database (Denmark)

    Hansen, Jacob B.; Zhang, H; Rasmussen, T H;

    2001-01-01

    The peroxisome proliferator-activated receptor gamma (PPARgamma) is a key regulator of terminal adipocyte differentiation. PPARdelta is expressed in preadipocytes, but the importance of this PPAR subtype in adipogenesis has been a matter of debate. Here we present a critical evaluation of the role...... of PPARdelta in adipocyte differentiation. We demonstrate that treatment of NIH-3T3 fibroblasts overexpressing PPARdelta with standard adipogenic inducers led to induction of PPARgamma2 expression and terminal adipocyte differentiation in a manner that was strictly dependent on simultaneous administration...... expression of PPARgamma and ALBP/aP2, but only modestly promoted terminal differentiation as determined by lipid accumulation. Finally, we provide evidence that synergistic activation of PPARdelta promotes mitotic clonal expansion in 3T3-L1 cells with or without forced expression of PPARdelta. In conclusion...

  13. The E92K melanocortin 1 receptor mutant induces cAMP production and arrestin recruitment but not ERK activity indicating biased constitutive signaling

    DEFF Research Database (Denmark)

    Benned-Jensen, Tau; Mokrosinski, Jacek; Rosenkilde, Mette M

    2011-01-01

    The melanocortin 1 receptor (MC1R) constitutes a key regulator of melanism. Consequently, many naturally-occurring MC1R mutations are associated with a change in color. An example is the Glu-to-Lys substitution found at position II:20/2.60 in the top of transmembrane helix II which has been...... identified in melanic mice and several other species. This mutation induces a pronounced increase in MC1R constitutive activity suggesting a link between constitutive activity and melanism which is corroborated by the attenuation of a-melanocyte stimulating hormone (aMSH) induced activation. However......, the mechanism by which the mutation induces constitutive activity is currently not known....

  14. 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

  15. Identification and Characterization of Pheromone Receptors and Interplay between Receptors and Pheromone Binding Proteins in the Diamondback Moth, Plutella xyllostella

    OpenAIRE

    Sun, Mengjing; Liu, Yang; Walker, William B.; Liu, Chengcheng; Lin, Kejian; Gu, Shaohua; ZHANG Yongjun; Zhou, Jingjiang; Wang, Guirong

    2013-01-01

    Moths depend on olfactory cues such as sex pheromones to find and recognize mating partners. Pheromone receptors (PRs) and Pheromone binding proteins (PBPs) are thought to be associated with olfactory signal transduction of pheromonal compounds in peripheral olfactory reception. Here six candidate pheromone receptor genes in the diamondback moth, Plutella xyllostella were identified and cloned. All of the six candidate PR genes display male-biased expression, which is a typical characteristic...

  16. BDNF downregulates 5-HT(2A) receptor protein levels in hippocampal cultures

    DEFF Research Database (Denmark)

    Trajkovska, V; Santini, M A; Marcussen, Anders Bue;

    2009-01-01

    Both brain-derived neurotrophic factor (BDNF) and the serotonin receptor 2A (5-HT(2A)) have been related to depression pathology. Specific 5-HT(2A) receptor changes seen in BDNF conditional mutant mice suggest that BDNF regulates the 5-HT(2A) receptor level. Here we show a direct effect of BDNF...... on 5-HT(2A) receptor protein levels in primary hippocampal neuronal and mature hippocampal organotypic cultures exposed to different BDNF concentrations for either 1, 3, 5 or 7 days. In vivo effects of BDNF on hippocampal 5-HT(2A) receptor levels were further corroborated in (BDNF +/-) mice...... with reduced BDNF levels. In primary neuronal cultures, 7 days exposure to 25 and 50ng/mL BDNF resulted in downregulation of 5-HT(2A), but not of 5-HT(1A), receptor protein levels. The BDNF-associated downregulation of 5-HT(2A) receptor levels was also observed in mature hippocampal organotypic cultures...

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

    Directory of Open Access Journals (Sweden)

    Michael S. Parker

    2014-03-01

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

  18. Summer Camp as Therapeutic Context: The Camp Logan Program.

    Science.gov (United States)

    McCammon, Susan; And Others

    These symposium papers describe various aspects of the Camp Logan, South Carolina, program, a therapeutic summer residential program for children, ages 8-14, who have significant behavior problems. The philosophy and advantages of the therapeutic camping model are discussed, e.g., structure during the summer, controlled though informal…

  19. G Protein-Coupled Receptors: Extranuclear Mediators for the Non-Genomic Actions of Steroids

    Directory of Open Access Journals (Sweden)

    Chen Wang

    2014-09-01

    Full Text Available Steroids hormones possess two distinct actions, a delayed genomic effect and a rapid non-genomic effect. Rapid steroid-triggered signaling is mediated by specific receptors localized most often to the plasma membrane. The nature of these receptors is of great interest and accumulated data suggest that G protein-coupled receptors (GPCRs are appealing candidates. Increasing evidence regarding the interaction between steroids and specific membrane proteins, as well as the involvement of G protein and corresponding downstream signaling, have led to identification of physiologically relevant GPCRs as steroid extranuclear receptors. Examples include G protein-coupled receptor 30 (GPR30 for estrogen, membrane progestin receptor for progesterone, G protein-coupled receptor family C group 6 member A (GPRC6A and zinc transporter member 9 (ZIP9 for androgen, and trace amine associated receptor 1 (TAAR1 for thyroid hormone. These receptor-mediated biological effects have been extended to reproductive development, cardiovascular function, neuroendocrinology and cancer pathophysiology. However, although great progress have been achieved, there are still important questions that need to be answered, including the identities of GPCRs responsible for the remaining steroids (e.g., glucocorticoid, the structural basis of steroids and GPCRs’ interaction and the integration of extranuclear and nuclear signaling to the final physiological function. Here, we reviewed the several significant developments in this field and highlighted a hypothesis that attempts to explain the general interaction between steroids and GPCRs.

  20. The adapter protein APPL1 links FSH receptor to inositol 1,4,5-trisphosphate production and is implicated in intracellular Ca(2+) mobilization.

    Science.gov (United States)

    Thomas, Richard M; Nechamen, Cheryl A; Mazurkiewicz, Joseph E; Ulloa-Aguirre, Alfredo; Dias, James A

    2011-04-01

    FSH binds to its receptor (FSHR) on target cells in the ovary and testis, to regulate oogenesis and spermatogenesis, respectively. The signaling cascades activated after ligand binding are extremely complex and have been shown to include protein kinase A, mitogen-activated protein kinase, phosphatidylinositol 3-kinase/protein kinase B, and inositol 1,4,5-trisphosphate-mediated calcium signaling pathways. The adapter protein APPL1 (Adapter protein containing Pleckstrin homology domain, Phosphotyrosine binding domain and Leucine zipper motif), which has been linked to an assortment of other signaling proteins, was previously identified as an interacting protein with FSHR. Thus, alanine substitution mutations in the first intracellular loop of FSHR were generated to determine which residues are essential for FSHR-APPL1 interaction. Three amino acids were essential; when any one of them was altered, APPL1 association with FSHR mutants was abrogated. Two of the mutants (L377A and F382A) that displayed poor cell-surface expression were not studied further. Substitution of FSHR-K376A did not affect FSH binding or agonist-stimulated cAMP production in either transiently transfected human embryonic kidney cells or virally transduced human granulosa cells (KGN). In the KGN line, as well as primary cultures of rat granulosa cells transduced with wild type or mutant receptor, FSH-mediated progesterone or estradiol production was not affected by the mutation. However, in human embryonic kidney cells inositol 1,4,5-trisphosphate production was curtailed and KGN cells transduced with FSHR-K376A evidenced reduced Ca(2+) mobilization from intracellular stores after FSH treatment.

  1. Regulation of G protein-coupled receptors by palmitoylation and cholesterol

    OpenAIRE

    2012-01-01

    Abstract Due to their membrane location, G protein-coupled receptors (GPCRs) are subject to regulation by soluble and integral membrane proteins as well as membrane components, including lipids and sterols. GPCRs also undergo a variety of post-translational modifications, including palmitoylation. A recent article by Zheng et al. in BMC Cell Biology demonstrates cooperative roles for receptor palmitoylation and cholesterol binding in GPCR dimerization and G protein coupling, underlining the c...

  2. Transcriptional activation of peroxisome proliferator-activated receptor-{gamma} requires activation of both protein kinase A and Akt during adipocyte differentiation

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Sang-pil [Department of Thoracic and Cardiovascular Surgery, Pusan National University School of Medicine (Korea, Republic of); Ha, Jung Min; Yun, Sung Ji; Kim, Eun Kyoung [MRC for Ischemic Tissue Regeneration, Medical Research Institute, and Department of Pharmacology, Pusan National University School of Medicine (Korea, Republic of); Chung, Sung Woon [Department of Thoracic and Cardiovascular Surgery, Pusan National University School of Medicine (Korea, Republic of); Hong, Ki Whan; Kim, Chi Dae [MRC for Ischemic Tissue Regeneration, Medical Research Institute, and Department of Pharmacology, Pusan National University School of Medicine (Korea, Republic of); Bae, Sun Sik, E-mail: sunsik@pusan.ac.kr [MRC for Ischemic Tissue Regeneration, Medical Research Institute, and Department of Pharmacology, Pusan National University School of Medicine (Korea, Republic of)

    2010-08-13

    Research highlights: {yields} Elevated cAMP activates both PKA and Epac. {yields} PKA activates CREB transcriptional factor and Epac activates PI3K/Akt pathway via Rap1. {yields} Akt modulates PPAR-{gamma} transcriptional activity in concert with CREB. -- Abstract: Peroxisome proliferator-activated receptor-{gamma} (PPAR-{gamma}) is required for the conversion of pre-adipocytes. However, the mechanism underlying activation of PPAR-{gamma} is unclear. Here we showed that cAMP-induced activation of protein kinase A (PKA) and Akt is essential for the transcriptional activation of PPAR-{gamma}. Hormonal induction of adipogenesis was blocked by a phosphatidylinositol 3-kinase (PI3K) inhibitor (LY294002), by a protein kinase A (PKA) inhibitor (H89), and by a Rap1 inhibitor (GGTI-298). Transcriptional activity of PPAR-{gamma} was markedly enhanced by 3-isobutyl-1-methylxanthine (IBMX), but not insulin and dexamethasone. In addition, IBMX-induced PPAR-{gamma} transcriptional activity was blocked by PI3K/Akt, PKA, or Rap1 inhibitors. 8-(4-Chlorophenylthio)-2'-O-methyl-cAMP (8-pCPT-2'-O-Me-cAMP) which is a specific agonist for exchanger protein directly activated by cAMP (Epac) significantly induced the activation of Akt. Furthermore, knock-down of Akt1 markedly attenuated PPAR-{gamma} transcriptional activity. These results indicate that both PKA and Akt signaling pathways are required for transcriptional activation of PPAR-{gamma}, suggesting post-translational activation of PPAR-{gamma} might be critical step for adipogenic gene expression.

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

    DEFF Research Database (Denmark)

    Nørskov-Lauritsen, Lenea; Bräuner-Osborne, Hans

    2015-01-01

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

  4. GATA Factor-G-Protein-Coupled Receptor Circuit Suppresses Hematopoiesis

    Directory of Open Access Journals (Sweden)

    Xin Gao

    2016-03-01

    Full Text Available Hematopoietic stem cells (HSCs originate from hemogenic endothelium within the aorta-gonad-mesonephros (AGM region of the mammalian embryo. The relationship between genetic circuits controlling stem cell genesis and multi-potency is not understood. A Gata2 cis element (+9.5 enhances Gata2 expression in the AGM and induces the endothelial to HSC transition. We demonstrated that GATA-2 rescued hematopoiesis in +9.5−/− AGMs. As G-protein-coupled receptors (GPCRs are the most common targets for FDA-approved drugs, we analyzed the GPCR gene ensemble to identify GATA-2-regulated GPCRs. Of the 20 GATA-2-activated GPCR genes, four were GATA-1-activated, and only Gpr65 expression resembled Gata2. Contrasting with the paradigm in which GATA-2-activated genes promote hematopoietic stem and progenitor cell genesis/function, our mouse and zebrafish studies indicated that GPR65 suppressed hematopoiesis. GPR65 established repressive chromatin at the +9.5 site, restricted occupancy by the activator Scl/TAL1, and repressed Gata2 transcription. Thus, a Gata2 cis element creates a GATA-2-GPCR circuit that limits positive regulators that promote hematopoiesis.

  5. Complete Reversible Refolding of a G-Protein Coupled Receptor on a Solid Support.

    Directory of Open Access Journals (Sweden)

    Natalie Di Bartolo

    Full Text Available The factors defining the correct folding and stability of integral membrane proteins are poorly understood. Folding of only a few select membrane proteins has been scrutinised, leaving considerable deficiencies in knowledge for large protein families, such as G protein coupled receptors (GPCRs. Complete reversible folding, which is problematic for any membrane protein, has eluded this dominant receptor family. Moreover, attempts to recover receptors from denatured states are inefficient, yielding at best 40-70% functional protein. We present a method for the reversible unfolding of an archetypal family member, the β1-adrenergic receptor, and attain 100% recovery of the folded, functional state, in terms of ligand binding, compared to receptor which has not been subject to any unfolding and retains its original, folded structure. We exploit refolding on a solid support, which could avoid unwanted interactions and aggregation that occur in bulk solution. We determine the changes in structure and function upon unfolding and refolding. Additionally, we employ a method that is relatively new to membrane protein folding; pulse proteolysis. Complete refolding of β1-adrenergic receptor occurs in n-decyl-β-D-maltoside (DM micelles from a urea-denatured state, as shown by regain of its original helical structure, ligand binding and protein fluorescence. The successful refolding strategy on a solid support offers a defined method for the controlled refolding and recovery of functional GPCRs and other membrane proteins that suffer from instability and irreversible denaturation once isolated from their native membranes.

  6. Protein-Protein Interactions in Crystals of the Human Receptor-Type Protein Tyrosine Phosphatase ICA512 Ectodomain

    Energy Technology Data Exchange (ETDEWEB)

    Primo M. E.; Jakoncic J.; Noguera M.E.; Risso V.A.; Sosa L.; Sica M.P.; Solimena M.; Poskus E. and Ermacora M.

    2011-09-15

    ICA512 (or IA-2) is a transmembrane protein-tyrosine phosphatase located in secretory granules of neuroendocrine cells. Initially, it was identified as one of the main antigens of autoimmune diabetes. Later, it was found that during insulin secretion, the cytoplasmic domain of ICA512 is cleaved and relocated to the nucleus, where it stimulates the transcription of the insulin gene. The role of the other parts of the receptor in insulin secretion is yet to be unveiled. The structures of the intracellular pseudocatalytic and mature extracellular domains are known, but the transmembrane domain and several intracellular and extracellular parts of the receptor are poorly characterized. Moreover the overall structure of the receptor remains to be established. We started to address this issue studying by X-ray crystallography the structure of the mature ectodomain of ICA512 (ME ICA512) and variants thereof. The variants and crystallization conditions were chosen with the purpose of exploring putative association interfaces, metal binding sites and all other structural details that might help, in subsequent works, to build a model of the entire receptor. Several structural features were clarified and three main different association modes of ME ICA512 were identified. The results provide essential pieces of information for the design of new experiments aimed to assess the structure in vivo.

  7. Protein-protein interactions in crystals of the human receptor-type protein tyrosine phosphatase ICA512 ectodomain.

    Directory of Open Access Journals (Sweden)

    María E Primo

    Full Text Available ICA512 (or IA-2 is a transmembrane protein-tyrosine phosphatase located in secretory granules of neuroendocrine cells. Initially, it was identified as one of the main antigens of autoimmune diabetes. Later, it was found that during insulin secretion, the cytoplasmic domain of ICA512 is cleaved and relocated to the nucleus, where it stimulates the transcription of the insulin gene. The role of the other parts of the receptor in insulin secretion is yet to be unveiled. The structures of the intracellular pseudocatalytic and mature extracellular domains are known, but the transmembrane domain and several intracellular and extracellular parts of the receptor are poorly characterized. Moreover the overall structure of the receptor remains to be established. We started to address this issue studying by X-ray crystallography the structure of the mature ectodomain of ICA512 (ME ICA512 and variants thereof. The variants and crystallization conditions were chosen with the purpose of exploring putative association interfaces, metal binding sites and all other structural details that might help, in subsequent works, to build a model of the entire receptor. Several structural features were clarified and three main different association modes of ME ICA512 were identified. The results provide essential pieces of information for the design of new experiments aimed to assess the structure in vivo.

  8. Multi-Component Protein - Protein Docking Based Protocol with External Scoring for Modeling Dimers of G Protein-Coupled Receptors.

    Science.gov (United States)

    Kaczor, Agnieszka A; Guixà-González, Ramon; Carrió, Pau; Poso, Antti; Dove, Stefan; Pastor, Manuel; Selent, Jana

    2015-04-01

    In order to apply structure-based drug design techniques to GPCR complexes, it is essential to model their 3D structure. For this purpose, a multi-component protocol was derived based on protein-protein docking which generates populations of dimers compatible with membrane integration, considering all reasonable interfaces. At the next stage, we applied a scoring procedure based on up to eleven different parameters including shape or electrostatics complementarity. Two methods of consensus scoring were performed: (i) average scores of 100 best scored dimers with respect to each interface, and (ii) frequencies of interfaces among 100 best scored dimers. In general, our multi-component protocol gives correct indications for dimer interfaces that have been observed in X-ray crystal structures of GPCR dimers (opsin dimer, chemokine CXCR4 and CCR5 dimers, κ opioid receptor dimer, β1 adrenergic receptor dimer and smoothened receptor dimer) but also suggests alternative dimerization interfaces. Interestingly, at times these alternative interfaces are scored higher than the experimentally observed ones suggesting them to be also relevant in the life cycle of studied GPCR dimers. Further results indicate that GPCR dimer and higher-order oligomer formation may involve transmembrane helices (TMs) TM1-TM2-TM7, TM3-TM4-TM5 or TM4-TM5-TM6 but not TM1-TM2-TM3 or TM2-TM3-TM4 which is in general agreement with available experimental and computational data.

  9. Summer Camp, July 2016

    CERN Multimedia

    Staff Association

    2016-01-01

    During the month of July, the Staff Association’s Children’s Day-Care Centre and School EVEE held a summer camp for 4- to 6-year-olds. 24 children altogether joined in on the adventures. On the summer camp, the children got to “travel” to a different continent of the world every week. Day after day, they would pass through make-believe Customs upon arrival and get their passports stamped by a “customs officer”. For the first week, we went on a trip to Africa. In the spirit of the theme, the children got to do plenty of crafts and coloring, make their own little bindles and play various games. They even had the chance to visit the Museum of Ethnography in Geneva (MEG), learn to play the balafon and make musical instruments with Sterrenlab. For the second week, we set off to discover the Americas, exploring both the South and the North. Alongside different workshops (singing, dancing, storytelling, crafts), the children could enjoy several special ac...

  10. Stabilizing effects of G protein on the active conformation of adenosine A1 receptor differ depending on G protein type.

    Science.gov (United States)

    Tateyama, Michihiro; Kubo, Yoshihiro

    2016-10-05

    G protein coupled receptors (GPCRs) trigger various cellular and physiological responses upon the ligand binding. The ligand binding induces conformational change in GPCRs which allows G protein to interact with the receptor. The interaction of G protein also affects the active conformation of GPCRs. In this study, we have investigated the effects of Gαi1, Gαo and chimeric Gαqi5 on the active conformation of the adenosine A1 receptor, as each Gα showed difference in the interaction with adenosine A1 receptor. The conformational changes in the adenosine A1 receptor were detected as the agonist-induced decreases in efficiency of Förster resonance energy transfer (FRET) between fluorescent proteins (FPs) fused at the two intracellular domains of the adenosine A1 receptor. Amplitudes of the agonist-induced FRET decreases were subtle when the FP-tagged adenosine A1 receptor was expressed alone, whereas they were significantly enhanced when co-expressed with Gαi1Gβ1Gγ22 (Gi1) or Gαqi5Gβ1Gγ22 (Gqi5) but not with GαοGβ1Gγ22 (Go). The enhancement of the agonist-induced FRET decrease in the presence of Gqi5 was significantly larger than that of Gi1. Furthermore, the FRET recovery upon the agonist removal in the presence of Gqi5 was significantly slower than that of Gi1. From these results it was revealed that the agonist-bound active conformation of adenosine A1 receptor is unstable without the binding of G protein and that the stabilizing effects of G protein differ depending on the types of G protein.

  11. The cAMP Response Element Binding protein (CREB) is activated by Insulin-like Growth Factor-1 (IGF-1) and regulates myostatin gene expression in skeletal myoblast

    Energy Technology Data Exchange (ETDEWEB)

    Zuloaga, R. [Facultad de Ciencias Biológicas, Universidad Andres Bello, Santiago (Chile); Fuentes, E.N.; Molina, A. [Facultad de Ciencias Biológicas, Universidad Andres Bello, Santiago (Chile); Interdisciplinary Center for Aquaculture Research (INCAR), Víctor Lamas 1290, PO Box 160-C, Concepción (Chile); Valdés, J.A., E-mail: jvaldes@unab.cl [Facultad de Ciencias Biológicas, Universidad Andres Bello, Santiago (Chile); Interdisciplinary Center for Aquaculture Research (INCAR), Víctor Lamas 1290, PO Box 160-C, Concepción (Chile)

    2013-10-18

    Highlights: •IGF-1 induces the activation of CREB via IGF-1R/PI3K/PLC signaling pathway. •Calcium dependent signaling pathways regulate myostatin gene expression. •IGF-1 regulates myostatin gene expression via CREB transcription in skeletal myoblast. -- Abstract: Myostatin, a member of the Transforming Growth Factor beta (TGF-β) superfamily, plays an important role as a negative regulator of skeletal muscle growth and differentiation. We have previously reported that IGF-1 induces a transient myostatin mRNA expression, through the activation of the Nuclear Factor of Activated T cells (NFAT) in an IP{sub 3}/calcium-dependent manner. Here we examined the activation of CREB transcription factor as downstream targets of IGF-1 during myoblast differentiation and its role as a regulator of myostatin gene expression. In cultured skeletal myoblast, IGF-1 induced the phosphorylation and transcriptional activation of CREB via IGF-1 Receptor/Phosphatidylinositol 3-Kinase (PI3K)/Phospholipase C gamma (PLC γ), signaling pathways. Also, IGF-1 induced calcium-dependent molecules such as Calmodulin Kinase II (CaMK II), Extracellular signal-regulated Kinases (ERK), Protein Kinase C (PKC). Additionally, we examined myostatin mRNA levels and myostatin promoter activity in differentiated myoblasts stimulated with IGF-1. We found a significant increase in mRNA contents of myostatin and its reporter activity after treatment with IGF-1. The expression of myostatin in differentiated myoblast was downregulated by the transfection of siRNA–CREB and by pharmacological inhibitors of the signaling pathways involved in CREB activation. By using pharmacological and genetic approaches together these data demonstrate that IGF-1 regulates the myostatin gene expression via CREB transcription factor during muscle cell differentiation.

  12. Streptococcus pyogenes CAMP factor attenuates phagocytic activity of RAW 264.7 cells.

    Science.gov (United States)

    Kurosawa, Mie; Oda, Masataka; Domon, Hisanori; Saitoh, Issei; Hayasaki, Haruaki; Terao, Yutaka

    2016-02-01

    Streptococcus pyogenes produces molecules that inhibit the function of human immune system, thus allowing the pathogen to grow and spread in tissues. It is known that S. pyogenes CAMP factor increases erythrocytosis induced by Staphylococcus aureus β-hemolysin. However, the effects of CAMP factor for immune cells are unclear. In this study, we investigated the effects of CAMP factor to macrophages. Western blotting analysis demonstrated that all examined strains expressed CAMP factor protein. In the presence of calcium or magnesium ion, CAMP factor was significantly released in the supernatant. In addition, both culture supernatant from S. pyogenes strain SSI-9 and recombinant CAMP factor dose-dependently induced vacuolation in RAW 264.7 cells, but the culture supernatant from Δcfa isogenic mutant strain did not. CAMP factor formed oligomers in RAW 264.7 cells in a time-dependent manner. CAMP factor suppressed cell proliferation via G2 phase cell cycle arrest without inducing cell death. Furthermore, CAMP factor reduced the uptake of S. pyogenes and phagocytic activity indicator by RAW 264.7 cells. These results suggest that CAMP factor works as a macrophage dysfunction factor. Therefore, we conclude that CAMP factor allows S. pyogenes to escape the host immune system, and contribute to the spread of streptococcal infection.

