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Sample records for putative g-protein mapping

  1. YAC-assisted cloning of a putative G-protein mapping to the MHC class I region

    SciTech Connect

    Denizot, F. CBM-CNRS, Marseille ); Mattei, M.G. ); Vernet, C.; Pontarotti, P.; Chimini, G. )

    1992-12-01

    The authors report the successful use of whole yeast artificial chromosomes (YACs) as probes for direct positional cloning of novel expressed sequences in a given genomic fragment. The class I region of the human major histocompatibility complex, in particular the chromosomal fragment spanning the HLA-E locus, was investigated. The screening of a cDNA library with a 210- kb-long YAC clone led to the identification of a new gene, positionally conserved in the major histocompatibility complex of the mouse genome and encoding a putative GTP binding protein. Although its precise function remains unknown, the interspecies conservation of both sequence and map position suggests a regulatory or functional link with the histocompatibility cluster. 27 refs., 6 figs.

  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. A Putative G Protein-Coupled Receptor, RDC1, Is a Novel Coreceptor for Human and Simian Immunodeficiency Viruses

    PubMed Central

    Shimizu, Nobuaki; Soda, Yasushi; Kanbe, Katsuaki; Liu, Hui-yu; Mukai, Ryozaburo; Kitamura, Toshio; Hoshino, Hiroo

    2000-01-01

    More than 10 G protein-coupled receptors (GPCRs) have been shown to act as coreceptors for infection of human immunodeficiency virus type 1 (HIV-1), HIV-2, and simian immunodeficiency virus (SIV). We have isolated HIV-1 variants infectious to primary brain-derived CD4-positive cells (BT-3 and BT-20/N) and U87/CD4 glioma cells that are resistant to T-cell line-tropic (T-tropic), macrophage-tropic (M-tropic), and T- and M-tropic (dualtropic) (X4, R5, and R5X4) HIV-1 strains. These primary brain-derived cells were also highly susceptible to HIV-2ROD, HIV-2SBL6669, and SIVmndGB-1. A factor or coreceptor that determines the susceptibility of these brain-derived cells to these HIV and SIV strains has not been fully identified. To identify this coreceptor, we examined amino acid sequences of all known HIV and SIV coreceptors and noticed that tyrosine residues are well conserved in their extracellular amino-terminal domains. By this criterion, we selected 18 GPCRs as candidates of coreceptors for HIV and SIV strains infectious to these brain-derived cells. mRNA expression of an orphan GPCR, RDC1, was detected in the brain-derived cells, the C8166 T-cell line, and peripheral blood lymphocytes, all of which are susceptible to HIV-1 variants, but not in macrophages, which are resistant to them. When a CD4-expressing cell line, NP-2/CD4, which shows strict resistance to infection not only with HIV-1 but also with HIV-2 or SIV, was transduced with the RDC1 gene, the cells became highly susceptible to HIV-2 and SIVmnd strains but to neither M- nor T-tropic HIV-1 strains. The cells also acquired a low susceptibility to the HIV-1 variants. These findings indicate that RDC1 is a novel coreceptor for several HIV-1, HIV-2, and SIV strains which infect brain-derived cells. PMID:10623723

  7. 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.

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

    PubMed Central

    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. PMID:26623315

  9. 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.

  10. 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

  11. GIPC and GAIP form a complex with TrkA: a putative link between G protein and receptor tyrosine kinase pathways.

    PubMed

    Lou, X; Yano, H; Lee, F; Chao, M V; Farquhar, M G

    2001-03-01

    NGF initiates the majority of its neurotrophic effects by promoting the activation of the tyrosine kinase receptor TrkA. Here we describe a novel interaction between TrkA and GIPC, a PDZ domain protein. GIPC binds to the juxtamembrane region of TrkA through its PDZ domain. The PDZ domain of GIPC also interacts with GAIP, an RGS (regulators of G protein signaling) protein. GIPC and GAIP are components of a G protein-coupled signaling complex thought to be involved in vesicular trafficking. In transfected HEK 293T cells GIPC, GAIP, and TrkA form a coprecipitable protein complex. Both TrkA and GAIP bind to the PDZ domain of GIPC, but their binding sites within the PDZ domain are different. The association of endogenous GIPC with the TrkA receptor was confirmed by coimmunoprecipitation in PC12 (615) cells stably expressing TrkA. By immunofluorescence GIPC colocalizes with phosphorylated TrkA receptors in retrograde transport vesicles located in the neurites and cell bodies of differentiated PC12 (615) cells. These results suggest that GIPC, like other PDZ domain proteins, serves to cluster transmembrane receptors with signaling molecules. When GIPC is overexpressed in PC12 (615) cells, NGF-induced phosphorylation of mitogen-activated protein (MAP) kinase (Erk1/2) decreases; however, there is no effect on phosphorylation of Akt, phospholipase C-gamma1, or Shc. The association of TrkA receptors with GIPC and GAIP plus the inhibition of MAP kinase by GIPC suggests that GIPC may provide a link between TrkA and G protein signaling pathways.

