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Sample records for putative g-protein mapping

  1. Partial cloning of putative G-proteins modulating mechanotransduction in the ciliate stentor.

    PubMed

    Marino, M J; Sherman, T G; Wood, D C

    2001-01-01

    Signal transduction systems known to utilize G-proteins in higher eukaryotes undoubtedly evolved prior to the development of metazoa. Pharmacological evidence indicates that the ciliates Paramecium, Stentor, and Tetrahymena all utilize signaling systems similar to those found in mammals. However, there has been relatively little direct evidence for the existence of G-proteins in ciliates. Since highly conserved heterotrimeric G-proteins form the basis of receptor-coupled signal transduction systems in a wide variety of metazoa, it is of interest to know if these important signaling molecules were early to evolve and are present and functionally important in a wide variety of unicellular organisms. We have previously shown that mechanotransduction in Stentor is modulated by opiates in a manner that may involve pertussis toxin-sensitive G-proteins. Here we utilize drugs known to interact with G-proteins to further test for the involvement of these important signaling molecules in Stentor mechanotransduction. We present behavioral and electrophysiological data demonstrating that putative G-proteins in Stentor decrease mechanical sensitivity by modulating the mechanotransduction process. In addition, we report the partial cloning of 4 G-protein alpha-subunits from Stentor. We confirm that these clones are of Stentor origin and are transcribed. Furthermore, we employ antisense oligodeoxynucleotide-mediated knockout to demonstrate that these ciliate G-proteins exert a modulatory influence on Stentor behavior, and that a G1/G0-like clone mediates the inhibitory action of beta-endorphin on mechanotransduction.

  2. Acetylcholine regulation of nicotinic receptor channels through a putative G protein in chick myotubes.

    PubMed Central

    Eusebi, F; Grassi, F; Molinaro, M; Zani, B M

    1987-01-01

    1. Single-channel currents induced by acetylcholine (ACh) were recorded from unstriated and non-innervated embryonic chick myotubes using the cell-attached patch-clamp technique. 2. ACh applied to the non-patched membrane decreased both channel opening probability and conductance. These ACh-induced effects occurred also when the non-patched membrane was exposed to nominally Ca2+-free extracellular medium, but were absent when it was treated with curare. 3. ACh-induced membrane current recorded under whole-cell patch-clamp conditions decreased in amplitude and time course when myotubes were intracellularly loaded with guanosine-5'-O-(3-thiotriphosphate) GTP gamma S), but not with guanosine-5'-O-(2-thiodiphosphate) (GDP beta S) or cyclic adenosine-5'-monophosphate (cyclic AMP). Internal perfusion of GTP gamma S affected the ACh-induced openings in a similar manner to the non-patch ACh application. 4. These results suggest that ACh, in addition to its direct effect, acts indirectly on the nicotinic receptor channels by delivering an intracellular messenger and through the activation of a putative G protein. PMID:2451747

  3. A putative G protein-coupled receptor involved in innate immune defense of Procambarus clarkii against bacterial infection.

    PubMed

    Dong, Chaohua; Zhang, Peng

    2012-02-01

    The immune functions of G protein-coupled receptor (GPCR) were widely investigated in mammals. However, limited researches on immune function of GPCRs were reported in invertebrates. In the present study, the immune functions of HP1R gene, a putative GPCR identified from red swamp crayfish Procambarus clarkii were reported. Expression of HP1R gene was significant up-regulated in response to heat-killed Aeromonas hydrophila challenge. HP1R gene silencing mediated by RNA interference significantly enhanced the susceptibility of red swamp crayfish to A. hydrophila and Vibrio alginolyticus, indicating that HP1R was required for red swamp crayfish to defend against bacterial challenge. In HP1R-silenced crayfish, increased bacterial burden and decreased THC in response to bacterial challenge were observed when compared with control crayfish. No significant difference of proPO gene expression was observed between HP1R-silenced and control crayfish after challenge with heat-killed A. hydrophila. However, PO activity in response to bacterial challenge was significantly reduced in HP1R-silenced crayfish. The results collectively indicated that HP1R was an important immune molecule which was required for red swamp crayfish to defend against bacterial infection.

  4. Characterization of gprK Encoding a Putative Hybrid G-Protein-Coupled Receptor in Aspergillus fumigatus

    PubMed Central

    Jung, Mun-Gu; Kim, Sung Su; Yu, Jae-Hyuk; Shin, Kwang-Soo

    2016-01-01

    The G-protein-coupled receptor (GPCR) family represents the largest and most varied collection of membrane embedded proteins that are sensitized by ligand binding and interact with heterotrimeric G proteins. Despite their presumed critical roles in fungal biology, the functions of the GPCR family members in the opportunistic human pathogen Aspergillus fumigatus are largely unknown, as only two (GprC and GprD) of the 15 predicted GPCRs have been studied. Here, we characterize the gprK gene, which is predicted to encode a hybrid GPCR with both 7-transmembrane and regulator of G-protein signaling (RGS) domains. The deletion of gprK causes severely impaired asexual development coupled with reduced expression of key developmental activators. Moreover, ΔgprK results in hyper-activation of germination even in the absence of carbon source, and elevated expression and activity of the protein kinase A PkaC1. Furthermore, proliferation of the ΔgprK mutant is restricted on the medium when pentose is the sole carbon source, suggesting that GprK may function in external carbon source sensing. Notably, the absence of gprK results in reduced tolerance to oxidative stress and significantly lowered mRNA levels of the stress-response associated genes sakA and atfA. Activities of catalases and SODs are severely decreased in the ΔgprK mutant, indicating that GprK may function in proper activation of general stress response. The ΔgprK mutant is also defective in gliotoxin (GT) production and slightly less virulent toward the greater wax moth, Galleria mellonella. Transcriptomic studies reveal that a majority of transporters are down-regulated by ΔgprK. In summary, GprK is necessary for proper development, GT production, and oxidative stress response, and functions in down-regulating the PKA-germination pathway. PMID:27584150

  5. D-GPCR: a novel putative G protein-coupled receptor overexpressed in prostate cancer and prostate.

    PubMed

    Weigle, Bernd; Fuessel, Susanne; Ebner, Reinhard; Temme, Achim; Schmitz, Marc; Schwind, Sandra; Kiessling, Andrea; Rieger, Michael A; Meye, Axel; Bachmann, Michael; Wirth, Manfred P; Rieber, E Peter

    2004-09-10

    The use of molecular targets in novel strategies of tumor treatment largely depends on the identification of proteins with a tumor- or tissue-restricted expression. We identified the novel protein D-GPCR that is selectively overexpressed in human prostate cancer and prostate and belongs to the subfamily of odorant-like orphan G protein-coupled receptors. Quantification of D-GPCR transcripts in different human tissues by real-time PCR demonstrated 27-fold overexpression in prostate compared to skeletal muscle, the organ with second highest transcript numbers in males. Investigation of tumor/normal cDNA pairs obtained from 241 cancer patients including four prostate tumors confirmed the preferential expression in prostate. When comparing the mean transcript level of 15 prostate cancer tissues to their non-tumorous counterparts, D-GPCR was almost 6-fold upregulated. Coupled in vitro transcription and translation of D-GPCR cDNA produced a protein band of approximately 28 kDa. Recombinant, His-tagged protein was expressed in transfected HEK293 cells and gave rise to a 30 kDa band specifically detected by anti-His antibody. These data provide the basis for future studies evaluating the diagnostic potential of D-GPCR and its utility as a novel target in immunotherapy of prostate cancer.

  6. Glucocorticoid acts on a putative G protein-coupled receptor to rapidly regulate the activity of NMDA receptors in hippocampal neurons.

    PubMed

    Zhang, Yanmin; Sheng, Hui; Qi, Jinshun; Ma, Bei; Sun, Jihu; Li, Shaofeng; Ni, Xin

    2012-04-01

    Glucocorticoids (GCs) have been demonstrated to act through both genomic and nongenomic mechanisms. The present study demonstrated that corticosterone rapidly suppressed the activity of N-methyl-D-aspartate (NMDA) receptors in cultured hippocampal neurons. The effect was maintained with corticosterone conjugated to bovine serum albumin and blocked by inhibition of G protein activity with intracellular GDP-β-S application. Corticosterone increased GTP-bound G(s) protein and cyclic AMP (cAMP) production, activated phospholipase Cβ(3) (PLC-β(3)), and induced inositol-1,4,5-triphosphate (IP(3)) production. Blocking PLC and the downstream cascades with PLC inhibitor, IP(3) receptor antagonist, Ca(2+) chelator, and protein kinase C (PKC) inhibitors prevented the actions of corticosterone. Blocking adenylate cyclase (AC) and protein kinase A (PKA) caused a decrease in NMDA-evoked currents. Application of corticosterone partly reversed the inhibition of NMDA currents caused by blockage of AC and PKA. Intracerebroventricular administration of corticosterone significantly suppressed long-term potentiation (LTP) in the CA1 region of the hippocampus within 30 min in vivo, implicating the possibly physiological significance of rapid effects of GC on NMDA receptors. Taken together, our results indicate that GCs act on a putative G protein-coupled receptor to activate multiple signaling pathways in hippocampal neurons, and the rapid suppression of NMDA activity by GCs is dependent on PLC and downstream signaling.

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

    PubMed

    Agbedanu, P N; Brewer, M T; Day, T A; Kimber, M J; Anderson, K L; Rasmussen, S K; Rasmussen, M A; Carlson, S A

    2013-01-01

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

  8. A molecular map of G protein alpha chains in microdissected rat nephron segments.

    PubMed Central

    Senkfor, S I; Johnson, G L; Berl, T

    1993-01-01

    Membrane-associated guanine nucleotide binding proteins regulate many receptor-mediated signals. Heterogeneity of biochemical and functional properties in nephron segments could be due to differences in G protein expression. To ascertain whether such heterogeneity of G proteins is present in various nephron segments, this study examines the distribution and relative abundance of G protein alpha chains in microdissected medullary thick ascending limb, cortical collecting tubules, outer medullary collecting tubules, proximal inner medullary tubules, and distal inner medullary tubules. Reverse transcription and polymerase chain reactions were employed using oligonucleotides encoding highly conserved regions of all known alpha chains. The cDNA was sequenced for alpha chain identification. The alpha i2 versus alpha s distribution was different in the outer medullary collecting tubules, when compared with the medullary thick ascending limb (P < 0.001) or the cortical collecting tubule, the proximal inner medullary tubules, and the distal inner medullary tubules (P < 0.05). These latter four segments did not significantly differ from each other. A similar analysis was applied to the frequently used line of kidney cells, LLC-PK1, whose exact cellular origin remains unclear. Interestingly, we detected both alpha i2 and alpha i3, while only alpha i2 was detected in the rat distal nephron. No alpha o or alpha z reverse transcription PCR products were detected. In contrast alpha 11 and alpha 14 members of the more recently described alpha q family were detected in the outer medullary collecting tubules and the proximal inner medullary tubules, respectively. We conclude that the majority of nephron segments have a relatively constant distribution of G protein alpha chains. Images PMID:8349818

  9. Activating mutation of the stimulatory G protein (gsp) as a putative cause of ovarian and testicular human stromal Leydig cell tumors.

    PubMed

    Fragoso, M C; Latronico, A C; Carvalho, F M; Zerbini, M C; Marcondes, J A; Araujo, L M; Lando, V S; Frazzatto, E T; Mendonca, B B; Villares, S M

    1998-06-01

    Activating mutations of the G protein genes have been associated with the development of several endocrine neoplasms. Such activating mutations, gip2, affecting the alpha-subunit of the G alpha i2 protein were previously described by a single group in 30% of ovarian sex cord stromal tumors. Other activating mutations of the alpha-subunit of the Gs (gsp) have been identified in GH-secreting and nonfunctioning pituitary tumors, autonomous thyroid adenomas, and all affected McCune-Albright tissues, but not in sex cord stromal tumors. In the present study, we investigated the presence of gip2 and gsp mutations in 14 human sex cord stromal tumors. Six Leydig cell tumors (4 ovaries and 2 testes), 2 thecomas, 2 granulosa cell tumors, 3 androblastomas, and 1 gonadoblastoma (sex cord and germ cell) were included in this study. Genomic DNA was obtained from either fresh-frozen tumor tissues or paraffin-embedded sections and in some cases from blood samples. Using PCR, denaturing gradient gel electrophoresis, and direct sequencing, we detected 4 tumors (66.6%) with the gsp mutation (R201C) in our series of ovarian and testicular Leydig cell tumors. In contrast, no gip2 mutations were found in any of the sex cord stromal tumors studied. In conclusion, our findings suggest that the putative oncogene gsp may play a significant role in the molecular mechanism of these tumors.

  10. The Gα4 G protein subunit interacts with the MAP kinase ERK2 using a D-motif that regulates developmental morphogenesis in Dictyostelium

    PubMed Central

    Nguyen, Hoai-Nghia; Hadwiger, Jeffrey A.

    2009-01-01

    G protein Gα subunits contribute to the specificity of different signal transduction pathways in Dictyostelium discoideum but Gα subunit-effector interactions have not been previously identified. The requirement of the Dictyostelium Gα4 subunit for MAP kinase (MAPK) activation and the identification of a putative MAPK docking site (D-motif) in this subunit suggested a possible interaction between the Gα4 subunit and MAPKs. In vivo association of the Gα4 subunit and ERK2 was demonstrated by pull-down and co-immunoprecipitation assays. Alteration of the D-motif reduced Gα4 subunit-ERK2 interactions but only slightly altered MAPK activation in response to folate. Expression of the Gα4 subunit with the altered D-motif in gα4− cells allowed for slug formation but not the morphogenesis associated with culmination. Expression of this mutant Gα4 subunit was sufficient to rescue chemotactic movement to folate. Alteration of the D-motif also reduced the aggregation defect associated with constitutively active Gα4 subunits. These results suggest Gα4 subunit-MAPK interactions are necessary for developmental morphogenesis but not for chemotaxis to folate. PMID:19765570

  11. Palmitoylation of muscarinic acetylcholine receptor m2 subtypes: reduction in their ability to activate G proteins by mutation of a putative palmitoylation site, cysteine 457, in the carboxyl-terminal tail.

    PubMed

    Hayashi, M K; Haga, T

    1997-04-15

    A putative palmitoylation site, Cys457, of muscarinic acetylcholine receptor m2 subtype (m2 receptor) was eliminated by conversion to alanine or stop codon by site-directed mutagenesis. The mutant m2 receptor C457A was not metabolically labeled with [3H] palmitic acid when expressed in Sf9 cells, whereas the wild-type m2 receptor was labeled under the same conditions. These results confirm that the Cys457 is the palmitoylation site. The rate of palmitoylation was markedly accelerated by addition of agonist, indicating that the palmitoylation reaction is affected by conformational changes of the receptor induced by agonist binding. The m2 receptor mutants without palmitoylation were purified and reconstituted with G proteins into phospholipid vesicles. Both mutants were good substrates of G protein-coupled receptor kinase 2 and the phosphorylation was stimulated by agonist and G protein beta gamma subunits, as was the case for wild-type receptors. The mutant receptors interacted with and activate Gi2 and G(o). However, the rate of [35S] GTP gamma S binding to Gi2 was half as much for the mutants as that for the wild type, and the proportion of guanine nucleotide-sensitive high-affinity agonist binding sites was significantly less for mutants (42-42%) compared to wild type (62%). These results indicate that the palmitoylation of m2 receptors is not an absolute requirement for their interaction with G proteins but enhances the ability of the receptors to interact with G proteins.

  12. Mapping physiological G protein-coupled receptor signaling pathways reveals a role for receptor phosphorylation in airway contraction

    PubMed Central

    Bradley, Sophie J.; 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.

    2016-01-01

    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. PMID:27071102

  13. Mapping physiological G protein-coupled receptor signaling pathways reveals a role for receptor phosphorylation in airway contraction.

    PubMed

    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.

  14. Human homologs of the putative G protein-coupled membrane progestin receptors (mPRalpha, beta, and gamma) localize to the endoplasmic reticulum and are not activated by progesterone.

    PubMed

    Krietsch, Tom; Fernandes, Maria Sofia; Kero, Jukka; Lösel, Ralf; Heyens, Maria; Lam, Eric W-F; Huhtaniemi, Ilpo; Brosens, Jan J; Gellersen, Birgit

    2006-12-01

    The steroid hormone progesterone exerts pleiotrophic functions in many cell types. Although progesterone controls transcriptional activation through binding to its nuclear receptors, it also initiates rapid nongenomic signaling events. Recently, three putative membrane progestin receptors (mPRalpha, beta, and gamma) with structural similarity to G protein-coupled receptors have been identified. These mPR isoforms are expressed in a tissue-specific manner and belong to the larger, highly conserved family of progestin and adiponectin receptors found in plants, eubacteria, and eukaryotes. The fish mPRalpha has been reported to mediate progesterone-dependent MAPK activation and inhibition of cAMP production through coupling to an inhibitory G protein. To functionally characterize the human homologs, we established human embryonic kidney 293 and MDA-MB-231 cell lines that stably express human mPRalpha, beta, or gamma. For comparison, we also established cell lines expressing the mPRalpha cloned from the spotted seatrout (Cynoscion nebulosus) and Japanese pufferfish (Takifugu rubripes). Surprisingly, we found no evidence that human or fish mPRs regulate cAMP production or MAPK (ERK1/2 or p38) activation upon progesterone stimulation. Furthermore, the mPRs did not couple to a highly promiscuous G protein subunit, Galpha(q5i), in transfection studies or provoke Ca(2+) mobilization in response to progesterone. Finally, we demonstrate that transfected mPRs, as well as endogenous human mPRalpha, localize to the endoplasmic reticulum, and that their expression does not lead to increased progestin binding either in membrane preparations or in intact cells. Our results therefore do not support the concept that mPRs are plasma membrane receptors involved in transducing nongenomic progesterone actions.

  15. Structural Mapping and Functional Characterization of Zebrafish Class B G-Protein Coupled Receptor (GPCR) with Dual Ligand Selectivity towards GLP-1 and Glucagon

    PubMed Central

    Oren, Deena A.; Wei, Yang; Skrabanek, Luce; Chow, Billy K. C.; Mommsen, Thomas; Mojsov, Svetlana

    2016-01-01

    GLP-1 and glucagon regulate glucose metabolism through a network of metabolic pathways initiated upon binding to their specific receptors that belong to class B G-protein coupled receptors (GPCRs). The therapeutic potential of glucagon is currently being evaluated, while GLP-1 is already used in the treatment of type 2 diabetes and obesity. Development of a second generation of GLP-1 based therapeutics depends on a molecular and structural understanding of the interactions between the GLP-1 receptor (GLP-1R) and its ligand GLP-1. There is considerable sequence conservation between GLP-1 and glucagon and between the hGLP-1R and human glucagon receptor (hGCGR), yet each receptor recognizes only its own specific ligand. Glucagon receptors in fish and frogs also exhibit ligand selectivity only towards glucagon and not GLP-1. Based on competitive binding experiments and assays of increase in intracellular cAMP, we demonstrate here that a GPCR in zebrafish (Danio rerio) exhibits dual ligand selectivity towards GLP-1 and glucagon, a characteristic not found in mammals. Further, many structural features found in hGLP-1R and hGCGR are also found in this zebrafish GPCR (zfGPCR). We show this by mapping of its sequence and structural features onto the hGLP-1R and hGCGR based on their partial and complementary crystal structures. Thus, we propose that zfGPCR represents a dual GLP-1R/GCGR. The main differences between the three receptors are in their stalk regions that connect their N-terminal extracellular domains (NECDs) with their transmembrane domains and the absence of loop 3 in the NECD in zfGLP-1R/GCGR. These observations suggest that the interactions between GLP-1 and glucagon with loop 3 and the stalk regions may induce different conformational changes in hGLP-1R and hGCGR upon ligand binding and activation that lead to selective recognition of their native ligands. PMID:27930690

  16. Maps of context-dependent putative regulatory regions and genomic signal interactions.

    PubMed

    Diamanti, Klev; Umer, Husen M; Kruczyk, Marcin; Dąbrowski, Michał J; Cavalli, Marco; Wadelius, Claes; Komorowski, Jan

    2016-11-02

    Gene transcription is regulated mainly by transcription factors (TFs). ENCODE and Roadmap Epigenomics provide global binding profiles of TFs, which can be used to identify regulatory regions. To this end we implemented a method to systematically construct cell-type and species-specific maps of regulatory regions and TF-TF interactions. We illustrated the approach by developing maps for five human cell-lines and two other species. We detected ∼144k putative regulatory regions among the human cell-lines, with the majority of them being ∼300 bp. We found ∼20k putative regulatory elements in the ENCODE heterochromatic domains suggesting a large regulatory potential in the regions presumed transcriptionally silent. Among the most significant TF interactions identified in the heterochromatic regions were CTCF and the cohesin complex, which is in agreement with previous reports. Finally, we investigated the enrichment of the obtained putative regulatory regions in the 3D chromatin domains. More than 90% of the regions were discovered in the 3D contacting domains. We found a significant enrichment of GWAS SNPs in the putative regulatory regions. These significant enrichments provide evidence that the regulatory regions play a crucial role in the genomic structural stability. Additionally, we generated maps of putative regulatory regions for prostate and colorectal cancer human cell-lines.

  17. Role of Protein Kinase C, PI3-kinase and Tyrosine Kinase in Activation of MAP Kinase by Glucose and Agonists of G-protein Coupled Receptors in INS-1 Cells

    PubMed Central

    Böcker, Dietmar

    2001-01-01

    MAP (mitogen-activated protein) kinase (also called Erk 1/2) plays a crucial role in cell proliferation and differentiation. Its impact on secretory events is less well established. The interplay of protein kinase C (PKC), PI3-kinase nd cellular tyrosine kinase with MAP kinase activity using inhibitors and compounds such as glucose, phorbol 12-myristate 13-acetate (PMA) and agonists of G-protein coupled receptors like gastrin releasing peptide (GRP), oxytocin (OT) and glucose-dependent insulinotropic peptide (GIP) was investigated in INS-1 cells, an insulin secreting cell line. MAP kinase activity was determined by using a peptide derived from the EGF receptor as a MAP kinase substrate and [ P 32 ]ATP. Glucose as well as GRP, OT and GIP exhibited a time-dependent increase in MAP kinase activity with a maximum at time point 2.5 min. All further experiments were performed using 2.5 min incubations. The flavone PD 098059 is known to bind to the inactive forms of MEK1 (MAPK/ERK-Kinase) thus preventing activation by upstream activators. 20 μM PD 098059 ( IC 50 =51 μM) inhibited MAP kinase stimulated by either glucose, GRP, OT, GIP or PMA. Inhibiton (“downregulation”) of PKC by a long term (22h) pretreatment with 1 μM PMA did not influence MAP kinase activity when augmented by either of the above mentioned compound. To investigate whether PI3-kinase and cellular tyrosine kinase are involved in G-protein mediated effects on MAP kinase, inhibitors were used: 100 nM wortmannin (PI3-kinase inhibitor) reduced the effects of GRP, OT and GIP but not that of PMA; 100 μM genistein (tyrosine kinase inhibitor) inhibited the stimulatory effect of either above mentioned compound on MAP kinase activation. Inhibition of MAP kinase by 20 μM PD 098059 did not influence insulin secretion modulated by either compound (glucose, GRP, OT or GIP). [ H 3 ]Thymidine incorporation, however, was severely inhibited by PD 098059. Thus MAP kinase is important for INS-1 cell proliferation but

  18. Mutational mapping of the transmembrane binding site of the G-protein coupled receptor TGR5 and binding mode prediction of TGR5 agonists.

    PubMed

    Gertzen, Christoph G W; Spomer, Lina; Smits, Sander H J; Häussinger, Dieter; Keitel, Verena; Gohlke, Holger

    2015-11-02

    TGR5 (Gpbar-1, M-Bar) is a class A G-protein coupled bile acid-sensing receptor predominately expressed in brain, liver and gastrointestinal tract, and a promising drug target for the treatment of metabolic disorders. Due to the lack of a crystal structure of TGR5, the development of TGR5 agonists has been guided by ligand-based approaches so far. Three binding mode models of bile acid derivatives have been presented recently. However, they differ from one another in terms of overall orientation or with respect to the location and interactions of the cholane scaffold, or cannot explain all results from mutagenesis experiments. Here, we present an extended binding mode model based on an iterative and integrated computational and biological approach. An alignment of 68 TGR5 agonists based on this binding mode leads to a significant and good structure-based 3D QSAR model, which constitutes the most comprehensive structure-based 3D-QSAR study of TGR5 agonists undertaken so far and suggests that the binding mode model is a close representation of the "true" binding mode. The binding mode model is further substantiated in that effects predicted for eight mutations in the binding site agree with experimental analyses on the impact of these TGR5 variants on receptor activity. In the binding mode, the hydrophobic cholane scaffold of taurolithocholate orients towards the interior of the orthosteric binding site such that rings A and B are in contact with TM5 and TM6, the taurine side chain orients towards the extracellular opening of the binding site and forms a salt bridge with R79(EL1), and the 3-hydroxyl group forms hydrogen bonds with E169(5.44) and Y240(6.51). The binding mode thus differs in important aspects from the ones recently presented. These results are highly relevant for the development of novel, more potent agonists of TGR5 and should be a valuable starting point for the development of TGR5 antagonists, which could show antiproliferative effects in tumor

  19. Molecular mapping of the putative gonadoblastoma locus on the Y chromosome.

    PubMed

    Salo, P; Kääriäinen, H; Petrovic, V; Peltomäki, P; Page, D C; de la Chapelle, A

    1995-11-01

    Based on the high incidence of gonadoblastoma in females with XY gonadal dysgenesis or 45,X/46,XY mosaicism, the existence of a susceptibility locus on the Y chromosome (GBY) has been postulated. We attempted to map GBY by making use of a recently developed dense map of Y-chromosomal sequence-tagged sites (STSs). In two female patients with gonadoblastoma, small marker chromosomes contained portions of the Y chromosome, and a single region of overlap could be defined extending from probe pDP97 in interval 4B, which contains the centromere, to marker sY182 in interval 5E of the proximal long arm. This interval is contained in a YAC contig that comprises approximately 4 Mb of DNA. Our findings confirm the previous localization of GBY and greatly refine it. The localization of GBY overlaps with the region to which a putative growth determinant, GCY, was recently assigned.

  20. G Protein-Coupled Receptors in Anopheles gambiae

    NASA Astrophysics Data System (ADS)

    Hill, Catherine A.; Fox, A. Nicole; Pitts, R. Jason; Kent, Lauren B.; Tan, Perciliz L.; Chrystal, Mathew A.; Cravchik, Anibal; Collins, Frank H.; Robertson, Hugh M.; Zwiebel, Laurence J.

    2002-10-01

    We used bioinformatic approaches to identify a total of 276 G protein-coupled receptors (GPCRs) from the Anopheles gambiae genome. These include GPCRs that are likely to play roles in pathways affecting almost every aspect of the mosquito's life cycle. Seventy-nine candidate odorant receptors were characterized for tissue expression and, along with 76 putative gustatory receptors, for their molecular evolution relative to Drosophila melanogaster. Examples of lineage-specific gene expansions were observed as well as a single instance of unusually high sequence conservation.

  1. A microsatellite-based genetic linkage map and putative sex-determining genomic regions in Lake Victoria cichlids.

    PubMed

    Kudo, Yu; Nikaido, Masato; Kondo, Azusa; Suzuki, Hikoyu; Yoshida, Kohta; Kikuchi, Kiyoshi; Okada, Norihiro

    2015-04-15

    Cichlid fishes in East Africa have undergone extensive adaptive radiation, which has led to spectacular diversity in their morphology and ecology. To date, genetic linkage maps have been constructed for several tilapias (riverine), Astatotilapia burtoni (Lake Tanganyika), and hybrid lines of Lake Malawi cichlids to facilitate genome-wide comparative analyses. In the present study, we constructed a genetic linkage map of the hybrid line of Lake Victoria cichlids, so that maps of cichlids from all the major areas of East Africa will be available. The genetic linkage map shown here is derived from the F2 progeny of an interspecific cross between Haplochromis chilotes and Haplochromis sauvagei and is based on 184 microsatellite and two single-nucleotide polymorphism (SNP) markers. Most of the microsatellite markers used in the present study were originally designed for other genetic linkage maps, allowing us to directly compare each linkage group (LG) among different cichlid groups. We found 25 LGs, the total length of which was 1133.2cM with an average marker spacing of about 6.09cM. Our subsequent linkage mapping analysis identified two putative sex-determining loci in cichlids. Interestingly, one of these two loci is located on cichlid LG5, on which the female heterogametic ZW locus and several quantitative trait loci (QTLs) related to adaptive evolution have been reported in Lake Malawi cichlids. We also found that V1R1 and V1R2, candidate genes for the fish pheromone receptor, are located very close to the recently detected sex-determining locus on cichlid LG5. The genetic linkage map study presented here may provide a valuable foundation for studying the chromosomal evolution of East African cichlids and the possible role of sex chromosomes in generating their genomic diversity.

  2. Mapping the flow of information within the putative mirror neuron system during gesture observation.

    PubMed

    Schippers, Marleen B; Keysers, Christian

    2011-07-01

    The putative mirror neuron system may either function as a strict feed-forward system or as a dynamic control system. A strict feed-forward system would predict that action observation leads to a predominantly temporal→parietal→premotor flow of information in which a visual representation is transformed into motor-programs which contribute to action understanding. Instead, a dynamic feedback control system would predict that the reverse direction of information flow predominates because of a combination of inhibitory forward and excitatory inverse models. Here we test which of these conflicting predictions best matches the information flow within the putative mirror neuron system (pMNS) and between the pMNS and the rest of the brain during the observation of comparatively long naturalistic stretches of communicative gestures. We used Granger causality to test the dominant direction of influence. Our results fit the predictions of the dynamic feedback control system: we found predominantly an information flow within the pMNS from premotor to parietal and middle temporal cortices. This is more pronounced during an active guessing task than while passively reviewing the same gestures. In particular, the ventral premotor cortex sends significantly more information to other pMNS areas than it receives during active guessing than during passive observation.

  3. G proteins in carotid body chemoreception.

    PubMed

    Prabhakar, N R; Kou, Y R; Kumar, G K

    1995-01-01

    G proteins are signal coupling molecules that play major roles in mediating the effects of transmitters as well as certain sensory signals. In the present study we examined whether oxygen chemoreception in the carotid body is coupled to G proteins. Experiments were performed on carotid bodies isolated from anesthetized cats. Presence of G proteins was examined with ADP-ribosylation of the carotid body membranes. Pertussis toxin (PTX), which inactivates G proteins in neuronal tissues, ADP-ribosylated a single band of carotid body protein with a molecular mass of 41 kDa. With cholera toxin (CTX) only a faint band of protein corresponding to approximately 45 kDa was evident. Perfusing the isolated carotid bodies with PTX (2.5 micrograms/min; 60 min) attenuated the sensory response to hypoxia by 52% of the controls. Perfusion with CTX (50 micrograms/min; for 60 min), on the other hand, increased baseline activity and potentiated the hypoxic response by 125% of controls. Heat-inactivated toxins, however, had no influence on the carotid body sensory response to hypoxia. These results suggest that G proteins are present in the chemoreceptor tissue and they seem to be coupled to the transduction and/or to the transmission of the hypoxic stimulus.

  4. Orexin-stimulated MAP kinase cascades are activated through multiple G-protein signalling pathways in human H295R adrenocortical cells: diverse roles for orexins A and B.

    PubMed

    Ramanjaneya, Manjunath; Conner, Alex C; Chen, Jing; Kumar, Prashanth; Brown, James E P; Jöhren, Olaf; Lehnert, Hendrik; Stanfield, Peter R; Randeva, Harpal S

    2009-08-01

    Orexins A and B (ORA and ORB) are neuropeptide hormones found throughout the central nervous system and periphery. They are required for a host of physiological processes including mitogen-activated protein kinase (MAPK) regulation, steroidogenesis, appetite control and energy regulation. While some signalling mechanisms have been proposed for individual recombinant orexin receptors in generic mammalian cell types, it is clear that the peripheral effects of orexin are spatially and temporally complex. This study dissects the different G-protein signalling and MAPK pathways activated in a pluripotent human adrenal H295R cell line capable of all the physiological steps involved in steroidogenesis. Both extracellular receptor kinase 1/2 (ERK1/2) and p38 were phosphorylated rapidly with a subsequent decline, in a time- and dose-dependent manner, in response to both ORA and ORB. Conversely, there was little or no direct activation of the ERK5 or JNK pathway. Analysis using signalling and MAPK inhibitors as well as receptor-specific antagonists determined the precise mediators of the orexin response in these cells. Both ERK1/2 and p38 activation were predominantly G(q)- and to a lesser extent G(s)-mediated; p38 activation even had a small G(i)-component. Effects were broadly comparable for both orexin sub-types ORA and ORB and although most of the effects were transmitted through the orexin receptor-1 subtype, we did observe a role for orexin receptor-2-mediated activation of both ERK1/2 and p38. Cortisol secretion also differed in response to ORA and ORB. These data suggest multiple roles for orexin-mediated MAPK activation in an adrenal cell-line, this complexity may help to explain the diverse biological actions of orexins with wide-ranging consequences for our understanding of the mechanisms initiated by these steroidogenic molecules.

  5. The emerging mutational landscape of G proteins and G-protein-coupled receptors in cancer.

    PubMed

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

    2013-06-01

    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 tumours carry activating mutations in GNAS (encoding Gαs), and that oncogenic activating mutations 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 tumours harbour mutations in GPCRs. Many human cancer-associated viruses also express constitutively active viral GPCRs. These studies indicate that G proteins, GPCRs and their linked signalling circuitry represent novel therapeutic targets for cancer prevention and treatment.

  6. G protein antagonists. A novel hydrophobic peptide competes with receptor for G protein binding.

    PubMed

    Mukai, H; Munekata, E; Higashijima, T

    1992-08-15

    A substance P (SP) analog, [D-Pro4,D-Trp7,9,10] SP4-11, is known to inhibit the actions of various structurally unrelated messenger molecules as well as SP. Our studies on the effects of this peptide on the regulation of purified G proteins by receptor showed that at least some of the biological effects of the peptide can be explained by the ability of the peptide to block the activation of G proteins by receptors. Here we report that a novel truncated SP-related peptide, pGlu-Gln-D-Trp-Phe-D-Trp-D-Trp-Met-NH2, inhibited the activation of G(i) or G(o) by M2 muscarinic cholinergic receptor (M2 mAChR) or of Gs by beta-adrenergic receptor in the reconstituted phospholipid vesicles, assayed by receptor-promoted GTP hydrolysis. The inhibition by the peptide was apparently reversible and competitive with respect to receptor binding to G proteins; the inhibition could be overcome by increasing the concentration of receptor in the vesicles and was not altered by changes in the concentration of G protein. The competing effects of the peptide were used to analyze the effect of agonist on receptor-G protein interaction. The concentration change of muscarinic agonist did not alter the inhibitory effects of the peptide on M2 mAChR-promoted GTPase by G(o), which is consistent with the idea that agonist increases the regulatory efficiency of the receptor but does not alter its affinity for G proteins. This new group of compounds (G protein antagonists) is a promising tool to study receptor-G protein interaction quantitatively.

  7. G-protein-coupled receptors and cancer.

    PubMed

    Dorsam, Robert T; Gutkind, J Silvio

    2007-02-01

    G-protein-coupled receptors (GPCRs), the largest family of cell-surface molecules involved in signal transmission, have recently emerged as crucial players in tumour growth and metastasis. Malignant cells often hijack the normal physiological functions of GPCRs to survive, proliferate autonomously, evade the immune system, increase their blood supply, invade their surrounding tissues and disseminate to other organs. This Review will address our current understanding of the many roles of GPCRs and their signalling circuitry in tumour progression and metastasis. We will also discuss how interfering with GPCRs might provide unique opportunities for cancer prevention and treatment.

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

    PubMed Central

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

    2014-01-01

    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-associated viruses also express constitutively active viral GPCRs. These studies indicate that G proteins, GPCRs and their linked signaling circuitry represent novel therapeutic targets for cancer prevention and treatment. PMID:23640210

  9. G Protein-Coupled Receptor Biased Agonism

    PubMed Central

    Hodavance, Sima Y.; Gareri, Clarice; Torok, Rachel D.; Rockman, Howard A.

    2016-01-01

    G protein-coupled receptors (GPCR) are the largest family of targets for current therapeutics. The classic model of their activation was binary, where agonist binding induced an active conformation and subsequent downstream signaling. Subsequently, the revised concept of biased agonism emerged, where different ligands at the same GPCR selectively activate one downstream pathway versus another. Advances in understanding the mechanism of biased agonism has led to the development of novel ligands, which have the potential for improved therapeutic and safety profiles. In this review, we summarize the theory and most recent breakthroughs in understanding biased signaling, examine recent laboratory investigations concerning biased ligands across different organ systems, and discuss the promising clinical applications of biased agonism. PMID:26751266

  10. G-protein-coupled receptors and melanoma.

    PubMed

    Lee, Hwa Jin; Wall, Brian; Chen, Suzie

    2008-08-01

    G-protein-coupled receptors (GPCR) are the largest family of receptors with over 500 members. Evaluation of GPCR gene expression in primary human tumors identified over-expression of GPCR in several tumor types. Analysis of cancer samples in different disease stages also suggests that some GPCR may be involved in early tumor progression and others may play a critical role in tumor invasion and metastasis. Currently, >50% of drug targets to various human diseases are based on GPCR. In this review, the relationships between several GPCR and melanoma development and/or progression will be discussed. Finally, the possibility of using one or more of these GPCR as therapeutic targets in melanoma will be summarized.

  11. Heterotrimeric G Proteins and Apoptosis: Intersecting Signaling Pathways Leading to Context Dependent Phenotypes

    PubMed Central

    Yanamadala, Vijay; Negoro, Hideyuki; Denker, Bradley M.

    2010-01-01

    Apoptosis, a programmed cell death mechanism, is a fundamental process during the normal development and somatic maintenance of all multicellular organisms and thus is highly conserved and tightly regulated through numerous signaling pathways. Apoptosis is of particular clinical importance as its dysregulation contributes significantly to numerous human diseases, primarily through changes in the expression and activation of key apoptotic regulators. Each of the four families of heterotrimeric G proteins (Gs, Gi/o, Gq/11 and G12/13) has been implicated in numerous cellular signaling processes, including proliferation, transformation, migration, differentiation, and apoptosis. Heterotrimeric G protein signaling is an important but not widely studied mechanism regulating apoptosis. G protein Signaling and Apoptosis broadly cover two large bodies of literature and share numerous signaling pathways. Examination of the intersection between these two areas is the focus of this review. Several studies have implicated signaling through each of the four heterotrimeric G protein families to regulate apoptosis within numerous disease contexts, but the mechanism(s) are not well defined. Each G protein family has been shown to stimulate and/or inhibit apoptosis in a context-dependent fashion through regulating numerous downstream effectors including the Bcl-2 family, NF-κB, PI3 Kinase, MAP Kinases, and small GTPases. These cell-type specific and G protein coupled receptor dependent effects have led to a complex body of literature of G protein regulation of apoptosis. Here, we review the literature and summarize apoptotic signaling through each of the four heterotrimeric G protein families (and the relevant G protein coupled receptors), and discuss limitations and future directions for research on regulating apoptosis through G protein coupled mechanisms. Continued investigation in this field is essential for the identification of important targets for pharmacological

  12. Crystallization of G Protein-Coupled Receptors

    PubMed Central

    Salom, David; Padayatti, Pius S.; Palczewski, Krzysztof

    2015-01-01

    Oligomerization is one of several mechanisms that can regulate the activity of G protein-coupled receptors (GPCRs), but little is known about the structure of GPCR oligomers. Crystallography and NMR are the only methods able to reveal the details of receptor–receptor interactions at an atomic level, and several GPCR homodimers already have been described from crystal structures. Two clusters of symmetric interfaces have been identified from these structures that concur with biochemical data, one involving helices I, II, and VIII and the other formed mainly by helices V and VI. In this chapter, we describe the protocols used in our laboratory for the crystallization of rhodopsin and the β2-adrenergic receptor (β2-AR). For bovine rhodopsin, we developed a new purification strategy including a (NH4)2SO4-induced phase separation that proved essential to obtain crystals of photoactivated rhodopsin containing parallel dimers. Crystallization of native bovine rhodopsin was achieved by the classic vapor-diffusion technique. For β2-AR, we developed a purification strategy based on previously published protocols employing a lipidic cubic phase to obtain diffracting crystals of a β2-AR/T4-lysozyme chimera bound to the antagonist carazolol. PMID:24143992

  13. G Protein-Coupled Receptors in Cancer

    PubMed Central

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

    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 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. PMID:27529230

  14. G Protein-Coupled Receptors in Cancer.

    PubMed

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

    2016-08-12

    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.

  15. Homologous and unique G protein alpha subunits in the nematode Caenorhabditis elegans.

    PubMed Central

    Lochrie, M A; Mendel, J E; Sternberg, P W; Simon, M I

    1991-01-01

    A cDNA corresponding to a known G protein alpha subunit, the alpha subunit of Go (Go alpha), was isolated and sequenced. The predicted amino acid sequence of C. elegans Go alpha is 80-87% identical to other Go alpha sequences. An mRNA that hybridizes to the C. elegans Go alpha cDNA can be detected on Northern blots. A C. elegans protein that crossreacts with antibovine Go alpha antibody can be detected on immunoblots. A cosmid clone containing the C. elegans Go alpha gene (goa-1) was isolated and mapped to chromosome I. The genomic fragments of three other C. elegans G protein alpha subunit genes (gpa-1, gpa-2, and gpa-3) have been isolated using the polymerase chain reaction. The corresponding cosmid clones were isolated and mapped to disperse locations on chromosome V. The sequences of two of the genes, gpa-1 and gpa-3, were determined. The predicted amino acid sequences of gpa-1 and gpa-3 are only 48% identical to each other. Therefore, they are likely to have distinct functions. In addition they are not homologous enough to G protein alpha subunits in other organisms to be classified. Thus C. elegans has G proteins that are identifiable homologues of mammalian G proteins as well as G proteins that appear to be unique to C. elegans. Study of identifiable G proteins in C. elegans may result in a further understanding of their function in other organisms, whereas study of the novel G proteins may provide an understanding of unique aspects of nematode physiology. Images PMID:1907494

  16. Novel insights into G protein and G protein-coupled receptor signaling in cancer.

    PubMed

    O'Hayre, Morgan; Degese, Maria S; Gutkind, J Silvio

    2014-04-01

    G protein-coupled receptors (GPCRs) play a central role in signal transmission, thereby controlling many facets of cellular function. Overwhelming evidence now implicates GPCRs, G proteins and their downstream signaling targets in cancer initiation and progression, where they can influence aberrant cell growth and survival, largely through activation of AKT/mTOR, MAPKs, and Hippo signaling pathways. GPCRs also play critical roles in the invasion and metastasis of cancer cells via activation of Rho GTPases and cytoskeletal changes, and angiogenesis to supply the tumor with nutrients and provide routes for metastasis. Lastly, GPCRs contribute to the establishment and maintenance of a permissive tumor microenvironment. Understanding GPCR involvement in cancer malignancy may help identify novel therapeutic opportunities for cancer prevention and treatment.

  17. Regulation, Signaling, and Physiological Functions of G-Proteins.

    PubMed

    Syrovatkina, Viktoriya; Alegre, Kamela O; Dey, Raja; Huang, Xin-Yun

    2016-09-25

    Heterotrimeric guanine-nucleotide-binding regulatory proteins (G-proteins) mainly relay the information from G-protein-coupled receptors (GPCRs) on the plasma membrane to the inside of cells to regulate various biochemical functions. Depending on the targeted cell types, tissues, and organs, these signals modulate diverse physiological functions. The basic schemes of heterotrimeric G-proteins have been outlined. In this review, we briefly summarize what is known about the regulation, signaling, and physiological functions of G-proteins. We then focus on a few less explored areas such as the regulation of G-proteins by non-GPCRs and the physiological functions of G-proteins that cannot be easily explained by the known G-protein signaling pathways. There are new signaling pathways and physiological functions for G-proteins to be discovered and further interrogated. With the advancements in structural and computational biological techniques, we are closer to having a better understanding of how G-proteins are regulated and of the specificity of G-protein interactions with their regulators.

  18. Crystal structure of non-phosphorylated MAP2K6 in a putative auto-inhibition state.

    PubMed

    Matsumoto, Takashi; Kinoshita, Takayoshi; Matsuzaka, Hitomi; Nakai, Ryoko; Kirii, Yasuyuki; Yokota, Koichi; Tada, Toshiji

    2012-05-01

    Mitogen-activated protein kinase kinase 6 (MAP2K6) plays a crucial role in the p38 MAP kinase signal cascade that regulates various stress-induced responses and is associated with pathological conditions. The crystal structure of human non-phosphorylated MAP2K6 (npMAP2K6) complexed with an ATP analogue was determined at 2.6 Å resolution and represents an auto-inhibition state of MAP2K6. Three characteristics of short α-helices configured in the activation loop region, termed activation helices (AH1, AH2 and AH3), are important in controlling the auto-inhibition mechanism. AH1 displaces the αC-helix, a component essential for forming the active configuration, away from the active site. AH1 and AH2 were found to enclose the γ-phosphate, the leaving group of ATP. A comparison with the related enzymes, MAP2K1 and MAP2K4 reveals that MAP2K6 has the unique auto-inhibition mechanism mediated by the three activation helices.

  19. Purine inhibitors of protein kinases, G proteins and polymerases

    DOEpatents

    Gray, Nathanael S.; Schultz, Peter; Kim, Sung-Hou; Meijer, Laurent

    2001-07-03

    The present invention relates to purine analogs that inhibit, inter alia, protein kinases, G-proteins and polymerases. In addition, the present invention relates to methods of using such purine analogs to inhibit protein kinases, G-proteins, polymerases and other cellular processes and to treat cellular proliferative diseases.

  20. Accessory proteins for heterotrimeric G-proteins in the kidney

    PubMed Central

    Park, Frank

    2015-01-01

    Heterotrimeric G-proteins play a fundamentally important role in regulating signal transduction pathways in the kidney. Accessory proteins are being identified as direct binding partners for heterotrimeric G-protein α or βγ subunits to promote more diverse mechanisms by which G-protein signaling is controlled. In some instances, accessory proteins can modulate the signaling magnitude, localization, and duration following the activation of cell membrane-associated receptors. Alternatively, accessory proteins complexed with their G-protein α or βγ subunits can promote non-canonical models of signaling activity within the cell. In this review, we will highlight the expression profile, localization and functional importance of these newly identified accessory proteins to control the function of select G-protein subunits under normal and various disease conditions observed in the kidney. PMID:26300785

  1. G proteins: critical control points for transmembrane signals.

    PubMed Central

    Neer, E. J.

    1994-01-01

    Heterotrimeric GTP-binding proteins (G proteins) that are made up of alpha and beta gamma subunits couple many kinds of cell-surface receptors to intracellular effector enzymes or ion channels. Every cell contains several types of receptors, G proteins, and effectors. The specificity with which G protein subunits interact with receptors and effectors defines the range of responses a cell is able to make to an external signal. Thus, the G proteins act as a critical control point that determines whether a signal spreads through several pathways or is focused to a single pathway. In this review, I will summarize some features of the structure and function of mammalian G protein subunits, discuss the role of both alpha and beta gamma subunits in regulation of effectors, the role of the beta gamma subunit in macromolecular assembly, and the mechanisms that might make some responses extremely specific and others rather diffuse. PMID:8142895

  2. Interaction between G-protein beta and gamma subunit types is selective.

    PubMed Central

    Pronin, A N; Gautam, N

    1992-01-01

    Signal-transducing guanine nucleotide-binding proteins (G proteins) are made up of three subunits, alpha, beta, and gamma. Each of these subunits comprises a family of proteins. The rules for association between members of one family with members of another to form a multimer are not known; it is not clear whether associations are specific or nonspecific. Other than transducin (Gt), the G protein in rod photoreceptors, most purified G proteins contain more than one subtype of beta or gamma subunits. The Gt alpha subunit is associated only with beta 1 and gamma 1. It is not known whether this specificity is due to the differential expression of these subunit types in a cell type or due to intrinsically different affinities between different beta and gamma subunit types. We have used a transfected cell assay system to examine the association of the beta 1, beta 2, and beta 3 proteins with the gamma 1 and gamma 2 proteins. Results show that gamma 1 does not associate with beta 2 and that beta 3 does not associate with gamma 1 or gamma 2. Differences in affinities between types of G protein subunits will impose restrictions on the formation of certain heterotrimers and determine which G protein will be active in a cell. A chimeric molecule of beta 1 and beta 2 was used to broadly map the regions on these subunits that determine specificity of association. Images PMID:1631113

  3. Role of antibodies in developing drugs that target G-protein-coupled receptor dimers.

    PubMed

    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.

  4. G protein modulation of recombinant P/Q-type calcium channels by regulators of G protein signalling proteins.

    PubMed

    Mark, M D; Wittemann, S; Herlitze, S

    2000-10-01

    1. Fast synaptic transmission is triggered by the activation of presynaptic Ca2+ channels which can be inhibited by Gbetagamma subunits via G protein-coupled receptors (GPCR). Regulators of G protein signalling (RGS) proteins are GTPase-accelerating proteins (GAPs), which are responsible for >100-fold increases in the GTPase activity of G proteins and might be involved in the regulation of presynaptic Ca2+ channels. In this study we investigated the effects of RGS2 on G protein modulation of recombinant P/Q-type channels expressed in a human embryonic kidney (HEK293) cell line using whole-cell recordings. 2. RGS2 markedly accelerates transmitter-mediated inhibition and recovery from inhibition of Ba2+ currents (IBa) through P/Q-type channels heterologously expressed with the muscarinic acetylcholine receptor M2 (mAChR M2). 3. Both RGS2 and RGS4 modulate the prepulse facilitation properties of P/Q-type Ca2+ channels. G protein reinhibition is accelerated, while release from inhibition is slowed. These kinetics depend on the availability of G protein alpha and betagamma subunits which is altered by RGS proteins. 4. RGS proteins unmask the Ca2+ channel beta subunit modulation of Ca2+ channel G protein inhibition. In the presence of RGS2, P/Q-type channels containing the beta2a and beta3 subunits reveal significantly altered kinetics of G protein modulation and increased facilitation compared to Ca2+ channels coexpressed with the beta1b or beta4 subunit.

  5. Heterotrimeric G protein-coupled signaling in plants.

    PubMed

    Urano, Daisuke; Jones, Alan M

    2014-01-01

    Investigators studying G protein-coupled signaling--often called the best-understood pathway in the world owing to intense research in medical fields--have adopted plants as a new model to explore the plasticity and evolution of G signaling. Much research on plant G signaling has not disappointed. Although plant cells have most of the core elements found in animal G signaling, differences in network architecture and intrinsic properties of plant G protein elements make G signaling in plant cells distinct from the animal paradigm. In contrast to animal G proteins, plant G proteins are self-activating, and therefore regulation of G activation in plants occurs at the deactivation step. The self-activating property also means that plant G proteins do not need and therefore do not have typical animal G protein-coupled receptors. Targets of activated plant G proteins, also known as effectors, are unlike effectors in animal cells. The simpler repertoire of G signal elements in Arabidopsis makes G signaling easier to manipulate in a multicellular context.

  6. Identification of G protein-coupled receptor signaling pathway proteins in marine diatoms using comparative genomics

    PubMed Central

    2013-01-01

    Background The G protein-coupled receptor (GPCR) signaling pathway plays an essential role in signal transmission and response to external stimuli in mammalian cells. Protein components of this pathway have been characterized in plants and simpler eukaryotes such as yeast, but their presence and role in unicellular photosynthetic eukaryotes have not been determined. We use a comparative genomics approach using whole genome sequences and gene expression libraries of four diatoms (Pseudo-nitzschia multiseries, Thalassiosira pseudonana, Phaeodactylum tricornutum and Fragilariopsis cylindrus) to search for evidence of GPCR signaling pathway proteins that share sequence conservation to known GPCR pathway proteins. Results The majority of the core components of GPCR signaling were well conserved in all four diatoms, with protein sequence similarity to GPCRs, human G protein α- and β-subunits and downstream effectors. There was evidence for the Gγ-subunit and thus a full heterotrimeric G protein only in T. pseudonana. Phylogenetic analysis of putative diatom GPCRs indicated similarity but deep divergence to the class C GPCRs, with branches basal to the GABAB receptor subfamily. The extracellular and intracellular regions of these putative diatom GPCR sequences exhibited large variation in sequence length, and seven of these sequences contained the necessary ligand binding domain for class C GPCR activation. Transcriptional data indicated that a number of the putative GPCR sequences are expressed in diatoms under various stress conditions in culture, and that many of the GPCR-activated signaling proteins, including the G protein, are also expressed. Conclusions The presence of sequences in all four diatoms that code for the proteins required for a functional mammalian GPCR pathway highlights the highly conserved nature of this pathway and suggests a complex signaling machinery related to environmental perception and response in these unicellular organisms. The lack of

  7. The relationship between cell proliferation and differentiation and mapping of putative dental pulp stem/progenitor cells during mouse molar development by chasing BrdU-labeling.

    PubMed

    Ishikawa, Yuko; Ida-Yonemochi, Hiroko; Nakakura-Ohshima, Kuniko; Ohshima, Hayato

    2012-04-01

    Human dental pulp contains adult stem cells. Our recent study demonstrated the localization of putative dental pulp stem/progenitor cells in the rat developing molar by chasing 5-bromo-2'-deoxyuridine (BrdU)-labeling. However, there are no available data on the localization of putative dental pulp stem/progenitor cells in the mouse molar. This study focuses on the mapping of putative dental pulp stem/progenitor cells in addition to the relationship between cell proliferation and differentiation in the developing molar using BrdU-labeling. Numerous proliferating cells appeared in the tooth germ and the most active cell proliferation in the mesenchymal cells occurred in the prenatal stages, especially on embryonic Day 15 (E15). Cell proliferation in the pulp tissue dramatically decreased in number by postnatal Day 3 (P3) when nestin-positive odontoblasts were arranged in the cusped areas and disappeared after postnatal Week 1 (P1W). Root dental papilla included numerous proliferating cells during P5 to P2W. Three to four intraperitoneal injections of BrdU were given to pregnant ICR mice and revealed slow-cycling long-term label-retaining cells (LRCs) in the mature tissues of postnatal animals. Numerous dense LRCs postnatally decreased in number and reached a plateau after P1W when they mainly resided in the center of the dental pulp, associating with blood vessels. Furthermore, numerous dense LRCs co-expressed mesenchymal stem cell markers such as STRO-1 and CD146. Thus, dense LRCs in mature pulp tissues were believed to be dental pulp stem/progenitor cells harboring in the perivascular niche surrounding the endothelium.

  8. Role of polarized G protein signaling in tracking pheromone gradients

    PubMed Central

    McClure, Allison W.; Minakova, Maria; Dyer, Jayme M.; Zyla, Trevin R.; Elston, Timothy C.; Lew, Daniel J.

    2015-01-01

    Summary Yeast cells track gradients of pheromones to locate mating partners. Intuition suggests that uniform distribution of pheromone receptors over the cell surface would yield optimal gradient sensing. However, yeast cells display polarized receptors. The benefit of such polarization was unknown. During gradient tracking, cell growth is directed by a patch of polarity regulators that wanders around the cortex. Patch movement is sensitive to pheromone dose, with wandering reduced on the up-gradient side of the cell, resulting in net growth in that direction. Mathematical modeling suggests that active receptors and associated G proteins lag behind the polarity patch and act as an effective drag on patch movement. In vivo, the polarity patch is trailed by a G protein-rich domain, and this polarized distribution of G proteins is required to constrain patch wandering. Our findings explain why G protein polarization is beneficial, and illuminate a novel mechanism for gradient tracking. PMID:26609960

  9. Purine inhibitors of protein kinases, G proteins and polymerases

    DOEpatents

    Gray, Nathanael S.; Schultz, Peter; Kim, Sung-Hou; Meijer, Laurent

    2004-10-12

    The present invention relates to 2-N-substituted 6-(4-methoxybenzylamino)-9-isopropylpurines that inhibit, inter alia, protein kinases, G-proteins and polymerases. In addition, the present invention relates to methods of using such 2-N-substituted 6-(4-methoxybenzylamino)-9-isopropylpurines to inhibit protein kinases, G-proteins, polymerases and other cellular processes and to treat cellular proliferative diseases.

  10. The G protein-coupled receptor GPRC5B contributes to neurogenesis in the developing mouse neocortex.

    PubMed

    Kurabayashi, Nobuhiro; Nguyen, Minh Dang; Sanada, Kamon

    2013-11-01

    Neural progenitor cells in the developing brain give rise to neurons and glia. Multiple extrinsic signalling molecules and their cognate membrane receptors have been identified to control neural progenitor fate. However, a role for G protein-coupled receptors in cell fate decisions in the brain remains largely putative. Here we show that GPRC5B, which encodes an orphan G protein-coupled receptor, is present in the ventricular surface of cortical progenitors in the mouse developing neocortex and is required for their neuronal differentiation. GPRC5B-depleted progenitors fail to adopt a neuronal fate and ultimately become astrocytes. Furthermore, GPRC5B-mediated signalling is associated with the proper regulation of β-catenin signalling, a pathway crucial for progenitor fate decision. Our study uncovers G protein-coupled receptor signalling in the neuronal fate determination of cortical progenitors.

  11. Engineering a minimal G protein to facilitate crystallisation of G protein-coupled receptors in their active conformation

    PubMed Central

    Carpenter, Byron; Tate, Christopher G.

    2016-01-01

    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. PMID:27672048

  12. Engineering a minimal G protein to facilitate crystallisation of G protein-coupled receptors in their active conformation.

    PubMed

    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.

  13. Regulators of G protein signalling proteins in the human myometrium.

    PubMed

    Ladds, Graham; Zervou, Sevasti; Vatish, Manu; Thornton, Steven; Davey, John

    2009-05-21

    The contractile state of the human myometrium is controlled by extracellular signals that promote relaxation or contraction. Many of these signals function through G protein-coupled receptors at the cell surface, stimulating heterotrimeric G proteins and leading to changes in the activity of effector proteins responsible for bringing about the response. G proteins can interact with multiple receptors and many different effectors and are key players in the response. Regulators of G protein signalling (RGS) proteins are GTPase activating proteins for heterotrimeric G proteins and help terminate the signal. Little is known about the function of RGS proteins in human myometrium and we have therefore analysed transcript levels for RGS proteins at various stages of pregnancy (non-pregnant, preterm, term non-labouring, term labouring). RGS2 and RGS5 were the most abundantly expressed isolates in each of the patient groups. The levels of RGS4 and RGS16 (and to a lesser extent RGS2 and RGS14) increased in term labouring samples relative to the other groups. Yeast two-hybrid analysis and co-immunoprecipitation in myometrial cells revealed that both RGS2 and RGS5 interact directly with the cytoplasmic tail of the oxytocin receptor, suggesting they might help regulate signalling through this receptor.

  14. G proteins as regulators in ethylene-mediated hypoxia signaling.

    PubMed

    Steffens, Bianka; Sauter, Margret

    2010-04-01

    Waterlogging or flooding are frequently or constitutively encountered by many plant species. The resulting reduction in endogenous O2 concentration poses a severe threat. Numerous adaptations at the anatomical, morphological and metabolic level help plants to either escape low oxygen conditions or to endure them. Formation of aerenchyma or rapid shoot elongation are escape responses, as is the formation of adventitious roots. The metabolic shift from aerobic respiration to anaerobic fermentation contributes to a basal energy supply at low oxygen conditions. Ethylene plays a central role in hypoxic stress signaling, and G proteins have been recognized as crucial signal transducers in various hypoxic signaling pathways. The programmed death of parenchyma cells that results in hypoxia-induced aerenchyma formation is an ethylene response. In maize, aerenchyma are induced in the absence of ethylene when G proteins are constitutively activated. Similarly, ethylene induced death of epidermal cells that cover adventitious roots at the stem node of rice is strictly dependent on heterotrimeric G protein activity. Knock down of the unique Gα gene RGA1 in rice prevents epidermal cell death. Finally, in Arabidopsis, induction of alcohol dehydrogenase with resulting increased plant survival relies on the balanced activities of a small Rop G protein and its deactivating protein RopGAP4. Identifying the general mechanisms of G protein signaling in hypoxia adaptation of plants is one of the tasks ahead.

  15. Identification of putative candidate genes for red rot resistance in sugarcane (Saccharum species hybrid) using LD-based association mapping.

    PubMed

    Singh, Ram K; Banerjee, Nandita; Khan, M S; Yadav, Sonia; Kumar, Sanjeev; Duttamajumder, S K; Lal, Ram Ji; Patel, Jinesh D; Guo, H; Zhang, Dong; Paterson, Andrew H

    2016-06-01

    Red rot is a serious disease of sugarcane caused by the fungus Colletotrichum falcatum that has a colossal damage potential. The fungus, prevalent mainly in the Indian sub-continent, keeps on producing new pathogenic strains leading to breakdown of resistance in newly released varieties and hence the deployment of linked markers for marker-assisted selection for resistance to this disease can fine tune the breeding programme. This study based on a panel of 119 sugarcane genotypes fingerprinted for 944 SSR alleles was undertaken with an aim to identify marker-trait associations (MTAs) for resistance to red rot. Mixed linear model containing population structure and kinship as co-factor detected four MTAs that were able to explain 10-16 % of the trait variation, individually. Among the four MTAs, EST sequences diagnostic of three could be BLAST searched to the sorghum genome with significant sequence homology. Several genes encoding important plant defence related proteins, viz., cytochrome P450, Glycerol-3-phosphate transporter-1, MAP Kinase-4, Serine/threonine-protein kinase, Ring finger domain protein and others were localized to the vicinity of these MTAs. These positional candidate genes are worth of further investigation and possibly these could contribute directly to red rot resistance, and may find a potential application in marker-assisted sugarcane breeding.

  16. Oligomeric forms of G protein-coupled receptors (GPCRs)

    PubMed Central

    Palczewski, Krzysztof

    2010-01-01

    Oligomerization is a general characteristic of cell membrane receptors that is shared by G protein-coupled receptors (GPCRs) together with their G protein partners. Recent studies of these complexes, both in vivo and in purified reconstituted forms, unequivocally support this contention for GPCRs, perhaps with only rare exceptions. As evidence has evolved from experimental cell lines to more relevant in vivo studies and from indirect biophysical approaches to well defined isolated complexes of dimeric receptors alone and complexed with G proteins, there is an expectation that the structural basis of oligomerization and the functional consequences for membrane signaling will be elucidated. Oligomerization of cell membrane receptors is fully supported by both thermodynamic calculations and the selectivity and duration of signaling required to reach targets located in various cellular compartments. PMID:20538466

  17. Expression of rabies virus G protein in carrots (Daucus carota).

    PubMed

    Rojas-Anaya, Edith; Loza-Rubio, Elizabeth; Olivera-Flores, Maria Teresa; Gomez-Lim, Miguel

    2009-12-01

    Antigens derived from various pathogens can readily be synthesized at high levels in plants in their authentic forms. Such antigens administered orally can induce an immune response and, in some cases, result in protection against a subsequent challenge. We here report the expression of rabies virus G protein into carrots. The G gene was subcloned into the pUCpSSrabG vector and then used to transform carrot embryogenic cells by particle bombardment. The carrot cells were selected in liquid medium, a method previously unreported. The presence of the transgene was verified by PCR, and by RT-PCR. By western blot, G protein transgene was identified in 93.3% of adult carrot roots. The G protein was quantified by densitometric analysis (range 0.4-1.2%). The expressed protein was antigenic in mice. This confirms that the carrot is an adequate system for antigen expression.

  18. Photosensory transduction in ciliates. II. Possible role of G-protein and cGMP in Stentor coeruleus.

    PubMed

    Fabczak, H; Park, P B; Fabczak, S; Song, P S

    1993-04-01

    The heterotrichous ciliate, Stentor coeruleus, exhibits a well-defined photophobic response to a sudden increase in the intensity of visible light. The phobic reactions usually appear with a latency period (i.e. a time delay between the onset of the stimulus and the stop response). This latency of phobic response was significantly increased when the cells were incubated with 8-bromo-guanosine 3',5'-cyclic monophosphate. In the presence of this nucleotide, a reduction of cell responsiveness (i.e. the number of photophobically responding cells) was also observed. Similar effects were observed when cells were treated with pertussis toxin, a G-protein activity modulator, and 3'-isobutyl-methylxanthine, an inhibitor of guanosine 3',5'-cyclic monophosphate (cGMP) phosphodiesterase. The G-protein activator fluoroaluminate and 6-anilino-5,8-quinolinedione (LY 83583) (an effective agent for lowering cellular cGMP levels) showed opposite effects on the cell photophobic response. These results indirectly suggest that the level of cytoplasmic cGMP, possibly modulated by a G-protein-coupled cGMP phosphodiesterase, plays a phototransducing role in Stentor. In addition, using an antiserum raised against bovine transducin, a cross-reacting protein with an apparent molecular mass of 39 kDa was detected on immunoblots. The alpha-subunit of a Stentor G-protein has also been partially cloned and sequenced. However, the possible coupling between the G-protein and the putative phosphodiesterase remains to be established.

  19. A Knockout Mutation of a Constitutive GPCR in Tetrahymena Decreases Both G-Protein Activity and Chemoattraction

    PubMed Central

    Lampert, Thomas J.; Coleman, Kevin D.; Hennessey, Todd M.

    2011-01-01

    Although G-protein coupled receptors (GPCRs) are a common element in many chemosensory transduction pathways in eukaryotic cells, no GPCR or regulated G-protein activity has yet been shown in any ciliate. To study the possible role for a GPCR in the chemoresponses of the ciliate Tetrahymena, we have generated a number of macronuclear gene knockouts of putative GPCRs found in the Tetrahymena Genome database. One of these knockout mutants, called G6, is a complete knockout of a gene that we call GPCR6 (TTHERM_00925490). Based on sequence comparisons, the Gpcr6p protein belongs to the Rhodopsin Family of GPCRs. Notably, Gpcr6p shares highest amino acid sequence homologies to GPCRs from Paramecium and several plants. One of the phenotypes of the G6 mutant is a decreased responsiveness to the depolarizing ions Ba2+ and K+, suggesting a decrease in basal excitability (decrease in Ca2+ channel activity). The other major phenotype of G6 is a loss of chemoattraction to lysophosphatidic acid (LPA) and proteose peptone (PP), two known chemoattractants in Tetrahymena. Using microsomal [35S]GTPγS binding assays, we found that wild-type (CU427) have a prominent basal G-protein activity. This activity is decreased to the same level by pertussis toxin (a G-protein inhibitor), addition of chemoattractants, or the G6 mutant. Since the basal G-protein activity is decreased by the GPCR6 knockout, it is likely that this gene codes for a constitutively active GPCR in Tetrahymena. We propose that chemoattractants like LPA and PP cause attraction in Tetrahymena by decreasing the basal G-protein stimulating activity of Gpcr6p. This leads to decreased excitability in wild-type and longer runs of smooth forward swimming (less interrupted by direction changes) towards the attractant. Therefore, these attractants may work as inverse agonists through the constitutively active Gpcr6p coupled to a pertussis-sensitive G-protein. PMID:22140501

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

    PubMed

    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.

  1. Alpha-Bulges in G Protein-Coupled Receptors

    PubMed Central

    van der Kant, Rob; Vriend, Gert

    2014-01-01

    Agonist binding is related to a series of motions in G protein-coupled receptors (GPCRs) that result in the separation of transmembrane helices III and VI at their cytosolic ends and subsequent G protein binding. A large number of smaller motions also seem to be associated with activation. Most helices in GPCRs are highly irregular and often contain kinks, with extensive literature already available about the role of prolines in kink formation and the precise function of these kinks. GPCR transmembrane helices also contain many α-bulges. In this article we aim to draw attention to the role of these α-bulges in ligand and G-protein binding, as well as their role in several aspects of the mobility associated with GPCR activation. This mobility includes regularization and translation of helix III in the extracellular direction, a rotation of the entire helix VI, an inward movement of the helices near the extracellular side, and a concerted motion of the cytosolic ends of the helices that makes their orientation appear more circular and that opens up space for the G protein to bind. In several cases, α-bulges either appear or disappear as part of the activation process. PMID:24806342

  2. G-protein-coupled receptors: past, present and future

    PubMed Central

    Hill, Stephen J

    2006-01-01

    The G-protein-coupled receptor (GPCR) family represents the largest and most versatile group of cell surface receptors. Drugs active at these receptors have therapeutic actions across a wide range of human diseases ranging from allergic rhinitis to pain, hypertension and schizophrenia. This review provides a brief historical overview of the properties and signalling characteristics of this important family of receptors. PMID:16402114

  3. Fine-Scale Mapping of the FGFR2 Breast Cancer Risk Locus: Putative Functional Variants Differentially Bind FOXA1 and E2F1

    PubMed Central

    Meyer, Kerstin B.; O’Reilly, Martin; Michailidou, Kyriaki; Carlebur, Saskia; Edwards, Stacey L.; French, Juliet D.; Prathalingham, Radhika; Dennis, Joe; Bolla, Manjeet K.; Wang, Qin; de Santiago, Ines; Hopper, John L.; Tsimiklis, Helen; Apicella, Carmel; Southey, Melissa C.; Schmidt, Marjanka K.; Broeks, Annegien; Van ’t Veer, Laura J.; Hogervorst, Frans B.; Muir, Kenneth; Lophatananon, Artitaya; Stewart-Brown, Sarah; Siriwanarangsan, Pornthep; Fasching, Peter A.; Lux, Michael P.; Ekici, Arif B.; Beckmann, Matthias W.; Peto, Julian; dos Santos Silva, Isabel; Fletcher, Olivia; Johnson, Nichola; Sawyer, Elinor J.; Tomlinson, Ian; Kerin, Michael J.; Miller, Nicola; Marme, Federick; Schneeweiss, Andreas; Sohn, Christof; Burwinkel, Barbara; Guénel, Pascal; Truong, Thérèse; Laurent-Puig, Pierre; Menegaux, Florence; Bojesen, Stig E.; Nordestgaard, Børge G.; Nielsen, Sune F.; Flyger, Henrik; Milne, Roger L.; Zamora, M. Pilar; Arias, Jose I.; Benitez, Javier; Neuhausen, Susan; Anton-Culver, Hoda; Ziogas, Argyrios; Dur, Christina C.; Brenner, Hermann; Müller, Heiko; Arndt, Volker; Stegmaier, Christa; Meindl, Alfons; Schmutzler, Rita K.; Engel, Christoph; Ditsch, Nina; Brauch, Hiltrud; Brüning, Thomas; Ko, Yon-Dschun; Nevanlinna, Heli; Muranen, Taru A.; Aittomäki, Kristiina; Blomqvist, Carl; Matsuo, Keitaro; Ito, Hidemi; Iwata, Hiroji; Yatabe, Yasushi; Dörk, Thilo; Helbig, Sonja; Bogdanova, Natalia V.; Lindblom, Annika; Margolin, Sara; Mannermaa, Arto; Kataja, Vesa; Kosma, Veli-Matti; Hartikainen, Jaana M.; Chenevix-Trench, Georgia; Wu, Anna H.; Tseng, Chiu-chen; Van Den Berg, David; Stram, Daniel O.; Lambrechts, Diether; Thienpont, Bernard; Christiaens, Marie-Rose; Smeets, Ann; Chang-Claude, Jenny; Rudolph, Anja; Seibold, Petra; Flesch-Janys, Dieter; Radice, Paolo; Peterlongo, Paolo; Bonanni, Bernardo; Bernard, Loris; Couch, Fergus J.; Olson, Janet E.; Wang, Xianshu; Purrington, Kristen; Giles, Graham G.; Severi, Gianluca; Baglietto, Laura; McLean, Catriona; Haiman, Christopher A.; Henderson, Brian E.; Schumacher, Fredrick; Le Marchand, Loic; Simard, Jacques; Goldberg, Mark S.; Labrèche, France; Dumont, Martine; Teo, Soo-Hwang; Yip, Cheng-Har; Phuah, Sze-Yee; Kristensen, Vessela; Grenaker Alnæs, Grethe; Børresen-Dale, Anne-Lise; Zheng, Wei; Deming-Halverson, Sandra; Shrubsole, Martha; Long, Jirong; Winqvist, Robert; Pylkäs, Katri; Jukkola-Vuorinen, Arja; Kauppila, Saila; Andrulis, Irene L.; Knight, Julia A.; Glendon, Gord; Tchatchou, Sandrine; Devilee, Peter; Tollenaar, Robert A.E.M.; Seynaeve, Caroline M.; García-Closas, Montserrat; Figueroa, Jonine; Chanock, Stephen J.; Lissowska, Jolanta; Czene, Kamila; Darabi, Hartef; Eriksson, Kimael; Hooning, Maartje J.; Martens, John W.M.; van den Ouweland, Ans M.W.; van Deurzen, Carolien H.M.; Hall, Per; Li, Jingmei; Liu, Jianjun; Humphreys, Keith; Shu, Xiao-Ou; Lu, Wei; Gao, Yu-Tang; Cai, Hui; Cox, Angela; Reed, Malcolm W.R.; Blot, William; Signorello, Lisa B.; Cai, Qiuyin; Pharoah, Paul D.P.; Ghoussaini, Maya; Harrington, Patricia; Tyrer, Jonathan; Kang, Daehee; Choi, Ji-Yeob; Park, Sue K.; Noh, Dong-Young; Hartman, Mikael; Hui, Miao; Lim, Wei-Yen; Buhari, Shaik A.; Hamann, Ute; Försti, Asta; Rüdiger, Thomas; Ulmer, Hans-Ulrich; Jakubowska, Anna; Lubinski, Jan; Jaworska, Katarzyna; Durda, Katarzyna; Sangrajrang, Suleeporn; Gaborieau, Valerie; Brennan, Paul; McKay, James; Vachon, Celine; Slager, Susan; Fostira, Florentia; Pilarski, Robert; Shen, Chen-Yang; Hsiung, Chia-Ni; Wu, Pei-Ei; Hou, Ming-Feng; Swerdlow, Anthony; Ashworth, Alan; Orr, Nick; Schoemaker, Minouk J.; Ponder, Bruce A.J.; Dunning, Alison M.; Easton, Douglas F.