  13. DMPD: G-protein-coupled receptor expression, function, and signaling in macrophages. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 17456803 G-protein-coupled receptor expression, function, and signaling in macropha...2007 Apr 24. (.png) (.svg) (.html) (.csml) Show G-protein-coupled receptor expression, function, and signali...ng in macrophages. PubmedID 17456803 Title G-protein-coupled receptor expression,

  14. Kids Camping Takes the Challenge.

    Science.gov (United States)

    James, Vickie L.; Hohnbaum, Claudia

    2002-01-01

    A Wisconsin Girl Scout camp integrated The Healthy Kids Challenge into its program. The camp evaluated policies related to meals, snacks, physical activities, team building, and self-esteem. Staff inservice training resulted in healthier meals on the same budget and developed ownership of the program. Campers and families had opportunities to…

  15. Encountering Child Abuse at Camp.

    Science.gov (United States)

    Durall, John K.

    1997-01-01

    Defines child abuse, including the three categories: physical, sexual, and psychological. Presents characteristics and behaviors of each type of abuse, and long-term effects. Discusses how to handle abuse that occurs at camp, and the effects on the camp. Sidebars present abuse statistics, 15 activities that promote psychological wellness, and 8…

  16. Does protein binding modulate the effect of angiotensin II receptor antagonists?

    Directory of Open Access Journals (Sweden)

    Marc P Maillard

    2001-03-01

    Full Text Available IntroductionAngiotensin II AT 1-receptor antagonists are highly bound to plasma proteins (≥ 99%. With some antagonists, such as DuP-532, the protein binding was such that no efficacy of the drug could be demonstrated clinically. Whether protein binding interferes with the efficacy of other antagonists is not known. We have therefore investigated in vitro how plasma proteins may affect the antagonistic effect of different AT1-receptor antagonists.MethodsA radio-receptor binding assay was used to analyse the interaction between proteins and the ability of various angiotensin II (Ang II antagonists to block AT1-receptors. In addition, the Biacore technology, a new technique which enables the real-time monitoring of binding events between two molecules, was used to evaluate the dissociation rate constants of five AT1-receptor antagonists from human serum albumin.ResultsThe in vitro AT 1-antagonistic effects of different Ang II receptor antagonists were differentially affected by the presence of human plasma, with rightward shifts of the IC50 ranging from one to several orders of magnitude. The importance of the shift correlates with the dissociation rate constants of these drugs from albumin. Our experiments also show that the way that AT1-receptor antagonists bind to proteins differs from one compound to another. These results suggest that the interaction with plasma proteins appears to modulate the efficacy of some Ang II antagonists.ConclusionAlthough the high binding level of Ang II receptor antagonist to plasma proteins appears to be a feature common to this class of compounds, the kinetics and characteristics of this binding is of great importance. With some antagonists, protein binding interferes markedly with their efficacy to block AT1-receptors.

  17. Mechanistic pathways and biological roles for receptor-independent activators of G-protein signaling.

    Science.gov (United States)

    Blumer, Joe B; Smrcka, Alan V; Lanier, Stephen M

    2007-03-01

    Signal processing via heterotrimeric G-proteins in response to cell surface receptors is a central and much investigated aspect of how cells integrate cellular stimuli to produce coordinated biological responses. The system is a target of numerous therapeutic agents and plays an important role in adaptive processes of organs; aberrant processing of signals through these transducing systems is a component of various disease states. In addition to G-protein coupled receptor (GPCR)-mediated activation of G-protein signaling, nature has evolved creative ways to manipulate and utilize the Galphabetagamma heterotrimer or Galpha and Gbetagamma subunits independent of the cell surface receptor stimuli. In such situations, the G-protein subunits (Galpha and Gbetagamma) may actually be complexed with alternative binding partners independent of the typical heterotrimeric Galphabetagamma. Such regulatory accessory proteins include the family of regulator of G-protein signaling (RGS) proteins that accelerate the GTPase activity of Galpha and various entities that influence nucleotide binding properties and/or subunit interaction. The latter group of proteins includes receptor-independent activators of G-protein signaling (AGS) proteins that play surprising roles in signal processing. This review provides an overview of our current knowledge regarding AGS proteins. AGS proteins are indicative of a growing number of accessory proteins that influence signal propagation, facilitate cross talk between various types of signaling pathways, and provide a platform for diverse functions of both the heterotrimeric Galphabetagamma and the individual Galpha and Gbetagamma subunits.

  18. A robust and rapid method of producing soluble, stable, and functional G-protein coupled receptors.

    Directory of Open Access Journals (Sweden)

    Karolina Corin

    Full Text Available Membrane proteins, particularly G-protein coupled receptors (GPCRs, are notoriously difficult to express. Using commercial E. coli cell-free systems with the detergent Brij-35, we could rapidly produce milligram quantities of 13 unique GPCRs. Immunoaffinity purification yielded receptors at >90% purity. Secondary structure analysis using circular dichroism indicated that the purified receptors were properly folded. Microscale thermophoresis, a novel label-free and surface-free detection technique that uses thermal gradients, showed that these receptors bound their ligands. The secondary structure and ligand-binding results from cell-free produced proteins were comparable to those expressed and purified from HEK293 cells. Our study demonstrates that cell-free protein production using commercially available kits and optimal detergents is a robust technology that can be used to produce sufficient GPCRs for biochemical, structural, and functional analyses. This robust and simple method may further stimulate others to study the structure and function of membrane proteins.

  19. Direct Modulation of Heterotrimeric G Protein-coupled Signaling by a Receptor Kinase Complex.

    Science.gov (United States)

    Tunc-Ozdemir, Meral; Urano, Daisuke; Jaiswal, Dinesh Kumar; Clouse, Steven D; Jones, Alan M

    2016-07-01

    Plants and some protists have heterotrimeric G protein complexes that activate spontaneously without canonical G protein-coupled receptors (GPCRs). In Arabidopsis, the sole 7-transmembrane regulator of G protein signaling 1 (AtRGS1) modulates the G protein complex by keeping it in the resting state (GDP-bound). However, it remains unknown how a myriad of biological responses is achieved with a single G protein modulator. We propose that in complete contrast to G protein activation in animals, plant leucine-rich repeat receptor-like kinases (LRR RLKs), not GPCRs, provide this discrimination through phosphorylation of AtRGS1 in a ligand-dependent manner. G protein signaling is directly activated by the pathogen-associated molecular pattern flagellin peptide 22 through its LRR RLK, FLS2, and co-receptor BAK1.

  20. Conformational transitions and interactions underlying the function of membrane embedded receptor protein kinases.

    Science.gov (United States)

    Bocharov, Eduard V; Sharonov, Georgy V; Bocharova, Olga V; Pavlov, Konstantin V

    2017-01-25

    Among membrane receptors, the single-span receptor protein kinases occupy a broad but specific functional niche determined by distinctive features of the underlying transmembrane signaling mechanisms that are briefly overviewed on the basis of some of the most representative examples, followed by a more detailed discussion of several hierarchical levels of organization and interactions involved. All these levels, including single-molecule interactions (e.g., dimerization, liganding, chemical modifications), local processes (e.g. lipid membrane perturbations, cytoskeletal interactions), and larger scale phenomena (e.g., effects of membrane surface shape or electrochemical potential gradients) appear to be closely integrated to achieve the observed diversity of the receptor functioning. Different species of receptor protein kinases meet their specific functional demands through different structural features defining their responses to stimulation, but certain common patterns exist. Signaling by receptor protein kinases is typically associated with the receptor dimerization and clustering, ligand-induced rearrangements of receptor domains through allosteric conformational transitions with involvement of lipids, release of the sequestered lipids, restriction of receptor diffusion, cytoskeleton and membrane shape remodeling. Understanding of complexity and continuity of the signaling processes can help identifying currently neglected opportunities for influencing the receptor signaling with potential therapeutic implications. This article is part of a Special Issue entitled: Interactions between membrane receptors in cellular membranes edited by Kalina Hristova.

  1. Biased and g protein-independent signaling of chemokine receptors

    DEFF Research Database (Denmark)

    Steen, Anne; Larsen, Olav; Thiele, Stefanie;

    2014-01-01

    Biased signaling or functional selectivity occurs when a 7TM-receptor preferentially activates one of several available pathways. It can be divided into three distinct forms: ligand bias, receptor bias, and tissue or cell bias, where it is mediated by different ligands (on the same receptor...... not be absolute, i.e., full versus no activation. Here we discuss biased signaling in the chemokine system, including the structural basis for biased signaling in chemokine receptors, as well as in class A 7TM receptors in general. This includes overall helical movements and the contributions of micro...... a single chemokine may bind to several receptors - in both cases with the same functional outcome. The ubiquitous biased signaling confers a hitherto unknown specificity to the chemokine system with a complex interaction pattern that is better described as promiscuous with context-defined roles...

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

    DEFF Research Database (Denmark)

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

    2012-01-01

    , GPCR oligomerization has been extensively studied using methods like bioluminescence resonance energy transfer (BRET) and today, receptor-receptor interactions within the GPCR superfamily is a well-established phenomenon. Evidence of the impact of GPCR oligomerization on, e.g., ligand binding, receptor...

  3. Ric-8A, a Gα protein guanine nucleotide exchange factor potentiates taste receptor signaling

    Directory of Open Access Journals (Sweden)

    Claire J Fenech

    2009-10-01

    Full Text Available Taste receptors for sweet, bitter and umami tastants are G-protein coupled receptors (GPCRs. While much effort has been devoted to understanding G-protein-receptor interactions and identifying the components of the signalling cascade downstream of these receptors, at the level of the G-protein the modulation of receptor signal transduction remains relatively unexplored. In this regard a taste-specific regulator of G-protein signaling (RGS, RGS21, has recently been identified. To study whether guanine nucleotide exchange factors (GEFs are involved in the transduction of the signal downstream of the taste GPCRs we investigated the expression of Ric-8A and Ric-8B in mouse taste cells and their interaction with G-protein subunits found in taste buds. Mammalian Ric-8 proteins were initially identified as potent GEFs for a range of Gα subunits and Ric-8B has recently been shown to amplify olfactory signal transduction. We find that both Ric-8A and Ric-8B are expressed in a large portion of taste bud cells and that most of these cells contain IP3R-3 a marker for sweet, umami and bitter taste receptor cells. Ric-8A interacts with Gα-gustducin and Gαi2 through which it amplifies the signal transduction of hTas2R16, a receptor for bitter compounds. Overall, these findings are consistent with a role for Ric-8 in mammalian taste signal transduction.

  4. Pharmacological activation of CB1 receptor modulates long term potentiation by interfering with protein synthesis.

    Science.gov (United States)

    Navakkode, Sheeja; Korte, Martin

    2014-04-01

    Cognitive impairment is one of the most important side effects associated with cannabis drug abuse, as well as the serious issue concerning the therapeutic use of cannabinoids. Cognitive impairments and neuropsychiatric symptoms are caused by early synaptic dysfunctions, such as loss of synaptic connections in different brain structures including the hippocampus, a region that is believed to play an important role in certain forms of learning and memory. We report here that metaplastic priming of synapses with a cannabinoid type 1 receptor (CB1 receptor) agonist, WIN55,212-2 (WIN55), significantly impaired long-term potentiation in the apical dendrites of CA1 pyramidal neurons. Interestingly, the CB1 receptor exerts its effect by altering the balance of protein synthesis machinery towards higher protein production. Therefore the activation of CB1 receptor, prior to strong tetanization, increased the propensity to produce new proteins. In addition, WIN55 priming resulted in the expression of late-LTP in a synaptic input that would have normally expressed early-LTP, thus confirming that WIN55 priming of LTP induces new synthesis of plasticity-related proteins. Furthermore, in addition to the effects on protein translation, WIN55 also induced synaptic deficits due to the ability of CB1 receptors to inhibit the release of acetylcholine, mediated by both muscarinic and nicotinic acetylcholine receptors. Taken together this supports the notion that the modulation of cholinergic activity by CB1 receptor activation is one mechanism that regulates the synthesis of plasticity-related proteins.

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

    DEFF Research Database (Denmark)

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

    2011-01-01

    G protein-coupled receptors (GPCRs) are responsible for the majority of cellular responses to hormones and neurotransmitters as well as the senses of sight, olfaction and taste. The paradigm of GPCR signalling is the activation of a heterotrimeric GTP binding protein (G protein) by an agonist...

  6. Association of coatomer proteins with the beta-receptor for platelet-derived growth factor

    DEFF Research Database (Denmark)

    Hansen, Klaus; Rönnstrand, L; Rorsman, C

    1997-01-01

    of intracellular vesicle transport. In order to explore the functional significance of the interaction between alpha- and beta'-COP and the PDGF receptor, a receptor mutant was made in which the conserved histidine residue 928 was mutated to an alanine residue. The mutant receptor, which was unable to bind alpha......The nonreceptor tyrosine kinase Src binds to and is activated by the beta-receptor for platelet-derived growth factor (PDGF). The interaction leads to Src phosphorylation of Tyr934 in the kinase domain of the receptor. In the course of the functional characterization of this phosphorylation, we...... noticed that components of 136 and 97 kDa bound to a peptide from this region of the receptor in a phosphorylation-independent manner. These components have now been purified and identified as alpha- and beta'-coatomer proteins (COPs), respectively. COPs are a family of proteins involved in the regulation...

  7. Basic evidence for class A G-protein-coupled receptor heteromerization

    Directory of Open Access Journals (Sweden)

    Rafael eFranco

    2016-03-01

    Full Text Available Cell membrane receptors rarely work on isolation, often they form oligomeric complexes with other receptor molecules and they may directly interact with different proteins of the signal transduction machinery. For a variety of reasons, rhodopsin-like class A G-protein-coupled receptors (GPCRs seem an exception to the general rule of receptor-receptor direct interaction. In fact, controversy surrounds their potential to form homo- hetero-dimers/oligomers with other class A GPCRs; in a sense, the field is going backwards instead of forward. This review focuses on the convergent, complementary and telling evidence showing that homo- and heteromers of class A GPCRs exist in transfected cells and, more importantly, in natural sources. It is time to decide between questioning the occurrence of heteromers or, alternatively, facing the vast scientific and technical challenges that class A receptor-dimer/oligomer existence pose to Pharmacology and to Drug Discovery.

  8. A Computational Modeling and Simulation Approach to Investigate Mechanisms of Subcellular cAMP Compartmentation.

    Science.gov (United States)

    Yang, Pei-Chi; Boras, Britton W; Jeng, Mao-Tsuen; Docken, Steffen S; Lewis, Timothy J; McCulloch, Andrew D; Harvey, Robert D; Clancy, Colleen E

    2016-07-01

    Subcellular compartmentation of the ubiquitous second messenger cAMP has been widely proposed as a mechanism to explain unique receptor-dependent functional responses. How exactly compartmentation is achieved, however, has remained a mystery for more than 40 years. In this study, we developed computational and mathematical models to represent a subcellular sarcomeric space in a cardiac myocyte with varying detail. We then used these models to predict the contributions of various mechanisms that establish subcellular cAMP microdomains. We used the models to test the hypothesis that phosphodiesterases act as functional barriers to diffusion, creating discrete cAMP signaling domains. We also used the models to predict the effect of a range of experimentally measured diffusion rates on cAMP compartmentation. Finally, we modeled the anatomical structures in a cardiac myocyte diad, to predict the effects of anatomical diffusion barriers on cAMP compartmentation. When we incorporated experimentally informed model parameters to reconstruct an in silico subcellular sarcomeric space with spatially distinct cAMP production sites linked to caveloar domains, the models predict that under realistic conditions phosphodiesterases alone were insufficient to generate significant cAMP gradients. This prediction persisted even when combined with slow cAMP diffusion. When we additionally considered the effects of anatomic barriers to diffusion that are expected in the cardiac myocyte dyadic space, cAMP compartmentation did occur, but only when diffusion was slow. Our model simulations suggest that additional mechanisms likely contribute to cAMP gradients occurring in submicroscopic domains. The difference between the physiological and pathological effects resulting from the production of cAMP may be a function of appropriate compartmentation of cAMP signaling. Therefore, understanding the contribution of factors that are responsible for coordinating the spatial and temporal

  9. Non-CB1, non-CB2 receptors for endocannabinoids, plant cannabinoids, and synthetic cannabimimetics: focus on G-protein-coupled receptors and transient receptor potential channels.

    Science.gov (United States)

    De Petrocellis, Luciano; Di Marzo, Vincenzo

    2010-03-01

    The molecular mechanism of action of Delta(9)-tetrahydrocannabinol (THC), the psychotropic constituent of Cannabis, has been a puzzle during the three decades separating its characterization, in 1964, and the cloning, in the 1990s, of cannabinoid CB1 and CB2 receptors. However, while these latter proteins do mediate most of the pharmacological actions of THC, they do not seem to act as receptors for other plant cannabinoids (phytocannabinoids), nor are they the unique targets of the endogenous lipids that were originally identified in animals as agonists of CB1 and CB2 receptors, and named endocannabinoids. Over the last decade, several potential alternative receptors for phytocannabinoids, endocannabinoids, and even synthetic cannabimimetics, have been proposed, often based uniquely on pharmacological evidence obtained in vitro. In particular, the endocannabinoid anandamide, and the other most abundant Cannabis constituent, cannabidiol, seem to be the most "promiscuous" of these compounds. In this article, we review the latest data on the non-CB1, non-CB2 receptors suggested so far for endocannabinoids and plant or synthetic cannabinoids, and lay special emphasis on uncharacterized or orphan G-protein-coupled receptors as well as on transient receptor potential channels.

  10. The arylpiperazine derivatives N-(4-cyanophenylmethyl)-4-(2-diphenyl)-1-piperazinehexanamide and N-benzyl-4-(2-diphenyl)-1-piperazinehexanamide exert a long-lasting inhibition of human serotonin 5-HT7 receptor binding and cAMP signaling.

    Science.gov (United States)

    Atanes, Patricio; Lacivita, Enza; Rodríguez, Javier; Brea, José; Burgueño, Javier; Vela, José Miguel; Cadavid, María Isabel; Loza, María Isabel; Leopoldo, Marcello; Castro, Marián

    2013-12-01

    We performed a detailed in vitro pharmacological characterization of two arylpiperazine derivatives, compound N-(4-cyanophenylmethyl)-4-(2-diphenyl)-1-piperazinehexanamide (LP-211) previously identified as a high-affinity brain penetrant ligand for 5-hydroxytryptamine (serotonin) type 7 (5-HT7) receptors, and its analog N-benzyl-4-(2-diphenyl)-1-piperazinehexanamide (MEL-9). Both ligands exhibited competitive displacement of [(3)H]-(2R)-1-[(3-hydroxyphenyl)sulfonyl]-2-[2-(4-methyl-1-piperidinyl)ethyl]pyrrolidine ([(3)H]-SB-269970) radioligand binding and insurmountable antagonism of 5-carboxamidotryptamine (5-CT)-stimulated cyclic adenosine monophosphate (cAMP) signaling in human embryonic kidney (HEK293) cells stably expressing human 5-HT7 receptors. They also inhibited forskolin-stimulated adenylate cyclase activity in 5-HT7-expressing HEK293 cells but not in the parental cell line. The compounds elicited long-lasting (at least 24 h) concentration-dependent inhibition of radioligand binding at 5-HT7-binding sites in whole-cell radioligand binding assays, after pretreatment of the cells with the compounds and subsequent compound removal. In cAMP assays, pretreatment of cells with the compounds rendered 5-HT7 receptors unresponsive to 5-CT and also rendered 5-HT7-expressing HEK293 cells unresponsive to forskolin. Compound 1-(2-biphenyl)piperazine (RA-7), a known active metabolite of LP-211 present in vivo, was able to partially inhibit 5-HT7 radioligand binding in a long-lasting irreversible manner. Hence, LP-211 and MEL-9 were identified as high-affinity long-acting inhibitors of human 5-HT7 receptor binding and function in cell lines. The detailed in vitro characterization of these two pharmacological tools targeting 5-HT7 receptors may benefit the study of 5-HT7 receptor function and it may lead to the development of novel selective pharmacological tools with defined functional properties at 5-HT7 receptors.

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

    Science.gov (United States)

    Alvaro, Christopher G; Thorner, Jeremy

    2016-04-08

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

  12. INSIGHTS INTO THE REGULATION OF 5-HT2A RECEPTORS BY SCAFFOLDING PROTEINS AND KINASES

    OpenAIRE

    Roth, Bryan L.; Allen, John A.; Yadav, Prem N.

    2008-01-01

    5-HT2A serotonin receptors are essential molecular targets for the actions of LSD-like hallucinogens and atypical antipsychotic drugs. 5-HT2A serotonin receptors also mediate a variety of physiological processes in peripheral and central nervous systems including platelet aggregation, smooth muscle contraction, and the modulation of mood and perception. Scaffolding proteins have emerged as important regulators of 5-HT2A receptors and our recent studies suggest multiple scaffolds exist for 5-H...

  13. G-protein coupled receptors of the renin-angiotensin system: new targets against breast cancer?

    OpenAIRE

    Rodrigues-Ferreira, Sylvie; Nahmias, Clara

    2015-01-01

    G-protein coupled receptors (GPCRs) constitute the largest family of membrane receptors, with high potential for drug discovery. These receptors can be activated by a panel of different ligands including ions, hormones, small molecules, and vasoactive peptides. Among those, angiotensins [angiotensin II (AngII) and angiotensin 1–7] are the major biologically active products of the classical and alternative renin-angiotensin system (RAS). These peptides bind and activate three different subtype...

  14. Fucosylation and protein glycosylation create functional receptors for cholera toxin

    DEFF Research Database (Denmark)

    Wands, Amberlyn M; Fujita, Akiko; McCombs, Janet E

    2015-01-01

    Cholera toxin (CT) enters and intoxicates host cells after binding cell surface receptors using its B subunit (CTB). The ganglioside (glycolipid) GM1 is thought to be the sole CT receptor; however, the mechanism by which CTB binding to GM1 mediates internalization of CT remains enigmatic. Here we...

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

    DEFF Research Database (Denmark)

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

    2002-01-01

    -type and mutant beta2-adrenergic receptors purified from Sf-9 insect cells. Our studies have also raised important questions regarding kinetics of receptors activation. These questions should be addressed in the future by application of techniques that will allow for simultaneous measurement of conformational...

  16. Protein kinase A regulation of P2X(4) receptors: requirement for a specific motif in the C-terminus.

    Science.gov (United States)

    Brown, David A; Yule, David I

    2010-02-01

    The P2X purinergic receptor sub-family of ligand-gated ion channels are subject to protein kinase modulation. We have previously demonstrated that P2X(4)R signaling can be positively regulated by increasing intracellular cAMP levels. The molecular mechanism underlying this effect was, however, unknown. The present study initially addressed whether protein kinase A (PKA) activation was required. Subsequently a mutational approach was utilized to determine which region of the receptor was required for this potentiation. In both DT-40 3KO and HEK-293 cells transiently expressing P2X(4)R, forskolin treatment enhanced ATP-mediated signaling. Specific PKA inhibitors prevented the forskolin-induced enhancement of ATP-mediated inward currents in P2X(4)R expressing HEK-293 cells. To define which region of the P2X(4)R was required for the potentiation, mutations were generated in the cytoplasmic C-terminal tail. It was determined that a limited region of the C-terminus, consisting of a non-canonical tyrosine based sorting motif, was required for the effects of PKA. Of note, this region does not harbor any recognizable PKA phosphorylation motifs, and no direct phosphorylation of P2X(4)R was detected, suggesting that PKA phosphorylation of an accessory protein interacts with the endocytosis motif in the C-terminus of the P2X(4)R. In support of this notion, using Total Internal Reflection Fluorescence Microscopy (TIRF)\\ P2X(4)-EGFP was shown to accumulate at/near the plasma membrane following forskolin treatment. In addition, disrupting the endocytosis machinery using a dominant-negative dynamin construct also prevented the PKA-mediated enhancement of ATP-stimulated Ca(2+) signals. Our results are consistent with a novel mechanism of P2XR regulation, whereby PKA activity, without directly phosphorylating P2X(4)R, markedly enhances ATP-stimulated P2X(4)R currents and hence cytosolic Ca(2+) signals. This may occur at least in part, by altering the trafficking of a population of

  17. Molecular identification of a Drosophila G protein-coupled receptor specific for crustacean cardioactive peptide

    DEFF Research Database (Denmark)

    Cazzamali, Giuseppe; Hauser, Frank; Kobberup, Sune

    2003-01-01

    The Drosophila Genome Project website (www.flybase.org) contains the sequence of an annotated gene (CG6111) expected to code for a G protein-coupled receptor. We have cloned this receptor and found that its gene was not correctly predicted, because an annotated neighbouring gene (CG14547) was als...