  12. GIPC and GAIP Form a Complex with TrkA: A Putative Link between G Protein and Receptor Tyrosine Kinase Pathways

    PubMed Central

    Lou, Xiaojing; Yano, Hiroko; Lee, Francis; Chao, Moses V.; Farquhar, Marilyn Gist

    2001-01-01

    NGF initiates the majority of its neurotrophic effects by promoting the activation of the tyrosine kinase receptor TrkA. Here we describe a novel interaction between TrkA and GIPC, a PDZ domain protein. GIPC binds to the juxtamembrane region of TrkA through its PDZ domain. The PDZ domain of GIPC also interacts with GAIP, an RGS (regulators of G protein signaling) protein. GIPC and GAIP are components of a G protein-coupled signaling complex thought to be involved in vesicular trafficking. In transfected HEK 293T cells GIPC, GAIP, and TrkA form a coprecipitable protein complex. Both TrkA and GAIP bind to the PDZ domain of GIPC, but their binding sites within the PDZ domain are different. The association of endogenous GIPC with the TrkA receptor was confirmed by coimmunoprecipitation in PC12 (615) cells stably expressing TrkA. By immunofluorescence GIPC colocalizes with phosphorylated TrkA receptors in retrograde transport vesicles located in the neurites and cell bodies of differentiated PC12 (615) cells. These results suggest that GIPC, like other PDZ domain proteins, serves to cluster transmembrane receptors with signaling molecules. When GIPC is overexpressed in PC12 (615) cells, NGF-induced phosphorylation of mitogen-activated protein (MAP) kinase (Erk1/2) decreases; however, there is no effect on phosphorylation of Akt, phospholipase C-γ1, or Shc. The association of TrkA receptors with GIPC and GAIP plus the inhibition of MAP kinase by GIPC suggests that GIPC may provide a link between TrkA and G protein signaling pathways. PMID:11251075

  13. 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.

  14. 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

  15. Inhibition of small G proteins by clostridium sordellii lethal toxin activates cdc2 and MAP kinase in Xenopus oocytes.

    PubMed

    Rime, H; Talbi, N; Popoff, M R; Suziedelis, K; Jessus, C; Ozon, R

    1998-12-15

    The lethal toxin (LT) from Clostridium sordellii is a glucosyltransferase that modifies and inhibits small G proteins of the Ras family, Ras and Rap, as well as Rac proteins. LT induces cdc2 kinase activation and germinal vesicle breakdown (GVBD) when microinjected into full-grown Xenopus oocytes. Toxin B from Clostridium difficile, that glucosylates and inactivates Rac proteins, does not induce cdc2 activation, indicating that proteins of the Ras family, Ras and/or Rap, negatively regulate cdc2 kinase activation in Xenopus oocyte. In oocyte extracts, LT catalyzes the incorporation of [14C]glucose into a group of proteins of 23 kDa and into one protein of 27 kDa. The 23-kDa proteins are recognized by anti-Rap1 and anti-Rap2 antibodies, whereas the 27-kDa protein is recognized by several anti-Ras antibodies and probably corresponds to K-Ras. Microinjection of LT into oocytes together with UDP-[14C]glucose results in a glucosylation pattern similar to the in vitro glucosylation, indicating that the 23- and 27-kDa proteins are in vivo substrates of LT. In vivo time-course analysis reveals that the 27-kDa protein glucosylation is completed within 2 h, well before cdc2 kinase activation, whereas the 23-kDa proteins are partially glucosylated at GVBD. This observation suggests that the 27-kDa Ras protein could be the in vivo target of LT allowing cdc2 kinase activation. Interestingly, inactivation of Ras proteins does not prevent the phosphorylation of c-Raf1 and the activation of MAP kinase that occurs normally around GVBD. Copyright 1998 Academic Press.

  16. 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.

  17. 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

  18. 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.

  19. 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.