    2013-01-01

    The 10q26 locus in the second intron of FGFR2 is the locus most strongly associated with estrogen-receptor-positive breast cancer in genome-wide association studies. We conducted fine-scale mapping in case-control studies genotyped with a custom chip (iCOGS), comprising 41 studies (n = 89,050) of European ancestry, 9 Asian ancestry studies (n = 13,983), and 2 African ancestry studies (n = 2,028) from the Breast Cancer Association Consortium. We identified three statistically independent risk signals within the locus. Within risk signals 1 and 3, genetic analysis identified five and two variants, respectively, highly correlated with the most strongly associated SNPs. By using a combination of genetic fine mapping, data on DNase hypersensitivity, and electrophoretic mobility shift assays to study protein-DNA binding, we identified rs35054928, rs2981578, and rs45631563 as putative functional SNPs. Chromatin immunoprecipitation showed that FOXA1 preferentially bound to the risk-associated allele (C) of rs2981578 and was able to recruit ERα to this site in an allele-specific manner, whereas E2F1 preferentially bound the risk variant of rs35054928. The risk alleles were preferentially found in open chromatin and bound by Ser5 phosphorylated RNA polymerase II, suggesting that the risk alleles are associated with changes in transcription. Chromatin conformation capture demonstrated that the risk region was able to interact with the promoter of FGFR2, the likely target gene of this risk region. A role for FOXA1 in mediating breast cancer susceptibility at this locus is consistent with the finding that the FGFR2 risk locus primarily predisposes to estrogen-receptor-positive disease. PMID:24290378

  4. G-protein coupled receptor-mediated nutrient sensing and developmental control in Aspergillus nidulans.

    PubMed

    Brown, Neil Andrew; Dos Reis, Thaila Fernanda; Ries, Laure Nicolas Annick; Caldana, Camila; Mah, Jae-Hyung; Yu, Jae-Hyuk; Macdonald, Jeffrey M; Goldman, Gustavo Henrique

    2015-10-01

    Nutrient sensing and utilisation are fundamental for all life forms. As heterotrophs, fungi have evolved a diverse range of mechanisms for sensing and taking up various nutrients. Despite its importance, only a limited number of nutrient receptors and their corresponding ligands have been identified in fungi. G-protein coupled receptors (GPCRs) are the largest family of transmembrane receptors. The Aspergillus nidulans genome encodes 16 putative GPCRs, but only a few have been functionally characterised. Our previous study showed the increased expression of an uncharacterised putative GPCR, gprH, during carbon starvation. GprH appears conserved throughout numerous filamentous fungi. Here, we reveal that GprH is a putative receptor involved in glucose and tryptophan sensing. The absence of GprH results in a reduction in cAMP levels and PKA activity upon adding glucose or tryptophan to starved cells. GprH is pre-formed in conidia and is increasingly active during carbon starvation, where it plays a role in glucose uptake and the recovery of hyphal growth. GprH also represses sexual development under conditions favouring sexual fruiting and during carbon starvation in submerged cultures. In summary, the GprH nutrient-sensing system functions upstream of the cAMP-PKA pathway, influences primary metabolism and hyphal growth, while represses sexual development in A. nidulans.

  5. Molecular cloning and characterisation of a novel GABAB-related G-protein coupled receptor.

    PubMed

    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.

  6. An in vitro selected binding protein (affibody) shows conformation-dependent recognition of the respiratory syncytial virus (RSV) G protein.

    PubMed

    Hansson, M; Ringdahl, J; Robert, A; Power, U; Goetsch, L; Nguyen, T N; Uhlén, M; Ståhl, S; Nygren, P A

    1999-03-01

    Using phage-display technology, a novel binding protein (Z-affibody) showing selective binding to the RSV (Long strain) G protein was selected from a combinatorial library of a small alpha-helical protein domain (Z), derived from staphylococcal protein A (SPA). Biopanning of the Z-library against a recombinant fusion protein comprising amino acids 130-230 of the G protein from RSV-subgroup A, resulted in the selection of a Z-affibody (Z(RSV1)) which showed G protein specific binding. Using biosensor technology, the affinity (K(D)) between Z(RSV1) and the recombinant protein was determined to be in the micromolar range (10(-6) M). Interestingly, the Z(RSV1) affibody was demonstrated to also recognize the partially (54%) homologous G protein of RSV subgroup B with similar affinity. Using different recombinant RSV G protein derived fragments, the binding was found to be dependent on the presence of the cysteinyl residues proposed to be involved in the formation of an intramolecular disulfide-constrained loop structure, indicating a conformation-dependent binding. Results from epitope mapping studies, employing a panel of monoclonal antibodies directed to different RSV G protein subfragments, suggest that the Z(RSV1) affibody binding site is located within the region of amino acids 164-186 of the G protein. This region contains a 13 amino acid residue sequence which is totally conserved between subgroups A and B of RSV and extends into the cystein loop region (amino acids 173-186). The potential use of the RSV G protein-specific Z(RSV1) affibody in diagnostic and therapeutic applications is discussed.

  7. The Role of Inhibitory G Proteins and Regulators of G Protein Signaling in the in vivo Control of Heart Rate and Predisposition to Cardiac Arrhythmias

    PubMed Central

    Ang, Richard; Opel, Aaisha; Tinker, Andrew

    2012-01-01

    Inhibitory heterotrimeric G proteins and the control of heart rate. The activation of cell signaling pathways involving inhibitory heterotrimeric G proteins acts to slow the heart rate via modulation of ion channels. A large number of Regulators of G protein signalings (RGSs) can act as GTPase accelerating proteins to inhibitory G proteins and thus it is important to understand the network of RGS\\G-protein interaction. We will review our recent findings on in vivo heart rate control in mice with global genetic deletion of various inhibitory G protein alpha subunits. We will discuss potential central and peripheral contributions to the phenotype and the controversies in the literature. PMID:22783193

  8. Discovery of G Protein-Biased EP2 Receptor Agonists

    PubMed Central

    2016-01-01

    To identify G protein-biased and highly subtype-selective EP2 receptor agonists, a series of bicyclic prostaglandin analogues were designed and synthesized. Structural hybridization of EP2/4 dual agonist 5 and prostacyclin analogue 6, followed by simplification of the ω chain enabled us to discover novel EP2 agonists with a unique prostacyclin-like scaffold. Further optimization of the ω chain was performed to improve EP2 agonist activity and subtype selectivity. Phenoxy derivative 18a showed potent agonist activity and excellent subtype selectivity. Furthermore, a series of compounds were identified as G protein-biased EP2 receptor agonists. These are the first examples of biased ligands of prostanoid receptors. PMID:26985320

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

    PubMed

    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. GRK mythology: G-protein receptor kinases in cardiovascular disease.

    PubMed

    Dorn, Gerald W

    2009-05-01

    G-protein receptor kinases (GRKs) are indispensable for terminating signaling of G-protein coupled receptors (GPCR) through receptor desensitization and downregulation. Increased neurohormone levels in heart failure and the adverse consequences of constant neurohormonal stimulation suggest an important protective role for mechanisms that desensitize neurohormone receptor responses. For that reason, GRK2, the first GRK identified in the heart, has been extensively studied in heart failure, cardiac hypertrophy, and myocardial infarction. However, our understanding of the roles of GRKs in general, and the differential effects of cardiac receptor phosphorylation by individual cardiac-expressed GRKs, have evolved considerably in the last few years. Here, recent developments are reviewed, with an emphasis on novel GRK functions and signaling pathways.

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

    SciTech Connect

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

    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.

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

    PubMed

    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.

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

    PubMed

    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. G protein-coupled receptor kinase GRK5 phosphorylates moesin and regulates metastasis in prostate cancer.

    PubMed

    Chakraborty, Prabir Kumar; Zhang, Yushan; Coomes, Alexandra S; Kim, Wan-Ju; Stupay, Rachel; Lynch, Lauren D; Atkinson, Tamieka; Kim, Jae I; Nie, Zhongzhen; Daaka, Yehia

    2014-07-01

    G protein-coupled receptor kinases (GRK) regulate diverse cellular functions ranging from metabolism to growth and locomotion. Here, we report an important contributory role for GRK5 in human prostate cancer. Inhibition of GRK5 kinase activity attenuated the migration and invasion of prostate cancer cells and, concordantly, increased cell attachment and focal adhesion formation. Mass spectrometric analysis of the phosphoproteome revealed the cytoskeletal-membrane attachment protein moesin as a putative GRK5 substrate. GRK5 regulated the subcellular distribution of moesin and colocalized with moesin at the cell periphery. We identified amino acid T66 of moesin as a principal GRK5 phosphorylation site and showed that enforcing the expression of a T66-mutated moesin reduced cell spreading. In a xenograft model of human prostate cancer, GRK5 silencing reduced tumor growth, invasion, and metastasis. Taken together, our results established GRK5 as a key contributor to the growth and metastasis of prostate cancer.

  15. Insights into G protein structure, function, and regulation.

    PubMed

    Cabrera-Vera, Theresa M; Vanhauwe, Jurgen; Thomas, Tarita O; Medkova, Martina; Preininger, Anita; Mazzoni, Maria R; Hamm, Heidi E

    2003-12-01

    In multicellular organisms from Caenorhabditis elegans to Homo sapiens, the maintenance of homeostasis is dependent on the continual flow and processing of information through a complex network of cells. Moreover, in order for the organism to respond to an ever-changing environment, intercellular signals must be transduced, amplified, and ultimately converted to the appropriate physiological response. The resolution of the molecular events underlying signal response and integration forms the basis of the signal transduction field of research. An evolutionarily highly conserved group of molecules known as heterotrimeric guanine nucleotide-binding proteins (G proteins) are key determinants of the specificity and temporal characteristics of many signaling processes and are the topic of this review. Numerous hormones, neurotransmitters, chemokines, local mediators, and sensory stimuli exert their effects on cells by binding to heptahelical membrane receptors coupled to heterotrimeric G proteins. These highly specialized transducers can modulate the activity of multiple signaling pathways leading to diverse biological responses. In vivo, specific combinations of G alpha- and G beta gamma-subunits are likely required for connecting individual receptors to signaling pathways. The structural determinants of receptor-G protein-effector specificity are not completely understood and, in addition to involving interaction domains of these primary acting proteins, also require the participation of scaffolding and regulatory proteins.

  16. Chromosomal Distribution of PcG Proteins during Drosophila Development

    PubMed Central

    Nègre, Nicolas; Hennetin, Jérôme; Sun, Ling V; Lavrov, Sergey; Bellis, Michel; White, Kevin P

    2006-01-01

    Polycomb group (PcG) proteins are able to maintain the memory of silent transcriptional states of homeotic genes throughout development. In Drosophila, they form multimeric complexes that bind to specific DNA regulatory elements named PcG response elements (PREs). To date, few PREs have been identified and the chromosomal distribution of PcG proteins during development is unknown. We used chromatin immunoprecipitation (ChIP) with genomic tiling path microarrays to analyze the binding profile of the PcG proteins Polycomb (PC) and Polyhomeotic (PH) across 10 Mb of euchromatin. We also analyzed the distribution of GAGA factor (GAF), a sequence-specific DNA binding protein that is found at most previously identified PREs. Our data show that PC and PH often bind to clustered regions within large loci that encode transcription factors which play multiple roles in developmental patterning and in the regulation of cell proliferation. GAF co-localizes with PC and PH to a limited extent, suggesting that GAF is not a necessary component of chromatin at PREs. Finally, the chromosome-association profile of PC and PH changes during development, suggesting that the function of these proteins in the regulation of some of their target genes might be more dynamic than previously anticipated. PMID:16613483

  17. Heterodimerization and Surface Localization of G Protein Coupled Receptors

    PubMed Central

    Minneman, Kenneth P.

    2007-01-01

    G protein coupled receptors (GPCRs) are one of the largest human gene families, and are targets for many important therapeutic drugs. Over the last few years, there has been a major paradigm shift in our understanding of how these receptors function. Formerly, GPCRs were thought to exist as monomers that, upon agonist occupation, activated a heterotrimeric G protein to alter the concentrations of specific second messengers. Until recently, this relatively linear cascade has been the standard paradigm for signaling by these molecules. However, it is now clear that this model is not adequate to explain many aspects of GPCR function. We now know that many, if not most, GPCRs form homo- and/or hetero-oligomeric complexes and interact directly with intracellular proteins in addition to G proteins. It now appears that many GPCRs may not function independently, but might more accurately be described as subunits of large multi-protein signaling complexes. These observations raise many important new questions; some of which include: 1) How many functionally and pharmacologically distinct receptor subtypes exist in vivo? 2) Which GPCRs physically associate, and in what stochiometries? 3) What are the roles of individual subunits in binding ligand and activating responses? 4) Are the pharmacological or signaling properties of GPCR heterodimers different from monomers? Since these receptors are the targets for a large number of clinically useful compounds, such information is likely to be of direct therapeutic importance, both in understanding how existing drugs work, but also in discovering novel compounds to treat disease. PMID:17011524

  18. Discovery of G Protein-Biased Dopaminergics with a Pyrazolo[1,5-a]pyridine Substructure.

    PubMed

    Möller, Dorothee; Banerjee, Ashutosh; Uzuneser, Taygun C; Skultety, Marika; Huth, Tobias; Plouffe, Bianca; Hübner, Harald; Alzheimer, Christian; Friedland, Kristina; Müller, Christian P; Bouvier, Michel; Gmeiner, Peter

    2017-03-01

    1,4-Disubstituted aromatic piperazines are privileged structural motifs recognized by aminergic G protein-coupled receptors. Connection of a lipophilic moiety to the arylpiperazine core by an appropriate linker represents a promising concept to increase binding affinity and to fine-tune functional properties. In particular, incorporation of a pyrazolo[1,5-a]pyridine heterocyclic appendage led to a series of high affinity dopamine receptor partial agonists. Comprehensive pharmacological characterization involving BRET biosensors, binding studies, electrophysiology and complementation-based assays revealed compounds favoring activation of G proteins (preferably Go) over β-arrestin recruitment at dopamine D2 receptors. The feasibility to design G protein-biased partial agonists as putative novel therapeutics was demonstrated for the representative 2-methoxyphenylpiperazine 16c, which unequivocally displayed antipsychotic activity in vivo. Moreover, combination of the pyrazolo[1,5-a]pyridine appendage with a 5-hydroxy-N-propyl-2-aminotetraline unit led to balanced or G protein-biased dopaminergic ligands depending on the stereochemistry of the head group, illustrating the complex structure-functional selectivity relationships at dopamine D2 receptors.

  19. Heterotrimeric G protein subunit Gγ13 is critical to olfaction

    PubMed Central

    Li, Feng; Ponissery-Saidu, Samsudeen; Yee, Karen; Wang, Hong; Chen, Meng-Ling; Iguchi, Naoko; Zhang, Genhua; Jiang, Ping; Reisert, Johannes; Huang, Liquan

    2013-01-01

    The activation of G-protein-coupled olfactory receptors on the olfactory sensory neurons (OSNs) triggers a signaling cascade, which is mediated by a heterotrimeric G protein consisting of α, β and γ subunits. Although its α subunit, Gαolf, has been identified and well characterized, the identities of its β and γ subunits and their function in olfactory signal transduction, however, have not been well established yet. We and others have found the expression of Gγ13 in the olfactory epithelium, particularly in the cilia of the OSNs. In this study, we generated a conditional gene knockout mouse line to specifically nullify Gγ13 expression in the olfactory marker protein-expressing OSNs. Immunohistochemical and Western blot results showed that Gγ13 subunit was indeed eliminated in the mutant mice’s olfactory epithelium. Intriguingly, Gαolf, β1 subunits, Ric-8B and CEP290 proteins were also absent in the epithelium whereas the presence of the effector enzyme adenylyl cyclase III remained largely unaltered. Electro-olfactogram studies showed that the mutant animals had greatly reduced responses to a battery of odorants including three presumable pheromones. Behavioral tests indicated that the mutant mice had a remarkably reduced ability to perform an odor-guided search task although their motivation and agility seemed normal. Our results indicate that Gαolf exclusively forms a functional heterotrimeric G protein with Gβ1 and Gγ13 in OSNs, mediating olfactory signal transduction. The identification of the olfactory G protein’s βγ moiety has provided a novel approach to understanding the feedback regulation of olfactory signal transduction pathways as well as the control of subcellular structures of OSNs. PMID:23637188

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

    PubMed

    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

  1. Regulating Rap small G-proteins in time and space.

    PubMed

    Gloerich, Martijn; Bos, Johannes L

    2011-10-01

    Signaling by the small G-protein Rap is under tight regulation by its GEFs and GAPs. These are multi-domain proteins that are themselves controlled by distinct upstream pathways, and thus couple different extra- and intracellular cues to Rap. The individual RapGEFs and RapGAPs are, in addition, targeted to specific cellular locations by numerous anchoring mechanisms and, consequently, may control different pools of Rap. Here, we review the various activating signals and targeting mechanisms of these proteins and discuss their contribution to the spatiotemporal regulation and biological functions of the Rap proteins.

  2. Mechanism of the intrinsic arginine finger in heterotrimeric G proteins

    PubMed Central

    Mann, Daniel; Teuber, Christian; Tennigkeit, Stefan A.; Schröter, Grit; Gerwert, Klaus

    2016-01-01

    Heterotrimeric G proteins are crucial molecular switches that maintain a large number of physiological processes in cells. The signal is encoded into surface alterations of the Gα subunit that carries GTP in its active state and GDP in its inactive state. The ability of the Gα subunit to hydrolyze GTP is essential for signal termination. Regulator of G protein signaling (RGS) proteins accelerates this process. A key player in this catalyzed reaction is an arginine residue, Arg178 in Gαi1, which is already an intrinsic part of the catalytic center in Gα in contrast to small GTPases, at which the corresponding GTPase-activating protein (GAP) provides the arginine “finger.” We applied time-resolved FTIR spectroscopy in combination with isotopic labeling and site-directed mutagenesis to reveal the molecular mechanism, especially of the role of Arg178 in the intrinsic Gαi1 mechanism and the RGS4-catalyzed mechanism. Complementary biomolecular simulations (molecular mechanics with molecular dynamics and coupled quantum mechanics/molecular mechanics) were performed. Our findings show that Arg178 is bound to γ-GTP for the intrinsic Gαi1 mechanism and pushed toward a bidentate α-γ-GTP coordination for the Gαi1·RGS4 mechanism. This movement induces a charge shift toward β-GTP, increases the planarity of γ-GTP, and thereby catalyzes the hydrolysis. PMID:27911799

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

    PubMed

    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.

  4. Covalent agonists for studying G protein-coupled receptor activation

    PubMed Central

    Weichert, Dietmar; Kruse, Andrew C.; Manglik, Aashish; Hiller, Christine; Zhang, Cheng; Hübner, Harald; Kobilka, Brian K.; Gmeiner, Peter

    2014-01-01

    Structural studies on G protein-coupled receptors (GPCRs) provide important insights into the architecture and function of these important drug targets. However, the crystallization of GPCRs in active states is particularly challenging, requiring the formation of stable and conformationally homogeneous ligand-receptor complexes. Native hormones, neurotransmitters, and synthetic agonists that bind with low affinity are ineffective at stabilizing an active state for crystallogenesis. To promote structural studies on the pharmacologically highly relevant class of aminergic GPCRs, we here present the development of covalently binding molecular tools activating Gs-, Gi-, and Gq-coupled receptors. The covalent agonists are derived from the monoamine neurotransmitters noradrenaline, dopamine, serotonin, and histamine, and they were accessed using a general and versatile synthetic strategy. We demonstrate that the tool compounds presented herein display an efficient covalent binding mode and that the respective covalent ligand-receptor complexes activate G proteins comparable to the natural neurotransmitters. A crystal structure of the β2-adrenoreceptor in complex with a covalent noradrenaline analog and a conformationally selective antibody (nanobody) verified that these agonists can be used to facilitate crystallogenesis. PMID:25006259

  5. Mechanism of the intrinsic arginine finger in heterotrimeric G proteins.

    PubMed

    Mann, Daniel; Teuber, Christian; Tennigkeit, Stefan A; Schröter, Grit; Gerwert, Klaus; Kötting, Carsten

    2016-12-13

    Heterotrimeric G proteins are crucial molecular switches that maintain a large number of physiological processes in cells. The signal is encoded into surface alterations of the Gα subunit that carries GTP in its active state and GDP in its inactive state. The ability of the Gα subunit to hydrolyze GTP is essential for signal termination. Regulator of G protein signaling (RGS) proteins accelerates this process. A key player in this catalyzed reaction is an arginine residue, Arg178 in Gαi1, which is already an intrinsic part of the catalytic center in Gα in contrast to small GTPases, at which the corresponding GTPase-activating protein (GAP) provides the arginine "finger." We applied time-resolved FTIR spectroscopy in combination with isotopic labeling and site-directed mutagenesis to reveal the molecular mechanism, especially of the role of Arg178 in the intrinsic Gαi1 mechanism and the RGS4-catalyzed mechanism. Complementary biomolecular simulations (molecular mechanics with molecular dynamics and coupled quantum mechanics/molecular mechanics) were performed. Our findings show that Arg178 is bound to γ-GTP for the intrinsic Gαi1 mechanism and pushed toward a bidentate α-γ-GTP coordination for the Gαi1·RGS4 mechanism. This movement induces a charge shift toward β-GTP, increases the planarity of γ-GTP, and thereby catalyzes the hydrolysis.

  6. G protein-coupled receptors as promising cancer targets.

    PubMed

    Liu, Ying; An, Su; Ward, Richard; Yang, Yang; Guo, Xiao-Xi; Li, Wei; Xu, Tian-Rui

    2016-07-01

    G protein-coupled receptors (GPCRs) regulate an array of fundamental biological processes, such as growth, metabolism and homeostasis. Specifically, GPCRs are involved in cancer initiation and progression. However, compared with the involvement of the epidermal growth factor receptor in cancer, that of GPCRs have been largely ignored. Recent findings have implicated many GPCRs in tumorigenesis, tumor progression, invasion and metastasis. Moreover, GPCRs contribute to the establishment and maintenance of a microenvironment which is permissive for tumor formation and growth, including effects upon surrounding blood vessels, signaling molecules and the extracellular matrix. Thus, GPCRs are considered to be among the most useful drug targets against many solid cancers. Development of selective ligands targeting GPCRs may provide novel and effective treatment strategies against cancer and some anticancer compounds are now in clinical trials. Here, we focus on tumor related GPCRs, such as G protein-coupled receptor 30, the lysophosphatidic acid receptor, angiotensin receptors 1 and 2, the sphingosine 1-phosphate receptors and gastrin releasing peptide receptor. We also summarize their tissue distributions, activation and roles in tumorigenesis and discuss the potential use of GPCR agonists and antagonists in cancer therapy.

  7. Regulation of G protein-coupled receptor export trafficking

    PubMed Central

    Dong, Chunmin; Filipeanu, Catalin M.; Duvernay, Matthew T.; Wu, Guangyu

    2007-01-01

    G protein-coupled receptors (GPCRs) constitute a superfamily of cell-surface receptors which share a common topology of seven transmembrane domains and modulate a variety of cell functions through coupling to heterotrimeric G proteins by responding to a vast array of stimuli. The magnitude of cellular response elicited by a given signal is dictated by the level of GPCR expression at the plasma membrane, which is the balance of elaborately regulated endocytic and exocytic trafficking. This review will cover recent advances in understanding the molecular mechanism underlying anterograde transport of the newly synthesized GPCRs from the endoplasmic reticulum (ER) through the Golgi to the plasma membrane. We will focus on recently identified motifs involved in GPCR exit from the ER and the Golgi, GPCR folding in the ER and the rescue of misfolded receptors from within, GPCR-interacting proteins that modulate receptor cell-surface targeting, pathways that mediate GPCR traffic, and the functional role of export in controlling GPCR signaling. PMID:17074298

  8. Heterotrimeric G proteins regulate nitrogen-use efficiency in rice.

    PubMed

    Sun, Hongying; Qian, Qian; Wu, Kun; Luo, Jijing; Wang, Shuansuo; Zhang, Chengwei; Ma, Yanfei; Liu, Qian; Huang, Xianzhong; Yuan, Qingbo; Han, Ruixi; Zhao, Meng; Dong, Guojun; Guo, Longbiao; Zhu, Xudong; Gou, Zhiheng; Wang, Wen; Wu, Yuejin; Lin, Hongxuan; Fu, Xiangdong

    2014-06-01

    The drive toward more sustainable agriculture has raised the profile of crop plant nutrient-use efficiency. Here we show that a major rice nitrogen-use efficiency quantitative trait locus (qNGR9) is synonymous with the previously identified gene DEP1 (DENSE AND ERECT PANICLES 1). The different DEP1 alleles confer different nitrogen responses, and genetic diversity analysis suggests that DEP1 has been subjected to artificial selection during Oryza sativa spp. japonica rice domestication. The plants carrying the dominant dep1-1 allele exhibit nitrogen-insensitive vegetative growth coupled with increased nitrogen uptake and assimilation, resulting in improved harvest index and grain yield at moderate levels of nitrogen fertilization. The DEP1 protein interacts in vivo with both the Gα (RGA1) and Gβ (RGB1) subunits, and reduced RGA1 or enhanced RGB1 activity inhibits nitrogen responses. We conclude that the plant G protein complex regulates nitrogen signaling and modulation of heterotrimeric G protein activity provides a strategy for environmentally sustainable increases in rice grain yield.

  9. Quantifying agonist activity at G protein-coupled receptors.

    PubMed

    Ehlert, Frederick J; Suga, Hinako; Griffin, Michael T

    2011-12-26

    When an agonist activates a population of G protein-coupled receptors (GPCRs), it elicits a signaling pathway that culminates in the response of the cell or tissue. This process can be analyzed at the level of a single receptor, a population of receptors, or a downstream response. Here we describe how to analyze the downstream response to obtain an estimate of the agonist affinity constant for the active state of single receptors. Receptors behave as quantal switches that alternate between active and inactive states (Figure 1). The active state interacts with specific G proteins or other signaling partners. In the absence of ligands, the inactive state predominates. The binding of agonist increases the probability that the receptor will switch into the active state because its affinity constant for the active state (K(b)) is much greater than that for the inactive state (K(a)). The summation of the random outputs of all of the receptors in the population yields a constant level of receptor activation in time. The reciprocal of the concentration of agonist eliciting half-maximal receptor activation is equivalent to the observed affinity constant (K(obs)), and the fraction of agonist-receptor complexes in the active state is defined as efficacy (ε) (Figure 2). Methods for analyzing the downstream responses of GPCRs have been developed that enable the estimation of the K(obs) and relative efficacy of an agonist. In this report, we show how to modify this analysis to estimate the agonist K(b) value relative to that of another agonist. For assays that exhibit constitutive activity, we show how to estimate K(b) in absolute units of M(-1). Our method of analyzing agonist concentration-response curves consists of global nonlinear regression using the operational model. We describe a procedure using the software application, Prism (GraphPad Software, Inc., San Diego, CA). The analysis yields an estimate of the product of K(obs) and a parameter proportional to efficacy (

  10. G protein-coupled receptors and the regulation of autophagy

    PubMed Central

    Wauson, Eric M.; Dbouk, Hashem A.; Ghosh, Anwesha B.; Cobb, Melanie H.

    2014-01-01

    Autophagy is an important catabolic cellular process that eliminates damaged and unnecessary cytoplasmic proteins and organelles. Basal autophagy occurs during normal physiological conditions, but the activity of this process can be significantly altered in human diseases. Thus, defining the regulatory inputs and signals that control autophagy is essential. Nutrients are key modulators of autophagy. While autophagy is generally accepted to be regulated in a cell autonomous fashion, recent studies suggest nutrients can modulate autophagy in a systemic manner by inducing the secretion of hormones and neurotransmitters that regulate G protein-coupled receptors (GPCRs). Emerging studies show that GPCRs also regulate autophagy by directly detecting extracellular nutrients. We review the role of GPCRs in autophagy regulation, highlighting their potential as therapeutic drug targets. PMID:24751357

  11. How Can Mutations Thermostabilize G-Protein-Coupled Receptors?

    PubMed

    Vaidehi, Nagarajan; Grisshammer, Reinhard; Tate, Christopher G

    2016-01-01

    Structures of over 30 different G-protein-coupled receptors (GPCRs) have advanced our understanding of cell signaling and have provided a foundation for structure-guided drug design. This exciting progress has required the development of three complementary methods to facilitate GPCR crystallization, one of which is the thermostabilization of receptors by systematic mutagenesis. However, the reason why a particular mutation, or combination of mutations, stabilizes the receptor is not always evident from a static crystal structure. Molecular dynamics (MD) simulations have been used to identify and estimate the energetic factors that affect thermostability through comparing the dynamics of the thermostabilized receptors with structure-based models of the wild-type receptor. The data indicate that receptors are stabilized through a combination of factors, including an increase in receptor rigidity, a decrease in collective motion, reduced stress at specific residues, and the presence of ordered water molecules. Predicting thermostabilizing mutations computationally represents a major challenge for the field.

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

    NASA Astrophysics Data System (ADS)

    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.

  13. Function and Regulation of Heterotrimeric G Proteins during Chemotaxis.

    PubMed

    Kamp, Marjon E; Liu, Youtao; Kortholt, Arjan

    2016-01-14

    Chemotaxis, or directional movement towards an extracellular gradient of chemicals, is necessary for processes as diverse as finding nutrients, the immune response, metastasis and wound healing. Activation of G-protein coupled receptors (GPCRs) is at the very base of the chemotactic signaling pathway. Chemotaxis starts with binding of the chemoattractant to GPCRs at the cell-surface, which finally leads to major changes in the cytoskeleton and directional cell movement towards the chemoattractant. Many chemotaxis pathways that are directly regulated by Gβγ have been identified and studied extensively; however, whether Gα is just a handle that regulates the release of Gβγ or whether Gα has its own set of distinct chemotactic effectors, is only beginning to be understood. In this review, we will discuss the different levels of regulation in GPCR signaling and the downstream pathways that are essential for proper chemotaxis.

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

    PubMed

    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.

  15. Cell-free expression of G-protein-coupled receptors.

    PubMed

    Orbán, Erika; Proverbio, Davide; Haberstock, Stefan; Dötsch, Volker; Bernhard, Frank

    2015-01-01

    Cell-free expression has emerged as a new standard for the production of membrane proteins. The reduction of expression complexity in cell-free systems eliminates central bottlenecks and allows the reliable and efficient synthesis of many different types of membrane proteins. Furthermore, the open accessibility of cell-free reactions enables the co-translational solubilization of cell-free expressed membrane proteins in a large variety of supplied additives. Hydrophobic environments can therefore be adjusted according to the requirements of individual membrane protein targets. We present different approaches for the preparative scale cell-free production of G-protein-coupled receptors using the extracts of Escherichia coli cells. We exemplify expression conditions implementing detergents, nanodiscs, or liposomes. The generated protein samples could be directly used for further functional characterization.

  16. Cross-Pharmacology Analysis of G Protein-Coupled Receptors

    PubMed Central

    Briansó, Ferran; Carrascosa, Maria C.; Oprea, Tudor I.; Mestres, Jordi

    2013-01-01

    The degree of applicability of chemogenomic approaches to protein families depends on the accuracy and completeness of pharmacological data and the corresponding level of pharmacological similarity observed among their protein members. The recent public domain availability of pharmacological data for thousands of small molecules on 204 G protein-coupled receptors (GPCRs) provides a firm basis for an in-depth cross-pharmacology analysis of this superfamily. The number of protein targets included in the cross-pharmacology profile of the different GPCRs changes significantly upon varying the ligand similarity and binding affinity criteria. However, with the exception of muscarinic receptors, aminergic GPCRs distinguish themselves from the rest of the members in the family by their remarkably high levels of pharmacological similarity among them. Clusters of non-GPCR targets related by cross-pharmacology with particular GPCRs are identified and the implications for unwanted side-effects, as well as for repurposing opportunities, discussed. PMID:21851335

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

    PubMed

    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.

  18. Crystal Structure of a Lipid G Protein-Coupled Receptor

    SciTech Connect

    Hanson, Michael A; Roth, Christopher B; Jo, Euijung; Griffith, Mark T; Scott, Fiona L; Reinhart, Greg; Desale, Hans; Clemons, Bryan; Cahalan, Stuart M; Schuerer, Stephan C; Sanna, M Germana; Han, Gye Won; Kuhn, Peter; Rosen, Hugh; Stevens, Raymond C

    2012-03-01

    The lyso-phospholipid sphingosine 1-phosphate modulates lymphocyte trafficking, endothelial development and integrity, heart rate, and vascular tone and maturation by activating G protein-coupled sphingosine 1-phosphate receptors. Here, we present the crystal structure of the sphingosine 1-phosphate receptor 1 fused to T4-lysozyme (S1P1-T4L) in complex with an antagonist sphingolipid mimic. Extracellular access to the binding pocket is occluded by the amino terminus and extracellular loops of the receptor. Access is gained by ligands entering laterally between helices I and VII within the transmembrane region of the receptor. This structure, along with mutagenesis, agonist structure-activity relationship data, and modeling, provides a detailed view of the molecular recognition and requirement for hydrophobic volume that activates S1P1, resulting in the modulation of immune and stromal cell responses.

  19. GPCRDB: an information system for G protein-coupled receptors.

    PubMed Central

    Horn, F; Weare, J; Beukers, M W; Hörsch, S; Bairoch, A; Chen, W; Edvardsen, O; Campagne, F; Vriend, G

    1998-01-01

    The GPCRDB is a G protein-coupled receptor (GPCR) database system aimed at the collection and dissemination of GPCR related data. It holds sequences, mutant data and ligand binding constants as primary (experimental) data. Computationally derived data such as multiple sequence alignments, three dimensional models, phylogenetic trees and two dimensional visualization tools are added to enhance the database's usefulness. The GPCRDB is an EU sponsored project aimed at building a generic molecular class specific database capable of dealing with highly heterogeneous data. GPCRs were chosen as test molecules because of their enormous importance for medical sciences and due to the availability of so much highly heterogeneous data. The GPCRDB is available via the WWW at http://www.gpcr.org/7tm PMID:9399852

  20. Serial Femtosecond Crystallography of G Protein-Coupled Receptors

    PubMed Central

    Liu, Wei; Wacker, Daniel; Gati, Cornelius; Han, Gye Won; James, Daniel; Wang, Dingjie; Nelson, Garrett; Weierstall, Uwe; Katritch, Vsevolod; Barty, Anton; Zatsepin, Nadia A.; Li, Dianfan; Messerschmidt, Marc; Boutet, Sébastien; Williams, Garth J.; Koglin, Jason E.; Seibert, M. Marvin; Wang, Chong; Shah, Syed T.A.; Basu, Shibom; Fromme, Raimund; Kupitz, Christopher; Rendek, Kimberley N.; Grotjohann, Ingo; Fromme, Petra; Kirian, Richard A.; Beyerlein, Kenneth R.; White, Thomas A.; Chapman, Henry N.; Caffrey, Martin; Spence, John C.H.; Stevens, Raymond C.; Cherezov, Vadim

    2014-01-01

    X-ray crystallography of G protein-coupled receptors and other membrane proteins is hampered by difficulties associated with growing sufficiently large crystals that withstand radiation damage and yield high-resolution data at synchrotron sources. Here we used an x-ray free-electron laser (XFEL) with individual 50-fs duration x-ray pulses to minimize radiation damage and obtained a high-resolution room temperature structure of a human serotonin receptor using sub-10 µm microcrystals grown in a membrane mimetic matrix known as lipidic cubic phase. Compared to the structure solved by traditional microcrystallography from cryo-cooled crystals of about two orders of magnitude larger volume, the room temperature XFEL structure displays a distinct distribution of thermal motions and conformations of residues that likely more accurately represent the receptor structure and dynamics in a cellular environment. PMID:24357322

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

    PubMed

    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.

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

    PubMed Central

    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

  3. Peptide drugs to target G protein-coupled receptors.

    PubMed

    Bellmann-Sickert, Kathrin; Beck-Sickinger, Annette G

    2010-09-01

    Major indications for use of peptide-based therapeutics include endocrine functions (especially diabetes mellitus and obesity), infectious diseases, and cancer. Whereas some peptide pharmaceuticals are drugs, acting as agonists or antagonists to directly treat cancer, others (including peptide diagnostics and tumour-targeting pharmaceuticals) use peptides to 'shuttle' a chemotherapeutic agent or a tracer to the tumour and allow sensitive imaging or targeted therapy. Significant progress has been made in the last few years to overcome disadvantages in peptide design such as short half-life, fast proteolytic cleavage, and low oral bioavailability. These advances include peptide PEGylation, lipidisation or multimerisation; the introduction of peptidomimetic elements into the sequences; and innovative uptake strategies such as liposomal, capsule or subcutaneous formulations. This review focuses on peptides targeting G protein-coupled receptors that are promising drug candidates or that have recently entered the pharmaceutical market.

  4. G-protein-coupled receptors, Hedgehog signaling and primary cilia.

    PubMed

    Mukhopadhyay, Saikat; Rohatgi, Rajat

    2014-09-01

    The Hedgehog (Hh) pathway has become an important model to study the cell biology of primary cilia, and reciprocally, the study of ciliary processes provides an opportunity to solve longstanding mysteries in the mechanism of vertebrate Hh signal transduction. The cilium is emerging as an unique compartment for G-protein-coupled receptor (GPCR) signaling in many systems. Two members of the GPCR family, Smoothened and Gpr161, play important roles in the Hh pathway. We review the current understanding of how these proteins may function to regulate Hh signaling and also highlight some of the critical unanswered questions being tackled by the field. Uncovering GPCR-regulated mechanisms important in Hh signaling may provide therapeutic strategies against the Hh pathway that plays important roles in development, regeneration and cancer.

  5. Lysophospholipid activation of G protein-coupled receptors.

    PubMed

    Mutoh, Tetsuji; Chun, Jerold

    2008-01-01

    One of the major lipid biology discoveries in last decade was the broad range of physiological activities of lysophospholipids that have been attributed to the actions of lysophospholipid receptors. The most well characterized lysophospholipids are lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P). Documented cellular effects of these lipid mediators include growth-factor-like effects on cells, such as proliferation, survival, migration, adhesion, and differentiation. The mechanisms for these actions are attributed to a growing family of 7-transmembrane, G protein-coupled receptors (GPCRs). Their pathophysiological actions include immune modulation, neuropathic pain modulation, platelet aggregation, wound healing, vasopressor activity, and angiogenesis. Here we provide a brief introduction to receptor-mediated lysophospholipid signaling and physiology, and then discuss potential therapeutic roles in human diseases.

  6. Lysophospholipids and their G protein-coupled receptors in atherosclerosis.

    PubMed

    Li, Ya-Feng; Li, Rong-Shan; Samuel, Sonia B; Cueto, Ramon; Li, Xin-Yuan; Wang, Hong; Yang, Xiao-Feng

    2016-01-01

    Lysophospholipids (LPLs) are bioactive lipid-derived signaling molecules generated by the enzymatic and chemical processes of regiospecific phospholipases on substrates such as membrane phospholipids (PLs) and sphingolipids (SLs). They play a major role as extracellular mediators by activating G-protein coupled receptors (GPCRs) and stimulating diverse cellular responses from their signaling pathways. LPLs are involved in various pathologies of the vasculature system including coronary heart disease and hypertension. Many studies suggest the importance of LPLs in their association with the development of atherosclerosis, a chronic and severe vascular disease. This paper focuses on the pathophysiological effects of different lysophospholipids on atherosclerosis, which may promote the pathogenesis of myocardial infarction and strokes. Their atherogenic biological activities take place in vascular endothelial cells, vascular smooth muscle cells, fibroblasts, monocytes and macrophages, dendritic cells, T-lymphocytes, platelets, etc.

  7. Therapeutic antibodies directed at G protein-coupled receptors.

    PubMed

    Hutchings, Catherine J; Koglin, Markus; Marshall, Fiona H

    2010-01-01

    G protein-coupled receptors (GPCRs) are one of the most important classes of targets for small molecule drug discovery, but many current GPCRs of interest are proving intractable to small molecule discovery and may be better approached with bio-therapeutics. GPCRs are implicated in a wide variety of diseases where antibody therapeutics are currently used. These include inflammatory diseases such as rheumatoid arthritis and Crohn disease, as well as metabolic disease and cancer. Raising antibodies to GPCRs has been difficult due to problems in obtaining suitable antigen because GPCRs are often expressed at low levels in cells and are very unstable when purified. A number of new developments in over-expressing receptors, as well as formulating stable pure protein, are contributing to the growing interest in targeting GPCRs with antibodies. This review discusses the opportunities for targeting GPCRs with antibodies using these approaches and describes the therapeutic antibodies that are currently in clinical development.

  8. Lysophospholipids and their G protein-coupled receptors in atherosclerosis

    PubMed Central

    Li, Ya-Feng; Li, Rong-Shan; Samuel, Sonia B.; Cueto, Ramon; Li, Xin-Yuan; Wang, Hong; Yang, Xiao-Feng

    2015-01-01

    Lysophospholipids (LPLs) are bioactive lipid-derived signaling molecules generated by the enzymatic and chemical processes of regiospecific phospholipases on substrates such as membrane phospholipids (PLs) and sphingolipids (SLs). They play a major role as extracellular mediators by activating G-protein coupled receptors (GPCRs) and stimulating diverse cellular responses from their signaling pathways. LPLs are involved in various pathologies of the vasculature system including coronary heart disease and hypertension. Many studies suggest the importance of LPLs in their association with the development of atherosclerosis, a chronic and severe vascular disease. This paper focuses on the pathophysiological effects of different lysophospholipids on atherosclerosis, which may promote the pathogenesis of myocardial infarction and strokes. Their atherogenic biological activities take place in vascular endothelial cells, vascular smooth muscle cells, fibroblasts, monocytes and macrophages, dendritic cells, T-lymphocytes, platelets, etc. PMID:26594106

  9. Therapeutic antibodies directed at G protein-coupled receptors

    PubMed Central

    Hutchings, Catherine J; Koglin, Markus

    2010-01-01

    G protein-coupled receptors (GPCRs) are one of the most important classes of targets for small molecule drug discovery, but many current GPCRs of interest are proving intractable to small molecule discovery and may be better approached with bio-therapeutics. GPCRs are implicated in a wide variety of diseases where antibody therapeutics are currently used. These include inflammatory diseases such as rheumatoid arthritis and Crohn disease, as well as metabolic disease and cancer. Raising antibodies to GPCRs has been difficult due to problems in obtaining suitable antigen because GPCRs are often expressed at low levels in cells and are very unstable when purified. A number of new developments in overexpressing receptors, as well as formulating stable pure protein, are contributing to the growing interest in targeting GPCRs with antibodies. This review discusses the opportunities for targeting GPCRs with antibodies using these approaches and describes the therapeutic antibodies that are currently in clinical development. PMID:20864805

  10. Anatomical profiling of G protein-coupled receptor expression

    PubMed Central

    Regard, Jean B.; Sato, Isaac T.; Coughlin, Shaun R.

    2008-01-01

    Summary G protein-coupled receptors (GPCRs) comprise the largest family of transmembrane signaling molecules and regulate a host of physiological and disease processes. To better understand the functions of GPCRs in vivo, we quantified transcript levels of 353 non-odorant GPCRs in 41 adult mouse tissues. Cluster analysis placed many GPCRs into anticipated anatomical and functional groups and predicted novel roles for less studied receptors. From one such prediction, we showed that the Gpr91 ligand succinate can regulate lipolysis in white adipose tissue suggesting that signaling by this citric acid cycle intermediate may regulate energy homeostasis. We also showed that pairwise analysis of GPCR expression across tissues may help predict drug side effects. This resource will aid studies to understand GPCR function in vivo and may assist in the identification of therapeutic targets. PMID:18984166

  11. Enhancement of G Protein-Coupled Receptor Surface Expression

    PubMed Central

    Dunham, Jill H.; Hall, Randy A.

    2009-01-01

    G protein-coupled receptors (GPCRs) mediate physiological responses to a diverse array of stimuli and are the molecular targets for numerous therapeutic drugs. GPCRs primarily signal from the plasma membrane, but when expressed in heterologous cells many GPCRs exhibit poor trafficking to the cell surface. Multiple approaches have been taken to enhance GPCR surface expression in heterologous cells, including addition/deletion of receptor sequences, co-expression with interacting proteins, and treatment with pharmacological chaperones. In addition to allowing for enhanced surface expression of certain GPCRs in heterologous cells, these approaches have also shed light on the control of GPCR trafficking in vivo and in some cases have led to new therapeutic approaches for treating human diseases that result from defects in GPCR trafficking. PMID:19679364

  12. Presynaptic G Protein-Coupled Receptors: Gatekeepers of Addiction?

    PubMed Central

    Johnson, Kari A.; Lovinger, David M.

    2016-01-01

    Drug abuse and addiction cause widespread social and public health problems, and the neurobiology underlying drug actions and drug use and abuse is an area of intensive research. Drugs of abuse alter synaptic transmission, and these actions contribute to acute intoxication as well as the chronic effects of abused substances. Transmission at most mammalian synapses involves neurotransmitter activation of two receptor subtypes, ligand-gated ion channels that mediate fast synaptic responses and G protein-coupled receptors (GPCRs) that have slower neuromodulatory actions. The GPCRs represent a large proportion of neurotransmitter receptors involved in almost all facets of nervous system function. In addition, these receptors are targets for many pharmacotherapeutic agents. Drugs of abuse directly or indirectly affect neuromodulation mediated by GPCRs, with important consequences for intoxication, drug taking and responses to prolonged drug exposure, withdrawal and addiction. Among the GPCRs are several subtypes involved in presynaptic inhibition, most of which are coupled to the Gi/o class of G protein. There is increasing evidence that these presynaptic Gi/o-coupled GPCRs have important roles in the actions of drugs of abuse, as well as behaviors related to these drugs. This topic will be reviewed, with particular emphasis on receptors for three neurotransmitters, Dopamine (DA; D1- and D2-like receptors), Endocannabinoids (eCBs; CB1 receptors) and glutamate (group II metabotropic glutamate (mGlu) receptors). The focus is on recent evidence from laboratory animal models (and some evidence in humans) implicating these receptors in the acute and chronic effects of numerous abused drugs, as well as in the control of drug seeking and taking. The ability of drugs targeting these receptors to modify drug seeking behavior has raised the possibility of using compounds targeting these receptors for addiction pharmacotherapy. This topic is also discussed, with emphasis on

  13. Identification and genetic mapping of the putative Thinopyrum intermedium-derived dominant powdery mildew resistance gene PmL962 on wheat chromosome arm 2BS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Powdery mildew, caused by Blumeria graminis f. sp. tritici (Bgt), is a destructive disease affecting the production of wheat (Triticum aestivum). Powdery mildew resistance was putatively transferred from Thinopyrum intermedium to the common wheat line L962, which conferred resistance to multiple Ch...

  14. Chemical biology methods for investigating G protein-coupled receptor signaling.

    PubMed

    Huber, Thomas; Sakmar, Thomas P

    2014-09-18

    G protein-coupled receptors (GPCRs) are targets for a quarter of prescription drugs. Despite recent progress in structural biology of GPCRs, only few key conformational states in the signal transduction process have been elucidated. Agonist ligands frequently display functional selectivity where activated receptors are biased to either G protein- or arrestin-mediated downstream signaling pathways. Selective manipulation of individual steps in the GPCR activation scheme requires precise information about the kinetics of ligand binding and the dynamics of downstream signaling. One approach is to obtain time-resolved information using receptors tagged with fluorescent or structural probes. Recent advances allow for site-specific introduction of genetically encoded unnatural amino acids into expressed GPCRs. We describe how bioorthogonal functional groups on GPCRs enable the mapping of receptor-ligand interactions and how bioorthogonal chemical reactions can be used to introduce fluorescent labels for single-molecule fluorescence applications to study the kinetics and conformational dynamics of GPCR signaling complexes ("signalosomes").

  15. An expressed sequence tag (EST) data mining strategy succeeding in the discovery of new G-protein coupled receptors.

    PubMed

    Wittenberger, T; Schaller, H C; Hellebrand, S

    2001-03-30

    We have developed a comprehensive expressed sequence tag database search method and used it for the identification of new members of the G-protein coupled receptor superfamily. Our approach proved to be especially useful for the detection of expressed sequence tag sequences that do not encode conserved parts of a protein, making it an ideal tool for the identification of members of divergent protein families or of protein parts without conserved domain structures in the expressed sequence tag database. At least 14 of the expressed sequence tags found with this strategy are promising candidates for new putative G-protein coupled receptors. Here, we describe the sequence and expression analysis of five new members of this receptor superfamily, namely GPR84, GPR86, GPR87, GPR90 and GPR91. We also studied the genomic structure and chromosomal localization of the respective genes applying in silico methods. A cluster of six closely related G-protein coupled receptors was found on the human chromosome 3q24-3q25. It consists of four orphan receptors (GPR86, GPR87, GPR91, and H963), the purinergic receptor P2Y1, and the uridine 5'-diphosphoglucose receptor KIAA0001. It seems likely that these receptors evolved from a common ancestor and therefore might have related ligands. In conclusion, we describe a data mining procedure that proved to be useful for the identification and first characterization of new genes and is well applicable for other gene families.

  16. Visualization of arrestin recruitment by a G Protein-Coupled Receptor

    PubMed Central

    Reis, Rosana I.; Huang, Li-Yin; Tripathi-Shukla, Prachi; Qian, Jiang; Li, Sheng; Blanc, Adi; Oleskie, Austin N.; Dosey, Anne M.; Su, Min; Liang, Cui-Rong; Gu, Ling-Ling; Shan, Jin-Ming; Chen, Xin; Hanna, Rachel; Choi, Minjung; Yao, Xiao Jie; Klink, Bjoern U.; Kahsai, Alem W.; Sidhu, Sachdev S.; Koide, Shohei; Penczek, Pawel A.; Kossiakoff, Anthony A.; Jr, Virgil L. Woods; Kobilka, Brian K.; Skiniotis, Georgios; Lefkowitz, Robert J.

    2014-01-01

    G Protein Coupled Receptors (GPCRs) are critically regulated by β-arrestins (βarrs), which not only desensitize G protein signaling but also initiate a G protein independent wave of signaling1-5. A recent surge of structural data on a number of GPCRs, including the β2 adrenergic receptor (β2AR)-G protein complex, has provided novel insights into the structural basis of receptor activation6-11. Lacking however has been complementary information on recruitment of βarrs to activated GPCRs primarily due to challenges in obtaining stable receptor-βarr complexes for structural studies. Here, we devised a strategy for forming and purifying a functional β2AR-βarr1 complex that allowed us to visualize its architecture by single particle negative stain electron microscopy (EM) and to characterize the interactions between β2AR and βarr1 using hydrogen-deuterium exchange mass spectrometry (HDXMS) and chemical cross-linking. EM 2D averages and 3D reconstructions reveal bimodal binding of βarr1 to the β2AR, involving two separate sets of interactions, one with the phosphorylated carboxy-terminus of the receptor and the other with its seven-transmembrane core. Areas of reduced HDX together with identification of cross-linked residues suggest engagement of the finger loop of βarr1 with the seven-transmembrane core of the receptor. In contrast, focal areas of increased HDX indicate regions of increased dynamics in both N and C domains of βarr1 when coupled to the β2AR. A molecular model of the β2AR-βarr signaling complex was made by docking activated βarr1 and β2AR crystal structures into the EM map densities with constraints provided by HDXMS and cross-linking, allowing us to obtain valuable insights into the overall architecture of a receptor-arrestin complex. The dynamic and structural information presented herein provides a framework for better understanding the basis of GPCR regulation by arrestins. PMID:25043026

  17. Phylogenetic analysis of fungal heterotrimeric G protein-encoding genes and their expression during dimorphism in Mucor circinelloides.

    PubMed

    Valle-Maldonado, Marco Iván; Jácome-Galarza, Irvin Eduardo; Díaz-Pérez, Alma Laura; Martínez-Cadena, Guadalupe; Campos-García, Jesús; Ramírez-Díaz, Martha Isela; Reyes-De la Cruz, Homero; Riveros-Rosas, Héctor; Díaz-Pérez, César; Meza-Carmen, Víctor

    2015-12-01

    In fungi, heterotrimeric G proteins are key regulators of biological processes such as mating, virulence, morphology, among others. Mucor circinelloides is a model organism for many biological processes, and its genome contains the largest known repertoire of genes that encode putative heterotrimeric G protein subunits in the fungal kingdom: twelve Gα (McGpa1-12), three Gβ (McGpb1-3), and three Gγ (McGpg1-3). Phylogenetic analysis of fungal Gα showed that they are divided into four distinct groups as reported previously. Fungal Gβ and Gγ are also divided into four phylogenetic groups, and to our understanding this is the first report of a phylogenetic classification for fungal Gβ and Gγ subunits. Almost all genes that encode putative heterotrimeric G subunits in M. circinelloides are differentially expressed during dimorphic growth, except for McGpg1 (Gγ) that showed very low mRNA levels at all developmental stages. Moreover, several of the subunits are expressed in a similar pattern and at the same level, suggesting that they constitute discrete complexes. For example, McGpb3 (Gβ), and McGpg2 (Gγ), are co-expressed during mycelium growth, and McGpa1, McGpb2, and McGpg2, are co-expressed during yeast development. These findings provide the conceptual framework to study the biological role of these genes during M. circinelloides morphogenesis.

  18. Rap G protein signal in normal and disordered lymphohematopoiesis.

    PubMed

    Minato, Nagahiro

    2013-09-10

    Rap proteins (Rap1, Rap2a, b, c) are small molecular weight GTPases of the Ras family. Rap G proteins mediate diverse cellular events such as cell adhesion, proliferation, and gene activation through various signaling pathways. Activation of Rap signal is regulated tightly by several specific regulatory proteins including guanine nucleotide exchange factors and GTPase-activating proteins. Beyond cell biological studies, increasing attempts have been made in the past decade to define the roles of Rap signal in specific functions of normal tissue systems as well as in cancer. In the immune and hematopoietic systems, Rap signal plays crucial roles in the development and function of essentially all lineages of lymphocytes and hematopoietic cells, and importantly, deregulated Rap signal may lead to unique pathological conditions depending on the affected cell types, including various types of leukemia and autoimmunity. The phenotypical studies have unveiled novel, even unexpected functional aspects of Rap signal in cells from a variety of tissues, providing potentially important clues for controlling human diseases, including malignancy.

  19. Differential Localization of G Protein βγ Subunits

    PubMed Central

    2015-01-01

    G protein βγ subunits play essential roles in regulating cellular signaling cascades, yet little is known about their distribution in tissues or their subcellular localization. While previous studies have suggested specific isoforms may exhibit a wide range of distributions throughout the central nervous system, a thorough investigation of the expression patterns of both Gβ and Gγ isoforms within subcellular fractions has not been conducted. To address this, we applied a targeted proteomics approach known as multiple-reaction monitoring to analyze localization patterns of Gβ and Gγ isoforms in pre- and postsynaptic fractions isolated from cortex, cerebellum, hippocampus, and striatum. Particular Gβ and Gγ subunits were found to exhibit distinct regional and subcellular localization patterns throughout the brain. Significant differences in subcellular localization between pre- and postsynaptic fractions were observed within the striatum for most Gβ and Gγ isoforms, while others exhibited completely unique expression patterns in all four brain regions examined. Such differences are a prerequisite for understanding roles of individual subunits in regulating specific signaling pathways throughout the central nervous system. PMID:24568373

  20. Multifactorial Regulation of G Protein-Coupled Receptor Endocytosis

    PubMed Central

    Zhang, Xiaohan; Kim, Kyeong-Man

    2017-01-01

    Endocytosis is a process by which cells absorb extracellular materials via the inward budding of vesicles formed from the plasma membrane. Receptor-mediated endocytosis is a highly selective process where receptors with specific binding sites for extracellular molecules internalize via vesicles. G protein-coupled receptors (GPCRs) are the largest single family of plasma-membrane receptors with more than 1000 family members. But the molecular mechanisms involved in the regulation of GPCRs are believed to be highly conserved. For example, receptor phosphorylation in collaboration with β-arrestins plays major roles in desensitization and endocytosis of most GPCRs. Nevertheless, a number of subsequent studies showed that GPCR regulation, such as that by endocytosis, occurs through various pathways with a multitude of cellular components and processes. This review focused on i) functional interactions between homologous and heterologous pathways, ii) methodologies applied for determining receptor endocytosis, iii) experimental tools to determine specific endocytic routes, iv) roles of small guanosine triphosphate-binding proteins in GPCR endocytosis, and v) role of post-translational modification of the receptors in endocytosis. PMID:28035080

  1. G protein-coupled estrogen receptor protects from atherosclerosis.

    PubMed

    Meyer, Matthias R; Fredette, Natalie C; Howard, Tamara A; Hu, Chelin; Ramesh, Chinnasamy; Daniel, Christoph; Amann, Kerstin; Arterburn, Jeffrey B; Barton, Matthias; Prossnitz, Eric R

    2014-12-23

    Coronary atherosclerosis and myocardial infarction in postmenopausal women have been linked to inflammation and reduced nitric oxide (NO) formation. Natural estrogen exerts protective effects on both processes, yet also displays uterotrophic activity. Here, we used genetic and pharmacologic approaches to investigate the role of the G protein-coupled estrogen receptor (GPER) in atherosclerosis. In ovary-intact mice, deletion of gper increased atherosclerosis progression, total and LDL cholesterol levels and inflammation while reducing vascular NO bioactivity, effects that were in some cases aggravated by surgical menopause. In human endothelial cells, GPER was expressed on intracellular membranes and mediated eNOS activation and NO formation, partially accounting for estrogen-mediated effects. Chronic treatment with G-1, a synthetic, highly selective small molecule agonist of GPER, reduced postmenopausal atherosclerosis and inflammation without uterotrophic effects. In summary, this study reveals an atheroprotective function of GPER and introduces selective GPER activation as a novel therapeutic approach to inhibit postmenopausal atherosclerosis and inflammation in the absence of uterotrophic activity.

  2. Structural organization of G-protein-coupled receptors

    NASA Astrophysics Data System (ADS)

    Lomize, Andrei L.; Pogozheva, Irina D.; Mosberg, Henry I.

    1999-07-01

    Atomic-resolution structures of the transmembrane 7-α-helical domains of 26 G-protein-coupled receptors (GPCRs) (including opsins, cationic amine, melatonin, purine, chemokine, opioid, and glycoprotein hormone receptors and two related proteins, retinochrome and Duffy erythrocyte antigen) were calculated by distance geometry using interhelical hydrogen bonds formed by various proteins from the family and collectively applied as distance constraints, as described previously [Pogozheva et al., Biophys. J., 70 (1997) 1963]. The main structural features of the calculated GPCR models are described and illustrated by examples. Some of the features reflect physical interactions that are responsible for the structural stability of the transmembrane α-bundle: the formation of extensive networks of interhelical H-bonds and sulfur-aromatic clusters that are spatially organized as 'polarity gradients' the close packing of side-chains throughout the transmembrane domain; and the formation of interhelical disulfide bonds in some receptors and a plausible Zn2+ binding center in retinochrome. Other features of the models are related to biological function and evolution of GPCRs: the formation of a common 'minicore' of 43 evolutionarily conserved residues; a multitude of correlated replacements throughout the transmembrane domain; an Na+-binding site in some receptors, and excellent complementarity of receptor binding pockets to many structurally dissimilar, conformationally constrained ligands, such as retinal, cyclic opioid peptides, and cationic amine ligands. The calculated models are in good agreement with numerous experimental data.

  3. Peptide ligand recognition by G protein-coupled receptors

    PubMed Central

    Krumm, Brian E.

    2015-01-01

    The past few years have seen spectacular progress in the structure determination of G protein-coupled receptors (GPCRs). We now have structural representatives from classes A, B, C, and F. Within the rhodopsin-like class A, most structures belong to the α group, whereas fewer GPCR structures are available from the β, γ, and δ groups, which include peptide GPCRs such as the receptors for neurotensin (β group), opioids, chemokines (γ group), and protease-activated receptors (δ group). Structural information on peptide GPCRs is restricted to complexes with non-peptidic drug-like antagonists with the exception of the chemokine receptor CXCR4 that has been crystallized in the presence of a cyclic peptide antagonist. Notably, the neurotensin receptor 1 is to date the only peptide GPCR whose structure has been solved in the presence of a peptide agonist. Although limited in number, the current peptide GPCR structures reveal great diversity in shape and electrostatic properties of the ligand binding pockets, features that play key roles in the discrimination of ligands. Here, we review these aspects of peptide GPCRs in view of possible models for peptide agonist binding. PMID:25852552

  4. Rap G protein signal in normal and disordered lymphohematopoiesis

    SciTech Connect

    Minato, Nagahiro

    2013-09-10

    Rap proteins (Rap1, Rap2a, b, c) are small molecular weight GTPases of the Ras family. Rap G proteins mediate diverse cellular events such as cell adhesion, proliferation, and gene activation through various signaling pathways. Activation of Rap signal is regulated tightly by several specific regulatory proteins including guanine nucleotide exchange factors and GTPase-activating proteins. Beyond cell biological studies, increasing attempts have been made in the past decade to define the roles of Rap signal in specific functions of normal tissue systems as well as in cancer. In the immune and hematopoietic systems, Rap signal plays crucial roles in the development and function of essentially all lineages of lymphocytes and hematopoietic cells, and importantly, deregulated Rap signal may lead to unique pathological conditions depending on the affected cell types, including various types of leukemia and autoimmunity. The phenotypical studies have unveiled novel, even unexpected functional aspects of Rap signal in cells from a variety of tissues, providing potentially important clues for controlling human diseases, including malignancy.

  5. Structure and Function of Serotonin G protein Coupled Receptors

    PubMed Central

    McCorvy, John D.; Roth, Bryan L.

    2015-01-01

    Serotonin receptors are prevalent throughout the nervous system and the periphery, and remain one of the most lucrative and promising drug discovery targets for disorders ranging from migraine headaches to neuropsychiatric disorders such as schizophrenia and depression. There are 14 distinct serotonin receptors, of which 13 are G protein coupled receptors (GPCRs), which are targets for approximately 40% of the approved medicines. Recent crystallographic and biochemical evidence has provided a converging understanding of the basic structure and functional mechanics of GPCR activation. Currently, two GPCR crystal structures exist for the serotonin family, the 5-HT1B and 5-HT2B receptor, with the antimigraine and valvulopathic drug ergotamine bound. The first serotonin crystal structures not only provide the first evidence of serotonin receptor topography but also provide mechanistic explanations into functional selectivity or biased agonism. This review will detail the findings of these crystal structures from a molecular and mutagenesis perspective for driving rational drug design for novel therapeutics incorporating biased signaling. PMID:25601315

  6. G-Protein-Coupled Receptors in Adult Neurogenesis

    PubMed Central

    Doze, Van A.

    2012-01-01

    The importance of adult neurogenesis has only recently been accepted, resulting in a completely new field of investigation within stem cell biology. The regulation and functional significance of adult neurogenesis is currently an area of highly active research. G-protein-coupled receptors (GPCRs) have emerged as potential modulators of adult neurogenesis. GPCRs represent a class of proteins with significant clinical importance, because approximately 30% of all modern therapeutic treatments target these receptors. GPCRs bind to a large class of neurotransmitters and neuromodulators such as norepinephrine, dopamine, and serotonin. Besides their typical role in cellular communication, GPCRs are expressed on adult neural stem cells and their progenitors that relay specific signals to regulate the neurogenic process. This review summarizes the field of adult neurogenesis and its methods and specifies the roles of various GPCRs and their signal transduction pathways that are involved in the regulation of adult neural stem cells and their progenitors. Current evidence supporting adult neurogenesis as a model for self-repair in neuropathologic conditions, adult neural stem cell therapeutic strategies, and potential avenues for GPCR-based therapeutics are also discussed. PMID:22611178

  7. G Protein-coupled Estrogen Receptor Protects from Atherosclerosis

    PubMed Central

    Meyer, Matthias R.; Fredette, Natalie C.; Howard, Tamara A.; Hu, Chelin; Ramesh, Chinnasamy; Daniel, Christoph; Amann, Kerstin; Arterburn, Jeffrey B.; Barton, Matthias; Prossnitz, Eric R.

    2014-01-01

    Coronary atherosclerosis and myocardial infarction in postmenopausal women have been linked to inflammation and reduced nitric oxide (NO) formation. Natural estrogen exerts protective effects on both processes, yet also displays uterotrophic activity. Here, we used genetic and pharmacologic approaches to investigate the role of the G protein-coupled estrogen receptor (GPER) in atherosclerosis. In ovary-intact mice, deletion of gper increased atherosclerosis progression, total and LDL cholesterol levels and inflammation while reducing vascular NO bioactivity, effects that were in some cases aggravated by surgical menopause. In human endothelial cells, GPER was expressed on intracellular membranes and mediated eNOS activation and NO formation, partially accounting for estrogen-mediated effects. Chronic treatment with G-1, a synthetic, highly selective small molecule agonist of GPER, reduced postmenopausal atherosclerosis and inflammation without uterotrophic effects. In summary, this study reveals an atheroprotective function of GPER and introduces selective GPER activation as a novel therapeutic approach to inhibit postmenopausal atherosclerosis and inflammation in the absence of uterotrophic activity. PMID:25532911

  8. Adhesion family of G protein-coupled receptors and cancer.

    PubMed

    Lin, Hsi-Hsien

    2012-01-01

    The adhesion-class G protein-coupled receptors (adhesion-GPCRs) constitute the second largest GPCR sub-family in humans. Adhesion-GPCRs are defined by the chimeric structure of an unusually large extracellular cell-adhesion domain and a GPCR-like seven-pass transmembrane domain. Adhesion-GPCRs are hence expected to display both cellular adhesion and signaling functions in many biological systems. Adhesion-GPCRs are normally expressed in the central nervous, immune, and reproductive systems in a cell type- or tissue-restricted fashion. However, aberrant expression of distinct adhesion-GPCR molecules has been identified in various human cancers with some of the receptors closely associated with cancer development. Tumor-associated adhesion-GPCRs are thought to involve in tumorigenesis by affecting the growth of tumor cells, angiogenesis, tumor cell migration, invasion and metastasis either positively or negatively. Furthermore, some adhesion-GPCRs are considered potential biomarkers for specific types of cancers. In this review article, the expressional characteristics and functional role of cancer-associated adhesion-GPCRs are discussed in depth.

  9. Minireview: Nutrient Sensing by G Protein-Coupled Receptors

    PubMed Central

    Wauson, Eric M.; Lorente-Rodríguez, Andrés

    2013-01-01

    G protein-coupled receptors (GPCRs) are membrane proteins that recognize molecules in the extracellular milieu and transmit signals inside cells to regulate their behaviors. Ligands for many GPCRs are hormones or neurotransmitters that direct coordinated, stereotyped adaptive responses. Ligands for other GPCRs provide information to cells about the extracellular environment. Such information facilitates context-specific decision making that may be cell autonomous. Among ligands that are important for cellular decisions are amino acids, required for continued protein synthesis, as metabolic starting materials and energy sources. Amino acids are detected by a number of class C GPCRs. One cluster of amino acid-sensing class C GPCRs includes umami and sweet taste receptors, GPRC6A, and the calcium-sensing receptor. We have recently found that the umami taste receptor heterodimer T1R1/T1R3 is a sensor of amino acid availability that regulates the activity of the mammalian target of rapamycin. This review focuses on an array of findings on sensing amino acids and sweet molecules outside of neurons by this cluster of class C GPCRs and some of the physiologic processes regulated by them. PMID:23820899

  10. G protein-coupled receptors participate in cytokinesis.

    PubMed

    Zhang, Xin; Bedigian, Anne V; Wang, Wenchao; Eggert, Ulrike S

    2012-10-01

    Cytokinesis, the last step during cell division, is a highly coordinated process that involves the relay of signals from both the outside and inside of the cell. We have a basic understanding of how cells regulate internal events, but how cells respond to extracellular cues is less explored. In a systematic RNAi screen of G protein-coupled receptors (GPCRs) and their effectors, we found that some GPCRs are involved in cytokinesis. RNAi knockdown of these GPCRs caused increased binucleated cell formation, and live cell imaging showed that most formed midbodies but failed at the abscission stage. OR2A4 (olfactory receptor, family 2, subfamily A, member 4) localized to cytokinetic structures in cells and its knockdown caused cytokinesis failure at an earlier stage, likely due to effects on the actin cytoskeleton. Identifying the downstream components that transmit GPCR signals during cytokinesis will be the next step and we show that GIPC1 (GIPC PDZ domain containing family, member 1), an adaptor protein for GPCRs, may play a part. RNAi knockdown of GIPC1 significantly increased binucleated cell formation. Understanding the molecular details of GPCRs and their interaction proteins in cytokinesis regulation will give us important clues about GPCRs signaling as well as how cells communicate with their environment during division.