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

    DEFF Research Database (Denmark)

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

    2016-01-01

    and in Lophotrochozoa. Furthermore, we have cloned and deorphanized two G protein-coupled receptors (GPCRs) from the oyster Crassostrea gigas (Mollusca) that are activated by low nanomolar concentrations of oyster AKH (pQVSFSTNWGSamide). Our discovery of functional AKH receptors in molluscs is especially significant...

  19. 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...

  20. A genome-wide functional investigation into the roles of receptor-like proteins in Arabidopsis

    NARCIS (Netherlands)

    Guodong Wang, G.; Ellendorff, U.; Kemp, B.; Mansfield, J.W.; Forsyth, A.; Mitchell, K.; Bastas, K.; Liu, C.M.; Woods-Tör, A.; Zipfel, C.; Wit, de P.J.G.M.; Jones, J.D.G.; Tör, M.; Thomma, B.P.H.J.

    2008-01-01

    Receptor-like proteins (RLPs) are cell surface receptors that typically consist of an extracellular leucine-rich repeat domain, a transmembrane domain, and a short cytoplasmatic tail. In several plant species, RLPs have been found to play a role in disease resistance, such as the tomato (Solanum lyc

  1. Low density lipoprotein receptor related protein 1 variant interacts with saturated fatty acids in Puerto Ricans

    Science.gov (United States)

    Low density lipoprotein related receptor protein 1 (LRP1) is a multi-functional endocytic receptor that is highly expressed in adipocytes and the hypothalamus. Animal models and in vitro studies support a role for LRP1 in adipocyte metabolism and leptin signaling, but genetic polymorphisms have not ...

  2. The Role of Rab Proteins in Neuronal Cells and in the Trafficking of Neurotrophin Receptors

    Directory of Open Access Journals (Sweden)

    Cecilia Bucci

    2014-10-01

    Full Text Available Neurotrophins are a family of proteins that are important for neuronal development, neuronal survival and neuronal functions. Neurotrophins exert their role by binding to their receptors, the Trk family of receptor tyrosine kinases (TrkA, TrkB, and TrkC and p75NTR, a member of the tumor necrosis factor (TNF receptor superfamily. Binding of neurotrophins to receptors triggers a complex series of signal transduction events, which are able to induce neuronal differentiation but are also responsible for neuronal maintenance and neuronal functions. Rab proteins are small GTPases localized to the cytosolic surface of specific intracellular compartments and are involved in controlling vesicular transport. Rab proteins, acting as master regulators of the membrane trafficking network, play a central role in both trafficking and signaling pathways of neurotrophin receptors. Axonal transport represents the Achilles' heel of neurons, due to the long-range distance that molecules, organelles and, in particular, neurotrophin-receptor complexes have to cover. Indeed, alterations of axonal transport and, specifically, of axonal trafficking of neurotrophin receptors are responsible for several human neurodegenerative diseases, such as Huntington’s disease, Alzheimer’s disease, amyotrophic lateral sclerosis and some forms of Charcot-Marie-Tooth disease. In this review, we will discuss the link between Rab proteins and neurotrophin receptor trafficking and their influence on downstream signaling pathways.

  3. G protein-coupled receptor 39 deficiency is associated with pancreatic islet dysfunction

    DEFF Research Database (Denmark)

    Holst, Birgitte; Egerod, Kristoffer L; Jin, Chunyu;

    2009-01-01

    G protein-coupled receptor (GPR)-39 is a seven-transmembrane receptor expressed mainly in endocrine and metabolic tissues that acts as a Zn(++) sensor signaling mainly through the G(q) and G(12/13) pathways. The expression of GPR39 is regulated by hepatocyte nuclear factor (HNF)-1alpha and HNF-4...

  4. Transcriptional and Functional Characterization of the G Protein-Coupled Receptor Repertoire of Gastric Somatostatin Cells

    DEFF Research Database (Denmark)

    Egerod, Kristoffer L; Engelstoft, Maja S; Lund, Mari L;

    2015-01-01

    characterized the G protein-coupled receptors expressed in gastric Sst-RFP-positive cells and probed their effects on SST secretion in primary cell cultures. Surprisingly, besides SST, amylin and PYY were also highly enriched in the SST cells. Several receptors found to regulate SST secretion were highly...

  5. Differential palmitoylation directs the AMPA receptor-binding protein ABP to spines or to intracellular clusters.

    Science.gov (United States)

    DeSouza, Sunita; Fu, Jie; States, Bradley A; Ziff, Edward B

    2002-05-01

    Long-term changes in excitatory synapse strength are thought to reflect changes in synaptic abundance of AMPA receptors mediated by receptor trafficking. AMPA receptor-binding protein (ABP) and glutamate receptor-interacting protein (GRIP) are two similar PDZ (postsynaptic density 95/Discs large/zona occludens 1) proteins that interact with glutamate receptors 2 and 3 (GluR2 and GluR3) subunits. Both proteins have proposed roles during long-term potentiation and long-term depression in the delivery and anchorage of AMPA receptors at synapses. Here we report a variant of ABP-L (seven PDZ form of ABP) called pABP-L that is palmitoylated at a cysteine residue at position 11 within a novel 18 amino acid N-terminal leader sequence encoded through differential splicing. In cultured hippocampal neurons, nonpalmitoylated ABP-L localizes with internal GluR2 pools expressed from a Sindbis virus vector, whereas pABP-L is membrane targeted and associates with surface-localized GluR2 receptors at the plasma membrane in spines. Mutation of Cys-11 to alanine blocks the palmitoylation of pABP-L and targets the protein to intracellular clusters, confirming that targeting the protein to spines is dependent on palmitoylation. Non-palmitoylated GRIP is primarily intracellular, but a chimera with the pABP-L N-terminal palmitoylation sequence linked to the body of the GRIP protein is targeted to spines. We suggest that pABP-L and ABP-L provide, respectively, synaptic and intracellular sites for the anchorage of AMPA receptors during receptor trafficking to and from the synapse.

  6. A family of plasmodesmal proteins with receptor-like properties for plant viral movement proteins.

    Science.gov (United States)

    Amari, Khalid; Boutant, Emmanuel; Hofmann, Christina; Schmitt-Keichinger, Corinne; Fernandez-Calvino, Lourdes; Didier, Pascal; Lerich, Alexander; Mutterer, Jérome; Thomas, Carole L; Heinlein, Manfred; Mély, Yves; Maule, Andrew J; Ritzenthaler, Christophe

    2010-09-23

    Plasmodesmata (PD) are essential but poorly understood structures in plant cell walls that provide symplastic continuity and intercellular communication pathways between adjacent cells and thus play fundamental roles in development and pathogenesis. Viruses encode movement proteins (MPs) that modify these tightly regulated pores to facilitate their spread from cell to cell. The most striking of these modifications is observed for groups of viruses whose MPs form tubules that assemble in PDs and through which virions are transported to neighbouring cells. The nature of the molecular interactions between viral MPs and PD components and their role in viral movement has remained essentially unknown. Here, we show that the family of PD-located proteins (PDLPs) promotes the movement of viruses that use tubule-guided movement by interacting redundantly with tubule-forming MPs within PDs. Genetic disruption of this interaction leads to reduced tubule formation, delayed infection and attenuated symptoms. Our results implicate PDLPs as PD proteins with receptor-like properties involved the assembly of viral MPs into tubules to promote viral movement.

  7. An ATIPical family of angiotensin II AT2 receptor-interacting proteins.

    Science.gov (United States)

    Rodrigues-Ferreira, Sylvie; Nahmias, Clara

    2010-11-01

    AT2, the second subtype of angiotensin II receptors, is a major component of the renin-angiotensin system involved in cardiovascular and neuronal functions. AT2 belongs to the superfamily of G protein-coupled receptors, but its intracellular signaling pathways have long remained elusive. Over the past few years, efforts to characterize this atypical receptor have led to the identification of novel molecular scaffolds that directly bind to its intracellular tail. The present review focuses on a family of AT2 receptor-interacting proteins (ATIPs) involved in neuronal differentiation, vascular remodeling and tumor suppression. Recent findings that ATIPs and ATIP-related proteins associate with microtubules suggest that they might constitute a novel family of multifunctional proteins regulating a wide range of physiopathological functions.

  8. 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.

  9. New insights into the role of cAMP in the production and function of the incretin hormone glucagon-like peptide-1 (GLP-1).

    Science.gov (United States)

    Yu, Zhiwen; Jin, Tianru

    2010-01-01

    The proglucagon gene (gcg) encodes both glucagon and glucagon-like peptide-1 (GLP-1), produced in pancreatic alpha cells and intestinal endocrine L cells, respectively. The incretin hormone GLP-1 stimulates insulin secretion and pro-insulin gene transcription. GLP-1 also enhances pancreatic beta-cell proliferation, inhibits cell apoptosis, and has been utilized in the trans-differentiation of insulin producing cells. A long-term effective GLP-1 receptor agonist, Byetta, has now been developed as the drug in treating type II diabetes and potentially other metabolic disorders. The expression of gcg and the production of GLP-1 can be activated by the elevation of the second messenger cyclic AMP (cAMP). Recent studies suggest that in addition to protein kinase A (PKA), exchange protein activated by cAMP (Epac), another effector of cAMP, and the crosstalk between PKA and the Wnt signaling pathway, are involved in cAMP-stimulated gcg transcription and GLP-1 production as well. Finally, functions of GLP-1 in pancreatic beta cells are also mediated by PKA, Epac, as well as the effector of the Wnt signaling pathway. Together, these novel findings bring us a new insight into the role of cAMP in the production and function of the incretin hormone GLP-1.

  10. Microvesicle and tunneling nanotube mediated intercellular transfer of g-protein coupled receptors in cell cultures

    Energy Technology Data Exchange (ETDEWEB)

    Guescini, M. [Department of Biomolecular Sciences, University of Urbino ' Carlo Bo' , 61029 Urbino (Italy); Leo, G.; Genedani, S. [Department Biomedical Sciences, University of Modena and Reggio Emilia (Italy); Carone, C. [Department Biomedical Sciences, University of Modena and Reggio Emilia (Italy); IRCCS San Camillo Lido, Venezia (Italy); Pederzoli, F. [Department Biomedical Sciences, University of Modena and Reggio Emilia (Italy); Ciruela, F. [Departament Patologia i Terapeutica Experimental, Universitat de Barcelona (Spain); Guidolin, D. [Department of Human Anatomy and Physiology, University of Padua (Italy); Stocchi, V.; Mantuano, M. [Department of Biomolecular Sciences, University of Urbino ' Carlo Bo' , 61029 Urbino (Italy); Borroto-Escuela, D.O.; Fuxe, K. [Department of Neuroscience, Karolinska Institutet, Stockholm (Sweden); Agnati, L.F., E-mail: luigiagnati@tin.it [IRCCS San Camillo Lido, Venezia (Italy)

    2012-03-10

    Recent evidence shows that cells exchange collections of signals via microvesicles (MVs) and tunneling nano-tubes (TNTs). In this paper we have investigated whether in cell cultures GPCRs can be transferred by means of MVs and TNTs from a source cell to target cells. Western blot, transmission electron microscopy and gene expression analyses demonstrate that A{sub 2A} and D{sub 2} receptors are present in released MVs. In order to further demonstrate the involvement of MVs in cell-to-cell communication we created two populations of cells (HEK293T and COS-7) transiently transfected with D{sub 2}R-CFP or A{sub 2A}R-YFP. These two types of cells were co-cultured, and FRET analysis demonstrated simultaneously positive cells to the D{sub 2}R-CFP and A{sub 2A}R-YFP. Fluorescence microscopy analysis also showed that GPCRs can move from one cell to another also by means of TNTs. Finally, recipient cells pre-incubated for 24 h with A{sub 2A}R positive MVs were treated with the adenosine A{sub 2A} receptor agonist CGS-21680. The significant increase in cAMP accumulation clearly demonstrated that A{sub 2A}Rs were functionally competent in target cells. These findings demonstrate that A{sub 2A} receptors capable of recognizing and decoding extracellular signals can be safely transferred via MVs from source to target cells.

  11. Orphan nuclear receptor Errγ induces C-reactive protein gene expression through induction of ER-bound Bzip transmembrane transcription factor CREBH.

    Directory of Open Access Journals (Sweden)

    Jagannath Misra

    Full Text Available The orphan nuclear receptor estrogen-related receptor-γ (ERRγ is a constitutively active transcription factor regulating genes involved in several important cellular processes, including hepatic glucose metabolism, alcohol metabolism, and the endoplasmic reticulum (ER stress response. cAMP responsive element-binding protein H (CREBH is an ER-bound bZIP family transcription factor that is activated upon ER stress and regulates genes encoding acute-phase proteins whose expression is increased in response to inflammation. Here, we report that ERRγ directly regulates CREBH gene expression in response to ER stress. ERRγ bound to the ERRγ response element (ERRE in the CREBH promoter. Overexpression of ERRγ by adenovirus significantly increased expression of CREBH as well as C-reactive protein (CRP, whereas either knockdown of ERRγ or inhibition of ERRγ by ERRγ specific inverse agonist, GSK5182, substantially inhibited ER stress-mediated induction of CREBH and CRP. The transcriptional coactivator PGC1α was required for ERRγ mediated induction of the CREBH gene as demonstrated by the chromatin immunoprecipitation (ChIP assay showing binding of both ERRγ and PGC1α on the CREBH promoter. The ChIP assay also revealed that histone H3 and H4 acetylation occurred at the ERRγ and PGC1α binding site. Moreover, chronic alcoholic hepatosteatosis, as well as the diabetic obese condition significantly increased CRP gene expression, and this increase was significantly attenuated by GSK5182 treatment. We suggest that orphan nuclear receptor ERRγ directly regulates the ER-bound transcription factor CREBH in response to ER stress and other metabolic conditions.

  12. Role of antibodies in developing drugs that target G-protein-coupled receptor dimers.

    Science.gov (United States)

    Hipser, Chris; Bushlin, Ittai; Gupta, Achla; Gomes, Ivone; Devi, Lakshmi A

    2010-01-01

    G-protein-coupled receptors are important molecular targets in drug discovery. These receptors play a pivotal role in physiological signaling pathways and are targeted by nearly 50% of currently available drugs. Mounting evidence suggests that G-protein-coupled receptors form dimers, and various studies have shown that dimerization is necessary for receptor maturation, signaling, and trafficking. However, the physiological implications of dimerization in vivo have not been well explored because detection of GPCR dimers in endogenous systems has been a challenging task. One exciting new approach to this challenge is the generation of antibodies against specific G-protein-coupled receptor dimers. Such antibodies could be used as tools for characterization of heteromer-specific function; as reagents for their purification, tissue localization, and regulation in vivo; and as probes for mapping their functional domains. In addition, such antibodies could serve as alternative ligands for G-protein-coupled receptor heteromers. Thus, heteromer-specific antibodies represent novel tools for the exploration and manipulation of G-protein-coupled receptor-dimer pharmacology.

  13. Functional cyclic AMP response element in the breast cancer resistance protein (BCRP/ABCG2) promoter modulates epidermal growth factor receptor pathway- or androgen withdrawal-mediated BCRP/ABCG2 transcription in human cancer cells.

    Science.gov (United States)

    Xie, Yi; Nakanishi, Takeo; Natarajan, Karthika; Safren, Lowell; Hamburger, Anne W; Hussain, Arif; Ross, Douglas D

    2015-03-01

    Phosphorylated cyclic-AMP (cAMP) response element binding protein (p-CREB) is a downstream effector of a variety of important signaling pathways. We investigated whether the human BCRP promoter contains a functional cAMP response element (CRE). 8Br-cAMP, a cAMP analogue, increased the activity of a BCRP promoter reporter construct and BCRP mRNA in human carcinoma cells. Epidermal growth factor receptor (EGFR) pathway activation also led to an increase in p-CREB and in BCRP promoter reporter activity via two major downstream EGFR signaling pathways: the phosphotidylinositol-3-kinase (PI3K)/AKT pathway and the mitogen-activated protein kinase (MAPK) pathway. EGF treatment increased the phosphorylation of EGFR, AKT, ERK and CREB, while simultaneously enhancing BCRP mRNA and functional protein expression. EGF-stimulated CREB phosphorylation and BCRP induction were diminished by inhibition of EGFR, PI3K/AKT or RAS/MAPK signaling. CREB silencing using RNA interference reduced basal levels of BCRP mRNA and diminished the induction of BCRP by EGF. Chromatin immunoprecipitation assays confirmed that a putative CRE site on the BCRP promoter bound p-CREB by a point mutation of the CRE site abolished EGF-induced stimulation of BCRP promoter reporter activity. Furthermore, the CREB co-activator, cAMP-regulated transcriptional co-activator (CRTC2), is involved in CREB-mediated BCRP transcription: androgen depletion of LNCaP human prostate cancer cells increased both CREB phosphorylation and CRTC2 nuclear translocation, and enhanced BCRP expression. Silencing CREB or CRTC2 reduced basal BCRP expression and BCRP induction under androgen-depletion conditions. This novel CRE site plays a central role in mediating BCRP gene expression in several human cancer cell lines following activation of multiple cancer-relevant signaling pathways.

  14. Day Camp Manual: Administration. Book I.

    Science.gov (United States)

    Babcock, William

    The first book in a 5-book manual on day camping focuses on summer day camp administration. The book defines day camps as organized group experiences in outdoor living on a day-by-day basis and under trained leadership. It includes a philosophy of day camping, noting benefits to the campers. The book is divided into further chapters that describe…

  15. Slave Labor Camps of the Third Reich.

    Science.gov (United States)

    Stone, Adolf

    1983-01-01

    Describes the ground rules used by Nazi architects in choosing the sites for slave labor camps. While some, like Auschwitz, became extermination camps, others also produced armaments. One camp, Theresienstadt, became a "model" camp to show to reporters and Red Cross representatives. (CS)

  16. Ultrastructural relationship between the mu opioid receptor and its interacting protein, GPR177, in striatal neurons

    OpenAIRE

    Reyes, Arith-Ruth S.; Levenson, Robert; Berrettini, Wade; Van Bockstaele, Elisabeth J.

    2010-01-01

    GPR177, the mammalian ortholog of Drosophila Wntless/Evi/Sprinter, was recently identified as a novel mu-opioid receptor (MOR) interacting protein. GPR177 is a trans-membrane protein pivotal to mediating the secretion of Wnt signaling proteins. Wnt proteins, in turn, are essential in regulating neuronal development, a phenomenon inhibited upon chronic exposure to MOR agonists such as morphine and heroin. We previously showed that GPR177 and MOR are co-localized in the mouse dorsolateral stria...

  17. Comparison of work motivation in camp supervisors and camp counselors in Greek private camps

    Directory of Open Access Journals (Sweden)

    George Costa

    2008-06-01

    Full Text Available The purpose of this study was twofold. First, the study sought to better understand the work motivators that led camp supervisors and counselors to accept their job. Second, the study sought to better understand the ranking and rating of 20 work motivators from supervisors and camp counselors. Responders to the research questionnaire (n=121 were camp supervisory staff and counselors, age 15-55, working in seven private camps in Greece. Two instruments were used to collect data. The first instrument collected demographic data while the second instrument focused on ranking and rating 20 work motivators. The study suggested that Herzberg's theory (Motivator / Hygiene does not apply on a full scale. The results suggested that supervisory staff indicated good working conditions, meeting other people and carrying out personal growth are important. In addition, they don't like travel and don't consider working in a camp, as a stable job. The camp counselors want to have fun in their job and the opportunity to work with youth. They also don't consider working in a camp, as a stable job and they don't accept the responsibility in proportion to their position.

  18. Induction of drug metabolism by forskolin: the role of the pregnane X receptor and the protein kinase a signal transduction pathway.

    Science.gov (United States)

    Ding, Xunshan; Staudinger, Jeff L

    2005-02-01

    An extract of the plant Coleus forskohlii has been used for centuries in Ayurvedic medicine to treat various diseases such as hypothyroidism, heart disease, and respiratory disorders. Additionally, complex herbal mixtures containing this extract are gaining popularity in United States for their putative "fat-burning" properties. The active ingredient in C. forskohlii extract is the diterpene compound forskolin. Forskolin is a widely used biochemical tool that activates adenyl cyclase, thereby increasing intracellular concentration of cAMP and thus activating the protein kinase A (PKA) signal transduction pathway. We show herein that both forskolin and its nonadenyl cyclase-activating analog 1,9 dideoxyforskolin induce CYP3A gene expression in primary hepatocytes by functioning as agonists of the pregnane X receptor (PXR). We show that activation of PKA signaling potentiates PXR-mediated induction of CYP3A gene expression in cultured hepatocytes and increases the strength of PXR-coactivator protein-protein interaction in cell-based assays. Kinase assays show that PXR can serve as a substrate for catalytically active PKA in vitro. Our data provide important insights into the molecular mechanism of both the PKA-dependent and -independent effects of forskolin on the expression of drug-metabolizing enzymes in liver. Finally, our data suggest that herbal therapy with C. forskohlii extract should be approached cautiously due to the potential for herb-drug interactions in patients on combination therapy.

  19. Assessment and Challenges of Ligand Docking into Comparative Models of G-Protein Coupled Receptors

    DEFF Research Database (Denmark)

    Nguyen, E.D.; Meiler, J.; Norn, C.;

    2013-01-01

    The rapidly increasing number of high-resolution X-ray structures of G-protein coupled receptors (GPCRs) creates a unique opportunity to employ comparative modeling and docking to provide valuable insight into the function and ligand binding determinants of novel receptors, to assist in virtual...... screening and to design and optimize drug candidates. However, low sequence identity between receptors, conformational flexibility, and chemical diversity of ligands present an enormous challenge to molecular modeling approaches. It is our hypothesis that rapid Monte-Carlo sampling of protein backbone...

  20. Receptor-like kinase SOBIR1/EVR interacts with receptor-like proteins in plant immunity against fungal infection.

    Science.gov (United States)

    Liebrand, Thomas W H; van den Berg, Grardy C M; Zhang, Zhao; Smit, Patrick; Cordewener, Jan H G; America, Antoine H P; America, Antione H P; Sklenar, Jan; Jones, Alexandra M E; Tameling, Wladimir I L; Robatzek, Silke; Thomma, Bart P H J; Joosten, Matthieu H A J

    2013-06-11

    The plant immune system is activated by microbial patterns that are detected as nonself molecules. Such patterns are recognized by immune receptors that are cytoplasmic or localized at the plasma membrane. Cell surface receptors are represented by receptor-like kinases (RLKs) that frequently contain extracellular leucine-rich repeats and an intracellular kinase domain for activation of downstream signaling, as well as receptor-like proteins (RLPs) that lack this signaling domain. It is therefore hypothesized that RLKs are required for RLPs to activate downstream signaling. The RLPs Cf-4 and Ve1 of tomato (Solanum lycopersicum) mediate resistance to the fungal pathogens Cladosporium fulvum and Verticillium dahliae, respectively. Despite their importance, the mechanism by which these immune receptors mediate downstream signaling upon recognition of their matching ligand, Avr4 and Ave1, remained enigmatic. Here we show that the tomato ortholog of the Arabidopsis thaliana RLK Suppressor Of BIR1-1/Evershed (SOBIR1/EVR) and its close homolog S. lycopersicum (Sl)SOBIR1-like interact in planta with both Cf-4 and Ve1 and are required for the Cf-4- and Ve1-mediated hypersensitive response and immunity. Tomato SOBIR1/EVR interacts with most of the tested RLPs, but not with the RLKs FLS2, SERK1, SERK3a, BAK1, and CLV1. SOBIR1/EVR is required for stability of the Cf-4 and Ve1 receptors, supporting our observation that these RLPs are present in a complex with SOBIR1/EVR in planta. We show that SOBIR1/EVR is essential for RLP-mediated immunity and propose that the protein functions as a regulatory RLK of this type of cell-surface receptors.