  20. Evolutionary hierarchy of vertebrate-like heterotrimeric G protein families.

    PubMed

    Krishnan, Arunkumar; Mustafa, Arshi; Almén, Markus Sällman; Fredriksson, Robert; Williams, Michael J; Schiöth, Helgi B

    2015-10-01

    Heterotrimeric G proteins perform a crucial role as molecular switches controlling various cellular responses mediated by G protein-coupled receptor (GPCR) signaling pathway. Recent data have shown that the vertebrate-like G protein families are found across metazoans and their closest unicellular relatives. However, an overall evolutionary hierarchy of vertebrate-like G proteins, including gene family annotations and in particular mapping individual gene gain/loss events across diverse holozoan lineages is still incomplete. Here, with more expanded invertebrate taxon sampling, we have reconstructed phylogenetic trees for each of the G protein classes/families and provide a robust classification and hierarchy of vertebrate-like heterotrimeric G proteins. Our results further extend the evidence that the common ancestor (CA) of holozoans had at least five ancestral Gα genes corresponding to all major vertebrate Gα classes and contain a total of eight genes including two Gβ and one Gγ. Our results also indicate that the GNAI/O-like gene likely duplicated in the last CA of metazoans to give rise to GNAI- and GNAO-like genes, which are conserved across invertebrates. Moreover, homologs of GNB1-4 paralogon- and GNB5 family-like genes are found in most metazoans and that the unicellular holozoans encode two ancestral Gβ genes. Similarly, most bilaterian invertebrates encode two Gγ genes which include a representative of the GNG gene cluster and a putative homolog of GNG13. Interestingly, our results also revealed key evolutionary events such as the Drosophila melanogaster eye specific Gβ subunit that is found conserved in most arthropods and several previously unidentified species specific expansions within Gαi/o, Gαs, Gαq, Gα12/13 classes and the GNB1-4 paralogon. Also, we provide an overall proposed evolutionary scenario on the expansions of all G protein families in vertebrate tetraploidizations. Our robust classification/hierarchy is essential to further

  1. 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

  2. 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.

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

    PubMed Central

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

    2016-01-01

    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. PMID:27625394

  4. 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

  5. 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

  6. Escherichia coli-induced productions of pro-inflammatory cytokines are regulated by MAP kinases and G-protein but not by Akt: Relationship with phylogenetic groups and resistance patterns.

    PubMed

    Auger, Gabriel; Corvec, Stéphane; Roquilly, Antoine; Segain, Jean Pierre; Lepelletier, Didier; Reynaud, Alain; Asehnoune, Karim

    2011-11-01

    We investigated the role of PI3-K, MAP kinases, and heterotrimeric G proteins in inducing cytokines production in human whole blood cultures stimulated by viable Escherichia coli (E. coli) clinical strains. We used eight E. coli strains that belong to different phylogenetic groups and presented by different antibiotic resistance patterns. Whole blood from healthy volunteers was incubated at 37°C for 150min, with lipopolysaccharide (LPS) from E. coli O111:B4 or selected viable E. coli clinical strains, with or without SB202190 (p38 inhibitor), PD98059 (ERK inhibitor), PTX (pertussis toxin; heterotrimeric G proteins inhibitor), wortmaninn (PI3-K inhibitor). The TNF-α, IL-1β, IL-10 and IFN-γ concentrations were measured in culture supernatants (ELISA). IL-10 and IFN-γ were not detectable. Susceptible strains induced higher TNF-α and IL-1β productions than β-lactam resistant strains (p<0.05), with no difference between phylogenetic groups. A transformed strain carrying a plasmid-mediated AmpC-β-lactamase gene (CMY-2) induced lower TNF-α and IL-1β production than the parent wild type strain (p<0.05). SB202190 (p38 inhibitor) and PD98059 (ERK inhibitor) reduced TNF-α concentrations by, respectively, 80% (p<0.05) and 50% (p<0.05). Wortmaninn (PI3-K inhibitor) had no significant effect. PTX (heterotrimeric G proteins inhibitor) altered TNF-α production after viable bacteria stimulation (1.7-fold increase; p<0.05) but not after LPS (TLR-4) stimulation. Regarding IL-1β, wortmaninn, SB202190 and PTX had no significant effect whereas PD98059 significantly decreased production in whole cell cultures (p<0.05). Susceptible strains induce greater TNF-α and IL-1β productions than resistant strains. ERK kinase plays a major role in viable E. coli strains inducing TNF-α and IL-1β production. E. coli exerts an effect on the pertussis toxin-sensitive G-protein through a TLR-4-independent mechanism. Copyright © 2011 Elsevier Ltd. All rights reserved.

  7. 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.

  8. Membrane androgen receptor characteristics of human ZIP9 (SLC39A) zinc transporter in prostate cancer cells: Androgen-specific activation and involvement of an inhibitory G protein in zinc and MAP kinase signaling.