  11. Crystal structure of the Bacillus-conserved MazG protein, a nucleotide pyrophosphohydrolase.

    PubMed

    Kim, Meong Il; Hong, Minsun

    2016-03-25

    BA1544 from Bacillus anthracis was previously annotated as a transcription factor for the gene cluster ba1554 - ba1558, but has not been experimentally characterized. B. anthracis is an obligate pathogen causing fatal inhalational anthrax, and BA1544 is absolutely conserved in Bacillus species, including Bacillus cereus, Bacillus thuringiensis and Bacillus mycoides, with 100% sequence identity. To address the function of BA1544, we performed structural and biochemical studies, which revealed that BA1544 is a MazG protein. Thus, herein, the protein is defined as Bacillus-conserved MazG (BcMazG). Like other MazG structures, BcMazG assembles into a tetrameric architecture. Each monomer adopts a four-α-helix bundle that accommodates a metal ion using four acidic residues, and presents one putative substrate-binding site. Enzymatic characterization demonstrated that BcMazG is a nucleoside triphosphate (NTP) pyrophosphohydrolase and prefers adenosine triphosphate as a substrate among canonical NTPs. Moreover, structural comparison of BcMazG with its homologues revealed a potential regulation mechanism whereby the enzymatic activity of BcMazG is regulated by its C-terminal region.

  12. G protein coupled receptor 18: A potential role for endocannabinoid signaling in metabolic dysfunction.

    PubMed

    Rajaraman, Gayathri; Simcocks, Anna; Hryciw, Deanne H; Hutchinson, Dana S; McAinch, Andrew J

    2016-01-01

    Endocannabinoids are products of dietary fatty acids that are modulated by an alteration in food intake levels. Overweight and obese individuals have substantially higher circulating levels of the arachidonic acid derived endocannabinoids, anandamide and 2-arachidonoyl glycerol, and show an altered pattern of cannabinoid receptor expression. These cannabinoid receptors are part of a large family of G protein coupled receptors (GPCRs). GPCRs are major therapeutic targets for various diseases within the cardiovascular, neurological, gastrointestinal, and endocrine systems, as well as metabolic disorders such as obesity and type 2 diabetes mellitus. Obesity is considered a state of chronic low-grade inflammation elicited by an immunological response. Interestingly, the newly deorphanized GPCR (GPR18), which is considered to be a putative cannabinoid receptor, is proposed to have an immunological function. In this review, the current scientific knowledge on GPR18 is explored including its localization, signaling pathways, and pharmacology. Importantly, the involvement of nutritional factors and potential dietary regulation of GPR18 and its (patho)physiological roles are described. Further research on this receptor and its regulation will enable a better understanding of the complex mechanisms of GPR18 and its potential as a novel therapeutic target for treating metabolic disorders.

  13. Interaction of G protein coupled receptors and cholesterol.

    PubMed

    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.

  14. G-protein coupled receptor expression patterns delineate medulloblastoma subgroups

    PubMed Central

    2013-01-01

    Background Medulloblastoma is the most common malignant brain tumor in children. Genetic profiling has identified four principle tumor subgroups; each subgroup is characterized by different initiating mutations, genetic and clinical profiles, and prognoses. The two most well-defined subgroups are caused by overactive signaling in the WNT and SHH mitogenic pathways; less is understood about Groups 3 and 4 medulloblastoma. Identification of tumor subgroup using molecular classification is set to become an important component of medulloblastoma diagnosis and staging, and will likely guide therapeutic options. However, thus far, few druggable targets have emerged. G-protein coupled receptors (GPCRs) possess characteristics that make them ideal targets for molecular imaging and therapeutics; drugs targeting GPCRs account for 30-40% of all current pharmaceuticals. While expression patterns of many proteins in human medulloblastoma subgroups have been discerned, the expression pattern of GPCRs in medulloblastoma has not been investigated. We hypothesized that analysis of GPCR expression would identify clear subsets of medulloblastoma and suggest distinct GPCRs that might serve as molecular targets for both imaging and therapy. Results Our study found that medulloblastoma tumors fall into distinct clusters based solely on GPCR expression patterns. Normal cerebellum clustered separately from the tumor samples. Further, two of the tumor clusters correspond with high fidelity to the WNT and SHH subgroups of medulloblastoma. Distinct over-expressed GPCRs emerge; for example, LGR5 and GPR64 are significantly and uniquely over-expressed in the WNT subgroup of tumors, while PTGER4 is over-expressed in the SHH subgroup. Uniquely under-expressed GPCRs were also observed. Our key findings were independently validated using a large international dataset. Conclusions Our results identify GPCRs with potential to act as imaging and therapeutic targets. Elucidating tumorigenic pathways

  15. G-protein-coupled receptor kinase 2 terminates G-protein-coupled receptor function in steroid hormone 20-hydroxyecdysone signaling

    PubMed Central

    Zhao, Wen-Li; Wang, Di; Liu, Chun-Yan; Zhao, Xiao-Fan

    2016-01-01

    G-protein-coupled receptors (GPCRs) transmit extracellular signals across the cell membrane. GPCR kinases (GRKs) desensitize GPCR signals in the cell membrane. However, the role and mechanism of GRKs in the desensitization of steroid hormone signaling are unclear. In this study, we propose that GRK2 is phosphorylated by protein kinase C (PKC) in response to induction by the steroid hormone 20-hydroxyecdysone (20E), which determines its translocation to the cell membrane of the lepidopteran Helicoverpa armigera. GRK2 protein expression is increased during the metamorphic stage because of induction by 20E. Knockdown of GRK2 in larvae causes accelerated pupation, an increase in 20E-response gene expression, and advanced apoptosis and metamorphosis. 20E induces translocation of GRK2 from the cytoplasm to the cell membrane via steroid hormone ecdysone-responsive GPCR (ErGPCR-2). GRK2 is phosphorylated by PKC on serine 680 after induction by 20E, which leads to the translocation of GRK2 to the cell membrane. GRK2 interacts with ErGPCR-2. These data indicate that GRK2 terminates the ErGPCR-2 function in 20E signaling in the cell membrane by a negative feedback mechanism. PMID:27412951

  16. G-Protein/β-Arrestin-Linked Fluctuating Network of G-Protein-Coupled Receptors for Predicting Drug Efficacy and Bias Using Short-Term Molecular Dynamics Simulation

    PubMed Central

    Ichikawa, Osamu; Fujimoto, Kazushi; Yamada, Atsushi; Okazaki, Susumu; Yamazaki, Kazuto

    2016-01-01

    The efficacy and bias of signal transduction induced by a drug at a target protein are closely associated with the benefits and side effects of the drug. In particular, partial agonist activity and G-protein/β-arrestin-biased agonist activity for the G-protein-coupled receptor (GPCR) family, the family with the most target proteins of launched drugs, are key issues in drug discovery. However, designing GPCR drugs with appropriate efficacy and bias is challenging because the dynamic mechanism of signal transduction induced by ligand—receptor interactions is complicated. Here, we identified the G-protein/β-arrestin-linked fluctuating network, which initiates large-scale conformational changes, using sub-microsecond molecular dynamics (MD) simulations of the β2-adrenergic receptor (β2AR) with a diverse collection of ligands and correlation analysis of their G protein/β-arrestin efficacy. The G-protein-linked fluctuating network extends from the ligand-binding site to the G-protein-binding site through the connector region, and the β-arrestin-linked fluctuating network consists of the NPxxY motif and adjacent regions. We confirmed that the averaged values of fluctuation in the fluctuating network detected are good quantitative indexes for explaining G protein/β-arrestin efficacy. These results indicate that short-term MD simulation is a practical method to predict the efficacy and bias of any compound for GPCRs. PMID:27187591

  17. Genetic mapping reveals that sinefungin resistance in Toxoplasma gondii is controlled by a putative amino acid transporter locus that can be used as a negative selectable marker.

    PubMed

    Behnke, Michael S; Khan, Asis; Sibley, L David

    2015-02-01

    Quantitative trait locus (QTL) mapping studies have been integral in identifying and understanding virulence mechanisms in the parasite Toxoplasma gondii. In this study, we interrogated a different phenotype by mapping sinefungin (SNF) resistance in the genetic cross between type 2 ME49-FUDR(r) and type 10 VAND-SNF(r). The genetic map of this cross was generated by whole-genome sequencing of the progeny and subsequent identification of single nucleotide polymorphisms (SNPs) inherited from the parents. Based on this high-density genetic map, we were able to pinpoint the sinefungin resistance phenotype to one significant locus on chromosome IX. Within this locus, a single nonsynonymous SNP (nsSNP) resulting in an early stop codon in the TGVAND_290860 gene was identified, occurring only in the sinefungin-resistant progeny. Using CRISPR/CAS9, we were able to confirm that targeted disruption of TGVAND_290860 renders parasites sinefungin resistant. Because disruption of the SNR1 gene confers resistance, we also show that it can be used as a negative selectable marker to insert either a positive drug selection cassette or a heterologous reporter. These data demonstrate the power of combining classical genetic mapping, whole-genome sequencing, and CRISPR-mediated gene disruption for combined forward and reverse genetic strategies in T. gondii.

  18. Molecular cloning and characterization of two novel retinoic acid-inducible orphan G-protein-coupled receptors (GPRC5B and GPRC5C).

    PubMed

    Robbins, M J; Michalovich, D; Hill, J; Calver, A R; Medhurst, A D; Gloger, I; Sims, M; Middlemiss, D N; Pangalos, M N

    2000-07-01

    Using homology searching of public databases with a metabotropic glutamate receptor sequence from Caenorhabditis elegans, two novel protein sequences (named RAIG-2 (HGMW-approved symbol GPRC5B) and RAIG-3 (HGMW-approved symbol GPRC5C) were identified containing seven putative transmembrane domains characteristic of G-protein-coupled receptors (GPCRs). RAIG-2 and RAIG-3 encode open reading frames of 403 and 442 amino acid polypeptides, respectively, and show 58% similarity to the recently identified retinoic acid-inducible gene-1 (RAIG-1, HGMW-approved symbol RAI3). Analysis of the three protein sequences places them within the type 3 GPCR family, which includes metabotropic glutamate receptors, GABA(B) receptors, calcium-sensing receptors, and pheromone receptors. However, in contrast to other type 3 GPCRs, RAIG-1, RAIG-2, and RAIG-3 have only short N-terminal domains. RAIG-2 and RAIG-3 cDNA sequences were cloned into the mammalian expression vector pcDNA3 with c-myc or HA epitope tags inserted at their N-termini, respectively. Transient transfection experiments in HEK239T cells using these constructs demonstrated RAIG-2 and RAIG-3 expression at the cell surface. Distribution profiles of mRNA expression obtained by semiquantitative Taq-Man PCR analysis showed RAIG-2 to be predominantly expressed in human brain areas and RAIG-3 to be predominantly expressed in peripheral tissues. In addition, expression of RAIG-2 and RAIG-3 mRNA was increased following treatment with all-trans-retinoic acid in a manner similar to that previously described for RAIG-1. Finally, RAIG-2 was mapped to chromosome 16p12 (D16S405-D16S3045) and RAIG-3 to chromosome 17q25 (D17S1352-D17S785). These results suggest that RAIG-1, RAIG-2, and RAIG-3 represent a novel family of retinoic acid-inducible receptors, most closely related to the type 3 GPCR subfamily, and provide further evidence for a linkage between retinoic acid and G-protein-coupled receptor signal transduction pathways.

  19. Novel antigen design for the generation of antibodies to G-protein-coupled receptors.

    PubMed

    Larsson, K; Hofström, C; Lindskog, C; Hansson, M; Angelidou, P; Hökfelt, T; Uhlén, M; Wernérus, H; Gräslund, T; Hober, S

    2011-07-29

    Antibodies are important tools for the study of G-protein-coupled receptors, key proteins in cellular signaling. Due to their large hydrophobic membrane spanning regions and often very short loops exposed on the surface of the cells, generation of antibodies able to recognize the receptors in the endogenous environment has been difficult. Here, we describe an antigen-design method where the extracellular loops and N-terminus are combined to a single antigen for generation of antibodies specific to three selected GPCRs: NPY5R, B2ARN and GLP1R. The design strategy enabled straightforward antigen production and antibody generation. Binding of the antibodies to intact receptors was analyzed using flow cytometry and immunofluorescence based confocal microscopy on A-431 cells overexpressing the respective GPCR. The antibody-antigen interactions were characterized using epitope mapping, and the antibodies were applied in immunohistochemical staining of human tissues. Most of the antibodies showed specific binding to their respective overexpressing cell line but not to the non-transfected cells, thus indicating binding to their respective target receptor. The epitope mapping showed that sub-populations within the purified antibody pool recognized different regions of the antigen. Hence, the genetic combination of several different epitopes enables efficient generation of specific antibodies with potential use in several applications for the study of endogenous receptors.

  20. Third-Party Bioluminescence Resonance Energy Transfer Indicates Constitutive Association of Membrane Proteins: Application to Class A G-Protein-Coupled Receptors and G-Proteins

    PubMed Central

    Kuravi, Sudhakiranmayi; Lan, Tien-Hung; Barik, Arnab; Lambert, Nevin A.

    2010-01-01

    Abstract Many of the molecules that mediate G-protein signaling are thought to constitutively associate with each other in variably stable signaling complexes. Much of the evidence for signaling complexes has come from Förster resonance energy transfer and bioluminescence resonance energy transfer (BRET) studies. However, detection of constitutive protein association with these methods is hampered by nonspecific energy transfer that occurs when donor and acceptor molecules are in close proximity by chance. We show that chemically-induced recruitment of local third-party BRET donors or acceptors reliably separates nonspecific and specific BRET. We use this method to reexamine the constitutive association of class A G-protein-coupled receptors (GPCRs) with other GPCRs and with heterotrimeric G-proteins. We find that β2 adrenoreceptors constitutively associate with each other and with several other class A GPCRs. In contrast, GPCRs and G-proteins are unlikely to exist in stable constitutive preassembled complexes. PMID:20483349

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

    PubMed Central

    Lynch, Jennifer R.; Wang, Jenny Yingzi

    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 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. PMID:27187360

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

    PubMed

    Lynch, Jennifer R; Wang, Jenny Yingzi

    2016-05-11

    G protein-coupled receptors (GPCRs) are a large superfamily of cell-surface signaling proteins that bind extracellular ligands and transduce signals into cells via heterotrimeric G proteins. GPCRs are highly tractable drug targets. Aberrant expression of GPCRs and G proteins has been observed in various cancers and their importance in cancer stem cells has begun to be appreciated. We have recently reported essential roles for G protein-coupled receptor 84 (GPR84) and G protein subunit Gαq in the maintenance of cancer stem cells in acute myeloid leukemia. This review will discuss how GPCRs and G proteins regulate stem cells with a focus on cancer stem cells, as well as their implications for the development of novel targeted cancer therapies.

  3. Recent Progress in Understanding the Conformational Mechanism of Heterotrimeric G Protein Activation

    PubMed Central

    Duc, Nguyen Minh; Kim, Hee Ryung; Chung, Ka Young

    2017-01-01

    Heterotrimeric G proteins are key intracellular coordinators that receive signals from cells through activation of cognate G protein-coupled receptors (GPCRs). The details of their atomic interactions and structural mechanisms have been described by many biochemical and biophysical studies. Specifically, a framework for understanding conformational changes in the receptor upon ligand binding and associated G protein activation was provided by description of the crystal structure of the β2-adrenoceptor-Gs complex in 2011. This review focused on recent findings in the conformational dynamics of G proteins and GPCRs during activation processes. PMID:28035078

  4. Polycomb Group (PcG) Proteins and Human Cancers: Multifaceted Functions and Therapeutic Implications

    PubMed Central

    Wang, Wei; Qin, Jiang-Jiang; Voruganti, Sukesh; Nag, Subhasree; Zhou, Jianwei; Zhang, Ruiwen

    2016-01-01

    Polycomb group (PcG) proteins are transcriptional repressors that regulate several crucial developmental and physiological processes in the cell. More recently, they have been found to play important roles in human carcinogenesis and cancer development and progression. The deregulation and dysfunction of PcG proteins often lead to blocking or inappropriate activation of developmental pathways, enhancing cellular proliferation, inhibiting apoptosis, and increasing the cancer stem cell population. Genetic and molecular investigations of PcG proteins have long been focused on their PcG functions. However, PcG proteins have recently been shown to exert non-polycomb functions, contributing to the regulation of diverse cellular functions. We and others have demonstrated that PcG proteins regulate the expression and function of several oncogenes and tumor suppressor genes in a PcG-independent manner, and PcG proteins are associated with the survival of patients with cancer. In this review, we summarize the recent advances in the research on PcG proteins, including both the polycomb-repressive and non-polycomb functions. We specifically focus on the mechanisms by which PcG proteins play roles in cancer initiation, development, and progression. Finally, we discuss the potential value of PcG proteins as molecular biomarkers for the diagnosis and prognosis of cancer, and as molecular targets for cancer therapy. PMID:26227500

  5. Novel Agonist Bioisosteres and Common Structure-Activity Relationships for The Orphan G Protein-Coupled Receptor GPR139

    PubMed Central

    Shehata, Mohamed A.; Nøhr, Anne C.; Lissa, Delphine; Bisig, Christoph; Isberg, Vignir; Andersen, Kirsten B.; Harpsøe, Kasper; Björkling, Fredrik; Bräuner-Osborne, Hans; Gloriam, David E.

    2016-01-01

    GPR139 is an orphan class A G protein-coupled receptor found mainly in the central nervous system. It has its highest expression levels in the hypothalamus and striatum, regions regulating metabolism and locomotion, respectively, and has therefore been suggested as a potential target for obesity and Parkinson’s disease. The two aromatic amino acids L-Trp and L-Phe have been proposed as putative endogenous agonists, and three structurally related benzohydrazide, glycine benzamide, and benzotriazine surrogate agonist series have been published. Herein, we assayed 158 new analogues selected from a pharmacophore model, and identified 12 new GPR139 agonists, containing previously untested bioisosteres. Furthermore, we present the first combined structure-activity relationships, and a refined pharmacophore model to serve as a rationale for future ligand identification and optimization. PMID:27830715

  6. Evidence for a bacterial lipopolysaccharide-recognizing G-protein-coupled receptor in the bacterial engulfment by Entamoeba histolytica.

    PubMed

    Brewer, Matthew T; Agbedanu, Prince N; Zamanian, Mostafa; Day, Tim A; Carlson, Steve A

    2013-11-01

    Entamoeba histolytica is the causative agent of amoebic dysentery, a worldwide protozoal disease that results in approximately 100,000 deaths annually. The virulence of E. histolytica may be due to interactions with the host bacterial flora, whereby trophozoites engulf colonic bacteria as a nutrient source. The engulfment process depends on trophozoite recognition of bacterial epitopes that activate phagocytosis pathways. E. histolytica GPCR-1 (EhGPCR-1) was previously recognized as a putative G-protein-coupled receptor (GPCR) used by Entamoeba histolytica during phagocytosis. In the present study, we attempted to characterize EhGPCR-1 by using heterologous GPCR expression in Saccharomyces cerevisiae. We discovered that bacterial lipopolysaccharide (LPS) is an activator of EhGPCR-1 and that LPS stimulates EhGPCR-1 in a concentration-dependent manner. Additionally, we demonstrated that Entamoeba histolytica prefers to engulf bacteria with intact LPS and that this engulfment process is sensitive to suramin, which prevents the interactions of GPCRs and G-proteins. Thus, EhGPCR-1 is an LPS-recognizing GPCR that is a potential drug target for treatment of amoebiasis, especially considering the well-established drug targeting to GPCRs.

  7. The repertoire of G protein-coupled receptors in the sea squirt Ciona intestinalis

    PubMed Central

    2008-01-01

    Background G protein-coupled receptors (GPCRs) constitute a large family of integral transmembrane receptor proteins that play a central role in signal transduction in eukaryotes. The genome of the protochordate Ciona intestinalis has a compact size with an ancestral complement of many diversified gene families of vertebrates and is a good model system for studying protochordate to vertebrate diversification. An analysis of the Ciona repertoire of GPCRs from a comparative genomic perspective provides insight into the evolutionary origins of the GPCR signalling system in vertebrates. Results We have identified 169 gene products in the Ciona genome that code for putative GPCRs. Phylogenetic analyses reveal that Ciona GPCRs have homologous representatives from the five major GRAFS (Glutamate, Rhodopsin, Adhesion, Frizzled and Secretin) families concomitant with other vertebrate GPCR repertoires. Nearly 39% of Ciona GPCRs have unambiguous orthologs of vertebrate GPCR families, as defined for the human, mouse, puffer fish and chicken genomes. The Rhodopsin family accounts for ~68% of the Ciona GPCR repertoire wherein the LGR-like subfamily exhibits a lineage specific gene expansion of a group of receptors that possess a novel domain organisation hitherto unobserved in metazoan genomes. Conclusion Comparison of GPCRs in Ciona to that in human reveals a high level of orthology of a protochordate repertoire with that of vertebrate GPCRs. Our studies suggest that the ascidians contain the basic ancestral complement of vertebrate GPCR genes. This is evident at the subfamily level comparisons since Ciona GPCR sequences are significantly analogous to vertebrate GPCR subfamilies even while exhibiting Ciona specific genes. Our analysis provides a framework to perform future experimental and comparative studies to understand the roles of the ancestral chordate versions of GPCRs that predated the divergence of the urochordates and the vertebrates. PMID:18452600

  8. Arabidopsis G-protein interactome reveals connections to cell wall carbohydrates and morphogenesis

    PubMed Central

    Klopffleisch, Karsten; Phan, Nguyen; Augustin, Kelsey; Bayne, Robert S; Booker, Katherine S; Botella, Jose R; Carpita, Nicholas C; Carr, Tyrell; Chen, Jin-Gui; Cooke, Thomas Ryan; Frick-Cheng, Arwen; Friedman, Erin J; Fulk, Brandon; Hahn, Michael G; Jiang, Kun; Jorda, Lucia; Kruppe, Lydia; Liu, Chenggang; Lorek, Justine; McCann, Maureen C; Molina, Antonio; Moriyama, Etsuko N; Mukhtar, M Shahid; Mudgil, Yashwanti; Pattathil, Sivakumar; Schwarz, John; Seta, Steven; Tan, Matthew; Temp, Ulrike; Trusov, Yuri; Urano, Daisuke; Welter, Bastian; Yang, Jing; Panstruga, Ralph; Uhrig, Joachim F; Jones, Alan M

    2011-01-01

    The heterotrimeric G-protein complex is minimally composed of Gα, Gβ, and Gγ subunits. In the classic scenario, the G-protein complex is the nexus in signaling from the plasma membrane, where the heterotrimeric G-protein associates with heptahelical G-protein-coupled receptors (GPCRs), to cytoplasmic target proteins called effectors. Although a number of effectors are known in metazoans and fungi, none of these are predicted to exist in their canonical forms in plants. To identify ab initio plant G-protein effectors and scaffold proteins, we screened a set of proteins from the G-protein complex using two-hybrid complementation in yeast. After deep and exhaustive interrogation, we detected 544 interactions between 434 proteins, of which 68 highly interconnected proteins form the core G-protein interactome. Within this core, over half of the interactions comprising two-thirds of the nodes were retested and validated as genuine in planta. Co-expression analysis in combination with phenotyping of loss-of-function mutations in a set of core interactome genes revealed a novel role for G-proteins in regulating cell wall modification. PMID:21952135

  9. Heterotrimeric G-protein shuttling via Gip1 extends the dynamic range of eukaryotic chemotaxis

    PubMed Central

    Kamimura, Yoichiro; Miyanaga, Yukihiro; Ueda, Masahiro

    2016-01-01

    Chemotactic eukaryote cells can sense chemical gradients over a wide range of concentrations via heterotrimeric G-protein signaling; however, the underlying wide-range sensing mechanisms are only partially understood. Here we report that a novel regulator of G proteins, G protein-interacting protein 1 (Gip1), is essential for extending the chemotactic range of Dictyostelium cells. Genetic disruption of Gip1 caused severe defects in gradient sensing and directed cell migration at high but not low concentrations of chemoattractant. Also, Gip1 was found to bind and sequester G proteins in cytosolic pools. Receptor activation induced G-protein translocation to the plasma membrane from the cytosol in a Gip1-dependent manner, causing a biased redistribution of G protein on the membrane along a chemoattractant gradient. These findings suggest that Gip1 regulates G-protein shuttling between the cytosol and the membrane to ensure the availability and biased redistribution of G protein on the membrane for receptor-mediated chemotactic signaling. This mechanism offers an explanation for the wide-range sensing seen in eukaryotic chemotaxis. PMID:27044073

  10. The interaction of acetylcholine receptors in porcine atrial membranes with three kinds of G proteins.

    PubMed

    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.

  11. Plant G-proteins come of age: Breaking the bond with animal models

    NASA Astrophysics Data System (ADS)

    Botella, Jimmy; Trusov, Yuri

    2016-05-01

    G-proteins are universal signal transducers mediating many cellular responses. Plant G-protein signaling has been modeled on the well-established animal paradigm but accumulated experimental evidence indicates that G-protein-dependent signaling in plants has taken a very different evolutionary path. Here we review the differences between plant and animal G-proteins reported over past two decades. Most importantly, while in animal systems the G-protein signaling cycle is activated by seven transmembrane-spanning G-protein coupled receptors, the existence of these type of receptors in plants is highly controversial. Instead plant G-proteins have been proven to be functionally associated with atypical receptors such as the Arabidopsis RGS1 and a number of receptor-like kinases. We propose that, instead of the GTP/GDP cycle used in animals, plant G-proteins are activated/de-activated by phosphorylation/de-phosphorylation. We discuss the need of a fresh new look at these signaling molecules and provide a hypothetical model that departs fromthe accepted animal paradigm.

  12. Plant G-Proteins Come of Age: Breaking the Bond with Animal Models

    PubMed Central

    Trusov, Yuri; Botella, José R.

    2016-01-01

    G-proteins are universal signal transducers mediating many cellular responses. Plant G-protein signaling has been modeled on the well-established animal paradigm but accumulated experimental evidence indicates that G-protein-dependent signaling in plants has taken a very different evolutionary path. Here we review the differences between plant and animal G-proteins reported over past two decades. Most importantly, while in animal systems the G-protein signaling cycle is activated by seven transmembrane-spanning G-protein coupled receptors, the existence of these type of receptors in plants is highly controversial. Instead plant G-proteins have been proven to be functionally associated with atypical receptors such as the Arabidopsis RGS1 and a number of receptor-like kinases. We propose that, instead of the GTP/GDP cycle used in animals, plant G-proteins are activated/de-activated by phosphorylation/de-phosphorylation. We discuss the need of a fresh new look at these signaling molecules and provide a hypothetical model that departs from the accepted animal paradigm. PMID:27252940

  13. Structural basis of GDP release and gating in G protein coupled Fe2+ transport

    PubMed Central

    Guilfoyle, Amy; Maher, Megan J; Rapp, Mikaela; Clarke, Ronald; Harrop, Stephen; Jormakka, Mika

    2009-01-01

    G proteins are key molecular switches in the regulation of membrane protein function and signal transduction. The prokaryotic membrane protein FeoB is involved in G protein coupled Fe2+ transport, and is unique in that the G protein is directly tethered to the membrane domain. Here, we report the structure of the soluble domain of FeoB, including the G protein domain, and its assembly into an unexpected trimer. Comparisons between nucleotide free and liganded structures reveal the closed and open state of a central cytoplasmic pore, respectively. In addition, these data provide the first observation of a conformational switch in the nucleotide-binding G5 motif, defining the structural basis for GDP release. From these results, structural parallels are drawn to eukaryotic G protein coupled membrane processes. PMID:19629046

  14. Model-Based Discovery of Synthetic Agonists for the Zn(2+)-Sensing G-Protein-Coupled Receptor 39 (GPR39) Reveals Novel Biological Functions.

    PubMed

    Frimurer, Thomas M; Mende, Franziska; Graae, Anne-Sofie; Engelstoft, Maja S; Egerod, Kristoffer L; Nygaard, Rie; Gerlach, Lars-Ole; Hansen, Jakob Bondo; Schwartz, Thue W; Holst, Birgitte

    2017-02-09

    The G-protein-coupled receptor 39 (GPR39) is a G-protein-coupled receptor activated by Zn(2+). We used a homology model-based approach to identify small-molecule pharmacological tool compounds for the receptor. The method focused on a putative binding site in GPR39 for synthetic ligands and knowledge of ligand binding to other receptors with similar binding pockets to select iterative series of minilibraries. These libraries were cherry-picked from all commercially available synthetic compounds. A total of only 520 compounds were tested in vitro, making this method broadly applicable for tool compound development. The compounds of the initial library were inactive when tested alone, but lead compounds were identified using Zn(2+) as an allosteric enhancer. Highly selective, highly potent Zn(2+)-independent GPR39 agonists were found in subsequent minilibraries. These agonists identified GPR39 as a novel regulator of gastric somatostatin secretion.

  15. Surface density of the Hendra G protein modulates Hendra F protein-promoted membrane fusion: Role for Hendra G protein trafficking and degradation

    SciTech Connect

    Whitman, Shannon D.; Dutch, Rebecca Ellis . E-mail: rdutc2@uky.edu

    2007-07-05

    Hendra virus, like most paramyxoviruses, requires both a fusion (F) and attachment (G) protein for promotion of cell-cell fusion. Recent studies determined that Hendra F is proteolytically processed by the cellular protease cathepsin L after endocytosis. This unique cathepsin L processing results in a small percentage of Hendra F on the cell surface. To determine how the surface densities of the two Hendra glycoproteins affect fusion promotion, we performed experiments that varied the levels of glycoproteins expressed in transfected cells. Using two different fusion assays, we found a marked increase in fusion when expression of the Hendra G protein was increased, with a 1:1 molar ratio of Hendra F:G on the cell surface resulting in optimal membrane fusion. Our results also showed that Hendra G protein levels are modulated by both more rapid protein turnover and slower protein trafficking than is seen for Hendra F.

  16. Signalling functions and biochemical properties of pertussis toxin-resistant G-proteins.

    PubMed Central

    Fields, T A; Casey, P J

    1997-01-01

    Pertussis toxin (PTX) has been widely used as a reagent to characterize the involvement of heterotrimeric G-proteins in signalling. This toxin catalyses the ADP-ribosylation of specific G-protein alpha subunits of the Gi family, and this modification prevents the occurrence of the receptor-G-protein interaction. This review focuses on the biochemical properties and signalling of those G-proteins historically classified as 'PTX-resistant' due to the inability of the toxin to influence signalling through them. These G-proteins include members of the Gq and G12 families and one Gi family member, i.e. Gz. Signalling pathways controlled by these G-proteins are well characterized only for Gq family members, which activate specific isoforms of phospholipase C, resulting in increases in intracellular calcium and activation of protein kinase C (PKC), among other responses. While members of the G12 family have been implicated in processes that regulate cell growth, and Gz has been shown to inhibit adenylate cyclase, the specific downstream targets to these G-proteins in vivo have not been clearly established. Since two of these proteins, G12 alpha and Gz alpha, are excellent substrates for PKC, there is the potential for cross-talk between their signalling and Gq-dependent processes leading to activation of PKC. In tissues that express these G-proteins, a number of guanine-nucleotide-dependent, PTX-resistant, signalling pathways have been defined for which the G-protein involved has not been identified. This review summarizes these pathways and discusses the evidence both for the participation of specific PTX-resistant G-proteins in them and for the regulation of these processes by PKC. PMID:9032437

  17. Ligand-Dependent Modulation of G Protein Conformation Alters Drug Efficacy.

    PubMed

    Furness, Sebastian George Barton; Liang, Yi-Lynn; Nowell, Cameron James; Halls, Michelle Louise; Wookey, Peter John; Dal Maso, Emma; Inoue, Asuka; Christopoulos, Arthur; Wootten, Denise; Sexton, Patrick Michael

    2016-10-20

    G protein-coupled receptor (GPCR) signaling, mediated by hetero-trimeric G proteins, can be differentially controlled by agonists. At a molecular level, this is thought to occur principally via stabilization of distinct receptor conformations by individual ligands. These distinct conformations control subsequent recruitment of transducer and effector proteins. Here, we report that ligand efficacy at the calcitonin GPCR (CTR) is also correlated with ligand-dependent alterations to G protein conformation. We observe ligand-dependent differences in the sensitivity of the G protein ternary complex to disruption by GTP, due to conformational differences in the receptor-bound G protein hetero-trimer. This results in divergent agonist-dependent receptor-residency times for the hetero-trimeric G protein and different accumulation rates for downstream second messengers. This study demonstrates that factors influencing efficacy extend beyond receptor conformation(s) and expands understanding of the molecular basis for how G proteins control/influence efficacy. This has important implications for the mechanisms that underlie ligand-mediated biased agonism. VIDEO ABSTRACT.

  18. Connecting G protein signaling to chemoattractant-mediated cell polarity and cytoskeletal reorganization.

    PubMed

    Liu, Youtao; Lacal, Jesus; Firtel, Richard A; Kortholt, Arjan

    2016-10-07

    The directional movement towards extracellular chemical gradients, a process called chemotaxis, is an important property of cells. Central to eukaryotic chemotaxis is the molecular mechanism by which chemoattractant-mediated activation of G-protein coupled receptors (GPCRs) induces symmetry breaking in the activated downstream signaling pathways. Studies with mainly Dictyostelium and mammalian neutrophils as experimental systems have shown that chemotaxis is mediated by a complex network of signaling pathways. Recently, several labs have used extensive and efficient proteomic approaches to further unravel this dynamic signaling network. Together these studies showed the critical role of the interplay between heterotrimeric G-protein subunits and monomeric G proteins in regulating cytoskeletal rearrangements during chemotaxis. Here we highlight how these proteomic studies have provided greater insight into the mechanisms by which the heterotrimeric G protein cycle is regulated, how heterotrimeric G proteins-induced symmetry breaking is mediated through small G protein signaling, and how symmetry breaking in G protein signaling subsequently induces cytoskeleton rearrangements and cell migration.

  19. Lamin A/C sustains PcG protein architecture, maintaining transcriptional repression at target genes.

    PubMed

    Cesarini, Elisa; Mozzetta, Chiara; Marullo, Fabrizia; Gregoretti, Francesco; Gargiulo, Annagiusi; Columbaro, Marta; Cortesi, Alice; Antonelli, Laura; Di Pelino, Simona; Squarzoni, Stefano; Palacios, Daniela; Zippo, Alessio; Bodega, Beatrice; Oliva, Gennaro; Lanzuolo, Chiara

    2015-11-09

    Beyond its role in providing structure to the nuclear envelope, lamin A/C is involved in transcriptional regulation. However, its cross talk with epigenetic factors--and how this cross talk influences physiological processes--is still unexplored. Key epigenetic regulators of development and differentiation are the Polycomb group (PcG) of proteins, organized in the nucleus as microscopically visible foci. Here, we show that lamin A/C is evolutionarily required for correct PcG protein nuclear compartmentalization. Confocal microscopy supported by new algorithms for image analysis reveals that lamin A/C knock-down leads to PcG protein foci disassembly and PcG protein dispersion. This causes detachment from chromatin and defects in PcG protein-mediated higher-order structures, thereby leading to impaired PcG protein repressive functions. Using myogenic differentiation as a model, we found that reduced levels of lamin A/C at the onset of differentiation led to an anticipation of the myogenic program because of an alteration of PcG protein-mediated transcriptional repression. Collectively, our results indicate that lamin A/C can modulate transcription through the regulation of PcG protein epigenetic factors.

  20. Heterotrimeric G proteins in green algae: an early innovation in the evolution of the plant lineage.

    PubMed

    Hackenberg, Dieter; Pandey, Sona

    2014-01-01

    Heterotrimeric G-proteins (G-proteins, hereafter) are important signaling components in all eukaryotes. The absence of these proteins in the sequenced genomes of Chlorophyaceaen green algae has raised questions about their evolutionary origin and prevalence in the plant lineage. The existence of G-proteins has often been correlated with the acquisition of embryophytic life-cycle and/or terrestrial habitats of plants which occurred around 450 million years ago. Our discovery of functional G-proteins in Chara braunii, a representative of the Charophycean green algae, establishes the existence of this conserved signaling pathway in the most basal plants and dates it even further back to 1-1.5 billion years ago. We have now identified the sequence homologs of G-proteins in additional algal families and propose that green algae represent a model system for one of the most basal forms of G-protein signaling known to exist to date. Given the possible differences that exist between plant and metazoan G-protein signaling mechanisms, such basal organisms will serve as important resources to trace the evolutionary origin of proposed mechanistic differences between the systems as well as their plant-specific functions.

  1. The G Protein α Chaperone Ric-8 as a Potential Therapeutic Target

    PubMed Central

    Papasergi, Makaía M.; Patel, Bharti R.

    2015-01-01

    Resistance to inhibitors of cholinesterase (Ric-8)A and Ric-8B are essential genes that encode positive regulators of heterotrimeric G protein α subunits. Controversy persists surrounding the precise way(s) that Ric-8 proteins affect G protein biology and signaling. Ric-8 proteins chaperone nucleotide-free Gα-subunit states during biosynthetic protein folding prior to G protein heterotrimer assembly. In organisms spanning the evolutionary window of Ric-8 expression, experimental perturbation of Ric-8 genes results in reduced functional abundances of G proteins because G protein α subunits are misfolded and degraded rapidly. Ric-8 proteins also act as Gα-subunit guanine nucleotide exchange factors (GEFs) in vitro. However, Ric-8 GEF activity could strictly be an in vitro phenomenon stemming from the ability of Ric-8 to induce partial Gα unfolding, thereby enhancing GDP release. Ric-8 GEF activity clearly differs from the GEF activity of G protein–coupled receptors (GPCRs). G protein βγ is inhibitory to Ric-8 action but obligate for receptors. It remains an open question whether Ric-8 has dual functions in cells and regulates G proteins as both a molecular chaperone and GEF. Clearly, Ric-8 has a profound influence on heterotrimeric G protein function. For this reason, we propose that Ric-8 proteins are as yet untested therapeutic targets in which pharmacological inhibition of the Ric-8/Gα protein–protein interface could serve to attenuate the effects of disease-causing G proteins (constitutively active mutants) and/or GPCR signaling. This minireview will chronicle the understanding of Ric-8 function, provide a comparative discussion of the Ric-8 molecular chaperoning and GEF activities, and support the case for why Ric-8 proteins should be considered potential targets for development of new therapies. PMID:25319541

  2. Allosteric mechanisms of G protein coupled receptor signaling: a structural perspective

    PubMed Central

    Thaker, Tarjani M.; Kaya, Ali I.; Preininger, Anita M.; Hamm, Heidi E.; Iverson, T.M.

    2012-01-01

    G protein-Coupled Receptors (GPCRs) use a complex series of intramolecular conformational changes to couple agonist binding to the binding and activation of cognate heterotrimeric G protein (Gαβγ). The mechanisms underlying this long-range activation have been identified using a variety of biochemical and structural approaches and have primarily used visual signal transduction via the GPCR rhodopsin and cognate heterotrimeric G protein transducin (Gt) as a model system. In this chapter, we will review the methods that have revealed allosteric signaling through rhodopsin and transducin. These methods can be applied to a variety of GPCR-mediated signaling pathways. PMID:22052489

  3. Functional Gene Group Analysis Indicates No Role for Heterotrimeric G Proteins in Cognitive Ability

    PubMed Central

    Davies, Gail; Liewald, David Cherry McLachlan; Payton, Anthony; Craig, Leone C. A.; Whalley, Lawrence J.; Horan, Mike; Ollier, William; Starr, John M.; Pendleton, Neil; Posthuma, Danielle; Bates, Timothy C.; Deary, Ian J.

    2014-01-01

    Previous functional gene group analyses implicated common single nucleotide polymorphisms (SNPs) in heterotrimeric G protein coding genes as being associated with differences in human intelligence. Here, we sought to replicate this finding using five independent cohorts of older adults including current IQ and childhood IQ, and using both gene- and SNP-based analytic strategies. No significant associations were found between variation in heterotrimeric G protein genes and intelligence in any cohort at either of the two time points. These results indicate that, whereas G protein systems are important in cognition, common genetic variation in these genes is unlikely to be a substantial influence on human intelligence differences. PMID:24626473

  4. Effects of transient oxygen-glucose deprivation on G-proteins and G-protein-coupled receptors in rat CA3 pyramidal cells in vitro.

    PubMed

    Tanabe, M; Gähwiler, B H; Gerber, U

    1998-06-01

    The role of guanosine triphosphate-binding proteins (G-proteins) in the generation of the outward current during transient oxygen-glucose deprivation (OGD) was investigated in CA3 pyramidal cells in rat hippocampal organotypic slice cultures using the single-electrode voltage-clamp technique with KMeSO4-filled microelectrodes. To simulate ischaemia, brief chemical OGD (2 mM 2-deoxyglucose and 3 mM NaN3 for 4-9 min) was used, which induced an outward K+ current associated with an increase in input conductance. OGD failed to induce the outward current under conditions where G-protein function was disrupted by loading cells with guanosine 5'-O-(2-thiodiphosphate) [GDPbetaS] or after prolonged injection of guanosine 5'-O(3-thiotdphosphate) [GTPgammaS]. However, in slices treated with pertussis toxin (PTX), OGD still elicited the outward current, indicating that PTX-insensitive G-proteins are involved. Consistent with this insensitivity to PTX, neither adenosine receptors nor GABA(B) (gamma-aminobutyric acid) receptors, which operate via PTX-sensitive G-proteins, mediate the OGD-induced outward current. When adenosine receptors or GABA(B) receptors were blocked with 1,3-dipropyl-8-psulphophenylxanthine (DPSPX, 5 microM) or CGP 52 432 (10 microM), respectively, the OGD-induced response was not modified. The response also persisted following pretreatment of slice cultures with tetanus toxin to prevent vesicular release of neurotransmitters and neuromodulators from presynaptic terminals. Both PTX-sensitive and PTX-insensitive G-protein-mediated responses were suppressed during OGD. The inward current induced by the metabotropic glutamate receptor agonist 1 S, 3R-1-aminocyclopentane-1,3-dicarboxylate (1S,3R-ACPD) and the outward current elicited by adenosine or baclofen were strongly or completely attenuated. In contrast, the ionotropic alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) response was not affected. These findings suggest that during OGD there is

  5. Mapping of the mutations present in the genome of the Rift Valley fever virus attenuated MP12 strain and their putative role in attenuation.

    PubMed

    Vialat, P; Muller, R; Vu, T H; Prehaud, C; Bouloy, M

    1997-11-01

    The MP12 attenuated strain of Rift Valley fever virus was obtained by 12 serial passages of a virulent isolate ZH548 in the presence of 5-fluorouracil (Caplen et al., 1985. Mutagen-directed attenuation of Rift Valley fever virus as a method for vaccine development. J. Gen. Virol., 66, 2271-2277). The comparison of the M segment of the two strains has already been reported by Takehara et al. (Takehara et al., 1989. Identification of mutations in the M RNA of a candidate vaccine strain of Rift Valley fever virus. Virology 169, 452-457). We have completed the comparison and found that altogether a total of nine, 12 and four nucleotides were changed in the L, M and S segments of the two strains, respectively. Three mutations induced amino acid changes in the L protein but none of them was located in the recognized motifs conserved among RNA dependent polymerases. In the S segment, a single change modified an amino acid in the NSs protein and in the M segment, seven of the mutations resulted in amino acid changes in each of the four encoded G1, G2, 14 kDa and 78 kDa proteins. Characterization of the MP12 virus indicated that determinants for attenuation were present in each segment and that they were introduced progressively during the 12 passages in the presence of the mutagen (Saluzzo and Smith, 1990. Use of reassortant viruses to map attenuating and temperature-sensitive mutations of the Rift Valley fever virus MP-12 vaccine. Vaccine 8, 369-375). Passages 4 and 7-9 were found to be essential for introduction of temperature-sensitive lesions and attenuation. In an attempt to correlate some of the mutations with the attenuated or temperature-sensitive phenotypes, we determined by sequencing the passage level at which the different mutations appeared. This work should help to address the question of the role of the viral gene products in Rift Valley fever pathogenesis.

  6. Activation of nematode G protein GOA-1 by the human muscarinic acetylcholine receptor M2 subtype. Functional coupling of G-protein-coupled receptor and G protein originated from evolutionarily distant animals.

    PubMed

    Minaba, Masaomi; Ichiyama, Susumu; Kojima, Katsura; Ozaki, Mamiko; Kato, Yusuke

    2006-12-01

    Signal transduction mediated by heterotrimeric G proteins regulates a wide variety of physiological functions. We are interested in the manipulation of G-protein-mediating signal transduction using G-protein-coupled receptors, which are derived from evolutionarily distant organisms and recognize unique ligands. As a model, we tested the functionally coupling GOA-1, G alpha(i/o) ortholog in the nematode Caenorhabditis elegans, with the human muscarinic acetylcholine receptor M2 subtype (M2), which is one of the mammalian G alpha(i/o)-coupled receptors. GOA-1 and M2 were prepared as a fusion protein using a baculovirus expression system. The affinity of the fusion protein for GDP was decreased by addition of a muscarinic agonist, carbamylcholine and the guanosine 5'-[3-O-thio]triphosphate ([35S]GTPgammaS) binding was increased with an increase in the carbamylcholine concentrations in a dose-dependent manner. These effects evoked by carbamylcholine were completely abolished by a full antagonist, atropine. In addition, the affinity for carbamylcholine decreased under the presence of GTP as reported for M2-G alpha(i/o) coupling. These results indicate that the M2 activates GOA-1 as well as G alpha(i/o).

  7. Monoclonal antibodies against G protein of spring viremia of carp virus.

    PubMed

    Luo, Peixiao; Ruan, Xindi; Zhang, Qi; Li, Zeming; Wang, Min; Liu, Xueqin

    2014-10-01

    Part of the G protein (3094-4170 bp) of spring viremia of carp virus (SVCV) was expressed in Escherichia coli and purified by dialysis in our study. Two clones of monoclonal antibodies (MAbs 1H11 and 4B8) against G protein were generated by fusion of mouse myeloma cell line SP2/0 and spleen lymphocytes from part of G protein (3094-4170 bp) immunized mice. The results of ELISA (enzyme-linked immunosorbent assay), IFA (indirect immunofluorescent assay), and Western blot assay further demonstrated the characterizations of the two MAbs. Both 1H11 and 4B8 were specific to SVCV G protein. Ten pairs of synthesized overlapping peptides were used to identify the epitope of the MAbs. The MAbs are useful in the development of SVCV diagnostic methods.

  8. Isolation of Drosophila genes encoding G protein-coupled receptor kinases.

    PubMed Central

    Cassill, J A; Whitney, M; Joazeiro, C A; Becker, A; Zuker, C S

    1991-01-01

    G protein-coupled receptors are regulated via phosphorylation by a variety of protein kinases. Recently, termination of the active state of two such receptors, the beta-adrenergic receptor and rhodopsin, has been shown to be mediated by agonist- or light-dependent phosphorylation of the receptor by members of a family of protein-serine/threonine kinases (here referred to as G protein-coupled receptor kinases). We now report the isolation of a family of genes encoding a set of Drosophila protein kinases that appear to code for G protein-coupled receptor kinases. These proteins share a high degree of sequence homology with the bovine beta-adrenergic receptor kinase. The presence of a conserved family of G protein-coupled receptor kinases in vertebrates and invertebrates points to the central role of these kinases in signal transduction cascades. Images PMID:1662381

  9. X-ray structure of the mammalian GIRK2-βγ G-protein complex

    SciTech Connect

    Whorton, Matthew R.; MacKinnon, Roderick

    2013-07-30

    G-protein-gated inward rectifier K+ (GIRK) channels allow neurotransmitters, through G-protein-coupled receptor stimulation, to control cellular electrical excitability. In cardiac and neuronal cells this control regulates heart rate and neural circuit activity, respectively. Here we present the 3.5Å resolution crystal structure of the mammalian GIRK2 channel in complex with βγ G-protein subunits, the central signalling complex that links G-protein-coupled receptor stimulation to K+ channel activity. Short-range atomic and long-range electrostatic interactions stabilize four βγ G-protein subunits at the interfaces between four K+ channel subunits, inducing a pre-open state of the channel. The pre-open state exhibits a conformation that is intermediate between the closed conformation and the open conformation of the constitutively active mutant. The resultant structural picture is compatible with ‘membrane delimited’ activation of GIRK channels by G proteins and the characteristic burst kinetics of channel gating. The structures also permit a conceptual understanding of how the signalling lipid phosphatidylinositol-4,5-bisphosphate (PIP2) and intracellular Na+ ions participate in multi-ligand regulation of GIRK channels.

  10. Roles for Regulator of G Protein Signaling Proteins in Synaptic Signaling and Plasticity

    PubMed Central

    Gerber, Kyle J.; Squires, Katherine E.

    2016-01-01

    The regulator of G protein signaling (RGS) family of proteins serves critical roles in G protein-coupled receptor (GPCR) and heterotrimeric G protein signal transduction. RGS proteins are best understood as negative regulators of GPCR/G protein signaling. They achieve this by acting as GTPase activating proteins (GAPs) for Gα subunits and accelerating the turnoff of G protein signaling. Many RGS proteins also bind additional signaling partners that either regulate their functions or enable them to regulate other important signaling events. At neuronal synapses, GPCRs, G proteins, and RGS proteins work in coordination to regulate key aspects of neurotransmitter release, synaptic transmission, and synaptic plasticity, which are necessary for central nervous system physiology and behavior. Accumulating evidence has revealed key roles for specific RGS proteins in multiple signaling pathways at neuronal synapses, regulating both pre- and postsynaptic signaling events and synaptic plasticity. Here, we review and highlight the current knowledge of specific RGS proteins (RGS2, RGS4, RGS7, RGS9-2, and RGS14) that have been clearly demonstrated to serve critical roles in modulating synaptic signaling and plasticity throughout the brain, and we consider their potential as future therapeutic targets. PMID:26655302

  11. Activation of G Proteins by Guanine Nucleotide Exchange Factors Relies on GTPase Activity

    PubMed Central

    Stanley, Rob J.; Thomas, Geraint M. H.

    2016-01-01

    G proteins are an important family of signalling molecules controlled by guanine nucleotide exchange and GTPase activity in what is commonly called an ‘activation/inactivation cycle’. The molecular mechanism by which guanine nucleotide exchange factors (GEFs) catalyse the activation of monomeric G proteins is well-established, however the complete reversibility of this mechanism is often overlooked. Here, we use a theoretical approach to prove that GEFs are unable to positively control G protein systems at steady-state in the absence of GTPase activity. Instead, positive regulation of G proteins must be seen as a product of the competition between guanine nucleotide exchange and GTPase activity—emphasising a central role for GTPase activity beyond merely signal termination. We conclude that a more accurate description of the regulation of G proteins via these processes is as a ‘balance/imbalance’ mechanism. This result has implications for the understanding of intracellular signalling processes, and for experimental strategies that rely on modulating G protein systems. PMID:26986850

  12. Diverse activation pathways in class A GPCRs converge near the G protein-coupling region

    PubMed Central

    Venkatakrishnan, A. J.; Deupi, Xavier; Lebon, Guillaume; Heydenreich, Franziska M.; Flock, Tilman; Miljus, Tamara; Balaji, Santhanam; Bouvier, Michel; Veprintsev, Dmitry B.; Tate, Christopher G.; Schertler, Gebhard F. X.; Babu, M. Madan

    2016-01-01

    Class A G protein coupled receptors (GPCRs) are a large family of membrane proteins that mediate a wide variety of physiological functions (e.g. vision, neurotransmission, and immune response)1–4. Not surprisingly, they are the targets of nearly one-third of all prescribed medicinal drugs5 (e.g. beta blockers, antipsychotics). GPCR activation is facilitated by extracellular ligands, and leads to the recruitment of intracellular G proteins3,6. Structural rearrangements of residue contacts in the transmembrane domain serve as ‘activation pathways’ that connect the ligand-binding pocket to the G protein-coupling region within the receptor. How similar are these activation pathways across different class A GPCRs? Here, we analysed 27 GPCRs from diverse subgroups for which structures of active and/or inactive states are available. We show that despite the diversity in activation pathways between receptors, the pathways converge near the G protein-coupling region. This convergence is mediated by a strikingly conserved structural rearrangement of residue contacts between transmembrane helices 3, 6, and 7 that releases G protein-contacting residues. The convergence of activation pathways may explain how the activation steps initiated by diverse ligands confer GPCRs the ability to bind a common repertoire of G proteins. PMID:27525504

  13. Phosphorylation-Dependent Regulation of G-Protein Cycle during Nodule Formation in Soybean[OPEN

    PubMed Central

    2015-01-01

    Signaling pathways mediated by heterotrimeric G-protein complexes comprising Gα, Gβ, and Gγ subunits and their regulatory RGS (Regulator of G-protein Signaling) protein are conserved in all eukaryotes. We have shown that the specific Gβ and Gγ proteins of a soybean (Glycine max) heterotrimeric G-protein complex are involved in regulation of nodulation. We now demonstrate the role of Nod factor receptor 1 (NFR1)-mediated phosphorylation in regulation of the G-protein cycle during nodulation in soybean. We also show that during nodulation, the G-protein cycle is regulated by the activity of RGS proteins. Lower or higher expression of RGS proteins results in fewer or more nodules, respectively. NFR1 interacts with RGS proteins and phosphorylates them. Analysis of phosphorylated RGS protein identifies specific amino acids that, when phosphorylated, result in significantly higher GTPase accelerating activity. These data point to phosphorylation-based regulation of G-protein signaling during nodule development. We propose that active NFR1 receptors phosphorylate and activate RGS proteins, which help maintain the Gα proteins in their inactive, trimeric conformation, resulting in successful nodule development. Alternatively, RGS proteins might also have a direct role in regulating nodulation because overexpression of their phospho-mimic version leads to partial restoration of nodule formation in nod49 mutants. PMID:26498905

  14. Targeted Elimination of G Proteins and Arrestins Defines Their Specific Contributions to Both Intensity and Duration of G Protein-coupled Receptor Signaling.

    PubMed

    Alvarez-Curto, Elisa; Inoue, Asuka; Jenkins, Laura; Raihan, Sheikh Zahir; Prihandoko, Rudi; Tobin, Andrew B; Milligan, Graeme

    2016-12-30

    G protein-coupled receptors (GPCRs) can initiate intracellular signaling cascades by coupling to an array of heterotrimeric G proteins and arrestin adaptor proteins. Understanding the contribution of each of these coupling options to GPCR signaling has been hampered by a paucity of tools to selectively perturb receptor function. Here we employ CRISPR/Cas9 genome editing to eliminate selected G proteins (Gαq and Gα11) or arrestin2 and arrestin3 from HEK293 cells together with the elimination of receptor phosphorylation sites to define the relative contribution of G proteins, arrestins, and receptor phosphorylation to the signaling outcomes of the free fatty acid receptor 4 (FFA4). A lack of FFA4-mediated elevation of intracellular Ca(2+) in Gαq/Gα11-null cells and agonist-mediated receptor internalization in arrestin2/3-null cells confirmed previously reported canonical signaling features of this receptor, thereby validating the genome-edited HEK293 cells. FFA4-mediated ERK1/2 activation was totally dependent on Gq/11 but intriguingly was substantially enhanced for FFA4 receptors lacking sites of regulated phosphorylation. This was not due to a simple lack of desensitization of Gq/11 signaling because the Gq/11-dependent calcium response was desensitized by both receptor phosphorylation and arrestin-dependent mechanisms, whereas a substantially enhanced ERK1/2 response was only observed for receptors lacking phosphorylation sites and not in arrestin2/3-null cells. In conclusion, we validate CRISPR/Cas9 engineered HEK293 cells lacking Gq/11 or arrestin2/3 as systems for GPCR signaling research and employ these cells to reveal a previously unappreciated interplay of signaling pathways where receptor phosphorylation can impact on ERK1/2 signaling through a mechanism that is likely independent of arrestins.

  15. Targeted Elimination of G Proteins and Arrestins Defines Their Specific Contributions to Both Intensity and Duration of G Protein-coupled Receptor Signaling*

    PubMed Central

    Alvarez-Curto, Elisa; Inoue, Asuka; Jenkins, Laura; Raihan, Sheikh Zahir; Prihandoko, Rudi; Tobin, Andrew B.

    2016-01-01

    G protein-coupled receptors (GPCRs) can initiate intracellular signaling cascades by coupling to an array of heterotrimeric G proteins and arrestin adaptor proteins. Understanding the contribution of each of these coupling options to GPCR signaling has been hampered by a paucity of tools to selectively perturb receptor function. Here we employ CRISPR/Cas9 genome editing to eliminate selected G proteins (Gαq and Gα11) or arrestin2 and arrestin3 from HEK293 cells together with the elimination of receptor phosphorylation sites to define the relative contribution of G proteins, arrestins, and receptor phosphorylation to the signaling outcomes of the free fatty acid receptor 4 (FFA4). A lack of FFA4-mediated elevation of intracellular Ca2+ in Gαq/Gα11-null cells and agonist-mediated receptor internalization in arrestin2/3-null cells confirmed previously reported canonical signaling features of this receptor, thereby validating the genome-edited HEK293 cells. FFA4-mediated ERK1/2 activation was totally dependent on Gq/11 but intriguingly was substantially enhanced for FFA4 receptors lacking sites of regulated phosphorylation. This was not due to a simple lack of desensitization of Gq/11 signaling because the Gq/11-dependent calcium response was desensitized by both receptor phosphorylation and arrestin-dependent mechanisms, whereas a substantially enhanced ERK1/2 response was only observed for receptors lacking phosphorylation sites and not in arrestin2/3-null cells. In conclusion, we validate CRISPR/Cas9 engineered HEK293 cells lacking Gq/11 or arrestin2/3 as systems for GPCR signaling research and employ these cells to reveal a previously unappreciated interplay of signaling pathways where receptor phosphorylation can impact on ERK1/2 signaling through a mechanism that is likely independent of arrestins. PMID:27852822

  16. G-protein stimulatory subunit alpha and Gq/11α G-proteins are both required to maintain quiescent stem-like chondrocytes

    PubMed Central

    Chagin, Andrei S.; Vuppalapati, Karuna K.; Kobayashi, Tatsuya; Guo, Jun; Hirai, Takao; Chen, Min; Offermanns, Stefan; Weinstein, Lee S.; Kronenberg, Henry M.

    2014-01-01

    Round chondrocytes in the resting zone of the growth plate provide precursors for columnar chondrocytes and have stem-like properties. Here we demonstrate that these stem-like chondrocytes undergo apoptosis in the absence of the receptor (PPR) for parathyroid hormone-related protein. We examine the possible roles of heterotrimeric G-proteins activated by the PPR. Inactivation of the G-protein stimulatory α-subunit (Gsα) leads to accelerated differentiation of columnar chondrocytes, as seen in the PPR knockout, but a remnant of growth cartilage remains, in contrast to disappearance of the growth cartilage in the PPR knockout. Stem-like chondrocytes lose their quiescence and proliferate upon Gsα ablation. Inactivation of Gsα in mice with a mutant PPR that cannot activate G proteins, Gq and G11, leads to a PPR knockout-like phenotype. Thus, Gsα is the major mediator of the anti-differentiation action of the PPR, while activation of both Gsα and Gq/11α is required for quiescence of stem-like chondrocytes. PMID:24781502

  17. Induction of RAGE Shedding by Activation of G Protein-Coupled Receptors

    PubMed Central

    Metz, Verena V.; Kojro, Elzbieta; Rat, Dorothea; Postina, Rolf

    2012-01-01

    The multiligand Receptor for Advanced Glycation End products (RAGE) is involved in various pathophysiological processes, including diabetic inflammatory conditions and Alzheimes disease. Full-length RAGE, a cell surface-located type I membrane protein, can proteolytically be converted by metalloproteinases ADAM10 and MMP9 into a soluble RAGE form. Moreover, administration of recombinant soluble RAGE suppresses activation of cell surface-located RAGE by trapping RAGE ligands. Therefore stimulation of RAGE shedding might have a therapeutic value regarding inflammatory diseases. We aimed to investigate whether RAGE shedding is inducible via ligand-induced activation of G protein-coupled receptors (GPCRs). We chose three different GPCRs coupled to distinct signaling cascades: the V2 vasopressin receptor (V2R) activating adenylyl cyclase, the oxytocin receptor (OTR) linked to phospholipase Cβ, and the PACAP receptor (subtype PAC1) coupled to adenylyl cyclase, phospholipase Cβ, calcium signaling and MAP kinases. We generated HEK cell lines stably coexpressing an individual GPCR and full-length RAGE and then investigated GPCR ligand-induced activation of RAGE shedding. We found metalloproteinase-mediated RAGE shedding on the cell surface to be inducible via ligand-specific activation of all analyzed GPCRs. By using specific inhibitors we have identified Ca2+ signaling, PKCα/PKCβI, CaMKII, PI3 kinases and MAP kinases to be involved in PAC1 receptor-induced RAGE shedding. We detected an induction of calcium signaling in all our cell lines coexpressing RAGE and different GPCRs after agonist treatment. However, we did not disclose a contribution of adenylyl cyclase in RAGE shedding induction. Furthermore, by using a selective metalloproteinase inhibitor and siRNA-mediated knock-down approaches, we show that ADAM10 and/or MMP9 are playing important roles in constitutive and PACAP-induced RAGE shedding. We also found that treatment of mice with PACAP increases the amount of

  18. Induction of RAGE shedding by activation of G protein-coupled receptors.

    PubMed

    Metz, Verena V; Kojro, Elzbieta; Rat, Dorothea; Postina, Rolf

    2012-01-01

    The multiligand Receptor for Advanced Glycation End products (RAGE) is involved in various pathophysiological processes, including diabetic inflammatory conditions and Alzheimers disease. Full-length RAGE, a cell surface-located type I membrane protein, can proteolytically be converted by metalloproteinases ADAM10 and MMP9 into a soluble RAGE form. Moreover, administration of recombinant soluble RAGE suppresses activation of cell surface-located RAGE by trapping RAGE ligands. Therefore stimulation of RAGE shedding might have a therapeutic value regarding inflammatory diseases. We aimed to investigate whether RAGE shedding is inducible via ligand-induced activation of G protein-coupled receptors (GPCRs). We chose three different GPCRs coupled to distinct signaling cascades: the V2 vasopressin receptor (V2R) activating adenylyl cyclase, the oxytocin receptor (OTR) linked to phospholipase Cβ, and the PACAP receptor (subtype PAC1) coupled to adenylyl cyclase, phospholipase Cβ, calcium signaling and MAP kinases. We generated HEK cell lines stably coexpressing an individual GPCR and full-length RAGE and then investigated GPCR ligand-induced activation of RAGE shedding. We found metalloproteinase-mediated RAGE shedding on the cell surface to be inducible via ligand-specific activation of all analyzed GPCRs. By using specific inhibitors we have identified Ca(2+) signaling, PKCα/PKCβI, CaMKII, PI3 kinases and MAP kinases to be involved in PAC1 receptor-induced RAGE shedding. We detected an induction of calcium signaling in all our cell lines coexpressing RAGE and different GPCRs after agonist treatment. However, we did not disclose a contribution of adenylyl cyclase in RAGE shedding induction. Furthermore, by using a selective metalloproteinase inhibitor and siRNA-mediated knock-down approaches, we show that ADAM10 and/or MMP9 are playing important roles in constitutive and PACAP-induced RAGE shedding. We also found that treatment of mice with PACAP increases the amount

  19. Evolutionary Conservation of a GPCR-Independent Mechanism of Trimeric G Protein Activation

    PubMed Central

    Coleman, Brantley D.; Marivin, Arthur; Parag-Sharma, Kshitij; DiGiacomo, Vincent; Kim, Seongseop; Pepper, Judy S.; Casler, Jason; Nguyen, Lien T.; Koelle, Michael R.; Garcia-Marcos, Mikel

    2016-01-01

    Trimeric G protein signaling is a fundamental mechanism of cellular communication in eukaryotes. The core of this mechanism consists of activation of G proteins by the guanine-nucleotide exchange factor (GEF) activity of G protein coupled receptors. However, the duration and amplitude of G protein-mediated signaling are controlled by a complex network of accessory proteins that appeared and diversified during evolution. Among them, nonreceptor proteins with GEF activity are the least characterized. We recently found that proteins of the ccdc88 family possess a Gα-binding and activating (GBA) motif that confers GEF activity and regulates mammalian cell behavior. A sequence similarity-based search revealed that ccdc88 genes are highly conserved across metazoa but the GBA motif is absent in most invertebrates. This prompted us to investigate whether the GBA motif is present in other nonreceptor proteins in invertebrates. An unbiased bioinformatics search in Caenorhabditis elegans identified GBAS-1 (GBA and SPK domain containing-1) as a GBA motif-containing protein with homologs only in closely related worm species. We demonstrate that GBAS-1 has GEF activity for the nematode G protein GOA-1 and that the two proteins are coexpressed in many cells of living worms. Furthermore, we show that GBAS-1 can activate mammalian Gα-subunits and provide structural insights into the evolutionarily conserved determinants of the GBA–G protein interface. These results demonstrate that the GBA motif is a functional GEF module conserved among highly divergent proteins across evolution, indicating that the GBA-Gα binding mode is strongly constrained under selective pressure to mediate receptor-independent G protein activation in metazoans. PMID:26659249

  20. delta-Opioid receptors exhibit high efficiency when activating trimeric G proteins in membrane domains.

    PubMed

    Bourova, Lenka; Kostrnova, Alexandra; Hejnova, Lucie; Moravcova, Zuzana; Moon, Hyo-Eun; Novotny, Jiri; Milligan, Graeme; Svoboda, Petr

    2003-04-01

    Low-density membrane fragments (domains) were separated from the bulk of plasma membranes of human embryonic kidney (HEK)293 cells expressing a delta-opioid (DOP) receptor-Gi1alpha fusion protein by drastic homogenization and flotation on equilibrium sucrose density gradients. The functional activity of trimeric G proteins and capacity of the DOP receptor to stimulate both the fusion protein-linked Gi1alpha and endogenous pertussis-toxin sensitive G proteins was measured as d-Ala2, d-Leu5-enkephalin stimulated high-affinity GTPase or guanosine-5'-[gamma-35S]triphosphate ([35S]GTPgammaS) binding. The maximum d-Ala2-d-Leu5 enkephalin (DADLE)-stimulated GTPase was two times higher in low-density membrane fragments than in bulk of plasma membranes; 58 and 27 pmol/mg/min, respectively. The same difference was obtained for [35S]GTPgammaS binding. Contrarily, the low-density domains contained no more than half the DOP receptor binding sites (Bmax = 6.6 pmol/mg versus 13.6 pmol/mg). Thus, when corrected for expression levels of the receptor, low-density domains exhibited four times higher agonist-stimulated GTPase and [35S]GTPgammaS binding than the bulk plasma membranes. The regulator of G protein signaling RGS1, enhanced further the G protein functional activity but did not remove the difference between domain-bound and plasma membrane pools of G protein. The potency of the agonist in functional studies and the affinity of specific [3H]DADLE binding to the receptor were, however, the same in both types of membranes - EC50 = 4.5 +/- 0.1 x 10(-8) and 3.2 +/- 1.4 x 10(-8) m for GTPase; Kd = 1.2 +/- 0.1 and 1.3 +/- 0.1 nm for [3H]DADLE radioligand binding assay. Similar results were obtained when sodium bicarbonate was used for alkaline isolation of membrane domains. By contrast, detergent-insensitive membrane domains isolated following treatment of cells with Triton X100 exhibited no DADLE-stimulated GTPase or GTPgammaS binding. Functional coupling between the DOP receptor

  1. Domain-Opening and Dynamic Coupling in the α-Subunit of Heterotrimeric G Proteins

    PubMed Central

    Yao, Xin-Qiu; Grant, Barry J.

    2013-01-01

    Heterotrimeric G proteins are conformational switches that turn on intracellular signaling cascades in response to the activation of G-protein-coupled receptors. Receptor activation by extracellular stimuli promotes a cycle of GTP binding and hydrolysis on the G protein α-subunit (Gα). Important conformational transitions occurring during this cycle have been characterized from extensive crystallographic studies of Gα. However, the link between the observed conformations and the mechanisms involved in G-protein activation and effector interaction remain unclear. Here we describe a comprehensive principal component analysis of available Gα crystallographic structures supplemented with extensive unbiased conventional and accelerated molecular dynamics simulations that together characterize the response of Gα to GTP binding and hydrolysis. Our studies reveal details of activating conformational changes as well as the intrinsic flexibility of the α-helical domain that includes a large-scale 60° domain opening under nucleotide-free conditions. This result is consistent with the recently reported open crystal structure of Gs, the stimulatory G protein for adenylyl cyclase, in complex with the α2 adrenergic receptor. Sets of unique interactions potentially important for the conformational transition are also identified. Moreover simulations reveal nucleotide-dependent dynamical couplings of distal regions and residues potentially important for the allosteric link between functional sites. PMID:23870276

  2. Lamin A/C sustains PcG protein architecture, maintaining transcriptional repression at target genes

    PubMed Central

    Cesarini, Elisa; Mozzetta, Chiara; Marullo, Fabrizia; Gregoretti, Francesco; Gargiulo, Annagiusi; Columbaro, Marta; Cortesi, Alice; Antonelli, Laura; Di Pelino, Simona; Squarzoni, Stefano; Palacios, Daniela; Zippo, Alessio; Bodega, Beatrice; Oliva, Gennaro

    2015-01-01

    Beyond its role in providing structure to the nuclear envelope, lamin A/C is involved in transcriptional regulation. However, its cross talk with epigenetic factors—and how this cross talk influences physiological processes—is still unexplored. Key epigenetic regulators of development and differentiation are the Polycomb group (PcG) of proteins, organized in the nucleus as microscopically visible foci. Here, we show that lamin A/C is evolutionarily required for correct PcG protein nuclear compartmentalization. Confocal microscopy supported by new algorithms for image analysis reveals that lamin A/C knock-down leads to PcG protein foci disassembly and PcG protein dispersion. This causes detachment from chromatin and defects in PcG protein–mediated higher-order structures, thereby leading to impaired PcG protein repressive functions. Using myogenic differentiation as a model, we found that reduced levels of lamin A/C at the onset of differentiation led to an anticipation of the myogenic program because of an alteration of PcG protein–mediated transcriptional repression. Collectively, our results indicate that lamin A/C can modulate transcription through the regulation of PcG protein epigenetic factors. PMID:26553927

  3. Biased G Protein-Coupled Receptor Signaling: New Player in Modulating Physiology and Pathology

    PubMed Central

    Bologna, Zuzana; Teoh, Jian-peng; Bayoumi, Ahmed S.; Tang, Yaoliang; Kim, Il-man

    2017-01-01

    G protein-coupled receptors (GPCRs) are a family of cell-surface proteins that play critical roles in regulating a variety of pathophysiological processes and thus are targeted by almost a third of currently available therapeutics. It was originally thought that GPCRs convert extracellular stimuli into intracellular signals through activating G proteins, whereas β-arrestins have important roles in internalization and desensitization of the receptor. Over the past decade, several novel functional aspects of β-arrestins in regulating GPCR signaling have been discovered. These previously unanticipated roles of β-arrestins to act as signal transducers and mediators of G protein-independent signaling have led to the concept of biased agonism. Biased GPCR ligands are able to engage with their target receptors in a manner that preferentially activates only G protein- or β-arrestin-mediated downstream signaling. This offers the potential for next generation drugs with high selectivity to therapeutically relevant GPCR signaling pathways. In this review, we provide a summary of the recent studies highlighting G protein- or β-arrestin-biased GPCR signaling and the effects of biased ligands on disease pathogenesis and regulation. PMID:28035079

  4. Human T-cell leukemia virus type-1 Tax oncoprotein regulates G-protein signaling.

    PubMed

    Twizere, Jean-Claude; Springael, Jean-Yves; Boxus, Mathieu; Burny, Arsène; Dequiedt, Franck; Dewulf, Jean-François; Duchateau, Julie; Portetelle, Daniel; Urbain, Patrice; Van Lint, Carine; Green, Patrick L; Mahieux, Renaud; Parmentier, Marc; Willems, Luc; Kettmann, Richard

    2007-02-01

    Human T-cell leukemia virus type-1 (HTLV-1) is associated with adult T-cell leukemia (ATL) and neurological syndromes. HTLV-1 encodes the oncoprotein Tax-1, which modulates viral and cellular gene expression leading to T-cell transformation. Guanine nucleotide-binding proteins (G proteins) and G protein-coupled receptors (GPCRs) constitute the largest family of membrane proteins known and are involved in the regulation of most biological functions. Here, we report an interaction between HTLV-1 Tax oncoprotein and the G-protein beta subunit. Interestingly, though the G-protein beta subunit inhibits Tax-mediated viral transcription, Tax-1 perturbs G-protein beta subcellular localization. Functional evidence for these observations was obtained using conditional Tax-1-expressing transformed T-lymphocytes, where Tax expression correlated with activation of the SDF-1/CXCR4 axis. Our data indicated that HTLV-1 developed a strategy based on the activation of the SDF-1/CXCR4 axis in the infected cell; this could have tremendous implications for new therapeutic strategies.