  1. The Par3 polarity protein is an exocyst receptor essential for mammary cell survival

    Science.gov (United States)

    Ahmed, Syed Mukhtar; Macara, Ian G.

    2017-01-01

    The exocyst is an essential component of the secretory pathway required for delivery of basolateral proteins to the plasma membranes of epithelial cells. Delivery occurs adjacent to tight junctions (TJ), suggesting that it recognizes a receptor at this location. However, no such receptor has been identified. The Par3 polarity protein associates with TJs but has no known function in membrane traffic. We now show that, unexpectedly, Par3 is essential for mammary cell survival. Par3 silencing causes apoptosis, triggered by phosphoinositide trisphosphate depletion and decreased Akt phosphorylation, resulting from failure of the exocyst to deliver basolateral proteins to the cortex. A small region of PAR3 binds directly to Exo70 and is sufficient for exocyst docking, membrane-protein delivery and cell survival. PAR3 lacking this domain can associate with the cortex but cannot support exocyst function. We conclude that Par3 is the long-sought exocyst receptor required for targeted membrane-protein delivery. PMID:28358000

  2. A urokinase receptor-associated protein with specific collagen binding properties

    DEFF Research Database (Denmark)

    Behrendt, N; Jensen, Ole Nørregaard; Engelholm, L H

    2000-01-01

    membrane-bound lectin with hitherto unknown function. The human cDNA was cloned and sequenced. The protein, designated uPARAP, is a member of the macrophage mannose receptor protein family and contains a putative collagen-binding (fibronectin type II) domain in addition to 8 C-type carbohydrate recognition......The plasminogen activation cascade system, directed by urokinase and the urokinase receptor, plays a key role in extracellular proteolysis during tissue remodeling. To identify molecular interaction partners of these trigger proteins on the cell, we combined covalent protein cross-linking with mass...... spectrometry based methods for peptide mapping and primary structure analysis of electrophoretically isolated protein conjugates. A specific tri-molecular complex was observed upon addition of pro-urokinase to human U937 cells. This complex included the urokinase receptor, pro-urokinase, and an unknown, high...

  3. Some properties of human neuronal α7 nicotinic acetylcholine receptors fused to the green fluorescent protein

    Science.gov (United States)

    Palma, Eleonora; Mileo, Anna M.; Martínez-Torres, Ataúlfo; Eusebi, Fabrizio; Miledi, Ricardo

    2002-01-01

    The functional properties and cellular localization of the human neuronal α7 nicotinic acetylcholine (AcCho) receptor (α7 AcChoR) and its L248T mutated (mut) form were investigated by expressing them alone or as gene fusions with the enhanced version of the green fluorescent protein (GFP). Xenopus oocytes injected with wild-type (wt), mutα7, or the chimeric subunit cDNAs expressed receptors that gated membrane currents when exposed to AcCho. As already known, AcCho currents generated by wtα7 receptors decay much faster than those elicited by the mutα7 receptors. Unexpectedly, the fusion of GFP to the wt and mutated α7 receptors led to opposite results: the AcCho-current decay of the wt receptors became slower, whereas that of the mutated receptors was accelerated. Furthermore, repetitive applications of AcCho led to a considerable “run-down” of the AcCho currents generated by mutα7-GFP receptors, whereas those of the wtα7-GFP receptors remained stable or increased in amplitude. The AcCho-current run-down of mutα7-GFP oocytes was accompanied by a marked decrease of α-bungarotoxin binding activity. Fluorescence, caused by the chimeric receptors expressed, was seen over the whole oocyte surface but was more intense and abundant in the animal hemisphere, whereas it was much weaker in the vegetal hemisphere. We conclude that fusion of GFP to wtα7 and mutα7 receptors provides powerful tools to study the distribution and function of α7 receptors. We also conclude that fused genes do not necessarily recapitulate all of the properties of the original receptors. This fact must be borne close in mind whenever reporter genes are attached to proteins. PMID:11891308

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

    Directory of Open Access Journals (Sweden)

    Mohammed Akli eAYOUB

    2012-06-01

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

  5. Antibodies to probe endogenous G protein-coupled receptor heteromer expression, regulation and function.

    Directory of Open Access Journals (Sweden)

    Ivone eGomes

    2014-12-01

    Full Text Available Over the last decade an increasing number of studies have focused on the ability of G protein-coupled receptors to form heteromers and explored how receptor heteromerization modulates the binding, signaling and trafficking properties of individual receptors. Most of these studies were carried out in heterologous cells expressing epitope tagged receptors. Very little information is available about the in vivo physiological role of G protein-coupled receptor heteromers due to a lack of tools to detect their presence in endogenous tissue. Recent advances such as the generation of mouse models expressing fluorescently labeled receptors, of TAT based peptides that can disrupt a given heteromer pair, or of heteromer-selective antibodies that recognize the heteromer in endogenous tissue have begun to elucidate the physiological and pathological roles of receptor heteromers. In this review we have focused on heteromer-selective antibodies and describe how a subtractive immunization strategy can be successfully used to generate antibodies that selectively recognize a desired heteromer pair. We also describe the uses of these antibodies to detect the presence of heteromers, to study their properties in endogenous tissues, and to monitor changes in heteromer levels under pathological conditions. Together, these findings suggest that G protein-coupled receptor heteromers represent unique targets for the development of drugs with reduced side-effects.

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

    DEFF Research Database (Denmark)

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

    2007-01-01

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

  7. Cell-surface protein-protein interaction analysis with time-resolved FRET and snap-tag technologies: application to G protein-coupled receptor oligomerization.

    Science.gov (United States)

    Comps-Agrar, Laëtitia; Maurel, Damien; Rondard, Philippe; Pin, Jean-Philippe; Trinquet, Eric; Prézeau, Laurent

    2011-01-01

    G protein-coupled receptors (GPCRs) are key players in cell-cell communication, the dysregulation of which has often deleterious effects leading to pathologies such as psychiatric and neurological diseases. Consequently, GPCRs represent excellent drug targets, and as such are the object of intense research in drug discovery for therapeutic application. Recently, the GPCR field has been revolutionized by the demonstration that GPCRs are part of large protein complexes that control their pharmacology, activity, and signaling. Moreover, in these complexes, one GPCR can either associate with itself, forming homodimers or homooligomers, or with other receptor types, forming heterodimeric or heterooligomeric receptor entities that display new receptor features. These features include alterations in ligand cooperativity and selectivity, the activation of novel signaling pathways, and novel processes of desensitization. Thus, it has become necessary to identify GPCR-associated protein complexes of interest at the cell surface, and to determine the state of oligomerization of these receptors and their interactions with their partner proteins. This is essential to understand the function of GPCRs in their native environment, as well as ways to either modulate or control receptor activity with appropriate pharmacological tools, and to develop new therapeutic strategies. This requires the development of technologies to precisely address protein-protein interactions between oligomers at the cell surface. In collaboration with Cisbio Bioassay, we have developed such a technology, which combines TR-FRET detection with a new labeling method called SnapTag. This technology has allowed us to address the oligomeric state of many GPCRs.

  8. Basic Pharmacological and Structural Evidence for Class A G-Protein-Coupled Receptor Heteromerization

    Science.gov (United States)

    Franco, Rafael; Martínez-Pinilla, Eva; Lanciego, José L.; Navarro, Gemma

    2016-01-01

    Cell membrane receptors rarely work on isolation, often they form oligomeric complexes with other receptor molecules and they may directly interact with different proteins of the signal transduction machinery. For a variety of reasons, rhodopsin-like class A G-protein-coupled receptors (GPCRs) seem an exception to the general rule of receptor–receptor direct interaction. In fact, controversy surrounds their potential to form homo- hetero-dimers/oligomers with other class A GPCRs; in a sense, the field is going backward instead of forward. This review focuses on the convergent, complementary and telling evidence showing that homo- and heteromers of class A GPCRs exist in transfected cells and, more importantly, in natural sources. It is time to decide between questioning the occurrence of heteromers or, alternatively, facing the vast scientific and technical challenges that class A receptor-dimer/oligomer existence pose to Pharmacology and to Drug Discovery. PMID:27065866

  9. Registration Day-Camp 2016

    CERN Multimedia

    Nursery School

    2016-01-01

    Registration for the CERN SA Day-camp are open for children from 4 to 6 years old From March 14 to 25 for children already enrolled in CERN SA EVE and School From April 4 to 15 for the children of CERN members of the personnel (MP) From April 18 for other children More information on the website: http://nurseryschool.web.cern.ch/. The day-camp is open to all children. An inscription per week is proposed, cost 480.-CHF/week, lunch included The camp will be open weeks 27, 28, 29 and 30, from 8:30 am to 5:30 pm. For further questions, thanks you for contacting us by email at Summer.Camp@cern.ch.

  10. A prospective cross-screening study on G-protein-coupled receptors: lessons learned in virtual compound library design.

    NARCIS (Netherlands)

    Sanders, M.P.A.; Roumen, L.; Horst, E. van der; Lane, J.R.; Vischer, H.F.; Offenbeek, J. van; Vries, H. de; Verhoeven, S.; Chow, K.Y.; Verkaar, F.; Beukers, M.W.; McGuire, R.; Leurs, R.; IJzerman, A.P.; Vlieg, J. de; Esch, I.J. de; Zaman, G.J.; Klomp, J.P.G.; Bender, A.; Graaf, C. de

    2012-01-01

    We present the systematic prospective evaluation of a protein-based and a ligand-based virtual screening platform against a set of three G-protein-coupled receptors (GPCRs): the beta-2 adrenoreceptor (ADRB2), the adenosine A(2A) receptor (AA2AR), and the sphingosine 1-phosphate receptor (S1PR1). Nov

  11. Organization and Dynamics of Receptor Proteins in a Plasma Membrane.

    Science.gov (United States)

    Koldsø, Heidi; Sansom, Mark S P

    2015-11-25

    The interactions of membrane proteins are influenced by their lipid environment, with key lipid species able to regulate membrane protein function. Advances in high-resolution microscopy can reveal the organization and dynamics of proteins and lipids within living cells at resolutions membranes of in vivo-like complexity. We explore the dynamics of proteins and lipids in crowded and complex plasma membrane models, thereby closing the gap in length and complexity between computations and experiments. Our simulations provide insights into the mutual interplay between lipids and proteins in determining mesoscale (20-100 nm) fluctuations of the bilayer, and in enabling oligomerization and clustering of membrane proteins.

  12. Avr4 promotes Cf-4 receptor-like protein association with the BAK1/SERK3 receptor-like kinase to initiate receptor endocytosis and plant immunity

    NARCIS (Netherlands)

    Postma, Jelle; Liebrand, Thomas; Bi, Guozhi; Evrard, Alexandre; Bye, Ruby R.; Mbengue, Malick; Kuhn, Hannah; Joosten, Matthieu H.A.J.; Robatzek, Silke

    2016-01-01

    The first layer of plant immunity is activated by cell surface receptor-like kinases (RLKs) and proteins (RLPs) that detect infectious pathogens. Constitutive interaction with the SUPPRESSOR OF BIR1 (SOBIR1) RLK contributes to RLP stability and kinase activity. As RLK activation requires transpho

  13. Structure-based drug design for G protein-coupled receptors.

    Science.gov (United States)

    Congreve, Miles; Dias, João M; Marshall, Fiona H

    2014-01-01

    Our understanding of the structural biology of G protein-coupled receptors has undergone a transformation over the past 5 years. New protein-ligand complexes are described almost monthly in high profile journals. Appreciation of how small molecules and natural ligands bind to their receptors has the potential to impact enormously how medicinal chemists approach this major class of receptor targets. An outline of the key topics in this field and some recent examples of structure- and fragment-based drug design are described. A table is presented with example views of each G protein-coupled receptor for which there is a published X-ray structure, including interactions with small molecule antagonists, partial and full agonists. The possible implications of these new data for drug design are discussed.

  14. The daf-4 gene encodes a bone morphogenetic protein receptor controlling C. elegans dauer larva development.

    Science.gov (United States)

    Estevez, M; Attisano, L; Wrana, J L; Albert, P S; Massagué, J; Riddle, D L

    1993-10-14

    The bone morphogenetic protein (BMP) family is a conserved group of signalling molecules within the transforming growth factor-beta (TGF-beta) superfamily. This group, including the Drosophila decapentaplegic (dpp) protein and the mammalian BMPs, mediates cellular interactions and tissue differentiation during development. Here we show that a homologue of human BMPs controls a developmental switch in the life cycle of the free-living soil nematode Caenorhabditis elegans. Starvation and overcrowding induce C. elegans to form a developmentally arrested, third-stage dauer larva. The daf-4 gene, which acts to inhibit dauer larva formation and promote growth, encodes a receptor protein kinase similar to the daf-1, activin and TGF-beta receptor serine/threonine kinases. When expressed in monkey COS cells, the daf-4 receptor binds human BMP-2 and BMP-4. The daf-4 receptor is the first to be identified for any growth factor in the BMP family.

  15. G Protein-Coupled Receptor Signaling in Stem Cells and Cancer

    Directory of Open Access Journals (Sweden)

    Jennifer R. Lynch

    2016-05-01

    Full Text Available G protein-coupled receptors (GPCRs are a large superfamily of cell-surface signaling proteins that bind extracellular ligands and transduce signals into cells via heterotrimeric G proteins. GPCRs are highly tractable drug targets. Aberrant expression of GPCRs and G proteins has been observed in various cancers and their importance in cancer stem cells has begun to be appreciated. We have recently reported essential roles for G protein-coupled receptor 84 (GPR84 and G protein subunit Gαq in the maintenance of cancer stem cells in acute myeloid leukemia. This review will discuss how GPCRs and G proteins regulate stem cells with a focus on cancer stem cells, as well as their implications for the development of novel targeted cancer therapies.

  16. Three-dimensional measurement of cAMP gradients using hyperspectral confocal microscopy

    Science.gov (United States)

    Rich, Thomas C.; Annamdevula, Naga; Britain, Andrea L.; Mayes, Samuel; Favreau, Peter F.; Leavesley, Silas J.

    2016-03-01

    Cyclic AMP (cAMP) is a ubiquitous second messenger known to differentially regulate many cellular functions over a wide range of timescales. Several lines of evidence have suggested that the distribution of cAMP within cells is not uniform, and that cAMP compartmentalization is largely responsible for signaling specificity within the cAMP signaling pathway. However, to date, no studies have experimentally measured three dimensional (3D) cAMP distributions within cells. Here we use both 2D and 3D hyperspectral microscopy to visualize cAMP gradients in endothelial cells from the pulmonary microvasculature (PMVECs). cAMP levels were measured using a FRETbased cAMP sensor comprised of a cAMP binding domain from EPAC sandwiched between FRET donors and acceptors -- Turquoise and Venus fluorescent proteins. Data were acquired using either a Nikon A1R spectral confocal microscope or custom spectral microscopy system. Analysis of hyperspectral image stacks from a single confocal slice or from summed images of all slices (2D analysis) indicated little or no cAMP gradients were formed within PMVECs under basal conditions or following agonist treatment. However, analysis of hyperspectral image stacks from 3D cellular geometries (z stacks) demonstrate marked cAMP gradients from the apical to basolateral membrane of PMVECs. These results strongly suggest that 2D imaging studies of cAMP compartmentalization -- whether epifluorescence or confocal microscopy -- may lead to erroneous conclusions about the existence of cAMP gradients, and that 3D studies are required to assess mechanisms of signaling specificity.

  17. Chlorella intake attenuates reduced salivary SIgA secretion in kendo training camp participants

    Directory of Open Access Journals (Sweden)

    Otsuki Takeshi

    2012-12-01

    Full Text Available Abstract Background The green alga Chlorella contains high levels of proteins, vitamins, and minerals. We previously reported that a chlorella-derived multicomponent supplement increased the secretion rate of salivary secretory immunoglobulin A (SIgA in humans. Here, we investigated whether intake of this chlorella-derived supplement attenuated the reduced salivary SIgA secretion rate during a kendo training camp. Methods Ten female kendo athletes participated in inter-university 6-day spring and 4-day summer camps. They were randomized into two groups; one took placebo tablets during the spring camp and chlorella tablets during the summer camp, while the other took chlorella tablets during the spring camp and placebo tablets during the summer camp. Subjects took these tablets starting 4 weeks before the camp until post-camp saliva sampling. Salivary SIgA concentrations were measured by ELISA. Results All subjects participated in nearly all training programs, and body-mass changes and subjective physical well-being scores during the camps were comparable between the groups. However, salivary SIgA secretion rate changes were different between these groups. Salivary SIgA secretion rates decreased during the camp in the placebo group (before vs. second, middle, and final day of camp, and after the camp: 146 ± 89 vs. 87 ± 56, 70 ± 45, 94 ± 58, and 116 ± 71 μg/min, whereas no such decreases were observed in the chlorella group (121 ± 53 vs. 113 ± 68, 98 ± 69,115 ± 80, and 128 ± 59 μg/min. Conclusion Our results suggest that a use of a chlorella-derived dietary supplement attenuates reduced salivary SIgA secretion during a training camp for a competitive sport.

  18. Mechanisms of Cellular Uptake of Thrombin-Antithrombin II Complexes Role of the Low-Density Lipoprotein Receptor-Related Protein as a Serpin-Enzyme Complex Receptor.

    Science.gov (United States)

    Strickland, D K; Kounnas, M Z

    1997-01-01

    Serine proteinase inhibitors (serpins) such as antithrombin III inhibit target proteinases by forming a stable complexwith the enzyme. Once formed, several serpin-enzyme complexes (SECs) are removed from the circulation by a receptor, termed the SEC receptor, that is present in the liver. Until recently, the identity of this clearance receptor remained unknown; however, data are now available that strongly implicates one member of the low-density lipoprotein (LDL) receptor family as a candidate for the SEC receptor. This receptor, known as the LDL receptor-related protein (LRP), is a prominent liver receptor that is known to bind numerous ligands that include proteinase-inhibitor complexes, matrix proteins, and certain apolipoprotein E- and lipoprotein lipase-enriched lipoproteins. © 1997, Elsevier Science Inc. (Trends Cardiovasc Med 1997;7:9-16).

  19. Transactivation of Epidermal Growth Factor Receptor by G Protein-Coupled Receptors: Recent Progress, Challenges and Future Research

    Directory of Open Access Journals (Sweden)

    Zhixiang Wang

    2016-01-01

    Full Text Available Both G protein-coupled receptors (GPCRs and receptor-tyrosine kinases (RTKs regulate large signaling networks, control multiple cell functions and are implicated in many diseases including various cancers. Both of them are also the top therapeutic targets for disease treatment. The discovery of the cross-talk between GPCRs and RTKs connects these two vast signaling networks and complicates the already complicated signaling networks that regulate cell signaling and function. In this review, we focus on the transactivation of epidermal growth factor receptor (EGFR, a subfamily of RTKs, by GPCRs. Since the first report of EGFR transactivation by GPCR, significant progress has been made including the elucidation of the mechanisms underlying the transactivation. Here, we first provide a basic picture for GPCR, EGFR and EGFR transactivation by GPCR. We then discuss the progress made in the last five years and finally provided our view of the future challenge and future researches needed to overcome these challenges.

  20. Computational Exploration of a Protein Receptor Binding Space with Student Proposed Peptide Ligands

    Science.gov (United States)

    King, Matthew D.; Phillips, Paul; Turner, Matthew W.; Katz, Michael; Lew, Sarah; Bradburn, Sarah; Andersen, Tim; McDougal, Owen M.

    2016-01-01

    Computational molecular docking is a fast and effective "in silico" method for the analysis of binding between a protein receptor model and a ligand. The visualization and manipulation of protein to ligand binding in three-dimensional space represents a powerful tool in the biochemistry curriculum to enhance student learning. The…

  1. The Arabidopsis kinase-associated protein phosphatase controls internalization of the somatic embryogenesis receptor kinase 1

    NARCIS (Netherlands)

    Shah, K.; Russinova, E.; Gadella, T.W.J.; Willemse, J.; Vries, de S.C.

    2002-01-01

    The AtSERK1 protein is a plasma membrane-located LRR receptor-like serine threonine kinase that is transiently expressed during plant embryogenesis. Our results show that AtSERK1 interacts with the kinase-associated protein phosphatase (KAPP) in vitro. The kinase interaction (KI) domain of KAPP does

  2. Receptor-like protein-tyrosine phosphatase alpha specifically inhibits insulin-increased prolactin gene expression

    DEFF Research Database (Denmark)

    Jacob, K K; Sap, J; Stanley, F M

    1998-01-01

    A physiologically relevant response to insulin, stimulation of prolactin promoter activity in GH4 pituitary cells, was used as an assay to study the specificity of protein-tyrosine phosphatase function. Receptor-like protein-tyrosine phosphatase alpha (RPTPalpha) blocks the effect of insulin to i...

  3. Adenovirus Core Protein pVII Is Translocated into the Nucleus by Multiple Import Receptor Pathways†

    Science.gov (United States)

    Wodrich, Harald; Cassany, Aurelia; D'Angelo, Maximiliano A.; Guan, Tinglu; Nemerow, Glen; Gerace, Larry

    2006-01-01

    Adenoviruses are nonenveloped viruses with an ∼36-kb double-stranded DNA genome that replicate in the nucleus. Protein VII, an abundant structural component of the adenovirus core that is strongly associated with adenovirus DNA, is imported into the nucleus contemporaneously with the adenovirus genome shortly after virus infection and may promote DNA import. In this study, we evaluated whether protein VII uses specific receptor-mediated mechanisms for import into the nucleus. We found that it contains potent nuclear localization signal (NLS) activity by transfection of cultured cells with protein VII fusion constructs and by microinjection of cells with recombinant protein VII fusions. We identified three NLS-containing regions in protein VII by deletion mapping and determined important NLS residues by site-specific mutagenesis. We found that recombinant protein VII and its NLS-containing domains strongly and specifically bind to importin α, importin β, importin 7, and transportin, which are among the most abundant cellular nuclear import receptors. Moreover, these receptors can mediate the nuclear import of protein VII fusions in vitro in permeabilized cells. Considered together, these data support the hypothesis that protein VII is a major NLS-containing adaptor for receptor-mediated import of adenovirus DNA and that multiple import pathways are utilized to promote efficient nuclear entry of the viral genome. PMID:16973564

  4. Adenovirus core protein pVII is translocated into the nucleus by multiple import receptor pathways.

    Science.gov (United States)

    Wodrich, Harald; Cassany, Aurelia; D'Angelo, Maximiliano A; Guan, Tinglu; Nemerow, Glen; Gerace, Larry

    2006-10-01

    Adenoviruses are nonenveloped viruses with an approximately 36-kb double-stranded DNA genome that replicate in the nucleus. Protein VII, an abundant structural component of the adenovirus core that is strongly associated with adenovirus DNA, is imported into the nucleus contemporaneously with the adenovirus genome shortly after virus infection and may promote DNA import. In this study, we evaluated whether protein VII uses specific receptor-mediated mechanisms for import into the nucleus. We found that it contains potent nuclear localization signal (NLS) activity by transfection of cultured cells with protein VII fusion constructs and by microinjection of cells with recombinant protein VII fusions. We identified three NLS-containing regions in protein VII by deletion mapping and determined important NLS residues by site-specific mutagenesis. We found that recombinant protein VII and its NLS-containing domains strongly and specifically bind to importin alpha, importin beta, importin 7, and transportin, which are among the most abundant cellular nuclear import receptors. Moreover, these receptors can mediate the nuclear import of protein VII fusions in vitro in permeabilized cells. Considered together, these data support the hypothesis that protein VII is a major NLS-containing adaptor for receptor-mediated import of adenovirus DNA and that multiple import pathways are utilized to promote efficient nuclear entry of the viral genome.

  5. 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

    G protein-coupled receptors (GPCRs) constitute a large class of seven transmembrane proteins, which bind selectively agonists or antagonists with important consequences for cellular signaling and function. Comprehension of the molecular details of ligand binding is important for the understanding...