    PubMed

    Thomas, Peter; Pang, Yefei; Dong, Jing

    2017-05-15

    Characteristics of novel human membrane androgen receptor (mAR), ZIP9 (SLC39A9), were investigated in ZIP9-transfected PC-3 cells (PC3-ZIP9). Ligand blot analysis showed plasma membrane [(3)H]-T binding corresponds to the position of ZIP9 on Western blots which suggests ZIP9 can bind [(3)H]-T alone, without a protein partner. Progesterone antagonized testosterone actions, blocking increases in zinc, Erk phosphorylation and apoptosis, further evidence that ZIP9 is specifically activated by androgens. Pre-treatment with GTPγS and pertussis toxin decreased plasma membrane [(3)H]-T binding and blocked testosterone-induced increases in Erk phosphorylation and intracellular zinc, indicating ZIP9 is coupled to an inhibitory G protein (Gi) that mediates both MAP kinase and zinc signaling. Testosterone treatment of nuclei and mitochondria which express ZIP9 decreased their zinc contents, suggesting ZIP9 also regulates free zinc through releasing it from these intracellular organelles. The results show ZIP9 is a specific Gi coupled-mAR mediating testosterone-induced MAP kinase and zinc signaling in PC3-ZIP9 cells. Copyright © 2017 Elsevier B.V. All rights reserved.

  9. RINGdb: an integrated database for G protein-coupled receptors and regulators of G protein signaling.

    PubMed

    Fang, Yu-Ching; Sun, Wei-Hsin; Wu, Li-Cheng; Huang, Hsien-Da; Juan, Hsueh-Fen; Horng, Jorng-Tzong

    2006-12-16

    Many marketed therapeutic agents have been developed to modulate the function of G protein-coupled receptors (GPCRs). The regulators of G-protein signaling (RGS proteins) are also being examined as potential drug targets. To facilitate clinical and pharmacological research, we have developed a novel integrated biological database called RINGdb to provide comprehensive and organized RGS protein and GPCR information. RINGdb contains information on mutations, tissue distributions, protein-protein interactions, diseases/disorders and other features, which has been automatically collected from the Internet and manually extracted from the literature. In addition, RINGdb offers various user-friendly query functions to answer different questions about RGS proteins and GPCRs such as their possible contribution to disease processes, the putative direct or indirect relationship between RGS proteins and GPCRs. RINGdb also integrates organized database cross-references to allow users direct access to detailed information. The database is now available at http://ringdb.csie.ncu.edu.tw/ringdb/. RINGdb is the only integrated database on the Internet to provide comprehensive RGS protein and GPCR information. This knowledge base will be useful for clinical research, drug discovery and GPCR signaling pathway research.

  10. 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.

  11. 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. Copyright © 2015 Elsevier B.V. All rights reserved.

  12. 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.

  13. Heterotrimeric G-proteins: a short history.

    PubMed

    Milligan, Graeme; Kostenis, Evi

    2006-01-01

    Some 865 genes in man encode G-protein-coupled receptors (GPCRs). The heterotrimeric guanine nucleotide-binding proteins (G-proteins) function to transduce signals from this vast panoply of receptors to effector systems including ion channels and enzymes that alter the rate of production, release or degradation of intracellular second messengers. However, it was not until the 1970s that the existence of such transducing proteins was even seriously suggested. Combinations of bacterial toxins that mediate their effects via covalent modification of the alpha-subunit of certain G-proteins and mutant cell lines that fail to generate cyclic AMP in response to agonists because they either fail to express or express a malfunctional G-protein allowed their identification and purification. Subsequent to initial cloning efforts, cloning by homology has defined the human G-proteins to derive from 35 genes, 16 encoding alpha-subunits, five beta and 14 gamma. All function as guanine nucleotide exchange on-off switches and are mechanistically similar to other proteins that are enzymic GTPases. Although not readily accepted initially, it is now well established that beta/gamma complexes mediate as least as many functions as the alpha-subunits. The generation of chimeras between different alpha-subunits defined the role of different sections of the primary/secondary sequence and crystal structures and cocrystals with interacting proteins have given detailed understanding of their molecular structure and basis of function. Finally, further modifications of such chimeras have generated a range of G-protein alpha-subunits with greater promiscuity to interact across GPCR classes and initiated the use of such modified G-proteins in drug discovery programmes.

  14. 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.

  15. 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.

  16. 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.

  17. G protein βγ subunits: Central mediators of G protein-coupled receptor signaling

    PubMed Central

    Smrcka, A. V.