  5. Visualization of a radical B12 enzyme with its G-protein chaperone

    SciTech Connect

    Jost, Marco; Cracan, Valentin; Hubbard, Paul A.; Banerjee, Ruma; Drennan, Catherine L.

    2015-02-09

    G-protein metallochaperones ensure fidelity during cofactor assembly for a variety of metalloproteins, including adenosylcobalamin (AdoCbl)-dependent methylmalonyl-CoA mutase and hydrogenase, and thus have both medical and biofuel development applications. In this paper, we present crystal structures of IcmF, a natural fusion protein of AdoCbl-dependent isobutyryl-CoA mutase and its corresponding G-protein chaperone, which reveal the molecular architecture of a G-protein metallochaperone in complex with its target protein. These structures show that conserved G-protein elements become ordered upon target protein association, creating the molecular pathways that both sense and report on the cofactor loading state. Structures determined of both apo- and holo-forms of IcmF depict both open and closed enzyme states, in which the cofactor-binding domain is alternatively positioned for cofactor loading and for catalysis. Finally and notably, the G protein moves as a unit with the cofactor-binding domain, providing a visualization of how a chaperone assists in the sequestering of a precious cofactor inside an enzyme active site.

  6. Visualization of a radical B12 enzyme with its G-protein chaperone

    DOE PAGES

    Jost, Marco; Cracan, Valentin; Hubbard, Paul A.; ...

    2015-02-09

    G-protein metallochaperones ensure fidelity during cofactor assembly for a variety of metalloproteins, including adenosylcobalamin (AdoCbl)-dependent methylmalonyl-CoA mutase and hydrogenase, and thus have both medical and biofuel development applications. In this paper, we present crystal structures of IcmF, a natural fusion protein of AdoCbl-dependent isobutyryl-CoA mutase and its corresponding G-protein chaperone, which reveal the molecular architecture of a G-protein metallochaperone in complex with its target protein. These structures show that conserved G-protein elements become ordered upon target protein association, creating the molecular pathways that both sense and report on the cofactor loading state. Structures determined of both apo- and holo-forms ofmore » IcmF depict both open and closed enzyme states, in which the cofactor-binding domain is alternatively positioned for cofactor loading and for catalysis. Finally and notably, the G protein moves as a unit with the cofactor-binding domain, providing a visualization of how a chaperone assists in the sequestering of a precious cofactor inside an enzyme active site.« less

  7. Autocrine selection of a GLP-1R G-protein biased agonist with potent antidiabetic effects

    PubMed Central

    Zhang, Hongkai; Sturchler, Emmanuel; Zhu, Jiang; Nieto, Ainhoa; Cistrone, Philip A.; Xie, Jia; He, LinLing; Yea, Kyungmoo; Jones, Teresa; Turn, Rachel; Di Stefano, Peter S.; Griffin, Patrick R.; Dawson, Philip E.; McDonald, Patricia H.; Lerner, Richard A.

    2015-01-01

    Glucagon-like peptide-1 (GLP-1) receptor (GLP-1R) agonists have emerged as treatment options for type 2 diabetes mellitus (T2DM). GLP-1R signals through G-protein-dependent, and G-protein-independent pathways by engaging the scaffold protein β-arrestin; preferential signalling of ligands through one or the other of these branches is known as ‘ligand bias'. Here we report the discovery of the potent and selective GLP-1R G-protein-biased agonist, P5. We identified P5 in a high-throughput autocrine-based screening of large combinatorial peptide libraries, and show that P5 promotes G-protein signalling comparable to GLP-1 and Exendin-4, but exhibited a significantly reduced β-arrestin response. Preclinical studies using different mouse models of T2DM demonstrate that P5 is a weak insulin secretagogue. Nevertheless, chronic treatment of diabetic mice with P5 increased adipogenesis, reduced adipose tissue inflammation as well as hepatic steatosis and was more effective at correcting hyperglycaemia and lowering haemoglobin A1c levels than Exendin-4, suggesting that GLP-1R G-protein-biased agonists may provide a novel therapeutic approach to T2DM. PMID:26621478

  8. Structural Evidence for a Sequential Release Mechanism for Activation of Heterotrimeric G Proteins

    SciTech Connect

    Kapoor, Neeraj; Menon, Santosh T.; Chauhan, Radha; Sachdev, Pallavi; Sakmar, Thomas P.

    2010-01-12

    Heptahelical G-protein (heterotrimeric guanine nucleotide-binding protein)-coupled receptors couple to heterotrimeric G proteins to relay extracellular signals to intracellular signaling networks, but the molecular mechanism underlying guanosine 5'-diphosphate (GDP) release by the G protein {alpha}-subunit is not well understood. Amino acid substitutions in the conserved {alpha}5 helix of Gi, which extends from the C-terminal region to the nucleotide-binding pocket, cause dramatic increases in basal (receptor-independent) GDP release rates. For example, mutant G{alpha}{sub i1}-T329A shows an 18-fold increase in basal GDP release rate and, when expressed in culture, it causes a significant decrease in forskolin-stimulated cAMP accumulation. The crystal structure of G{alpha}{sub i1}-T329A {center_dot} GDP shows substantial conformational rearrangement of the switch I region and additional striking alterations of side chains lining the catalytic pocket that disrupt the Mg{sup +2} coordination sphere and dislodge bound Mg{sup +2}. We propose a 'sequential release' mechanism whereby a transient conformational change in the {alpha}5 helix alters switch I to induce GDP release. Interestingly, this mechanistic model for heterotrimeric G protein activation is similar to that suggested for the activation of the plant small G protein Rop4 by RopGEF8.

  9. Exon capture analysis of G protein-coupled receptors identifies activating mutations in GRM3 in melanoma.

    PubMed

    Prickett, Todd D; Wei, Xiaomu; Cardenas-Navia, Isabel; Teer, Jamie K; Lin, Jimmy C; Walia, Vijay; Gartner, Jared; Jiang, Jiji; Cherukuri, Praveen F; Molinolo, Alfredo; Davies, Michael A; Gershenwald, Jeffrey E; Stemke-Hale, Katherine; Rosenberg, Steven A; Margulies, Elliott H; Samuels, Yardena

    2011-09-25

    G protein-coupled receptors (GPCRs), the largest human gene family, are important regulators of signaling pathways. However, knowledge of their genetic alterations is limited. In this study, we used exon capture and massively parallel sequencing methods to analyze the mutational status of 734 GPCRs in melanoma. This investigation revealed that one family member, GRM3, was frequently mutated and that one of its mutations clustered within one position. Biochemical analysis of GRM3 alterations revealed that mutant GRM3 selectively regulated the phosphorylation of MEK, leading to increased anchorage-independent growth and migration. Melanoma cells expressing mutant GRM3 had reduced cell growth and cellular migration after short hairpin RNA-mediated knockdown of GRM3 or treatment with a selective MEK inhibitor, AZD-6244, which is currently being used in phase 2 clinical trials. Our study yields the most comprehensive map of genetic alterations in the GPCR gene family.

  10. Dynamic Coupling and Allosteric Networks in the α Subunit of Heterotrimeric G Proteins*

    PubMed Central

    Yao, Xin-Qiu; Malik, Rabia U.; Griggs, Nicholas W.; Skjærven, Lars; Traynor, John R.; Sivaramakrishnan, Sivaraj; Grant, Barry J.

    2016-01-01

    G protein α subunits cycle between active and inactive conformations to regulate a multitude of intracellular signaling cascades. Important structural transitions occurring during this cycle have been characterized from extensive crystallographic studies. However, the link between observed conformations and the allosteric regulation of binding events at distal sites critical for signaling through G proteins remain unclear. Here we describe molecular dynamics simulations, bioinformatics analysis, and experimental mutagenesis that identifies residues involved in mediating the allosteric coupling of receptor, nucleotide, and helical domain interfaces of Gαi. Most notably, we predict and characterize novel allosteric decoupling mutants, which display enhanced helical domain opening, increased rates of nucleotide exchange, and constitutive activity in the absence of receptor activation. Collectively, our results provide a framework for explaining how binding events and mutations can alter internal dynamic couplings critical for G protein function. PMID:26703464

  11. Chemical genetics reveals an RGS/G-protein role in the action of a compound.

    PubMed

    Fitzgerald, Kevin; Tertyshnikova, Svetlana; Moore, Lisa; Bjerke, Lynn; Burley, Ben; Cao, Jian; Carroll, Pamela; Choy, Robert; Doberstein, Steve; Dubaquie, Yves; Franke, Yvonne; Kopczynski, Jenny; Korswagen, Hendrik; Krystek, Stanley R; Lodge, Nicholas J; Plasterk, Ronald; Starrett, John; Stouch, Terry; Thalody, George; Wayne, Honey; van der Linden, Alexander; Zhang, Yongmei; Walker, Stephen G; Cockett, Mark; Wardwell-Swanson, Judi; Ross-Macdonald, Petra; Kindt, Rachel M

    2006-04-01

    We report here on a chemical genetic screen designed to address the mechanism of action of a small molecule. Small molecules that were active in models of urinary incontinence were tested on the nematode Caenorhabditis elegans, and the resulting phenotypes were used as readouts in a genetic screen to identify possible molecular targets. The mutations giving resistance to compound were found to affect members of the RGS protein/G-protein complex. Studies in mammalian systems confirmed that the small molecules inhibit muscarinic G-protein coupled receptor (GPCR) signaling involving G-alphaq (G-protein alpha subunit). Our studies suggest that the small molecules act at the level of the RGS/G-alphaq signaling complex, and define new mutations in both RGS and G-alphaq, including a unique hypo-adapation allele of G-alphaq. These findings suggest that therapeutics targeted to downstream components of GPCR signaling may be effective for treatment of diseases involving inappropriate receptor activation.

  12. Histaminylation of glutamine residues is a novel posttranslational modification implicated in G-protein signaling.

    PubMed

    Vowinckel, Jakob; Stahlberg, Silke; Paulmann, Nils; Bluemlein, Katharina; Grohmann, Maik; Ralser, Markus; Walther, Diego J

    2012-11-02

    Posttranslational modifications (PTM) have been shown to be essential for protein function and signaling. Here we report the identification of a novel modification, protein transfer of histamine, and provide evidence for its function in G protein signaling. Histamine, known as neurotransmitter and mediator of the inflammatory response, was found incorporated into mastocytoma proteins. Histaminylation was dependent on transglutaminase II. Mass spectrometry confirmed histamine modification of the small and heterotrimeric G proteins Cdc42, Gαo1 and Gαq. The modification was specific for glutamine residues in the catalytic core, and triggered their constitutive activation. TGM2-mediated histaminylation is thus a novel PTM that functions in G protein signaling. Protein αmonoaminylations, thus including histaminylation, serotonylation, dopaminylation and norepinephrinylation, hence emerge as a novel class of regulatory PTMs.

  13. Two Neuronal G Proteins Are Involved in Chemosensation of the Caenorhabditis Elegans Dauer-Inducing Pheromone

    PubMed Central

    Zwaal, R. R.; Mendel, J. E.; Sternberg, P. W.; Plasterk, RHA.

    1997-01-01

    Caenorhabditis elegans uses chemosensation to determine its course of development. Young larvae can arrest as dauer larvae in response to increasing population density, which they measure by a nematode-excreted pheromone, and decreasing food supply. Dauer larvae can resume development in response to a decrease in pheromone and increase in food concentration. We show here that two novel G protein alpha subunits (GPA-2 and GPA-3) show promoter activity in subsets of chemosensory neurons and are involved in the decision to form dauer larvae primarily through the response to dauer pheromone. Dominant activating mutations in these G proteins result in constitutive, pheromone-independent dauer formation, whereas inactivation results in reduced sensitivity to pheromone, and, under certain conditions, an alteration in the response to food. Interactions between gpa-2, gpa-3 and other genes controlling dauer formation suggest that these G proteins may act in parallel to regulate the neuronal decision making that precedes dauer formation. PMID:9055081

  14. Immunoglobulin-sulfated polysaccharide interactions. Binding of agaropectin and heparin by human IgG proteins

    PubMed Central

    1981-01-01

    The interaction of immunoglobulins with certain acidic polysaccharides was demonstrated by the binding of the sulfated glycans agaropectin and heparin by certain human IgG proteins. Heparin-binding IgG proteins can distinguish between the molecular forms of heparin derived from porcine intestine, bovine lung, and rat skin. The major specificity of these proteins is for native and certain high molecular weight subunit components of rat skin heparin. The interactions with multi-chain and single chain rat skin heparin are stable under physiological conditions and involve the Fab and, more specifically, the Fv region of the IgG molecule. These reactions occur as a result of an electrostatic interaction between cationic sites on certain IgG proteins and anionic sulfate resides of agaropectin or heparin. The characteristics of heparin-IgG interaction resemble those of heparin with other plasma proteins, the interactions of which have biological significance. PMID:7252414

  15. A G protein alpha null mutation confers prolificacy potential in maize

    DOE PAGES

    Urano, Daisuke; Jackson, David; Jones, Alan M.

    2015-05-06

    Plasticity in plant development is controlled by environmental signals through largely unknown signalling networks. Signalling coupled by the heterotrimeric G protein complex underlies various developmental pathways in plants. The morphology of two plastic developmental pathways, root system architecture and female inflorescence formation, was quantitatively assessed in a mutant compact plant 2 (ct2) lacking the alpha subunit of the heterotrimeric G protein complex in maize. The ct2 mutant partially compensated for a reduced shoot height by increased total leaf number, and had far more ears, even in the presence of pollination signals. Lastly, the maize heterotrimeric G protein complex is importantmore » in some plastic developmental traits in maize. In particular, the maize Gα subunit is required to dampen the overproduction of female inflorescences.« less

  16. Small G proteins in the cardiovascular system: physiological and pathological aspects.

    PubMed

    Loirand, Gervaise; Sauzeau, Vincent; Pacaud, Pierre

    2013-10-01

    Small G proteins exist in eukaryotes from yeast to human and constitute the Ras superfamily comprising more than 100 members. This superfamily is structurally classified into five families: the Ras, Rho, Rab, Arf, and Ran families that control a wide variety of cell and biological functions through highly coordinated regulation processes. Increasing evidence has accumulated to identify small G proteins and their regulators as key players of the cardiovascular physiology that control a large panel of cardiac (heart rhythm, contraction, hypertrophy) and vascular functions (angiogenesis, vascular permeability, vasoconstriction). Indeed, basal Ras protein activity is required for homeostatic functions in physiological conditions, but sustained overactivation of Ras proteins or spatiotemporal dysregulation of Ras signaling pathways has pathological consequences in the cardiovascular system. The primary object of this review is to provide a comprehensive overview of the current progress in our understanding of the role of small G proteins and their regulators in cardiovascular physiology and pathologies.

  17. G protein-coupled receptors provide survival signals in prostate cancer.

    PubMed

    Yowell, Charles W; Daaka, Yehia

    2002-12-01

    Prostate cancer is the leading cause for noncutaneous cancer-related deaths among men in the United States. The disease is biologically characterized as being either androgen dependent or androgen independent. Whereas androgen-dependent prostate cancer can be successfully treated with androgen ablative therapy, to date no cure exists for androgen-independent disease. Mechanisms involved in the progression of prostate cancer to androgen independence are not known. Here we present evidence that in addition to growth factor receptor tyrosine kinases, G protein- coupled receptors can mediate survival signals in prostate cancer cells. The G protein- coupled receptors exert their effects by activating multiple intracellular signal transduction networks that promote prostate cancer cell survival, including the activation of c-Jun N-terminal kinase, protein kinase B (Akt) and nuclear factor-kB. Prostate-expressed G protein- coupled receptors and their downstream effectors may prove to be effective targets in the treatment of advanced prostate cancer.

  18. A G protein alpha null mutation confers prolificacy potential in maize

    SciTech Connect

    Urano, Daisuke; Jackson, David; Jones, Alan M.

    2015-05-06

    Plasticity in plant development is controlled by environmental signals through largely unknown signalling networks. Signalling coupled by the heterotrimeric G protein complex underlies various developmental pathways in plants. The morphology of two plastic developmental pathways, root system architecture and female inflorescence formation, was quantitatively assessed in a mutant compact plant 2 (ct2) lacking the alpha subunit of the heterotrimeric G protein complex in maize. The ct2 mutant partially compensated for a reduced shoot height by increased total leaf number, and had far more ears, even in the presence of pollination signals. Lastly, the maize heterotrimeric G protein complex is important in some plastic developmental traits in maize. In particular, the maize Gα subunit is required to dampen the overproduction of female inflorescences.

  19. A G protein alpha null mutation confers prolificacy potential in maize

    PubMed Central

    Urano, Daisuke; Jackson, David; Jones, Alan M.

    2015-01-01

    Plasticity in plant development is controlled by environmental signals through largely unknown signalling networks. Signalling coupled by the heterotrimeric G protein complex underlies various developmental pathways in plants. The morphology of two plastic developmental pathways, root system architecture and female inflorescence formation, was quantitatively assessed in a mutant compact plant 2 (ct2) lacking the alpha subunit of the heterotrimeric G protein complex in maize. The ct2 mutant partially compensated for a reduced shoot height by increased total leaf number, and had far more ears, even in the presence of pollination signals. The maize heterotrimeric G protein complex is important in some plastic developmental traits in maize. In particular, the maize Gα subunit is required to dampen the overproduction of female inflorescences. PMID:25948706

  20. Approaches to Assess Functional Selectivity in GPCRs: Evaluating G Protein Signaling in an Endogenous Environment

    PubMed Central

    Bohn, Laura M.; Zhou, Lei; Ho, Jo-Hao

    2016-01-01

    Ligand-directed signaling, biased agonism, and functional selectivity are terms that describe the propensity of a ligand to drive signaling toward one GPCR pathway over another. Most of the early examples demonstrated to date examine the divergence between GPCR signaling to G protein coupling and βarrestin2 recruitment. As biased agonists begin to become available based on cell-based screening criteria, a need arises to determine if G protein signaling biases will be maintained in the endogenous setting, wherein receptors are functioning to control relevant biological responses. This report presents our method and offers tips for evaluating G protein signaling in endogenous tissues. Predominately, brain tissues are discussed here; optimization points that can be applied to any tissues are highlighted. PMID:26260601

  1. Identification and expression profiles of neuropeptides and their G protein-coupled receptors in the rice stem borer Chilo suppressalis.

    PubMed

    Xu, Gang; Gu, Gui-Xiang; Teng, Zi-Wen; Wu, Shun-Fan; Huang, Jia; Song, Qi-Sheng; Ye, Gong-Yin; Fang, Qi

    2016-06-29

    In insects, neuropeptides play important roles in the regulation of multiple physiological processes by binding to their corresponding receptors, which are primarily G protein-coupled receptors (GPCRs). The genes encoding neuropeptides and their associated GPCRs in the rice stem borer Chilo suppressalis were identified by a transcriptomic analysis and were used to identify potential targets for the disruption of physiological processes and the protection of crops. Forty-three candidate genes were found to encode the neuropeptide precursors for all known insect neuropeptides except for arginine-vasopressin-like peptide (AVLP), CNMamide, neuropeptide-like precursors 2-4 (NPLP2-4), and proctolin. In addition, novel alternative splicing variants of three neuropeptide genes (allatostatin CC, CCHamide 1, and short neuropeptide F) are reported for the first time, and 51 putative neuropeptide GPCRs were identified. Phylogenetic analyses demonstrated that 44 of these GPCRs belong to the A-family (or rhodopsin-like), 5 belong to the B-family (or secretin-like), and 2 are leucine-rich repeat-containing GPCRs. These GPCRs and their likely ligands were also described. qRT-PCR analyses revealed the expression profiles of the neuropeptide precursors and GPCR genes in various tissues of C. suppressalis. Our study provides fundamental information that may further our understanding of neuropeptidergic signaling systems in Lepidoptera and aid in the design of peptidomimetics, pseudopeptides or small molecules capable of disrupting the physiological processes regulated by these signaling molecules and their receptors.

  2. An evolutionary comparison of leucine-rich repeat containing G protein-coupled receptors reveals a novel LGR subtype.

    PubMed

    Van Hiel, Matthias B; Vandersmissen, Hans Peter; Van Loy, Tom; Vanden Broeck, Jozef

    2012-03-01

    Leucine-rich repeat containing G protein-coupled receptors or LGRs are receptors with important functions in development and reproduction. Belonging to this evolutionarily conserved group of receptors are the well-studied glycoprotein hormone receptors and relaxin receptors in mammals, as well as the bursicon receptor, which triggers cuticle hardening and tanning in freshly enclosed insects. In this study, the numerous LGR sequences in different animal phyla are analyzed and compared. Based on these data a phylogenetic tree was generated. This information sheds new light on structural and evolutionary aspects regarding this receptor group. Apart from vertebrates and insects, LGRs are also present in early chordates (Urochordata, Cephalochordata and Hyperoartia) and other arthropods (Arachnida and Branchiopoda) as well as in Mollusca, Echinodermata, Hemichordata, Nematoda, and even in ancient animal life forms, such as Cnidaria and Placozoa. Three distinct types of LGR exist, distinguishable by their number of leucine-rich repeats (LRRs), their type-specific hinge region and the presence or absence of an LDLa motif. Type C LGRs containing only one LDLa (C1 subtype) appear to be present in nearly all animal phyla. We here describe a second subtype, C2, containing multiple LDLa motifs, which was discovered in echinoderms, mollusks and in one insect species (Pediculus humanis corporis). In addition, eight putative LGRs can be predicted from the genome data of the placozoan species Trichoplax adhaerens. They may represent an ancient form of the LGRs, however, more genomic data will be required to confirm this hypothesis.

  3. Discovery and Cardioprotective Effects of the First Non-Peptide Agonists of the G Protein-Coupled Prokineticin Receptor-1

    PubMed Central

    Urayama, Kyoji; Nishi, Toshishide; Kurose, Hitoshi; Tafi, Andrea; Ribeiro, Nigel; Désaubry, Laurent; Nebigil, Canan G.

    2015-01-01

    Prokineticins are angiogenic hormones that activate two G protein-coupled receptors: PKR1 and PKR2. PKR1 has emerged as a critical mediator of cardiovascular homeostasis and cardioprotection. Identification of non-peptide PKR1 agonists that contribute to myocardial repair and collateral vessel growth hold promises for treatment of heart diseases. Through a combination of in silico studies, medicinal chemistry, and pharmacological profiling approaches, we designed, synthesized, and characterized the first PKR1 agonists, demonstrating their cardioprotective activity against myocardial infarction (MI) in mice. Based on high throughput docking protocol, 250,000 compounds were computationally screened for putative PKR1 agonistic activity, using a homology model, and 10 virtual hits were pharmacologically evaluated. One hit internalizes PKR1, increases calcium release and activates ERK and Akt kinases. Among the 30 derivatives of the hit compound, the most potent derivative, IS20, was confirmed for its selectivity and specificity through genetic gain- and loss-of-function of PKR1. Importantly, IS20 prevented cardiac lesion formation and improved cardiac function after MI in mice, promoting proliferation of cardiac progenitor cells and neovasculogenesis. The preclinical investigation of the first PKR1 agonists provides a novel approach to promote cardiac neovasculogenesis after MI. PMID:25831128

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

    PubMed

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

    2015-08-01

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

  5. Identification and expression profiles of neuropeptides and their G protein-coupled receptors in the rice stem borer Chilo suppressalis

    PubMed Central

    Xu, Gang; Gu, Gui-Xiang; Teng, Zi-Wen; Wu, Shun-Fan; Huang, Jia; Song, Qi-Sheng; Ye, Gong-Yin; Fang, Qi

    2016-01-01

    In insects, neuropeptides play important roles in the regulation of multiple physiological processes by binding to their corresponding receptors, which are primarily G protein-coupled receptors (GPCRs). The genes encoding neuropeptides and their associated GPCRs in the rice stem borer Chilo suppressalis were identified by a transcriptomic analysis and were used to identify potential targets for the disruption of physiological processes and the protection of crops. Forty-three candidate genes were found to encode the neuropeptide precursors for all known insect neuropeptides except for arginine-vasopressin-like peptide (AVLP), CNMamide, neuropeptide-like precursors 2-4 (NPLP2-4), and proctolin. In addition, novel alternative splicing variants of three neuropeptide genes (allatostatin CC, CCHamide 1, and short neuropeptide F) are reported for the first time, and 51 putative neuropeptide GPCRs were identified. Phylogenetic analyses demonstrated that 44 of these GPCRs belong to the A-family (or rhodopsin-like), 5 belong to the B-family (or secretin-like), and 2 are leucine-rich repeat-containing GPCRs. These GPCRs and their likely ligands were also described. qRT-PCR analyses revealed the expression profiles of the neuropeptide precursors and GPCR genes in various tissues of C. suppressalis. Our study provides fundamental information that may further our understanding of neuropeptidergic signaling systems in Lepidoptera and aid in the design of peptidomimetics, pseudopeptides or small molecules capable of disrupting the physiological processes regulated by these signaling molecules and their receptors. PMID:27353701

  6. Regulation of G protein signaling by the 70kDa heat shock protein.

    PubMed

    Lim, William K; Kanelakis, Kimon C; Neubig, Richard R

    2013-02-01

    G protein-coupled receptors (GPCRs) transduce extracellular signals to the interior of the cell by activating membrane-bound guanine nucleotide-binding regulatory proteins (G proteins). An increasing number of proteins have been reported to bind to and regulate GPCRs. We report a novel regulation of the alpha(2A) adrenergic receptor (α(2A)-R) by the ubiquitous stress-inducible 70kDa heat shock protein, hsp70. Hsp70, but not hsp90, attenuated G protein-dependent high affinity agonist binding to the α(2A)-R in Sf9 membranes. Antagonist binding was unchanged, suggesting that hsp70 uncouples G proteins from the receptor. As hsp70 did not bind G proteins but complexed with the α(2A)-R in intact cells, a direct interaction with the receptor seems likely. In the presence of hsp70, α(2A)-R-catalyzed [(35)S]GTPγS binding was reduced by approximately 70%. In contrast, approximately 50-fold higher concentrations of hsp70 were required to reduce agonist binding to the stress-inducible 5-hydroxytryptamine(1A) receptor (5-HT(1A)-R). In heat-stressed CHO cells, the α(2A)-R was significantly uncoupled from G proteins, coincident with an increased localization of hsp70 at the membrane. The contrasting effect of hsp70 on the α(2A)-R compared to the 5-HT(1A)-R suggests that during stress, upregulation of hsp70 may attenuate signaling from specific GPCRs as part of the stress response to foster survival.

  7. Regulator of G-protein signaling (RGS) proteins in cancer biology.

    PubMed

    Hurst, Jillian H; Hooks, Shelley B

    2009-11-15

    The regulator of G-protein signaling (RGS) family is a diverse group of multifunctional proteins that regulate cellular signaling events downstream of G-protein coupled receptors (GPCRs). In recent years, GPCRs have been linked to the initiation and progression of multiple cancers; thus, regulators of GPCR signaling are also likely to be important to the pathophysiology of cancer. This review highlights recent studies detailing changes in RGS transcript expression during oncogenesis, single nucleotide polymorphisms in RGS proteins linked to lung and bladder cancers, and specific roles for RGS proteins in multiple cancer types.

  8. Sodium tungstate activates glycogen synthesis through a non-canonical mechanism involving G-proteins.

    PubMed

    Zafra, Delia; Nocito, Laura; Domínguez, Jorge; Guinovart, Joan J

    2013-01-31

    Tungstate treatment ameliorates experimental diabetes by increasing liver glycogen deposition through an as yet unidentified mechanism. The signalling mechanism of tungstate was studied in CHOIR cells and primary cultured hepatocytes. This compound exerted its pro-glycogenic effects through a new G-protein-dependent and Tyr-Kinase Receptor-independent mechanism. Chemical or genetic disruption of G-protein signalling prevented the activation of the Ras/ERK cascade and the downstream induction of glycogen synthesis caused by tungstate. Thus, these findings unveil a novel non-canonical signalling pathway that leads to the activation of glycogen synthesis and that could be exploited as an approach to treat diabetes.

  9. [Roles of G protein-coupled estrogen receptor in the male reproductive system].

    PubMed

    Chen, Kai-hong; Zhang, Xian; Jiang, Xue-wu

    2016-02-01

    The G protein-coupled estrogen receptor (GPER), also known as G protein-coupled receptor 30 (GPR30), was identified in the recent years as a functional membrane receptor different from the classical nuclear estrogen receptors. This receptor is widely expressed in the cortex, cerebellum, hippocampus, heart, lung, liver, skeletal muscle, and the urogenital system. It is responsible for the mediation of nongenomic effects associated with estrogen and its derivatives, participating in the physiological activities of the body. The present study reviews the molecular structure, subcellular localization, signaling pathways, distribution, and function of GPER in the male reproductive system.

  10. Effects of central galanin administration on muscarinic cholinergic and galanin receptor G protein coupling.

    PubMed

    Barreda-Gómez, G; Giralt, M T; Rodríguez-Puertas, R

    2005-06-01

    The neuropeptide galanin is expressed in the mammalian central nervous system and has been implicated in neurotrophic actions. Central galanin administration induces cognitive deficits in rodents and inhibits the release of acetylcholine in the hippocampus. In addition, a galanin hyperinnervation of the basal forebrain cholinergic cells in Alzheimer's disease patients has been reported. To evaluate the effect of galanin treatment on galanin and muscarinic cholinergic receptor G protein coupling, galanin was administered into the lateral ventricle of rats via an implanted cannula. Galanin or muscarinic receptor functional coupling to G proteins was quantified by galanin or carbachol stimulation of guanosine 5'-(gamma-[35S]thio)triphosphate binding in rat brain slices. Guanosine 5'-(gamma-[35S]thio)triphosphate basal binding in nucleus basalis of Meynert and thalamic nuclei was increased in the vehicle treated group. This effect was reverted by galanin treatment and indicates that the surgery increased receptor functional coupling to G proteins, which is restored by a possible neurotrophic action mediated by galanin. In addition, in galanin administered animals, galanin-stimulated binding was increased in the amygdala but decreased in the diagonal band, whilst binding stimulation mediated by carbachol was found to be increased in the amygdala, thalamic nuclei and diagonal band. These findings indicate that galanin treatment modulates the coupling of galanin and muscarinic cholinergic receptors to G proteins in specific regions of the rat central nervous system.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  12. Activation of G proteins mediates flow-induced prostaglandin E2 production in osteoblasts

    NASA Technical Reports Server (NTRS)

    Reich, K. M.; McAllister, T. N.; Gudi, S.; Frangos, J. A.

    1997-01-01

    Interstitial fluid flow may play a role in load-induced bone remodeling. Previously, we have shown that fluid flow stimulates osteoblast production of cAMP inositol trisphosphate (IP3), and PGE2. Flow-induced increases in cAMP and IP3 were shown to be a result of PG production. Thus, PGE2 production appears to be an important component in fluid flow induced signal transduction. In the present study, we investigated the mechanism of flow-induced PGE2 synthesis. Flow-induced a 20-fold increase in PGE2 production in osteoblasts. Increases were also observed with ALF4-(10mM) (98-fold), an activator of guanidine nucleotide-binding proteins (G proteins), and calcium ionophore A23187 (2 microM) (100-fold) in stationary cells. We then investigated whether flow stimulation is mediated by G proteins and increases in intracellular calcium. Flow-induced PGE2 production was inhibited by the G protein inhibitors GDP beta S (100 microM) and pertussis toxin (1 microgram/ml) by 83% and 72%, respectively. Chelation of extracellular calcium by EGTA (2 mM) and intracellular calcium by quin-2/AM (30 microM) blocked flow stimulation by 87% and 67%, respectively. These results suggest that G proteins and calcium play an important role in mediating mechanochemical signal transduction in osteoblasts.

  13. Structure and function of regulator of G protein signaling homology domains.

    PubMed

    Tesmer, John J G

    2009-01-01

    All regulator of G protein signaling (RGS) proteins contain a conserved domain of approximately 130 amino acids that binds to activated heterotrimeric G protein α subunits (Gα) and accelerates their rate of GTP hydrolysis. Homologous domains are found in at least six other protein families, including a family of Rho guanine nucleotide exchange factors (RhoGEFs) and the G protein-coupled receptor kinases (GRKs). Although some of the RhoGEF and GRK RGS-like domains can also bind to activated Gα subunits, they do so in distinct ways and with much lower levels of GTPase activation. In other protein families, the domains have as of yet no obvious relationship to heterotrimeric G protein signaling. These RGS homology (RH) domains are now recognized as mediators of extraordinarily diverse protein-protein interactions. Through these interactions, they play roles that range from enzyme to molecular scaffold to signal transducing module. In this review, the atomic structures of RH domains from RGS proteins, Axins, RhoGEFs, and GRKs are compared in light of what is currently known about their functional roles.

  14. Heterotrimeric G-protein Signaling Is Critical to Pathogenic Processes in Entamoeba histolytica

    PubMed Central

    Muller, Robin E.; Giguère, Patrick M.; Machius, Mischa; Willard, Francis S.; Temple, Brenda R. S.; Siderovski, David P.

    2012-01-01

    Heterotrimeric G-protein signaling pathways are vital components of physiology, and many are amenable to pharmacologic manipulation. Here, we identify functional heterotrimeric G-protein subunits in Entamoeba histolytica, the causative agent of amoebic colitis. The E. histolytica Gα subunit EhGα1 exhibits conventional nucleotide cycling properties and is seen to interact with EhGβγ dimers and a candidate effector, EhRGS-RhoGEF, in typical, nucleotide-state-selective fashions. In contrast, a crystal structure of EhGα1 highlights unique features and classification outside of conventional mammalian Gα subfamilies. E. histolytica trophozoites overexpressing wildtype EhGα1 in an inducible manner exhibit an enhanced ability to kill host cells that may be wholly or partially due to enhanced host cell attachment. EhGα1-overexpressing trophozoites also display enhanced transmigration across a Matrigel barrier, an effect that may result from altered baseline migration. Inducible expression of a dominant negative EhGα1 variant engenders the converse phenotypes. Transcriptomic studies reveal that modulation of pathogenesis-related trophozoite behaviors by perturbed heterotrimeric G-protein expression includes transcriptional regulation of virulence factors and altered trafficking of cysteine proteases. Collectively, our studies suggest that E. histolytica possesses a divergent heterotrimeric G-protein signaling axis that modulates key aspects of cellular processes related to the pathogenesis of this infectious organism. PMID:23166501

  15. Sporophyte Formation and Life Cycle Completion in Moss Requires Heterotrimeric G-Proteins1[OPEN

    PubMed Central

    Hackenberg, Dieter; Quatrano, Ralph

    2016-01-01

    In this study, we report the functional characterization of heterotrimeric G-proteins from a nonvascular plant, the moss Physcomitrella patens. In plants, G-proteins have been characterized from only a few angiosperms to date, where their involvement has been shown during regulation of multiple signaling and developmental pathways affecting overall plant fitness. In addition to its unparalleled evolutionary position in the plant lineages, the P. patens genome also codes for a unique assortment of G-protein components, which includes two copies of Gβ and Gγ genes, but no canonical Gα. Instead, a single gene encoding an extra-large Gα (XLG) protein exists in the P. patens genome. Here, we demonstrate that in P. patens the canonical Gα is biochemically and functionally replaced by an XLG protein, which works in the same genetic pathway as one of the Gβ proteins to control its development. Furthermore, the specific G-protein subunits in P. patens are essential for its life cycle completion. Deletion of the genomic locus of PpXLG or PpGβ2 results in smaller, slower growing gametophores. Normal reproductive structures develop on these gametophores, but they are unable to form any sporophyte, the only diploid stage in the moss life cycle. Finally, the mutant phenotypes of ΔPpXLG and ΔPpGβ2 can be complemented by the homologous genes from Arabidopsis, AtXLG2 and AtAGB1, respectively, suggesting an overall conservation of their function throughout the plant evolution. PMID:27550997

  16. MECHANISTIC PATHWAYS AND BIOLOGICAL ROLES FOR RECEPTOR-INDEPENDENT ACTIVATORS OF G-PROTEIN SIGNALING

    PubMed Central

    Blumer, Joe B.; Smrcka, Alan V.; Lanier, S.M.

    2007-01-01

    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, plays an important role in adaptive processes of organs, and aberrant processing of signals through these transducing systems is a component of various disease states. In addition to GPCR-mediated activation of G-protein signaling, nature has evolved creative ways to manipulate and utilize the Gαβγ heterotrimer or Gα and Gαβγ subunits independent of the cell surface receptor stimuli. In such situations, the G-protein subunits (Gα and Gαβγ) may actually be complexed with alternative binding partners independent of the typical heterotrimeric Gαβγ. Such regulatory accessory proteins include the family of RGS proteins that accelerate the GTPase activity of Gα 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 or 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 Gαβγ and the individual Gα and Gαβγ subunits. PMID:17240454

  17. Possible Mechanism of Action of the Electromagnetic Fields of Ultralow Frequency on G-protein

    SciTech Connect

    Nava, J. J. Godina; Segura, M. A. Rodriguez; Garcia, M. N. Jimenez; Cadena, M. S. Reyes

    2008-08-11

    Based in several clinical achievements and mathematical simulation of the immune sytem, previously studied, permit us to establish that a possible Mechanism of Action of ultralow frequency Electromagnetic Fields (ELF) is on G-protein as it has been proposed in specialized literature.

  18. Recent Advances on the Role of G Protein-Coupled Receptors in Hypoxia-Mediated Signaling.

    PubMed

    Lappano, Rosamaria; Rigiracciolo, Damiano; De Marco, Paola; Avino, Silvia; Cappello, Anna Rita; Rosano, Camillo; Maggiolini, Marcello; De Francesco, Ernestina Marianna

    2016-03-01

    G protein-coupled receptors (GPCRs) are cell surface proteins mainly involved in signal transmission; however, they play a role also in several pathophysiological conditions. Chemically heterogeneous molecules like peptides, hormones, lipids, and neurotransmitters activate second messengers and induce several biological responses by binding to these seven transmembrane receptors, which are coupled to heterotrimeric G proteins. Recently, additional molecular mechanisms have been involved in GPCR-mediated signaling, leading to an intricate network of transduction pathways. In this regard, it should be mentioned that diverse GPCR family members contribute to the adaptive cell responses to low oxygen tension, which is a distinguishing feature of several illnesses like neoplastic and cardiovascular diseases. For instance, the G protein estrogen receptor, namely G protein estrogen receptor (GPER)/GPR30, has been shown to contribute to relevant biological effects induced by hypoxia via the hypoxia-inducible factor (HIF)-1α in diverse cell contexts, including cancer. Likewise, GPER has been found to modulate the biological outcome of hypoxic/ischemic stress in both cardiovascular and central nervous systems. Here, we describe the role exerted by GPCR-mediated signaling in low oxygen conditions, discussing, in particular, the involvement of GPER by a hypoxic microenvironment.

  19. G protein-coupled receptors: extranuclear mediators for the non-genomic actions of steroids.

    PubMed

    Wang, Chen; Liu, Yi; Cao, Ji-Min

    2014-09-01

    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 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. Total synthesis and structure-activity relationship studies of a series of selective G protein inhibitors

    NASA Astrophysics Data System (ADS)

    Xiong, Xiao-Feng; Zhang, Hang; Underwood, Christina R.; Harpsøe, Kasper; Gardella, Thomas J.; Wöldike, Mie F.; Mannstadt, Michael; Gloriam, David E.; Bräuner-Osborne, Hans; Strømgaard, Kristian

    2016-11-01

    G proteins are key mediators of G protein-coupled receptor signalling, which facilitates a plethora of important physiological processes. The cyclic depsipeptides YM-254890 and FR900359 are the only known specific inhibitors of the Gq subfamily of G proteins; however, no synthetic route has been reported previously for these complex natural products and they are not easily isolated from natural sources. Here we report the first total synthesis of YM-254890 and FR900359, as well as of two known analogues, YM-385780 and YM-385781. The versatility of the synthetic approach also enabled the design and synthesis of ten analogues, which provided the first structure-activity relationship study for this class of compounds. Pharmacological characterization of all the compounds at Gq-, Gi- and Gs-mediated signalling provided succinct information on the structural requirements for inhibition, and demonstrated that both YM-254890 and FR900359 are highly potent inhibitors of Gq signalling, with FR900359 being the most potent. These natural products and their analogues represent unique tools for explorative studies of G protein inhibition.

  1. The c-terminus of GRK3 indicates rapid dissociation of G protein heterotrimers

    PubMed Central

    Hollins, Bettye; Kuravi, Sudhakiranmayi; Digby, Gregory J.; Lambert, Nevin A.

    2009-01-01

    Signals mediated by heterotrimeric G proteins often develop over the course of tens of milliseconds, and could require either conformational rearrangement or complete physical dissociation of Gα βγ heterotrimers. Although it is known that some active heterotrimers are dissociated (into Gα and Gβγ) at steady-state, it is not clear that dissociation occurs quickly enough to participate in rapid signaling. Here we show that fusion proteins containing the c-terminus of GPCR kinase 3 (GRK3ct) and either the fluorescent protein cerulean or Renilla luciferase bind to venus-labeled Gβγ dimers (Gβγ-V), resulting in Förster or bioluminescence resonance energy transfer (FRET or BRET). GRK3ct fusion proteins are freely-diffusible, and do not form preassembled complexes with G proteins. GRK3ct fusion proteins bind to free Gβγ-V dimers but not to rearranged heterotrimers, and thus can report G protein dissociation with high temporal resolution. We find that heterotrimer dissociation can occur in living cells in less than 100 milliseconds. Under the conditions of these experiments diffusion and collision of masGRK3ct fusion proteins and Gβγ-V were not rate-limiting. These results indicate that G protein heterotrimers can dissociate quickly enough to participate in rapid signaling. PMID:19258039

  2. Structure and Function of Vps15 in the Endosomal G Protein Signaling Pathway

    SciTech Connect

    Heenan, Erin J.; Vanhooke, Janeen L.; Temple, Brenda R.; Betts, Laurie; Sondek, John E.; Dohlman, Henrik G.

    2009-09-11

    G protein-coupled receptors mediate cellular responses to a wide variety of stimuli, including taste, light, and neurotransmitters. In the yeast Saccharomyces cerevisiae, activation of the pheromone pathway triggers events leading to mating. The view had long been held that the G protein-mediated signal occurs principally at the plasma membrane. Recently, it has been shown that the G protein {alpha} subunit Gpa1 can promote signaling at endosomes and requires two components of the sole phosphatidylinositol-3-kinase in yeast, Vps15 and Vps34. Vps15 contains multiple WD repeats and also binds to Gpa1 preferentially in the GDP-bound state; these observations led us to hypothesize that Vps15 may function as a G protein {beta} subunit at the endosome. Here we show an X-ray crystal structure of the Vps15 WD domain that reveals a seven-bladed propeller resembling that of typical G{beta} subunits. We show further that the WD domain is sufficient to bind Gpa1 as well as to Atg14, a potential G{gamma} protein that exists in a complex with Vps15. The Vps15 kinase domain together with the intermediate domain (linking the kinase and WD domains) also contributes to Gpa1 binding and is necessary for Vps15 to sustain G protein signaling. These findings reveal that the Vps15 G{beta}-like domain serves as a scaffold to assemble Gpa1 and Atg14, whereas the kinase and intermediate domains are required for proper signaling at the endosome.

  3. Hypocretin-1 causes G protein activation and increases ACh release in rat pons.

    PubMed

    Bernard, René; Lydic, Ralph; Baghdoyan, Helen A

    2003-10-01

    The effects of the arousal-promoting peptide hypocretin on brain stem G protein activation and ACh release were examined using 16 adult Sprague-Dawley rats. In vitro[35S]GTPgammaS autoradiography was used to test the hypothesis that hypocretin-1-stimulated G protein activation is concentration-dependent and blocked by the hypocretin receptor antagonist SB-334867. Activated G proteins were quantified in dorsal raphe nucleus (DR), locus coeruleus (LC) and pontine reticular nucleus oral part (PnO) and caudal part (PnC). Concentration-response data revealed a significant (P < 0.001) effect of hypocretin-1 (2-2000 nm) in all brain regions examined. Maximal increases over control levels of [35S]GTPgammaS binding were 37% (DR), 58% (LC), 52% (PnO) and 44% (PnC). SB-334867 (2 micro m) significantly (P < 0.002) blocked hypocretin-1 (200 nm)-stimulated [35S]GTPgammaS binding in all four nuclei. This is the first autoradiographic demonstration that hypocretin-1 activates G proteins in arousal-related brain stem nuclei as a result of specific receptor interactions. This finding suggests that some hypocretin receptors in brain stem couple to inhibitory G proteins. In vivo microdialysis was used to test the hypothesis that PnO administration of hypocretin-1 increases ACh release in PnO. Dialysis delivery of hypocretin-1 (100 micro m) significantly (P < 0.002) increased (87%) ACh release. This finding is consistent with the interpretation that one mechanism by which hypocretin promotes arousal is by enhancing cholinergic neurotransmission in the pontine reticular formation.

  4. Noribogaine is a G-protein biased κ-opioid receptor agonist.

    PubMed

    Maillet, Emeline L; Milon, Nicolas; Heghinian, Mari D; Fishback, James; Schürer, Stephan C; Garamszegi, Nandor; Mash, Deborah C

    2015-12-01

    Noribogaine is the long-lived human metabolite of the anti-addictive substance ibogaine. Noribogaine efficaciously reaches the brain with concentrations up to 20 μM after acute therapeutic dose of 40 mg/kg ibogaine in animals. Noribogaine displays atypical opioid-like components in vivo, anti-addictive effects and potent modulatory properties of the tolerance to opiates for which the mode of action remained uncharacterized thus far. Our binding experiments and computational simulations indicate that noribogaine may bind to the orthosteric morphinan binding site of the opioid receptors. Functional activities of noribogaine at G-protein and non G-protein pathways of the mu and kappa opioid receptors were characterized. Noribogaine was a weak mu antagonist with a functional inhibition constants (Ke) of 20 μM at the G-protein and β-arrestin signaling pathways. Conversely, noribogaine was a G-protein biased kappa agonist 75% as efficacious as dynorphin A at stimulating GDP-GTP exchange (EC50=9 μM) but only 12% as efficacious at recruiting β-arrestin, which could contribute to the lack of dysphoric effects of noribogaine. In turn, noribogaine functionally inhibited dynorphin-induced kappa β-arrestin recruitment and was more potent than its G-protein agonistic activity with an IC50 of 1 μM. This biased agonist/antagonist pharmacology is unique to noribogaine in comparison to various other ligands including ibogaine, 18-MC, nalmefene, and 6'-GNTI. We predict noribogaine to promote certain analgesic effects as well as anti-addictive effects at effective concentrations>1 μM in the brain. Because elevated levels of dynorphins are commonly observed and correlated with anxiety, dysphoric effects, and decreased dopaminergic tone, a therapeutically relevant functional inhibition bias to endogenously released dynorphins by noribogaine might be worthy of consideration for treating anxiety and substance related disorders.

  5. Ric-8 Proteins Are Molecular Chaperones That Direct Nascent G Protein α Subunit Membrane Association

    PubMed Central

    Gabay, Meital; Pinter, Mary E.; Wright, Forrest A.; Chan, PuiYee; Murphy, Andrew J.; Valenzuela, David M.; Yancopoulos, George D.; Tall, Gregory G.

    2013-01-01

    Ric-8A (resistance to inhibitors of cholinesterase 8A) and Ric-8B are guanine nucleotide exchange factors that enhance different heterotrimeric guanine nucleotide–binding protein (G protein) signaling pathways by unknown mechanisms. Because transgenic disruption of Ric-8A or Ric-8B in mice caused early embryonic lethality, we derived viable Ric-8A– or Ric-8B–deleted embryonic stem (ES) cell lines from blastocysts of these mice. We observed pleiotropic G protein signaling defects in Ric-8A−/− ES cells, which resulted from reduced steady-state amounts of Gαi, Gαq, and Gα13 proteins to <5% of those of wild-type cells. The amounts of Gαs and total Gβ protein were partially reduced in Ric-8A−/− cells compared to those in wild-type cells, and only the amount of Gαs was reduced substantially in Ric-8B−/− cells. The abundances of mRNAs encoding the G protein α subunits were largely unchanged by loss of Ric-8A or Ric-8B. The plasma membrane residence of G proteins persisted in the absence of Ric-8 but was markedly reduced compared to that in wild-type cells. Endogenous Gαi and Gαq were efficiently translated in Ric-8A−/− cells but integrated into endomembranes poorly; however, the reduced amounts of G protein α subunits that reached the membrane still bound to nascent Gβγ. Finally, Gαi, Gαq, and Gβ1 proteins exhibited accelerated rates of degradation in Ric-8A−/− cells compared to those in wild-type cells. Together, these data suggest that Ric-8 proteins are molecular chaperones required for the initial association of nascent Gα subunits with cellular membranes. PMID:22114146

  6. Lipid modulation of early G protein-coupled receptor signalling events.

    PubMed

    Dijkman, Patricia M; Watts, Anthony

    2015-11-01

    Upon binding of extracellular ligands, G protein coupled-receptors (GPCRs) initiate signalling cascades by activating heterotrimeric G proteins through direct interactions with the α subunit. While the lipid dependence of ligand binding has previously been studied for one class A GPCR, the neurotensin receptor 1 (NTS1), the role the lipid environment plays in the interaction of activated GPCRs with G proteins is less well understood. It is therefore of interest to understand the balance of lipid interactions required to support both ligand binding and G protein activation, not least since some receptors have multiple locations, and may experience different membrane environments when signalling in the plasma membrane or during endocytosis. Here, using the sensitive biophysical technique of microscale thermophoresis in conjunction with nanodisc lipid bilayer reconstitution, we show that in more native lipid environments rich in phosphatidyl ethanolamine (PE), the Gαi1 subunit has a ~4-fold higher affinity for NTS1 than in the absence of native lipids. The G protein-receptor affinity was further shown to be dependent on the ligand-binding state of the receptor, with potential indication of biased signalling for the known antagonist SR142948A. Gαi1 also showed preferential interaction with empty nanodiscs of native lipid mixtures rich in PE by around 2- to 4-fold over phosphatidyl choline (PC)/phosphatidyl glycerol (PG) lipid mixtures. The lipid environment may therefore play a role in creating favourable micro-environments for efficient GPCR signalling. Our approach combining nanodiscs with microscale thermophoresis will be useful in future studies to elucidate further the complexity of the GPCR interactome.

  7. Pluto's Putative Cryovolcanic Constructs

    NASA Astrophysics Data System (ADS)

    Singer, K. N.; White, O. L.; Schenk, P. M.; Moore, J. M.; Spencer, J. R.; McKinnon, W. B.; Howard, A. D.; Stern, A. S.; Cook, J. C.; Grundy, W. M.; Cruikshank, D. P.; Beyer, R. A.; Umurhan, O.; Howett, C. J. A.; Parker, A. H.; Protopapa, S.; Lauer, T. R.; Weaver, H. A.; Young, L. A.; Olkin, C. B.; Ennico, K.

    2016-06-01

    New Horizons imaged two large mounds with deep central depressions on Pluto. Both features appear constructional, and have relatively young surfaces. This mapping is part of effort to characterize and assess the age and origin of the mounds.

  8. G protein-coupled receptor kinase 6/β-arrestin 2 system in a rat model of dopamine supersensitivity psychosis.

    PubMed

    Oda, Yasunori; Tadokoro, Shigenori; Takase, Masayuki; Kanahara, Nobuhisa; Watanabe, Hiroyuki; Shirayama, Yukihiko; Hashimoto, Kenji; Iyo, Masaomi

    2015-12-01

    In humans, long-term antipsychotic treatment is known to induce movement disorders and a psychosis, called dopamine supersensitivity psychosis (DSP). The mechanism by which chronic administration of antipsychotic(s) causes DSP may be the treatment-induced up-regulation of dopamine D2 receptors (DRD2). G protein-coupled receptor kinase 6 (GRK6) and beta-arrestin 2 (ARRB2) play important roles in the trafficking of DRD2 by phosphorylation and internalization. We investigated the effects of chronic continuous treatment with mini-pump-administered haloperidol (HAL) on the sensitivity of Wistar rats to dopamine, as measured by the locomotor response to methamphetamine (MAP) and the density of striatal DRD2. Chronic continuous treatment with HAL resulted in significantly higher locomotor response to MAP and significantly higher striatal DRD2 density compared with those in rats administered vehicle (VEH). Enzyme-linked immunosorbent assays revealed that striatal ARRB2 in DSP model rats tended to decrease in comparison with that in the VEH group. In addition, the ratio of GRK6/ARRB2 in DSP model rats was significantly higher than that in controls. Our results suggest that alterations of the GRK6 and ARRB2 system could induce both DRD2 up-regulation and impairment of the dopamine signaling pathway, resulting potentially in the development of DSP.

  9. The regulator of G protein signaling (RGS) domain of G protein-coupled receptor kinase 5 (GRK5) regulates plasma membrane localization and function.

    PubMed

    Xu, Hua; Jiang, Xiaoshan; Shen, Ke; Fischer, Christopher C; Wedegaertner, Philip B

    2014-07-01

    The G protein-coupled receptor (GPCR) kinases (GRKs) phosphorylate activated GPCRs at the plasma membrane (PM). Here GRK5/GRK4 chimeras and point mutations in GRK5 identify a short sequence within the regulator of G protein signaling (RGS) domain in GRK5 that is critical for GRK5 PM localization. This region of the RGS domain of GRK5 coincides with a region of GRK6 and GRK1 shown to form a hydrophobic dimeric interface (HDI) in crystal structures. Coimmunoprecipitation (coIP) and acceptor photobleaching fluorescence resonance energy transfer assays show that expressed GRK5 self-associates in cells, whereas GRK5-M165E/F166E (GRK5-EE), containing hydrophilic mutations in the HDI region of the RGS domain, displays greatly decreased coIP interactions. Both forcing dimerization of GRK5-EE, via fusion to leucine zipper motifs, and appending an extra C-terminal membrane-binding region to GRK5-EE (GRK5-EE-CT) recover PM localization. In addition, GRK5-EE displays a decreased ability to inhibit PAR1-induced calcium release compared with GRK5 wild type (wt). In contrast, PM-localized GRK5-EE-CaaX (appending a C-terminal prenylation and polybasic motif from K-ras) or GRK5-EE-CT shows comparable ability to GRK5 wt to inhibit PAR1-induced calcium release. The results suggest a novel model in which GRK5 dimerization is important for its plasma membrane localization and function.

  10. Differential distribution of G-protein beta-subunits in brain: an immunocytochemical analysis.

    PubMed

    Brunk, I; Pahner, I; Maier, U; Jenner, B; Veh, R W; Nürnberg, B; Ahnert-Hilger, G

    1999-05-01

    Heterotrimeric G proteins play central roles in signal transduction of neurons and other cells. The variety of their alpha-, beta-, and gamma-subunits allows numerous combinations thereby confering specificity to receptor-G-protein-effector interactions. Using antisera against individual G-protein beta-subunits we here present a regional and subcellular distribution of Gbeta1, Gbeta2, and Gbeta5 in rat brain. Immunocytochemical specificity of the subtype-specific antisera is revealed in Sf9 cells infected with various G-protein beta-subunits. Since Gbeta-subunits together with a G-protein gamma-subunit affect signal cascades we include a distribution of the neuron-specific Ggamma2- and Ggamma3-subunits in selected brain areas. Gbeta1, Gbeta2, and Gbeta5 are preferentially distributed in the neuropil of hippocampus, cerebellum and spinal cord. Gbeta2 is highly concentrated in the mossy fibres of dentate gyrus neurons ending in the stratum lucidum of hippocampal CA3-area. High amounts of Gbeta2 also occur in interneurons innervating spinal cord alpha-motoneurons. Gbeta5 is differentially distributed in all brain areas studied. It is found in the pyramidal cells of hippocampal CA1-CA3 as well as in the granule cell layer of dentate gyrus and in some interneurons. In the spinal cord Gbeta5 in contrast to Gbeta2 concentrates around alpha-motoneurons. In cultivated mouse hippocampal and hypothalamic neurons Gbeta2 and Gbeta5 are found in different subcellular compartments. Whereas Gbeta5 is restricted to the perikarya, Gbeta2 is also found in processes and synaptic contacts where it partially colocalizes with the synaptic vesicle protein synaptobrevin. An antiserum recognizing Ggamma2 and Ggamma3 reveals that these subunits are less expressed in hippocampus and cerebellum. Presumably this antiserum specifically recognizes Ggamma2 and Ggamma3 in combinations with certain G alphas and/or Gbetas. The widespread but regionally and cellularly rather different distribution of

  11. Role of APP Interactions with Heterotrimeric G Proteins: Physiological Functions and Pathological Consequences.

    PubMed

    Copenhaver, Philip F; Kögel, Donat

    2017-01-01

    Following the discovery that the amyloid precursor protein (APP) is the source of β-amyloid peptides (Aβ) that accumulate in Alzheimer's disease (AD), structural analyses suggested that the holoprotein resembles a transmembrane receptor. Initial studies using reconstituted membranes demonstrated that APP can directly interact with the heterotrimeric G protein Gαo (but not other G proteins) via an evolutionarily G protein-binding motif in its cytoplasmic domain. Subsequent investigations in cell culture showed that antibodies against the extracellular domain of APP could stimulate Gαo activity, presumably mimicking endogenous APP ligands. In addition, chronically activating wild type APP or overexpressing mutant APP isoforms linked with familial AD could provoke Go-dependent neurotoxic responses, while biochemical assays using human brain samples suggested that the endogenous APP-Go interactions are perturbed in AD patients. More recently, several G protein-dependent pathways have been implicated in the physiological roles of APP, coupled with evidence that APP interacts both physically and functionally with Gαo in a variety of contexts. Work in insect models has demonstrated that the APP ortholog APPL directly interacts with Gαo in motile neurons, whereby APPL-Gαo signaling regulates the response of migratory neurons to ligands encountered in the developing nervous system. Concurrent studies using cultured mammalian neurons and organotypic hippocampal slice preparations have shown that APP signaling transduces the neuroprotective effects of soluble sAPPα fragments via modulation of the PI3K/Akt pathway, providing a mechanism for integrating the stress and survival responses regulated by APP. Notably, this effect was also inhibited by pertussis toxin, indicating an essential role for Gαo/i proteins. Unexpectedly, C-terminal fragments (CTFs) derived from APP have also been found to interact with Gαs, whereby CTF-Gαs signaling can promote neurite outgrowth

  12. Role of APP Interactions with Heterotrimeric G Proteins: Physiological Functions and Pathological Consequences

    PubMed Central

    Copenhaver, Philip F.; Kögel, Donat

    2017-01-01

    Following the discovery that the amyloid precursor protein (APP) is the source of β-amyloid peptides (Aβ) that accumulate in Alzheimer’s disease (AD), structural analyses suggested that the holoprotein resembles a transmembrane receptor. Initial studies using reconstituted membranes demonstrated that APP can directly interact with the heterotrimeric G protein Gαo (but not other G proteins) via an evolutionarily G protein-binding motif in its cytoplasmic domain. Subsequent investigations in cell culture showed that antibodies against the extracellular domain of APP could stimulate Gαo activity, presumably mimicking endogenous APP ligands. In addition, chronically activating wild type APP or overexpressing mutant APP isoforms linked with familial AD could provoke Go-dependent neurotoxic responses, while biochemical assays using human brain samples suggested that the endogenous APP-Go interactions are perturbed in AD patients. More recently, several G protein-dependent pathways have been implicated in the physiological roles of APP, coupled with evidence that APP interacts both physically and functionally with Gαo in a variety of contexts. Work in insect models has demonstrated that the APP ortholog APPL directly interacts with Gαo in motile neurons, whereby APPL-Gαo signaling regulates the response of migratory neurons to ligands encountered in the developing nervous system. Concurrent studies using cultured mammalian neurons and organotypic hippocampal slice preparations have shown that APP signaling transduces the neuroprotective effects of soluble sAPPα fragments via modulation of the PI3K/Akt pathway, providing a mechanism for integrating the stress and survival responses regulated by APP. Notably, this effect was also inhibited by pertussis toxin, indicating an essential role for Gαo/i proteins. Unexpectedly, C-terminal fragments (CTFs) derived from APP have also been found to interact with Gαs, whereby CTF-Gαs signaling can promote neurite

  13. Signal Activation and Inactivation by the Gα Helical Domain: A Long-Neglected Partner in G Protein Signaling

    PubMed Central

    Dohlman, Henrik G.; Jones, Janice C.

    2013-01-01

    Heterotrimeric guanine nucleotide–binding proteins (G proteins) are positioned at the top of many signal transduction pathways. The G protein α subunit is composed of two domains, one that resembles Ras and another that is composed entirely of α helices. Historically, most attention has focused on the Ras-like domain, but emerging evidence reveals that the helical domain is an active participant in G protein signaling. PMID:22649098

  14. The unconventional G-protein cycle of LRRK2 and Roco proteins.

    PubMed

    Terheyden, Susanne; Nederveen-Schippers, Laura M; Kortholt, Arjan

    2016-12-15

    Mutations in the human leucine-rich repeat kinase 2 (LRRK2) are the most frequent cause of hereditary Parkinson's disease (PD). LRRK2 belongs to the Roco family of proteins, which are characterized by the presence of a Ras of complex proteins domain (Roc), a C-terminal of Roc domain (COR) and a kinase domain. Despite intensive research, much remains unknown about activity and the effect of PD-associated mutations. Recent biochemical and structural studies suggest that LRRK2 and Roco proteins are noncanonical G-proteins that do not depend on guanine nucleotide exchange factors or GTPase-activating proteins for activation. In this review, we will discuss the unusual G-protein cycle of LRRK2 in the context of the complex intramolecular LRRK2 activation mechanism.

  15. GAP-43 augments G protein-coupled receptor transduction in Xenopus laevis oocytes.

    PubMed Central

    Strittmatter, S M; Cannon, S C; Ross, E M; Higashijima, T; Fishman, M C

    1993-01-01

    The neuronal protein GAP-43 is thought to play a role in determining growth-cone motility, perhaps as an intracellular regulator of signal transduction, but its molecular mechanism of action has remained unclear. We find that GAP-43, when microinjected into Xenopus laevis oocytes, increases the oocyte response to G protein-coupled receptor agonists by 10- to 100-fold. Higher levels of GAP-43 cause a transient current flow, even without receptor stimulation. The GAP-43-induced current, like receptor-stimulated currents, is mediated by a calcium-activated chloride channel and can be desensitized by injection of inositol 1,4,5-trisphosphate. This suggests that neuronal GAP-43 may serve as an intracellular signal to greatly enhance the sensitivity of G protein-coupled receptor transduction. Images Fig. 1 Fig. 2 PMID:7685122

  16. GRK2: multiple roles beyond G protein-coupled receptor desensitization

    PubMed Central

    Evron, Tama; Daigle, Tanya L.; Caron, Marc G.

    2012-01-01

    G protein-coupled receptor kinases (GRKs) regulate numerous G protein-coupled receptors (GPCRs) by phosphorylating the intracellular domain of the active receptor, resulting in receptor desensitization and internalization. GRKs also regulate GPCR trafficking in a phosphorylation-independent manner via direct protein-protein interactions. Emerging evidence suggests that GRK2, the most widely studied member of this family of kinases, modulates multiple cellular responses in various physiological contexts by either phosphorylating non-receptor substrates or by directly interacting with signaling molecules. In this review, we discuss traditional and newly discovered roles of GRK2 in receptor internalization and signaling as well as its impact on non-receptor substrates. We also discuss novel exciting roles of GRK2 in the regulation of dopamine receptor signaling and in the activation and trafficking of the atypical GPCR, Smoothened (Smo). PMID:22277298

  17. Structure-based drug design for G protein-coupled receptors.

    PubMed

    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.

  18. The Molecular Pharmacology of G Protein Signaling Then and Now: A Tribute to Alfred G. Gilman.

    PubMed

    Sunahara, Roger K; Insel, Paul A

    2016-05-01

    The recent, unfortunate death of Alfred G. ("Al") Gilman, M.D., Ph.D., represents a sad signpost for an era spanning over 40 years in molecular pharmacology. Gilman's discoveries, influence, and persona were dominant forces in research and training in pharmacology. Here, we review the progression of ideas and knowledge that spawned early work by Gilman and collaborators (among them, one of the authors) and later efforts (including those of the other author) that have recently yielded a comprehensive and precise structural understanding of fundamental topics in pharmacology: the binding of ligands to G protein-coupled receptors (GPCRs) and the interaction of GPCRs with heterotrimeric G proteins and effector molecules. Those data provide new and important insights into the molecular basis that underlies affinity and efficacy, two of the most important features of drug action, which represent the latest chapter in the saga that Al Gilman's work helped launch.

  19. Alcohol modulation of G-protein-gated inwardly rectifying potassium channels: from binding to therapeutics

    PubMed Central

    Bodhinathan, Karthik; Slesinger, Paul A.

    2014-01-01

    Alcohol (ethanol)-induced behaviors may arise from direct interaction of alcohol with discrete protein cavities within brain proteins. Recent structural and biochemical studies have provided new insights into the mechanism of alcohol-dependent activation of G protein-gated inwardly rectifying potassium (GIRK) channels, which regulate neuronal responses in the brain reward circuit. GIRK channels contain an alcohol binding pocket formed at the interface of two adjacent channel subunits. Here, we discuss the physiochemical properties of the alcohol pocket and the roles of G protein βγ subunits and membrane phospholipid PIP2 in regulating the alcohol response of GIRK channels. Some of the features of alcohol modulation of GIRK channels may be common to other alcohol-sensitive brain proteins. We discuss the possibility of alcohol-selective therapeutics that block alcohol access to the pocket. Understanding alcohol recognition and modulation of brain proteins is essential for development of therapeutics for alcohol abuse and addiction. PMID:24611054

  20. Emerging concepts for G protein-gated inwardly rectifying potassium (GIRK) channels in health and disease

    PubMed Central

    Lüscher, Christian; Slesinger, Paul A.

    2010-01-01

    G protein-gated inwardly rectifying potassium (GIRK) channels hyperpolarize neurons in response to the activation of many G-protein coupled receptors and thus control the excitability of neurons through GIRK-mediated self-inhibition, slow synaptic potentials and volume transmission. GIRK channel function and trafficking are highly dependent on their subunit composition. Pharmacological investigations of GIRK channels and studies in animal models suggest that GIRK activity has an important role in physiological responses, including pain perception and memory modulation. Moreover, abnormal GIRK function has been implicated in altering neuronal excitability and cell death that may be important in the pathophysiology of human diseases such as epilepsy, Down’s syndrome, Parkinson’s disease and drug addiction. GIRK channels may therefore prove to be a valuable new therapeutic target for treating these health problems. PMID:20389305

  1. Lighting up G protein-coupled purinergic receptors with engineered fluorescent ligands

    PubMed Central

    Ciruela, Francisco; Fernández-Dueñas, Víctor; Jacobson, Kenneth A.

    2015-01-01

    The use of G protein-coupled receptors fluorescent ligands is undergoing continuous expansion. In line with this, fluorescent agonists and antagonists of high affinity for G protein-coupled adenosine and P2Y receptors have been shown to be useful pharmacological probe compounds. Fluorescent ligands for A1R, A2AR, and A3R (adenosine receptors) and P2Y2R, P2Y4R, P2Y6R, and P2Y14R (nucleotide receptors) have been reported. Such ligands have been successfully applied to drug discovery and to GPCR characterization by flow cytometry, fluorescence correlation spectroscopy, fluorescence microscopy, fluorescence polarization, fluorescence resonance energy transfer and scanning confocal microscopy. Here we summarize recently reported and readily available representative fluorescent ligands of purinergic receptors. In addition, we pay special attention on the use of this family of fluorescent ligands revealing two main aspects of purinergic receptor biology, namely ligand binding and receptor oligomerization. PMID:25890205

  2. Role and therapeutic potential of G-protein coupled receptors in breast cancer progression and metastases.

    PubMed

    Singh, Anukriti; Nunes, Jessica J; Ateeq, Bushra

    2015-09-15

    G-protein-coupled receptors (GPCRs) comprise a large family of cell-surface receptors, which have recently emerged as key players in tumorigenesis, angiogenesis and metastasis. In this review, we discussed our current understanding of the many roles played by GPCRs in general, and particularly Angiotensin II type I receptor (AGTR1), a member of the seven-transmembrane-spanning G-protein coupled receptor superfamily, and its significance in breast cancer progression and metastasis. We have also discussed different strategies for targeting AGTR1, and its ligand Angiotension II (Ang II), which might unravel unique opportunities for breast cancer prevention and treatment. For example, AGTR1 blockers (ARBs) which are already in clinical use for treating hypertension, merit further investigation as a therapeutic strategy for AGTR1-positive cancer patients and may have the potential to prevent Ang II-AGTR1 signalling mediated cancer pathogenesis and metastases.

  3. G Protein-Coupled Receptors in cancer: biochemical interactions and drug design.

    PubMed

    Audigier, Yves; Picault, François-Xavier; Chaves-Almagro, Carline; Masri, Bernard

    2013-01-01

    G Protein-Coupled Receptors (GPCRs) share the same topology made of seven-transmembrane segments and represent the largest family of membrane receptors. Initially associated with signal transduction in differentiated cells, GPCRs and heterotrimeric G proteins were shown to behave as proto-oncogenes whose overexpression or activating mutations confer transforming properties. The first part of this review focuses on the link between biochemical interactions of a GPCR with other receptors, such as dimerization or multiprotein complexes, and their oncogenic properties. Alteration of these interactions or deregulation of transduction cascades can promote uncontrolled cell proliferation or cell transformation that leads to tumorigenicity and malignancy. The second part concerns the design of drugs specifically targeting these complex interactions and their promise in cancer therapy.