  6. Sorting by the cytoplasmic domain of the amyloid precursor protein binding receptor SorLA

    DEFF Research Database (Denmark)

    Nielsen, Morten S; Gustafsen, Camilla; Madsen, Peder

    2007-01-01

    -formation with the amyloid precursor protein it downregulates generation of Alzheimer's disease-associated Abeta-peptide. The receptor is mainly located in vesicles, suggesting a function in protein sorting and transport. Here we examined SorLA's trafficking using full-length and chimeric receptors and find that its...... established that the AP-1 adaptor complex is essential to SorLA's transport between Golgi membranes and endosomes. Our results further implicate the GGA proteins in SorLA trafficking and provide evidence that SNX1 and Vps35, as parts of the retromer complex or possibly in a separate context, are engaged...

  7. Role of Cbl-associated protein/ponsin in receptor tyrosine kinase signaling and cell adhesion

    Directory of Open Access Journals (Sweden)

    Ritva Tikkanen

    2012-10-01

    Full Text Available The Cbl-associated protein/ponsin (CAP is an adaptor protein that contains a so-called Sorbin homology (SoHo domain and three Src homology 3 (SH3 domains which are engaged in diverse protein-protein interactions. CAP has been shown to function in the regulation of the actin cytoskeleton and cell adhesion and to be involved in the differentiation of muscle cells and adipocytes. In addition, it participates in signaling pathways through several receptor tyrosine kinases such as insulin and neurotrophin receptors. In the last couple of years, several studies have shed light on the details of these processes and identified novel interaction partners of CAP. In this review, we summarize these recent findings and provide an overview on the function of CAP especially in cell adhesion and membrane receptor signaling.

  8. Allosteric regulation of G protein-coupled receptor activity by phospholipids.

    Science.gov (United States)

    Dawaliby, Rosie; Trubbia, Cataldo; Delporte, Cédric; Masureel, Matthieu; Van Antwerpen, Pierre; Kobilka, Brian K; Govaerts, Cédric

    2016-01-01

    Lipids are emerging as key regulators of membrane protein structure and activity. These effects can be attributed either to the modification of bilayer properties (thickness, curvature and surface tension) or to the binding of specific lipids to the protein surface. For G protein-coupled receptors (GPCRs), the effects of phospholipids on receptor structure and activity remain poorly understood. Here we reconstituted purified β2-adrenergic receptor (β2R) in high-density lipoparticles to systematically characterize the effect of biologically relevant phospholipids on receptor activity. We observed that the lipid headgroup type affected ligand binding (agonist and antagonist) and receptor activation. Specifically, phosphatidylgycerol markedly favored agonist binding and facilitated receptor activation, whereas phosphatidylethanolamine favored antagonist binding and stabilized the inactive state of the receptor. We then showed that these effects could be recapitulated with detergent-solubilized lipids, demonstrating that the functional modulation occurred in the absence of a bilayer. Our data suggest that phospholipids act as direct allosteric modulators of GPCR activity.

  9. Amphipols in G protein-coupled receptor pharmacology: what are they good for?

    Science.gov (United States)

    Mary, Sophie; Damian, Marjorie; Rahmeh, Rita; Mouillac, Bernard; Marie, Jacky; Granier, Sébastien; Banères, Jean-Louis

    2014-10-01

    G protein-coupled receptors are at a central node of all cell communications. Investigating their molecular functioning is therefore crucial for both academic purposes and drug design. However, getting the receptors as isolated, stable and purified proteins for such studies still stumbles over their instability out of the membrane environment. Different membrane-mimicking environments have been developed so far to increase the stability of purified receptors. Among them are amphipols. These polymers not only preserve the native fold of receptors purified from membrane fractions but they also allow specific applications such as folding receptors purified from inclusion bodies back to their native state. Of importance, amphipol-trapped G protein-coupled receptors essentially maintain their pharmacological properties so that they are perfectly adapted to further investigate the molecular mechanisms underlying signaling processes. We review here how amphipols have been used to refold and stabilize detergent-solubilized purified receptors and what are the main subsequent molecular pharmacology analyses that were performed using this strategy.

  10. Delayed toxicity associated with soluble anthrax toxin receptor decoy-Ig fusion protein treatment.

    Directory of Open Access Journals (Sweden)

    Diane Thomas

    Full Text Available Soluble receptor decoy inhibitors, including receptor-immunogloubulin (Ig fusion proteins, have shown promise as candidate anthrax toxin therapeutics. These agents act by binding to the receptor-interaction site on the protective antigen (PA toxin subunit, thereby blocking toxin binding to cell surface receptors. Here we have made the surprising observation that co-administration of receptor decoy-Ig fusion proteins significantly delayed, but did not protect, rats challenged with anthrax lethal toxin. The delayed toxicity was associated with the in vivo assembly of a long-lived complex comprised of anthrax lethal toxin and the receptor decoy-Ig inhibitor. Intoxication in this system presumably results from the slow dissociation of the toxin complex from the inhibitor following their prolonged circulation. We conclude that while receptor decoy-Ig proteins represent promising candidates for the early treatment of B. anthracis infection, they may not be suitable for therapeutic use at later stages when fatal levels of toxin have already accumulated in the bloodstream.

  11. Putative hAPN receptor binding sites in SARS_CoV spike protein

    Institute of Scientific and Technical Information of China (English)

    YUXiao-Jing; LUOCheng; LinJian-Cheng; HAOPei; HEYou-Yu; GUOZong-Ming; QINLei; SUJiong; LIUBo-Shu; HUANGYin; NANPeng; LIChuan-Song; XIONGBin; LUOXiao-Min; ZHAOGuo-Ping; PEIGang; CHENKai-Xian; SHENXu; SHENJian-Hua; ZOUJian-Ping; HEWei-Zhong; SHITie-Liu; ZHONGYang; JIANGHua-Liang; LIYi-Xue

    2003-01-01

    AIM:To obtain the information of ligand-receptor binding between thd S protein of SARS_CoV and CD13, identify the possible interacting domains or motifs related to binding sites, and provide clues for studying the functions of SARS proteins and designing anti-SARS drugs and vaccines. METHODS: On the basis of comparative genomics, the homology search, phylogenetic analyses, and multi-sequence alignment were used to predict CD13 related interacting domains and binding sites sites in the S protein of SARS_CoV. Molecular modeling and docking simulation methods were employed to address the interaction feature between CD13 and S protein of SARS_CoV in validating the bioinformatics predictions. RESULTS:Possible binding sites in the SARS_CoV S protein to CD13 have been mapped out by using bioinformatics analysis tools. The binding for one protein-protein interaction pair (D757-R761 motif of the SARS_CoV S protein to P585-A653 domain of CD13) has been simulated by molecular modeling and docking simulation methods. CONCLUSION:CD13 may be a possible receptor of the SARS_CoV S protein which may be associated with the SARS infection. This study also provides a possible strategy for mapping the possible binding receptors of the proteins in a genome.

  12. Coordinate regulation of G protein signaling via dynamic interactions of receptor and GAP.

    Directory of Open Access Journals (Sweden)

    Marc Turcotte

    Full Text Available Signal output from receptor-G-protein-effector modules is a dynamic function of the nucleotide exchange activity of the receptor, the GTPase-accelerating activity of GTPase-activating proteins (GAPs, and their interactions. GAPs may inhibit steady-state signaling but may also accelerate deactivation upon removal of stimulus without significantly inhibiting output when the receptor is active. Further, some effectors (e.g., phospholipase C-beta are themselves GAPs, and it is unclear how such effectors can be stimulated by G proteins at the same time as they accelerate G protein deactivation. The multiple combinations of protein-protein associations and interacting regulatory effects that allow such complex behaviors in this system do not permit the usual simplifying assumptions of traditional enzyme kinetics and are uniquely subject to systems-level analysis. We developed a kinetic model for G protein signaling that permits analysis of both interactive and independent G protein binding and regulation by receptor and GAP. We evaluated parameters of the model (all forward and reverse rate constants by global least-squares fitting to a diverse set of steady-state GTPase measurements in an m1 muscarinic receptor-G(q-phospholipase C-beta1 module in which GTPase activities were varied by approximately 10(4-fold. We provide multiple tests to validate the fitted parameter set, which is consistent with results from the few previous pre-steady-state kinetic measurements. Results indicate that (1 GAP potentiates the GDP/GTP exchange activity of the receptor, an activity never before reported; (2 exchange activity of the receptor is biased toward replacement of GDP by GTP; (3 receptor and GAP bind G protein with negative cooperativity when G protein is bound to either GTP or GDP, promoting rapid GAP binding and dissociation; (4 GAP indirectly stabilizes the continuous binding of receptor to G protein during steady-state GTPase hydrolysis, thus further

  13. Evidence that the newly cloned low-density-lipoprotein receptor related protein (LRP) is the alpha 2-macroglobulin receptor

    DEFF Research Database (Denmark)

    Kristensen, T; Moestrup, Søren Kragh; Gliemann, Jørgen;

    1990-01-01

    these polypeptides, and analysis of a 1772 bp cDNA encoding part of the 500 kDa polypeptide provide evidence that the 500 kDa and 85 kDa chains are the alpha- and beta-subunits, respectively, of a recently cloned hepatic membrane protein, termed the low density lipoprotein receptor related protein (LRP) (Herz, J......The human placental receptor (alpha 2MR) for alpha 2-macroglobulin-proteinase complexes contains 3 polypeptides of approx. 500 kDa, 85 kDa, and 40 kDa. N-terminal sequence analysis of the 500 kDa and 85 kDa polypeptides, analysis of a random selection of peptides convering 536 residues from...

  14. Increases in cAMP, MAPK Activity and CREB Phosphorylation during REM Sleep: Implications for REM Sleep and Memory Consolidation

    OpenAIRE

    Luo, Jie; Phan, Trongha X.; Yang, Yimei; Garelick, Michael G.; Storm, Daniel R.

    2013-01-01

    The cyclic adenosine monophosphate (cAMP), mitogen-activated protein kinase (MAPK) and cAMP response element-binding protein (CREB) transcriptional pathway is required for consolidation of hippocampus-dependent memory. In mice, this pathway undergoes a circadian oscillation required for memory persistence that reaches a peak during the daytime. Since mice exhibit polyphasic sleep patterns during the day, this suggested the interesting possibility that cAMP, MAPK activity and CREB phosphorylat...

  15. RGS proteins destroy spare receptors: Effects of GPCR-interacting proteins and signal deamplification on measurements of GPCR agonist potency.

    Science.gov (United States)

    Chidiac, Peter

    2016-01-01

    Many GPCRs are able to activate multiple distinct signaling pathways, and these may include biochemical cascades activated via either heterotrimeric G proteins or by β-arrestins. The relative potencies and/or efficacies among a series of agonists that act on a common receptor can vary depending upon which signaling pathway is being activated. This phenomenon is known as biased signaling or functional selectivity, and is presumed to reflect underlying differences in ligand binding affinities for alternate conformational states of the receptor. The first part of this review discusses how various cellular GPCR interacting proteins (GIPs) can influence receptor conformation and thereby affect ligand-receptor interactions and contribute to signaling bias. Upon activation, receptors trigger biochemical cascades that lead to altered cellular function, and measuring points along the cascade (e.g., second messenger production) conveys information about receptor activity. As a signal continues along its way, the observed concentration dependence of a GPCR ligand may change due to amplification and saturation of biochemical steps. The second part of this review considers additional cellular factors that affect signal processing, focusing mainly on structural elements and deamplification mechanisms, and discusses the relevance of these to measurements of potency and functional selectivity.

  16. Marlin-1, a novel RNA-binding protein associates with GABA receptors.

    Science.gov (United States)

    Couve, Andrés; Restituito, Sophie; Brandon, Julia M; Charles, Kelly J; Bawagan, Hinayana; Freeman, Katie B; Pangalos, Menelas N; Calver, Andrew R; Moss, Stephen J

    2004-04-02

    GABA(B) receptors are heterodimeric G protein-coupled receptors that mediate slow synaptic inhibition in the central nervous system. Whereas heterodimerization between GABA(B) receptor GABA(B)R1 and GABA(B)R2 subunits is essential for functional expression, how neurons coordinate the assembly of these critical receptors remains to be established. Here we have identified Marlin-1, a novel GABA(B) receptor-binding protein that associates specifically with the GABA(B)R1 subunit in yeast, tissue culture cells, and neurons. Marlin-1 is expressed in the brain and exhibits a granular distribution in cultured hippocampal neurons. Marlin-1 binds different RNA species including the 3'-untranslated regions of both the GABA(B)R1 and GABA(B)R2 mRNAs in vitro and also associates with RNA in cultured neurons. Inhibition of Marlin-1 expression via small RNA interference technology results in enhanced intracellular levels of the GABA(B)R2 receptor subunit without affecting the level of GABA(B)R1. Together our results suggest that Marlin-1 functions to regulate the cellular levels of GABA(B) R2 subunits, which may have significant effects on the production of functional GABA(B) receptor heterodimers. Therefore, our observations provide an added level of regulation for the control of GABA(B) receptor expression and for the efficacy of inhibitory synaptic transmission.

  17. Peripheral-type benzodiazepine receptor: a protein of mitochondrial outer membranes utilizing porphyrins as endogenous ligands

    Energy Technology Data Exchange (ETDEWEB)

    Snyder, S.H.; Verma, A.; Trifiletti, R.R.

    1987-10-01

    The peripheral-type benzodiazepine receptor is a site identified by its nanomolar affinity for (/sup 3/H)diazepam, similar to the affinity of diazepam for the central-type benzodiazepine receptor in the brain. The peripheral type benzodiazepine receptor occurs in many peripheral tissues but has discrete localizations as indicated by autoradiographic studies showing uniquely high densities of the receptors in the adrenal cortex and in Leydig cells of the testes. Subcellular localization studies reveal a selective association of the receptors with the outer membrane of mitochondria. Photoaffinity labeling of the mitochondrial receptor with (/sup 3/H)flunitrazepam reveals two discrete labeled protein bands of 30 and 35 kDa, respectively. The 35-kDa band appears to be identical with the voltage-dependent anion channel protein porin. Fractionation of numerous peripheral tissues reveals a single principal endogenous ligand for the receptor, consisting of porphyrins, which display nanomolar affinity. Interactions of porphyrins with the mitochondrial receptor may clarify its physiological role and account for many pharmacological actions of benzodiazepines.

  18. Characterization of receptor proteins using affinity cross-linking with biotinylated ligands.

    Science.gov (United States)

    Shinya, Tomonori; Osada, Tomohiko; Desaki, Yoshitake; Hatamoto, Masahiro; Yamanaka, Yuko; Hirano, Hisashi; Takai, Ryota; Che, Fang-Sik; Kaku, Hanae; Shibuya, Naoto

    2010-02-01

    The plant genome encodes a wide range of receptor-like proteins but the function of most of these proteins is unknown. We propose the use of affinity cross-linking of biotinylated ligands for a ligand-based survey of the corresponding receptor molecules. Biotinylated ligands not only enable the analysis of receptor-ligand interactions without the use of radioactive compounds but also the isolation and identification of receptor molecules by a simple affinity trapping method. We successfully applied this method for the characterization, isolation and identification of the chitin elicitor binding protein (CEBiP). A biocytin hydrazide conjugate of N-acetylchitooctaose (GN8-Bio) was synthesized and used for the detection of CEBiP in the plasma or microsomal membrane preparations from rice and carrot cells. Binding characteristics of CEBiP analyzed by inhibition studies were in good agreement with the previous results obtained with the use of a radiolabeled ligand. The biotin-tagged CEBiP could be purified by avidin affinity chromatography and identified by LC-MALDI-MS/MS after tryptic digestion. We also used this method to detect OsFLS2, a rice receptor-like kinase for the perception of the peptide elicitor flg22, in membrane preparations from rice cells overexpressing OsFLS2. This work demonstrates the applicability of this method to the purification and identification of plant receptor proteins.

  19. Definition of the G protein-coupled receptor transmembrane bundle binding pocket and calculation of receptor similarities for drug design

    DEFF Research Database (Denmark)

    Gloriam, David Erik Immanuel; Foord, Steven M; Blaney, Frank E;

    2009-01-01

    Recent advances in structural biology for G-protein-coupled receptors (GPCRs) have provided new opportunities to improve the definition of the transmembrane binding pocket. Here a reference set of 44 residue positions accessible for ligand binding was defined through detailed analysis of all curr...... the pharmacology/selectivity profile of ligands at Family A GPCRs. This has wide applicability to GPCR drug design problems across many disease areas....

  20. cAMP Signals in Drosophila Motor Neurons Are Confined to Single Synaptic Boutons

    Directory of Open Access Journals (Sweden)

    Isabella Maiellaro

    2016-10-01

    Full Text Available The second messenger cyclic AMP (cAMP plays an important role in synaptic plasticity. Although there is evidence for local control of synaptic transmission and plasticity, it is less clear whether a similar spatial confinement of cAMP signaling exists. Here, we suggest a possible biophysical basis for the site-specific regulation of synaptic plasticity by cAMP, a highly diffusible small molecule that transforms the physiology of synapses in a local and specific manner. By exploiting the octopaminergic system of Drosophila, which mediates structural synaptic plasticity via a cAMP-dependent pathway, we demonstrate the existence of local cAMP signaling compartments of micrometer dimensions within single motor neurons. In addition, we provide evidence that heterogeneous octopamine receptor localization, coupled with local differences in phosphodiesterase activity, underlies the observed differences in cAMP signaling in the axon, cell body, and boutons.

  1. Mapping physiological G protein-coupled receptor signaling pathways reveals a role for receptor phosphorylation in airway contraction.

    Science.gov (United States)

    Bradley, Sophie J; Wiegman, Coen H; Iglesias, Max Maza; Kong, Kok Choi; Butcher, Adrian J; Plouffe, Bianca; Goupil, Eugénie; Bourgognon, Julie-Myrtille; Macedo-Hatch, Timothy; LeGouill, Christian; Russell, Kirsty; Laporte, Stéphane A; König, Gabriele M; Kostenis, Evi; Bouvier, Michel; Chung, Kian Fan; Amrani, Yassine; Tobin, Andrew B

    2016-04-19

    G protein-coupled receptors (GPCRs) are known to initiate a plethora of signaling pathways in vitro. However, it is unclear which of these pathways are engaged to mediate physiological responses. Here, we examine the distinct roles of Gq/11-dependent signaling and receptor phosphorylation-dependent signaling in bronchial airway contraction and lung function regulated through the M3-muscarinic acetylcholine receptor (M3-mAChR). By using a genetically engineered mouse expressing a G protein-biased M3-mAChR mutant, we reveal the first evidence, to our knowledge, of a role for M3-mAChR phosphorylation in bronchial smooth muscle contraction in health and in a disease state with relevance to human asthma. Furthermore, this mouse model can be used to distinguish the physiological responses that are regulated by M3-mAChR phosphorylation (which include control of lung function) from those responses that are downstream of G protein signaling. In this way, we present an approach by which to predict the physiological/therapeutic outcome of M3-mAChR-biased ligands with important implications for drug discovery.

  2. Structure of metabotropic glutamate receptor C-terminal domains in contact with interacting proteins

    OpenAIRE

    Enz, Ralf

    2012-01-01

    Metabotropic glutamate receptors (mGluRs) regulate intracellular signal pathways that control several physiological tasks, including neuronal excitability, learning, and memory. This is achieved by the formation of synaptic signal complexes, in which mGluRs assemble with functionally related proteins such as enzymes, scaffolds, and cytoskeletal anchor proteins. Thus, mGluR associated proteins actively participate in the regulation of glutamatergic neurotransmission. Importantly, dysfunction o...

  3. LDL receptor-GFP fusion proteins: new tools for the characterization of disease-causing mutations in the LDL receptor gene

    DEFF Research Database (Denmark)

    Holst, Henrik Uffe; Dagnæs-Hansen, Frederik; Corydon, Thomas Juhl;

    2001-01-01

    The function of a series of LDL receptor GFP fusion proteins with different, flexible, unstructured spacer regions was analysed. An optimised version of the fusion protein was used to analyse the effect of a LDL receptor mutation (W556S) found in FH patients and characterized as transport defective....... In cultured liver cells this mutation was found to inhibit the transport of LDL receptor GFP fusion protein to the cell surface, thus leading to impaired internalisation of fluorescent labelled LDL. Co-locallisation studies confirmed the retention of the mutant protein in the endoplasmic reticulum....

  4. Active-state models of ternary GPCR complexes: determinants of selective receptor-G-protein coupling.

    Directory of Open Access Journals (Sweden)

    Ralf C Kling

    Full Text Available Based on the recently described crystal structure of the β2 adrenergic receptor--Gs-protein complex, we report the first molecular-dynamics simulations of ternary GPCR complexes designed to identify the selectivity determinants for receptor-G-protein binding. Long-term molecular dynamics simulations of agonist-bound β2AR-Gαs and D2R-Gαi complexes embedded in a hydrated bilayer environment and computational alanine-scanning mutagenesis identified distinct residues of the N-terminal region of intracellular loop 3 to be crucial for coupling selectivity. Within the G-protein, specific amino acids of the α5-helix, the C-terminus of the Gα-subunit and the regions around αN-β1 and α4-β6 were found to determine receptor recognition. Knowledge of these determinants of receptor-G-protein binding selectivity is essential for designing drugs that target specific receptor/G-protein combinations.

  5. Dimerization of TOC receptor GTPases and its implementation for the control of protein import into chloroplasts.

    Science.gov (United States)

    Aronsson, Henrik; Jarvis, Paul

    2011-06-01

    Pre-protein import into chloroplasts is facilitated by multiprotein translocon complexes in the envelope membranes. Major components of the TOC (translocon at the outer envelope membrane of chloroplasts) complex are the receptor proteins Toc33 and Toc159. These two receptors are related GTPases, and they are predicted to engage in homodimerization and/or heterodimerization. Although such dimerization has been studied extensively, its exact function in vivo remains elusive. In this issue of the Biochemical Journal, Oreb et al. present evidence that homodimerization of Toc33 prevents nucleotide exchange, thereby locking the receptor in the GDP-loaded state and preventing further activity. Pre-protein arrival is proposed to release this lock, through disruption of the dimer and subsequent nucleotide exchange. The Toc33-bound pre-protein is then able to progress to downstream steps in the translocation mechanism, with GTP hydrolysis defining another important control point as well as preparing the receptor for the next pre-protein client. These new results are discussed in the context of previous findings pertaining to TOC receptor dimerization and function.

  6. The interaction of acetylcholine receptors in porcine atrial membranes with three kinds of G proteins.

    Science.gov (United States)

    Haga, T; Ikegaya, T; Haga, K

    1990-09-01

    We developed a simple procedure to detect the interaction of muscarinic receptors in atrial membranes with exogenous GTP-binding proteins (G proteins). The procedure consists of mixing atrial membranes with G proteins in the presence of sodium cholate, diluting the mixture with a salt buffer and then measuring the ligand binding activity. The displacement by carbachol of [3H] QNB binding to muscarinic receptors in the atrial membranes was not affected by guanine nucleotides when the membranes had been treated at 60 degrees C for 30 min or with N-ethylmeleimide (NEM) and became affected by them after mixing the heat- or NEM-treated membranes with G proteins. The displacement curves in the presence of GTP were essentially the same irrespective of the presence or absence of G proteins. Those in the absence of GTP shifted to a lower concentration of carbachol with addition of a higher concentration of G proteins, indicating an increase in GTP-sensitive high affinity agonist binding sites. The highest affinity for carbachol was detected with membranes treated with NEM and then mixed with G proteins. The GTP-sensitive high affinity agonist binding could be detected with any one of three kinds of G proteins (Gi, Go, Gn) which were purified from porcine cerebrum, indicating that the muscarinic receptor m2 subtype may interact with and possibly activate these three kinds of G proteins.

  7. Distinct protein domains and expression patterns confer divergent axon guidance functions for Drosophila Robo receptors.

    Science.gov (United States)

    Spitzweck, Bettina; Brankatschk, Marko; Dickson, Barry J

    2010-02-05

    The orthogonal array of axon pathways in the Drosophila CNS is constructed in part under the control of three Robo family axon guidance receptors: Robo1, Robo2 and Robo3. Each of these receptors is responsible for a distinct set of guidance decisions. To determine the molecular basis for these functional specializations, we used homologous recombination to create a series of 9 "robo swap" alleles: expressing each of the three Robo receptors from each of the three robo loci. We demonstrate that the lateral positioning of longitudinal axon pathways relies primarily on differences in gene regulation, not distinct combinations of Robo proteins as previously thought. In contrast, specific features of the Robo1 and Robo2 proteins contribute to their distinct functions in commissure formation. These specializations allow Robo1 to prevent crossing and Robo2 to promote crossing. These data demonstrate how diversification of expression and structure within a single family of guidance receptors can shape complex patterns of neuronal wiring.