    2008-01-01

    G protein βγ subunits are central participants in G protein-coupled receptor signaling pathways. They interact with receptors, G protein α subunits and downstream targets to coordinate multiple, different GPCR functions. Much is known about the biology of Gβγ subunits but mysteries remain. Here, we will review what is known about general aspects of structure and function of Gβγ as well as discuss emerging mechanisms for regulation of Gβγ signaling. Recent data suggest that Gβγ is a potential therapeutic drug target. Thus, a thorough understanding of the molecular and physiological functions of Gβγ has significant implications. PMID:18488142

  18. G protein defects in signal transduction.

    PubMed

    Spiegel, A M

    2000-01-01

    G proteins couple receptors for many hormones to effectors that regulate second messenger metabolism. Several endocrine disorders have been shown to be caused by either loss- or gain-of-function mutations in G proteins or G protein-coupled receptors. In pseudohypoparathyroidism type Ia (PHP Ia), there are generalized hormone resistance (parathyroid hormone [PTH], thyroid-stimulating hormone, gonadotropins) and associated abnormal physical features, Albright hereditary osteodystrophy. Subjects with PHP Ib are normal in appearance and show renal resistance to PTH. In McCune-Albright syndrome (MAS), subjects show autonomous endocrine hyperfunction associated with fibrous dysplasia of bone and skin hyperpigmentation. Germline loss-of-function mutations have been identified in the G(s)-alpha gene in PHP Ia, and recent evidence suggests that the G(s)-alpha gene is paternally imprinted in a tissue-specific manner. Abnormal imprinting of the G(s)-alpha gene may be the cause of PHP Ib. MAS, in contrast, is caused by gain-of-function missense mutations of the G(s)-alpha gene. Copyright 2000 S. Karger AG, Basel

  19. Oligomerization of G protein-coupled receptors: A reality

    PubMed Central

    Ferré, Sergi; Franco, Rafael

    2009-01-01

    As reviewed in the present issue, we have now an important amount of experimental evidence that indicates that G protein-coupled receptor (GPCR) oligomerization, including homo- and heteromerization, is a general phenomenon. Receptor heteromers possess unique biochemical characteristics that are demonstrably different from those of its individual components (protomers). Those properties include allosteric modulations between protomers, changes in ligand recognition, G protein-coupling and trafficking. The discovery of GPCR oligomers have been related to the parallel discovery and application of a variety of resonance energy transfer (RET) techniques, such as bioluminescence, fluorescence and sequential RET (BRET, FRET and SRET, respectively), time resolved FRET (T-FRET) and fluorescence recovery after photobleaching (FRAP) microscopy. However, RET techniques are difficult to implement in native tissues. For receptor heteromers, indirect approaches, such as the determination of a unique biochemical characteristic (‘biochemical fingerprint’), are allowing their identification in native tissues and their use as targets for drug development. Dopamine and opioid receptor heteromers are being the focus of intense research, which is related to the possible multiple applications of their putative ligands in pathological conditions, which include basal ganglia disorders, schizophrenia, drug addiction and pain. PMID:20015687

  20. 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.

  1. Inherited diseases involving g proteins and g protein-coupled receptors.

    PubMed

    Spiegel, Allen M; Weinstein, Lee S

    2004-01-01

    Heterotrimeric G proteins couple seven-transmembrane receptors for diverse extracellular signals to effectors that generate intracellular signals altering cell function. Mutations in the gene encoding the alpha subunit of the G protein-coupling receptors to stimulation of adenylyl cyclase cause developmental abnormalities of bone, as well as hormone resistance (pseudohypoparathyroidism caused by loss-of-function mutations) and hormone hypersecretion (McCune-Albright syndrome caused by gain-of-function mutations). Loss- and gain-of-function mutations in genes encoding G protein-coupled receptors (GPCRs) have been identified as the cause of an increasing number of retinal, endocrine, metabolic, and developmental disorders. GPCRs comprise an evolutionarily conserved gene superfamily ( 1 ). By coupling to heterotrimeric G proteins, GPCRs transduce a wide variety of extracellular signals including monoamine, amino acid, and nucleoside neurotransmitters, as well as photons, chemical odorants, divalent cations, hormones, lipids, peptides and proteins. Following a brief overview of G protein-coupled signal transduction, we review the growing body of evidence that mutations in genes encoding GPCRs and G proteins are an important cause of human disease.

  2. 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.

  3. Fine mapping of shattering locus Br2 reveals a putative chromosomal inversion polymorphism between the two lineages of Aegilops tauschii.