  4. Role and therapeutic potential of G-protein coupled receptors in breast cancer progression and metastases

    PubMed Central

    Singh, Anukriti; Nunes, Jessica J.; Ateeq, Bushra

    2015-01-01

    G-protein-coupled receptors (GPCRs) comprise a large family of cell-surface receptors, which have recently emerged as key players in tumorigenesis, angiogenesis and metastasis. In this review, we discussed our current understanding of the many roles played by GPCRs in general, and particularly Angiotensin II type I receptor (AGTR1), a member of the seven-transmembrane-spanning G-protein coupled receptor superfamily, and its significance in breast cancer progression and metastasis. We have also discussed different strategies for targeting AGTR1, and its ligand Angiotension II (Ang II), which might unravel unique opportunities for breast cancer prevention and treatment. For example, AGTR1 blockers (ARBs) which are already in clinical use for treating hypertension, merit further investigation as a therapeutic strategy for AGTR1-positive cancer patients and may have the potential to prevent Ang II-AGTR1 signalling mediated cancer pathogenesis and metastases. PMID:25981295

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

    PubMed Central

    Leyva-Illades, Dinorah; DeMorrow, Sharon

    2013-01-01

    G protein-coupled receptors (GPCRs) modulate a vast array of cellular processes. The current review gives an overview of the general characteristics of GPCRs and their role in physiological conditions. In addition, it describes the current knowledge of the physiological and pathophysiological functions of GPR55, an orphan GPCR, and how it can be exploited as a therapeutic target to combat various cancers. PMID:23869178

  6. Mechanism of membrane fusion induced by vesicular stomatitis virus G protein.

    PubMed

    Kim, Irene S; Jenni, Simon; Stanifer, Megan L; Roth, Eatai; Whelan, Sean P J; van Oijen, Antoine M; Harrison, Stephen C

    2017-01-03

    The glycoproteins (G proteins) of vesicular stomatitis virus (VSV) and related rhabdoviruses (e.g., rabies virus) mediate both cell attachment and membrane fusion. The reversibility of their fusogenic conformational transitions differentiates them from many other low-pH-induced viral fusion proteins. We report single-virion fusion experiments, using methods developed in previous publications to probe fusion of influenza and West Nile viruses. We show that a three-stage model fits VSV single-particle fusion kinetics: (i) reversible, pH-dependent, G-protein conformational change from the known prefusion conformation to an extended, monomeric intermediate; (ii) reversible trimerization and clustering of the G-protein fusion loops, leading to an extended intermediate that inserts the fusion loops into the target-cell membrane; and (iii) folding back of a cluster of extended trimers into their postfusion conformations, bringing together the viral and cellular membranes. From simulations of the kinetic data, we conclude that the critical number of G-protein trimers required to overcome membrane resistance is 3 to 5, within a contact zone between the virus and the target membrane of 30 to 50 trimers. This sequence of conformational events is similar to those shown to describe fusion by influenza virus hemagglutinin (a "class I" fusogen) and West Nile virus envelope protein ("class II"). Our study of VSV now extends this description to "class III" viral fusion proteins, showing that reversibility of the low-pH-induced transition and architectural differences in the fusion proteins themselves do not change the basic mechanism by which they catalyze membrane fusion.

  7. Mechanism of membrane fusion induced by vesicular stomatitis virus G protein

    PubMed Central

    Kim, Irene S.; Jenni, Simon; Stanifer, Megan L.; Roth, Eatai; Whelan, Sean P. J.; van Oijen, Antoine M.; Harrison, Stephen C.

    2017-01-01

    The glycoproteins (G proteins) of vesicular stomatitis virus (VSV) and related rhabdoviruses (e.g., rabies virus) mediate both cell attachment and membrane fusion. The reversibility of their fusogenic conformational transitions differentiates them from many other low-pH-induced viral fusion proteins. We report single-virion fusion experiments, using methods developed in previous publications to probe fusion of influenza and West Nile viruses. We show that a three-stage model fits VSV single-particle fusion kinetics: (i) reversible, pH-dependent, G-protein conformational change from the known prefusion conformation to an extended, monomeric intermediate; (ii) reversible trimerization and clustering of the G-protein fusion loops, leading to an extended intermediate that inserts the fusion loops into the target-cell membrane; and (iii) folding back of a cluster of extended trimers into their postfusion conformations, bringing together the viral and cellular membranes. From simulations of the kinetic data, we conclude that the critical number of G-protein trimers required to overcome membrane resistance is 3 to 5, within a contact zone between the virus and the target membrane of 30 to 50 trimers. This sequence of conformational events is similar to those shown to describe fusion by influenza virus hemagglutinin (a “class I” fusogen) and West Nile virus envelope protein (“class II”). Our study of VSV now extends this description to “class III” viral fusion proteins, showing that reversibility of the low-pH-induced transition and architectural differences in the fusion proteins themselves do not change the basic mechanism by which they catalyze membrane fusion. PMID:27974607

  8. Strategic Research Institute G-Protein-Coupled Receptors Drug Discovery World Summit.

    PubMed

    Felder, Christian C

    2004-08-01

    The Strategic Research Institute provided a well-organised 2-day summit that offered presentations and posters on new assay technology, structure-based small-molecule discovery and examples of clinical candidates targeted to G-protein-coupled receptor (GPCR) targets. A wide variety of topics were presented providing recent advances in GPCR target selection, bioassay-enabling technology and medicinal chemistry targeted to GPCR-relevant chemical libraries. GPCRs continue to be an attractive platform for drug discovery.

  9. The association between glycosylphosphatidylinositol-anchored proteins and heterotrimeric G protein alpha subunits in lymphocytes.

    PubMed Central

    Solomon, K R; Rudd, C E; Finberg, R W

    1996-01-01

    Glycosylphosphatidylinositol (GPI)-anchored proteins are nonmembrane spanning cell surface proteins that have been demonstrated to be signal transduction molecules. Because these proteins do not extend into the cytoplasm, the mechanism by which cross-linking of these molecules leads to intracellular signal transduction events is obscure. Previous analysis has indicated that these proteins are associated with src family member tyrosine kinases; however, the role this interaction plays in the generation of intracellular signals is not clear. Here we show that GPI-anchored proteins are associated with alpha subunits of heterotrimeric GTP binding proteins (G proteins) in both human and murine lymphocytes. When the GPI-anchored proteins CD59, CD48, and Thy-1 were immunoprecipitated from various cell lines or freshly isolated lymphocytes, all were found to be associated with a 41-kDa phosphoprotein that we have identified, by using specific antisera, as a mixture of tyrosine phosphorylated G protein alpha subunits: a small amount of Gialpha1, and substantial amounts of Gialpha2 and Gialpha3. GTP binding assays performed with immunoprecipitations of CD59 indicated that there was GTP-binding activity associated with this molecule. Thus, we have shown by both immunochemical and functional criteria that GPI-anchored proteins are physically associated with G proteins. These experiments suggest a potential role of G proteins in the transduction of signals generated by GPI-anchored molecules expressed on lymphocytes of both mouse and human. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 PMID:8650218

  10. G protein-linked signaling pathways in bipolar and major depressive disorders

    PubMed Central

    Tomita, Hiroaki; Ziegler, Mary E.; Kim, Helen B.; Evans, Simon J.; Choudary, Prabhakara V.; Li, Jun Z.; Meng, Fan; Dai, Manhong; Myers, Richard M.; Neal, Charles R.; Speed, Terry P.; Barchas, Jack D.; Schatzberg, Alan F.; Watson, Stanley J.; Akil, Huda; Jones, Edward G.; Bunney, William E.; Vawter, Marquis P.

    2013-01-01

    The G-protein linked signaling system (GPLS) comprises a large number of G-proteins, G protein-coupled receptors (GPCRs), GPCR ligands, and downstream effector molecules. G-proteins interact with both GPCRs and downstream effectors such as cyclic adenosine monophosphate (cAMP), phosphatidylinositols, and ion channels. The GPLS is implicated in the pathophysiology and pharmacology of both major depressive disorder (MDD) and bipolar disorder (BPD). This study evaluated whether GPLS is altered at the transcript level. The gene expression in the dorsolateral prefrontal (DLPFC) and anterior cingulate (ACC) were compared from MDD, BPD, and control subjects using Affymetrix Gene Chips and real time quantitative PCR. High quality brain tissue was used in the study to control for confounding effects of agonal events, tissue pH, RNA integrity, gender, and age. GPLS signaling transcripts were altered especially in the ACC of BPD and MDD subjects. Transcript levels of molecules which repress cAMP activity were increased in BPD and decreased in MDD. Two orphan GPCRs, GPRC5B and GPR37, showed significantly decreased expression levels in MDD, and significantly increased expression levels in BPD. Our results suggest opposite changes in BPD and MDD in the GPLS, “activated” cAMP signaling activity in BPD and “blunted” cAMP signaling activity in MDD. GPRC5B and GPR37 both appear to have behavioral effects, and are also candidate genes for neurodegenerative disorders. In the context of the opposite changes observed in BPD and MDD, these GPCRs warrant further study of their brain effects. PMID:24391664

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

    PubMed

    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. Ligand-Based Peptide Design and Combinatorial Peptide Libraries to Target G Protein-Coupled Receptors

    PubMed Central

    Gruber, Christian W.; Muttenthaler, Markus; Freissmuth, Michael

    2016-01-01

    G protein-coupled receptors (GPCRs) are considered to represent the most promising drug targets; it has been repeatedly said that a large fraction of the currently marketed drugs elicit their actions by binding to GPCRs (with cited numbers varying from 30–50%). Closer scrutiny, however, shows that only a modest fraction of (~60) GPCRs are, in fact, exploited as drug targets, only ~20 of which are peptide-binding receptors. The vast majority of receptors in the humane genome have not yet been explored as sites of action for drugs. Given the drugability of this receptor class, it appears that opportunities for drug discovery abound. In addition, GPCRs provide for binding sites other than the ligand binding sites (referred to as the “orthosteric site”). These additional sites include (i) binding sites for ligands (referred to as “allosteric ligands”) that modulate the affinity and efficacy of orthosteric ligands, (ii) the interaction surface that recruits G proteins and arrestins, (iii) the interaction sites of additional proteins (GIPs, GPCR interacting proteins that regulate G protein signaling or give rise to G protein-independent signals). These sites can also be targeted by peptides. Combinatorial and natural peptide libraries are therefore likely to play a major role in identifying new GPCR ligands at each of these sites. In particular the diverse natural peptide libraries such as the venom peptides from marine cone-snails and plant cyclotides have been established as a rich source of drug leads. High-throughput screening and combinatorial chemistry approaches allow for progressing from these starting points to potential drug candidates. This will be illustrated by focusing on the ligand-based drug design of oxytocin (OT) and vasopressin (AVP) receptor ligands using natural peptide leads as starting points. PMID:20687879

  13. Therapeutic effects of cell-permeant peptides that activate G proteins downstream of growth factors

    PubMed Central

    Ma, Gary S.; Aznar, Nicolas; Kalogriopoulos, Nicholas; Midde, Krishna K.; Lopez-Sanchez, Inmaculada; Sato, Emi; Dunkel, Ying; Gallo, Richard L.; Ghosh, Pradipta

    2015-01-01

    In eukaryotes, receptor tyrosine kinases (RTKs) and trimeric G proteins are two major signaling hubs. Signal transduction via trimeric G proteins has long been believed to be triggered exclusively by G protein-coupled receptors (GPCRs). This paradigm has recently been challenged by several studies on a multimodular signal transducer, Gα-Interacting Vesicle associated protein (GIV/Girdin). We recently demonstrated that GIV’s C terminus (CT) serves as a platform for dynamic association of ligand-activated RTKs with Gαi, and for noncanonical transactivation of G proteins. However, exogenous manipulation of this platform has remained beyond reach. Here we developed cell-permeable GIV-CT peptides by fusing a TAT-peptide transduction domain (TAT-PTD) to the minimal modular elements of GIV that are necessary and sufficient for activation of Gi downstream of RTKs, and used them to engineer signaling networks and alter cell behavior. In the presence of an intact GEF motif, TAT-GIV-CT peptides enhanced diverse processes in which GIV’s GEF function has previously been implicated, e.g., 2D cell migration after scratch-wounding, invasion of cancer cells, and finally, myofibroblast activation and collagen production. Furthermore, topical application of TAT-GIV-CT peptides enhanced the complex, multireceptor-driven process of wound repair in mice in a GEF-dependent manner. Thus, TAT-GIV peptides provide a novel and versatile tool to manipulate Gαi activation downstream of growth factors in a diverse array of pathophysiologic conditions. PMID:25926659

  14. Coupling of G Proteins to Reconstituted Monomers and Tetramers of the M2 Muscarinic Receptor*

    PubMed Central

    Redka, Dar'ya S.; Morizumi, Takefumi; Elmslie, Gwendolynne; Paranthaman, Pranavan; Shivnaraine, Rabindra V.; Ellis, John; Ernst, Oliver P.; Wells, James W.

    2014-01-01

    G protein-coupled receptors can be reconstituted as monomers in nanodiscs and as tetramers in liposomes. When reconstituted with G proteins, both forms enable an allosteric interaction between agonists and guanylyl nucleotides. Both forms, therefore, are candidates for the complex that controls signaling at the level of the receptor. To identify the biologically relevant form, reconstituted monomers and tetramers of the purified M2 muscarinic receptor were compared with muscarinic receptors in sarcolemmal membranes for the effect of guanosine 5′-[β,γ-imido]triphosphate (GMP-PNP) on the inhibition of N-[3H]methylscopolamine by the agonist oxotremorine-M. With monomers, a stepwise increase in the concentration of GMP-PNP effected a lateral, rightward shift in the semilogarithmic binding profile (i.e. a progressive decrease in the apparent affinity of oxotremorine-M). With tetramers and receptors in sarcolemmal membranes, GMP-PNP effected a vertical, upward shift (i.e. an apparent redistribution of sites from a state of high affinity to one of low affinity with no change in affinity per se). The data were analyzed in terms of a mechanistic scheme based on a ligand-regulated equilibrium between uncoupled and G protein-coupled receptors (the “ternary complex model”). The model predicts a rightward shift in the presence of GMP-PNP and could not account for the effects at tetramers in vesicles or receptors in sarcolemmal membranes. Monomers present a special case of the model in which agonists and guanylyl nucleotides interact within a complex that is both constitutive and stable. The results favor oligomers of the M2 receptor over monomers as the biologically relevant state for coupling to G proteins. PMID:25023280

  15. Chemokines integrate JAK/STAT and G-protein pathways during chemotaxis and calcium flux responses.

    PubMed

    Soriano, Silvia F; Serrano, Antonio; Hernanz-Falcón, Patricia; Martín de Ana, Ana; Monterrubio, María; Martínez, Carlos; Rodríguez-Frade, J Miguel; Mellado, Mario

    2003-05-01

    The JAK/STAT (Janus kinase / signaling transducer and activator of transcription) signaling pathway is implicated in converting stationary epithelial cells to migratory cells. In mammals, migratory responses are activated by chemoattractant proteins, including chemokines. We found that by binding to seven-transmembrane G-protein-coupled receptors, chemokines activate the JAK/STAT pathway to trigger chemotactic responses. We show that chemokine-mediated JAK/STAT activation is critical for G-protein induction and for phospholipase C-beta dependent Ca(2+) flux; in addition, pharmacological inhibition of JAK or mutation of the JAK kinase domain causes defects in both responses. Furthermore, G alpha(i) association with the receptor is dependent on JAK activation, and the chemokine-mediated Ca(2+) flux that requires phospholipase C-beta activity takes place downstream of JAK kinases. The chemokines thus employ a mechanism that links heterologous signaling pathways--G proteins and tyrosine kinases--in a network that may be essential for mediating their pleiotropic responses.

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

    SciTech Connect

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

    2010-01-14

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

  17. A G-Protein Subunit Translocation Embedded Network Motif Underlies GPCR Regulation of Calcium Oscillations

    PubMed Central

    Giri, Lopamudra; Patel, Anilkumar K.; Karunarathne, W.K. Ajith; Kalyanaraman, Vani; Venkatesh, K.V.; Gautam, N.

    2014-01-01

    G-protein βγ subunits translocate reversibly from the plasma membrane to internal membranes on receptor activation. Translocation rates differ depending on the γ subunit type. There is limited understanding of the role of the differential rates of Gβγ translocation in modulating signaling dynamics in a cell. Bifurcation analysis of the calcium oscillatory network structure predicts that the translocation rate of a signaling protein can regulate the damping of system oscillation. Here, we examined whether the Gβγ translocation rate regulates calcium oscillations induced by G-protein-coupled receptor activation. Oscillations in HeLa cells expressing γ subunit types with different translocation rates were imaged and quantitated. The results show that differential Gβγ translocation rates can underlie the diversity in damping characteristics of calcium oscillations among cells. Mathematical modeling shows that a translocation embedded motif regulates damping of G-protein-mediated calcium oscillations consistent with experimental data. The current study indicates that such a motif may act as a tuning mechanism to design oscillations with varying damping patterns by using intracellular translocation of a signaling component. PMID:24988358

  18. Using siRNA to define functional interactions between melanopsin and multiple G Protein partners.

    PubMed

    Hughes, Steven; Jagannath, Aarti; Hickey, Doron; Gatti, Silvia; Wood, Matthew; Peirson, Stuart N; Foster, Russell G; Hankins, Mark W

    2015-01-01

    Melanopsin expressing photosensitive retinal ganglion cells (pRGCs) represent a third class of ocular photoreceptors and mediate a range of non-image forming responses to light. Melanopsin is a G protein coupled receptor (GPCR) and existing data suggest that it employs a membrane bound signalling cascade involving Gnaq/11 type G proteins. However, to date the precise identity of the Gα subunits involved in melanopsin phototransduction remains poorly defined. Here we show that Gnaq, Gna11 and Gna14 are highly co-expressed in pRGCs of the mouse retina. Furthermore, using RNAi based gene silencing we show that melanopsin can signal via Gnaq, Gna11 or Gna14 in vitro, and demonstrate that multiple members of the Gnaq/11 subfamily, including Gna14 and at least Gnaq or Gna11, can participate in melanopsin phototransduction in vivo and contribute to the pupillary light responses of mice lacking rod and cone photoreceptors. This diversity of G protein interactions suggests additional complexity in the melanopsin phototransduction cascade and may provide a basis for generating the diversity of light responses observed from pRGC subtypes.

  19. Development of benzimidazole derivatives to inhibit HIV-1 replication through protecting APOBEC3G protein.

    PubMed

    Pan, Ting; He, Xin; Chen, Bing; Chen, Hui; Geng, Guannan; Luo, Haihua; Zhang, Hui; Bai, Chuan

    2015-05-05

    Human APOBEC3G (apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3G, A3G) is a potent restriction factor against human immunodeficiency virus type 1 (HIV-1) by inducing hypermutation of G to A in viral genome after its incorporation into virions. HIV-1 Vif (Virion Infectivity Factor) counteracts A3G by inducing ubiquitination and proteasomal degradation of A3G protein. Vif-A3G axis therefore is a promising therapeutic target of HIV-1. Here we report the screening, synthesis and SAR studies of benzimidazole derivatives as potent inhibitors against HIV-1 replication via protecting A3G protein. Based on the steep SAR of the benzimidazole scaffold, we identified compound 14 and 26 which provided the best potency, with IC50 values of 3.45 nM and 58.03 nM respectively in the anti-HIV-1 replication assay in H9 cells. Compound 14 and 26 also afforded protective effects on A3G protein level. Both compounds have been proved to be safe in acute toxicological studies. Taken together, we suggest that these two benzimidazole derivatives can be further developed as a new category of anti-HIV-1 leads.

  20. Regulation of Airway Inflammation by G-protein Regulatory Motif Peptides of AGS3 protein

    PubMed Central

    Choi, IL-Whan; Ahn, Do Whan; Choi, Jang-Kyu; Cha, Hee-Jae; Ock, Mee Sun; You, EunAe; Rhee, SangMyung; Kim, Kwang Chul; Choi, Yung Hyun; Song, Kyoung Seob

    2016-01-01

    Respiratory diseases such as asthma, chronic obstructive pulmonary disease (COPD), and lung infections have critical consequences on mortality and morbidity in humans. The aims of the present study were to examine the mechanisms by which CXCL12 affects MUC1 transcription and airway inflammation, which depend on activator of G-protein signaling (AGS) 3 and to identify specific molecules that suppress CXCL12-induced airway inflammation by acting on G-protein-coupled receptors. Herein, AGS3 suppresses CXCL12-mediated upregulation of MUC1 and TNFα by regulating Gαi. We found that the G-protein regulatory (GPR) motif peptide in AGS3 binds to Gαi and downregulates MUC1 expression; in contrast, this motif upregulates TNFα expression. Mutated GPR Q34A peptide increased the expression of MUC1 and TGFβ but decreased the expression of TNFα and IL-6. Moreover, CXCR4-induced dendritic extensions in 2D and 3D matrix cultures were inhibited by the GPR Q34A peptide compared with a wild-type GPR peptide. The GPR Q34A peptide also inhibited CXCL12-induced morphological changes and inflammatory cell infiltration in the mouse lung, and production of inflammatory cytokines in bronchoalveolar lavage (BAL) fluid and the lungs. Our data indicate that the GPR motif of AGS3 is critical for regulating MUC1/Muc1 expression and cytokine production in the inflammatory microenvironment. PMID:27270970

  1. Regulation of Chemokine Signal Integration by Activator of G-Protein Signaling 4 (AGS4)

    PubMed Central

    Robichaux, William G.; Branham-O’Connor, Melissa; Hwang, Il-Young; Vural, Ali; Kehrl, Johne H.

    2017-01-01

    Activator of G-protein signaling 4 (AGS4)/G-protein signaling modulator 3 (Gpsm3) contains three G-protein regulatory (GPR) motifs, each of which can bind Gαi-GDP free of Gβγ. We previously demonstrated that the AGS4-Gαi interaction is regulated by seven transmembrane-spanning receptors (7-TMR), which may reflect direct coupling of the GPR-Gαi module to the receptor analogous to canonical Gαβγ heterotrimer. We have demonstrated that the AGS4-Gαi complex is regulated by chemokine receptors in an agonist-dependent manner that is receptor-proximal. As an initial approach to investigate the functional role(s) of this regulated interaction in vivo, we analyzed leukocytes, in which AGS4/Gpsm3 is predominantly expressed, from AGS4/Gpsm3-null mice. Loss of AGS4/Gpsm3 resulted in mild but significant neutropenia and leukocytosis. Dendritic cells, T lymphocytes, and neutrophils from AGS4/Gpsm3-null mice also exhibited significant defects in chemoattractant-directed chemotaxis and extracellular signal-regulated kinase activation. An in vivo peritonitis model revealed a dramatic reduction in the ability of AGS4/Gpsm3-null neutrophils to migrate to primary sites of inflammation. Taken together, these data suggest that AGS4/Gpsm3 is required for proper chemokine signal processing in leukocytes and provide further evidence for the importance of the GPR-Gαi module in the regulation of leukocyte function. PMID:28062526

  2. Role of Regulators of G Protein Signaling Proteins in Bone Physiology and Pathophysiology

    PubMed Central

    Jules, Joel; Yang, Shuying; Chen, Wei; Li, Yi-Ping

    2016-01-01

    Regulators of G protein signaling (RGS) proteins enhance the intrinsic GTPase activity of α subunits of the heterotrimeric G protein complex of G protein-coupled receptors (GPCRs) and thereby inactivate signal transduction initiated by GPCRs. The RGS family consists of nearly 37 members with a conserved RGS homology domain which is critical for their GTPase accelerating activity. RGS proteins are expressed in most tissues, including heart, lung, brain, kidney, and bone and play essential roles in many physiological and pathological processes. In skeletal development and bone homeostasis as well as in many bone disorders, RGS proteins control the functions of various GPCRs, including the parathyroid hormone receptor type 1 and calcium-sensing receptor and also regulate various critical signaling pathways, such as Wnt and calcium oscillations. This chapter will discuss the current findings on the roles of RGS proteins in regulating signaling of key GPCRs in skeletal development and bone homeostasis. We also will examine the current updates of RGS proteins’ regulation of calcium oscillations in bone physiology and highlight the roles of RGS proteins in selected bone pathological disorders. Despite the recent advances in bone and mineral research, RGS proteins remain understudied in the skeletal system. Further understanding of the roles of RGS proteins in bone should not only provide great insights into the molecular basis of various bone diseases but also generate great therapeutic drug targets for many bone diseases. PMID:26123302

  3. Light-dependent activation of G proteins by two isoforms of chicken melanopsins.

    PubMed

    Torii, Masaki; Kojima, Daisuke; Nishimura, Akiyuki; Itoh, Hiroshi; Fukada, Yoshitaka

    2015-11-01

    In the chicken pineal gland, light stimuli trigger signaling pathways mediated by two different subtypes, Gt and G11. These G proteins may be activated by any of the three major pineal opsins, pinopsin, OPN4-1 and OPN4-2, but biochemical evidence for the coupling has been missing except for functional coupling between pinopsin and Gt. Here we investigated the relative expression levels and the functional difference among the three pineal opsins. In the chicken pineal gland, the pinopsin mRNA level was significantly more abundant than the others, of which the OPN4-2 mRNA level was higher than that of OPN4-1. In G protein activation assays, Gt was strongly activated by pinopsin in a light-dependent manner, being consistent with previous studies, and weakly activated by OPN4-2. Unexpectedly, illuminated OPN4-2 more efficiently activated G protein(s) that was endogenously expressed in HEK293T cells in culture. On the other hand, Gq, the closest analogue of G11, was activated only by OPN4-1 although the activity was relatively weak under these conditions. These results suggest that OPN4-1 and OPN4-2 couple with Gq and Gt, respectively. Two melanopsins, OPN4-1 and OPN4-2, appear to have acquired mutually different functions through the evolution.

  4. Biophysical characterization of G protein ectodomain of group B human respiratory syncytial virus from E. coli.

    PubMed

    Khan, Wajihul Hasan; Srungaram, V L N Raghuram; Islam, Asimul; Beg, Ilyas; Haider, Md Shakir H; Ahmad, Faizan; Broor, Shobha; Parveen, Shama

    2016-07-03

    Human respiratory syncytial virus (hRSV) is an important pathogen of acute respiratory tract infection. The G protein of hRSV is a transmembrane glycoprotein that is a neutralizing antigen and is thus a vaccine candidate. In this study, synthetic codon optimized ectodomain G protein [G(ΔTM)] of BA genotype of group B hRSV was cloned, expressed, and characterized using biophysical techniques. The molar absorption coefficient and mean residue ellipticity at 222 nm ([θ]222) of G (ΔTM) was found to be 7950 M(-1) cm(-1) and -19701.7 deg cm(2) dmol(-1) respectively. It was concluded that G(ΔTM) mainly consist of α-helix (74.9%) with some amount of β-sheet (4%). The protein was stable up to 85°C without any transition curve. However, heat-induced denaturation of G(ΔTM) resulted in total loss of β-sheet whereas not much change was observed in the α-helix part of the secondary structure. It was concluded that G(ΔTM) is an α-helical protein and it is highly stable at high temperature, but could be easily denatured using high concentrations of GdmCl/urea or acidic condition. This is the first investigation of cloning, expression, and characterization of G(ΔTM) of BA viruses from India. Structural characterization of G protein will assist in drug designing and vaccine development for hRSV.

  5. Taste receptors and gustatory associated G proteins in channel catfish, Ictalurus punctatus.

    PubMed

    Gao, Sen; Liu, Shikai; Yao, Jun; Zhou, Tao; Li, Ning; Li, Qi; Dunham, Rex; Liu, Zhanjiang

    2017-03-01

    Taste sensation plays a pivotal role in nutrient identification and acquisition. This is particularly true for channel catfish (Ictalurus punctatus) that live in turbid waters with limited visibility. This biological process is mainly mediated by taste receptors expressed in taste buds that are distributed in several organs and tissues, including the barbels and skin. In the present study, we identified a complete repertoire of taste receptor and gustatory associated G protein genes in the channel catfish genome. A total of eight taste receptor genes were identified, including five type I and three type II taste receptor genes. Their genomic locations, phylogenetic relations, orthologies and expression were determined. Phylogenetic and collinear analyses provided understanding of the evolution dynamics of this gene family. Furthermore, the motif and dN/dS analyses indicated that selection pressures of different degrees were imposed on these receptors. Additionally, four genes of gustatory associated G proteins were also identified. It was indicated that expression patterns of catfish taste receptors and gustatory associated G proteins across organs mirror the distribution of taste buds across organs. Finally, the expression comparison between catfish and zebrafish organs provided evidence of potential roles of catfish skin and gill involved in taste sensation.

  6. R4 Regulator of G Protein Signaling (RGS) Proteins in Inflammation and Immunity.

    PubMed

    Xie, Zhihui; Chan, Eunice C; Druey, Kirk M

    2016-03-01

    G protein-coupled receptors (GPCRs) have important functions in both innate and adaptive immunity, with the capacity to bridge interactions between the two arms of the host responses to pathogens through direct recognition of secreted microbial products or the by-products of host cells damaged by pathogen exposure. In the mid-1990s, a large group of intracellular proteins was discovered, the regulator of G protein signaling (RGS) family, whose main, but not exclusive, function appears to be to constrain the intensity and duration of GPCR signaling. The R4/B subfamily--the focus of this review--includes RGS1-5, 8, 13, 16, 18, and 21, which are the smallest RGS proteins in size, with the exception of RGS3. Prominent roles in the trafficking of B and T lymphocytes and macrophages have been described for RGS1, RGS13, and RGS16, while RGS18 appears to control platelet and osteoclast functions. Additional G protein independent functions of RGS13 have been uncovered in gene expression in B lymphocytes and mast cell-mediated allergic reactions. In this review, we discuss potential physiological roles of this RGS protein subfamily, primarily in leukocytes having central roles in immune and inflammatory responses. We also discuss approaches to target RGS proteins therapeutically, which represents a virtually untapped strategy to combat exaggerated immune responses leading to inflammation.

  7. Structural prerequisites for G-protein activation by the neurotensin receptor

    DOE PAGES

    Krumm, Brian E.; White, Jim F.; Shah, Priyanka; ...

    2015-07-24

    We previously determined the structure of neurotensin receptor NTSR1 in an active-like conformation with six thermostabilizing mutations bound to the peptide agonist neurotensin. This receptor was unable to activate G proteins, indicating that the mutations restricted NTSR1 to relate agonist binding to G-protein activation. Here we analyse the effect of three of those mutations (E166A3.49, L310A6.37, F358A7.42) and present two structures of NTSR1 able to catalyse nucleotide exchange at Gα. The presence of F3587.42 causes the conserved W3216.48 to adopt a side chain orientation parallel to the lipid bilayer sealing the collapsed Na+ ion pocket and linking the agonist withmore » residues in the lower receptor part implicated in GPCR activation. In the intracellular receptor half, the bulkier L3106.37 side chain dictates the position of R1673.50 of the highly conserved D/ERY motif. These residues, together with the presence of E1663.49 provide determinants for G-protein activation by NTSR1.« less

  8. Knockout of G protein β5 impairs brain development and causes multiple neurologic abnormalities in mice

    PubMed Central

    Zhang, Jian-Hua; Pandey, Mritunjay; Seigneur, Erica M.; Panicker, Leelamma M.; Koo, Lily; Schwartz, Owen M.; Chen, Weiping; Chen, Ching-Kang; Simonds, William F.

    2011-01-01

    Gβ5 is a divergent member of the signal-transducing G protein β subunit family encoded by GNB5 and expressed principally in brain and neuronal tissue. Among heterotrimeric Gβ isoforms, Gβ5 is unique in its ability to heterodimerize with members of the R7 subfamily of the regulator of G protein signaling (RGS) proteins that contain G protein-γ like domains. Previous studies employing Gnb5 knockout (KO) mice have shown that Gβ5 is an essential stabilizer of such RGS proteins and regulates the deactivation of retinal phototransduction and the proper functioning of retinal bipolar cells. However, little is known of the function of Gβ5 in the brain outside the visual system. We show here that mice lacking Gβ5 have a markedly abnormal neurologic phenotype that includes impaired development, tiptoe-walking, motor learning and coordination deficiencies, and hyperactivity. We further show that Gβ5-deficient mice have abnormalities of neuronal development in cerebellum and hippocampus. We find that the expression of both mRNA and protein from multiple neuronal genes is dysregulated in Gnb5 KO mice. Taken together with previous observations from Gnb5 KO mice, our findings suggest a model in which Gβ5 regulates dendritic arborization and/or synapse formation during development, in part by effects on gene expression. PMID:21883221

  9. Structural prerequisites for G-protein activation by the neurotensin receptor

    PubMed Central

    Krumm, Brian E.; White, Jim F.; Shah, Priyanka; Grisshammer, Reinhard

    2015-01-01

    We previously determined the structure of neurotensin receptor NTSR1 in an active-like conformation with six thermostabilizing mutations bound to the peptide agonist neurotensin. This receptor was unable to activate G proteins, indicating that the mutations restricted NTSR1 to relate agonist binding to G-protein activation. Here we analyse the effect of three of those mutations (E166A3.49, L310A6.37, F358A7.42) and present two structures of NTSR1 able to catalyse nucleotide exchange at Gα. The presence of F3587.42 causes the conserved W3216.48 to adopt a side chain orientation parallel to the lipid bilayer sealing the collapsed Na+ ion pocket and linking the agonist with residues in the lower receptor part implicated in GPCR activation. In the intracellular receptor half, the bulkier L3106.37 side chain dictates the position of R1673.50 of the highly conserved D/ERY motif. These residues, together with the presence of E1663.49 provide determinants for G-protein activation by NTSR1. PMID:26205105

  10. Progestin, estrogen and androgen G-protein coupled receptors in fish gonads.

    PubMed

    Thomas, Peter; Dressing, Gwen; Pang, Yefei; Berg, Hakan; Tubbs, Christopher; Benninghoff, Abby; Doughty, Kelly

    2006-04-01

    The identities of the membrane receptors mediating the majority of rapid, cell surface-initiated, nongenomic (i.e. nonclassical) steroid actions described to date are unclear. Two novel 7-transmembrane spanning proteins, representing two distinct classes of steroid membrane receptors, membrane progestin receptor alpha (mPRalpha) and a membrane estrogen receptor (mER), GPR30, have recently been identified in several vertebrate species. Evidence that both receptors activate G-proteins and function as G-protein coupled receptors (GPCRs) is briefly reviewed. New data on progestin actions on fish gametes suggest a widespread involvement of mPRalpha in oocyte maturation and sperm hyperactivity in this vertebrate group. Information on the second messenger pathways activated upon estrogen binding to a membrane estrogen receptor in croaker gonads and preliminary evidence for the presence of a GPR30-like protein in fish gonads are discussed. Finally, initial characterization of the ligand binding, G-protein activation and molecular size of a membrane androgen receptor (mAR) in croaker ovaries suggests the presence of a third unique steroid receptor in fish gonads that also may function as a GPCR.

  11. G protein-coupled receptors stimulation and the control of cell migration.

    PubMed

    Cotton, Mathieu; Claing, Audrey

    2009-07-01

    Cell migration is a fundamental biological process involved in normal physiology. Altered motile phenotypes are however often associated with the development and progression of diseases such as cancer and atherosclerosis. Remodeling of the actin cytoskeleton is required for cell shape changes and is controlled by a broad variety of cellular proteins. Interestingly, several extracellular stimuli can promote actin reorganization and result in enhanced cell migration. Namely, G protein-coupled receptors (GPCRs), which are activated by factors ranging from small amines, to hormones, and chemokines, initiate signalling cascades resulting in cell shape changes, formation of a migrating front (leading edge) and altered adhesion. GPCRs are heptahelical membrane proteins, which classically transmit signal via the activation of heterotrimeric G proteins. Sustained stimulation leads to the activation of G protein-coupled receptor kinases (GRKs) and the recruitment of arrestin proteins, which engage alternative signalling pathways. In this review, we will discuss the role of GPCR mediated signal transduction and review their importance in the regulation of actin remodeling leading to cell migration.

  12. The nature and origin of spontaneous noise in G protein-gated ion channels

    PubMed Central

    1991-01-01

    Arrival of agonist is generally thought to initiate the signal transduction process in G protein-receptor coupled systems. However, the muscarinic atrial K+ (K+[ACh]) channel opens spontaneously in the absence of applied agonist, giving a noisy appearance to the current records. We investigated the nature and origin of the noise by measuring single channel currents in cell-attached or excised, inside- out membrane patches. Guanosine triphosphate (GTP) produced identical single channel currents in a concentration- and Mg(2+)-dependent manner in the presence or absence of carbachol, but the requirements for GTP were greater in the absence of agonist. Hence the agonist-independent currents appeared to be produced by an endogenous G protein, Gk. This prediction was confirmed when an affinity-purified, sequence-specific Gi-3 alpha antibody or pertussis toxin (PTX) blocked the agonist- independent currents. Candidate endogenous agonists were ruled out by the lack of effect of their corresponding antagonists. Thus agonist- independent currents had the same nature as agonist-dependent K+[ACh] currents and seemed to originate in the same way. We have developed a hypothesis in which agonist-free, empty receptors prime Gk with GTP and Gk activates atrial K+ [ACh] channels producing basal currents or noise. Agonist-independent activation by G proteins of effectors including ion channels appears to be a common occurrence. PMID:1651979

  13. Antibodies to probe endogenous G protein-coupled receptor heteromer expression, regulation, and function

    PubMed Central

    Gomes, Ivone; Gupta, Achla; Bushlin, Ittai; Devi, Lakshmi A.

    2014-01-01

    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. PMID:25520661

  14. Activator of G-Protein Signaling 3-Induced Lysosomal Biogenesis Limits Macrophage Intracellular Bacterial Infection.

    PubMed

    Vural, Ali; Al-Khodor, Souhaila; Cheung, Gordon Y C; Shi, Chong-Shan; Srinivasan, Lalitha; McQuiston, Travis J; Hwang, Il-Young; Yeh, Anthony J; Blumer, Joe B; Briken, Volker; Williamson, Peter R; Otto, Michael; Fraser, Iain D C; Kehrl, John H

    2016-01-15

    Many intracellular pathogens cause disease by subverting macrophage innate immune defense mechanisms. Intracellular pathogens actively avoid delivery to or directly target lysosomes, the major intracellular degradative organelle. In this article, we demonstrate that activator of G-protein signaling 3 (AGS3), an LPS-inducible protein in macrophages, affects both lysosomal biogenesis and activity. AGS3 binds the Gi family of G proteins via its G-protein regulatory (GoLoco) motif, stabilizing the Gα subunit in its GDP-bound conformation. Elevated AGS3 levels in macrophages limited the activity of the mammalian target of rapamycin pathway, a sensor of cellular nutritional status. This triggered the nuclear translocation of transcription factor EB, a known activator of lysosomal gene transcription. In contrast, AGS3-deficient macrophages had increased mammalian target of rapamycin activity, reduced transcription factor EB activity, and a lower lysosomal mass. High levels of AGS3 in macrophages enhanced their resistance to infection by Burkholderia cenocepacia J2315, Mycobacterium tuberculosis, and methicillin-resistant Staphylococcus aureus, whereas AGS3-deficient macrophages were more susceptible. We conclude that LPS priming increases AGS3 levels, which enhances lysosomal function and increases the capacity of macrophages to eliminate intracellular pathogens.

  15. Structural prerequisites for G-protein activation by the neurotensin receptor

    SciTech Connect

    Krumm, Brian E.; White, Jim F.; Shah, Priyanka; Grisshammer, Reinhard

    2015-07-24

    We previously determined the structure of neurotensin receptor NTSR1 in an active-like conformation with six thermostabilizing mutations bound to the peptide agonist neurotensin. This receptor was unable to activate G proteins, indicating that the mutations restricted NTSR1 to relate agonist binding to G-protein activation. Here we analyse the effect of three of those mutations (E166A3.49, L310A6.37, F358A7.42) and present two structures of NTSR1 able to catalyse nucleotide exchange at Gα. The presence of F3587.42 causes the conserved W3216.48 to adopt a side chain orientation parallel to the lipid bilayer sealing the collapsed Na+ ion pocket and linking the agonist with residues in the lower receptor part implicated in GPCR activation. In the intracellular receptor half, the bulkier L3106.37 side chain dictates the position of R1673.50 of the highly conserved D/ERY motif. These residues, together with the presence of E1663.49 provide determinants for G-protein activation by NTSR1.

  16. Inhibitory G proteins and their receptors: emerging therapeutic targets for obesity and diabetes.

    PubMed

    Kimple, Michelle E; Neuman, Joshua C; Linnemann, Amelia K; Casey, Patrick J

    2014-06-20

    The worldwide prevalence of obesity is steadily increasing, nearly doubling between 1980 and 2008. Obesity is often associated with insulin resistance, a major risk factor for type 2 diabetes mellitus (T2DM): a costly chronic disease and serious public health problem. The underlying cause of T2DM is a failure of the beta cells of the pancreas to continue to produce enough insulin to counteract insulin resistance. Most current T2DM therapeutics do not prevent continued loss of insulin secretion capacity, and those that do have the potential to preserve beta cell mass and function are not effective in all patients. Therefore, developing new methods for preventing and treating obesity and T2DM is very timely and of great significance. There is now considerable literature demonstrating a link between inhibitory guanine nucleotide-binding protein (G protein) and G protein-coupled receptor (GPCR) signaling in insulin-responsive tissues and the pathogenesis of obesity and T2DM. These studies are suggesting new and emerging therapeutic targets for these conditions. In this review, we will discuss inhibitory G proteins and GPCRs that have primary actions in the beta cell and other peripheral sites as therapeutic targets for obesity and T2DM, improving satiety, insulin resistance and/or beta cell biology.

  17. G protein-coupled receptor kinase and beta-arrestin-mediated desensitization of the angiotensin II type 1A receptor elucidated by diacylglycerol dynamics.

    PubMed

    Violin, Jonathan D; Dewire, Scott M; Barnes, William G; Lefkowitz, Robert J

    2006-11-24

    Receptor desensitization progressively limits responsiveness of cells to chronically applied stimuli. Desensitization in the continuous presence of agonist has been difficult to study with available assay methods. Here, we used a fluorescence resonance energy transfer-based live cell assay for the second messenger diacylglycerol to measure desensitization of a model seven-transmembrane receptor, the Gq-coupled angiotensin II type 1(A) receptor, expressed in human embryonic kidney 293 cells. In response to angiotensin II, we observed a transient diacylglycerol response reflecting activation and complete desensitization of the receptor within 2-5 min. By utilizing a variety of approaches including graded tetracycline-inducible receptor expression, mutated receptors, and overexpression or short interfering RNA-mediated silencing of putative components of the cellular desensitization machinery, we conclude that the rate and extent of receptor desensitization are critically determined by the following: receptor concentration in the plasma membrane; the presence of phosphorylation sites on the carboxyl terminus of the receptor; kinase activity of G protein-coupled receptor kinase 2, but not of G protein-coupled receptor kinases 3, 5, or 6; and stoichiometric expression of beta-arrestin. The findings introduce the use of the biosensor diacylglycerol reporter as a powerful means for studying Gq-coupled receptor desensitization and document that, at the levels of receptor overexpression commonly used in such studies, the properties of the desensitization process are markedly perturbed and do not reflect normal cellular physiology.

  18. Blunted apoptosis of erythrocytes in mice deficient in the heterotrimeric G-protein subunit Gαi2

    PubMed Central

    Bissinger, Rosi; Lang, Elisabeth; Ghashghaeinia, Mehrdad; Singh, Yogesh; Zelenak, Christine; Fehrenbacher, Birgit; Honisch, Sabina; Chen, Hong; Fakhri, Hajar; Umbach, Anja T.; Liu, Guilai; Rexhepaj, Rexhep; Liu, Guoxing; Schaller, Martin; Mack, Andreas F.; Lupescu, Adrian; Birnbaumer, Lutz; Lang, Florian; Qadri, Syed M.

    2016-01-01

    Putative functions of the heterotrimeric G-protein subunit Gαi2-dependent signaling include ion channel regulation, cell differentiation, proliferation and apoptosis. Erythrocytes may, similar to apoptosis of nucleated cells, undergo eryptosis, characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine (PS) exposure. Eryptosis may be triggered by increased cytosolic Ca2+ activity and ceramide. In the present study, we show that Gαi2 is expressed in both murine and human erythrocytes and further examined the survival of erythrocytes drawn from Gαi2-deficient mice (Gαi2−/−) and corresponding wild-type mice (Gαi2+/+). Our data show that plasma erythropoietin levels, erythrocyte maturation markers, erythrocyte counts, hematocrit and hemoglobin concentration were similar in Gαi2−/− and Gαi2+/+ mice but the mean corpuscular volume was significantly larger in Gαi2−/− mice. Spontaneous PS exposure of circulating Gαi2−/− erythrocytes was significantly lower than that of circulating Gαi2+/+ erythrocytes. PS exposure was significantly lower in Gαi2−/− than in Gαi2+/+ erythrocytes following ex vivo exposure to hyperosmotic shock, bacterial sphingomyelinase or C6 ceramide. Erythrocyte Gαi2 deficiency further attenuated hyperosmotic shock-induced increase of cytosolic Ca2+ activity and cell shrinkage. Moreover, Gαi2−/− erythrocytes were more resistant to osmosensitive hemolysis as compared to Gαi2+/+ erythrocytes. In conclusion, Gαi2 deficiency in erythrocytes confers partial protection against suicidal cell death. PMID:27499046

  19. Characterization of a family of endogenous neuropeptide ligands for the G protein-coupled receptors GPR7 and GPR8

    PubMed Central

    Tanaka, Hirokazu; Yoshida, Tetsuo; Miyamoto, Norimasa; Motoike, Toshiyuki; Kurosu, Hiroshi; Shibata, Kenji; Yamanaka, Akihiro; Williams, S. Clay; Richardson, James A.; Tsujino, Natsuko; Garry, Mary G.; Lerner, Michael R.; King, David S.; O'Dowd, Brian F.; Sakurai, Takeshi; Yanagisawa, Masashi

    2003-01-01

    GPR7 and GPR8 are orphan G protein-coupled receptors that are highly similar to each other. These receptors are expressed predominantly in brain, suggesting roles in central nervous system function. We have purified an endogenous peptide ligand for GPR7 from bovine hypothalamus extracts. This peptide, termed neuropeptide B (NPB), has a C-6-brominated tryptophan residue at the N terminus. It binds and activates human GPR7 or GPR8 with median effective concentrations (EC50) of 0.23 nM and 15.8 nM, respectively. In situ hybridization shows distinct localizations of the prepro-NPB mRNA in mouse brain, i.e., in paraventricular hypothalamic nucleus, hippocampus, and several nuclei in midbrain and brainstem. Intracerebroventricular (i.c.v.) injection of NPB in mice induces hyperphagia during the first 2 h, followed by hypophagia. Intracerebroventricular injection of NPB produces analgesia to s.c. formalin injection in rats. Through EST database searches, we identified a putative paralogous peptide. This peptide, termed neuropeptide W (NPW), also has an N-terminal tryptophan residue. Synthetic human NPW binds and activates human GPR7 or GPR8 with EC50 values of 0.56 nM and 0.51 nM, respectively. The expression of NPW mRNA in mouse brain is confined to specific nuclei in midbrain and brainstem. These findings suggest diverse physiological functions of NPB and NPW in the central nervous system, acting as endogenous ligands on GPR7 and/or GPR8. PMID:12719537

  20. Activation of G-protein-coupled receptor 30 is sufficient to enhance spatial recognition memory in ovariectomized rats.

    PubMed

    Hawley, Wayne R; Grissom, Elin M; Moody, Nicole M; Dohanich, Gary P; Vasudevan, Nandini

    2014-04-01

    In ovariectomized rats, administration of estradiol, or selective estrogen receptor agonists that activate either the α or β isoforms, have been shown to enhance spatial cognition on a variety of learning and memory tasks, including those that capitalize on the preference of rats to seek out novelty. Although the effects of the putative estrogen G-protein-coupled receptor 30 (GPR30) on hippocampus-based tasks have been reported using food-motivated tasks, the effects of activation of GPR30 receptors on tasks that depend on the preference of rats to seek out spatial novelty remain to be determined. Therefore, the aim of the current study was to determine if short-term treatment of ovariectomized rats with G-1, an agonist for GPR30, would mimic the effects on spatial recognition memory observed following short-term estradiol treatment. In Experiment 1, ovariectomized rats treated with a low dose (1 μg) of estradiol 48 h and 24 h prior to the information trial of a Y-maze task exhibited a preference for the arm associated with the novel environment on the retention trial conducted 48 h later. In Experiment 2, treatment of ovariectomized rats with G-1 (25 μg) 48 h and 24 h prior to the information trial of a Y-maze task resulted in a greater preference for the arm associated with the novel environment on the retention trial. Collectively, the results indicated that short-term treatment of ovariectomized rats with a GPR30 agonist was sufficient to enhance spatial recognition memory, an effect that also occurred following short-term treatment with a low dose of estradiol.

  1. Regulation of cardiomyocyte signaling by RGS proteins: differential selectivity towards G proteins and susceptibility to regulation.

    PubMed

    Hao, Jianming; Michalek, Christina; Zhang, Wei; Zhu, Ming; Xu, Xiaomei; Mende, Ulrike

    2006-07-01

    Many signals that regulate cardiomyocyte growth, differentiation and function are mediated via heterotrimeric G proteins, which are under the control of RGS proteins (Regulators of G protein Signaling). Several RGS proteins are expressed in the heart, but so far little is known about their function and regulation. Using adenoviral gene transfer, we conducted the first comprehensive analysis of the capacity and selectivity of the major cardiac RGS proteins (RGS2-RGS5) to regulate central G protein-mediated signaling pathways in adult ventricular myocytes (AVM). All four RGS proteins potently inhibited Gq/11-mediated phospholipase C beta stimulation and cell growth (assessed in neonatal myocytes). Importantly, RGS2 selectively inhibited Gq/11 signaling, whereas RGS3, RGS4 and RGS5 had the capacity to regulate both Gq/11 and Gi/o signaling (carbachol-induced cAMP inhibition). Gs signaling was unaffected, and, contrary to reports in other cell lines, RGS2-RGS5 did not appear to regulate adenylate cyclase directly in AVM. Since RGS proteins can be highly regulated in their expression by many different stimuli, we also tested the hypothesis that RGS expression is subject to G protein-mediated regulation in AVM and determined the specificity with which enhanced G protein signaling alters endogenous RGS expression in AVM. RGS2 mRNA and protein were markedly but transiently up-regulated by enhanced Gq/11 signaling (alpha1-adrenergic stimulation or Galphaq* overexpression), possibly by a negative feedback mechanism. In contrast, the other negative regulators of Gq/11 signaling (RGS3-RGS5) were unchanged. Endogenous RGS2 (but not RGS3-RGS5) expression was also up-regulated in cells with enhanced AC signaling (beta-adrenergic or forskolin stimulation). Taken together, these findings suggest diverse roles of RGS proteins in regulating myocyte signaling. RGS2 emerged as the only selective and highly regulated inhibitor of Gq/11 signaling that could potentially become a promising

  2. Characterization, localization and function of pertussis toxin-sensitive G proteins in the nervous systems of Aplysia and Loligo

    SciTech Connect

    Vogel, S.S.

    1989-01-01

    The author has characterized pertussis toxin-sensitive G proteins in the nervous systems of the gastropod mollusc Aplysia and the cephalopod Loligo using ({sup 32}P)ADP-ribosylation and immunoblotting with G protein specific antisera. As in vertebrates, this class of G protein is associated with membranes and enriched in nervous tissue in Aplysia. Analysis of dissected Aplysia ganglia reveal that it is enriched in neuropil, a region containing most of the central nervous system synapses. Because both Aplysia and Loligo synaptosomes are enriched in pertussis toxin-sensitive G proteins, it is likely that they are found in synaptic terminals. Fractionation of Aplysia synaptosomes into membrane and vesicle fractions reveals that, although the majority of G protein is recovered in the plasma membrane fraction, a small proportion is recovered in the vesicle fraction. He shows that G proteins are on intracellular membranes by ADP-ribosylating extruded axoplasm with pertussis toxin. A plausible explanation for vesicular localization of G protein in axoplasm is that G proteins are transported to terminals on vesicles. He has shown, using ligature experiments with Aplysia connectives and temperature block experiments in the giant axon of Loligo, that G proteins move by anterograde fast axonal transport. Injection of pertussis toxin into the identified Aplysia neuron L10 blocks histamine-induced presynaptic inhibition of transmitter release. This suggests that pertussis toxin sensitive G proteins play a role in modulating transmitter release at synaptic terminals. In the giant synapse of Loligo, he presents preliminary data that demonstrates that the activation of G proteins in the presynaptic terminal results in decreased transmitter release.

  3. Identification of the G-protein-coupled ORL1 receptor in the mouse spinal cord by [35S]-GTPgammaS binding and immunohistochemistry.

    PubMed

    Narita, M; Mizoguchi, H; Oji, D E; Dun, N J; Hwang, B H; Nagase, H; Tseng, L F

    1999-11-01

    1 Although the ORL1 receptor is clearly located within the spinal cord, the functional signalling mechanism of the ORL1 receptor in the spinal cord has not been clearly documented. The present study was then to investigate the guanine nucleotide binding protein (G-protein) activation mediated through by the ORL1 receptor in the mouse spinal cord, measuring the modulation of guanosine-5'-o-(3-[35S]-thio) triphosphate ([35S]-GTPgammaS) binding by the putative endogenous ligand nociceptin, also referred as orphanin FQ. We also studied the anatomical distribution of nociceptin-like immunoreactivity and nociceptin-stimulated [35S]-GTPgammaS autoradiography in the spinal cord. 2 Immunohistochemical staining of mouse spinal cord sections revealed a dense plexus of nociceptin-like immunoreactive fibres in the superficial layers of the dorsal horn throughout the entire length of the spinal cord. In addition, networks of fibres were seen projecting from the lateral border of the dorsal horn to the lateral grey matter and around the central canal. 3 In vitro [35S]-GTPgammaS autoradiography showed high levels of nociceptin-stimulated [35S]-GTPgammaS binding in the superficial layers of the mouse dorsal horn and around the central canal, corresponding to the areas where nociceptin-like immunoreactive fibres were concentrated. 4 In [35S]-GTPgammaS membrane assay, nociceptin increased [35S]-GTPgammaS binding of mouse spinal cord membranes in a concentration-dependent and saturable manner, affording maximal stimulation of 64.1+/-2.4%. This effect was markedly inhibited by the specific ORL1 receptor antagonist [Phe1Psi (CH2-NH) Gly2] nociceptin (1 - 13) NH2. None of the mu-, delta-, and kappa-opioid and other G-protein-coupled receptor antagonists had a significant effect on basal or nociceptin-stimulated [35S]-GTPgammaS binding. 5 These findings suggest that nociceptin-containing fibres terminate in the superficial layers of the dorsal horn and the central canal and that

  4. The evolution of regulators of G protein signalling proteins as drug targets - 20 years in the making: IUPHAR Review 21.

    PubMed

    Sjögren, B

    2017-03-01

    Regulators of G protein signalling (RGS) proteins are celebrating the 20th anniversary of their discovery. The unveiling of this new family of negative regulators of G protein signalling in the mid-1990s solved a persistent conundrum in the G protein signalling field, in which the rate of deactivation of signalling cascades in vivo could not be replicated in exogenous systems. Since then, there has been tremendous advancement in the knowledge of RGS protein structure, function, regulation and their role as novel drug targets. RGS proteins play an important modulatory role through their GTPase-activating protein (GAP) activity at active, GTP-bound Gα subunits of heterotrimeric G proteins. They also possess many non-canonical functions not related to G protein signalling. Here, an update on the status of RGS proteins as drug targets is provided, highlighting advances that have led to the inclusion of RGS proteins in the IUPHAR/BPS Guide to PHARMACOLOGY database of drug targets.

  5. In vivo stoichiometry monitoring of G protein coupled receptor oligomers using spectrally resolved two-photon microscopy

    NASA Astrophysics Data System (ADS)

    Stoneman, M. R.; Singh, D. R.; Raicu, V.

    2010-02-01

    Resonance Energy Transfer (RET) between a donor molecule in an electronically excited state and an acceptor molecule in close proximity has been frequently utilized for studies of protein-protein interactions in living cells. Typically, the cell under study is scanned a number of times in order to accumulate enough spectral information to accurately determine the RET efficiency for each region of interest within the cell. However, the composition of these regions may change during the course of the acquisition period, limiting the spatial determination of the RET efficiency to an average over entire cells. By means of a novel spectrally resolved two-photon microscope, we were able to obtain a full set of spectrally resolved images after only one complete excitation scan of the sample of interest. From this pixel-level spectral data, a map of RET efficiencies throughout the cell is calculated. By applying a simple theory of RET in oligomeric complexes to the experimentally obtained distribution of RET efficiencies throughout the cell, a single spectrally resolved scan reveals stoichiometric and structural information about the oligomer complex under study. This presentation will describe our experimental setup and data analysis procedure, as well as an application of the method to the determination of RET efficiencies throughout yeast cells (S. cerevisiae) expressing a G-protein-coupled receptor, Sterile 2 α factor protein (Ste2p), in the presence and absence of α-factor - a yeast mating pheromone.

  6. Deriving structural and functional insights from a ligand-based hierarchical classification of G protein-coupled receptors.

    PubMed

    Attwood, T K; Croning, M D R; Gaulton, A

    2002-01-01

    G protein-coupled receptors (GPCRs) constitute the largest known family of cell-surface receptors. With hundreds of members populating the rhodopsin-like GPCR superfamily and many more awaiting discovery in the human genome, they are of interest to the pharmaceutical industry because of the opportunities they afford for yielding potentially lucrative drug targets. Typical sequence analysis strategies for identifying novel GPCRs tend to involve similarity searches using standard primary database search tools. This will reveal the most similar sequence, generally without offering any insight into its family or superfamily relationships. Conversely, searches of most 'pattern' or family databases are likely to identify the superfamily, but not the closest matching subtype. Here we describe a diagnostic resource that allows identification of GPCRs in a hierarchical fashion, based principally upon their ligand preference. This resource forms part of the PRINTS database, which now houses approximately 250 GPCR-specific fingerprints (http://www.bioinf.man.ac.uk/dbbrowser/gpcrPRINTS/). This collection of fingerprints is able to provide more sensitive diagnostic opportunities than have been realized by related approaches and is currently the only diagnostic tool for assigning GPCR subtypes. Mapping such fingerprints on to three-dimensional GPCR models offers powerful insights into the structural and functional determinants of subtype specificity.

  7. Specific Subunits of Heterotrimeric G Proteins Play Important Roles during Nodulation in Soybean1[W][OA

    PubMed Central

    Choudhury, Swarup Roy; Pandey, Sona

    2013-01-01

    Heterotrimeric G proteins comprising Gα, Gβ, and Gγ subunits regulate many fundamental growth and development processes in all eukaryotes. Plants possess a relatively limited number of G-protein components compared with mammalian systems, and their detailed functional characterization has been performed mostly in Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa). However, the presence of single Gα and Gβ proteins in both these species has significantly undermined the complexity and specificity of response regulation in plant G-protein signaling. There is ample pharmacological evidence for the role of G proteins in regulation of legume-specific processes such as nodulation, but the lack of genetic data from a leguminous species has restricted its direct assessment. Our recent identification and characterization of an elaborate G-protein family in soybean (Glycine max) and the availability of appropriate molecular-genetic resources have allowed us to directly evaluate the role of G-protein subunits during nodulation. We demonstrate that all G-protein genes are expressed in nodules and exhibit significant changes in their expression in response to Bradyrhizobium japonicum infection and in representative supernodulating and nonnodulating soybean mutants. RNA interference suppression and overexpression of specific G-protein components results in lower and higher nodule numbers, respectively, validating their roles as positive regulators of nodule formation. Our data further show preferential usage of distinct G-protein subunits in the presence of an additional signal during nodulation. Interestingly, the Gα proteins directly interact with the soybean nodulation factor receptors NFR1α and NFR1β, suggesting that the plant G proteins may couple with receptors other than the canonical heptahelical receptors common in metazoans to modulate signaling. PMID:23569109

  8. Importance of voltage-dependent inactivation in N-type calcium channel regulation by G-proteins

    PubMed Central

    Weiss, Norbert; Tadmouri, Abir; Mikati, Mohamad; Ronjat, Michel; De Waard, Michel

    2007-01-01

    Direct regulation of N-type calcium channels by G-proteins is essential to control neuronal excitability and neurotransmitter release. Binding of the Gβγ dimer directly onto the channel is characterized by a marked current inhibition (“ON” effect), whereas the pore opening- and time-dependent dissociation of this complex from the channel produce a characteristic set of biophysical modifications (“OFF” effects). Although G-protein dissociation is linked to channel opening, the contribution of channel inactivation to G-protein regulation has been poorly studied. Here, the role of channel inactivation was assessed by examining time-dependent G-protein de-inhibition of Cav2.2 channels in the presence of various inactivation-altering β subunit constructs. G-protein activation was produced via μ-opioid receptor activation using the DAMGO agonist. Whereas the “ON” effect of G-protein regulation is independent of the type of β subunit, the “OFF” effects were critically affected by channel inactivation. Channel inactivation acts as a synergistic factor to channel activation for the speed of G-protein dissociation. However, fast inactivating channels also reduce the temporal window of opportunity for G-protein dissociation, resulting in a reduced extent of current recovery, whereas slow inactivating channels undergo a far more complete recovery from inhibition. Taken together, these results provide novel insights on the role of channel inactivation in N-type channel regulation by G-proteins and contribute to the understanding of the physiological consequence of channel inactivation in the modulation of synaptic activity by G-protein coupled receptors. PMID:17171365

  9. G alpha 16, a G protein alpha subunit specifically expressed in hematopoietic cells.

    PubMed Central

    Amatruda, T T; Steele, D A; Slepak, V Z; Simon, M I

    1991-01-01

    Signal-transduction pathways mediated by guanine nucleotide-binding regulatory proteins (G proteins) determine many of the responses of hematopoietic cells. A recently identified gene encoding a G protein alpha subunit, G alpha 16, is specifically expressed in human cells of the hematopoietic lineage. The G alpha 16 cDNA encodes a protein with predicted Mr of 43,500, which resembles the G q class of alpha subunits and does not include a pertussis toxin ADP-ribosylation site. In comparison with other G protein alpha subunits, the G alpha 16 predicted protein has distinctive amino acid sequences in the amino terminus, the region A guanine nucleotide-binding domain, and in the carboxyl-terminal third of the protein. Cell lines of myelomonocytic and T-cell phenotype express the G alpha 16 gene, but no expression is detectable in two B-cell lines or in nonhematopoietic cell lines. G alpha 16 gene expression is down-regulated in HL-60 cells induced to differentiate to neutrophils with dimethyl sulfoxide. Antisera generated from synthetic peptides that correspond to two regions of G alpha 16 specifically react with a protein of 42- to 43-kDa in bacterial strains that overexpress G alpha 16 and in HL-60 membranes. This protein is decreased in membranes from dimethyl sulfoxide-differentiated HL-60 cells and is not detectable in COS cell membranes. The restricted expression of this gene suggests that G alpha 16 regulates cell-type-specific signal-transduction pathways, which are not inhibited by pertussis toxin. Images PMID:1905813

  10. Glucocorticoids regulate arrestin gene expression and redirect the signaling profile of G protein-coupled receptors.

    PubMed

    Oakley, Robert H; Revollo, Javier; Cidlowski, John A

    2012-10-23

    G protein-coupled receptors (GPCRs) compose the largest family of cell surface receptors and are the most common target of therapeutic drugs. The nonvisual arrestins, β-arrestin-1 and β-arrestin-2, are multifunctional scaffolding proteins that play critical roles in GPCR signaling. On binding of activated GPCRs at the plasma membrane, β-arrestins terminate G protein-dependent responses (desensitization) and stimulate β-arrestin-dependent signaling pathways. Alterations in the cellular complement of β-arrestin-1 and β-arrestin-2 occur in many human diseases, and their genetic ablation in mice has severe consequences. Surprisingly, however, the factors that control β-arrestin gene expression are poorly understood. We demonstrate that glucocorticoids differentially regulate β-arrestin-1 and β-arrestin-2 gene expression in multiple cell types. Glucocorticoids act via the glucocorticoid receptor (GR) to induce the synthesis of β-arrestin-1 and repress the expression of β-arrestin-2. Glucocorticoid-dependent regulation involves the recruitment of ligand-activated glucocorticoid receptors to conserved and functional glucocorticoid response elements in intron-1 of the β-arrestin-1 gene and intron-11 of the β-arrestin-2 gene. In human lung adenocarcinoma cells, the increased expression of β-arrestin-1 after glucocorticoid treatment impairs G protein-dependent activation of inositol phosphate signaling while enhancing β-arrestin-1-dependent stimulation of the MAPK pathway by protease activated receptor 1. These studies demonstrate that glucocorticoids redirect the signaling profile of GPCRs via alterations in β-arrestin gene expression, revealing a paradigm for cross-talk between nuclear and cell surface receptors and a mechanism by which glucocorticoids alter the clinical efficacy of GPCR-based drugs.

  11. Molecular chaperoning function of Ric-8 is to fold nascent heterotrimeric G protein α subunits.

    PubMed

    Chan, Puiyee; Thomas, Celestine J; Sprang, Stephen R; Tall, Gregory G

    2013-03-05

    We have shown that resistance to inhibitors of cholinesterase 8 (Ric-8) proteins regulate an early step of heterotrimeric G protein α (Gα) subunit biosynthesis. Here, mammalian and plant cell-free translation systems were used to study Ric-8A action during Gα subunit translation and protein folding. Gα translation rates and overall produced protein amounts were equivalent in mock and Ric-8A-immunodepleted rabbit reticulocyte lysate (RRL). GDP-AlF4(-)-bound Gαi, Gαq, Gα13, and Gαs produced in mock-depleted RRL had characteristic resistance to limited trypsinolysis, showing that these G proteins were folded properly. Gαi, Gαq, and Gα13, but not Gαs produced from Ric-8A-depleted RRL were not protected from trypsinization and therefore not folded correctly. Addition of recombinant Ric-8A to the Ric-8A-depleted RRL enhanced GDP-AlF4(-)-bound Gα subunit trypsin protection. Dramatic results were obtained in wheat germ extract (WGE) that has no endogenous Ric-8 component. WGE-translated Gαq was gel filtered and found to be an aggregate. Ric-8A supplementation of WGE allowed production of Gαq that gel filtered as a ∼100 kDa Ric-8A:Gαq heterodimer. Addition of GTPγS to Ric-8A-supplemented WGE Gαq translation resulted in dissociation of the Ric-8A:Gαq heterodimer and production of functional Gαq-GTPγS monomer. Excess Gβγ supplementation of WGE did not support functional Gαq production. The molecular chaperoning function of Ric-8 is to participate in the folding of nascent G protein α subunits.

  12. Ca2+-dependent inhibition of G protein-coupled receptor kinase 2 by calmodulin.

    PubMed

    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.

  13. Synthetic FXR agonist GW4064 is a modulator of multiple G protein-coupled receptors.

    PubMed

    Singh, Nidhi; Yadav, Manisha; Singh, Abhishek Kumar; Kumar, Harish; Dwivedi, Shailendra Kumar Dhar; Mishra, Jay Sharan; Gurjar, Anagha; Manhas, Amit; Chandra, Sharat; Yadav, Prem Narayan; Jagavelu, Kumaravelu; Siddiqi, Mohammad Imran; Trivedi, Arun Kumar; Chattopadhyay, Naibedya; Sanyal, Sabyasachi

    2014-05-01

    The synthetic nuclear bile acid receptor (farnesoid X receptor [FXR]) agonist GW4064 is extensively used as a specific pharmacological tool to illustrate FXR functions. We noticed that GW4064 activated empty luciferase reporters in FXR-deficient HEK-293T cells. We postulated that this activity of GW4064 might be routed through as yet unknown cellular targets and undertook an unbiased exploratory approach to identify these targets. Investigations revealed that GW4064 activated cAMP and nuclear factor for activated T-cell response elements (CRE and NFAT-RE, respectively) present on these empty reporters. Whereas GW4064-induced NFAT-RE activation involved rapid intracellular Ca(2+) accumulation and NFAT nuclear translocation, CRE activation involved soluble adenylyl cyclase-dependent cAMP accumulation and Ca(2+)-calcineurin-dependent nuclear translocation of transducers of regulated CRE-binding protein 2. Use of dominant negative heterotrimeric G-protein minigenes revealed that GW4064 caused activation of Gαi/o and Gq/11 G proteins. Sequential pharmacological inhibitor-based screening and radioligand-binding studies revealed that GW4064 interacted with multiple G protein-coupled receptors. Functional studies demonstrated that GW4064 robustly activated H1 and H4 and inhibited H2 histamine receptor signaling events. We also found that MCF-7 breast cancer cells, reported to undergo GW4064-induced apoptosis in an FXR-dependent manner, did not express FXR, and the GW4064-mediated apoptosis, also apparent in HEK-293T cells, could be blocked by selective histamine receptor regulators. Taken together, our results demonstrate identification of histamine receptors as alternate targets for GW4064, which not only necessitates cautious interpretation of the biological functions attributed to FXR using GW4064 as a pharmacological tool but also provides a basis for the rational designing of new pharmacophores for histamine receptor modulation.

  14. Potent and Selective Peptide-based Inhibition of the G Protein Gαq.

    PubMed

    Charpentier, Thomas H; Waldo, Gary L; Lowery-Gionta, Emily G; Krajewski, Krzysztof; Strahl, Brian D; Kash, Thomas L; Harden, T Kendall; Sondek, John

    2016-12-02

    In contrast to G protein-coupled receptors, for which chemical and peptidic inhibitors have been extensively explored, few compounds are available that directly modulate heterotrimeric G proteins. Active Gαq binds its two major classes of effectors, the phospholipase C (PLC)-β isozymes and Rho guanine nucleotide exchange factors (RhoGEFs) related to Trio, in a strikingly similar fashion: a continuous helix-turn-helix of the effectors engages Gαq within its canonical binding site consisting of a groove formed between switch II and helix α3. This information was exploited to synthesize peptides that bound active Gαq in vitro with affinities similar to full-length effectors and directly competed with effectors for engagement of Gαq A representative peptide was specific for active Gαq because it did not bind inactive Gαq or other classes of active Gα subunits and did not inhibit the activation of PLC-β3 by Gβ1γ2 In contrast, the peptide robustly prevented activation of PLC-β3 or p63RhoGEF by Gαq; it also prevented G protein-coupled receptor-promoted neuronal depolarization downstream of Gαq in the mouse prefrontal cortex. Moreover, a genetically encoded form of this peptide flanked by fluorescent proteins inhibited Gαq-dependent activation of PLC-β3 at least as effectively as a dominant-negative form of full-length PLC-β3. These attributes suggest that related, cell-penetrating peptides should effectively inhibit active Gαq in cells and that these and genetically encoded sequences may find application as molecular probes, drug leads, and biosensors to monitor the spatiotemporal activation of Gαq in cells.