  8. cAMP response element binding protein1 is essential for activation of steroyl co-enzyme a desaturase 1 (Scd1 in mouse lung type II epithelial cells.

    Directory of Open Access Journals (Sweden)

    Nisha Antony

    Full Text Available Cyclic AMP Response Element-Binding Protein 1 (Creb1 is a transcription factor that mediates cyclic adenosine 3', 5'-monophosphate (cAMP signalling in many tissues. Creb1(-/- mice die at birth due to respiratory failure and previous genome-wide microarray analysis of E17.5 Creb1(-/- fetal mouse lung identified important Creb1-regulated gene targets during lung development. The lipogenic enzymes stearoyl-CoA desaturase 1 (Scd1 and fatty acid synthase (Fasn showed highly reduced gene expression in Creb1(-/- lungs. We therefore hypothesized that Creb1 plays a crucial role in the transcriptional regulation of genes involved in pulmonary lipid biosynthetic pathways during lung development. In this study we confirmed that Scd1 and Fasn mRNA levels were down regulated in the E17.5 Creb1(-/- mouse lung while the lipogenic-associated transcription factors SrebpF1, C/ebpα and Pparγ were increased. In vivo studies using germline (Creb1(-/- and lung epithelial-specific (Creb1(EpiΔ/Δ Creb1 knockout mice showed strongly reduced Scd1, but not Fasn gene expression and protein levels in lung epithelial cells. In vitro studies using mouse MLE-15 epithelial cells showed that forskolin-mediated activation of Creb1 increased both Scd1 gene expression and protein synthesis. Additionally, MLE15 cells transfected with a dominant-negative ACreb vector blocked forskolin-mediated stimulation of Scd1 gene expression. Lipid profiling in MLE15 cells showed that dominant-negative ACreb suppressed forskolin-induced desaturation of ether linked lipids to produce plasmalogens, as well as levels of phosphatidylethanolamine, ceramide and lysophosphatidylcholine. Taken together these results demonstrate that Creb1 is essential for the induction and maintenance of Scd1 in developing fetal mouse lung epithelial cells.

  9. Hydromania: Summer Science Camp Curriculum.

    Energy Technology Data Exchange (ETDEWEB)

    Moura, Joan

    1995-07-01

    In 1992, Bonneville Power Administration (BPA) and the US Department of Energy (DOE) began a collaborative pilot project with the Portland Parks and Recreation Community Schools Program and others to provide summer science camps to children in Grades 4--6. Camps run two weeks in duration between late June and mid-August. Sessions are five days per week, from 9 a.m. to 3 p.m. In addition to hands-on science and math curriculum, at least three field trips are incorporated into the educational learning experience. The purpose of the BPA/DOE summer camps is to make available opportunities for fun, motivating experiences in science to students who otherwise would have difficulty accessing them. This includes inner city, minority, rural and low income students. Public law 101-510, which Congress passed in 1990, authorizes DOE facilities to establish collaborative inner-city and rural partnership programs in science and math. A primary goal of the BPA summer hands on science camps is to bring affordable science camp experiences to students where they live. It uses everyday materials to engage students` minds and to give them a sense that they have succeeded through a fun hands-on learning environment.

  10. REEPs are membrane shaping adapter proteins that modulate specific g protein-coupled receptor trafficking by affecting ER cargo capacity.

    Science.gov (United States)

    Björk, Susann; Hurt, Carl M; Ho, Vincent K; Angelotti, Timothy

    2013-01-01

    Receptor expression enhancing proteins (REEPs) were identified by their ability to enhance cell surface expression of a subset of G protein-coupled receptors (GPCRs), specifically GPCRs that have proven difficult to express in heterologous cell systems. Further analysis revealed that they belong to the Yip (Ypt-interacting protein) family and that some REEP subtypes affect ER structure. Yip family comparisons have established other potential roles for REEPs, including regulation of ER-Golgi transport and processing/neuronal localization of cargo proteins. However, these other potential REEP functions and the mechanism by which they selectively enhance GPCR cell surface expression have not been clarified. By utilizing several REEP family members (REEP1, REEP2, and REEP6) and model GPCRs (α2A and α2C adrenergic receptors), we examined REEP regulation of GPCR plasma membrane expression, intracellular processing, and trafficking. Using a combination of immunolocalization and biochemical methods, we demonstrated that this REEP subset is localized primarily to ER, but not plasma membranes. Single cell analysis demonstrated that these REEPs do not specifically enhance surface expression of all GPCRs, but affect ER cargo capacity of specific GPCRs and thus their surface expression. REEP co-expression with α2 adrenergic receptors (ARs) revealed that this REEP subset interacts with and alter glycosidic processing of α2C, but not α2A ARs, demonstrating selective interaction with cargo proteins. Specifically, these REEPs enhanced expression of and interacted with minimally/non-glycosylated forms of α2C ARs. Most importantly, expression of a mutant REEP1 allele (hereditary spastic paraplegia SPG31) lacking the carboxyl terminus led to loss of this interaction. Thus specific REEP isoforms have additional intracellular functions besides altering ER structure, such as enhancing ER cargo capacity, regulating ER-Golgi processing, and interacting with select cargo proteins

  11. CGRP-RCP, a novel protein required for signal transduction at calcitonin gene-related peptide and adrenomedullin receptors.

    Science.gov (United States)

    Evans, B N; Rosenblatt, M I; Mnayer, L O; Oliver, K R; Dickerson, I M

    2000-10-06

    It is becoming clear that receptors that initiate signal transduction by interacting with G-proteins do not function as monomers, but often require accessory proteins for function. Some of these accessory proteins are chaperones, required for correct transport of the receptor to the cell surface, but the function of many accessory proteins remains unknown. We determined the role of an accessory protein for the receptor for calcitonin gene-related peptide (CGRP), a potent vasodilator neuropeptide. We have previously shown that this accessory protein, the CGRP-receptor component protein (RCP), is expressed in CGRP responsive tissues and that RCP protein expression correlates with the biological efficacy of CGRP in vivo. However, the function of RCP has remained elusive. In this study stable cell lines were made that express antisense RCP RNA, and CGRP- and adrenomedullin-mediated signal transduction were greatly reduced. However, the loss of RCP did not effect CGRP binding or receptor density, indicating that RCP did not behave as a chaperone but was instead coupling the CGRP receptor to downstream effectors. A candidate CGRP receptor named calcitonin receptor-like receptor (CRLR) has been identified, and in this study RCP co-immunoprecipitated with CRLR indicating that these two proteins interact directly. Since CGRP and adrenomedullin can both signal through CRLR, which has been previously shown to require a chaperone protein for function, we now propose that a functional CGRP or adrenomedullin receptor consists of at least three proteins: the receptor (CRLR), the chaperone protein (RAMP), and RCP that couples the receptor to the cellular signal transduction pathway.

  12. Insights into the structural biology of G-protein coupled receptors impacts drug design for central nervous system neurodegenerative processes

    OpenAIRE

    Dalet, Farfán-García Eunice; Guadalupe, Trujillo-Ferrara José; María del Carmen, Castillo-Hernández; Humberto, Guerra-Araiza Christian; Antonio, Soriano-Ursúa Marvin

    2013-01-01

    In the last few years, there have been important new insights into the structural biology of G-protein coupled receptors. It is now known that allosteric binding sites are involved in the affinity and selectivity of ligands for G-protein coupled receptors, and that signaling by these receptors involves both G-protein dependent and independent pathways. The present review outlines the physiological and pharmacological implications of this perspective for the design of new drugs to treat disord...

  13. Engineering a minimal G protein to facilitate crystallisation of G protein-coupled receptors in their active conformation.

    Science.gov (United States)

    Carpenter, Byron; Tate, Christopher G

    2016-12-01

    G protein-coupled receptors (GPCRs) modulate cytoplasmic signalling in response to extracellular stimuli, and are important therapeutic targets in a wide range of diseases. Structure determination of GPCRs in all activation states is important to elucidate the precise mechanism of signal transduction and to facilitate optimal drug design. However, due to their inherent instability, crystallisation of GPCRs in complex with cytoplasmic signalling proteins, such as heterotrimeric G proteins and β-arrestins, has proved challenging. Here, we describe the design of a minimal G protein, mini-Gs, which is composed solely of the GTPase domain from the adenylate cyclase stimulating G protein Gs Mini-Gs is a small, soluble protein, which efficiently couples GPCRs in the absence of Gβγ subunits. We engineered mini-Gs, using rational design mutagenesis, to form a stable complex with detergent-solubilised β1-adrenergic receptor (β1AR). Mini G proteins induce similar pharmacological and structural changes in GPCRs as heterotrimeric G proteins, but eliminate many of the problems associated with crystallisation of these complexes, specifically their large size, conformational dynamics and instability in detergent. They are therefore novel tools, which will facilitate the biochemical and structural characterisation of GPCRs in their active conformation.

  14. [The Cytoskelrtal Protein Zvxin Interacts with the Hedgehog Receptor Patched].

    Science.gov (United States)

    Martynova, N U; Ermolina, L V; Eroshkin, F M; Zarayskiy, A G

    2015-01-01

    Earlier, we demonstrated Zyxin influence upon Hedgehog (Hh)-signaling pathway during early patterning of the central neural system (CNS) anlage of the Xenopus laevis embryo. Now we show that Zyxin can physically interact with the transmembrane receptor of Hh, Patched2 (Ptc2). Binding of Hh by this receptor activates signaling pathway, which regulates many events, including numerous types of cell differentiation during the embryonic development. In particular, patterning of the CNS anlage. The ability of Zyxin to interact with Ptc2 have been confirmed by immunoprecipitation experiments, in which we tested mutual binding affinity of Zyxin and Ptc2, as well as mutual affinity of their deletion mutants. As a result, we have established that in Xenopus levis, Zyxin binding to Ptc2 is due to the interaction of Zyxin 2nd LIM-domain (530-590 aa) with the under-membrane region of the cytoplasmic C-terminus of Ptc2 (1159-1412 aa). We have also demonstrated that similar interaction is valid for the homologous regions of the human Zyxin and human Hh receptor, Ptc1. The data obtained allow to hypothesize existence of evolutionary conserved mechanism that modulates Hh-signaling and based on the interaction of Zyxin with Ptc.

  15. GPR99, a new G protein-coupled receptor with homology to a new subgroup of nucleotide receptors

    Directory of Open Access Journals (Sweden)

    Chica Schaller H

    2002-07-01

    Full Text Available Abstract Background Based on sequence similarity, the superfamily of G protein-coupled receptors (GPRs can be subdivided into several subfamilies, the members of which often share similar ligands. The sequence data provided by the human genome project allows us to identify new GPRs by in silico homology screening, and to predict their ligands. Results By searching the human genomic database with known nucleotide receptors we discovered the gene for GPR99, a new orphan GPR. The mRNA of GPR99 was found in kidney and placenta. Phylogenetic analysis groups GPR99 into the P2Y subfamily of GPRs. Based on the phylogenetic tree we propose a new classification of P2Y nucleotide receptors into two subgroups predicting a nucleotide ligand for GPR99. By assaying known nucleotide ligands on heterologously expressed GPR99, we could not identify specifically activating substances, indicating that either they are not agonists of GPR99 or that GPR99 was not expressed at the cell surface. Analysis of the chromosomal localization of all genes of the P2Y subfamily revealed that all members of subgroup "a" are encoded by less than 370 kb on chromosome 3q24, and that the genes of subgroup "b" are clustered on one hand to chromosome 11q13.5 and on the other on chromosome 3q24-25.1 close to the subgroup "a" position. Therefore, the P2Y subfamily is a striking example for local gene amplification. Conclusions We identified a new orphan receptor, GPR99, with homology to the family of G protein-coupled nucleotide receptors. Phylogenetic analysis separates this family into different subgroups predicting a nucleotide ligand for GPR99.

  16. G-protein-coupled receptors for free fatty acids: nutritional and therapeutic targets

    OpenAIRE

    Milligan, Graeme; Ulven, Trond; Murdoch, Hannah; Hudson, Brian D.

    2014-01-01

    It is becoming evident that nutrients and metabolic intermediates derived from such nutrients regulate cellular function by activating a number of cell-surface G-protein coupled receptors (GPCRs). Until now, members of the GPCR family have largely been considered as the molecular targets that communicate cellular signals initiated by hormones and neurotransmitters. Recently, based on tissue expression patterns of these receptors and the concept that they may elicit the production of a range o...

  17. Zinc Is Involved in Depression by Modulating G Protein-Coupled Receptor Heterodimerization.

    Science.gov (United States)

    Tena-Campos, Mercè; Ramon, Eva; Lupala, Cecylia S; Pérez, Juan J; Koch, Karl-W; Garriga, Pere

    2016-04-01

    5-Hydroxytryptamine 1A receptor and galanin receptor 1 belong to the G protein-coupled receptors superfamily, and they have been described to heterodimerize triggering an anomalous physiological state that would underlie depression. Zinc supplementation has been widely reported to improve treatment against major depressive disorder. Our work has focused on the study and characterization of these receptors and its relationships with zinc both under purified conditions and in cell culture. To this aim, we have designed a strategy to purify the receptors in a conformationally active state. We have used receptors tagged with the monoclonal Rho-1D4 antibody and employed ligand-assisted purification in order to successfully purify both receptors in a properly folded and active state. The interaction between both purified receptors has been analyzed by surface plasmon resonance in order to determine the kinetics of dimerization. Zinc effect on heteromer has also been tested using the same methodology but exposing the 5-hydroxytryptamine 1A receptor to zinc before the binding experiment. These results, combined with Förster resonance energy transfer (FRET) measurements, in the absence and presence of zinc, suggest that this ion is capable of disrupting this interaction. Moreover, molecular modeling suggests that there is a coincidence between zinc-binding sites and heterodimerization interfaces for the serotonin receptor. Our results establish a rational explanation for the role of zinc in the molecular processes associated with receptor-receptor interactions and its relationship with depression, in agreement with previously reported evidence for the positive effects of zinc in depression treatment, and the involvement of our target dimer in the same disease.

  18. Gc protein-derived macrophage-activating factor (GcMAF) stimulates cAMP formation in human mononuclear cells and inhibits angiogenesis in chick embryo chorionallantoic membrane assay.

    Science.gov (United States)

    Pacini, Stefania; Morucci, Gabriele; Punzi, Tiziana; Gulisano, Massimo; Ruggiero, Marco

    2011-04-01

    The effects of Gc protein-derived macrophage-activating factor (GcMAF) have been studied in cancer and other conditions where angiogenesis is deregulated. In this study, we demonstrate for the first time that the mitogenic response of human peripheral blood mononuclear cells (PBMCs) to GcMAF was associated with 3'-5'-cyclic adenosine monophosphate (cAMP) formation. The effect was dose dependent, and maximal stimulation was achieved using 0.1 ng/ml. Heparin inhibited the stimulatory effect of GcMAF on PBMCs. In addition, we demonstrate that GcMAF (1 ng/ml) inhibited prostaglandin E(1)- and human breast cancer cell-stimulated angiogenesis in chick embryo chorionallantoic membrane (CAM) assay. Finally, we tested different GcMAF preparations on CAM, and the assay proved to be a reliable, reproducible and inexpensive method to determine the relative potencies of different preparations and their stability; we observed that storage at room temperature for 15 days decreased GcMAF potency by about 50%. These data could prove useful for upcoming clinical trials on GcMAF.

  19. "cAMP sponge": a buffer for cyclic adenosine 3', 5'-monophosphate.

    Directory of Open Access Journals (Sweden)

    Konstantinos Lefkimmiatis

    Full Text Available BACKGROUND: While intracellular buffers are widely used to study calcium signaling, no such tool exists for the other major second messenger, cyclic AMP (cAMP. METHODS/PRINCIPAL FINDINGS: Here we describe a genetically encoded buffer for cAMP based on the high-affinity cAMP-binding carboxy-terminus of the regulatory subunit RIbeta of protein kinase A (PKA. Addition of targeting sequences permitted localization of this fragment to the extra-nuclear compartment, while tagging with mCherry allowed quantification of its expression at the single cell level. This construct (named "cAMP sponge" was shown to selectively bind cAMP in vitro. Its expression significantly suppressed agonist-induced cAMP signals and the downstream activation of PKA within the cytosol as measured by FRET-based sensors in single living cells. Point mutations in the cAMP-binding domains of the construct rendered the chimera unable to bind cAMP in vitro or in situ. Cyclic AMP sponge was fruitfully applied to examine feedback regulation of gap junction-mediated transfer of cAMP in epithelial cell couplets. CONCLUSIONS: This newest member of the cAMP toolbox has the potential to reveal unique biological functions of cAMP, including insight into the functional significance of compartmentalized signaling events.

  20. Cloning of human genes encoding novel G protein-coupled receptors

    Energy Technology Data Exchange (ETDEWEB)

    Marchese, A.; Docherty, J.M.; Heiber, M. [Univ. of Toronto, (Canada)] [and others

    1994-10-01

    We report the isolation and characterization of several novel human genes encoding G protein-coupled receptors. Each of the receptors contained the familiar seven transmembrane topography and most closely resembled peptide binding receptors. Gene GPR1 encoded a receptor protein that is intronless in the coding region and that shared identity (43% in the transmembrane regions) with the opioid receptors. Northern blot analysis revealed that GPR1 transcripts were expressed in the human hippocampus, and the gene was localized to chromosome 15q21.6. Gene GPR2 encoded a protein that most closely resembled an interleukin-8 receptor (51% in the transmembrane regions), and this gene, not expressed in the six brain regions examined, was localized to chromosome 17q2.1-q21.3. A third gene, GPR3, showed identity (56% in the transmembrane regions) with a previously characterized cDNA clone from rat and was localized to chromosome 1p35-p36.1. 31 refs., 5 figs., 1 tab.

  1. Role of CGRP-receptor component protein (RCP) in CLR/RAMP function.

    Science.gov (United States)

    Dickerson, Ian M

    2013-08-01

    The receptor for calcitonin gene-related peptide (CGRP) and adrenomedullin (AM) requires an intracellular peripheral membrane protein named CGRP-receptor component protein (RCP) for signaling. RCP is required for CGRP and AM receptor signaling, and it has recently been discovered that RCP enables signaling by binding directly to the receptor. RCP is present in most immortalized cell lines, but in vivo RCP expression is limited to specific subsets of cells, usually co-localizing with CGRP-containing neurons. RCP protein expression correlates with CGRP efficacy in vivo, suggesting that RCP regulates CGRP signaling in vivo as it does in cell culture. RCP is usually identified in cytoplasm or membranes of cells, but recently has been observed in nucleus of neurons, suggesting an additional transcriptional role for RCP in cell function. Together, these data support an essential role for RCP in CGRP and AM receptor function, in which RCP expression enhances signaling of the CGRP or AM receptor, and therefore increases the efficacy of CGRP and AM in vivo.

  2. SLAM family receptors and the SLAM-associated protein (SAP) modulate T cell functions.

    Science.gov (United States)

    Detre, Cynthia; Keszei, Marton; Romero, Xavier; Tsokos, George C; Terhorst, Cox

    2010-06-01

    One or more of the signaling lymphocytic activation molecule (SLAM) family (SLAMF) of cell surface receptors, which consists of nine transmembrane proteins, i.e., SLAMF1-9, are expressed on most hematopoietic cells. While most SLAMF receptors serve as self-ligands, SLAMF2 and SLAMF4 use each other as counter structures. Six of the receptors carry one or more copies of a unique intracellular tyrosine-based switch motif, which has high affinity for the single SH2-domain signaling molecules SLAM-associated protein and EAT-2. Whereas SLAMF receptors are costimulatory molecules on the surface of CD4+, CD8+, and natural killer (NK) T cells, they also involved in early phases of lineage commitment during hematopoiesis. SLAMF receptors regulate T lymphocyte development and function and modulate lytic activity, cytokine production, and major histocompatibility complex-independent cell inhibition of NK cells. Furthermore, they modulate B cell activation and memory generation, neutrophil, dendritic cell, macrophage and eosinophil function, and platelet aggregation. In this review, we will discuss the role of SLAM receptors and their adapters in T cell function, and we will examine the role of these receptors and their adapters in X-linked lymphoproliferative disease and their contribution to disease susceptibility in systemic lupus erythematosus.

  3. The SOL-2/Neto auxiliary protein modulates the function of AMPA-subtype ionotropic glutamate receptors.

    Science.gov (United States)

    Wang, Rui; Mellem, Jerry E; Jensen, Michael; Brockie, Penelope J; Walker, Craig S; Hoerndli, Frédéric J; Hauth, Linda; Madsen, David M; Maricq, Andres V

    2012-09-06

    The neurotransmitter glutamate mediates excitatory synaptic transmission by gating ionotropic glutamate receptors (iGluRs). AMPA receptors (AMPARs), a subtype of iGluR, are strongly implicated in synaptic plasticity, learning, and memory. We previously discovered two classes of AMPAR auxiliary proteins in C. elegans that modify receptor kinetics and thus change synaptic transmission. Here, we have identified another auxiliary protein, SOL-2, a CUB-domain protein that associates with both the related auxiliary subunit SOL-1 and with the GLR-1 AMPAR. In sol-2 mutants, behaviors dependent on glutamatergic transmission are disrupted, GLR-1-mediated currents are diminished, and GLR-1 desensitization and pharmacology are modified. Remarkably, a secreted variant of SOL-1 delivered in trans can rescue sol-1 mutants, and this rescue depends on in cis expression of SOL-2. Finally, we demonstrate that SOL-1 and SOL-2 have an ongoing role in the adult nervous system to control AMPAR-mediated currents.

  4. Sch proteins are localized on endoplasmic reticulum membranes and are redistributed after tyrosine kinase receptor activation

    DEFF Research Database (Denmark)

    Lotti, L V; Lanfrancone, L; Migliaccio, E;

    1996-01-01

    area of the cell and mostly associated with the cytosolic side of rough endoplasmic reticulum membranes. Upon epidermal growth factor treatment and receptor tyrosine kinase activation, the immunolabeling became peripheral and was found to be associated with the cytosolic surface of the plasma membrane......The intracellular localization of Shc proteins was analyzed by immunofluorescence and immunoelectron microscopy in normal cells and cells expressing the epidermal growth factor receptor or the EGFR/erbB2 chimera. In unstimulated cells, the immunolabeling was localized in the central perinuclear....... The rough endoplasmic reticulum localization of Shc proteins in unstimulated cells and their massive recruitment to the plasma membrane, endocytic structures, and peripheral cytosol following receptor tyrosine kinase activation could account for multiple putative functions of the adaptor protein....

  5. Present perspectives on the automated classification of the G-protein coupled receptors (GPCRs) at the protein sequence level

    DEFF Research Database (Denmark)

    Davies, Matthew N; Gloriam, David E; Secker, Andrew;

    2011-01-01

    The G-protein coupled receptors--or GPCRs--comprise simultaneously one of the largest and one of the most multi-functional protein families known to modern-day molecular bioscience. From a drug discovery and pharmaceutical industry perspective, the GPCRs constitute one of the most commercially...... and economically important groups of proteins known. The GPCRs undertake numerous vital metabolic functions and interact with a hugely diverse range of small and large ligands. Many different methodologies have been developed to efficiently and accurately classify the GPCRs. These range from motif-based techniques...

  6. Global and local missions of cAMP signaling in neural plasticity, learning and memory

    Directory of Open Access Journals (Sweden)

    Daewoo eLee

    2015-08-01

    Full Text Available The fruit fly Drosophila melanogaster has been a popular model to study cAMP signaling and resultant behaviors due to its powerful genetic approaches. All molecular components (AC, PDE, PKA, CREB, etc essential for cAMP signaling have been identified in the fly. Among them, adenylyl cyclase (AC gene rutabaga and phosphodiesterase (PDE gene dunce have been intensively studied to understand the role of cAMP signaling. Interestingly, these two mutant genes were originally identified on the basis of associative learning deficits. This commentary summarizes findings on the role of cAMP in Drosophila neuronal excitability, synaptic plasticity and memory. It mainly focuses on two distinct mechanisms (global versus local regulating excitatory and inhibitory synaptic plasticity related to cAMP homeostasis. This dual regulatory role of cAMP is to increase the strength of excitatory neural circuits on one hand, but to act locally on postsynaptic GABA receptors to decrease inhibitory synaptic plasticity on the other. Thus the action of cAMP could result in a global increase in the neural circuit excitability and memory. Implications of this cAMP signaling related to drug discovery for neural diseases are also described.