    PubMed

    Zhang, Zhengzhi; Zhu, Huilan; Gill, Bikram S; Li, Wanlong

    2015-04-01

    This work laid the foundation for cloning of shattering gene Br2 and provided first line of evidence that two major Aegilops tauschii lineages are differentiated by an inversion polymorphism. Chromosome inversions often accompany population differentiation and capture local adaptation during speciation. Aegilops tauschii, the D-genome donor species of hexaploid wheat, consists of two genetically isolated lineages, L1 and L2, but little is known about the genetic mechanisms underlying the population differentiation in this diploid species. During fine mapping of the shattering gene Br2 using a large F2 population derived from a cross between TA1604 (an L1 accession) and AL8/78 (an L2 accession), we found contrasting patterns of crossover distribution in the Br2 interval and neighboring regions despite the high local gene synteny with Brachypodium distachyon and rice. Br2 was localized in a 0.08-cM interval, and 13 marker loci formed a block, where single-crossovers were completely suppressed, but double-crossovers were enriched with a recombination rate of ~11 cM/Mb. In contrast, in a neighboring region no double-crossover was recovered, but single-crossover rate reached 24 cM/Mb, which is much higher than the genome-wide average. This result suggests a putative inversion polymorphism between the parental lines in the Br2 region. Genotyping using the markers from the Br2 region divided a collection of 55 randomly sampled A. tauschii accessions into two major groups, and they are largely genetically isolated. The two groups correspond to the L1 and L2 lineages based on their geographic distribution patterns. This provides first evidence that inversions may underlie the evolution of A. tauschii lineages. The presence of inter-lineage inversions may complicate map-based cloning in A. tauschii and transfer of useful traits to wheat.

  4. 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

  5. Hormone resistance caused by mutations in G proteins and G protein-coupled receptors.

    PubMed

    Spiegel, A M

    1999-04-01

    G proteins couple receptors for many hormones to effectors that regulate second messenger metabolism. Several endocrine disorders have been shown to be caused by either loss or gain of function mutations in G proteins or G protein-coupled receptors. Pseudohypoparathyroidism (PHP), the first described example of a hormone resistance disorder, is characterized by renal resistance to parathyroid hormone (PTH) proximal to generation of the second messenger, cAMP. In PHP Ia there is more generalized hormone resistance (PTH, TSH, gonadotropins) and associated abnormal physical features, Albright hereditary osteodystrophy (AHO). Subjects with PHP Ib are normal in appearance and resistant exclusively to PTH. Germline loss of function mutations have been identified in the Gs-alpha gene in PHP Ia, and recent evidence suggests that the Gs-alpha gene is paternally imprinted in a tissue-specific manner. In PHP Ib, several studies have excluded PTH receptor gene mutations, and the molecular basis has not yet been defined.

  6. 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.

  7. 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

  8. Mapping of putative ether-anesthesia resistance gene using C57BL/6J and MSM/Ms mouse strains.

    PubMed

    Megumi, Tsuneo; Song, Chang-Woo; Park, Yeong-Gwan; Tanaka, Yoshiharu; Uchida, Ichiro; Okumoto, Masaaki

    2004-01-01

    We attempted to identify the locations of major mouse genes responsible for sensitivity to diethylether (ether) anesthesia, using microsatellite linkage analyses including Quantitative Trait Locus (QTL) analysis. To determine the locations of ether anesthesia resistance genes on chromosomes, an ether anesthesia-resistant mouse strain, C57BL/6J (C57BL), and an ether anesthesia-sensitive mouse strain, MSM/Ms (MSM), were used. The sensitivity of mice to ether anesthesia was determined from the latency time required to lose the righting reflex during exposure to 4% ether vapor in air. The (C57BL x MSM) F(1) mice were found to be resistant to ether, showing that the resistant phenotype is genetically dominant. Twelve resistant and 12 sensitive mice were then selected from the 196 backcrossed F(2) mice (F(1) x MSM) at 11-16 weeks of age. Genomic DNA samples were extracted from the tails for mapping ether anesthesia-related genes using microsatellite linkage analyses. One major putative gene related to resistance to ether anesthesia was restricted in the region 23 to 37 cM from the centromere in chromosome 7 by primary and secondary linkage analyses. The QTL analysis narrowed the position of the gene to 29.0 cM, with a maximum logarithm of odds (LOD) score of 3.03, and it was termed Etan1 ( ether-anesthesia 1). Microsatellite linkage analyses, including QTL analysis, determined the location of the ether-resistance gene, Etan1, within a narrow range. Our findings should be helpful for further experiments, such as cloning of the gene governing the sensitivity to ether anesthesia in mice.