  15. Influence of G-protein β-Polypeptide 3 C825T Polymorphism on Antihypertensive Response to Telmisartan and Amlodipine in Chinese Patients

    PubMed Central

    Zhang, Zan-Lin; Li, Hui-Lan; Wen, Zhi-Peng; Yang, Guo-Ping; Zhang, Wei; Chen, Xiao-Ping

    2016-01-01

    Background: G-protein β-polypeptide 3 (GNB3) is a β subunit isoform of G-protein that plays important role in signal transduction of membrane G-protein coupled receptors (GPCRs). The GNB3 splice variant C825T (rs5443) is associated with risk for essential hypertension (EH) and efficacy of therapeutic drugs targeting GPCRs. It is unknown whether the polymorphism is associated with blood pressure (BP) response to telmisartan or amlodipine, two widely prescribed antihypertensive drugs. Methods: A total of 93 subjects initially diagnosed as EH were recruited and underwent a 4-week treatment with telmisartan (42 patients) or amlodipine (51 patients) monotherapy. Both baseline and after-treatment BP were measured. GNB3 C825T polymorphism was genotyped by polymerase chain reaction-restriction fragment length polymorphism. Results: Baseline systolic BP (SBP) and diastolic BP (DBP) were comparable among C825T genotypes in both telmisartan and amlodipine treatment groups. Patients with the CT or TT genotypes showed significantly lower body mass index (BMI) as compared with CC homozygotes in both groups (P < 0.05, respectively). GNB3 825TT homozygotes showed significantly higher after-treatment DBP and mean arterial pressure (MAP) than those carrying at least one 825C allele (P < 0.01) in the telmisartan treatment group. No difference in after-treatment SBP, DBP, and MAP levels among C825T genotypes was observed in the amlodipine treatment group. No significant difference in absolute changes in BP levels was observed among the genotypes in either treatment group. Conclusion: The GNB3 C825T splice variant is associated with the DBP-lowering effect of telmisartan but not amlodipine in Chinese EH patients. PMID:26712426

  16. Sequential Co-immunoprecipitation and Immunoblot Approach to Determine Oligomerisation of G-Protein-Coupled Receptors.

    PubMed

    Guest, Paul C

    2017-01-01

    G-protein-coupled receptors (GPCRs) play a major role in psychiatric disorders and are the targets of several current therapeutic approaches in this field. A number of studies have now shown that GPCRs can assemble as high molecular weight homo- and hetero-oligomers, which could affect ligand binding, intracellular signalling or trafficking. This information could be critical in design of new drugs to treat neurological and psychiatric disorders. This chapter describes a sequential co-immunoprecipitation and immunoblot protocol for determining oligomerisation of the 5-hydroxytryptamine (HT)1A receptor with other GPCRs in co-transfected HEK-293 cells.

  17. Large-scale production and protein engineering of G protein-coupled receptors for structural studies

    PubMed Central

    Milić, Dalibor; Veprintsev, Dmitry B.

    2015-01-01

    Structural studies of G protein-coupled receptors (GPCRs) gave insights into molecular mechanisms of their action and contributed significantly to molecular pharmacology. This is primarily due to technical advances in protein engineering, production and crystallization of these important receptor targets. On the other hand, NMR spectroscopy of GPCRs, which can provide information about their dynamics, still remains challenging due to difficulties in preparation of isotopically labeled receptors and their low long-term stabilities. In this review, we discuss methods used for expression and purification of GPCRs for crystallographic and NMR studies. We also summarize protein engineering methods that played a crucial role in obtaining GPCR crystal structures. PMID:25873898

  18. Loss of cAMP-specific phosphodiesterase rescues spore development in G protein mutant in dictyostelium.

    PubMed

    Schwebs, David J; Nguyen, Hoai-Nghia; Miller, Jamison A; Hadwiger, Jeffrey A

    2014-02-01

    Cyclic AMP (cAMP) is an important intracellular signaling molecule for many G protein-mediated signaling pathways but the specificity of cAMP signaling in cells with multiple signaling pathways is not well-understood. In Dictyostelium, at least two different G protein signaling pathways, mediated by the Gα2 and Gα4 subunits, are involved with cAMP accumulation, spore production, and chemotaxis and the stimulation of these pathways results in the activation of ERK2, a mitogen-activated protein kinase that can down regulate the cAMP-specific phosphodiesterase RegA. The regA gene was disrupted in gα2(−) and gα4(−) cells to determine if the absence of this phosphodiesterase rescues the development of these G protein mutants as it does for erk2(−) mutants. There gA(−) mutation had no major effects on developmental morphology but enriched the distribution of the Gα mutant cells to the prespore/prestalk border in chimeric aggregates. The loss of RegA function had no effect on Gα4- mediated folate chemotaxis. However, the regA gene disruption in gα4(−) cells, but not in gα2(−) cells, resulted in a substantial rescue and acceleration of spore production. This rescue in sporulation required cell autonomous signaling because the precocious sporulation could not be induced through intercellular signaling in chimeric aggregates. However, intercellular signals from regA(−) strains increased the expression of the prestalk gene ecmB and accelerated the vacuolization of stalk cells. Intercellular signaling from the gα4(−)regA(−) strain did not induce ecmA gene expression indicating cell-type specificity in the promotion of prestalk cell development. regA gene disruption in a Gα4(HC) (Gα4 overexpression) strain did not result in precocious sporulation or stalk cell development indicating that elevated Gα4 subunit expression can mask regA(−) associated phenotypes even when provided with wild-type intercellular signaling. These findings indicate that

  19. Kinetics of M1 muscarinic receptor and G protein signaling to phospholipase C in living cells

    PubMed Central

    Falkenburger, Björn H.; Jensen, Jill B.

    2010-01-01

    G protein–coupled receptors (GPCRs) mediate responses to external stimuli in various cell types. Early events, such as the binding of ligand and G proteins to the receptor, nucleotide exchange (NX), and GTPase activity at the Gα subunit, are common for many different GPCRs. For Gq-coupled M1 muscarinic (acetylcholine) receptors (M1Rs), we recently measured time courses of intermediate steps in the signaling cascade using Förster resonance energy transfer (FRET). The expression of FRET probes changes the density of signaling molecules. To provide a full quantitative description of M1R signaling that includes a simulation of kinetics in native (tsA201) cells, we now determine the density of FRET probes and construct a kinetic model of M1R signaling through Gq to activation of phospholipase C (PLC). Downstream effects on the trace membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP2) and PIP2-dependent KCNQ2/3 current are considered in our companion paper in this issue (Falkenburger et al. 2010. J. Gen. Physiol. doi:10.1085/jgp.200910345). By calibrating their fluorescence intensity, we found that we selected transfected cells for our experiments with ∼3,000 fluorescently labeled receptors, G proteins, or PLC molecules per µm2 of plasma membrane. Endogenous levels are much lower, 1–40 per µm2. Our kinetic model reproduces the time courses and concentration–response relationships measured by FRET and explains observed delays. It predicts affinities and rate constants that align well with literature values. In native tsA201 cells, much of the delay between ligand binding and PLC activation reflects slow binding of G proteins to receptors. With M1R and Gβ FRET probes overexpressed, 10% of receptors have G proteins bound at rest, rising to 73% in the presence of agonist. In agreement with previous work, the model suggests that binding of PLC to Gαq greatly speeds up NX and GTPase activity, and that PLC is maintained in the active state by cycles of

  20. Alternative Splicing of G-protein Coupled Receptors: Relevance to Pain Management

    PubMed Central

    Oladosu, Folabomi A.; Maixner, William; Nackley, Andrea G.

    2015-01-01

    Drugs that target G-protein coupled receptors (GPCRs) represent the primary treatment strategy for patients with acute and chronic pain; however, there is substantial individual variability in both the efficacy and adverse side effects associated with these drugs. Variability in drug responses is, in part, due to individuals’ diversity in alternative splicing of pain-relevant GPCRs. GPCR alternative splice variants often exhibit distinct tissue distribution patterns, drug binding properties, and signaling characteristics that may impact disease pathology as well as the size and direction of analgesic effects. Here, we review the importance of GPCRs and their known splice variants to the management of pain. PMID:26250730

  1. Understanding the added value of g-protein-coupled receptor heteromers.

    PubMed

    Franco, Nuria; Franco, Rafael

    2014-01-01

    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.

  2. Large-scale production and protein engineering of G protein-coupled receptors for structural studies.

    PubMed

    Milić, Dalibor; Veprintsev, Dmitry B

    2015-01-01

    Structural studies of G protein-coupled receptors (GPCRs) gave insights into molecular mechanisms of their action and contributed significantly to molecular pharmacology. This is primarily due to technical advances in protein engineering, production and crystallization of these important receptor targets. On the other hand, NMR spectroscopy of GPCRs, which can provide information about their dynamics, still remains challenging due to difficulties in preparation of isotopically labeled receptors and their low long-term stabilities. In this review, we discuss methods used for expression and purification of GPCRs for crystallographic and NMR studies. We also summarize protein engineering methods that played a crucial role in obtaining GPCR crystal structures.

  3. G protein-coupled receptors in child development, growth, and maturation.

    PubMed

    Latronico, Ana Claudia; Hochberg, Ze'ev

    2010-10-12

    G protein-coupled receptors (GPCRs) constitute a large family of cell membrane receptors that affect embryogenesis, development, and child physiology, and they are targets for approved drugs and those still in development. The sensitivity of GPCRs to their respective extracellular hormones, neurotransmitters, and environmental stimulants, as well as their interaction with other receptors and intracellular signaling proteins (such as receptor activity-modifying proteins), contribute to variations in child development, growth, and maturation. Here, we summarize current knowledge about the mechanisms of activation (in either the presence or absence of ligands) that lead to the sensitivities of GPCRs and their respective effects as seen throughout human developmental and maturational phases.

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

    PubMed

    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 and protease-activated receptors, both of which are GPCRs, are central to the metastatic melanoma phenotype and may serve as potential targets in novel therapies against melanoma and other cancers.

  5. G protein-coupled receptors: bridging the gap from the extracellular signals to the Hippo pathway.

    PubMed

    Zhou, Xin; Wang, Zhen; Huang, Wei; Lei, Qun-Ying

    2015-01-01

    The Hippo pathway is crucial in organ size control, whereas its dysregulation contributes to organ degeneration or tumorigenesis. The kinase cascade of MST1/2 and LATS1/2 and the coupling transcription co-activators YAP/TAZ represent the core components of the Hippo pathway. Extensive studies have identified a number of upstream regulators of the Hippo pathway, including contact inhibition, mechanic stress, extracellular matrix stiffness, cytoskeletal rearrangement, and some molecules of cell polarity and cell junction. However, how the diffuse extracellular signals regulate the Hippo pathway puzzles the researchers for a long time. Unexpectedly, recent elegant studies demonstrated that stimulation of some G protein-coupled receptors (GPCRs), such as lysophosphatidic acid receptor, sphingosine-1-phosphate receptor, and the protease activated receptor PAR1, causes potent YAP/TAZ dephosphorylation and activation by promoting actin cytoskeleton assemble. In this review, we briefly describe the components of the Hippo pathway and focus on the recent progress with respect to the regulation of the Hippo pathway by GPCRs and G proteins in cancer cells. In addition, we also discuss the potential therapeutic roles targeting the Hippo pathway in human cancers.

  6. Photosynthate Regulation of the Root System Architecture Mediated by the Heterotrimeric G Protein Complex in Arabidopsis

    PubMed Central

    Mudgil, Yashwanti; Karve, Abhijit; Teixeira, Paulo J. P. L.; Jiang, Kun; Tunc-Ozdemir, Meral; Jones, Alan M.

    2016-01-01

    Assimilate partitioning to the root system is a desirable developmental trait to control but little is known of the signaling pathway underlying partitioning. A null mutation in the gene encoding the Gβ subunit of the heterotrimeric G protein complex, a nexus for a variety of signaling pathways, confers altered sugar partitioning in roots. While fixed carbon rapidly reached the roots of wild type and agb1-2 mutant seedlings, agb1 roots had more of this fixed carbon in the form of glucose, fructose, and sucrose which manifested as a higher lateral root density. Upon glucose treatment, the agb1-2 mutant had abnormal gene expression in the root tip validated by transcriptome analysis. In addition, PIN2 membrane localization was altered in the agb1-2 mutant. The heterotrimeric G protein complex integrates photosynthesis-derived sugar signaling incorporating both membrane-and transcriptional-based mechanisms. The time constants for these signaling mechanisms are in the same range as photosynthate delivery to the root, raising the possibility that root cells are able to use changes in carbon fixation in real time to adjust growth behavior. PMID:27610112

  7. Membrane-Mediated Oligomerization of G Protein Coupled Receptors and Its Implications for GPCR Function

    PubMed Central

    Gahbauer, Stefan; Böckmann, Rainer A.

    2016-01-01

    The dimerization or even oligomerization of G protein coupled receptors (GPCRs) causes ongoing, controversial debates about its functional role and the coupled biophysical, biochemical or biomedical implications. A continously growing number of studies hints to a relation between oligomerization and function of GPCRs and strengthens the assumption that receptor assembly plays a key role in the regulation of protein function. Additionally, progress in the structural analysis of GPCR-G protein and GPCR-ligand interactions allows to distinguish between actively functional and non-signaling complexes. Recent findings further suggest that the surrounding membrane, i.e., its lipid composition may modulate the preferred dimerization interface and as a result the abundance of distinct dimeric conformations. In this review, the association of GPCRs and the role of the membrane in oligomerization will be discussed. An overview of the different reported oligomeric interfaces is provided and their capability for signaling discussed. The currently available data is summarized with regard to the formation of GPCR oligomers, their structures and dependency on the membrane microenvironment as well as the coupling of oligomerization to receptor function. PMID:27826255

  8. Regulation of longevity by regulator of G-protein signaling protein, Loco.

    PubMed

    Lin, Yuh-Ru; Kim, Keetae; Yang, Yanfei; Ivessa, Andreas; Sadoshima, Junichi; Park, Yongkyu

    2011-06-01

    Regulator of G-protein signaling (RGS) proteins contribute to G-protein signaling pathways as activators or repressors with GTPase-activating protein (GAP) activity. To characterize whether regulation of RGS proteins influences longevity in several species, we measured stress responses and lifespan of RGS-overexpressing and RGS-lacking mutants. Reduced expression of Loco, a RGS protein of Drosophila melanogaster, resulted in a longer lifespan for both male and female flies, also exhibiting stronger resistance to three different stressors (starvation, oxidation, and heat) and higher manganese-containing superoxide dismutase (MnSOD) activity. In addition, this reduction in Loco expression increased fat content and diminished cAMP levels. In contrast, overexpression of both genomic and cDNA loco gene significantly shortened the lifespan with weaker stress resistance and lower fat content. Deletion analysis of the Loco demonstrated that its RGS domain is required for the regulation of longevity. Consistently, when expression of RGS14, mammalian homologue of Loco, was reduced in rat fibroblast cells, the resistance to oxidative stress increased with higher MnSOD expression. The changes of yeast Rgs2 expression, which shares a conserved RGS domain with the fly Loco protein, also altered lifespan and stress resistance in Saccharomyces cerevisiae. Here, we provide the first evidence that RGS proteins with GAP activity affect both stress resistance and longevity in several species.

  9. The Concise Guide to Pharmacology 2013/14: G Protein-Coupled Receptors

    PubMed Central

    Alexander, Stephen PH; Benson, Helen E; Faccenda, Elena; Pawson, Adam J; Sharman, Joanna L; Spedding, Michael; Peters, John A; Harmar, Anthony J

    2013-01-01

    The Concise Guide to PHARMACOLOGY 2013/14 provides concise overviews of the key properties of over 2000 human drug targets with their pharmacology, plus links to an open access knowledgebase of drug targets and their ligands (www.guidetopharmacology.org), which provides more detailed views of target and ligand properties. The full contents can be found at http://onlinelibrary.wiley.com/doi/10.1111/bph.12444/full. G protein-coupled receptors are one of the seven major pharmacological targets into which the Guide is divided, with the others being G protein-coupled receptors, ligand-gated ion channels, ion channels, catalytic receptors, nuclear hormone receptors, transporters and enzymes. These are presented with nomenclature guidance and summary information on the best available pharmacological tools, alongside key references and suggestions for further reading. A new landscape format has easy to use tables comparing related targets. It is a condensed version of material contemporary to late 2013, which is presented in greater detail and constantly updated on the website www.guidetopharmacology.org, superseding data presented in previous Guides to Receptors and Channels. It is produced in conjunction with NC-IUPHAR and provides the official IUPHAR classification and nomenclature for human drug targets, where appropriate. It consolidates information previously curated and displayed separately in IUPHAR-DB and the Guide to Receptors and Channels, providing a permanent, citable, point-in-time record that will survive database updates. PMID:24517644

  10. Improvements in G protein-coupled receptor purification yield light stable rhodopsin crystals.

    PubMed

    Salom, David; Le Trong, Isolde; Pohl, Ehmke; Ballesteros, Juan A; Stenkamp, Ronald E; Palczewski, Krzysztof; Lodowski, David T

    2006-12-01

    G protein-coupled receptors (GPCRs) represent the largest family of transmembrane signaling proteins and are the target of approximately half of all therapeutic agents. Agonist ligands bind their cognate GPCRs stabilizing the active conformation that is competent to bind G proteins, thus initiating a cascade of intracellular signaling events leading to modification of the cell activity. Despite their biomedical importance, the only known GPCR crystal structures are those of inactive rhodopsin forms. In order to understand how GPCRs are able to transduce extracellular signals across the plasma membrane, it is critical to determine the structure of these receptors in their ligand-bound, active state. Here, we report a novel combination of purification procedures that allowed the crystallization of rhodopsin in two new crystal forms and can be applicable to the purification and crystallization of other membrane proteins. Importantly, these new crystals are stable upon photoactivation and the preliminary X-ray diffraction analysis of both photoactivated and ground state rhodopsin crystals are also reported.

  11. Regulator of G protein signaling proteins differentially modulate signaling of μ and δ opioid receptors

    PubMed Central

    Xie, Zhihua; Li, Zhisong; Guo, Lei; Ye, Caiying; Li, Juan; Yu, Xiaoli; Yang, Huifen; Wang, Yulin; Chen, Chongguang; Zhang, Dechang; Liu-Chen, Lee-Yuan

    2009-01-01

    Effects of regulator of G protein signaling (RGS) proteins on μ and δ opioid receptors were investigated in HEK293 cells. Co-expression of RGS1, RGS2, RGS4, RGS9, RGS10 or RGS19 (Gα-interacting protein (GAIP)) significantly reduced [Tyr-D-Ala-Gly-N-methyl-Phe-Gly-ol]-Enkephalin (DAMGO)-induced inhibition of adenylyl cyclase (AC) mediated by μ opioid receptor, but only RGS9 decreased the effects of [Tyr-D-Pen-Gly-p-Chloro-Phe-D-Pen]-Enkephalin (DPDPE) mediated by δ opioid receptor. When C-tails of the receptors were exchanged (μ/δC and δ/μC chimeras), RGS proteins decreased δ/μC-mediated AC inhibition, but none had significant effects on that via μ/δC receptor. Thus, the C-terminal domains of the receptors are critical for the differential effects of RGS proteins, which may be due to differences in receptor - G protein - RGS protein interactions in signaling complexes. PMID:17433292

  12. Amphipols in G protein-coupled receptor pharmacology: what are they good for?

    PubMed

    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.

  13. Regulation of Ca(V)2 calcium channels by G protein coupled receptors.

    PubMed

    Zamponi, Gerald W; Currie, Kevin P M

    2013-07-01

    Voltage gated calcium channels (Ca²⁺ channels) are key mediators of depolarization induced calcium influx into excitable cells, and thereby play pivotal roles in a wide array of physiological responses. This review focuses on the inhibition of Ca(V)2 (N- and P/Q-type) Ca²⁺-channels by G protein coupled receptors (GPCRs), which exerts important autocrine/paracrine control over synaptic transmission and neuroendocrine secretion. Voltage-dependent inhibition is the most widespread mechanism, and involves direct binding of the G protein βγ dimer (Gβγ) to the α1 subunit of Ca(V)2 channels. GPCRs can also recruit several other distinct mechanisms including phosphorylation, lipid signaling pathways, and channel trafficking that result in voltage-independent inhibition. Current knowledge of Gβγ-mediated inhibition is reviewed, including the molecular interactions involved, determinants of voltage-dependence, and crosstalk with other cell signaling pathways. A summary of recent developments in understanding the voltage-independent mechanisms prominent in sympathetic and sensory neurons is also included. This article is part of a Special Issue entitled: Calcium channels.

  14. The contribution of pathways initiated via the Gq\\11 G-protein family to atrial fibrillation.

    PubMed

    Tinker, Andrew; Finlay, Malcom; Nobles, Muriel; Opel, Aaisha

    2016-03-01

    Atrial fibrillation is the commonest cardiac arrhythmia and leads to significant clinical morbidity and mortality. It has a complex pathophysiology but is often initiated by atrial ectopic beats and because of atrial remodelling once it occurs it can become established. Thus therapeutic interventions designed to prevent the initial occurrence of the arrhythmia are particularly needed. At the cellular level, these ectopic beats arise because of abnormal calcium release events from the sarcoplasmic reticulum leading to an inward current mediated by the sodium-calcium exchanger. There has been considerable interest in this over the last few years largely focused on the ryanodine receptor and related signalling pathways. However, atrial myocytes also possess a well-developed inositol trisphosphate (IP3) dependent calcium release system and this has been less studied. In this review we focus on pathways and molecules that couple via the Gq\\11 family of G-proteins including regulators of G-protein signalling that may influence IP3 mediated calcium release and atrial fibrillation.

  15. Biased ligands for better cardiovascular drugs: dissecting G-protein-coupled receptor pharmacology.

    PubMed

    DeWire, Scott M; Violin, Jonathan D

    2011-07-08

    Drug discovery efforts targeting G-protein-coupled receptors (GPCR) have been immensely successful in creating new cardiovascular medicines. Currently marketed GPCR drugs are broadly classified as either agonists that activate receptors or antagonists that prevent receptor activation by endogenous stimuli. However, GPCR couple to a multitude of intracellular signaling pathways beyond classical G-protein signals, and these signals can be independently activated by biased ligands to vastly expand the potential for new drugs at these classic targets. By selectively engaging only a subset of a receptor's potential intracellular partners, biased ligands may deliver more precise therapeutic benefit with fewer side effects than current GPCR-targeted drugs. In this review, we discuss the history of biased ligand research, the current understanding of how biased ligands exert their unique pharmacology, and how research into GPCR signaling has uncovered previously unappreciated capabilities of receptor pharmacology. We focus on several receptors to illustrate the approaches taken and discoveries made, and how these are steadily illuminating the intricacies of GPCR pharmacology. Discoveries of biased ligands targeting the angiotensin II type 1 receptor and of separable pharmacology suggesting the potential value of biased ligands targeting the β-adrenergic receptors and nicotinic acid receptor GPR109a highlight the powerful clinical promise of this new category of potential therapeutics.

  16. Vibrational resonance, allostery, and activation in rhodopsin-like G protein-coupled receptors

    NASA Astrophysics Data System (ADS)

    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.

  17. GABA(B) receptors: altered coupling to G-proteins in rats sensitized to amphetamine.

    PubMed

    Zhang, K; Tarazi, F I; Campbell, A; Baldessarini, R J

    2000-01-01

    Modified dopamine and glutamate neurotransmission in discrete brain regions is implicated in stimulant-induced behavioral sensitization. Release of both neurotransmitters is influenced by GABA(B) metabotropic receptors for the principal inhibitory neurotransmitter GABA. Accordingly, GABA(B) receptors were examined in rats sensitized to amphetamine by measuring receptor density and coupling to G-proteins indicated as [(3)H]baclofen binding and baclofen-mediated [(35)S]GTP gamma S binding. Repeated treatment with (+)-amphetamine (5mg/kg per day, i.p., for five days) sensitized the rats to amphetamine challenge (1mg/kg) at 14 days, but not one day, later. GABA(B) receptor density was not altered at either time. Baclofen-mediated [(35)S]GTP gamma S binding, however, was selectively augmented in the prefrontal cortex and attenuated in the nucleus accumbens at 14 days, but not one day, after amphetamine treatment. Changes in GABA(B) receptor coupling to G-proteins in rats sensitized to amphetamine, but not in similarly treated but unsensitized rats, lead us to suggest that altered GABA(B) receptor functioning may contribute to the expression of amphetamine-induced behavioral sensitization.

  18. The Significance of G Protein-Coupled Receptor Crystallography for Drug Discovery

    PubMed Central

    Salon, John A.; Lodowski, David T.

    2011-01-01

    Crucial as molecular sensors for many vital physiological processes, seven-transmembrane domain G protein-coupled receptors (GPCRs) comprise the largest family of proteins targeted by drug discovery. Together with structures of the prototypical GPCR rhodopsin, solved structures of other liganded GPCRs promise to provide insights into the structural basis of the superfamily's biochemical functions and assist in the development of new therapeutic modalities and drugs. One of the greatest technical and theoretical challenges to elucidating and exploiting structure-function relationships in these systems is the emerging concept of GPCR conformational flexibility and its cause-effect relationship for receptor-receptor and receptor-effector interactions. Such conformational changes can be subtle and triggered by relatively small binding energy effects, leading to full or partial efficacy in the activation or inactivation of the receptor system at large. Pharmacological dogma generally dictates that these changes manifest themselves through kinetic modulation of the receptor's G protein partners. Atomic resolution information derived from increasingly available receptor structures provides an entrée to the understanding of these events and practically applying it to drug design. Supported by structure-activity relationship information arising from empirical screening, a unified structural model of GPCR activation/inactivation promises to both accelerate drug discovery in this field and improve our fundamental understanding of structure-based drug design in general. This review discusses fundamental problems that persist in drug design and GPCR structural determination. PMID:21969326

  19. G protein-coupled receptors show unusual patterns of intrinsic unfolding.

    PubMed

    Jaakola, Veli-Pekka; Prilusky, Jaime; Sussman, Joel L; Goldman, Adrian

    2005-02-01

    Intrinsically unstructured proteins (IUPs) or IUP-like regions often play key roles in controlling processes ranging from transcription to the cell cycle. In silico such proteins can be identified by their sequence properties; they have low hydrophobicity and high net charge. In this study, we applied the FoldIndex (http://bioportal.weizmann.ac.il/fldbin/findex) program to analyze human G protein-coupled receptors and compared them with membrane proteins of known structure and with IUPs. We show that human G protein-coupled receptor (GPCR) extramembranous domains include long (>50 residues) disordered segments, unlike membrane proteins of known structure. The predicted disorder occurred primarily in the N-terminal, C-terminal and third intracellular domain regions: 55, 69 and 56% of the human GPCRs were disordered in these regions, respectively. This increased flexibility may therefore be critical for GPCR function. Surprisingly, however, the kinds of residues used in GPCR unstructured regions were different than in hitherto-identified IUPs. The GPCR third intracellular loop domains contain very high percentages of Arg, Lys and His residues, especially Arg, but the percentage of Glu, Asp and Pro is no higher than in folded proteins. We propose that this has structural and functional consequences.

  20. The therapeutic potential of G-protein coupled receptors in Huntington's disease.

    PubMed

    Dowie, Megan J; Scotter, Emma L; Molinari, Emanuela; Glass, Michelle

    2010-11-01

    Huntington's disease is a late-onset autosomal dominant inherited neurodegenerative disease characterised by increased symptom severity over time and ultimately premature death. An expanded CAG repeat sequence in the huntingtin gene leads to a polyglutamine expansion in the expressed protein, resulting in complex dysfunctions including cellular excitotoxicity and transcriptional dysregulation. Symptoms include cognitive deficits, psychiatric changes and a movement disorder often referred to as Huntington's chorea, which involves characteristic involuntary dance-like writhing movements. Neuropathologically Huntington's disease is characterised by neuronal dysfunction and death in the striatum and cortex with an overall decrease in cerebral volume (Ho et al., 2001). Neuronal dysfunction begins prior to symptom presentation, and cells of particular vulnerability include the striatal medium spiny neurons. Huntington's is a devastating disease for patients and their families and there is currently no cure, or even an effective therapy for disease symptoms. G-protein coupled receptors are the most abundant receptor type in the central nervous system and are linked to complex downstream pathways, manipulation of which may have therapeutic application in many neurological diseases. This review will highlight the potential of G-protein coupled receptor drug targets as emerging therapies for Huntington's disease.

  1. Understanding Molecular Recognition by G protein βγ Subunits on the Path to Pharmacological Targeting

    PubMed Central

    Lin, Yuan

    2011-01-01

    Heterotrimeric G proteins, composed of Gα and Gβγ subunits, transduce extracellular signals via G-protein-coupled receptors to modulate many important intracellular responses. The Gβγ subunits hold a central position in this signaling system and have been implicated in multiple aspects of physiology and the pathophysiology of disease. The Gβ subunit belongs to a large family of WD40 repeat proteins with a circular β-bladed propeller structure. This structure allows Gβγ to interact with a broad range of proteins to play diverse roles. How Gβγ interacts with and regulates such a wide variety of partners yet maintains specificity is an interesting problem in protein-protein molecular recognition in signal transduction, where signal transfer by proteins is often driven by modular conserved recognition motifs. Evidence has accumulated that one mechanism for Gβγ multitarget recognition is through an intrinsically flexible protein surface or “hot spot” that accommodates multiple modes of binding. Because each target has a unique recognition mode for Gβγ subunits, it suggests that these interactions could be selectively manipulated with small molecules, which could have significant therapeutic potential. PMID:21737569

  2. The influence of GAP-43 on orientation of cell division through G proteins.

    PubMed

    Huang, Rui; Zhao, Junpeng; Ju, Lili; Wen, Yujun; Xu, Qunyuan

    2015-12-01

    Recent studies have shown that GAP-43 is highly expressed in horizontally dividing neural progenitor cells, and G protein complex are required for proper mitotic-spindle orientation of those progenitors in the mammalian developing cortex. In order to verify the hypothesis that GAP-43 may influence the orientation of cell division through interacting with G proteins during neurogenesis, the GAP-43 RNA from adult C57 mouse was cloned into the pEGFP-N1 vector, which was then transfected into Madin-Darby Canine Kidney (MDCK) cells cultured in a three-dimensional (3D) cell culture system. The interaction of GAP-43 with Gαi was detected by co-immunoprecipitation (co-IP), while cystogenesis of 3D morphogenesis of MDCK cells and expression of GAP-43 and Gαi were determined by immunofluorescence and Western blotting. The results showed are as follows: After being transfected by pEGFP-N1-GAP-43, GAP-43 was localized on the cell membrane and co-localized with Gαi, and this dramatically induced a defective cystogenesis in 3D morphogenesis of MDCK cells. The functional interaction between GAP-43 and Gαi proteins was proven by the co-IP assay. It can be considered from the results that the GAP-43 is involved in the orientation of cell division by interacting with Gαi and this should be an important mechanism for neurogenesis in the mammalian brain.

  3. Phosphorylation of G protein-coupled receptors: from the barcode hypothesis to the flute model.

    PubMed

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

    2017-02-28

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

  4. Acidic tumor microenvironment and pH-sensing G protein-coupled receptors.

    PubMed

    Justus, Calvin R; Dong, Lixue; Yang, Li V

    2013-12-05

    The tumor microenvironment is acidic due to glycolytic cancer cell metabolism, hypoxia, and deficient blood perfusion. It is proposed that acidosis in the tumor microenvironment is an important stress factor and selection force for cancer cell somatic evolution. Acidic pH has pleiotropic effects on the proliferation, migration, invasion, metastasis, and therapeutic response of cancer cells and the function of immune cells, vascular cells, and other stromal cells. However, the molecular mechanisms by which cancer cells and stromal cells sense and respond to acidic pH in the tumor microenvironment are poorly understood. In this article the role of a family of pH-sensing G protein-coupled receptors (GPCRs) in tumor biology is reviewed. Recent studies show that the pH-sensing GPCRs, including GPR4, GPR65 (TDAG8), GPR68 (OGR1), and GPR132 (G2A), regulate cancer cell metastasis and proliferation, immune cell function, inflammation, and blood vessel formation. Activation of the proton-sensing GPCRs by acidosis transduces multiple downstream G protein signaling pathways. Since GPCRs are major drug targets, small molecule modulators of the pH-sensing GPCRs are being actively developed and evaluated. Research on the pH-sensing GPCRs will continue to provide important insights into the molecular interaction between tumor and its acidic microenvironment and may identify new targets for cancer therapy and chemoprevention.

  5. Molecular mechanisms deployed by virally encoded G protein-coupled receptors in human diseases.

    PubMed

    Montaner, Silvia; Kufareva, Irina; Abagyan, Ruben; Gutkind, J Silvio

    2013-01-01

    G protein-coupled receptors (GPCRs) represent the largest family of cell surface molecules involved in signal transduction. Surprisingly, open reading frames for multiple GPCRs were hijacked in the process of coevolution between Herpesviridae family viruses and their human and mammalian hosts. Virally encoded GPCRs (vGPCRs) evolved as parts of viral genomes, and this evolution allowed the power of host GPCR signaling circuitries to be harnessed in order to ensure viral replicative success. Phylogenetically, vGPCRs are distantly related to human chemokine receptors, although they feature several unique characteristics. Here, we describe the molecular mechanisms underlying vGPCR-mediated viral pathogenesis. These mechanisms include constitutive activity, aberrant coupling to human G proteins and β-arrestins, binding and activation by human chemokines, and dimerization with other GPCRs expressed in infected cells. The likely structural basis for these molecular events is described for the two closest viral homologs of human GPCRs. This information may aid in the development of novel targeted therapeutic strategies against viral diseases.

  6. Expression of the G-protein alpha-subunit gustducin in mammalian spermatozoa.

    PubMed

    Fehr, Johanna; Meyer, Dorke; Widmayer, Patricia; Borth, Heike Claudia; Ackermann, Frauke; Wilhelm, Beate; Gudermann, Thomas; Boekhoff, Ingrid

    2007-01-01

    Although chemotaxis has been proposed to guide sperm to egg throughout the animal kingdom, sperm attractants released from mammalian eggs have not been identified. Since the G protein subunit alpha-gustducin is accepted as a marker of chemosensitive cells, attempts were made to explore whether alpha-gustducin is also expressed in spermatozoa of mammals. Immunohistochemical approaches using an anti-alpha-gustducin-specific antibody revealed the most intense immunoreactivity in differentiating spermatids. Further evidence for the alpha-gustducin expression was obtained analyzing testicular and sperm-derived tissue preparations in western blot analyses. To elucidate whether alpha-gustducin is retained in mature spermatozoa, epididymal mouse and rat sperm were subjected to immunocytochemistry as well as immunogold electron microscopy. A specific staining was obtained within the circumference of the midpiece-localized mitochondria, on the axoneme and the outer dense fibers surrounding the microtubules of this region, whereas no labeling was detectable in the end piece regions. The analysis of ejaculated bovine and human sperm revealed a comparable segmental distribution pattern for alpha-gustducin. Although a possible function for alpha-gustducin has yet to be determined, the axonemal-associated localization within the midpiece and principal piece of different mammalian spermatozoa raises the possibility that this G protein alpha-subunit may process intracellular signals controlling sperm motility.

  7. Recombinant VSV G proteins reveal a novel raft-dependent endocytic pathway in resorbing osteoclasts

    SciTech Connect

    Mulari, Mika T.K. Nars, Martin; Laitala-Leinonen, Tiina; Kaisto, Tuula; Metsikkoe, Kalervo; Sun Yi; Vaeaenaenen, H. Kalervo

    2008-05-01

    Transcytotic membrane flow delivers degraded bone fragments from the ruffled border to the functional secretory domain, FSD, in bone resorbing osteoclasts. Here we show that there is also a FSD-to-ruffled border trafficking pathway that compensates for the membrane loss during the matrix uptake process and that rafts are essential for this ruffled border-targeted endosomal pathway. Replacing the cytoplasmic tail of the vesicular stomatitis virus G protein with that of CD4 resulted in partial insolubility in Triton X-100 and retargeting from the peripheral non-bone facing plasma membrane to the FSD. Recombinant G proteins were subsequently endosytosed and delivered from the FSD to the peripheral fusion zone of the ruffled border, which were both rich in lipid rafts as suggested by viral protein transport analysis and visualizing the rafts with fluorescent recombinant cholera toxin. Cholesterol depletion by methyl-{beta}-cyclodextrin impaired the ruffled border-targeted vesicle trafficking pathway and inhibited bone resorption dose-dependently as quantified by measuring the CTX and TRACP 5b secreted to the culture medium and by measuring the resorbed area visualized with a bi-phasic labeling method using sulpho-NHS-biotin and WGA-lectin. Thus, rafts are vital for membrane recycling from the FSD to the late endosomal/lysosomal ruffled border and bone resorption.

  8. Vibrational resonance, allostery, and activation in rhodopsin-like G protein-coupled receptors.

    PubMed

    Woods, Kristina N; Pfeffer, Jürgen; Dutta, Arpana; Klein-Seetharaman, Judith

    2016-11-16

    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.

  9. Long term regulation of cardiac L-type calcium channel by small G proteins.

    PubMed

    Magyar, J; Jenes, A; Kistamás, K; Ruzsnavszky, F; Nánási, P P; Satin, J; Szentandrássy, N; Bányász, T

    2011-01-01

    Calcium ions are crucial elements of excitation-contraction coupling in cardiac myocytes. The intracellular Ca(2+ ) concentration changes continously during the cardiac cycle, but the Ca(2+ ) entering to the cell serves as an intracellular second messenger, as well. The Ca(2+ ) as a second messenger influences the activity of many intracellular signalling pathways and regulates gene expression. In cardiac myocytes the major pathway for Ca(2+ ) entry into cells is L-type calcium channel (LTCC). The precise control of LTCC function is essential for maintaining the calcium homeostasis of cardiac myocytes. Dysregulation of LTCC may result in different diseases like cardiac hypertrophy, arrhytmias, heart failure. The physiological and pathological structural changes in the heart are induced in part by small G proteins. These proteins are involved in wide spectrum of cell biological functions including protein transport, regulation of cell proliferation, migration, apoptosis, and cytoskeletal rearrangement. Understanding the crosstalk between small G proteins and LTCC may help to understand the pathomechanism of different cardiac diseases and to develop a new generation of genetically-encoded Ca(2+ ) channel inhibitors.

  10. Application of RGS box proteins to evaluate G-protein selectivity in receptor-promoted signaling.

    PubMed

    Hains, Melinda D; Siderovski, David P; Harden, T Kendall

    2004-01-01

    Regulator of G-protein signaling (RGS) domains bind directly to GTP-bound Galpha subunits and accelerate their intrinsic GTPase activity by up to several thousandfold. The selectivity of RGS proteins for individual Galpha subunits has been illustrated. Thus, the expression of RGS proteins can be used to inhibit signaling pathways activated by specific G protein-coupled receptors (GPCRs). This article describes the use of specific RGS domain constructs to discriminate among G(i/o), Gq-and G(12/13)-mediated activation of phospholipase C (PLC) isozymes in COS-7 cells. Overexpression of the N terminus of GRK2 (amino acids 45-178) or p115 RhoGEF (amino acids 1-240) elicited selective inhibition of Galphaq- or Galpha(12/13)-mediated signaling to PLC activation, respectively. In contrast, RGS2 overexpression was found to inhibit PLC activation by both G(i/o)- and Gq-coupled GPCRs. RGS4 exhibited dramatic receptor selectivity in its inhibitory actions; of the G(i/o)- and Gq-coupled GPCRs tested (LPA1, LPA2, P2Y1, S1P3), only the Gq-coupled lysophosphatidic acid-activated LPA2 receptor was found to be inhibited by RGS4 overexpression.

  11. Allosteric Activation of a G Protein-coupled Receptor with Cell-penetrating Receptor Mimetics*

    PubMed Central

    Zhang, Ping; Leger, Andrew J.; Baleja, James D.; Rana, Rajashree; Corlin, Tiffany; Nguyen, Nga; Koukos, Georgios; Bohm, Andrew; Covic, Lidija; Kuliopulos, Athan

    2015-01-01

    G protein-coupled receptors (GPCRs) are remarkably versatile signaling systems that are activated by a large number of different agonists on the outside of the cell. However, the inside surface of the receptors that couple to G proteins has not yet been effectively modulated for activity or treatment of diseases. Pepducins are cell-penetrating lipopeptides that have enabled chemical and physical access to the intracellular face of GPCRs. The structure of a third intracellular (i3) loop agonist, pepducin, based on protease-activated receptor-1 (PAR1) was solved by NMR and found to closely resemble the i3 loop structure predicted for the intact receptor in the on-state. Mechanistic studies revealed that the pepducin directly interacts with the intracellular H8 helix region of PAR1 and allosterically activates the receptor through the adjacent (D/N)PXXYYY motif through a dimer-like mechanism. The i3 pepducin enhances PAR1/Gα subunit interactions and induces a conformational change in fluorescently labeled PAR1 in a very similar manner to that induced by thrombin. As pepducins can potentially be made to target any GPCR, these data provide insight into the identification of allosteric modulators to this major drug target class. PMID:25934391

  12. Vibrational resonance, allostery, and activation in rhodopsin-like G protein-coupled receptors

    PubMed Central

    Woods, Kristina N.; Pfeffer, Jürgen; Dutta, Arpana; Klein-Seetharaman, Judith

    2016-01-01

    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. PMID:27849063

  13. Distinct profiles of functional discrimination among G proteins determine the actions of G protein–coupled receptors

    PubMed Central

    Masuho, Ikuo; Ostrovskaya, Olga; Kramer, Grant M.; Jones, Christopher D.; Xie, Keqiang; Martemyanov, Kirill A.

    2016-01-01

    Members of the G protein coupled receptor (GPCR) family play key roles in many physiological functions and have been extensively exploited pharmacologically to treat diseases. Individual GPCRs exert diverse and distinct effects on cellular physiology and transduce signals by activating heterotrimeric G proteins. Mammalian genomes encode 16 different G protein alpha subunits, and each one of them has distinct properties. Here, we developed a single-platform, optical strategy for the direct monitoring of G protein activation in live cells, and using it we profiled the activities of individual GPCRs across a range of different G proteins, simultaneously quantifying both magnitude of their signaling and activation rates. We report that GPCRs engage multiple G proteins with varying efficacy and kinetics, generating fingerprint-like profiles that define individual receptors. We found that different classes of GPCR ligands, including full and partial agonists, allosteric modulators, and antagonists distinctly affected these fingerprints to functionally bias GPCR signaling. Finally, we showed that intracellular signaling modulators further altered the G protein–coupling profiles of GPCRs, which suggests that their differential expression may alter signaling outcomes in a cell-specific manner. . These observations suggest that the diversity of the effects of GPCRs on cellular physiology may be determined by their differential engagement of multiple G proteins with varying signal magnitudes and activation kinetics, properties that may be exploited pharmacologically. PMID:26628681

  14. G-protein modulation of N-type calcium channel gating current in human embryonic kidney cells (HEK 293).

    PubMed Central

    Jones, L P; Patil, P G; Snutch, T P; Yue, D T

    1997-01-01

    1. Voltage-dependent inhibition of N-type calcium currents by G-proteins contributes importantly to presynaptic inhibition. To examine the effect of G-proteins on key intermediary transitions leading to channel opening, we measured both gating and ionic currents arising from recombinant N-type channels (alpha 1B, beta 1b and alpha 2) expressed in transiently transfected human embryonic kidney cells (HEK 293). Recombinant expression of a homogeneous population of channels provided a favourable system for rigorous examination of the mechanisms underlying G-protein modulation. 2. During intracellular dialysis with GTP gamma S to activate G-proteins, ionic currents demonstrated classic features of voltage-dependent inhibition, i.e. strong depolarizing prepulses increased ionic currents and produced hyperpolarizing shifts in the voltage-dependent activation of ionic current. No such effects were observed with GDP beta S present to minimize G-protein activity. 3. Gating currents were clearly resolved after ionic current blockade with 0.1 mM free La3+, enabling this first report of gating charge translocation arising exclusively from N-type channels. G-proteins decreased the amplitude of gating currents and produced depolarizing shifts in the voltage-dependent activation of gating charge movement. However, the greatest effect was to induce a approximately 20 mV separation between the voltage-dependent activation of gating charge movement and ionic current. Strong depolarizing prepulses largely reversed these effects. These modulatory features provide telling clues about the kinetic steps affected by G-proteins because gating currents arise from the movement of voltage sensors that trigger channel activation. 4. The mechanistic implications of concomitant G-protein-mediated changes in gating and ionic currents are discussed. We argue that G-proteins act to inhibit both voltage-sensor movement and the transduction of voltage-sensor activation into channel opening. Images

  15. Computational Simulation of the Activation Cycle of Gα Subunit in the G Protein Cycle Using an Elastic Network Model

    PubMed Central

    Kim, Min Hyeok; Kim, Young Jin; Kim, Hee Ryung; Jeon, Tae-Joon; Choi, Jae Boong; Chung, Ka Young; Kim, Moon Ki

    2016-01-01

    Agonist-activated G protein-coupled receptors (GPCRs) interact with GDP-bound G protein heterotrimers (Gαβγ) promoting GDP/GTP exchange, which results in dissociation of Gα from the receptor and Gβγ. The GTPase activity of Gα hydrolyzes GTP to GDP, and the GDP-bound Gα interacts with Gβγ, forming a GDP-bound G protein heterotrimer. The G protein cycle is allosterically modulated by conformational changes of the Gα subunit. Although biochemical and biophysical methods have elucidated the structure and dynamics of Gα, the precise conformational mechanisms underlying the G protein cycle are not fully understood yet. Simulation methods could help to provide additional details to gain further insight into G protein signal transduction mechanisms. In this study, using the available X-ray crystal structures of Gα, we simulated the entire G protein cycle and described not only the steric features of the Gα structure, but also conformational changes at each step. Each reference structure in the G protein cycle was modeled as an elastic network model and subjected to normal mode analysis. Our simulation data suggests that activated receptors trigger conformational changes of the Gα subunit that are thermodynamically favorable for opening of the nucleotide-binding pocket and GDP release. Furthermore, the effects of GTP binding and hydrolysis on mobility changes of the C and N termini and switch regions are elucidated. In summary, our simulation results enabled us to provide detailed descriptions of the structural and dynamic features of the G protein cycle. PMID:27483005

  16. The GAPs, GEFs, GDIs and…now, GEMs: New kids on the heterotrimeric G protein signaling block.

    PubMed

    Ghosh, Pradipta; Rangamani, Padmini; Kufareva, Irina

    2017-03-13

    The canonical process of activation of heterotrimeric G proteins by G protein coupled receptors (GPCRs) is well studied. Recently, a rapidly emerging paradigm has revealed the existence of a new, non-canonical set of cytosolic G protein modulators, guanine exchange modulators (GEMs). Among G proteins regulators, GEMs are uniquely capable of initiating pleiotropic signals: these bifunctional modulators can activate cAMP inhibitory (Gi) proteins and inhibit cAMP-stimulatory (Gs) proteins through a single short evolutionarily conserved module. A prototypical member of the GEM family, GIV/Girdin, integrates signals downstream of a myriad of cell surface receptors, e.g., growth factor RTKs, integrins, cytokine, GPCRs, etc., and translates these signals into G protein activation or inhibition. By their pleiotropic action, GIV and other GEMs modulate several key pathways within downstream signaling network. Unlike canonical G protein signaling that is finite and is triggered directly and exclusively by GPCRs, the temporal and spatial features of non-canonical activation of G protein via GIV-family of cytosolic GEMs are unusually relaxed. GIV uses this relaxed circuitry to integrate, reinforce and compartmentalize signals downstream of both growth factors and G proteins in a way that enables it to orchestrate cellular phenotypes in a sustained manner. Mounting evidence suggests the importance of GIV and other GEMs as disease modulators and their potential to serve as therapeutic targets; however, a lot remains unknown within the layers of the proverbial onion that must be systematically peeled. This perspective summarizes the key concepts of the GEM-dependent G protein signaling paradigm and discusses the multidisciplinary approaches that are likely to revolutionize our understanding of this paradigm from the atomic level to systems biology.

  17. Ligand-induced dynamics of heterotrimeric G protein-coupled receptor-like kinase complexes

    PubMed Central

    Tunc-Ozdemir, Meral; Jones, Alan M.

    2017-01-01

    Background Arabidopsis, 7-transmembrane Regulator of G signaling protein 1 (AtRGS1) modulates canonical G protein signaling by promoting the inactive state of heterotrimeric G protein complex on the plasma membrane. It is known that plant leucine-rich repeat receptor–like kinases (LRR RLKs) phosphorylate AtRGS1 in vitro but little is known about the in vivo interaction, molecular dynamics, or the cellular consequences of this interaction. Methods Therefore, a subset of the known RLKs that phosphorylate AtRGS1 were selected for elucidation, namely, BAK1, BIR1, FLS2. Several microscopies for both static and dynamic protein-protein interactions were used to follow in vivo interactions between the RLKs and AtRGS1 after the presentation of the Pathogen-associated Molecular Pattern, Flagellin 22 (Flg22). These microscopies included Förster Resonance Energy Transfer, Bimolecular Fluoresence Complementation, and Cross Number and Brightness Fluorescence Correlation Spectroscopy. In addition, reactive oxygen species and calcium changes in living cells were quantitated using luminometry and R-GECO1 microscopy. Results The LRR RLKs BAK1 and BIR1, interact with AtRGS1 at the plasma membrane. The RLK ligand flg22 sets BAK1 in motion toward AtRGS1 and BIR1 away, both returning to the baseline orientations by 10 minutes. The C-terminal tail of AtRGS1 is important for the interaction with BAK1 and for the tempo of the AtRGS1/BIR1 dynamics. This window of time corresponds to the flg22-induced transient production of reactive oxygen species and calcium release which are both attenuated in the rgs1 and the bak1 null mutants. Conclusions A temporal model of these interactions is proposed. flg22 binding induces nearly instantaneous dimerization between FLS2 and BAK1. Phosphorylated BAK1 interacts with and enables AtRGS1 to move away from BIR1 and AtRGS1 becomes phosphorylated leading to its endocytosis thus leading to de-repression by permitting AtGPA1 to exchange GDP for GTP

  18. Actions of Rho family small G proteins and p21-activated protein kinases on mitogen-activated protein kinase family members.

    PubMed Central

    Frost, J A; Xu, S; Hutchison, M R; Marcus, S; Cobb, M H

    1996-01-01

    The mitogen-activated protein (MAP) kinases are a family of serine/threonine kinases that are regulated by distinct extracellular stimuli. The currently known members include extracellular signal-regulated protein kinase 1 (ERK1), ERK2, the c-Jun N-terminal kinase/stress-activated protein kinases (JNK/SAPKs), and p38 MAP kinases. We find that overexpression of the Ste20-related enzymes p21-activated kinase 1 (PAK1) and PAK2 in 293 cells is sufficient to activate JNK/SAPK and to a lesser extent p38 MAP kinase but not ERK2. Rat MAP/ERK kinase kinase 1 can stimulate the activity of each of these MAP kinases. Although neither activated Rac nor the PAKs stimulate ERK2 activity, overexpression of either dominant negative Rac2 or the N-terminal regulatory domain of PAK1 inhibits Ras-mediated activation of ERK2, suggesting a permissive role for Rac in the control of the ERK pathway. Furthermore, constitutively active Rac2, Cdc42hs, and RhoA synergize with an activated form of Raf to increase ERK2 activity. These findings reveal a previously unrecognized connection between Rho family small G proteins and the ERK pathway. PMID:8668187

  19. Orphan G protein-coupled receptors (GPCRs): biological functions and potential drug targets

    PubMed Central

    Tang, Xiao-long; Wang, Ying; Li, Da-li; Luo, Jian; Liu, Ming-yao

    2012-01-01

    The superfamily of G protein-coupled receptors (GPCRs) includes at least 800 seven-transmembrane receptors that participate in diverse physiological and pathological functions. GPCRs are the most successful targets of modern medicine, and approximately 36% of marketed pharmaceuticals target human GPCRs. However, the endogenous ligands of more than 140 GPCRs remain unidentified, leaving the natural functions of those GPCRs in doubt. These are the so-called orphan GPCRs, a great source of drug targets. This review focuses on the signaling transduction pathways of the adhesion GPCR family, the LGR subfamily, and the PSGR subfamily, and their potential functions in immunology, development, and cancers. In this review, we present the current approaches and difficulties of orphan GPCR deorphanization and characterization. PMID:22367282

  20. Tethered agonists: a new mechanism underlying adhesion G protein-coupled receptor activation.

    PubMed

    Schöneberg, Torsten; Liebscher, Ines; Luo, Rong; Monk, Kelly R; Piao, Xianhua

    2015-06-01

    The family of adhesion G protein-coupled receptors (aGPCRs) comprises 33 members in the human genome, which are subdivided into nine subclasses. Many aGPCRs undergo an autoproteolytic process via their GPCR Autoproteolysis-INducing (GAIN) domain during protein maturation to generate an N- and a C-terminal fragments, NTF and CTF, respectively. The NTF and CTF are non-covalently reassociated on the plasma membrane to form a single receptor unit. How aGPCRs are activated upon ligand binding remains one of the leading questions in the field of aGPCR research. Recent work from our labs and others shows that ligand binding can remove the NTF from the plasma membrane-bound CTF, exposing a tethered agonist which potently activates downstream signaling.

  1. G protein-coupled receptors: potential therapeutic targets for diabetic nephropathy.

    PubMed

    Ding, Hai-Hua; Ni, Wei-Jian; Tang, Li-Qin; Wei, Wei

    2015-12-16

    Diabetic nephropathy, a lethal microvascular complication of diabetes mellitus, is characterized by progressive albuminuria, excessive deposition of extracellular matrix, thickened glomerular basement membrane, podocyte abnormalities, and podocyte loss. The G protein-coupled receptors (GPCRs) have attracted considerable attention in diabetic nephropathy, but the specific effects have not been elucidated yet. Likewise, abnormal signaling pathways are closely interrelated to the pathologic process of diabetic nephropathy, despite the fact that the mechanisms have not been explored clearly. Therefore, GPCRs and its mediated signaling pathways are essential for priority research, so that preventative strategies and potential targets might be developed for diabetic nephropathy. This article will give us comprehensive overview of predominant GPCR types, roles, and correlative signaling pathways in diabetic nephropathy.

  2. Discovery of functional monoclonal antibodies targeting G-protein-coupled receptors and ion channels.

    PubMed

    Wilkinson, Trevor C I

    2016-06-15

    The development of recombinant antibody therapeutics is a significant area of growth in the pharmaceutical industry with almost 50 approved monoclonal antibodies on the market in the US and Europe. Despite this growth, however, certain classes of important molecular targets have remained intractable to therapeutic antibodies due to complexity of the target molecules. These complex target molecules include G-protein-coupled receptors and ion channels which represent a large potential target class for therapeutic intervention with monoclonal antibodies. Although these targets have typically been addressed by small molecule approaches, the exquisite specificity of antibodies provides a significant opportunity to provide selective modulation of these target proteins. Given this opportunity, substantial effort has been applied to address the technical challenges of targeting these complex membrane proteins with monoclonal antibodies. In this review recent progress made in the strategies for discovery of functional monoclonal antibodies for these challenging membrane protein targets is addressed.

  3. Methuselah/Methuselah-like G protein-coupled receptors constitute an ancient metazoan gene family

    PubMed Central

    de Mendoza, Alexandre; Jones, Jeffery W.; Friedrich, Markus

    2016-01-01

    Inconsistent conclusions have been drawn regarding the phylogenetic age of the Methuselah/Methuselah-like (Mth/Mthl) gene family of G protein-coupled receptors, the founding member of which regulates development and lifespan in Drosophila. Here we report the results from a targeted homolog search of 39 holozoan genomes and phylogenetic analysis of the conserved seven transmembrane domain. Our findings reveal that the Mth/Mthl gene family is ancient, has experienced numerous extinction and expansion events during metazoan evolution, and acquired the current definition of the Methuselah ectodomain during its exceptional expansion in arthropods. In addition, our findings identify Mthl1, Mthl5, Mthl14, and Mthl15 as the oldest Mth/Mthl gene family paralogs in Drosophila. Future studies of these genes have the potential to define ancestral functions of the Mth/Mthl gene family. PMID:26915348

  4. Cell-free expression of G-protein coupled receptors: new pipelines for challenging targets.

    PubMed

    Rues, Ralf-Bernhardt; Orbán, Erika; Dötsch, Volker; Bernhard, Frank

    2014-12-01

    Based on their eminent importance for medical applications, G-protein coupled receptors are currently amongst the most frequently membrane protein targets analyzed by cell-free expression. The cell-free expression approach removes most bottlenecks known from conventional cell-based protein production pipelines and ensures fast access to a selected receptor target. In addition, receptors can be synthesized in presence of a large variety of artificial solubilization environments comprising detergents, lipids, nanodiscs and other amphiphilic compounds. The currently accumulated data based on a variety of analyzed receptors already opens promising perspectives for applications of cell-free synthesized samples in functional characterization and drug screening. Structural evaluation still suffers from high conformational dynamics causing sample instability and might be addressed in future by molecular engineering or immuno-stabilization approaches.

  5. From G Protein-coupled Receptor Structure Resolution to Rational Drug Design.

    PubMed

    Jazayeri, Ali; Dias, Joao M; Marshall, Fiona H

    2015-08-07

    A number of recent technical solutions have led to significant advances in G protein-coupled receptor (GPCR) structural biology. Apart from a detailed mechanistic view of receptor activation, the new structures have revealed novel ligand binding sites. Together, these insights provide avenues for rational drug design to modulate the activities of these important drug targets. The application of structural data to GPCR drug discovery ushers in an exciting era with the potential to improve existing drugs and discover new ones. In this review, we focus on technical solutions that have accelerated GPCR crystallography as well as some of the salient findings from structures that are relevant to drug discovery. Finally, we outline some of the approaches used in GPCR structure based drug design.

  6. Identification of G protein-coupled estrogen receptor in human and pig spermatozoa.

    PubMed

    Rago, V; Giordano, F; Brunelli, E; Zito, D; Aquila, S; Carpino, A

    2014-06-01

    Estrogens are known to influence functional properties of mammalian spermatozoa inducing rapid responses through the classical estrogen receptors (ERα and ERβ). Recently, the G protein-coupled estrogen receptor (GPER) has been identified as mediator of fast non-genomic estrogen effects in different cells. This work investigated the expression of GPER in human and pig spermatozoa using immunofluorescence, Western blot analysis and RT-PCR. GPER was found to be confined to the mid-piece of human sperm cells, whereas it was detected in the acrosomal region, the equatorial segment and the mid-piece of pig spermatozoa. Furthermore, in the male gametes of both species, the immunoblots of sperm extracts revealed a band at ~42 kDa, consistent with the GPER molecular weight, and RT-PCR detected the GPER transcripts. This is the first report demonstrating the expression of GPER in human and pig mature sperm cells and evidencing its species-specific cellular localization.

  7. Biased ligands at G-protein-coupled receptors: promise and progress.

    PubMed

    Violin, Jonathan D; Crombie, Aimee L; Soergel, David G; Lark, Michael W

    2014-07-01

    Drug discovery targeting G protein-coupled receptors (GPCRs) is no longer limited to seeking agonists or antagonists to stimulate or block cellular responses associated with a particular receptor. GPCRs are now known to support a diversity of pharmacological profiles, a concept broadly referred to as functional selectivity. In particular, the concept of ligand bias, whereby a ligand stabilizes subsets of receptor conformations to engender novel pharmacological profiles, has recently gained increasing prominence. This review discusses how biased ligands may deliver safer, better tolerated, and more efficacious drugs, and highlights several biased ligands that are in clinical development. Biased ligands targeting the angiotensin II type 1 receptor and the μ opioid receptor illustrate the translation of the biased ligand concept from basic biology to clinical drug development.

  8. G protein-coupled receptor 37 is a negative regulator of oligodendrocyte differentiation and myelination

    PubMed Central

    Yang, Hyun-Jeong; Vainshtein, Anna; Maik-Rachline, Galia; Peles, Elior

    2016-01-01

    While the formation of myelin by oligodendrocytes is critical for the function of the central nervous system, the molecular mechanism controlling oligodendrocyte differentiation remains largely unknown. Here we identify G protein-coupled receptor 37 (GPR37) as an inhibitor of late-stage oligodendrocyte differentiation and myelination. GPR37 is enriched in oligodendrocytes and its expression increases during their differentiation into myelin forming cells. Genetic deletion of Gpr37 does not affect the number of oligodendrocyte precursor cells, but results in precocious oligodendrocyte differentiation and hypermyelination. The inhibition of oligodendrocyte differentiation by GPR37 is mediated by suppression of an exchange protein activated by cAMP (EPAC)-dependent activation of Raf-MAPK-ERK1/2 module and nuclear translocation of ERK1/2. Our data suggest that GPR37 regulates central nervous system myelination by controlling the transition from early-differentiated to mature oligodendrocytes. PMID:26961174

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

    SciTech Connect

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

    2015-03-05

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

  10. The paradigm of G protein receptor transactivation: a mechanistic definition and novel example.

    PubMed

    Little, Peter J; Burch, Micah L; Al-aryahi, Sefaa; Zheng, Wenhua

    2011-03-22

    Seven transmembrane G protein-coupled receptors are among the most common in biology and they transduce cellular signals from a plethora of hormones. As well as their own well-characterized signaling pathways, they can also transactivate tyrosine kinase growth factor receptors to greatly expand their own cellular repertoire of responses. Recent data in vascular smooth muscle cells have expanded the breadth of transactivation to include serine/threonine kinase receptors, specifically the receptor for transforming growth factor beta (TGF-beta). Stimulation with endothelin and thrombin leads to the rapid formation of C-terminal phosphorylated Smad2, which is the immediate product of activation of the TGF-beta receptor. Additionally, it appears that no definition of transactivation based on mechanism is available, so we have provided a definition involving temporal aspects and the absence of de novo protein synthesis. The phenomenon of transactivation is an important biochemical mechanism and potential drug target in multiple diseases.

  11. Diverse Regulation of Temperature Sensation by Trimeric G-Protein Signaling in Caenorhabditis elegans

    PubMed Central

    Ujisawa, Tomoyo; Ohta, Akane; Uda-Yagi, Misato

    2016-01-01

    Temperature sensation by the nervous system is essential for life and proliferation of animals. The molecular-physiological mechanisms underlying temperature signaling have not been fully elucidated. We show here that diverse regulatory machinery underlies temperature sensation through trimeric G-protein signaling in the nematode Caenorhabditis elegans. Molecular-genetic studies demonstrated that cold tolerance is regulated by additive functions of three Gα proteins in a temperature-sensing neuron, ASJ, which is also known to be a light-sensing neuron. Optical recording of calcium concentration in ASJ upon temperature-changes demonstrated that three Gα proteins act in different aspects of temperature signaling. Calcium concentration changes in ASJ upon temperature change were unexpectedly decreased in a mutant defective in phosphodiesterase, which is well known as a negative regulator of calcium increase. Together, these data demonstrate commonalities and differences in the molecular components concerned with light and temperature signaling in a single sensory neuron. PMID:27788246

  12. Antibodies against G-protein coupled receptors: novel uses in screening and drug development.

    PubMed

    Gupta, Achla; Heimann, Andrea S; Gomes, Ivone; Devi, Lakshmi A

    2008-07-01

    Antibodies are components of the body's humoral immune system that are generated in response to foreign pathogens. Modern biomedical research has employed these very specific and efficient molecules designed by nature in the diagnosis of diseases, localization of gene products as well as in the rapid screening of targets for drug discovery and testing. In addition, the introduction of antibodies with fluorescent or enzymatic tags has significantly contributed to advances in imaging and microarray technology, which are revolutionizing disease research and the search for effective therapeutics. More recently antibodies have been used in the isolation of dimeric G protein-coupled receptor (GPCR) complexes. In this review, we discuss antibodies as powerful research tools for studying GPCRs, and their potential to be developed as drugs themselves.

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

    PubMed

    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.

  14. Denatured G-protein coupled receptors as immunogens to generate highly specific antibodies.

    PubMed

    Talmont, Franck; Moulédous, Lionel; Boué, Jérôme; Mollereau, Catherine; Dietrich, Gilles

    2012-01-01

    G-protein coupled receptors (GPCRs) play a major role in a number of physiological and pathological processes. Thus, GPCRs have become the most frequent targets for development of new therapeutic drugs. In this context, the availability of highly specific antibodies may be decisive to obtain reliable findings on localization, function and medical relevance of GPCRs. However, the rapid and easy generation of highly selective anti-GPCR antibodies is still a challenge. Herein, we report that highly specific antibodies suitable for detection of GPCRs in native and unfolded forms can be elicited by immunizing animals against purified full length denatured recombinant GPCRs. Contrasting with the currently admitted postulate, our study shows that an active and well-folded GPCR is not required for the production of specific anti-GPCR antibodies. This new immunizing strategy validated with three different human GPCR (μ-opioid, κ-opioid, neuropeptide FF2 receptors) might be generalized to other members of the GPCR family.

  15. G-protein coupling and nuclear translocation of the human abscisic acid receptor LANCL2

    PubMed Central

    Fresia, Chiara; Vigliarolo, Tiziana; Guida, Lucrezia; Booz, Valeria; Bruzzone, Santina; Sturla, Laura; Di Bona, Melody; Pesce, Mattia; Usai, Cesare; De Flora, Antonio; Zocchi, Elena

    2016-01-01

    Abscisic acid (ABA), a long known phytohormone, has been recently demonstrated to be present also in humans, where it targets cells of the innate immune response, mesenchymal and hemopoietic stem cells and cells involved in the regulation of systemic glucose homeostasis. LANCL2, a peripheral membrane protein, is the mammalian ABA receptor. We show that N-terminal glycine myristoylation causes LANCL2 localization to the plasmamembrane and to cytoplasmic membrane vesicles, where it interacts with the α subunit of a Gi protein and starts the ABA signaling pathway via activation of adenylate cyclase. Demyristoylation of LANCL2 by chemical or genetic means triggers its nuclear translocation. Nuclear enrichment of native LANCL2 is also induced by ABA treatment. Therefore human LANCL2 is a non-transmembrane G protein-coupled receptor susceptible to hormone-induced nuclear translocation. PMID:27222287

  16. Modulation of cellular signaling by herpesvirus-encoded G protein-coupled receptors

    PubMed Central

    de Munnik, Sabrina M.; Smit, Martine J.; Leurs, Rob; Vischer, Henry F.

    2015-01-01

    Human herpesviruses (HHVs) are widespread infectious pathogens that have been associated with proliferative and inflammatory diseases. During viral evolution, HHVs have pirated genes encoding viral G protein-coupled receptors (vGPCRs), which are expressed on infected host cells. These vGPCRs show highest homology to human chemokine receptors, which play a key role in the immune system. Importantly, vGPCRs have acquired unique properties such as constitutive activity and the ability to bind a broad range of human chemokines. This allows vGPCRs to hijack human proteins and modulate cellular signaling for the benefit of the virus, ultimately resulting in immune evasion and viral dissemination to establish a widespread and lifelong infection. Knowledge on the mechanisms by which herpesviruses reprogram cellular signaling might provide insight in the contribution of vGPCRs to viral survival and herpesvirus-associated pathologies. PMID:25805993

  17. EGFR Transactivation by Peptide G Protein-Coupled Receptors in Cancer.

    PubMed

    Moody, Terry W; Nuche-Berenguer, Bernardo; Nakamura, Taichi; Jensen, Robert T

    2016-01-01

    Lung cancer kills approximately 1.3 million citizens in the world annually. The tyrosine kinase inhibitors (TKI) erlotinib and gefitinib are effective anti-tumor agents especially in lung cancer patients with epidermal growth factor receptor (EGFR) mutations. The goal is to increase the potency of TKI in lung cancer patients with wild type EGFR. G protein-coupled receptors (GPCR) transactivate the wild type EGFR in lung cancer cells. The GPCR can be activated by peptide agonists causing phosphatidylinositol turnover or stimulation of adenylylcyclase. Recently, nonpeptide antagonists were found to inhibit the EGFR transactivation caused by peptides. Nonpeptide antagonists for bombesin (BB), neurotensin (NTS) and cholecystokinin (CCK) inhibit lung cancer growth and increase the cytotoxicity of gefitinib. The results suggest that GPCR transactivation of the EGFR may play an important role in cancer cell proliferation.

  18. Harnessing the genome for characterization of G-protein coupled receptors in cancer pathogenesis.

    PubMed

    Feigin, Michael E

    2013-10-01

    G-protein coupled receptors (GPCRs) mediate numerous physiological processes and represent the targets for a vast array of therapeutics for diseases ranging from depression to hypertension to reflux. Despite the recognition that GPCRs can act as oncogenes and tumour suppressors by regulating oncogenic signalling networks, few drugs targeting GPCRs are utilized in cancer therapy. Recent large-scale genome-wide analyses of multiple human tumours have uncovered novel GPCRs altered in cancer. However, work aiming to determine which GPCRs from these lists are the drivers of tumourigenesis, and hence valid therapeutic targets, comprises a formidable challenge. The present review highlights recent studies providing evidence that GPCRs are relevant targets for cancer therapy through their effects on known cancer signalling pathways, tumour progression, invasion and metastasis, and the microenvironment. Furthermore, the review also explores how genomic analysis is beginning to highlight GPCRs as therapeutic targets in the age of personalized medicine.

  19. G protein-coupled receptors: novel targets for drug discovery in cancer.

    PubMed

    Lappano, Rosamaria; Maggiolini, Marcello

    2011-01-01

    G protein-coupled receptors (GPCRs) belong to a superfamily of cell surface signalling proteins that have a pivotal role in many physiological functions and in multiple diseases, including the development of cancer and cancer metastasis. Current drugs that target GPCRs - many of which have excellent therapeutic benefits - are directed towards only a few GPCR members. Therefore, huge efforts are currently underway to develop new GPCR-based drugs, particularly for cancer. We review recent findings that present unexpected opportunities to interfere with major tumorigenic signals by manipulating GPCR-mediated pathways. We also discuss current data regarding novel GPCR targets that may provide promising opportunities for drug discovery in cancer prevention and treatment.

  20. Prediction of G Protein-Coupled Receptors with SVM-Prot Features and Random Forest

    PubMed Central

    Ju, Ying

    2016-01-01

    G protein-coupled receptors (GPCRs) are the largest receptor superfamily. In this paper, we try to employ physical-chemical properties, which come from SVM-Prot, to represent GPCR. Random Forest was utilized as classifier for distinguishing them from other protein sequences. MEME suite was used to detect the most significant 10 conserved motifs of human GPCRs. In the testing datasets, the average accuracy was 91.61%, and the average AUC was 0.9282. MEME discovery analysis showed that many motifs aggregated in the seven hydrophobic helices transmembrane regions adapt to the characteristic of GPCRs. All of the above indicate that our machine-learning method can successfully distinguish GPCRs from non-GPCRs. PMID:27529053

  1. G protein-coupled receptor 183 facilitates endothelial-to-hematopoietic transition via Notch1 inhibition.

    PubMed

    Zhang, Panpan; He, Qiuping; Chen, Dongbo; Liu, Weixiao; Wang, Lu; Zhang, Chunxia; Ma, Dongyuan; Li, Wei; Liu, Bing; Liu, Feng

    2015-10-01

    In vertebrates, embryonic hematopoietic stem and progenitor cells (HSPCs) are derived from a subset of endothelial cells, the hemogenic endothelium (HE), through the endothelial-to-hematopoietic transition (EHT). Notch signaling is essential for HSPC development during embryogenesis across vertebrates. However, whether and how it regulates EHT remains unclear. Here, we show that G protein-coupled receptor 183 (Gpr183) signaling serves as an indispensable switch for HSPC emergence by repressing Notch signaling before the onset of EHT. Inhibition of Gpr183 significantly upregulates Notch signaling and abolishes HSPC emergence. Upon activation by its ligand 7α-25-OHC, Gpr183 recruits β-arrestin1 and the E3 ligase Nedd4 to degrade Notch1 in specified HE cells and then facilitates the subsequent EHT. Importantly, 7α-25-OHC stimulation promotes HSPC emergence in vivo and in vitro, providing an attractive strategy for enhancing the in vitro generation of functional HSPCs.