  7. Long-Term Memory for Place Learning Is Facilitated by Expression of cAMP Response Element-Binding Protein in the Dorsal Hippocampus

    Science.gov (United States)

    Brightwell, Jennifer J.; Smith, Clayton A.; Neve, Rachael L.; Colombo, Paul J.

    2007-01-01

    Extensive research has shown that the hippocampus is necessary for consolidation of long-term spatial memory in rodents. We reported previously that rats using a place strategy to solve a cross maze task showed sustained phosphorylation of hippocampus cyclic AMP response element-binding protein (CREB), a transcription factor implicated in…

  8. Predicting the Coupling Specificity of G-protein Coupled Receptors to G-proteins by Support Vector Machines

    Institute of Scientific and Technical Information of China (English)

    Cui-Ping Guan; Zhen-Ran Jiang; Yan-Hong Zhou

    2005-01-01

    G-protein coupled receptors (GPCRs) represent one of the most important classes of drug targets for pharmaceutical industry and play important roles in cellular signal transduction. Predicting the coupling specificity of GPCRs to G-proteins is vital for further understanding the mechanism of signal transduction and the function of the receptors within a cell, which can provide new clues for pharmaceutical research and development. In this study, the features of amino acid compositions and physiochemical properties of the full-length GPCR sequences have been analyzed and extracted. Based on these features, classifiers have been developed to predict the coupling specificity of GPCRs to G-proteins using support vector machines. The testing results show that this method could obtain better prediction accuracy.

  9. Effects of dopamine 1 receptor agonists on the content of cAMP of mesenteric and pulmonary arteries in rat%DA1受体激动剂对大鼠离体肺动脉和肠动脉cAMP生成量的影响

    Institute of Scientific and Technical Information of China (English)

    朱琳; 刘爱华; 赵荣瑞

    2001-01-01

    Aim:To evaluate and compare the effects of DA1 receptor agonist, fenoldopam, on rat pulmonary and mesenteric arteries.Methods:Using radioimmunoassay. The effects of fenoldopam on the cAMP generating system in rat mesenteric and pulmonary arteries were determined. Result:The selective DA1-receptors agonist, fenoldopam, induced a dose-related increases cAMP formation in mesenteric and pulmonary arteries; however, the magnitude of increase in the mesenteric artery was remarkably greater than that in the pulmonary artery. The selective DA1-receptors antagonist, SCH23390, blocked fenoldopam-induced cAMP production, while the selective DA2-receptors antagonist, domperidone, was without effect on the increase of cAMP elicited by fenoldopam. Conclusion:The results showed the existence of DA1-receptors associated with stimulation of AC activity in rat mesenteric and pulmonary arteries. However, there are much fewer receptor sites in the pulmonary artery than in mesenteric artery, suggesting less physioloical importace of such receptors in the pulmonary artery than that in the mesenteric artery.%目的:对比分析多巴胺1(DA1)受体激动剂非诺多泮(FODA)对大鼠离体肺动脉和肠动脉cAMP含量的影响。方法:采用放射免疫测定法,测定FODA对肺动脉和肠系膜动脉cAMP生成量的影响以及DA受体拮抗剂对FODA诱发肠动脉、肺动脉血管cAMP变化的影响。结果:非诺多泮可剂量依赖性增加肠、肺动脉cAMP的生成量,肠动脉cAMP的生成量显著高于肺动脉cAMP的生成量。选择性多巴胺1(DA1)受体阻断剂SCH23390能够阻断非诺多泮所引起的肺动脉和肠动脉cAMP生成量增加,多巴胺2(DA2)受体阻断剂Domperidone则不影响非诺多泮的反应。结论:大鼠肺动脉和肠动脉均存在有刺激腺苷酸环化酶(AC)活性的DA1受体,但肺动脉DA1受体的位点数明显少于肠动脉DA1受体位点数,提示肺动脉DA1受体的生理反应弱于肠动脉。

  10. EGF receptor transactivation in angiotensin II and endothelin control of vascular protein synthesis in vivo.

    Science.gov (United States)

    Beaucage, Pierre; Moreau, Pierre

    2004-11-01

    Endothelin represents a necessary intermediate of angiotensin II-induced resistance artery remodeling in hypertension. Recent data suggest that epidermal growth factor receptors are rapidly transactivated by angiotensin II stimulation to mediate its growth-promoting effects. Because endothelin also transactivates epidermal growth factor receptors in vitro, we studied the contribution of epidermal growth factor receptor transactivation in the in vivo trophic actions of the upstream effector angiotensin II and its downstream mediator endothelin in rat mesenteric arteries. Twenty-six-hour infusion of angiotensin II (400 ng/kg per min) or endothelin (5 pmol/kg per min) via osmotic pumps significantly enhanced vascular protein synthesis. With angiotensin II, treatment with the inhibitor of epidermal growth factor receptor transactivation (AG1478, 0.5 mg/kg) produced a significant attenuation (P < 0.05) of protein synthesis. In contrast, AG1478 did not abrogate the elevation of protein synthesis induced by endothelin. In conclusion, angiotensin II-induced epidermal growth factor receptor transactivation seems to be involved in the recruitment of endothelin in the cascade leading to vascular protein synthesis, rather than in the effect of endothelin on small artery remodeling.

  11. A mechanism regulating G protein-coupled receptor signaling that requires cycles of protein palmitoylation and depalmitoylation.

    Science.gov (United States)

    Jia, Lixia; Chisari, Mariangela; Maktabi, Mohammad H; Sobieski, Courtney; Zhou, Hao; Konopko, Aaron M; Martin, Brent R; Mennerick, Steven J; Blumer, Kendall J

    2014-02-28

    Reversible attachment and removal of palmitate or other long-chain fatty acids on proteins has been hypothesized, like phosphorylation, to control diverse biological processes. Indeed, palmitate turnover regulates Ras trafficking and signaling. Beyond this example, however, the functions of palmitate turnover on specific proteins remain poorly understood. Here, we show that a mechanism regulating G protein-coupled receptor signaling in neuronal cells requires palmitate turnover. We used hexadecyl fluorophosphonate or palmostatin B to inhibit enzymes in the serine hydrolase family that depalmitoylate proteins, and we studied R7 regulator of G protein signaling (RGS)-binding protein (R7BP), a palmitoylated allosteric modulator of R7 RGS proteins that accelerate deactivation of Gi/o class G proteins. Depalmitoylation inhibition caused R7BP to redistribute from the plasma membrane to endomembrane compartments, dissociated R7BP-bound R7 RGS complexes from Gi/o-gated G protein-regulated inwardly rectifying K(+) (GIRK) channels and delayed GIRK channel closure. In contrast, targeting R7BP to the plasma membrane with a polybasic domain and an irreversibly attached lipid instead of palmitate rendered GIRK channel closure insensitive to depalmitoylation inhibitors. Palmitate turnover therefore is required for localizing R7BP to the plasma membrane and facilitating Gi/o deactivation by R7 RGS proteins on GIRK channels. Our findings broaden the scope of biological processes regulated by palmitate turnover on specific target proteins. Inhibiting R7BP depalmitoylation may provide a means of enhancing GIRK activity in neurological disorders.

  12. Characterization of rabbit ileal receptors for Clostridium difficile toxin A. Evidence for a receptor-coupled G protein

    Energy Technology Data Exchange (ETDEWEB)

    Pothoulakis, C.; LaMont, J.T.; Eglow, R.; Gao, N.; Rubins, J.B.; Theoharides, T.C.; Dickey, B.F. (Boston Univ. School of Medicine, MA (USA))

    1991-07-01

    The purpose of this study was to characterize the surface receptor for toxin A, the enterotoxin from Clostridium difficile, on rabbit intestinal brush borders (BB) and on rat basophilic leukemia (RBL) cells. Purified toxin A was radiolabeled using a modified Bolton-Hunter method to sp act 2 microCi/micrograms, with retention of full biologic activity. 3H-Toxin A bound specifically to a single class of receptors on rabbit BB and on RBL cells with dissociation constants of 5.4 x 10(-8) and 3.5 x 10(-8) M, respectively. RBL cells were highly sensitive to toxin A (cell rounding) and had 180,000 specific binding sites per cell, whereas IMR-90 fibroblasts were far less sensitive to toxin A and lacked detectable specific binding sites. Exposure of BB to trypsin or chymotrypsin significantly reduced 3H-toxin A specific binding. Preincubation of BB with Bandeirea simplicifolia (BS-1) lectin also reduced specific binding, and CHAPS-solubilized receptors could be immobilized with WGA-agarose. The addition of 100 nM toxin A accelerated the association of 35S-GTP gamma S with rabbit ileal BB, and preincubation of BB with the GTP analogues GTP gamma S or Gpp(NH)p, significantly reduced 3H-toxin A specific binding. Our data indicate that the membrane receptor for toxin A is a galactose and N-acetyl-glucosamine-containing glycoprotein which appears to be coupled to a G protein.

  13. Chromatin Modulatory Proteins and Olfactory Receptor Signaling in the Refinement and Maintenance of Fruitless Expression in Olfactory Receptor Neurons.

    Directory of Open Access Journals (Sweden)

    Catherine E Hueston

    2016-04-01

    Full Text Available During development, sensory neurons must choose identities that allow them to detect specific signals and connect with appropriate target neurons. Ultimately, these sensory neurons will successfully integrate into appropriate neural circuits to generate defined motor outputs, or behavior. This integration requires a developmental coordination between the identity of the neuron and the identity of the circuit. The mechanisms that underlie this coordination are currently unknown. Here, we describe two modes of regulation that coordinate the sensory identities of Drosophila melanogaster olfactory receptor neurons (ORNs involved in sex-specific behaviors with the sex-specific behavioral circuit identity marker fruitless (fru. The first mode involves a developmental program that coordinately restricts to appropriate ORNs the expression of fru and two olfactory receptors (Or47b and Ir84a involved in sex-specific behaviors. This regulation requires the chromatin modulatory protein Alhambra (Alh. The second mode relies on the signaling from the olfactory receptors through CamK and histone acetyl transferase p300/CBP to maintain ORN-specific fru expression. Our results highlight two feed-forward regulatory mechanisms with both developmentally hardwired and olfactory receptor activity-dependent components that establish and maintain fru expression in ORNs. Such a dual mechanism of fru regulation in ORNs might be a trait of neurons driving plastic aspects of sex-specific behaviors.

  14. Signaling governed by G proteins and cAMP is crucial for growth, secondary metabolism and sexual development in Fusarium fujikuroi.

    Directory of Open Access Journals (Sweden)

    Lena Studt

    Full Text Available The plant-pathogenic fungus Fusarium fujikuroi is a notorious rice pathogen causing hyper-elongation of infected plants due to the production of gibberellic acids (GAs. In addition to GAs, F. fujikuroi produces a wide range of other secondary metabolites, such as fusarins, fusaric acid or the red polyketides bikaverins and fusarubins. The recent availability of the fungal genome sequence for this species has revealed the potential of many more putative secondary metabolite gene clusters whose products remain to be identified. However, the complex regulation of secondary metabolism is far from being understood. Here we studied the impact of the heterotrimeric G protein and the cAMP-mediated signaling network, including the regulatory subunits of the cAMP-dependent protein kinase (PKA, to study their effect on colony morphology, sexual development and regulation of bikaverins, fusarubins and GAs. We demonstrated that fusarubin biosynthesis is negatively regulated by at least two Gα subunits, FfG1 and FfG3, which both function as stimulators of the adenylyl cyclase FfAC. Surprisingly, the primary downstream target of the adenylyl cyclase, the PKA, is not involved in the regulation of fusarubins, suggesting that additional, yet unidentified, cAMP-binding protein(s exist. In contrast, bikaverin biosynthesis is significantly reduced in ffg1 and ffg3 deletion mutants and positively regulated by FfAC and FfPKA1, while GA biosynthesis depends on the active FfAC and FfPKA2 in an FfG1- and FfG3-independent manner. In addition, we provide evidence that G Protein-mediated/cAMP signaling is important for growth in F. fujikuroi because deletion of ffg3, ffac and ffpka1 resulted in impaired growth on minimal and rich media. Finally, sexual crosses of ffg1 mutants showed the importance of a functional FfG1 protein for development of perithecia in the mating strain that carries the MAT1-1 idiomorph.

  15. Receptor protein tyrosine phosphatase alpha is essential for hippocampal neuronal migration and long-term potentiation

    DEFF Research Database (Denmark)

    Petrone, Angiola; Battaglia, Fortunato; Wang, Cheng;

    2003-01-01

    (RPTPalpha) regulates SRC family kinases, potassium channels and NMDA receptors. Here, we report that absence of RPTPalpha compromises correct positioning of pyramidal neurons during development of mouse hippocampus. Thus, RPTPalpha is a novel member of the functional class of genes that control radial......Despite clear indications of their importance in lower organisms, the contributions of protein tyrosine phosphatases (PTPs) to development or function of the mammalian nervous system have been poorly explored. In vitro studies have indicated that receptor protein tyrosine phosphatase alpha...

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

    Directory of Open Access Journals (Sweden)

    Nuria Franco

    2014-01-01

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

  17. Agonistic autoantibodies directed against G-protein-coupled receptors and their relationship to cardiovascular diseases.

    Science.gov (United States)

    Wallukat, Gerd; Schimke, Ingolf

    2014-05-01

    Agonistic autoantibodies (AABs) against G-protein-coupled receptor (GPCR) are present mainly in diseases of the cardiovascular system or in diseases associated with cardiovascular disturbances. The increasing knowledge about the role of autoantibodies against G-protein-coupled receptor (GPCR-AABs) as pathogenic drivers, the resulting development of strategies aimed at their removal or neutralization, and the evidenced patient benefit associated with such therapies have created the need for a summary of GPCR-AAB-associated diseases. Here, we summarize the present knowledge about GPCR-AABs in cardiovascular diseases. The identity of the GPCR-AABs and their prevalence in each of several specific cardiovascular diseases are documented. The structure of GPCR is also briefly discussed. Using this information, differences between classic agonists and GPCR-AABs in their GPCR binding and activation are presented and the resulting pathogenic consequences are discussed. Furthermore, treatment strategies that are currently under study, most of which are aimed at the removal and in vivo neutralization of GPCR-AABs, are indicated and their patient benefits discussed. In this context, immunoadsorption using peptides/proteins or aptamers as binders are introduced. The use of peptides or aptamers for in vivo neutralization of GPCR-AABs is also described. Particular attention is given to the GPCR-AABs directed against the adrenergic beta1-, beta2-, and α1-receptor as well as the muscarinic receptor M2, angiotensin II-angiotensin receptor type I, endothelin1 receptor type A, angiotensin (1-7) Mas-receptor, and 5-hydroxytryptamine receptor 4. Among the diseases associated with GPCR-AABs, special focus is given to idiopathic dilated cardiomyopathy, Chagas' cardiomyopathy, malignant and pulmonary hypertension, and kidney diseases. Relationships of GPCR-AABs are indicated to glaucoma, peripartum cardiomyopathy, myocarditis, pericarditis, preeclampsia, Alzheimer's disease, Sj

  18. Starting a Technology Camp for Children.

    Science.gov (United States)

    Litowitz, Len S.; Baylor, Steven C.

    1997-01-01

    Presents information for starting and maintaining a technology camp for students. Includes selecting topics, identifying participants, marketing, managing the camp, budgeting, and benefits to students and host institutions. (JOW)

  19. Eurasia Project—2007 Italian Camp Held

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    <正>From July 14 to 29,the Eurasia Project—2007 Italian Camp was held at the Castle Fusano Country Club in Rome. 52 high school students from Germany,Poland,Italy and China participated in the summer camp.

  20. Regulation of growth factor receptor degradation by ADP-ribosylation factor domain protein (ARD) 1.

    Science.gov (United States)

    Meza-Carmen, Victor; Pacheco-Rodriguez, Gustavo; Kang, Gi Soo; Kato, Jiro; Donati, Chiara; Zhang, Chun-Yi; Vichi, Alessandro; Payne, D Michael; El-Chemaly, Souheil; Stylianou, Mario; Moss, Joel; Vaughan, Martha

    2011-06-28

    ADP-ribosylation factor domain protein 1 (ARD1) is a 64-kDa protein containing a functional ADP-ribosylation factor (GTP hydrolase, GTPase), GTPase-activating protein, and E3 ubiquitin ligase domains. ARD1 activation by the guanine nucleotide-exchange factor cytohesin-1 was known. GTPase and E3 ligase activities of ARD1 suggest roles in protein transport and turnover. To explore this hypothesis, we used mouse embryo fibroblasts (MEFs) from ARD1-/- mice stably transfected with plasmids for inducible expression of wild-type ARD1 protein (KO-WT), or ARD1 protein with inactivating mutations in E3 ligase domain (KO-E3), or containing persistently active GTP-bound (KO-GTP), or inactive GDP-bound (KO-GDP) GTPase domains. Inhibition of proteasomal proteases in mifepristone-induced KO-WT, KO-GDP, or KO-GTP MEFs resulted in accumulation of these ARD1 proteins, whereas KO-E3 accumulated without inhibitors. All data were consistent with the conclusion that ARD1 regulates its own steady-state levels in cells by autoubiquitination. Based on reported growth factor receptor-cytohesin interactions, EGF receptor (EGFR) was investigated in induced MEFs. Amounts of cell-surface and total EGFR were higher in KO-GDP and lower in KO-GTP than in KO-WT MEFs, with levels in both mutants greater (p = 0.001) after proteasomal inhibition. Significant differences among MEF lines in content of TGF-β receptor III were similar to those in EGFR, albeit not as large. Differences in amounts of insulin receptor mirrored those in EGFR, but did not reach statistical significance. Overall, the capacity of ARD1 GTPase to cycle between active and inactive forms and its autoubiquitination both appear to be necessary for the appropriate turnover of EGFR and perhaps additional growth factor receptors.

  1. Ca2+-dependent inhibition of G protein-coupled receptor kinase 2 by calmodulin.

    Science.gov (United States)

    Haga, K; Tsuga, H; Haga, T

    1997-02-11

    Agonist- or light-dependent phosphorylation of muscarinic acetylcholine receptor m2 subtypes (m2 receptors) or rhodopsin by G protein-coupled receptor kinase 2 (GRK2) was found to be inhibited by calmodulin in a Ca2+-dependent manner. The phosphorylation was fully inhibited in the absence of G protein betagamma subunits and partially inhibited in the presence of betagamma subunits. The dose-response curve for stimulation by betagamma subunits of the m2 and rhodopsin phosphorylation was shifted to the higher concentration of betagamma subunits by addition of Ca2+-calmodulin. The phosphorylation by GRK2 of a glutathione S-transferase fusion protein containing a peptide corresponding to the central part of the third intracellular loop of m2 receptors (I3-GST) was not affected by Ca2+-calmodulin in the presence or absence of betagamma subunits, but the agonist-dependent stimulation of I3-GST phosphorylation by an I3-deleted m2 receptor mutant in the presence of betagamma subunits was suppressed by Ca2+-calmodulin. These results indicate that Ca2+-calmodulin does not directly interact with the catalytic site of GRK2 but inhibits the kinase activity of GRK2 by interfering with the activation of GRK2 by agonist-bound m2 receptors and G protein betagamma subunits. In agreement with the assumption that GRK2 activity is suppressed by the increase in intracellular Ca2+, the sequestration of m2 receptors expressed in Chinese hamster ovary cells was found to be attenuated by the treatment with a Ca2+ ionophore, A23187.

  2. Proteomic analysis of Cry2Aa-binding proteins and their receptor function in Spodoptera exigua

    Science.gov (United States)

    Qiu, Lin; Zhang, Boyao; Liu, Lang; Ma, Weihua; Wang, Xiaoping; Lei, Chaoliang; Chen, Lizhen

    2017-01-01

    The bacterium Bacillus thuringiensis produces Crystal (Cry) proteins that are toxic to a diverse range of insects. Transgenic crops that produce Bt Cry proteins are grown worldwide because of their improved resistance to insect pests. Although Bt “pyramid” cotton that produces both Cry1A and Cry2A is predicted to be more resistant to several lepidopteran pests, including Spodoptera exigua, than plants that produce Cry1Ac alone, the mechanisms responsible for the toxicity of Cry2Aa in S. exigua are not well understood. We identified several proteins that bind Cry2Aa (polycalin, V-ATPase subunits A and B, actin, 4-hydroxybutyrate CoA-transferase [4-HB-CoAT]), and a receptor for activated protein kinase C (Rack), in S. exigua. Recombinant, expressed versions of these proteins were able to bind the Cry2Aa toxin in vitro assays. RNA interference gene knockdown of the Se-V-ATPase subunit B significantly decreased the susceptibility of S. exigua larvae to Cry2Aa, whereas knockdown of the other putative binding proteins did not. Moreover, an in vitro homologous competition assay demonstrated that the Se-V-ATPase subunit B binds specifically to the Cry2Aa toxin, suggesting that this protein acts as a functional receptor of Cry2Aa in S. exigua. This the first Cry2Aa toxin receptor identified in S. exigua brush-border membrane vesicles. PMID:28067269

  3. Vibrational resonance, allostery, and activation in rhodopsin-like G protein-coupled receptors

    Science.gov (United States)

    Woods, Kristina N.; Pfeffer, Jürgen; Dutta, Arpana; Klein-Seetharaman, Judith

    2016-11-01

    G protein-coupled receptors are a large family of membrane proteins activated by a variety of structurally diverse ligands making them highly adaptable signaling molecules. Despite recent advances in the structural biology of this protein family, the mechanism by which ligands induce allosteric changes in protein structure and dynamics for its signaling function remains a mystery. Here, we propose the use of terahertz spectroscopy combined with molecular dynamics simulation and protein evolutionary network modeling to address the mechanism of activation by directly probing the concerted fluctuations of retinal ligand and transmembrane helices in rhodopsin. This approach allows us to examine the role of conformational heterogeneity in the selection and stabilization of specific signaling pathways in the photo-activation of the receptor. We demonstrate that ligand-induced shifts in the conformational equilibrium prompt vibrational resonances in the protein structure that link the dynamics of conserved interactions with fluctuations of the active-state ligand. The connection of vibrational modes creates an allosteric association of coupled fluctuations that forms a coherent signaling pathway from the receptor ligand-binding pocket to the G-protein activation region. Our evolutionary analysis of rhodopsin-like GPCRs suggest that specific allosteric sites play a pivotal role in activating structural fluctuations that allosterically modulate functional signals.

  4. Activation of EphA receptors mediates the recruitment of the adaptor protein Slap, contributing to the downregulation of N-methyl-D-aspartate receptors.

    Science.gov (United States)

    Semerdjieva, Sophia; Abdul-Razak, Hayder H; Salim, Sharifah S; Yáñez-Muñoz, Rafael J; Chen, Philip E; Tarabykin, Victor; Alifragis, Pavlos

    2013-04-01

    Regulation of the activity of N-methyl-d-aspartate receptors (NMDARs) at glutamatergic synapses is essential for certain forms of synaptic plasticity underlying learning and memory and is also associated with neurotoxicity and neurodegenerative diseases. In this report, we investigate the role of Src-like adaptor protein (Slap) in NMDA receptor signaling. We present data showing that in dissociated neuronal cultures, activation of ephrin (Eph) receptors by chimeric preclustered eph-Fc ligands leads to recruitment of Slap and NMDA receptors at the sites of Eph receptor activation. Interestingly, our data suggest that prolonged activation of EphA receptors is as efficient in recruiting Slap and NMDA receptors as prolonged activation of EphB receptors. Using established heterologous systems, we examined whether Slap is an integral part of NMDA receptor signaling. Our results showed that Slap does not alter baseline activity of NMDA receptors and does not affect Src-dependent potentiation of NMDA receptor currents in Xenopus oocytes. We also demonstrate that Slap reduces excitotoxic cell death triggered by activation of NMDARs in HEK293 cells. Finally, we present evidence showing reduced levels of NMDA receptors in the presence of Slap occurring in an activity-dependent manner, suggesting that Slap is part of a mechanism that homeostatically modulates the levels of NMDA receptors.