  9. 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

  10. Diversity of heterotrimeric G-protein γ subunits in plants.

    PubMed

    Trusov, Yuri; Chakravorty, David; Botella, José Ramón

    2012-10-31

    Heterotrimeric G-proteins, consisting of three subunits Gα, Gβ and Gγ are present in most eukaryotes and mediate signaling in numerous biological processes. In plants, Gγ subunits were shown to provide functional selectivity to G-proteins. Three unconventional Gγ subunits were recently reported in Arabidopsis, rice and soybean but no structural analysis has been reported so far. Their relationship with conventional Gγ subunits and taxonomical distribution has not been yet demonstrated. After an extensive similarity search through plant genomes, transcriptomes and proteomes we assembled over 200 non-redundant proteins related to the known Gγ subunits. Structural analysis of these sequences revealed that most of them lack the obligatory C-terminal prenylation motif (CaaX). According to their C-terminal structures we classified the plant Gγ subunits into three distinct types. Type A consists of Gγ subunits with a putative prenylation motif. Type B subunits lack a prenylation motif and do not have any cysteine residues in the C-terminal region, while type C subunits contain an extended C-terminal domain highly enriched with cysteines. Comparative analysis of C-terminal domains of the proteins, intron-exon arrangement of the corresponding genes and phylogenetic studies suggested a common origin of all plant Gγ subunits. Phylogenetic analyses suggest that types C and B most probably originated independently from type A ancestors. We speculate on a potential mechanism used by those Gγ subunits lacking isoprenylation motifs to anchor the Gβγ dimer to the plasma membrane and propose a new flexible nomenclature for plant Gγ subunits. Finally, in the light of our new classification, we give a word of caution about the interpretation of Gγ research in Arabidopsis and its generalization to other plant species.

  11. Mechanisms of disease: Mutations of G proteins and G-protein-coupled receptors in endocrine diseases.

    PubMed

    Lania, Andrea G; Mantovani, Giovanna; Spada, Anna

    2006-12-01

    G proteins and G-protein-coupled receptors (GPCRs) mediate the effects of a number of hormones. Genes that encode these molecules are subject to loss-of function or gain-of-function mutations that result in endocrine disorders. Loss-of-function mutations prevent signaling in response to the corresponding agonist and cause resistance to hormone actions, which mimics hormone deficiency. Gain-of-function mutations lead to constitutive, agonist-independent activation of signaling, which mimics hormone excess. Disease-causing mutations of GPCRs have been identified in patients with various disorders of the pituitary-thyroid, pituitary-gonadal and pituitary-adrenal axes, and in those with abnormalities in food intake, growth, water balance and mineral-ion turnover. The only mutational changes in G proteins unequivocally associated with endocrine disorders occur in GNAS (guanine nucleotide-binding protein G-stimulatory subunit alpha, or G(s)alpha). Heterozygous loss-of-function mutations of GNAS in the active, maternal allele cause resistance to hormones that act through G(s)alpha-coupled GPCRs, whereas somatic gain-of-function mutations cause proliferation of endocrine cells that recognize cyclic AMP as a mitogen. The study of mutations in G proteins and GPCRs has already had major implications for understanding the molecular basis of rare endocrine diseases, as well as susceptibility to multifactorial disorders that are associated with polymorphisms in these genes.

  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

    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.

  14. 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

  15. 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.

  16. 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

  17. Expression and Purification of Mini G Proteins from Escherichia coli.

    PubMed

    Carpenter, Byron; Tate, Christopher G

    2017-04-20

    Heterotrimeric G proteins modulate intracellular signalling by transducing information from cell surface G protein-coupled receptors (GPCRs) to cytoplasmic effector proteins. Structural and functional characterisation of GPCR-G protein complexes is important to fully decipher the mechanism of signal transduction. However, native G proteins are unstable and conformationally dynamic when coupled to a receptor. We therefore developed an engineered minimal G protein, mini-Gs, which formed a stable complex with GPCRs, and facilitated the crystallisation and structure determination of the human adenosine A2A receptor (A2AR) in its active conformation. Mini G proteins are potentially useful tools in a variety of applications, including characterising GPCR pharmacology, binding affinity and kinetic experiments, agonist drug discovery, and structure determination of GPCR-G protein complexes. Here, we describe a detailed protocol for the expression and purification of mini-Gs.

  18. G protein signaling in plants: minus times minus equals plus.

    PubMed

    Stateczny, Dave; Oppenheimer, Jara; Bommert, Peter

    2016-12-01

    Heterotrimeric G proteins are key regulators in the transduction of extracellular signals both in animals and plants. In plants, heterotrimeric G protein signaling plays essential roles in development and in response to biotic and abiotic stress. However, over the last decade it has become clear that plants have unique mechanisms of G protein signaling. Although plants share most of the core components of heterotrimeric G proteins, some of them exhibit unusual properties compared to their animal counterparts. In addition, plants do not share functional GPCRs. Therefore the well-established paradigm of the animal G protein signaling cycle is not applicable in plants. In this review, we summarize recent insights into these unique mechanisms of G protein signaling in plants with special focus on the evident potential of G protein signaling as a target to modify developmental and physiological parameters important for yield increase. Copyright © 2016 Elsevier Ltd. All rights reserved.