  2. Adhesion G-protein-coupled receptors: elusive hybrids come of age.

    PubMed

    Simundza, Julia; Cowin, Pamela

    2013-12-01

    Adhesion G-protein-coupled receptors (GPCRs) are the most recently identified and least understood subfamily of GPCRs. Adhesion GPCRs are characterized by unusually long ectodomains with adhesion-related repeats that facilitate cell- cell and cell-cell matrix contact, as well as a proteolytic cleavage site-containing domain that is a structural hallmark of the family. Their unusual chimeric structure of adhesion-related ectodomain with a seven-pass transmembrane domain and cytoplasmic signaling makes these proteins highly versatile in mediating cellular signaling in response to extracellular adhesion or cell motility events. The ligand binding and cytoplasmic signaling modes for members of this family are beginning to be elucidated, and recent studies have demonstrated critical roles for Adhesion GPCRs in planar polarity and other important cell-cell and cell-matrix interactions during development and morphogenesis, as well as heritable diseases and cancer.

  3. G-protein-coupled receptors in control of natural killer cell migration.

    PubMed

    Walzer, Thierry; Vivier, Eric

    2011-10-01

    Natural killer (NK) cells are highly motile cells that patrol lymphoid and non-lymphoid organs, and are poised to react to infectious or other inflammatory situations. Several NK cell subsets equipped with different sets of chemotactic G-protein-coupled receptors, and which display distinct distribution across lymphoid and non-lymphoid organs, have been described. These receptors detect various guidance cues including sphingosine-1-phosphate and chemokines that orchestrate NK cell trafficking. Here, we highlight recent advances regarding the receptors involved in NK cell migration, with a focus on bone marrow egress, entry into activated lymph nodes, extravasation into inflamed tissues, and motility within lymph nodes or tumors. Understanding NK cell migration could provide a rational basis for the design of novel therapies in various clinical conditions.

  4. Basic Pharmacological and Structural Evidence for Class A G-Protein-Coupled Receptor Heteromerization

    PubMed Central

    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

  5. Function of G-Protein-Coupled Estrogen Receptor-1 in Reproductive System Tumors

    PubMed Central

    Qian, Hongyan; Xuan, Jingxiu; Liu, Yuan; Shi, Guixiu

    2016-01-01

    The G-protein-coupled estrogen receptor-1 (GPER-1), also known as GPR30, is a novel estrogen receptor mediating estrogen receptor signaling in multiple cell types. The progress of estrogen-related cancer is promoted by GPER-1 activation through mitogen-activated protein kinases (MAPK), phosphoinositide 3-kinase (PI3K), and phospholipase C (PLC) signaling pathways. However, this promoting effect of GPER-1 is nonclassic estrogen receptor (ER) dependent manner. In addition, clinical evidences revealed that GPER-1 is associated with estrogen resistance in estrogen-related cancer patients. These give a hint that GPER-1 may be a novel therapeutic target for the estrogen-related cancers. However, preclinical studies also found that GPER-1 activation of its special agonist G-1 inhibits cancer cell proliferation. This review aims to summarize the characteristics and complex functions of GPER-1 in cancers. PMID:27314054

  6. Structure-based drug screening for G protein-coupled receptors

    PubMed Central

    Shoichet, Brian K.; Kobilka, Brian K.

    2012-01-01

    G protein-coupled receptors (GPCRs) represent a large family of signaling proteins that includes many therapeutic targets; however, progress in identifying new small molecule drugs has been disappointing. The past four years have seen remarkable progress in the structural biology of GPCRs, raising the possibility of applying structure-based approaches to GPCR drug discovery efforts. Of the various structure-based approaches that have been applied to soluble protein targets, such as proteases and kinases, in silico docking is among the most ready applicable to GPCRs. Early studies suggest that GPCR binding pockets are well suited to docking, and docking screens have identified potent and novel compounds for these targets. This review will focus on the current state of in silico docking for GPCRs. PMID:22503476

  7. Systematic and quantitative analysis of G protein-coupled receptor trafficking motifs.

    PubMed

    Hurt, Carl M; Ho, Vincent K; Angelotti, Timothy

    2013-01-01

    Plasma membrane expression of G protein-coupled receptors (GPCRs) is a dynamic process balancing anterograde and retrograde trafficking. Multiple interrelated cellular processes determine the final level of cell surface expression, including endoplasmic reticulum (ER) export/retention, receptor internalization, recycling, and degradation. These processes are highly regulated to achieve specific localization to subcellular domains (e.g., dendrites or basolateral membranes) and to affect receptor signaling. Analysis of potential ER trafficking motifs within GPCRs requires careful consideration of intracellular dynamics, such as protein folding, ER export and retention, and glycosylation. This chapter presents an approach and methods for qualitative and quantitative assessment of these processes to aid in accurate identification of GPCR trafficking motifs, utilizing the analysis of a hydrophobic extracellular trafficking motif in α2C adrenergic receptors as a model system.

  8. The helical domain of a G protein alpha subunit is a regulator of its effector.

    PubMed

    Liu, W; Northup, J K

    1998-10-27

    The alpha subunit (Galpha) of heterotrimeric G proteins is a major determinant of signaling selectivity. The Galpha structure essentially comprises a GTPase "Ras-like" domain (RasD) and a unique alpha-helical domain (HD). We used the vertebrate phototransduction model to test for potential functions of HD and found that the HD of the retinal transducin Galpha (Galphat) and the closely related gustducin (Galphag), but not Galphai1, Galphas, or Galphaq synergistically enhance guanosine 5'-gamma[-thio]triphosphate bound Galphat (GalphatGTPgammaS) activation of bovine rod cGMP phosphodiesterase (PDE). In addition, both HDt and HDg, but not HDi1, HDs, or HDq attenuate the trypsin-activated PDE. GalphatGDP and HDt attenuation of trypsin-activated PDE saturate with similar affinities and to an identical 38% of initial activity. These data suggest that interaction of intact Galphat with the PDE catalytic core may be caused by the HD moiety, and they indicate an independent site(s) for the HD moiety of Galphat within the PDE catalytic core in addition to the sites for the inhibitory Pgamma subunits. The HD moiety of GalphatGDP is an attenuator of the activated catalytic core, whereas in the presence of activated GalphatGTPgammaS the independently expressed HDt is a potent synergist. Rhodopsin catalysis of Galphat activation enhances the PDE activation produced by subsaturating levels of Galphat, suggesting a HD-moiety synergism from a transient conformation of Galphat. These results establish HD-selective regulations of vertebrate retinal PDE, and they provide evidence demonstrating that the HD is a modulatory domain. We suggest that the HD works in concert with the RasD, enhancing the efficiency of G protein signaling.

  9. Role of heterotrimeric G protein and calcium in cardiomyocyte hypertrophy induced by IGF-1.

    PubMed

    Carrasco, Loreto; Cea, Paola; Rocco, Paola; Peña-Oyarzún, Daniel; Rivera-Mejias, Pablo; Sotomayor-Flores, Cristian; Quiroga, Clara; Criollo, Alfredo; Ibarra, Cristian; Chiong, Mario; Lavandero, Sergio

    2014-04-01

    In the heart, insulin-like growth factor-1 (IGF-1) is a peptide with pro-hypertrophic and anti-apoptotic actions. The pro-hypertrophic properties of IGF-1 have been attributed to the extracellular regulated kinase (ERK) pathway. Recently, we reported that IGF-1 also increases intracellular Ca(2+) levels through a pertussis toxin (PTX)-sensitive G protein. Here we investigate whether this Ca(2+) signal is involved in IGF-1-induced cardiomyocyte hypertrophy. Our results show that the IGF-1-induced increase in Ca(2+) level is abolished by the IGF-1 receptor tyrosine kinase inhibitor AG538, PTX and the peptide inhibitor of Gβγ signaling, βARKct. Increases in the activities of Ca(2+) -dependent enzymes calcineurin, calmodulin kinase II (CaMKII), and protein kinase Cα (PKCα) were observed at 5 min after IGF-1 exposure. AG538, PTX, βARKct, and the dominant negative PKCα prevented the IGF-1-dependent phosphorylation of ERK1/2. Participation of calcineurin and CaMKII in ERK phosphorylation was discounted. IGF-1-induced cardiomyocyte hypertrophy, determined by cell size and β-myosin heavy chain (β-MHC), was prevented by AG538, PTX, βARKct, dominant negative PKCα, and the MEK1/2 inhibitor PD98059. Inhibition of calcineurin with CAIN did not abolish IGF-1-induced cardiac hypertrophy. We conclude that IGF-1 induces hypertrophy in cultured cardiomyocytes by activation of the receptor tyrosine kinase activity/βγ-subunits of a PTX-sensitive G protein/Ca(2+) /PKCα/ERK pathway without the participation of calcineurin.

  10. Molecular dissection of G protein preference using Gsalpha chimeras reveals novel ligand signaling of GPCRs.

    PubMed

    Hsu, Shih-Han; Luo, Ching-Wei

    2007-10-01

    Although only 16 genes have been identified in mammals, several Galpha subunits can be simultaneously activated by G protein-coupled receptors (GPCRs) to modulate their complicated functions. Current GPCR assays are limited in the evaluation of selective Galpha activation, thus not allowing a comprehensive pathway screening. Because adenylyl cyclases are directly activated by G(s)alpha and the carboxyl termini of the various Galpha proteins determine their receptor coupling specificity, we proposed a set of chimeric G(s)alpha where the COOH-terminal five amino acids are replaced by those of other Galpha proteins and used these to dissect the potential Galpha linked to a given GPCR. Unlike G(q)alpha, G(12)alpha, and G(i)alpha outputs, compounding the signals from several Galpha members, the chimeric G(s)alpha proteins provide a superior molecular approach that reflects the previously uncharacterized pathways of GPCRs under the same cAMP platform. This is, to our knowledge, the first time allowing verification of the whole spectrum of Galpha coupling preference of adenosine A1 receptor, reported to couple to multiple G proteins and modulate many physiological processes. Furthermore, we were able to distinguish the uncharacterized pathways between the two neuromedin U receptors (NMURs), which distribute differently but are stimulated by a common agonist. In contrast to the G(q) signals mainly conducted by NMUR1, NMUR2 routed preferentially to the G(i) pathways. Dissecting the potential Galpha coupling to these GPCRs will promote an understanding of their physiological roles and benefit the pharmaceutical development of agonists/antagonists by exploiting the selective affinity toward a certain Galpha subclass.

  11. Reciprocal Alterations in Regulator of G Protein Signaling 4 and microRNA16 in Schizophrenia.

    PubMed

    Kimoto, Sohei; Glausier, Jill R; Fish, Kenneth N; Volk, David W; Bazmi, H Holly; Arion, Dominique; Datta, Dibyadeep; Lewis, David A

    2016-03-01

    N-methyl-d-aspartate receptor (NMDAR) hypofunction in the dorsolateral prefrontal cortex (DLPFC) has been implicated in the pathology of schizophrenia. NMDAR activity is negatively regulated by some G protein-coupled receptors (GPCRs). Signaling through these GPCRs is reduced by Regulator of G protein Signaling 4 (RGS4). Thus, lower levels of RGS4 would enhance GPCR-mediated reductions in NMDAR activity and could contribute to NMDAR hypofunction in schizophrenia. In this study, we quantified RGS4 mRNA and protein levels at several levels of resolution in the DLPFC from subjects with schizophrenia and matched healthy comparison subjects. To investigate molecular mechanisms that could contribute to altered RGS4 levels, we quantified levels of small noncoding RNAs, known as microRNAs (miRs), which regulate RGS4 mRNA integrity after transcription. RGS4 mRNA and protein levels were significantly lower in schizophrenia subjects and were positively correlated across all subjects. The RGS4 mRNA deficit was present in pyramidal neurons of DLPFC layers 3 and 5 of the schizophrenia subjects. In contrast, levels of miR16 were significantly higher in the DLPFC of schizophrenia subjects, and higher miR16 levels predicted lower RGS4 mRNA levels. These findings provide convergent evidence of lower RGS4 mRNA and protein levels in schizophrenia that may result from increased expression of miR16. Given the role of RGS4 in regulating GPCRs, and consequently the strength of NMDAR signaling, these findings could contribute to the molecular substrate for NMDAR hypofunction in DLPFC pyramidal cells in schizophrenia.

  12. Graded activation and free energy landscapes of a muscarinic G-protein-coupled receptor.

    PubMed

    Miao, Yinglong; McCammon, J Andrew

    2016-10-25

    G-protein-coupled receptors (GPCRs) recognize ligands of widely different efficacies, from inverse to partial and full agonists, which transduce cellular signals at differentiated levels. However, the mechanism of such graded activation remains unclear. Using the Gaussian accelerated molecular dynamics (GaMD) method that enables both unconstrained enhanced sampling and free energy calculation, we have performed extensive GaMD simulations (∼19 μs in total) to investigate structural dynamics of the M2 muscarinic GPCR that is bound by the full agonist iperoxo (IXO), the partial agonist arecoline (ARC), and the inverse agonist 3-quinuclidinyl-benzilate (QNB), in the presence or absence of the G-protein mimetic nanobody. In the receptor-nanobody complex, IXO binding leads to higher fluctuations in the protein-coupling interface than ARC, especially in the receptor transmembrane helix 5 (TM5), TM6, and TM7 intracellular domains that are essential elements for GPCR activation, but less flexibility in the receptor extracellular region due to stronger binding compared with ARC. Two different binding poses are revealed for ARC in the orthosteric pocket. Removal of the nanobody leads to GPCR deactivation that is characterized by inward movement of the TM6 intracellular end. Distinct low-energy intermediate conformational states are identified for the IXO- and ARC-bound M2 receptor. Both dissociation and binding of an orthosteric ligand are observed in a single all-atom GPCR simulation in the case of partial agonist ARC binding to the M2 receptor. This study demonstrates the applicability of GaMD for exploring free energy landscapes of large biomolecules and the simulations provide important insights into the GPCR functional mechanism.

  13. G protein-coupled receptors function as logic gates for nanoparticle binding and cell uptake

    PubMed Central

    Hild, Wolfgang; Pollinger, Klaus; Caporale, Andrea; Cabrele, Chiara; Keller, Max; Pluym, Nicola; Buschauer, Armin; Rachel, Reinhard; Tessmar, Joerg; Breunig, Miriam; Goepferich, Achim

    2010-01-01

    More selective interactions of nanoparticles with cells would substantially increase their potential for diagnostic and therapeutic applications. Thus, it would not only be highly desirable that nanoparticles can be addressed to any cell with high target specificity and affinity, but that we could unequivocally define whether they rest immobilized on the cell surface as a diagnostic tag, or if they are internalized to serve as a delivery vehicle for drugs. To date no class of targets is known that would allow direction of nanoparticle interactions with cells alternatively into one of these mutually exclusive events. Using MCF-7 breast cancer cells expressing the human Y1-receptor, we demonstrate that G protein-coupled receptors provide us with this option. We show that quantum dots carrying a surface-immobilized antagonist remain with nanomolar affinity on the cell surface, and particles carrying an agonist are internalized upon receptor binding. The receptor functions like a logic “and-gate” that grants cell access only to those particles that carry a receptor ligand “and” where the ligand is an agonist. We found that agonist- and antagonist-modified nanoparticles bind to several receptor molecules at a time. This multiligand binding leads to five orders of magnitude increased-receptor affinities, compared with free ligand, in displacement studies. More than 800 G protein-coupled receptors in humans provide us with the paramount advantage that targeting of a plethora of cells is possible, and that switching from cell recognition to cell uptake is simply a matter of nanoparticle surface modification with the appropriate choice of ligand type. PMID:20498042

  14. Agonistic autoantibodies directed against G-protein-coupled receptors and their relationship to cardiovascular diseases.

    PubMed

    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

  15. Postnatal developmental expression of regulator of G protein signaling 14 (RGS14) in the mouse brain.

    PubMed

    Evans, Paul R; Lee, Sarah E; Smith, Yoland; Hepler, John R

    2014-01-01

    Regulator of G protein signaling 14 (RGS14) is a multifunctional scaffolding protein that integrates G protein and mitogen-activated protein kinase (MAPK) signaling pathways. In the adult mouse brain, RGS14 mRNA and protein are found almost exclusively in hippocampal CA2 neurons. We have shown that RGS14 is a natural suppressor of CA2 synaptic plasticity and hippocampal-dependent learning and memory. However, the protein distribution and spatiotemporal expression patterns of RGS14 in mouse brain during postnatal development are unknown. Here, using a newly characterized monoclonal anti-RGS14 antibody, we demonstrate that RGS14 protein immunoreactivity is undetectable at birth (P0), with very low mRNA expression in the brain. However, RGS14 protein and mRNA are upregulated during early postnatal development, with protein first detected at P7, and both increasing over time until reaching highest sustained levels throughout adulthood. Our immunoperoxidase data demonstrate that RGS14 protein is expressed in regions outside of hippocampal CA2 during development including the primary olfactory areas, the anterior olfactory nucleus and piriform cortex, and the olfactory associated orbital and entorhinal cortices. RGS14 is also transiently expressed in neocortical layers II/III and V during postnatal development. Finally, we show that RGS14 protein is first detected in the hippocampus at P7, with strongest immunoreactivity in CA2 and fasciola cinerea and sporadic immunoreactivity in CA1; labeling intensity in hippocampus increases until adulthood. These results show that RGS14 mRNA and protein are upregulated throughout postnatal mouse development, and RGS14 protein exhibits a dynamic localization pattern that is enriched in hippocampus and primary olfactory cortex in the adult mouse brain.

  16. Consequences of splice variation on Secretin family G protein-coupled receptor function

    PubMed Central

    Furness, Sebastian GB; Wootten, Denise; Christopoulos, Arthur; Sexton, Patrick M

    2012-01-01

    The Secretin family of GPCRs are endocrine peptide hormone receptors that share a common genomic organization and are the subject of a wide variety of alternative splicing. All GPCRs contain a central seven transmembrane domain responsible for transducing signals from the outside of the cell as well as extracellular amino and intracellular carboxyl termini. Members of the Secretin receptor family have a relatively large N-terminus and a variety of lines of evidence support a common mode of ligand binding and a common ligand binding fold. These receptors are best characterized as coupling to intracellular signalling pathways via Gαs and Gαq but are also reported to couple to a multitude of other signalling pathways. The intracellular loops are implicated in regulating the interaction between the receptor and heterotrimeric G protein complexes. Alternative splicing of exons encoding both the extracellular N-terminal domain as well as the extracellular loops of some family members has been reported and as expected these splice variants display altered ligand affinity as well as differential activation by endogenous ligands. Various forms of alternative splicing have also been reported to alter intracellular loops 1 and 3 as well as the C-terminus and as one might expect these display differences in signalling bias towards downstream effectors. These diverse pharmacologies require that the physiological role of these splice variants be addressed but should provide unique opportunities for drug design and development. LINKED ARTICLES This article is part of a themed section on Secretin Family (Class B) G Protein-Coupled Receptors. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2012.166.issue-1 PMID:21718310

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

    PubMed

    Grånäs, Charlotta; Lundholt, Betina Kerstin; Heydorn, Arne; Linde, Viggo; Pedersen, Hans-Christian; Krog-Jensen, Christian; Rosenkilde, Mette M; Pagliaro, Len

    2005-06-01

    G protein-coupled receptors (GPCRs) have been one of the most productive classes of drug targets for several decades, and new technologies for GPCR-based discovery promise to keep this field active for years to come. While molecular screens for GPCR receptor agonist- and antagonist-based drugs will continue to be valuable discovery tools, the most exciting developments in the field involve cell-based assays for GPCR function. Some cell-based discovery strategies, such as the use of beta-arrestin as a surrogate marker for GPCR function, have already been reduced to practice, and have been used as valuable discovery tools for several years. The application of high content cell-based screening to GPCR discovery has opened up additional possibilities, such as direct tracking of GPCRs, G proteins and other signaling pathway components using intracellular translocation assays. These assays provide 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 discovery is described, and proof-of-concept data from a pilot screen with a CXCR4 assay are presented. This chemokine receptor is a highly relevant drug target which plays an important role in the pathogenesis of inflammatory disease and also has been shown to be a co-receptor for entry of HIV into cells as well as to play a role in metastasis of certain cancer cells.

  18. G protein-coupled receptors function as logic gates for nanoparticle binding and cell uptake.

    PubMed

    Hild, Wolfgang; Pollinger, Klaus; Caporale, Andrea; Cabrele, Chiara; Keller, Max; Pluym, Nicola; Buschauer, Armin; Rachel, Reinhard; Tessmar, Joerg; Breunig, Miriam; Goepferich, Achim

    2010-06-08

    More selective interactions of nanoparticles with cells would substantially increase their potential for diagnostic and therapeutic applications. Thus, it would not only be highly desirable that nanoparticles can be addressed to any cell with high target specificity and affinity, but that we could unequivocally define whether they rest immobilized on the cell surface as a diagnostic tag, or if they are internalized to serve as a delivery vehicle for drugs. To date no class of targets is known that would allow direction of nanoparticle interactions with cells alternatively into one of these mutually exclusive events. Using MCF-7 breast cancer cells expressing the human Y(1)-receptor, we demonstrate that G protein-coupled receptors provide us with this option. We show that quantum dots carrying a surface-immobilized antagonist remain with nanomolar affinity on the cell surface, and particles carrying an agonist are internalized upon receptor binding. The receptor functions like a logic "and-gate" that grants cell access only to those particles that carry a receptor ligand "and" where the ligand is an agonist. We found that agonist- and antagonist-modified nanoparticles bind to several receptor molecules at a time. This multiligand binding leads to five orders of magnitude increased-receptor affinities, compared with free ligand, in displacement studies. More than 800 G protein-coupled receptors in humans provide us with the paramount advantage that targeting of a plethora of cells is possible, and that switching from cell recognition to cell uptake is simply a matter of nanoparticle surface modification with the appropriate choice of ligand type.

  19. G-protein-coupled estrogen receptor-30 gene polymorphisms are associated with uterine leiomyoma risk

    PubMed Central

    Kasap, Burcu; Turhan, Nilgün Öztürk; Edgünlü, Tuba; Duran, Müzeyyen; Akbaba, Eren; Öner, Gökalp

    2016-01-01

    The G-protein-coupled estrogen receptor, GPER-1) is a member of the G-protein-coupled receptor 1 family and is expressed significantly in uterine leiomyomas. To understand the relationship between GPR30 single nucleotide polymorphisms and the risk of leiomyoma, we measured the follicle-stimulating hormone (FSH) and estradiol (E2) levels of 78 perimenopausal healthy women and 111 perimenopausal women with leiomyomas. The participants’ leiomyoma number and volume were recorded. DNA was extracted from whole blood with a GeneJET Genomic DNA Purification Kit. An amplification-refractory mutation system polymerase chain reaction approach was used for genotyping of the GPR30 gene (rs3808350, rs3808351, and rs11544331). The differences in genotype and allele frequencies between the leiomyoma and control groups were calculated using the chi-square (χ2) and Fischer’s exact test. The median FSH level was higher in controls (63 vs. 10 IU/L, p=0.000), whereas the median E2 level was higher in the leiomyoma group (84 vs. 9.1 pg/mL, p=0.000). The G allele of rs3808351 and the GG genotype of both the rs3808350 and rs3808351 polymorphisms and the GGC haplotype increased the risk of developing leiomyoma. There was no significant difference in genotype frequencies or leiomyoma volume. However, the GG genotype of the GPR30 rs3808351 polymorphism and G allele of the GPR30 rs3808351 polymorphism were associated with the risk of having a single leiomyoma. Our results suggest that the presence of the GG genotype of the GPR30 rs3808351 polymorphism and the G allele of the GPR30 rs3808351 polymorphism affect the characteristics and development of leiomyomas in the Turkish population. PMID:26773178

  20. Targeting G-Protein Signaling for the Therapeutics of Prostate Tumor Bone Metastases and the Associated Chronic Bone Pain

    DTIC Science & Technology

    2013-07-01

    results in increased activity/expression of key pain-sensing receptor channels, such as TRPV1 , such that the channels are constitutively activated...Keywords: Prostate Cancer Bone Metastasis, Bone Cancer Pain, Heterotrimeric G protein betagamma subunits, G protein coupled receptors (GPCRs), TRPV1 ...cell growth, migration and invasion in vitro, as well as mediating GPCR-regulated TRPV1 channel function in cultured mouse sensory neurons (Aim 1

  1. MAPkinase: a second site of G-protein regulation of B-cell activation via the antigen receptors.

    PubMed Central

    Deehan, M R; Klaus, G G; Holman, M J; Harnett, W; Harnett, M M

    1998-01-01

    Ligation of the antigen receptors on B cells transduces transmembrane signals leading to the induction of DNA synthesis. We now show that a pertussis toxin-sensitive heterotrimeric G-protein(s) of the Gi class plays a key role in the regulation of surface immunoglobulin (sIg)-mediated DNA synthesis in B cells. This site of G-protein regulation is distinct from that we have previously reported to govern the coupling of the antigen receptors on B cells to the phospholipase C-mediated hydrolysis of phosphatidylinositol-4,5-bisphosphate. We have, moreover, identified a candidate target for this new G-protein regulation by showing that mitogen-activating protein kinase (MAPkinase) activity, which plays a key role in the transduction of sIg-mediated proliferative signals in B cells, is abrogated by pre-exposure to pertussis toxin that covalently modifies and inactivates heterotrimeric G-proteins of the Gi class. Furthermore, our data suggest that this pertussis toxin-sensitive G-protein couples the antigen receptors to MAPkinase activation, at least in part, by regulating sIg-coupling to Lyn, Syk and perhaps Blk and Fyn activity, results consistent with studies in other systems which show that classical G-protein-coupled receptors recruit such protein tyrosine kinases to tranduce MAPkinase activation. Interestingly, however, this G-protein plays no apparent role in the control of up-regulation of major histocompatibility complex class II expression on B cells, suggesting that such G-protein-regulated-tyrosine kinase and MAPkinase activation is not required for the induction of this biological response following antigen receptor ligation. Images Figure 5 PMID:9824472

  2. Functional Role of the C-Terminal Amphipathic Helix 8 of Olfactory Receptors and Other G Protein-Coupled Receptors

    PubMed Central

    Sato, Takaaki; Kawasaki, Takashi; Mine, Shouhei; Matsumura, Hiroyoshi

    2016-01-01

    G protein-coupled receptors (GPCRs) transduce various extracellular signals, such as neurotransmitters, hormones, light, and odorous chemicals, into intracellular signals via G protein activation during neurological, cardiovascular, sensory and reproductive signaling. Common and unique features of interactions between GPCRs and specific G proteins are important for structure-based design of drugs in order to treat GPCR-related diseases. Atomic resolution structures of GPCR complexes with G proteins have revealed shared and extensive interactions between the conserved DRY motif and other residues in transmembrane domains 3 (TM3), 5 and 6, and the target G protein C-terminal region. However, the initial interactions formed between GPCRs and their specific G proteins remain unclear. Alanine scanning mutagenesis of the murine olfactory receptor S6 (mOR-S6) indicated that the N-terminal acidic residue of helix 8 of mOR-S6 is responsible for initial transient and specific interactions with chimeric Gα15_olf, resulting in a response that is 2.2-fold more rapid and 1.7-fold more robust than the interaction with Gα15. Our mutagenesis analysis indicates that the hydrophobic core buried between helix 8 and TM1–2 of mOR-S6 is important for the activation of both Gα15_olf and Gα15. This review focuses on the functional role of the C-terminal amphipathic helix 8 based on several recent GPCR studies. PMID:27869740

  3. Antiproliferative effects of Bortezomib in endothelial cells transformed by viral G protein-coupled receptor associated to Kaposi's sarcoma.

    PubMed

    Suares, A; Mori Sequeiros Garcia, M; Paz, C; González-Pardo, V

    2017-04-01

    The Kaposi's Sarcoma-associated Herpes virus G Protein-Coupled Receptor (vGPCR) is a key molecule in the pathogenesis of Kaposi Sarcoma. We have previously demonstrated that the proteasome inhibitor Bortezomib inhibits NF-κB pathway, which is required for tumor maintenance in endothelial cells that express vGPCR (vGPCR cells). In this work, we further investigated Bortezomib anti-proliferative mechanism of action. We demonstrated that Bortezomib decreases vGPCR cell number in a dose-dependent manner and induces cell morphology changes. Bortezomib decreases ERK1/2 phosphorylation whereas induces the accumulation of MKP-3 - a specific ERK1/2 MAP kinase phosphatase - in time and concentration dependent manner (1.5-32h; 0.25-1nM). The transcription factor FOXO1 is activated by dephosphorylation and regulates p21 expression. Here, we demonstrated that Bortezomib increases FOXO1 protein and decreases its phosphorylation in a concentration dependent manner (0.25-1nM). Bortezomib (0.5nM, 24h) also increase nuclear FOXO1 protein, in line with FOXO1 dephosphorylation induced by the drug. Consistent with FOXO1 dephosphorylation/activation, p21 mRNA expression is increased by Bortezomib in a MKP-3-dependent way. Bortezomib (0.5nM, 24h) also decreases VEGF, an ERK1/2 -dependent effect. It is concluded that in vGPCR cells, Bortezomib decreases ERK1/2 and FOXO1 phosphorylation through MKP-3 accumulation, leading ERK1/2 deactivation and FOXO1 activation respectively and, consequently, to cell proliferation inhibition, p21 induction and VEGF repression. Taken together, all these events contribute to the anti-tumoral effect of Bortezomib.

  4. Evolution, expression differentiation and interaction specificity of heterotrimeric G-protein subunit gene family in the mesohexaploid Brassica rapa.

    PubMed

    Arya, Gulab C; Kumar, Roshan; Bisht, Naveen C

    2014-01-01

    Heterotrimeric G-proteins, comprising of Gα, Gβ, and Gγ subunits, are important signal transducers which regulate many aspects of fundamental growth and developmental processes in all eukaryotes. Initial studies in model plants Arabidopsis and rice suggest that the repertoire of plant G-protein is much simpler than that observed in metazoans. In order to assess the consequence of whole genome triplication events within Brassicaceae family, we investigated the multiplicity of G-protein subunit genes in mesohexaploid Brassica rapa, a globally important vegetable and oilseed crop. We identified one Gα (BraA.Gα1), three Gβ (BraA.Gβ1, BraA.Gβ2, and BraA.Gβ3), and five Gγ (BraA.Gγ1, BraA.Gγ2, BraA.Gγ3, BraA.Gγ4, and BraA.Gγ5) genes from B. rapa, with a possibility of 15 Gαβγ heterotrimer combinations. Our analysis suggested that the process of genome triplication coupled with gene-loss (gene-fractionation) phenomenon have shaped the quantitative and sequence diversity of G-protein subunit genes in the extant B. rapa genome. Detailed expression analysis using qRT-PCR assays revealed that the G-protein genes have retained ubiquitous but distinct expression profiles across plant development. The expression of multiple G-protein genes was differentially regulated during seed-maturation and germination stages, and in response to various phytohormone treatments and stress conditions. Yeast-based interaction analysis showed that G-protein subunits interacted in most of the possible combinations, with some degree of subunit-specific interaction specificity, to control the functional selectivity of G-protein heterotrimer in different cell and tissue-types or in response to different environmental conditions. Taken together, this research identifies a highly diverse G-protein signaling network known to date from B. rapa, and provides a clue about the possible complexity of G-protein signaling networks present across globally important Brassica species.

  5. Evolution, Expression Differentiation and Interaction Specificity of Heterotrimeric G-Protein Subunit Gene Family in the Mesohexaploid Brassica rapa

    PubMed Central

    Arya, Gulab C.; Kumar, Roshan; Bisht, Naveen C.

    2014-01-01

    Heterotrimeric G-proteins, comprising of Gα, Gβ, and Gγ subunits, are important signal transducers which regulate many aspects of fundamental growth and developmental processes in all eukaryotes. Initial studies in model plants Arabidopsis and rice suggest that the repertoire of plant G-protein is much simpler than that observed in metazoans. In order to assess the consequence of whole genome triplication events within Brassicaceae family, we investigated the multiplicity of G-protein subunit genes in mesohexaploid Brassica rapa, a globally important vegetable and oilseed crop. We identified one Gα (BraA.Gα1), three Gβ (BraA.Gβ1, BraA.Gβ2, and BraA.Gβ3), and five Gγ (BraA.Gγ1, BraA.Gγ2, BraA.Gγ3, BraA.Gγ4, and BraA.Gγ5) genes from B. rapa, with a possibility of 15 Gαβγ heterotrimer combinations. Our analysis suggested that the process of genome triplication coupled with gene-loss (gene-fractionation) phenomenon have shaped the quantitative and sequence diversity of G-protein subunit genes in the extant B. rapa genome. Detailed expression analysis using qRT-PCR assays revealed that the G-protein genes have retained ubiquitous but distinct expression profiles across plant development. The expression of multiple G-protein genes was differentially regulated during seed-maturation and germination stages, and in response to various phytohormone treatments and stress conditions. Yeast-based interaction analysis showed that G-protein subunits interacted in most of the possible combinations, with some degree of subunit-specific interaction specificity, to control the functional selectivity of G-protein heterotrimer in different cell and tissue-types or in response to different environmental conditions. Taken together, this research identifies a highly diverse G-protein signaling network known to date from B. rapa, and provides a clue about the possible complexity of G-protein signaling networks present across globally important Brassica species. PMID

  6. Heterotrimeric G protein-dependent WNT-5A signaling to ERK1/2 mediates distinct aspects of microglia proinflammatory transformation

    PubMed Central

    2012-01-01

    Background WNT-5A signaling in the central nervous system is important for morphogenesis, neurogenesis and establishment of functional connectivity; the source of WNT-5A and its importance for cellular communication in the adult brain, however, are mainly unknown. We have previously investigated the inflammatory effects of WNT/β-catenin signaling in microglia in Alzheimer's disease. WNT-5A, however, generally recruits β-catenin-independent signaling. Thus, we aim here to characterize the role of WNT-5A and downstream signaling pathways for the inflammatory transformation of the brain's macrophages, the microglia. Methods Mouse brain sections were used for immunohistochemistry. Primary isolated microglia and astrocytes were employed to characterize the WNT-induced inflammatory transformation and underlying intracellular signaling pathways by immunoblotting, quantitative mRNA analysis, proliferation and invasion assays. Further, measurements of G protein activation by [γ-35 S]GTP binding, examination of calcium fluxes and cyclic AMP production were used to define intracellular signaling pathways. Results Astrocytes in the adult mouse brain express high levels of WNT-5A, which could serve as a novel astroglia-microglia communication pathway. The WNT-5A-induced proinflammatory microglia response is characterized by increased expression of inducible nitric oxide synthase, cyclooxygenase-2, cytokines, chemokines, enhanced invasive capacity and proliferation. Mapping of intracellular transduction pathways reveals that WNT-5A activates heterotrimeric Gi/o proteins to reduce cyclic AMP levels and to activate a Gi/o protein/phospholipase C/calcium-dependent protein kinase/extracellular signal-regulated kinase 1/2 (ERK1/2) axis. We show further that WNT-5A-induced ERK1/2 signaling is responsible for distinct aspects of the proinflammatory transformation, such as matrix metalloprotease 9/13 expression, invasion and proliferation. Conclusions Thus, WNT-5A-induced and G

  7. The role of Arabidopsis G-protein subunits in MLO2 function and MAMP-triggered immunity

    PubMed Central

    Lorek, Justine; Griebel, Thomas; Jones, Alan M.; Panstruga, Ralph

    2016-01-01

    Heterotrimeric G-proteins are composed of Gα, Gβ and Gγ subunits and regulate many fundamental processes in plants. Nonetheless plants have a considerably simpler repertoire of G-protein signaling components than metazoans. In animals, ligand binding to 7 transmembrane (7TM) cell surface receptors designated GPCRs leads to G protein activation, however, activation of the plant G protein complex is constitutive, therefore the exact role of plant 7TM proteins is unclear. MLOs are the best characterized 7TM plant proteins. While genetic ablation of either MLO2 or G proteins alters resistance to pathogens, it is unknown if G proteins directly couple signaling through MLO2. Here we exploited two well-documented phenotypes of Arabidopsis mlo2 mutants, broad-spectrum powdery mildew resistance and spontaneous callose deposition, to assess the relationship of MLO2 proteins to the G protein complex. Although our data reveal modulation of antifungal defence responses by Gβ and Gγ subunits, our findings are overall inconsistent with a role of MLO2 as a canonical GPCR. We discovered that mutants defective in the Gβ subunit show delayed accumulation of a subset of defence-associated genes following exposure to the microbe-associated molecular pattern (MAMP), flg22. Moreover Gβ mutants were found to be hypersusceptible to spray-inoculation with the bacterial pathogen, Pseudomonas syringae. In sum, our data do not support a function for MLO proteins as GPCRs, but unravel a role for Gβ and Gγ subunits as modulators of basal defence against biotrophic and hemibiotrophic phytopathogens. PMID:23656333

  8. The human thyrotropin receptor: a heptahelical receptor capable of stimulating members of all four G protein families.

    PubMed Central

    Laugwitz, K L; Allgeier, A; Offermanns, S; Spicher, K; Van Sande, J; Dumont, J E; Schultz, G

    1996-01-01

    Thyrotropin is the primary hormone that, via one heptahelical receptor, regulates thyroid cell functions such as secretion, specific gene expression, and growth. In human thyroid, thyrotropin receptor activation leads to stimulation of the adenylyl cyclase and phospholipase C cascades. However, the G proteins involved in thyrotropin receptor action have been only partially defined. In membranes of human thyroid gland, we immunologically identified alpha subunits of the G proteins Gs short, Gs long, Gi1, Gi2, Gi3, G(o) (Go2 and another form of Go, presumably Go1), Gq, G11, G12, and G13. Activation of the thyrotropin (TSH) receptor by bovine TSH led to increased incorporation of the photoreactive GTP analogue [alpha-32P]GTP azidoanilide into immunoprecipitated alpha subunits of all G proteins detected in thyroid membranes. This effect was receptor-dependent and not due to direct G protein stimulation because it was mimicked by TSH receptor-stimulating antibodies of patients suffering from Grave disease and was abolished by a receptor-blocking antiserum from a patient with autoimmune hypothyroidism. The TSH-induced activation of individual G proteins occurred with EC50 values of 5-50 milliunits/ml, indicating that the activated TSH receptor coupled with similar potency to different G proteins. When human thyroid slices were pretreated with pertussis toxin, the TSH receptor-mediated accumulation of cAMP increased by approximately 35% with TSH at 1 milliunits/ml, indicating that the TSH receptor coupled to Gs and G(i). Taken together, these findings show that, at least in human thyroid membranes, in which the protein is expressed at its physiological levels, the TSH receptor resembles a naturally occurring example of a general G protein-activating receptor. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:8552586

  9. Antigenic and immunogenic analysis of group A and group B respiratory syncytial virus G proteins expressed from recombinant baculoviruses.

    PubMed

    Sullender, W M; Britt, W J

    1996-04-01

    The attachment glycoprotein G plays a major role in the antigenic variability of respiratory syncytial (RS) virus. We have expressed from recombinant baculoviruses antigenic group A and group B RS virus G proteins (designated bacAG for the group A and bacBG for the group B virus G protein). The insect cell-produced G proteins migrated more rapidly in SDS-PAGE as compared to HEp-2 cell derived G proteins owing to glycosylation differences. Antigenicity was tested by immunofluorescence; five or five group cross-reactive, five or six group A-specific, and six of six group B-specific MAbs reacted appropriately with bacAG and/or bacBG. In addition, bacAG and bacBG reacted with human polyclonal antibodies to RS virus. Cotton rats were immunized with bacAG, bacBG or a control lysate and challenged intranasally with a group A RS virus. The bacAG-immunized group had a statistically significant reduction in viral replication in the lungs (lung titres as mean log10 p.f.u./g +/- SD, bacAG = 3.1 +/- 1.2; control = 4.8 +/- 0.6, P = 0.013). The bacBG-immunized group showed less reduction in viral titres (bacBG lung titres = 4.1 +/- 0.6, P = 0.13 for bacBG compared to control). Thus, as expected, homologous protein (bacAG) immunization provided more protection against viral replication than immunization with the heterologous protein (bacBG). The G protein of RS virus expressed in insect cells had antigenic and immunogenic features which were similar to that of the G protein expressed in mammalian cells. The baculovirus-expressed G proteins should be useful for the study of immune responses to RS viruses.

  10. Mu and Delta opioid receptors activate the same G proteins in human neuroblastoma SH-SY5Y cells

    PubMed Central

    Alt, A; Clark, M J; Woods, J H; Traynor, J R

    2002-01-01

    There is evidence for interactions between mu and delta opioid systems both in vitro and in vivo. This work examines the hypothesis that interaction between these two receptors can occur intracellularly at the level of G protein in human neuroblastoma SH-SY5Y cells.The [35S]GTPγS binding assay was used to measure G protein activation following agonist occupation of opioid receptors. The agonists DAMGO (EC50, 45 nM) and SNC80 (EC50, 32 nM) were found to be completely selective for stimulation of [35S]-GTPγS binding through mu and delta opioid receptors respectively. Maximal stimulation of [35S]-GTPγS binding produced by SNC80 was 57% of that seen with DAMGO. When combined with a maximally effective concentration of DAMGO, SNC80 caused no additional [35S]-GTPγS binding. This effect was also seen when measured at the level of adenylyl cyclase.Receptor activation increased the dissociation of pre-bound [35S]-GTPγS. In addition, the delta agonist SNC80 promoted the dissociation of [35S]-GTPγS from G proteins initially labelled using the mu agonist DAMGO. Conversely, DAMGO promoted the dissociation of [35S]-GTPγS from G proteins initially labelled using SNC80.Tolerance to DAMGO and SNC80 in membranes from cells exposed to agonist for 18 h was homologous and there was no evidence for alteration in G protein activity.The findings support the hypothesis that mu- and delta-opioid receptors share a common G protein pool, possibly through a close organization of the two receptors and G protein at the plasma membrane. PMID:11786497

  11. Enhanced IPC by activation of pertussis toxin-sensitive and -insensitive G protein-coupled purinoceptors.

    PubMed

    Ninomiya, Hideki; Otani, Hajime; Lu, Kejie; Uchiyama, Takamichi; Kido, Masakuni; Imamura, Hiroji

    2002-05-01

    Extracellular ATP plays an important role in ischemic preconditioning (IPC) through the activation of P(2y) purinoceptors. This study examined whether ATP-stimulated P(2y) purinoceptors are coupled to pertussis toxin (PTX)-insensitive G protein and whether activation of this pathway enhances myocardial protection afforded by IPC. The rat was treated with PTX for 48 h, and the heart was then isolated and buffer perfused. The heart underwent IPC by three cycles of 5-min ischemia and 5-min reperfusion before 25 min of global ischemia. Isovolumic left ventricular function was measured, and functional recovery at 30 min after reperfusion was taken as an end point of myocardial protection. PTX pretreatment partially inhibited functional protection by IPC. Treatment with 100 microM 8-(p-sulfophenyl) theophylline (SPT) during IPC had no further effect on PTX-induced inhibition of functional protection by IPC, whereas suramin (300 microM) or reactive blue (RB) (10 microM) completely abolished myocardial protection in the preconditioned heart pretreated with PTX. Supplementation with adenosine (30 microM), ATP (30 microM), or UTP (50 microM) significantly enhanced IPC-induced functional protection, although preconditioning with these nucleotides without IPC had no protective effect. Adenosine-enhanced IPC was inhibited by pretreatment with PTX and SPT but not by suramin or RB, whereas ATP-enhanced IPC was inhibited by suramin or RB in combination with PTX pretreatment. On the other hand, UTP-enhanced IPC was not affected by PTX pretreatment but was inhibited by suramin or RB. Adenosine supplemented IPC without PTX pretreatment and ATP supplemented IPC with PTX pretreatment were not affected by nitric oxide synthase inhibitor N(omega)-nitro-L-arginine methyl ester (100 microM). Although the protein kinase C inhibitor Ro318425 (0.3 microM) or tyrosine kinase inhibitor genistein (50 microM) had no significant effect on the functional protection afforded by adenosine

  12. PcG Proteins, DNA Methylation, and Gene Repression by Chromatin Looping

    PubMed Central

    Tiwari, Vijay K; McGarvey, Kelly M; Licchesi, Julien D.F; Ohm, Joyce E; Herman, James G; Schübeler, Dirk; Baylin, Stephen B

    2008-01-01

    Many DNA hypermethylated and epigenetically silenced genes in adult cancers are Polycomb group (PcG) marked in embryonic stem (ES) cells. We show that a large region upstream (∼30 kb) of and extending ∼60 kb around one such gene, GATA-4, is organized—in Tera-2 undifferentiated embryonic carcinoma (EC) cells—in a topologically complex multi-loop conformation that is formed by multiple internal long-range contact regions near areas enriched for EZH2, other PcG proteins, and the signature PcG histone mark, H3K27me3. Small interfering RNA (siRNA)–mediated depletion of EZH2 in undifferentiated Tera-2 cells leads to a significant reduction in the frequency of long-range associations at the GATA-4 locus, seemingly dependent on affecting the H3K27me3 enrichments around those chromatin regions, accompanied by a modest increase in GATA-4 transcription. The chromatin loops completely dissolve, accompanied by loss of PcG proteins and H3K27me3 marks, when Tera-2 cells receive differentiation signals which induce a ∼60-fold increase in GATA-4 expression. In colon cancer cells, however, the frequency of the long-range interactions are increased in a setting where GATA-4 has no basal transcription and the loops encompass multiple, abnormally DNA hypermethylated CpG islands, and the methyl-cytosine binding protein MBD2 is localized to these CpG islands, including ones near the gene promoter. Removing DNA methylation through genetic disruption of DNA methyltransferases (DKO cells) leads to loss of MBD2 occupancy and to a decrease in the frequency of long-range contacts, such that these now more resemble those in undifferentiated Tera-2 cells. Our findings reveal unexpected similarities in higher order chromatin conformation between stem/precursor cells and adult cancers. We also provide novel insight that PcG-occupied and H3K27me3-enriched regions can form chromatin loops and physically interact in cis around a single gene in mammalian cells. The loops associate with a

  13. G protein activation by endomorphins in the mouse periaqueductal gray matter.

    PubMed

    Narita, M; Mizoguchi, H; Narita, M; Dun, N J; Hwang, B H; Endoh, T; Suzuki, T; Nagase, H; Suzuki, T; Tseng, L F

    2000-01-01

    The midbrain periaqueductal gray matter (PAG) is an important brain region for the coordination of mu-opioid-induced pharmacological actions. The present study was designed to determine whether newly isolated mu-opioid peptide endomorphins can activate G proteins through mu-opioid receptors in the PAG by monitoring the binding to membranes of the non-hydrolyzable analog of GTP, guanosine-5'-O-(3-[(35)S]thio)triphosphate ([(35)S]GTPgammaS). An autoradiographic [(35)S]GTPgammaS binding study showed that both endomorphin-1 and -2 produced similar anatomical distributions of activated G proteins in the mouse midbrain region. In the mouse PAG, endomorphin-1 and -2 at concentrations from 0.001 to 10 microM increased [(35)S]GTPgammaS binding in a concentration-dependent manner and reached a maximal stimulation of 74.6+/-3.8 and 72.3+/-4.0%, respectively, at 10 microM. In contrast, the synthetic selective mu-opioid receptor agonist [D-Ala(2),NHPhe(4), Gly-ol]enkephalin (DAMGO) had a much greater efficacy and produced a 112.6+/-5.1% increase of the maximal stimulation. The receptor specificity of endomorphin-stimulated [(35)S]GTPgammaS binding was verified by coincubating membranes with endomorphins in the presence of specific mu-, delta- or kappa-opioid receptor antagonists. Coincubation with selective mu-opioid receptor antagonists beta-funaltrexamine or D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Phe-Thr-NH(2) (CTOP) blocked both endomorphin-1 and-2-stimulated [(35)S]GTPgammaS binding. In contrast, neither delta- nor kappa-opioid receptor antagonist had any effect on the [(35)S]GTPgammaS binding stimulated by either endomorphin-1 or -2. These findings indicate that both endomorphin-1 and -2 increase [(35)S]GTPgammaS binding by selectively stimulating mu-opioid receptors with intrinsic activity less than that of DAMGO and suggest that these new endogenous ligands might be partial agonists for mu-opioid receptors in the mouse PAG.

  14. Heterotrimeric G protein mediates ethylene-induced stomatal closure via hydrogen peroxide synthesis in Arabidopsis.

    PubMed

    Ge, Xiao-Min; Cai, Hong-Li; Lei, Xue; Zhou, Xue; Yue, Ming; He, Jun-Min

    2015-04-01

    Heterotrimeric G proteins function as key players in hydrogen peroxide (H2O2) production in plant cells, but whether G proteins mediate ethylene-induced H2O2 production and stomatal closure are not clear. Here, evidences are provided to show the Gα subunit GPA1 as a missing link between ethylene and H2O2 in guard cell ethylene signalling. In wild-type leaves, ethylene-triggered H2O2 synthesis and stomatal closure were dependent on activation of Gα. GPA1 mutants showed the defect of ethylene-induced H2O2 production and stomatal closure, whereas wGα and cGα overexpression lines showed faster stomatal closure and H2O2 production in response to ethylene. Ethylene-triggered H2O2 generation and stomatal closure were impaired in RAN1, ETR1, ERS1 and EIN4 mutants but not impaired in ETR2 and ERS2 mutants. Gα activator and H2O2 rescued the defect of RAN1 and EIN4 mutants or etr1-3 in ethylene-induced H2O2 production and stomatal closure, but only rescued the defect of ERS1 mutants or etr1-1 and etr1-9 in ethylene-induced H2O2 production. Stomata of CTR1 mutants showed constitutive H2O2 production and stomatal closure, but which could be abolished by Gα inhibitor. Stomata of EIN2, EIN3 and ARR2 mutants did not close in responses to ethylene, Gα activator or H2O2, but do generate H2O2 following challenge of ethylene or Gα activator. The data indicate that Gα mediates ethylene-induced stomatal closure via H2O2 production, and acts downstream of RAN1, ETR1, ERS1, EIN4 and CTR1 and upstream of EIN2, EIN3 and ARR2. The data also show that ETR1 and ERS1 mediate both ethylene and H2O2 signalling in guard cells.

  15. Complexing receptor pharmacology: modulation of family B G protein-coupled receptor function by RAMPs.

    PubMed

    Sexton, Patrick M; Morfis, Maria; Tilakaratne, Nanda; Hay, Debbie L; Udawela, Madhara; Christopoulos, George; Christopoulos, Arthur

    2006-07-01

    The most well-characterized subgroup of family B G protein-coupledreceptors (GPCRs) comprises receptors for peptide hormones, such as secretin, calcitonin (CT), glucagon, and vasoactive intestinal peptide (VIP). Recent data suggest that many of these receptors can interact with a novel family of GPCR accessory proteins termed receptor activity modifying proteins (RAMPs). RAMP interaction with receptors can lead to a variety of actions that include chaperoning of the receptor protein to the cell surface as is the case for the calcitonin receptor-like receptor (CLR) and the generation of novel receptor phenotypes. RAMP heterodimerization with the CLR and related CT receptor is required for the formation of specific CT gene-related peptide, adrenomedullin (AM) or amylin receptors. More recent work has revealed that the specific RAMP present in a heterodimer may modulate other functions such as receptor internalization and recycling and also the strength of activation of downstream signaling pathways. In this article we review our current state of knowledge of the consequence of RAMP interaction with family B GPCRs.

  16. Fluorescent Approaches for Understanding Interactions of Ligands with G Protein Coupled Receptors

    PubMed Central

    Sridharan, Rajashri; Zuber, Jeffrey; Connelly, Sara M.; Mathew, Elizabeth; Dumont, Mark E.

    2014-01-01

    G Protein Coupled Receptors (GPCRs) are responsible for a wide variety of signaling responses in diverse cell types. Despite major advances in the determination of structures of this class of receptors, the underlying mechanisms by which binding of different types of ligands specifically elicits particular signaling responses remains unclear. The use of fluorescence spectroscopy can provide important information about the process of ligand binding and ligand dependent conformational changes in receptors, especially kinetic aspects of these processes, that can be difficult to extract from x-ray structures. We present an overview of the extensive array of fluorescent ligands that have been used in studies of GPCRs and describe spectroscopic approaches for assaying binding and probing the environment of receptor-bound ligands with particular attention to examples involving yeast pheromone receptors. In addition, we discuss the use of fluorescence spectroscopy for detecting and characterizing conformational changes in receptors induced by the binding of ligands. Such studies have provided strong evidence for diversity of receptor conformations elicited by different ligands, consistent with the idea that GPCRs are not simple on and off switches. This diversity of states constitutes an underlying mechanistic basis for biased agonism, the observation that different stimuli can produce different responses from a single receptor. It is likely that continued technical advances will allow fluorescence spectroscopy to play an important role in continued probing of structural transitions in GPCRs. PMID:24055822

  17. Kisspeptin/G protein-coupled receptor-54 system as an essential gatekeeper of pubertal development

    PubMed Central

    2013-01-01

    Puberty is the end-point of a complex series of developmental events, defined by the dynamic interaction between genetic factors and environmental cues, ultimately leading to the attainment of reproductive capacity. Kisspeptins, products of the KISS1 gene, were originally identified as metastasis suppressor peptides with the ability to bind G protein-coupled receptors (GPR54). In 2003, loss-of-function mutations of the GPR54 gene were found in patients with hypogonadotropic hypogonadism. This finding triggered study of the role of the kisspeptin/GPR54 system as an essential gatekeeper of control of reproduction and pubertal development. Kisspeptins are very potent elicitors of gonadotropin secretion, primarily through stimulation of gonadotropin-releasing hormone release. KISS1 also functions as an essential integrator for peripheral inputs, including gonadal steroids and nutritional signals, and for controlling GnRH and gonadotropin secretion. Whether the kisspeptin/GPR54 system is the trigger for puberty onset and/or it operates as integrator and effector of up-stream regulatory factors warrants further investigation. PMID:24904852

  18. Molecular recognition of parathyroid hormone by its G protein-coupled receptor

    SciTech Connect

    Pioszak, Augen A.; Xu, H. Eric

    2008-08-07

    Parathyroid hormone (PTH) is central to calcium homeostasis and bone maintenance in vertebrates, and as such it has been used for treating osteoporosis. It acts primarily by binding to its receptor, PTH1R, a member of the class B G protein-coupled receptor (GPCR) family that also includes receptors for glucagon, calcitonin, and other therapeutically important peptide hormones. Despite considerable interest and much research, determining the structure of the receptor-hormone complex has been hindered by difficulties in purifying the receptor and obtaining diffraction-quality crystals. Here, we present a method for expression and purification of the extracellular domain (ECD) of human PTH1R engineered as a maltose-binding protein (MBP) fusion that readily crystallizes. The 1.95-{angstrom} structure of PTH bound to the MBP-PTH1R-ECD fusion reveals that PTH docks as an amphipathic helix into a central hydrophobic groove formed by a three-layer {alpha}-{beta}-{beta}{alpha} fold of the PTH1R ECD, resembling a hot dog in a bun. Conservation in the ECD scaffold and the helical structure of peptide hormones emphasizes this hot dog model as a general mechanism of hormone recognition common to class B GPCRs. Our findings reveal critical insights into PTH actions and provide a rational template for drug design that targets this hormone signaling pathway.

  19. Regulation of bone formation and remodeling by G-protein-coupled receptor 48.

    PubMed

    Luo, Jian; Zhou, Wei; Zhou, Xin; Li, Dali; Weng, Jinsheng; Yi, Zhengfang; Cho, Sung Gook; Li, Chenghai; Yi, Tingfang; Wu, Xiushan; Li, Xiao-Ying; de Crombrugghe, Benoit; Höök, Magnus; Liu, Mingyao

    2009-08-01

    G-protein-coupled receptor (GPCR) 48 (Gpr48; Lgr4), a newly discovered member of the glycoprotein hormone receptor subfamily of GPCRs, is an orphan GPCR of unknown function. Using a knockout mouse model, we have characterized the essential roles of Gpr48 in bone formation and remodeling. Deletion of Gpr48 in mice results in a dramatic delay in osteoblast differentiation and mineralization, but not in chondrocyte proliferation and maturation, during embryonic bone formation. Postnatal bone remodeling is also significantly affected in Gpr48(-/-) mice, including the kinetic indices of bone formation rate, bone mineral density and osteoid formation, whereas the activity and number of osteoclasts are increased as assessed by tartrate-resistant acid phosphatase staining. Examination of the molecular mechanism of Gpr48 action in bone formation revealed that Gpr48 can activate the cAMP-PKA-CREB signaling pathway to regulate the expression level of Atf4 in osteoblasts. Furthermore, we show that Gpr48 significantly downregulates the expression levels of Atf4 target genes/proteins, such as osteocalcin (Ocn; Bglap2), bone sialoprotein (Bsp; Ibsp) and collagen. Together, our data demonstrate that Gpr48 regulates bone formation and remodeling through the cAMP-PKA-Atf4 signaling pathway.

  20. Prognostic value of regulator of G-protein signaling 6 in colorectal cancer.

    PubMed

    Luo, Yang; Qin, Shao-Lan; Yu, Min-Hao; Mu, Yi-Fei; Wang, Zheng-Shi; Zhong, Ming

    2015-12-01

    Reprogrammed metabolism is a hallmark of cancer cells. Regulator of G-protein signaling 6 (RGS6), which is frequently down-regulated in multiple human malignancies, has been demonstrated to play a critical function in energy metabolism, cell apoptosis and tumorigenesis. However, limited knowledge is known about the expression pattern and prognostic value of RGS6 in colorectal cancer (CRC). In this study, we first observed that RGS6 mRNA and protein is commonly downregulated in 32 paired CRC tissues compared with their normal counterparts. Furthermore, by a large scale of immunohistochemical analysis in a tissue microarray containing 310 cases of CRC specimens, we demonstrated that the protein expression of RGS6 expression is downregulated in 40.97% (127/310) samples and detected that decreasing RGS6 expression is closely correlated with enhanced tumor size, CEA level, T classification, TNM stage, and easier lymphatic and distant metastasis. Meanwhile, Kaplan-Meier survival analysis showed that CRC patients with a lower RGS6 expression have a poorer clinical outcome than those with a higher RGS6 expression. Multivariate Cox regression analysis revealed that RGS6, lymphatic metastasis and distant metastasis are the independent prognostic factors for overall survival rate of CRC patients. Taken together, our studies reveal the prognostic value of RGS6 in CRC and support that RGS6 may act as a molecular target for CRC treatment.

  1. G Protein-Coupled Receptor Kinase 2 Promotes Flaviviridae Entry and Replication

    PubMed Central

    Le Sommer, Caroline; Barrows, Nicholas J.; Bradrick, Shelton S.; Pearson, James L.; Garcia-Blanco, Mariano A.

    2012-01-01

    Flaviviruses cause a wide range of severe diseases ranging from encephalitis to hemorrhagic fever. Discovery of host factors that regulate the fate of flaviviruses in infected cells could provide insight into the molecular mechanisms of infection and therefore facilitate the development of anti-flaviviral drugs. We performed genome-scale siRNA screens to discover human host factors required for yellow fever virus (YFV) propagation. Using a 2×2 siRNA pool screening format and a duplicate of the screen, we identified a high confidence list of YFV host factors. To find commonalities between flaviviruses, these candidates were compared to host factors previously identified for West Nile virus (WNV) and dengue virus (DENV). This comparison highlighted a potential requirement for the G protein-coupled receptor kinase family, GRKs, for flaviviral infection. The YFV host candidate GRK2 (also known as ADRBK1) was validated both in siRNA-mediated knockdown HuH-7 cells and in GRK−/− mouse embryonic fibroblasts. Additionally, we showed that GRK2 was required for efficient propagation of DENV and Hepatitis C virus (HCV) indicating that GRK2 requirement is conserved throughout the Flaviviridae. Finally, we found that GRK2 participates in multiple distinct steps of the flavivirus life cycle by promoting both entry and RNA synthesis. Together, our findings identified GRK2 as a novel regulator of flavivirus infection and suggest that inhibition of GRK2 function may constitute a new approach for treatment of flavivirus associated diseases. PMID:23029581

  2. Isotopic labeling of mammalian G protein-coupled receptors (GPCRs) heterologously expressed in Caenorhabditis elegans*

    PubMed Central

    Salom, David; Cao, Pengxiu; Yuan, Yiyuan; Miyagi, Masaru; Feng, Zhaoyang; Palczewski, Krzysztof

    2015-01-01

    High-resolution structural determination and dynamic characterization of membrane proteins by nuclear magnetic resonance (NMR) require their isotopic labeling. Although a number of labeled eukaryotic membrane proteins have been successfully expressed in bacteria, they lack posttranslational modifications and usually need to be refolded from inclusion bodies. This shortcoming of bacterial expression systems is particularly detrimental for the functional expression of G protein-coupled receptors (GPCRs), the largest family of drug targets, due to their inherent instability. In this work we show that proteins expressed by a eukaryotic organism can be isotopically labeled and produced with a quality and quantity suitable for NMR characterization. Using our previously described expression system in Caenorhabditis elegans, we showed the feasibility of labeling proteins produced by these worms with 15N,13C by providing them with isotopically labeled bacteria. 2H labeling also was achieved by growing C. elegans in presence of 70% heavy water. Bovine rhodopsin, simultaneously expressed in muscular and neuronal worm tissues, was employed as the ‘test’ GPCR to demonstrate the viability of this approach. Although the worms’ cell cycle was slightly affected by the presence of heavy isotopes, the final protein yield and quality was appropriate for NMR structural characterization. PMID:25461480

  3. Use of Kaede fusions to visualize recycling of G protein-coupled receptors.

    PubMed

    Schmidt, Antje; Wiesner, Burkhard; Weisshart, Klaus; Schulz, Katharina; Furkert, Jens; Lamprecht, Björn; Rosenthal, Walter; Schülein, Ralf

    2009-01-01

    The heptahelical G protein-coupled receptors (GPCRs) are internalized following agonist treatment and either recycle rapidly to the plasma membrane or enter the lysosomal degradation pathway. Many conventional GPCR recycling assays suffer from the fact that receptors arriving from the secretory pathway may interfere with recycling receptors. In this study, we introduce a new methodology to study post-endocytotic GPCR trafficking using fusions with the recently cloned Kaede protein. In contrast to the widely used green fluorescent protein, the fluorescence of Kaede can be converted from green to red using ultraviolet irradiation. Our methodology allows to study recycling of GPCRs microscopically in real-time bypassing problems with secretory pathway receptors. Initially, receptors are internalized using an agonist. Fluorescence signals in endosomes are switched, and trafficking of the receptors to the plasma membrane can be easily visualized by monitoring their new fluorescence. Using this methodology, we show that the corticotropin-releasing factor receptor type 1 belongs to the family of recycling GPCRs. Moreover, we demonstrate by fluorescence correlation spectroscopy that Kaede does not oligomerize when fused to membrane proteins, representing an additional advantage of this technique. The Kaede technology may be a powerful tool to study membrane protein trafficking in general.

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

    PubMed Central

    Li, Shizhong; Hauser, Frank; Skadborg, Signe K.; Nielsen, Stine V.; Kirketerp-Møller, Nikolaj; Grimmelikhuijzen, Cornelis J. P.

    2016-01-01

    Most multicellular animals belong to two evolutionary lineages, the Proto– and Deuterostomia, which diverged 640–760 million years (MYR) ago. Neuropeptide signaling is abundant in animals belonging to both lineages, but it is often unclear whether there exist evolutionary relationships between the neuropeptide systems used by proto- or deuterostomes. An exception, however, are members of the gonadotropin-releasing hormone (GnRH) receptor superfamily, which occur in both evolutionary lineages, where GnRHs are the ligands in Deuterostomia and GnRH-like peptides, adipokinetic hormone (AKH), corazonin, and AKH/corazonin-related peptide (ACP) are the ligands in Protostomia. AKH is a well-studied insect neuropeptide that mobilizes lipids and carbohydrates from the insect fat body during flight. In our present paper, we show that AKH is not only widespread in insects, but also in other Ecdysozoa 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, because it traces the emergence of AKH signaling back to about 550 MYR ago and brings us closer to a more complete understanding of the evolutionary origins of the GnRH receptor superfamily. PMID:27628442

  5. G protein-coupled receptors as oncogenic signals in glioma: emerging therapeutic avenues

    PubMed Central

    Cherry, Allison E; Stella, Nephi

    2014-01-01

    Gliomas are the most common malignant intracranial tumors. Newly developed targeted therapies for these cancers aim to inhibit oncogenic signals, many of which emanate from receptor tyrosine kinases, including the epidermal growth factor receptor (EGFR) and the vascular endothelial growth factor receptor (VEGFR). Unfortunately, the first generation treatments targeting these oncogenic signals provide little survival benefit in both mouse xenograft models and human patients. The search for new treatment options has uncovered several G protein-coupled receptor (GPCR) candidates and generated a growing interest in this class of proteins as alternative therapeutic targets for the treatment of various cancers, including GBM. GPCRs constitute a large family of membrane receptors that influence oncogenic pathways through canonical and non-canonical signaling. Accordingly, evidence indicates that GPCRs display a unique ability to crosstalk with receptor tyrosine kinases, making them important molecular components controlling tumorigenesis. This review summarizes the current research on GPCR functionality in gliomas and explores the potential of modulating these receptors to treat this devastating disease. PMID:25158675

  6. G-protein coupled receptor 15 mediates angiogenesis and cytoprotective function of thrombomodulin.

    PubMed

    Pan, Bin; Wang, Xiangmin; Nishioka, Chie; Honda, Goichi; Yokoyama, Akihito; Zeng, Lingyu; Xu, Kailin; Ikezoe, Takayuki

    2017-04-06

    Thrombomodulin (TM) stimulates angiogenesis and protects vascular endothelial cells (ECs) via its fifth epidermal growth factor-like region (TME5); however, the cell surface receptor that mediates the pro-survival signaling activated by TM has remained unknown. We applied pull-down assay followed by MALDI-TOF MS and western blot analysis, and identified G-protein coupled receptor 15 (GPR15) as a binding partner of TME5. TME5 rescued growth inhibition and apoptosis caused by calcineurin inhibitor FK506 in vascular ECs isolated from wild type (WT) C57BL/6 mice. On the other hand, TME5 failed to protect ECs isolated from GPR15 knockout (GPR15 KO) mice from FK506-caused vascular injury. TME5 induced activation of extracellular signal-regulated kinase (ERK) and increased level of anti-apoptotic proteins in a GPR15 dependent manner. In addition, in vivo Matrigel plug angiogenesis assay found that TME5 stimulated angiogenesis in mice. TME5 promoted endothelial migration in vitro. Furthermore, TME5 increased production of NO in association with activated endothelial NO synthase (eNOS) in ECs. All these pro-angiogenesis functions of TME5 were abolished by knockout of GPR15. Our findings suggest that GPR15 plays an important role in mediating cytoprotective function as well as angiogenesis of TM.

  7. G Protein Coupled Receptor Kinase 3 Regulates Breast Cancer Migration, Invasion, and Metastasis

    PubMed Central

    Billard, Matthew J.; Fitzhugh, David J.; Parker, Joel S.; Brozowski, Jaime M.; McGinnis, Marcus W.; Timoshchenko, Roman G.; Serafin, D. Stephen; Lininger, Ruth; Klauber-Demore, Nancy; Sahagian, Gary; Truong, Young K.; Sassano, Maria F.; Serody, Jonathan S.; Tarrant, Teresa K.

    2016-01-01

    Triple negative breast cancer (TNBC) is a heterogeneous disease that has a poor prognosis and limited treatment options. Chemokine receptor interactions are important modulators of breast cancer metastasis; however, it is now recognized that quantitative surface expression of one important chemokine receptor, CXCR4, may not directly correlate with metastasis and that its functional activity in breast cancer may better inform tumor pathogenicity. G protein coupled receptor kinase 3 (GRK3) is a negative regulator of CXCR4 activity, and we show that GRK expression correlates with tumorigenicity, molecular subtype, and metastatic potential in human tumor microarray analysis. Using established human breast cancer cell lines and an immunocompetent in vivo mouse model, we further demonstrate that alterations in GRK3 expression levels in tumor cells directly affect migration and invasion in vitro and the establishment of distant metastasis in vivo. The effects of GRK3 modulation appear to be specific to chemokine-mediated migration behaviors without influencing tumor cell proliferation or survival. These data demonstrate that GRK3 dysregulation may play an important part in TNBC metastasis. PMID:27049755

  8. Minireview: Ubiquitination-regulated G Protein-Coupled Receptor Signaling and Trafficking

    PubMed Central

    Alonso, Verónica

    2013-01-01

    G protein-coupled receptors (GPCRs) are the largest and most diverse superfamily of membrane proteins and mediate most cellular responses to hormones and neurotransmitters. Posttranslational modifications are considered the main regulators of all GPCRs. In addition to phosphorylation, glycosylation, and palmitoylation, increasing evidence as reviewed here reveals that ubiquitination also regulates the magnitude and temporospatial aspects of GPCR signaling. Posttranslational protein modification by ubiquitin is a key molecular mechanism governing proteins degradation. Ubiquitination mediates the covalent conjugation of ubiquitin, a highly conserved polypeptide of 76 amino acids, to protein substrates. This process is catalyzed by 3 enzymes acting in tandem: an E1, ubiquitin-activating enzyme; an E2, ubiquitin-carrying enzyme; and an E3, ubiquitin ligase. Ubiquitination is counteracted by deubiquitinating enzymes that deconjugate ubiquitin-modified proteins and rescue the substrate from proteasomal degradation. Although ubiquitination is known to target many GPCRs for lysosomal or proteasomal degradation, emerging findings define novel roles for the basal status of ubiquitination and for rapid deubiquitination and transubiquitination controlling cell surface expression and cellular responsiveness of some GPCRs. In this review, we highlight the classical and novel roles of ubiquitin in the regulation of GPCR function, signaling, and trafficking. PMID:23471539

  9. G-protein-coupled receptor kinase 2 and endothelial dysfunction: molecular insights and pathophysiological mechanisms

    PubMed Central

    Taguchi, Kumiko; Matsumoto, Takayuki; Kobayashi, Tsuneo

    2015-01-01

    Smooth muscle cells (SMC) and endothelial cells are the major cell types in blood vessels. The principal function of vascular SMC in the body is to regulate blood flow and pressure through contraction and relaxation. The endothelium performs a crucial role in maintaining vascular integrity by achieving whole-organ metabolic homeostasis via the production of factors associated with vasoconstriction or vasorelaxation. In this review, we have focused on the production of nitric oxide (NO), a vasorelaxation factor. The extent of NO production represents a key marker in vascular health. A decrease in NO is capable of inducing pathological conditions associated with endothelial dysfunction, such as obesity, diabetes, cardiovascular disease, and atherosclerosis. Recent studies have strongly implicated the involvement of G-protein-coupled receptor kinase 2 (GRK2) in the progression of cardiovascular disease. Vasculature which is affected by insulin resistance and type 2 diabetes expresses high levels of GRK2, which may induce endothelial dysfunction by reducing intracellular NO. GRK2 activation also induces changes in the subcellular localization of GRK2 itself and also of β-arrestin 2, a downstream protein. In this review, we describe the pathophysiological mechanisms of insulin resistance and diabetes, focusing on the signal transduction for NO production via GRK2 and β-arrestin 2, providing novel insights into the potential field of translational investigation in the treatment of diabetic complications. PMID:26447102

  10. Citric acid cycle intermediates as ligands for orphan G-protein-coupled receptors.

    PubMed

    He, Weihai; Miao, Frederick J-P; Lin, Daniel C-H; Schwandner, Ralf T; Wang, Zhulun; Gao, Jinhai; Chen, Jin-Long; Tian, Hui; Ling, Lei

    2004-05-13

    The citric acid cycle is central to the regulation of energy homeostasis and cell metabolism. Mutations in enzymes that catalyse steps in the citric acid cycle result in human diseases with various clinical presentations. The intermediates of the citric acid cycle are present at micromolar concentration in blood and are regulated by respiration, metabolism and renal reabsorption/extrusion. Here we show that GPR91 (ref. 3), a previously orphan G-protein-coupled receptor (GPCR), functions as a receptor for the citric acid cycle intermediate succinate. We also report that GPR99 (ref. 4), a close relative of GPR91, responds to alpha-ketoglutarate, another intermediate in the citric acid cycle. Thus by acting as ligands for GPCRs, succinate and alpha-ketoglutarate are found to have unexpected signalling functions beyond their traditional roles. Furthermore, we show that succinate increases blood pressure in animals. The succinate-induced hypertensive effect involves the renin-angiotensin system and is abolished in GPR91-deficient mice. Our results indicate a possible role for GPR91 in renovascular hypertension, a disease closely linked to atherosclerosis, diabetes and renal failure.