  5. Prediction and Classification of Human G-protein Coupled Receptors Based on Support Vector Machines

    Institute of Scientific and Technical Information of China (English)

    Yun-Fei Wang; Huan Chen; Yan-Hong Zhou

    2005-01-01

    A computational system for the prediction and classification of human G-protein coupled receptors (GPCRs) has been developed based on the support vector machine (SVM) method and protein sequence information. The feature vectors used to develop the SVM prediction models consist of statistically significant features selected from single amino acid, dipeptide, and tripeptide compositions of protein sequences. Furthermore, the length distribution difference between GPCRsand non-GPCRs has also been exploited to improve the prediction performance.The testing results with annotated human protein sequences demonstrate that this system can get good performance for both prediction and classification of human GPCRs.

  6. Low-density lipoprotein receptor-related protein-1 facilitates heme scavenging after intracerebral hemorrhage in mice.

    Science.gov (United States)

    Wang, Gaiqing; Manaenko, Anatol; Shao, Anwen; Ou, Yibo; Yang, Peng; Budbazar, Enkhjargal; Nowrangi, Derek; Zhang, John H; Tang, Jiping

    2016-06-17

    Heme-degradation after erythrocyte lysis plays an important role in the pathophysiology of intracerebral hemorrhage. Low-density lipoprotein receptor-related protein-1 is a receptor expressed predominately at the neurovascular interface, which facilitates the clearance of the hemopexin and heme complex. In the present study, we investigated the role of low-density lipoprotein receptor-related protein-1 in heme removal and neuroprotection in a mouse model of intracerebral hemorrhage. Endogenous low-density lipoprotein receptor-related protein-1 and hemopexin were increased in ipsilateral brain after intracerebral hemorrhage, accompanied by increased hemoglobin levels, brain water content, blood-brain barrier permeability and neurological deficits. Exogenous human recombinant low-density lipoprotein receptor-related protein-1 protein reduced hematoma volume, brain water content surrounding hematoma, blood-brain barrier permeability and improved neurological function three days after intracerebral hemorrhage. The expression of malondialdehyde, fluoro-Jade C positive cells and cleaved caspase 3 was increased three days after intracerebral hemorrhage in the ipsilateral brain tissues and decreased with recombinant low-density lipoprotein receptor-related protein-1. Intracerebral hemorrhage decreased and recombinant low-density lipoprotein receptor-related protein-1 increased the levels of superoxide dismutase 1. Low-density lipoprotein receptor-related protein-1 siRNA reduced the effect of human recombinant low-density lipoprotein receptor-related protein-1 on all outcomes measured. Collectively, our findings suggest that low-density lipoprotein receptor-related protein-1 contributed to heme clearance and blood-brain barrier protection after intracerebral hemorrhage. The use of low-density lipoprotein receptor-related protein-1 as supplement provides a novel approach to ameliorating intracerebral hemorrhage brain injury via its pleiotropic neuroprotective effects.

  7. A novel human gene encoding a G-protein-coupled receptor (GPR15) is located on chromosome 3

    Energy Technology Data Exchange (ETDEWEB)

    Heiber, M.; Marchese, A.; O`Dowd, B.F. [Univ. of Toronto, Ontario (Canada)] [and others

    1996-03-05

    We used sequence similarities among G-protein-coupled receptor genes to discover a novel receptor gene. Using primers based on conserved regions of the opioid-related receptors, we isolated a PCR product that was used to locate the full-length coding region of a novel human receptor gene, which we have named GPR15. A comparison of the amino acid sequence of the receptor gene, which we have named GPR15. A comparison of the amino acid sequence of the receptor encoded by GPR15 with other receptors revealed that it shared sequence identity with the angiotensin II AT1 and AT2 receptors, the interleukin 8b receptor, and the orphan receptors GPR1 and AGTL1. GPR15 was mapped to human chromosome 3q11.2-q13.1. 12 refs., 2 figs.

  8. High-resolution crystal structure of an engineered human beta2-adrenergic G protein-coupled receptor

    DEFF Research Database (Denmark)

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

    2007-01-01

    Heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors constitute the largest family of eukaryotic signal transduction proteins that communicate across the membrane. We report the crystal structure of a human beta2-adrenergic receptor-T4 lysozyme fusion protein bound...... to the partial inverse agonist carazolol at 2.4 angstrom resolution. The structure provides a high-resolution view of a human G protein-coupled receptor bound to a diffusible ligand. Ligand-binding site accessibility is enabled by the second extracellular loop, which is held out of the binding cavity by a pair...

  9. Lectin receptor kinases participate in protein-protein interactions to mediate plasma membrane-cell wall adhesions in Arabidopsis.

    Science.gov (United States)

    Gouget, Anne; Senchou, Virginie; Govers, Francine; Sanson, Arnaud; Barre, Annick; Rougé, Pierre; Pont-Lezica, Rafael; Canut, Hervé

    2006-01-01

    Interactions between plant cell walls and plasma membranes are essential for cells to function properly, but the molecules that mediate the structural continuity between wall and membrane are unknown. Some of these interactions, which are visualized upon tissue plasmolysis in Arabidopsis (Arabidopsis thaliana), are disrupted by the RGD (arginine-glycine-aspartic acid) tripeptide sequence, a characteristic cell adhesion motif in mammals. In planta induced-O (IPI-O) is an RGD-containing protein from the plant pathogen Phytophthora infestans that can disrupt cell wall-plasma membrane adhesions through its RGD motif. To identify peptide sequences that specifically bind the RGD motif of the IPI-O protein and potentially play a role in receptor recognition, we screened a heptamer peptide library displayed in a filamentous phage and selected two peptides acting as inhibitors of the plasma membrane RGD-binding activity of Arabidopsis. Moreover, the two peptides also disrupted cell wall-plasma membrane adhesions. Sequence comparison of the RGD-binding peptides with the Arabidopsis proteome revealed 12 proteins containing amino acid sequences in their extracellular domains common with the two RGD-binding peptides. Eight belong to the receptor-like kinase family, four of which have a lectin-like extracellular domain. The lectin domain of one of these, At5g60300, recognized the RGD motif both in peptides and proteins. These results imply that lectin receptor kinases are involved in protein-protein interactions with RGD-containing proteins as potential ligands, and play a structural and signaling role at the plant cell surfaces.

  10. Lectin Receptor Kinases Participate in Protein-Protein Interactions to Mediate Plasma Membrane-Cell Wall Adhesions in Arabidopsis1

    Science.gov (United States)

    Gouget, Anne; Senchou, Virginie; Govers, Francine; Sanson, Arnaud; Barre, Annick; Rougé, Pierre; Pont-Lezica, Rafael; Canut, Hervé

    2006-01-01

    Interactions between plant cell walls and plasma membranes are essential for cells to function properly, but the molecules that mediate the structural continuity between wall and membrane are unknown. Some of these interactions, which are visualized upon tissue plasmolysis in Arabidopsis (Arabidopsis thaliana), are disrupted by the RGD (arginine-glycine-aspartic acid) tripeptide sequence, a characteristic cell adhesion motif in mammals. In planta induced-O (IPI-O) is an RGD-containing protein from the plant pathogen Phytophthora infestans that can disrupt cell wall-plasma membrane adhesions through its RGD motif. To identify peptide sequences that specifically bind the RGD motif of the IPI-O protein and potentially play a role in receptor recognition, we screened a heptamer peptide library displayed in a filamentous phage and selected two peptides acting as inhibitors of the plasma membrane RGD-binding activity of Arabidopsis. Moreover, the two peptides also disrupted cell wall-plasma membrane adhesions. Sequence comparison of the RGD-binding peptides with the Arabidopsis proteome revealed 12 proteins containing amino acid sequences in their extracellular domains common with the two RGD-binding peptides. Eight belong to the receptor-like kinase family, four of which have a lectin-like extracellular domain. The lectin domain of one of these, At5g60300, recognized the RGD motif both in peptides and proteins. These results imply that lectin receptor kinases are involved in protein-protein interactions with RGD-containing proteins as potential ligands, and play a structural and signaling role at the plant cell surfaces. PMID:16361528

  11. Marketing Camp to Parents and Children.

    Science.gov (United States)

    Cony, Steven R.

    1995-01-01

    An effective camp marketing strategy should address both parents' and children's concerns that influence decisions about camp. Includes strategies for developing a targeted message through print media or video that addresses these concerns and persuades families to choose camp. Stresses the importance of following up with parents and children. (LP)

  12. Extension Sustainability Camp: Design, Implementation, and Evaluation

    Science.gov (United States)

    Brain, Roslynn; Upton, Sally; Tingey, Brett

    2015-01-01

    Sustainability Camps provide an opportunity for Extension educators to be in the forefront of sustainability outreach and to meet the growing demand for sustainability education. This article shares development, implementation, and evaluation of an Extension Sustainability Camp for youth, grades 4-6. Camp impact was measured via daily pre-and…

  13. Molecular cloning and characterisation of a novel GABAB-related G-protein coupled receptor.

    Science.gov (United States)

    Calver, A R; Michalovich, D; Testa, T T; Robbins, M J; Jaillard, C; Hill, J; Szekeres, P G; Charles, K J; Jourdain, S; Holbrook, J D; Boyfield, I; Patel, N; Medhurst, A D; Pangalos, M N

    2003-02-20

    Using a homology-based bioinformatics approach we have analysed human genomic sequence and identified the human and rodent orthologues of a novel putative seven transmembrane G protein coupled receptor, termed GABA(BL). The amino acid sequence homology of these cDNAs compared to GABA(B1) and GABA(B2) led us to postulate that GABA(BL) was a putative novel GABA(B) receptor subunit. The C-terminal sequence of GABA(BL) contained a putative coiled-coil domain, di-leucine and several RXR(R) ER retention motifs, all of which have been shown to be critical in GABA(B) receptor subunit function. In addition, the distribution of GABA(BL) in the central nervous system was reminiscent of that of the other known GABA(B) subunits. However, we were unable to detect receptor function in response to any GABA(B) ligands when GABA(BL) was expressed in isolation or in the presence of either GABA(B1) or GABA(B2). Therefore, if GABA(BL) is indeed a GABA(B) receptor subunit, its partner is a potentially novel receptor subunit or chaperone protein which has yet to be identified.

  14. Scotopic vision in the monkey is modulated by the G protein-coupled receptor 55

    DEFF Research Database (Denmark)

    Bouskila, Joseph; Harrar, Vanessa; Javadi, Pasha

    2016-01-01

    The endogenous cannabinoid system plays important roles in the retina of mice and monkeys via their classic CB1 and CB2 receptors. We have previously reported that the G protein-coupled receptor 55 (GPR55), a putative cannabinoid receptor, is exclusively expressed in rod photoreceptors in the mon......The endogenous cannabinoid system plays important roles in the retina of mice and monkeys via their classic CB1 and CB2 receptors. We have previously reported that the G protein-coupled receptor 55 (GPR55), a putative cannabinoid receptor, is exclusively expressed in rod photoreceptors...... in the monkey retina, suggesting its possible role in scotopic vision. To test this hypothesis, we recorded full-field electroretinograms (ERGs) after the intravitreal injection of the GPR55 agonist lysophosphatidylglucoside (LPG) or the selective GPR55 antagonist CID16020046 (CID), under light- and dark......-adapted conditions. Thirteen vervet monkeys (Chlorocebus sabaeus) were used in this study: four controls (injected with the vehicle dimethyl sulfoxide, DMSO), four injected with LPG and five with CID. We analyzed amplitudes and latencies of the a-wave (photoreceptor responses) and the b-wave (rod and cone system...

  15. Nicotinic acetylcholine receptor α7 subunits with a C2 cytoplasmic loop yellow fluorescent protein insertion form functional receptors

    Institute of Scientific and Technical Information of China (English)

    Teresa A MURRAY; Qiang LIU; Paul WHITEAKER; Jie WU; Ronald J LUKAS

    2009-01-01

    Aim: Several nicotinic acetylcholine receptor (nAChR) subunits have been engineered as fluorescent protein (FP) fusions and exploited to illuminate features of nAChRs. The aim of this work was to create a FP fusion in the nAChR a.7 subunit without compromising formation of functional receptors.Methods: A gene construct was generated to introduce yellow fluorescent protein (YFP), in frame, into the otherwise unaltered, large, second cytoplamsic loop between the third and fourth transmembrane domains of the mouse nAChR al sub-unit (a7Y). SH-EP1 cells were transfected with mouse nAChR wild type a.7 subunits (a.7) or with a7Y subunits, alone or with the chaperone protein, hRJC-3. Receptor function was assessed using whole-cell current recording. Receptor expression was measured with 125I-labeled a-bungarotoxin (I-Bgt) binding, laser scanning confocal microscopy, and total internal reflectance fluorescence (TIRF) microscopy.Results: Whole-cell currents revealed that a7Y nAChRs and al nAChRs were functional with comparable EC50 values for the a7 nAChR-selective agonist, choline, and IC50 values for the a.7 nAChR-selective antagonist, methyllycaconitine. I-Bgt binding was detected only after co-expression with hRIC-3. Confocal microscopy revealed that a7Y had primarily intracel-lular rather than surface expression. TIRF microscopy confirmed that little a7Y localized to the plasma membrane, typical of a7 nAChRs.Conclusion: nAChRs composed as homooligomers of a7Y subunits containing cytoplasmic loop YFP have functional, ligand binding, and trafficking characteristics similar to those of a.7 nAChRs. a7Y nAChRs may be used to elucidate properties of a.7 nAChRs and to identify and develop novel probes for these receptors, perhaps in high-throughput fashion.

  16. GRK2 protein-mediated transphosphorylation contributes to loss of function of μ-opioid receptors induced by neuropeptide FF (NPFF2) receptors.

    Science.gov (United States)

    Moulédous, Lionel; Froment, Carine; Dauvillier, Stéphanie; Burlet-Schiltz, Odile; Zajac, Jean-Marie; Mollereau, Catherine

    2012-04-13

    Neuropeptide FF (NPFF) interacts with specific receptors to modulate opioid functions in the central nervous system. On dissociated neurons and neuroblastoma cells (SH-SY5Y) transfected with NPFF receptors, NPFF acts as a functional antagonist of μ-opioid (MOP) receptors by attenuating the opioid-induced inhibition of calcium conductance. In the SH-SY5Y model, MOP and NPFF(2) receptors have been shown to heteromerize. To understand the molecular mechanism involved in the anti-opioid activity of NPFF, we have investigated the phosphorylation status of the MOP receptor using phospho-specific antibody and mass spectrometry. Similarly to direct opioid receptor stimulation, activation of the NPFF(2) receptor by [D-Tyr-1-(NMe)Phe-3]NPFF (1DMe), an analog of NPFF, induced the phosphorylation of Ser-377 of the human MOP receptor. This heterologous phosphorylation was unaffected by inhibition of second messenger-dependent kinases and, contrarily to homologous phosphorylation, was prevented by inactivation of G(i/o) proteins by pertussis toxin. Using siRNA knockdown we could demonstrate that 1DMe-induced Ser-377 cross-phosphorylation and MOP receptor loss of function were mediated by the G protein receptor kinase GRK2. In addition, mass spectrometric analysis revealed that the phosphorylation pattern of MOP receptors was qualitatively similar after treatment with the MOP agonist Tyr-D-Ala-Gly (NMe)-Phe-Gly-ol (DAMGO) or after treatment with the NPFF agonist 1DMe, but the level of multiple phosphorylation was more intense after DAMGO. Finally, NPFF(2) receptor activation was sufficient to recruit β-arrestin2 to the MOP receptor but not to induce its internalization. These data show that NPFF-induced heterologous desensitization of MOP receptor signaling is mediated by GRK2 and could involve transphosphorylation within the heteromeric receptor complex.

  17. Human eosinophil major basic protein is an endogenous allosteric antagonist at the inhibitory muscarinic M2 receptor.

    OpenAIRE

    Jacoby, D B; Gleich, G J; Fryer, A. D.

    1993-01-01

    The effect of human eosinophil major basic protein (MBP) as well as other eosinophil proteins, on binding of [3H]N-methyl-scopolamine ([3H]NMS: 1 x 10(-10) M) to muscarinic M2 receptors in heart membranes and M3 receptors in submandibular gland membranes was studied. MBP inhibited specific binding of [3H]NMS to M2 receptors but not to M3 receptors. MBP also inhibited atropine-induced dissociation of [3H]NMS-receptor complexes in a dose-dependent fashion, demonstrating that the interaction of ...

  18. The repertoire of G-protein-coupled receptors in Xenopus tropicalis

    Directory of Open Access Journals (Sweden)

    Hu Yinghe

    2009-06-01

    Full Text Available Abstract Background The G-protein-coupled receptor (GPCR superfamily represents the largest protein family in the human genome. These proteins have a variety of physiological functions that give them well recognized roles in clinical medicine. In Xenopus tropicalis, a widely used animal model for physiology research, the repertoire of GPCRs may help link the GPCR evolutionary history in vertebrates from teleost fish to mammals. Results We have identified 1452 GPCRs in the X. tropicalis genome. Phylogenetic analyses classified these receptors into the following seven families: Glutamate, Rhodopsin, Adhesion, Frizzled, Secretin, Taste 2 and Vomeronasal 1. Nearly 70% of X. tropicalis GPCRs are represented by the following three types of receptors thought to receive chemosensory information from the outside world: olfactory, vomeronasal 1 and vomeronasal 2 receptors. Conclusion X. tropicalis shares a more similar repertoire of GPCRs with mammals than it does with fish. An examination of the three major groups of receptors related to olfactory/pheromone detection shows that in X. tropicalis, these groups have undergone lineage specific expansion. A comparison of GPCRs in X. tropicalis, teleost fish and mammals reveals the GPCR evolutionary history in vertebrates.

  19. Summer Science Camps Program (SSC).

    Science.gov (United States)

    National Science Foundation, Washington, DC. Directorate for Education and Human Resources.

    The Summer Science Camps (SSC) Program supports residential and commuter enrichment projects for seventh through ninth grade minority students who are underrepresented in science, engineering, and mathematics. Eligible organizations include school districts, museums, colleges, universities, and nonprofit youth-centered and/or community-based…

  20. The Emotional Benefits of Camping.

    Science.gov (United States)

    Johnson, Rebecca Cowan

    1991-01-01

    Regardless of participant background, age, or ethnic origin, camp can aid in the following key components of emotional maturity: open, positive and appropriate expression of feelings; self-acceptance; a sense of self; an awareness and acceptance of others and their feelings; the ability to develop relationships; and emotional stability. (LP)

  1. The NAO goes to camp

    NARCIS (Netherlands)

    Wigdor, N.; Fraaije, A.; Solms, L.; Greeff, J. de; Janssen, J.; Blanson Henkemans, O.A.

    2014-01-01

    ALIZ-E is a Europe-wide project focusing on long-term child-robot interaction, specifically as a means of educating diabetic children on their condition. This video showcases a recent field study at "SugarKidsClub", a camp devoted to helping 7-12 year-olds handle type-1 diabetes. A wide range of CRI

  2. Muscarinic 2 Receptors Modulate Cardiac Proteasome Function in a Protein Kinase G-dependent Manner

    OpenAIRE

    Ranek, Mark J.; Kost, Curtis K.; Hu, Chengjun; Martin, Douglas S.; Wang, Xuejun

    2014-01-01

    Proteasome function insufficiency and inadequate protein quality control are strongly implicated in a large subset of cardiovascular disease and may play an important role in their pathogenesis. Protein degradation by the ubiquitin proteasome system can be physiologically regulated. Cardiac muscarinic 2 (M2) receptors were pharmacologically interrogated in intact mice and cultured neonatal rat ventricular myocytes (NRVMs). Proteasome-mediated proteolysis was measured with a surrogate misfolde...

  3. Characterization of two patched receptors for the vertebrate hedgehog protein family

    OpenAIRE

    1998-01-01

    The multitransmembrane protein Patched (PTCH) is the receptor for Sonic Hedgehog (Shh), a secreted molecule implicated in the formation of embryonic structures and in tumorigenesis. Current models suggest that binding of Shh to PTCH prevents the normal inhibition of the seven-transmembrane-protein Smoothened (SMO) by PTCH. According to this model, the inhibition of SMO signaling is relieved after mutational inactivation of PTCH in the basal cell nevus syndrome. Recently, PTCH2, a molecule wit...

  4. Affinity Purification and Characterization of a G-Protein Coupled Receptor, Saccharomyces cerevisiae Ste2p

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Byung-Kwon [University of Tennessee, Knoxville (UTK); Jung, Kyung-Sik [University of Tennessee, Knoxville (UTK); Son, Cagdas D [ORNL; Kim, Heejung [University of Tennessee, Knoxville (UTK); Verberkmoes, Nathan C [ORNL; Arshava, Boris [College of Staten Island; Naider, Fred [College of Staten Island; Becker, Jeffrey Marvin [ORNL

    2007-01-01

    We present a rare example of a biologically active G protein coupled receptor (GPCR) whose purity and identity were verified by mass spectrometry after being purified to near homogeneity from its native system. An overexpression vector was constructed to encode the Saccharomyces cerevisiae GPCR -factor receptor (Ste2p, the STE2 gene product) containing a 9-amino acid sequence of rhodopsin that served as an epitope/affinity tag. In the construct, two glycosylation sites and two cysteine residues were removed to aid future structural and functional studies. The receptor was expressed in yeast cells and was detected as a single band in a western blot indicating the absence of glycosylation. Tests of the epitope-tagged, mutated receptor showed it maintained its full biological activity. For extraction of Ste2p, yeast membranes were solubilized with 0.5 % n-dodecyl maltoside (DM). Approximately 120 g of purified -factor receptor was obtained per liter of culture by single-step affinity chromatography using a monoclonal antibody to the rhodopsin epitope. The binding affinity (Kd) of the purified -factor receptor in DM micelles was 28 nM as compared to Kd = 12.7 nM for Ste2p in cell membranes, and approximately 40 % of the purified receptor was correctly folded as judged by ligand saturation binding. About 50 % of the receptor sequence was retrieved from MALDITOF and nanospray mass spectrometry after CNBr digestion of the purified receptor. The methods described will enable structural studies of the -factor receptor and may provide an efficient technique to purify other GPCRs that have been functionally expressed in yeast.

  5. A new crystal structure fragment-based pharmacophore method for G protein-coupled receptors

    DEFF Research Database (Denmark)

    Fidom, Kimberley; Isberg, Vignir; Hauser, Alexander Sebastian;

    2015-01-01

    We have developed a new method for the building of pharmacophores for G protein-coupled receptors, a major drug target family. The method is a combination of the ligand- and target-based pharmacophore methods and founded on the extraction of structural fragments, interacting ligand moiety...... for new targets. A validating retrospective virtual screening of histamine H1 and H3 receptor pharmacophores yielded area-under-the-curves of 0.88 and 0.82, respectively. The fragment-based method has the unique advantage that it can be applied to targets for which no (homologous) crystal structures...... or ligands are known. 47% of the class A G protein-coupled receptors can be targeted with at least four-element pharmacophores. The fragment libraries can also be used to grow known ligands or for rotamer refinement of homology models. Researchers can download the complete fragment library or a subset...

  6. Structural basis for cAMP-mediated allosteric control of the catabolite activator protein.

    Science.gov (United States)

    Popovych, Nataliya; Tzeng, Shiou-Ru; Tonelli, Marco; Ebright, Richard H; Kalodimos, Charalampos G

    2009-04-28

    The cAMP-mediated allosteric transition in the catabolite activator protein (CAP; also known as the cAMP receptor protein, CRP) is a textbook example of modulation of DNA-binding activity by small-molecule binding. Here we report the structure of CAP in the absence of cAMP, which, together with structures of CAP in the presence of cAMP, defines atomic details of the cAMP-mediated allosteric transition. The structural changes, and their relationship to cAMP binding and DNA binding, are remarkably clear and simple. Binding of cAMP results in a coil-to-helix transition that extends the coiled-coil dimerization interface of CAP by 3 turns of helix and concomitantly causes rotation, by approximately 60 degrees , and translation, by approximately 7 A, of the DNA-binding domains (DBDs) of CAP, positioning the recognition helices in the DBDs in the correct orientation to interact with DNA. The allosteric transition is stabilized further by expulsion of an aromatic residue from the cAMP-binding pocket upon cAMP binding. The results define the structural mechanisms that underlie allosteric control of this prototypic transcriptional regulatory factor and provide an illustrative example of how effector-mediated structural changes can control the activity of regulatory proteins.