  11. G-Protein-Coupled Receptor Kinase 2 as a Potential Modulator of the Hallmarks of Cancer.

    PubMed

    Nogués, Laura; Reglero, Clara; Rivas, Verónica; Neves, María; Penela, Petronila; Mayor, Federico

    2017-03-01

    Malignant features-such as sustained proliferation, refractoriness to growth suppressors, resistance to cell death or aberrant motility, and metastasis-can be triggered by a variety of mutations and signaling adaptations. Signaling nodes can act as cancer-associated factors by cooperating with oncogene-governed pathways or participating in compensatory transduction networks to strengthen tumor properties. G-protein-coupled receptor kinase 2 (GRK2) is arising as one of such nodes. Via its complex network of connections with other cellular proteins, GRK2 contributes to the modulation of basic cellular functions-such as cell proliferation, survival, or motility-and is involved in metabolic homeostasis, inflammation, or angiogenic processes. Moreover, altered GRK2 levels are starting to be reported in different tumoral contexts and shown to promote breast tumorigenesis or to trigger the tumoral angiogenic switch. The ability to modulate several of the hallmarks of cancer puts forward GRK2 as an oncomodifier, able to modulate carcinogenesis in a cell-type specific way.

  12. Volatile anesthetics interfere with muscarinic receptor-g protein interactions in rat heart

    SciTech Connect

    Anthony, B.L.

    1987-01-01

    The influence of halothane and enflurane (0.5-8%) on muscarinic receptor binding in rat atrium was studied using (/sup 3/H) methylscopolamine ((/sup 3/H)MS). Anesthetic-gas mixtures were blown over membrane suspensions for 20 min before and during the binding assays. Halothane and enflurane increased the affinity of cardiac muscarinic receptors for (/sup 3/H)MS by slowing the rate of dissociation. These anesthetics did not affect the affinity of the receptor for carbamylcholine, but significantly reduced the sensitivity of agonist binding to regulation by guanine nucleotides. For example, the fraction of receptors displaying high affinity agonist binding was decreased by a GTP analog from 0.64 to 0.43 in the absence, but only to 0.52 in the presence of 2% halothane. The binding of a radiolabeled agonist, (/sup 3/H)oxotremorine-M, was reduced by 50% by halothane, while its sensitivity to guanine nucleotides was reduced by at least 100 fold. The diminution of the guanine nucleotide effect may reflect a stabilization of the receptor-G proteincomplex due to either a direct action on the receptor complex or to an alteration of the physical state of the membrane. It is also possible that the ability of the G protein to bind guanine nucleotides is adversely affected by anesthetic agents.

  13. Study of G-protein-coupled receptor-protein interactions by bioluminescence resonance energy transfer.

    PubMed

    Kroeger, Karen M; Eidne, Karin A

    2004-01-01

    Complex networks of protein-protein interactions are key determinants of cellular function, including those regulated by G-protein-coupled receptors (GPCRs). Formation of either stable or transitory complexes are involved in regulating all aspects of receptor function, from ligand binding through to signal transduction, desensitization, resensitization and downregulation. Today, 50% of all recently launched drugs are targeted against GPCRs. This particular class of proteins is extremely useful as a drug target because the receptors are partly located outside the cell, simplifying bioavailability and delivery of drugs directed against them. However, being located within the cell membrane causes difficulties for the study of GPCR function and bioluminescence resonance energy transfer (BRET), a naturally occurring phenomenon, represents a newly emerging, powerful tool with which to investigate and monitor dynamic interactions involving this receptor class. BRET is a noninvasive, highly sensitive technique, performed as a simple homogeneous assay. involving the proximity-dependent transfer of energy from an energy donor to acceptor resulting in the emission of light. This technology has several advantages over alternative approaches as the detection occurs within live cells, in real time, and is not restricted to a particular cellular compartment. The use of such biophysical techniques as BRET, will not only increase our understanding of the nature of GPCR regulation and the protein complexes involved, but could also potentially lead to the development of novel therapeutics that modulate these interactions.

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

    Wang, Xiaoqiang; Zhang, Shuguang

    2011-01-01

    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.

  16. Regulator of G protein signaling 2 (RGS2) deficiency accelerates the progression of kidney fibrosis.

    PubMed

    Jang, Hee-Seong; Kim, Jee In; Noh, Mira; Rhee, Man Hee; Park, Kwon Moo

    2014-09-01

    The regulator of G protein signaling 2 (RGS2) is a potent negative regulator of Gq protein signals including the angiotensin II (AngII)/AngII receptor signal, which plays a critical role in the progression of fibrosis. However, the role of RGS2 on the progression of kidney fibrosis has not been assessed. Here, we investigated the role of RGS2 in kidney fibrosis induced by unilateral ureteral obstruction (UUO) in mice. UUO resulted in increased expression of RGS2 mRNA and protein in the kidney along with increases of AngII and its type 1 receptor (AT1R) signaling and fibrosis. Furthermore, UUO increased the levels of F4/80, Ly6G, myeloperoxidase, and CXCR4 in the kidneys. RGS2 deficiency significantly enhanced these changes in the kidney. RGS2 deletion in the bone marrow-derived cells by transplanting the bone marrow of RGS2 knock-out mice into wild type mice enhanced UUO-induced kidney fibrosis. Overexpression of RGS2 in HEK293 cells, a human embryonic kidney cell line, and RAW264.7 cells, a monocyte/macrophage line, inhibited the AngII-induced activation of ERK and increase of CXCR4 expression. These findings provide the first evidence that RGS2 negatively regulates the progression of kidney fibrosis following UUO, likely by suppressing fibrogenic and inflammatory responses through the inhibition of AngII/AT1R signaling.

  17. Molecular Mechanism of Selectivity among G Protein-Coupled Receptor Kinase 2 Inhibitors

    SciTech Connect

    Thal, David M.; Yeow, Raymond Y.; Schoenau, Christian; Huber, Jochen; Tesmer, John J.G.

    2012-07-11

    G protein-coupled receptors (GPCRs) are key regulators of cell physiology and control processes ranging from glucose homeostasis to contractility of the heart. A major mechanism for the desensitization of activated GPCRs is their phosphorylation by GPCR kinases (GRKs). Overexpression of GRK2 is strongly linked to heart failure, and GRK2 has long been considered a pharmaceutical target for the treatment of cardiovascular disease. Several lead compounds developed by Takeda Pharmaceuticals show high selectivity for GRK2 and therapeutic potential for the treatment of heart failure. To understand how these drugs achieve their selectivity, we determined crystal structures of the bovine GRK2-G{beta}{gamma} complex in the presence of two of these inhibitors. Comparison with the apoGRK2-G{beta}{gamma} structure demonstrates that the compounds bind in the kinase active site in a manner similar to that of the AGC kinase inhibitor balanol. Both balanol and the Takeda compounds induce a slight closure of the kinase domain, the degree of which correlates with the potencies of the inhibitors. Based on our crystal structures and homology modeling, we identified five amino acids surrounding the inhibitor binding site that we hypothesized could contribute to inhibitor selectivity. However, our results indicate that these residues are not major determinants of selectivity among GRK subfamilies. Rather, selectivity is achieved by the stabilization of a unique inactive conformation of the GRK2 kinase domain.

  18. Diverse roles of G-protein coupled receptors in the regulation of neurohypophyseal hormone secretion.

    PubMed

    Sladek, C D; Song, Z

    2012-04-01

    The magnocellular neurones in the supraoptic nucleus project to the neural lobe and release vasopressin and oxytocin into the peripheral circulation, where they act on the kidney to promote fluid retention or stimulate smooth muscles in the vasculature, uterus and mammary glands to support blood pressure, promote parturition or induce milk let-down, respectively. Hormone release is regulated by complex afferent pathways carrying information about plasma osmolality, blood pressure and volume, cervical stretch, and suckling. These afferent pathways utilise a broad array of neurotransmitters and peptides that activate both ligand-gated ion channels and G-protein coupled receptors (GPCRs). The ligand-gated ion channels induce rapid changes in membrane potential resulting in the generation of action potentials, initiation of exocytosis and the release of hormone into the periphery. By contrast, the GPCRs activate a host of diverse signalling cascades that modulate action potential firing and regulate other cellular functions required to support hormone release (e.g. hormone synthesis, processing, packaging and trafficking). The diversity of these actions is critical for integration of the distinct regulatory signals into a response appropriate for maintaining homeostasis. This review describes several diverse roles of GPCRs in magnocellular neurones, focusing primarily on adrenergic, purinergic and peptidergic (neurokinin and angiotensin) receptors.

  19. Pheromone action regulates G-protein alpha-subunit myristoylation in the yeast Saccharomyces cerevisiae.

    PubMed Central

    Dohlman, H G; Goldsmith, P; Spiegel, A M; Thorner, J

    1993-01-01

    Myristic acid (C14:0) is added to the N-terminal glycine residue of the alpha subunits of certain receptor-coupled guanine nucleotide-binding regulatory proteins (G proteins). The G alpha subunit (GPA1 gene product) coupled to yeast pheromone receptors exists as a pool of both myristoylated and unmyristolyated species. After treatment of MATa cells with alpha factor, the myristoylated form of Gpa1p increases dramatically, and the unmyristoylated form decreases concomitantly. This pheromone-stimulated shift depends on the function of STE2 (alpha-factor receptor), STE11 (a protein kinase in the response pathway), and NMT1 (myristoyl-CoA:protein N-myristoyltransferase) genes and uses the existing pool of fatty acids (is not blocked by cerulenin). Myristoylated Gpa1p persists long after pheromone is removed. Because myristoylation is essential for proper G alpha-G beta gamma association and receptor coupling, pheromone-dependent stimulation of Gpa1p myristoylation may be an important contributing factor in adaptation after signal transmission. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 PMID:8415763

  20. G-protein ligands inhibit in vitro reactions of vacuole inheritance

    PubMed Central

    1994-01-01

    During budding in Saccharomyces cerevisiae, maternal vacuole material is delivered into the growing daughter cell via tubular or vesicular structures. One of the late steps in vacuole inheritance is the fusion in the bud of vesicles derived from the maternal vacuole. This process has been reconstituted in vitro and requires isolated vacuoles, a physiological temperature, cytosolic factors, and ATP (Conradt, B., J. Shaw, T. Vida, S. Emr, and W. Wickner. 1992. J. Cell Biol. 119:1469- 1479). We now report a simple and reliable assay to quantify vacuole-to- vacuole fusion in vitro. This assay is based on the maturation and activation of vacuole membrane-bound pro-alkaline phosphatase by vacuolar proteinase A after vacuole-to-vacuole fusion. In vitro fusion allowed maturation of 30 to 60% of pro-alkaline phosphatase. Vacuoles prepared from a mutant defective in vacuole inheritance in vivo (vac2- 1) were inactive in this assay. Vacuole fusion in vitro required a vacuole membrane potential. Inhibition by nonhydrolyzable guanosine derivatives, mastoparans, and benzalkonium chloride suggest that GTP- hydrolyzing G proteins may play a key role in the in vitro fusion events. PMID:8027189

  1. G-protein-coupled receptors for free fatty acids: nutritional and therapeutic targets.

    PubMed

    Milligan, Graeme; Ulven, Trond; Murdoch, Hannah; Hudson, Brian D

    2014-06-01

    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 of appetite- and hunger-regulating peptides, such nutrient sensing GPCRs are attracting considerable attention due to their potential to modulate satiety, improve glucose homeostasis and supress the production of various pro-inflammatory mediators. Despite the developing interests in these nutrients sensing GPCR both as sensors of nutritional status, and targets for limiting the development of metabolic diseases, major challenges remain to exploit their potential for therapeutic purposes. Mostly, this is due to limited characterisation and validation of these receptors because of paucity of selective and high-potency/affinity pharmacological agents to define the detailed function and regulation of these receptors. However, ongoing clinical trials of agonists of free fatty acid receptor 1 suggest that this receptor and other receptors for free fatty acids may provide a successful strategy for controlling hyperglycaemia and providing novel approaches to treat diabetes. Receptors responsive to free fatty acid have been of particular interest, and some aspects of these are considered herein.

  2. Dynamic Cholesterol-Conditioned Dimerization of the G Protein Coupled Chemokine Receptor Type 4

    PubMed Central

    Kranz, Franziska

    2016-01-01

    G protein coupled receptors (GPCRs) allow for the transmission of signals across biological membranes. For a number of GPCRs, this signaling was shown to be coupled to prior dimerization of the receptor. The chemokine receptor type 4 (CXCR4) was reported before to form dimers and their functionality was shown to depend on membrane cholesterol. Here, we address the dimerization pattern of CXCR4 in pure phospholipid bilayers and in cholesterol-rich membranes. Using ensembles of molecular dynamics simulations, we show that CXCR4 dimerizes promiscuously in phospholipid membranes. Addition of cholesterol dramatically affects the dimerization pattern: cholesterol binding largely abolishes the preferred dimer motif observed for pure phospholipid bilayers formed mainly by transmembrane helices 1 and 7 (TM1/TM5-7) at the dimer interface. In turn, the symmetric TM3,4/TM3,4 interface is enabled first by intercalating cholesterol molecules. These data provide a molecular basis for the modulation of GPCR activity by its lipid environment. PMID:27812115

  3. G-protein coupled receptor BAI3 promotes myoblast fusion in vertebrates

    PubMed Central

    Hamoud, Noumeira; Tran, Viviane; Croteau, Louis-Philippe; Kania, Artur; Côté, Jean-François

    2014-01-01

    Muscle fibers form as a result of myoblast fusion, yet the cell surface receptors regulating this process are unknown in vertebrates. In Drosophila, myoblast fusion involves the activation of the Rac pathway by the guanine nucleotide exchange factor Myoblast City and its scaffolding protein ELMO, downstream of cell-surface cell-adhesion receptors. We previously showed that the mammalian ortholog of Myoblast City, DOCK1, functions in an evolutionarily conserved manner to promote myoblast fusion in mice. In search for regulators of myoblast fusion, we identified the G-protein coupled receptor brain-specific angiogenesis inhibitor (BAI3) as a cell surface protein that interacts with ELMO. In cultured cells, BAI3 or ELMO1/2 loss of function severely impaired myoblast fusion without affecting differentiation and cannot be rescued by reexpression of BAI3 mutants deficient in ELMO binding. The related BAI protein family member, BAI1, is functionally distinct from BAI3, because it cannot rescue the myoblast fusion defects caused by the loss of BAI3 function. Finally, embryonic muscle precursor expression of a BAI3 mutant unable to bind ELMO was sufficient to block myoblast fusion in vivo. Collectively, our findings provide a role for BAI3 in the relay of extracellular fusion signals to their intracellular effectors, identifying it as an essential transmembrane protein for embryonic vertebrate myoblast fusion. PMID:24567399

  4. A Practical Guide to Approaching Biased Agonism at G Protein Coupled Receptors.

    PubMed

    Gundry, Jaimee; Glenn, Rachel; Alagesan, Priya; Rajagopal, Sudarshan

    2017-01-01

    Biased agonism, the ability of a receptor to differentially activate downstream signaling pathways depending on binding of a "biased" agonist compared to a "balanced" agonist, is a well-established paradigm for G protein-coupled receptor (GPCR) signaling. Biased agonists have the promise to act as smarter drugs by specifically targeting pathogenic or therapeutic signaling pathways while avoiding others that could lead to side effects. A number of biased agonists targeting a wide array of GPCRs have been described, primarily based on their signaling in pharmacological assays. However, with the promise of biased agonists as novel therapeutics, comes the peril of not fully characterizing and understanding the activities of these compounds. Indeed, it is likely that some of the compounds that have been described as biased, may not be if quantitative approaches for bias assessment are used. Moreover, cell specific effects can result in "system bias" that cannot be accounted by current approaches for quantifying ligand bias. Other confounding includes kinetic effects which can alter apparent bias and differential propagation of biological signal that results in different levels of amplification of reporters downstream of the same effector. Moreover, the effects of biased agonists frequently cannot be predicted from their pharmacological profiles, and must be tested in the vivo physiological context. Thus, the development of biased agonists as drugs requires a detailed pharmacological characterization, involving both qualitative and quantitative approaches, and a detailed physiological characterization. With this understanding, we stand on the edge of a new era of smarter drugs that target GPCRs.

  5. G-protein coupled receptor-evoked glutamate exocytosis from astrocytes: role of prostaglandins.

    PubMed

    Cali, Corrado; Lopatar, Jan; Petrelli, Francesco; Pucci, Luca; Bezzi, Paola

    2014-01-01

    Astrocytes are highly secretory cells, participating in rapid brain communication by releasing glutamate. Recent evidences have suggested that this process is largely mediated by Ca(2+)-dependent regulated exocytosis of VGLUT-positive vesicles. Here by taking advantage of VGLUT1-pHluorin and TIRF illumination, we characterized mechanisms of glutamate exocytosis evoked by endogenous transmitters (glutamate and ATP), which are known to stimulate Ca(2+) elevations in astrocytes. At first we characterized the VGLUT1-pHluorin expressing vesicles and found that VGLUT1-positive vesicles were a specific population of small synaptic-like microvesicles containing glutamate but which do not express VGLUT2. Endogenous mediators evoked a burst of exocytosis through activation of G-protein coupled receptors. Subsequent glutamate exocytosis was reduced by about 80% upon pharmacological blockade of the prostaglandin-forming enzyme, cyclooxygenase. On the other hand, receptor stimulation was accompanied by extracellular release of prostaglandin E2 (PGE2). Interestingly, administration of exogenous PGE2 produced per se rapid, store-dependent burst exocytosis of glutamatergic vesicles in astrocytes. Finally, when PGE2-neutralizing antibody was added to cell medium, transmitter-evoked exocytosis was again significantly reduced (by about 50%). Overall these data indicate that cyclooxygenase products are responsible for a major component of glutamate exocytosis in astrocytes and that large part of such component is sustained by autocrine/paracrine action of PGE2.

  6. Targeting G protein coupled receptor-related pathways as emerging molecular therapies

    PubMed Central

    Ghanemi, Abdelaziz

    2013-01-01

    G protein coupled receptors (GPCRs) represent the most important targets in modern pharmacology because of the different functions they mediate, especially within brain and peripheral nervous system, and also because of their functional and stereochemical properties. In this paper, we illustrate, via a variety of examples, novel advances about the GPCR-related molecules that have been shown to play diverse roles in GPCR pathways and in pathophysiological phenomena. We have exemplified how those GPCRs’ pathways are, or might constitute, potential targets for different drugs either to stimulate, modify, regulate or inhibit the cellular mechanisms that are hypothesized to govern some pathologic, physiologic, biologic and cellular or molecular aspects both in vivo and in vitro. Therefore, influencing such pathways will, undoubtedly, lead to different therapeutical applications based on the related pharmacological implications. Furthermore, such new properties can be applied in different fields. In addition to offering fruitful directions for future researches, we hope the reviewed data, together with the elements found within the cited references, will inspire clinicians and researchers devoted to the studies on GPCR’s properties. PMID:25972730

  7. Amphipol-assisted in vitro folding of G protein-coupled receptors.

    PubMed

    Dahmane, Tassadite; Damian, Marjorie; Mary, Sophie; Popot, Jean-Luc; Banères, Jean-Louis

    2009-07-14

    G protein-coupled receptors (GPCRs) regulate numerous physiological functions. The primary difficulty presented by their study in vitro is to obtain them in sufficient amounts under a functional and stable form. Escherichia coli is a host of choice for producing recombinant proteins for structural studies. However, the insertion of GPCRs into its plasma membrane usually results in bacterial death. An alternative approach consists of targeting recombinant receptors to inclusion bodies, where they accumulate without affecting bacterial growth, and then folding them in vitro. This approach, however, stumbles over the very low folding yields typically achieved, whether in detergent solutions or in detergent-lipid mixtures. Here, we show that synthetic polymers known as amphipols provide a highly efficient medium for folding GPCRs. Using a generic protocol, we have folded four class A GPCRs to their functional state, as evidenced by the binding of their respective ligands. This strategy thus appears to have the potential to be generalized to a large number of GPCRs. These data are also of interest from a more fundamental point of view: they indicate that the structural information stored in the sequence of these four receptors allows them to reach their correct three-dimensional structure in an environment that bears no similarity, beyond the amphiphilic character, to lipid bilayers.

  8. Chaperoning G Protein-Coupled Receptors: From Cell Biology to Therapeutics

    PubMed Central

    Conn, P. Michael

    2014-01-01

    G protein-coupled receptors (GPCRs) are membrane proteins that traverse the plasma membrane seven times (hence, are also called 7TM receptors). The polytopic structure of GPCRs makes the folding of GPCRs difficult and complex. Indeed, many wild-type GPCRs are not folded optimally, and defects in folding are the most common cause of genetic diseases due to GPCR mutations. Both general and receptor-specific molecular chaperones aid the folding of GPCRs. Chemical chaperones have been shown to be able to correct the misfolding in mutant GPCRs, proving to be important tools for studying the structure-function relationship of GPCRs. However, their potential therapeutic value is very limited. Pharmacological chaperones (pharmacoperones) are potentially important novel therapeutics for treating genetic diseases caused by mutations in GPCR genes that resulted in misfolded mutant proteins. Pharmacoperones also increase cell surface expression of wild-type GPCRs; therefore, they could be used to treat diseases that do not harbor mutations in GPCRs. Recent studies have shown that indeed pharmacoperones work in both experimental animals and patients. High-throughput assays have been developed to identify new pharmacoperones that could be used as therapeutics for a number of endocrine and other genetic diseases. PMID:24661201

  9. Recurrent activating mutations of G-protein-coupled receptor CYSLTR2 in uveal melanoma

    PubMed Central

    Moore, Amanda R; Ceraudo, Emilie; Sher, Jessica J; Guan, Youxin; Shoushtari, Alexander N; Chang, Matthew T; Zhang, Jenny Q; Walczak, Edward G; Kazmi, Manija A; Taylor, Barry S; Huber, Thomas; Chi, Ping; Sakmar, Thomas P; Chen, Yu

    2016-01-01

    Uveal melanomas are molecularly distinct from cutaneous melanomas and lack mutations in BRAF, NRAS, KIT, and NF1. Instead, they are characterized by activating mutations in GNAQ and GNA11, two highly homologous α subunits of Gαq/11 heterotrimeric G proteins, and in PLCB4 (phospholipase C β4), the downstream effector of Gαq signaling 1–3. We analyzed genomics data from 136 uveal melanoma samples and found a recurrent mutation in CYSLTR2 (cysteinyl leukotriene receptor 2) encoding a p.Leu129Gln substitution in 4 of 9 samples that lacked mutations in GNAQ, GNA11, and PLCB4 but in 0 of 127 samples that harbored mutations in these genes. The Leu129Gln CysLT2R mutant protein constitutively activates endogenous Gαq and is unresponsive to stimulation by leukotriene. Expression of Leu129Gln CysLT2R in melanocytes enforces expression of a melanocyte-lineage signature, drives phorbol ester–independent growth in vitro, and promotes tumorigenesis in vivo. Our findings implicate CYSLTR2 as a uveal melanoma oncogene and highlight the critical role of Gαq signaling in uveal melanoma pathogenesis. PMID:27089179

  10. Opioid desensitization: interactions with G-protein-coupled receptors in the locus coeruleus.

    PubMed

    Fiorillo, C D; Williams, J T

    1996-02-15

    In rat locus coeruleus (LC) neurons, alpha 2 adrenoceptors, mu-opioid and somatostatin receptors all activate the same potassium conductance. Chronic treatment with morphine causes a loss of sensitivity that is specific to the mu-opioid response, with no change in the alpha 2 adrenoceptor-mediated response. Acute desensitization induced by opioid, somatostatin, and alpha 2-adrenoceptor agonists was studied in brain slices of rat LC using intracellular recording. A supramaximal concentration of the opioid agonist Met5-enkephalin induced a profound homologous desensitization but little heterologous desensitization to an alpha 2-adrenoceptor agonist (UK 14304) or somatostatin. All desensitized currents showed partial recovery. A supramaximal concentration of UK14304 caused a relatively small amount of desensitization. Although little interaction was observed among inhibitory G-protein-coupled receptors, activation of an excitatory receptor had marked effects on inhibitory responses. Muscarinic agonists, which produce an inward current in LC neurons, reduced the magnitude of agonist-induced outward currents and increased both the rate and amount of opioid desensitization. Muscarinic activation did not alter desensitization of alpha 2-adrenoceptor responses. Acute desensitization shares several characteristics with the tolerance induced by chronic morphine treatment of animals.

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

    SciTech Connect

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

    2009-01-15

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

  12. CARMA3 deficiency abrogates G protein-coupled receptor-induced NF-κB activation

    PubMed Central

    Grabiner, Brian C.; Blonska, Marzenna; Lin, Pei-Chun; You, Yun; Wang, Donghai; Sun, Jiyuan; Darnay, Bryant G.; Dong, Chen; Lin, Xin

    2007-01-01

    G protein-coupled receptors (GPCRs) play pivotal roles in regulating various cellular functions. Although many GPCRs induce NF-κB activation, the molecular mechanism of GPCR-induced NF-κB activation remains largely unknown. CARMA3 (CARD and MAGUK domain-containing protein 3) is a scaffold molecule with unknown biological functions. By generating CARMA3 knockout mice using the gene targeting approach, here we show CARMA3 is required for GPCR-induced NF-κB activation. Mechanistically, we found that CARMA3 deficiency impairs GPCR-induced IκB kinase (IKK) activation, although it does not affect GPCR-induced IKKα/β phosphorylation, indicating that inducible phosphorylation of IKKα/β alone is not sufficient to induce its kinase activity. We also found that CARMA3 is physically associated with NEMO/IKKγ, and induces polyubiquitination of an unknown protein(s) that associates with NEMO, likely by linking NEMO to TRAF6. Consistently, we found TRAF6 deficiency also abrogates GPCR-induced NF-κB activation. Together, our results provide the genetic evidence that CARMA3 is required for GPCR-induced NF-κB activation. PMID:17438001

  13. Immunohistochemical analysis for G protein in the olfactory organs of soft-shelled turtle, Pelodiscus sinensis

    PubMed Central

    NAKAMUTA, Shoko; YOKOSUKA, Makoto; TANIGUCHI, Kazumi; YAMAMOTO, Yoshio; NAKAMUTA, Nobuaki

    2015-01-01

    In turtles, the epithelia lining the upper and lower chambers of the nasal cavity project axons to the ventral and dorsal parts of the olfactory bulbs, respectively. In a semi-aquatic soft-shelled turtle, Pelodiscus sinensis, more than 1,000 odorant receptor genes have been found, but it is not known where they are expressed. In this study, we aimed to clarify the distribution of cells expressing these genes in the olfactory organs of soft-shelled turtles. Immunoreactions for the Gαolf, the α subunit of G protein coupled to the odorant receptors, were detected on the surface of epithelia lining both the upper and lower chambers of the nasal cavity. The receptor cells in the epithelium of both chambers possessed cilia on the tip of their dendrites, whereas microvillous, non-ciliated, receptor cells were not found. These data suggest that the odorant receptor genes are expressed by the ciliated receptor cells in the upper and lower chamber epithelia. Precise location of the vomeronasal epithelium is not known at present. PMID:26440778

  14. Orphan G-protein-coupled receptors: the next generation of drug targets?

    PubMed Central

    Wilson, Shelagh; Bergsma, Derk J; Chambers, Jon K; Muir, Alison I; Fantom, Kenneth G M; Ellis, Catherine; Murdock, Paul R; Herrity, Nicole C; Stadel, Jeffrey M

    1998-01-01

    The pharmaceutical industry has readily embraced genomics to provide it with new targets for drug discovery. Large scale DNA sequencing has allowed the identification of a plethora of DNA sequences distantly related to known G protein-coupled receptors (GPCRs), a superfamily of receptors that have a proven history of being excellent therapeutic targets. In most cases the extent of sequence homology is insufficient to assign these `orphan' receptors to a particular receptor subfamily. Consequently, reverse molecular pharmacological and functional genomic strategies are being employed to identify the activating ligands of the cloned receptors. Briefly, the reverse molecular pharmacological methodology includes cloning and expression of orphan GPCRs in mammalian cells and screening these cells for a functional response to cognate or surrogate agonists present in biological extract preparations, peptide libraries, and complex compound collections. The functional genomics approach involves the use of `humanized yeast cells, where the yeast GPCR transduction system is engineered to permit functional expression and coupling of human GPCRs to the endogenous signalling machinery. Both systems provide an excellent platform for identifying novel receptor ligands. Once activating ligands are identified they can be used as pharmacological tools to explore receptor function and relationship to disease. PMID:9884064

  15. G-protein-coupled receptor controls steroid hormone signaling in cell membrane

    PubMed Central

    Wang, Di; Zhao, Wen-Li; Cai, Mei-Juan; Wang, Jin-Xing; Zhao, Xiao-Fan

    2015-01-01

    G-protein-coupled receptors (GPCRs) are involved in animal steroid hormone signaling, but their mechanism is unclear. In this research, we report that a GPCR called ErGPCR-2 controls steroid hormone 20-hydroxyecdysone (20E) signaling in the cell membrane of the lepidopteran insect Helicoverpa armigera. ErGPCR-2 was highly expressed during molting and metamorphosis. 20E, via ErGPCR-2, regulated rapid intracellular calcium increase, protein phosphorylation, gene transcription, and insect metamorphosis. ErGPCR-2 was located in the cell surface and was internalized by 20E induction. GPCR kinase 2 participated in 20E-induced ErGPCR-2 phosphorylation and internalization. The internalized ErGPCR-2 was degraded by proteases to desensitize 20E signaling. ErGPCR-2 knockdown suppressed the entrance of 20E analog [3H] ponasterone A ([3H]Pon A) into the cells. ErGPCR-2 overexpression or blocking of ErGPCR-2 internalization increased the entrance of [3H]Pon A into the cells. However, ErGPCR-2 did not bind to [3H]Pon A. Results suggest that ErGPCR-2 transmits steroid hormone 20E signaling and controls 20E entrance into cells in the cell membrane. PMID:25728569

  16. Select Neuropeptides and their G-Protein Coupled Receptors in Caenorhabditis Elegans and Drosophila Melanogaster

    PubMed Central

    Bendena, William G.; Campbell, Jason; Zara, Lian; Tobe, Stephen S.; Chin-Sang, Ian D.

    2012-01-01

    The G-protein coupled receptor (GPCR) family is comprised of seven transmembrane domain proteins and play important roles in nerve transmission, locomotion, proliferation and development, sensory perception, metabolism, and neuromodulation. GPCR research has been targeted by drug developers as a consequence of the wide variety of critical physiological functions regulated by this protein family. Neuropeptide GPCRs are the least characterized of the GPCR family as genetic systems to characterize their functions have lagged behind GPCR gene discovery. Drosophila melanogaster and Caenorhabditis elegans are genetic model organisms that have proved useful in characterizing neuropeptide GPCRs. The strength of a genetic approach leads to an appreciation of the behavioral plasticity that can result from subtle alterations in GPCRs or regulatory proteins in the pathways that GPCRs control. Many of these invertebrate neuropeptides, GPCRs, and signaling pathway components serve as models for mammalian counterparts as they have conserved sequences and function. This review provides an overview of the methods to match neuropeptides to their cognate receptor and a state of the art account of neuropeptide GPCRs that have been characterized in D. melanogaster and C. elegans and the behaviors that have been uncovered through genetic manipulation. PMID:22908006

  17. Cross-Genome Clustering of Human and C. elegans G-Protein Coupled Receptors

    PubMed Central

    Nagarathnam, Balasubramanian; Kalaimathy, Singaravelu; Balakrishnan, Veluchamy; Sowdhamini, Ramanathan

    2012-01-01

    G-protein coupled receptors (GPCRs) are one of the largest groups of membrane proteins and are popular drug targets. The work reported here attempts to perform cross-genome phylogeny on GPCRs from two widely different taxa, human versus C. elegans genomes and to address the issues on evolutionary plasticity, to identify functionally related genes, orthologous relationship, and ligand binding properties through effective bioinformatic approaches. Through RPS blast around 1106 nematode GPCRs were given chance to associate with previously established 8 types of human GPCR profiles at varying E-value thresholds and resulted 32 clusters were illustrating co-clustering and class-specific retainsionship. In the significant thresholds, 81% of the C. elegans GPCRs were associated with 32 clusters and 27 C. elegans GPCRs (2%) inferred for orthology. 177 hypothetical proteins were observed in cluster association and could be reliably associated with one of 32 clusters. Several nematode-specific GPCR clades were observed suggesting lineage-specific functional recruitment in response to environment. PMID:22807621

  18. Structural modeling of G-protein coupled receptors: An overview on automatic web-servers.

    PubMed

    Busato, Mirko; Giorgetti, Alejandro

    2016-08-01

    Despite the significant efforts and discoveries during the last few years in G protein-coupled receptor (GPCR) expression and crystallization, the receptors with known structures to date are limited only to a small fraction of human GPCRs. The lack of experimental three-dimensional structures of the receptors represents a strong limitation that hampers a deep understanding of their function. Computational techniques are thus a valid alternative strategy to model three-dimensional structures. Indeed, recent advances in the field, together with extraordinary developments in crystallography, in particular due to its ability to capture GPCRs in different activation states, have led to encouraging results in the generation of accurate models. This, prompted the community of modelers to render their methods publicly available through dedicated databases and web-servers. Here, we present an extensive overview on these services, focusing on their advantages, drawbacks and their role in successful applications. Future challenges in the field of GPCR modeling, such as the predictions of long loop regions and the modeling of receptor activation states are presented as well.

  19. Class II G Protein-Coupled Receptors and Their Ligands in Neuronal Function and Protection

    PubMed Central

    Martin, Bronwen; de Maturana, Rakel Lopez; Brenneman, Randall; Walent, Tom; Mattson, Mark P.; Maudsley, Stuart

    2008-01-01

    G protein-coupled receptors (GPCRs) play pivotal roles in regulating the function and plasticity of neuronal circuits in the nervous system. Among the myriad of GPCRs expressed in neural cells, class II GPCRs which couples predominantly to the Gs–adenylate cyclase–cAMP signaling pathway, have recently received considerable attention for their involvement in regulating neuronal survival. Neuropeptides that activate class II GPCRs include secretin, glucagon-like peptides (GLP-1 and GLP-2), growth hormone-releasing hormone (GHRH), pituitary adenylate cyclase activating peptide (PACAP), corticotropin-releasing hormone (CRH), vasoactive intestinal peptide (VIP), parathyroid hormone (PTH), and calcitonin-related peptides. Studies of patients and animal and cell culture models, have revealed possible roles for class II GPCRs signaling in the pathogenesis of several prominent neurodegenerative conditions including stroke, Alzheimer's, Parkinson's, and Huntington's diseases. Many of the peptides that activate class II GPCRs promote neuron survival by increasing the resistance of the cells to oxidative, metabolic, and excitotoxic injury. A better understanding of the cellular and molecular mechanisms by which class II GPCRs signaling modulates neuronal survival and plasticity will likely lead to novel therapeutic interventions for neurodegenerative disorders. PMID:16052036

  20. Assembly and Intracellular Targeting of the βγ Subunits of Heterotrimeric G Proteins

    PubMed Central

    Rehm, Armin; Ploegh, Hidde L.

    1997-01-01

    The assembly in living cells of heterotrimeric guanine nucleotide binding proteins from their constituent α, β, and γ subunits is a complex process, compounded by the multiplicity of the genes that encode them, and the diversity of receptors and effectors with which they interact. Monoclonal anti-β antibodies (ARC5 and ARC9), raised against immunoaffinity purified βγ complexes, recognize β subunits when not associated with γ and can thus be used to monitor assembly of βγ complexes. Complex formation starts immediately after synthesis and is complete within 30 min. Assembly occurs predominantly in the cytosol, and association of βγ complexes with the plasma membrane fraction starts between 15–30 min of chase. Three pools of β subunits can be distinguished based on their association with γ subunits, their localization, and their detergent solubility. Association of β and α subunits with detergent-insoluble domains occurs within 1 min of chase, and increases to reach a plateau of near complete detergent resistance within 30 min of chase. Brefeldin A treatment does not interfere with delivery of βγ subunits to detergent-insoluble domains, suggesting that assembly of G protein subunits with their receptors occurs distally from the BFA-imposed block of intracellular membrane trafficking and may occur directly at the plasma membrane. PMID:9128244

  1. Interaction of a novel fluorescent GTP analogue with the small G-protein K-Ras.

    PubMed

    Iwata, Seigo; Masuhara, Kaori; Umeki, Nobuhisa; Sako, Yasushi; Maruta, Shinsaku

    2016-01-01

    A novel fluorescent guanosine 5'-triphosphate (GTP) analogue, 2'(3')-O-{6-(N-(7-nitrobenz-2-oxa-l,3-diazol-4-yl)amino) hexanoic}-GTP (NBD-GTP), was synthesized and utilized to monitor the effect of mutations in the functional region of mouse K-Ras. The effects of the G12S, A59T and G12S/A59T mutations on GTPase activity, nucleotide exchange rates were compared with normal Ras. Mutation at A59T resulted in reduction of the GTPase activity by 0.6-fold and enhancement of the nucleotide exchange rate by 2-fold compared with normal Ras. On the other hand, mutation at G12S only slightly affected the nucleotide exchange rate and did not affect the GTPase activity. We also used NBD-GTP to study the effect of these mutations on the interaction between Ras and SOS1, a guanine nucleotide exchange factor. The mutation at A59T abolished the interaction with SOS1. The results suggest that the fluorescent GTP analogue, NBD-GTP, is applicable to the kinetic studies for small G-proteins.

  2. Ligands Raise the Constraint That Limits Constitutive Activation in G Protein-coupled Opioid Receptors*

    PubMed Central

    Vezzi, Vanessa; Onaran, H. Ongun; Molinari, Paola; Guerrini, Remo; Balboni, Gianfranco; Calò, Girolamo; Costa, Tommaso

    2013-01-01

    Using a cell-free bioluminescence resonance energy transfer strategy we compared the levels of spontaneous and ligand-induced receptor-G protein coupling in δ (DOP) and μ (MOP) opioid receptors. In this assay GDP can suppress spontaneous coupling, thus allowing its quantification. The level of constitutive activity was 4–5 times greater at the DOP than at the MOP receptor. A series of opioid analogues with a common peptidomimetic scaffold displayed remarkable inversions of efficacy in the two receptors. Agonists that enhanced coupling above the low intrinsic level of the MOP receptor were inverse agonists in reducing the greater level of constitutive coupling of the DOP receptor. Yet the intrinsic activities of such ligands are identical when scaled over the GDP base line of both receptors. This pattern is in conflict with the predictions of the ternary complex model and the “two state” extensions. According to this theory, the order of spontaneous and ligand-induced coupling cannot be reversed if a shift of the equilibrium between active and inactive forms raises constitutive activation in one receptor type. We propose that constitutive activation results from a lessened intrinsic barrier that restrains spontaneous coupling. Any ligand, regardless of its efficacy, must enhance this constraint to stabilize the ligand-bound complexed form. PMID:23836900

  3. Developmental Expression of Orphan G Protein-Coupled Receptor 50 in the Mouse Brain

    PubMed Central

    2012-01-01

    Mental disorders have a complex etiology resulting from interactions between multiple genetic risk factors and stressful life events. Orphan G protein-coupled receptor 50 (GPR50) has been identified as a genetic risk factor for bipolar disorder and major depression in women, and there is additional genetic and functional evidence linking GPR50 to neurite outgrowth, lipid metabolism, and adaptive thermogenesis and torpor. However, in the absence of a ligand, a specific function has not been identified. Adult GPR50 expression has previously been reported in brain regions controlling the HPA axis, but its developmental expression is unknown. In this study, we performed extensive expression analysis of GPR50 and three protein interactors using rt-PCR and immunohistochemistry in the developing and adult mouse brain. Gpr50 is expressed at embryonic day 13 (E13), peaks at E18, and is predominantly expressed by neurons. Additionally we identified novel regions of Gpr50 expression, including brain stem nuclei involved in neurotransmitter signaling: the locus coeruleus, substantia nigra, and raphe nuclei, as well as nuclei involved in metabolic homeostasis. Gpr50 colocalizes with yeast-two-hybrid interactors Nogo-A, Abca2, and Cdh8 in the hypothalamus, amygdala, cortex, and selected brain stem nuclei at E18 and in the adult. With this study, we identify a link between GPR50 and neurotransmitter signaling and strengthen a likely role in stress response and energy homeostasis. PMID:22860215

  4. Molecular manipulation of G-protein-coupled receptors: a new avenue into drug discovery.

    PubMed

    Sautel, M; Milligan, G

    2000-09-01

    During the past 10 years or so, associated with the introduction of molecular biology techniques to G protein-coupled receptor (GPCR) research, outstanding progress has been made in understanding the mechanisms of action of these key proteins and their physiological functions. in-vivo manipulation of levels of GPCRs using transgenic and gene knock-out approaches have been particularly successful in assessing the roles of specific GPCRs in animal physiology. Drug discovery is aiming to produce highly specific compounds based on subtle definition of receptor subtypes which can best be studied using heterologous expression of wild type or mutated forms of cDNA or genes encoding these proteins. Furthermore, new therapeutic opportunities may be provided by investigation of orphan receptors, the natural ligands for which remain unidentified. Some human diseases have been shown to be associated with rare mutations of GPCRs and the possibility that widely distributed polymorphisms in GPCR genes may allow selective therapeutic strategies for population subgroups is driving the development of the science of pharmacogenetics.

  5. Structural basis for modulation of a G-protein-coupled receptor by allosteric drugs.

    PubMed

    Dror, Ron O; Green, Hillary F; Valant, Celine; Borhani, David W; Valcourt, James R; Pan, Albert C; Arlow, Daniel H; Canals, Meritxell; Lane, J Robert; Rahmani, Raphaël; Baell, Jonathan B; Sexton, Patrick M; Christopoulos, Arthur; Shaw, David E

    2013-11-14

    The design of G-protein-coupled receptor (GPCR) allosteric modulators, an active area of modern pharmaceutical research, has proved challenging because neither the binding modes nor the molecular mechanisms of such drugs are known. Here we determine binding sites, bound conformations and specific drug-receptor interactions for several allosteric modulators of the M2 muscarinic acetylcholine receptor (M2 receptor), a prototypical family A GPCR, using atomic-level simulations in which the modulators spontaneously associate with the receptor. Despite substantial structural diversity, all modulators form cation-π interactions with clusters of aromatic residues in the receptor extracellular vestibule, approximately 15 Å from the classical, 'orthosteric' ligand-binding site. We validate the observed modulator binding modes through radioligand binding experiments on receptor mutants designed, on the basis of our simulations, either to increase or to decrease modulator affinity. Simulations also revealed mechanisms that contribute to positive and negative allosteric modulation of classical ligand binding, including coupled conformational changes of the two binding sites and electrostatic interactions between ligands in these sites. These observations enabled the design of chemical modifications that substantially alter a modulator's allosteric effects. Our findings thus provide a structural basis for the rational design of allosteric modulators targeting muscarinic and possibly other GPCRs.

  6. Responsiveness of G protein-coupled odorant receptors is partially attributed to the activation mechanism

    PubMed Central

    Yu, Yiqun; de March, Claire A.; Ni, Mengjue J.; Adipietro, Kaylin A.; Golebiowski, Jérôme; Matsunami, Hiroaki; Ma, Minghong

    2015-01-01

    Mammals detect and discriminate numerous odors via a large family of G protein-coupled odorant receptors (ORs). However, little is known about the molecular and structural basis underlying OR response properties. Using site-directed mutagenesis and computational modeling, we studied ORs sharing high sequence homology but with different response properties. When tested in heterologous cells by diverse odorants, MOR256-3 responded broadly to many odorants, whereas MOR256-8 responded weakly to a few odorants. Out of 36 mutant MOR256-3 ORs, the majority altered the responses to different odorants in a similar manner and the overall response of an OR was positively correlated with its basal activity, an indication of ligand-independent receptor activation. Strikingly, a single mutation in MOR256-8 was sufficient to confer both high basal activity and broad responsiveness to this receptor. These results suggest that broad responsiveness of an OR is at least partially attributed to its activation likelihood. PMID:26627247

  7. Structural basis for modulation of a G-protein-coupled receptor by allosteric drugs

    NASA Astrophysics Data System (ADS)

    Dror, Ron O.; Green, Hillary F.; Valant, Celine; Borhani, David W.; Valcourt, James R.; Pan, Albert C.; Arlow, Daniel H.; Canals, Meritxell; Lane, J. Robert; Rahmani, Raphaël; Baell, Jonathan B.; Sexton, Patrick M.; Christopoulos, Arthur; Shaw, David E.

    2013-11-01

    The design of G-protein-coupled receptor (GPCR) allosteric modulators, an active area of modern pharmaceutical research, has proved challenging because neither the binding modes nor the molecular mechanisms of such drugs are known. Here we determine binding sites, bound conformations and specific drug-receptor interactions for several allosteric modulators of the M2 muscarinic acetylcholine receptor (M2 receptor), a prototypical family A GPCR, using atomic-level simulations in which the modulators spontaneously associate with the receptor. Despite substantial structural diversity, all modulators form cation-π interactions with clusters of aromatic residues in the receptor extracellular vestibule, approximately 15Å from the classical, `orthosteric' ligand-binding site. We validate the observed modulator binding modes through radioligand binding experiments on receptor mutants designed, on the basis of our simulations, either to increase or to decrease modulator affinity. Simulations also revealed mechanisms that contribute to positive and negative allosteric modulation of classical ligand binding, including coupled conformational changes of the two binding sites and electrostatic interactions between ligands in these sites. These observations enabled the design of chemical modifications that substantially alter a modulator's allosteric effects. Our findings thus provide a structural basis for the rational design of allosteric modulators targeting muscarinic and possibly other GPCRs.

  8. GPCR-ModSim: A comprehensive web based solution for modeling G-protein coupled receptors

    PubMed Central

    Esguerra, Mauricio; Siretskiy, Alexey; Bello, Xabier; Sallander, Jessica; Gutiérrez-de-Terán, Hugo

    2016-01-01

    GPCR-ModSim (http://open.gpcr-modsim.org) is a centralized and easy to use service dedicated to the structural modeling of G-protein Coupled Receptors (GPCRs). 3D molecular models can be generated from amino acid sequence by homology-modeling techniques, considering different receptor conformations. GPCR-ModSim includes a membrane insertion and molecular dynamics (MD) equilibration protocol, which can be used to refine the generated model or any GPCR structure uploaded to the server, including if desired non-protein elements such as orthosteric or allosteric ligands, structural waters or ions. We herein revise the main characteristics of GPCR-ModSim and present new functionalities. The templates used for homology modeling have been updated considering the latest structural data, with separate profile structural alignments built for inactive, partially-active and active groups of templates. We have also added the possibility to perform multiple-template homology modeling in a unique and flexible way. Finally, our new MD protocol considers a series of distance restraints derived from a recently identified conserved network of helical contacts, allowing for a smoother refinement of the generated models which is particularly advised when there is low homology to the available templates. GPCR- ModSim has been tested on the GPCR Dock 2013 competition with satisfactory results. PMID:27166369

  9. Peptide modifications differentially alter G protein-coupled receptor internalization and signaling bias.

    PubMed

    Mäde, Veronika; Babilon, Stefanie; Jolly, Navjeet; Wanka, Lizzy; Bellmann-Sickert, Kathrin; Diaz Gimenez, Luis E; Mörl, Karin; Cox, Helen M; Gurevich, Vsevolod V; Beck-Sickinger, Annette G

    2014-09-15

    Although G protein-coupled receptors (GPCRs) are targeted by more clinically used drugs than any other type of protein, their ligand development is particularly challenging. Humans have four neuropeptide Y receptors: hY1R and hY5R are orexigenic, while hY2R and hY4R are anorexigenic, and represent important anti-obesity drug targets. We show for the first time that PEGylation and lipidation, chemical modifications that prolong the plasma half-lives of peptides, confer additional benefits. Both modifications enhance pancreatic polypeptide preference for hY2R/hY4R over hY1R/hY5R. Lipidation biases the ligand towards arrestin recruitment and internalization, whereas PEGylation confers the opposite bias. These effects were independent of the cell system and modified residue. We thus provide novel insights into the mode of action of peptide modifications and open innovative venues for generating peptide agonists with extended therapeutic potential.

  10. Peptide Modifications Differentially Alter G Protein-Coupled Receptor Internalization and Signaling Bias**

    PubMed Central

    Mäde, Veronika; Babilon, Stefanie; Jolly, Navjeet; Wanka, Lizzy; Bellmann-Sickert, Kathrin; Diaz Gimenez, Luis E.; Mörl, Karin; Cox, Helen M.; Gurevich, Vsevolod V.; Beck-Sickinger, Annette G.

    2016-01-01

    Although G protein-coupled receptors (GPCRs) are targeted by more clinically used drugs than any other type of protein, their ligand development is particularly challenging. Humans have four neuropeptide Y receptors: hY1R and hY5R are orexigenic, while hY2R and hY4R are anorexigenic, and represent important anti-obesity drug targets. We show for the first time that PEGylation and lipidation, chemical modifications that prolong the plasma half-lives of peptides, confer additional benefits. Both modifications enhance pancreatic polypeptide preference for hY2R/hY4R over hY1R/hY5R. Lipidation biases the ligand towards arrestin recruitment and internalization, whereas PEGylation confers the opposite bias. These effects were independent of the cell system and modified residue. We thus provide novel insights into the mode of action of peptide modifications and open innovative venues for generating peptide agonists with extended therapeutic potential. PMID:25065900

  11. G-protein-coupled inward rectifier potassium channels involved in corticostriatal presynaptic modulation.

    PubMed

    Meneses, David; Mateos, Verónica; Islas, Gustavo; Barral, Jaime

    2015-09-01

    Presynaptic modulation has been associated mainly with calcium channels but recent data suggests that inward rectifier potassium channels (K(IR)) also play a role. In this work we set to characterize the role of presynaptic K(IR) channels in corticostriatal synaptic transmission. We elicited synaptic potentials in striatum by stimulating cortical areas and then determined the synaptic responses of corticostriatal synapsis by using paired pulse ratio (PPR) in the presence and absence of several potassium channel blockers. Unspecific potassium channels blockers Ba(2+) and Cs(+) reduced the PPR, suggesting that these channels are presynaptically located. Further pharmacological characterization showed that application of tertiapin-Q, a specific K(IR)3 channel family blocker, also induced a reduction of PPR, suggesting that K(IR)3 channels are present at corticostriatal terminals. In contrast, exposure to Lq2, a specific K(IR)1.1 inward rectifier potassium channel, did not induce any change in PPR suggesting the absence of these channels in the presynaptic corticostriatal terminals. Our results indicate that K(IR)3 channels are functionally expressed at the corticostriatal synapses, since blockage of these channels result in PPR decrease. Our results also help to explain how synaptic activity may become sensitive to extracellular signals mediated by G-protein coupled receptors. A vast repertoire of receptors may influence neurotransmitter release in an indirect manner through regulation of K(IR)3 channels.

  12. An algebra of dimerization and its implications for G-protein coupled receptor signaling.

    PubMed

    Woolf, Peter J; Linderman, Jennifer J

    2004-07-21

    Many species of receptors form dimers, but how can we use this information to make predictions about signal transduction? This problem is particularly difficult when receptors dimerize with many different species, leading to a combinatoric increase in the possible number of dimer pairs. As an example system, we focus on receptors in the G-protein coupled receptor (GPCR) family. GPCRs have been shown to reversibly form dimers, but this dimerization does not directly affect signal transduction. Here we present a new theoretical framework called a dimerization algebra. This algebra provides a systematic and rational way to represent, manipulate, and in some cases simplify large and often complicated networks of dimerization interactions. To compliment this algebra, Monte Carlo simulations are used to predict dimerization's effect on receptor organization on the membrane, signal transduction, and internalization. These simulation results are directly comparable to various experimental measures such as fluorescence resonance energy transfer (FRET), and as such provide a link between the dimerization algebra and experimental data. As an example, we show how the algebra and computational results can be used to predict the effects of dimerization on the dopamine D2 and somatastatin SSTR1 receptors. When these predictions were compared to experimental findings from the literature, good agreement was found, demonstrating the utility of our approach. Applications of this work to the development of a novel class of dimerization-modulating drugs are also discussed.

  13. G-protein based ELISA as a potency test for rabies vaccines.

    PubMed

    Chabaud-Riou, Martine; Moreno, Nadège; Guinchard, Fabien; Nicolai, Marie Claire; Niogret-Siohan, Elisabeth; Sève, Nicolas; Manin, Catherine; Guinet-Morlot, Françoise; Riou, Patrice

    2017-03-01

    The NIH test is currently used to assess the potency of rabies vaccine, a key criterion for vaccine release. This test is based on mice immunization followed by intracerebral viral challenge. As part of global efforts to reduce animal experimentation and in the framework of the development of Sanofi Pasteur next generation, highly-purified vaccine, produced without any material of human or animal origin, we developed an ELISA as an alternative to the NIH test. This ELISA is based on monoclonal antibodies recognizing specifically the native form of the viral G-protein, the major antigen that induces neutralizing antibody response to rabies virus. We show here that our ELISA is able to distinguish between potent and different types of sub-potent vaccine lots. Satisfactory agreement was observed between the ELISA and the NIH test in the determination of the vaccine titer and their capacity to discern conform from non-conform batches. Our ELISA meets the criteria for a stability-indicating assay and has been successfully used to develop the new generation of rabies vaccine candidates. After an EPAA international pre-collaborative study, this ELISA was selected as the assay of choice for the EDQM collaborative study aimed at replacing the rabies vaccine NIH in vivo potency test.

  14. Expression analysis of G Protein-Coupled Receptors in mouse macrophages

    PubMed Central

    Lattin, Jane E; Schroder, Kate; Su, Andrew I; Walker, John R; Zhang, Jie; Wiltshire, Tim; Saijo, Kaoru; Glass, Christopher K; Hume, David A; Kellie, Stuart; Sweet, Matthew J

    2008-01-01

    Background Monocytes and macrophages express an extensive repertoire of G Protein-Coupled Receptors (GPCRs) that regulate inflammation and immunity. In this study we performed a systematic micro-array analysis of GPCR expression in primary mouse macrophages to identify family members that are either enriched in macrophages compared to a panel of other cell types, or are regulated by an inflammatory stimulus, the bacterial product lipopolysaccharide (LPS). Results Several members of the P2RY family had striking expression patterns in macrophages; P2ry6 mRNA was essentially expressed in a macrophage-specific fashion, whilst P2ry1 and P2ry5 mRNA levels were strongly down-regulated by LPS. Expression of several other GPCRs was either restricted to macrophages (e.g. Gpr84) or to both macrophages and neural tissues (e.g. P2ry12, Gpr85). The GPCR repertoire expressed by bone marrow-derived macrophages and thioglycollate-elicited peritoneal macrophages had some commonality, but there were also several GPCRs preferentially expressed by either cell population. Conclusion The constitutive or regulated expression in macrophages of several GPCRs identified in this study has not previously been described. Future studies on such GPCRs and their agonists are likely to provide important insights into macrophage biology, as well as novel inflammatory pathways that could be future targets for drug discovery. PMID:18442421

  15. Systematic generation of in vivo G protein-coupled receptor mutants in the rat

    PubMed Central

    van Boxtel, R; Vroling, B; Toonen, P; Nijman, I J; van Roekel, H; Verheul, M; Baakman, C; Guryev, V; Vriend, G; Cuppen, E

    2011-01-01

    G-protein-coupled receptors (GPCRs) constitute a large family of cell surface receptors that are involved in a wide range of physiological and pathological processes, and are targets for many therapeutic interventions. However, genetic models in the rat, one of the most widely used model organisms in physiological and pharmacological research, are largely lacking. Here, we applied N-ethyl-N-nitrosourea (ENU)-driven target-selected mutagenesis to generate an in vivo GPCR mutant collection in the rat. A pre-selected panel of 250 human GPCR homologs was screened for mutations in 813 rats, resulting in the identification of 131 non-synonymous mutations. From these, seven novel potential rat gene knockouts were established as well as 45 lines carrying missense mutations in various genes associated with or involved in human diseases. We provide extensive in silico modeling results of the missense mutations and show experimental data, suggesting loss-of-function phenotypes for several models, including Mc4r and Lpar1. Taken together, the approach used resulted not only in a set of novel gene knockouts, but also in allelic series of more subtle amino acid variants, similar as commonly observed in human disease. The mutants presented here may greatly benefit studies to understand specific GPCR function and support the development of novel therapeutic strategies. PMID:20531371

  16. Systematic generation of in vivo G protein-coupled receptor mutants in the rat.

    PubMed

    van Boxtel, R; Vroling, B; Toonen, P; Nijman, I J; van Roekel, H; Verheul, M; Baakman, C; Guryev, V; Vriend, G; Cuppen, E

    2011-10-01

    G-protein-coupled receptors (GPCRs) constitute a large family of cell surface receptors that are involved in a wide range of physiological and pathological processes, and are targets for many therapeutic interventions. However, genetic models in the rat, one of the most widely used model organisms in physiological and pharmacological research, are largely lacking. Here, we applied N-ethyl-N-nitrosourea (ENU)-driven target-selected mutagenesis to generate an in vivo GPCR mutant collection in the rat. A pre-selected panel of 250 human GPCR homologs was screened for mutations in 813 rats, resulting in the identification of 131 non-synonymous mutations. From these, seven novel potential rat gene knockouts were established as well as 45 lines carrying missense mutations in various genes associated with or involved in human diseases. We provide extensive in silico modeling results of the missense mutations and show experimental data, suggesting loss-of-function phenotypes for several models, including Mc4r and Lpar1. Taken together, the approach used resulted not only in a set of novel gene knockouts, but also in allelic series of more subtle amino acid variants, similar as commonly observed in human disease. The mutants presented here may greatly benefit studies to understand specific GPCR function and support the development of novel therapeutic strategies.

  17. G protein-coupled receptor 56 regulates mechanical overload-induced muscle hypertrophy.

    PubMed

    White, James P; Wrann, Christiane D; Rao, Rajesh R; Nair, Sreekumaran K; Jedrychowski, Mark P; You, Jae-Sung; Martínez-Redondo, Vicente; Gygi, Steven P; Ruas, Jorge L; Hornberger, Troy A; Wu, Zhidan; Glass, David J; Piao, Xianhua; Spiegelman, Bruce M

    2014-11-04

    Peroxisome proliferator-activated receptor gamma coactivator 1-alpha 4 (PGC-1α4) is a protein isoform derived by alternative splicing of the PGC1α mRNA and has been shown to promote muscle hypertrophy. We show here that G protein-coupled receptor 56 (GPR56) is a transcriptional target of PGC-1α4 and is induced in humans by resistance exercise. Furthermore, the anabolic effects of PGC-1α4 in cultured murine muscle cells are dependent on GPR56 signaling, because knockdown of GPR56 attenuates PGC-1α4-induced muscle hypertrophy in vitro. Forced expression of GPR56 results in myotube hypertrophy through the expression of insulin-like growth factor 1, which is dependent on Gα12/13 signaling. A murine model of overload-induced muscle hypertrophy is associated with increased expression of both GPR56 and its ligand collagen type III, whereas genetic ablation of GPR56 expression attenuates overload-induced muscle hypertrophy and associated anabolic signaling. These data illustrate a signaling pathway through GPR56 which regulates muscle hypertrophy associated with resistance/loading-type exercise.

  18. Computational approaches for ligand discovery and design in class-A G protein- coupled receptors.

    PubMed

    Rodríguez, David; Gutiérrez-de-Terán, Hugo

    2013-01-01

    Our structural understanding of the superfamily of G-protein coupled receptors, a group of targets of utmost pharmacological importance, has improved dramatically in the last few years. This was directly translated in an increase of both the number and the relevance of computer-assisted drug design efforts devoted to these receptors. The field, which had been greatly influenced by ligand-based methods, has experienced a radical transformation with a number of successful structure-based ligand design and ligand discovery studies. This revolution has been accompanied by the exponential increase of computational resources, and as a result the scenario in GPCR structural and chemical studies is now more complex and richer than ever. Virtual screens, both structure- and ligand-based, co-exist with accurate computational characterizations of the receptor conformational dynamics and of the energy landscapes of receptor-ligand interactions. We here provide an integrated and updated view of the different computational techniques applied to the ligand design of GPCRs. Particular emphasis is put on the studies that take into account the novel structural information of GPCRs, together with those that consider the enormous amount of chemical information accumulated on these receptors in the last decades. Indeed, we propose that proper combinations of the different computational techniques: ligand-based, structure-based and molecular dynamics studies, should be performed to better integrate all available information whenever possible. With this in mind, a major impact of computational technologies in the ligand design on GPCRs is expected in the forthcoming years.

  19. Antibody fragments for stabilization and crystallization of G protein-coupled receptors and their signaling complexes.

    PubMed

    Shukla, Arun K; Gupta, Charu; Srivastava, Ashish; Jaiman, Deepika

    2015-01-01

    G protein-coupled receptors (GPCRs) are one of the key players in extracellular signal recognition and their subsequent communications with cellular signaling machinery. Crystallization and high-resolution structure determination of GPCRs has been one of the major advances in the area of GPCR biology over the last 7-8 years. There have primarily been three approaches to GPCR crystallization till date. These are fusion protein strategy, thermostabilization, and antibody fragment-mediated crystallization. Of these, antibody fragment-mediated crystallization has not only provided the first breakthrough in structure determination of a non-rhodopsin GPCR but it has also assisted in obtaining structures of fully active conformations of GPCRs. Antibody fragment approach has also been crucial in obtaining structural information on GPCR signaling complexes. Here, we highlight the specific examples of GPCR crystal structures that have utilized antibody fragments for promoting crystallogenesis and structure solution. We also discuss emerging powerful technologies such as the nanobody technology and the synthetic phage display libraries in the context of GPCR crystallization and underline how these tools are likely to propel key GPCR structural studies in future.

  20. Functionalization of SU-8 photoresist surfaces with IgG proteins

    NASA Astrophysics Data System (ADS)

    Blagoi, Gabriela; Keller, Stephan; Johansson, Alicia; Boisen, Anja; Dufva, Martin

    2008-12-01

    The negative epoxy-based photoresist SU-8 has a variety of applications within microelectromechanical systems (MEMS) and lab-on-a-chip systems. Here, several methods to functionalize SU-8 surfaces with IgG proteins were investigated. Fluorescent labeled proteins and fluorescent sandwich immunoassays were employed to characterize the binding efficiency of model proteins to bare SU-8 surface, SU-8 treated with cerium ammonium nitrate (CAN) etchant and CAN treated surfaces modified by aminosilanization. The highest binding capacity of antibodies was observed on bare SU-8. This explains why bare SU-8 in a functional fluorescent sandwich immunoassay detecting C-reactive protein (CRP) gave twice as high signal as compared with the other two surfaces. Immunoassays performed on bare SU-8 and CAN treated SU-8 resulted in detection limits of CRP of 30 and 80 ng/ml respectively which is sufficient for detecting CRP in clinical samples, where concentrations of 3-10 μg/ml are normal for healthy individuals. In conclusion, bare SU-8 and etched SU-8 can be modified with antibodies by a simple adsorption procedure which simplifies building lab-on-a-chip systems in SU-8. Additionally, we report the fabrication process and use of microwells created in a SU-8 layer with the same dimensions as a standard microscope glass slide that could fit into fluorescent scanners. The SU-8 microwells minimize the reagent consumption and are straightforward to handle compared to SU-8 coated microscope slides.

  1. G-Protein α-Subunit Gsα Is Required for Craniofacial Morphogenesis

    PubMed Central

    Wei, Yanxia; Chen, Min; Weinstein, Lee S.; Hong, Yang; Zhu, Minyan; Li, Hongchang; Li, Huashun

    2016-01-01

    The heterotrimeric G protein subunit Gsα couples receptors to activate adenylyl cyclase and is required for the intracellular cAMP response and protein kinase A (PKA) activation. Gsα is ubiquitously expressed in many cell types; however, the role of Gsα in neural crest cells (NCCs) remains unclear. Here we report that NCCs-specific Gsα knockout mice die within hours after birth and exhibit dramatic craniofacial malformations, including hypoplastic maxilla and mandible, cleft palate and craniofacial skeleton defects. Histological and anatomical analysis reveal that the cleft palate in Gsα knockout mice is a secondary defect resulting from craniofacial skeleton deficiencies. In Gsα knockout mice, the morphologies of NCCs-derived cranial nerves are normal, but the development of dorsal root and sympathetic ganglia are impaired. Furthermore, loss of Gsα in NCCs does not affect cranial NCCs migration or cell proliferation, but significantly accelerate osteochondrogenic differentiation. Taken together, our study suggests that Gsα is required for neural crest cells-derived craniofacial development. PMID:26859889

  2. Thematic Minireview Series: New Directions in G Protein-coupled Receptor Pharmacology.

    PubMed

    Dohlman, Henrik G

    2015-08-07

    Over the past half-century, The Journal of Biological Chemistry has been the venue for many landmark publications on the topic of G protein-coupled receptors (GPCRs, also known as seven-transmembrane receptors). The GPCR superfamily in humans is composed of about 800 members, and is the target of about one-third of all pharmaceuticals. Most of these drugs target a very small subset of GPCRs, and do so by mimicking or competing with endogenous hormones and neurotransmitters. This thematic minireview series examines some emerging trends in GPCR drug discovery. The first article describes efforts to systematically interrogate the human "GPCR-ome," including more than 150 uncharacterized "orphan" receptors. The second article describes recent efforts to target alternative receptor binding sites with drugs that act as allosteric modulators of orthosteric ligands. The third article describes how the recent expansion of GPCR structures is providing new opportunities for computer-guided drug discovery. Collectively, these three articles provide a roadmap for the most important emerging trends in GPCR pharmacology.

  3. Preparation, crystallization and preliminary X-ray analysis of YjcG protein from Bacillus subtilis

    SciTech Connect

    Li, Dan; Chan, Chiomui; Liang, Yu-He Zheng, Xiaofeng; Li, Lanfen; Su, Xiao-Dong

    2005-05-01

    B. subtilis YjcG protein was expressed, purified and crystallized. A complete diffraction data set was collected at BSRF beamline 3W1A and processed to 2.3 Å resolution. Bacillus subtilis YjcG is a functionally uncharacterized protein with 171 residues that has no structural homologue in the Protein Data Bank. However, it shows sequence homology to bacterial and archaeal 2′–5′ RNA ligases. In order to identify its exact function via structural studies, the yjcG gene was amplified from B. subtilis genomic DNA and cloned into the expression vector pET21-DEST. The protein was expressed in a soluble form in Escherichia coli and was purified to homogeneity. Crystals suitable for X-ray analysis were obtained that diffracted to 2.3 Å and belonged to space group C2, with unit-cell parameters a = 99.66, b = 73.93, c = 61.77 Å, β = 113.56°.

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

    PubMed

    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.

  5. G-protein-coupled receptor kinase 2 and endothelial dysfunction: molecular insights and pathophysiological mechanisms.

    PubMed

    Taguchi, Kumiko; Matsumoto, Takayuki; Kobayashi, Tsuneo

    2015-01-01

    Smooth muscle cells (SMC) and endothelial cells are the major cell types in blood vessels. The principal function of vascular SMC in the body is to regulate blood flow and pressure through contraction and relaxation. The endothelium performs a crucial role in maintaining vascular integrity by achieving whole-organ metabolic homeostasis via the production of factors associated with vasoconstriction or vasorelaxation. In this review, we have focused on the production of nitric oxide (NO), a vasorelaxation factor. The extent of NO production represents a key marker in vascular health. A decrease in NO is capable of inducing pathological conditions associated with endothelial dysfunction, such as obesity, diabetes, cardiovascular disease, and atherosclerosis. Recent studies have strongly implicated the involvement of G-protein-coupled receptor kinase 2 (GRK2) in the progression of cardiovascular disease. Vasculature which is affected by insulin resistance and type 2 diabetes expresses high levels of GRK2, which may induce endothelial dysfunction by reducing intracellular NO. GRK2 activation also induces changes in the subcellular localization of GRK2 itself and also of β-arrestin 2, a downstream protein. In this review, we describe the pathophysiological mechanisms of insulin resistance and diabetes, focusing on the signal transduction for NO production via GRK2 and β-arrestin 2, providing novel insights into the potential field of translational investigation in the treatment of diabetic complications.

  6. Endothelial nitric oxide synthase interactions with G-protein-coupled receptors.

    PubMed Central

    Marrero, M B; Venema, V J; Ju, H; He, H; Liang, H; Caldwell, R B; Venema, R C

    1999-01-01

    The endothelial nitric oxide synthase (eNOS) is activated in response to stimulation of endothelial cells by a number of vasoactive substances including, bradykinin (BK), angiotensin II (Ang II), endothelin-1 (ET-1) and ATP. In the present study we have used in vitro activity assays of purified eNOS and in vitro binding assays with glutathione S-transferase fusion proteins to show that the capacity to bind and inhibit eNOS is a common feature of membrane-proximal regions of intracellular domain 4 of the BK B2, the Ang II AT1 and the ET-1 ETB receptors, but not of the ATP P2Y2 receptor. Phosphorylation of serine or tyrosine residues in the eNOS-interacting region of the B2 receptor results in a loss of eNOS inhibition due to a decrease in the binding affinity of the receptor domain for the eNOS enzyme. Furthermore, the B2 receptor is transiently phosphorylated on tyrosine residues in cultured endothelial cells in response to BK stimulation. Phosphorylation occurs during the time in which eNOS transiently dissociates from the receptor accompanied by a transient increase in nitric oxide production. Taken together, these data support the hypotheses that eNOS is regulated in endothelial cells by reversible and inhibitory interactions with G-protein-coupled receptors and that these int