  19. 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.

  20. Dynamics of bovine opsin bound to G-protein fragments.

    PubMed

    Sugihara, Minoru; Suwa, Makiko; Bondar, Ana-Nicoleta

    2014-10-01

    G protein-coupled receptors (GPCRs) are a large class of membrane proteins that mediate communication of the cell with the outer environment. Upon activation by an agonist, GPCRs undergo large-scale conformational changes that enable binding of the G protein to the receptor. A key open question concerns the mechanism of the long-distance coupling between the agonist-binding site and the cytoplasmic site where G protein binds. Here we address this question by exploring the molecular dynamics of bovine opsin bound to three different fragments of G-proteins. We find that an extended network of hydrogen bonds connects the agonist retinal binding site to the G protein binding site via conserved amino acid residues. The dynamics of the hydrogen-bonding network inside opsin couples to interactions at the G protein binding site. Copyright © 2014 Elsevier Inc. All rights reserved.

  1. Biased and G Protein-Independent Signaling of Chemokine Receptors

    PubMed Central

    Steen, Anne; Larsen, Olav; Thiele, Stefanie; Rosenkilde, Mette M.

    2014-01-01

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

  2. 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.

  3. Detection of G protein-selective G protein-coupled receptor (GPCR) conformations in live cells.

    PubMed

    Malik, Rabia U; Ritt, Michael; DeVree, Brian T; Neubig, Richard R; Sunahara, Roger K; Sivaramakrishnan, Sivaraj

    2013-06-14

    Although several recent studies have reported that GPCRs adopt multiple conformations, it remains unclear how subtle conformational changes are translated into divergent downstream responses. In this study, we report on a novel class of FRET-based sensors that can detect the ligand/mutagenic stabilization of GPCR conformations that promote interactions with G proteins in live cells. These sensors rely on the well characterized interaction between a GPCR and the C terminus of a Gα subunit. We use these sensors to elucidate the influence of the highly conserved (E/D)RY motif on GPCR conformation. Specifically, Glu/Asp but not Arg mutants of the (E/D)RY motif are known to enhance basal GPCR signaling. Hence, it is unclear whether ionic interactions formed by the (E/D)RY motif (ionic lock) are necessary to stabilize basal GPCR states. We find that mutagenesis of the β2-AR (E/D)RY ionic lock enhances interaction with Gs. However, only Glu/Asp but not Arg mutants increase G protein activation. In contrast, mutagenesis of the opsin (E/D)RY ionic lock does not alter its interaction with transducin. Instead, opsin-specific ionic interactions centered on residue Lys-296 are both necessary and sufficient to promote interactions with transducin. Effective suppression of β2-AR basal activity by inverse agonist ICI 118,551 requires ionic interactions formed by the (E/D)RY motif. In contrast, the inverse agonist metoprolol suppresses interactions with Gs and promotes Gi binding, with concomitant pertussis toxin-sensitive inhibition of adenylyl cyclase activity. Taken together, these studies validate the use of the new FRET sensors while revealing distinct structural mechanisms for ligand-dependent GPCR function.

  4. Bio::Homology::InterologWalk--a Perl module to build putative protein-protein interaction networks through interolog mapping.

    PubMed

    Gallone, Giuseppe; Simpson, T Ian; Armstrong, J Douglas; Jarman, Andrew P

    2011-07-18

    Protein-protein interaction (PPI) data are widely used to generate network models that aim to describe the relationships between proteins in biological systems. The fidelity and completeness of such networks is primarily limited by the paucity of protein interaction information and by the restriction of most of these data to just a few widely studied experimental organisms. In order to extend the utility of existing PPIs, computational methods can be used that exploit functional conservation between orthologous proteins across taxa to predict putative PPIs or 'interologs'. To date most interolog prediction efforts have been restricted to specific biological domains with fixed underlying data sources and there are no software tools available that provide a generalised framework for 'on-the-fly' interolog prediction. We introduce Bio::Homology::InterologWalk, a Perl module to retrieve, prioritise and visualise putative protein-protein interactions through an orthology-walk method. The module uses orthology and experimental interaction data to generate putative PPIs and optionally collates meta-data into an Interaction Prioritisation Index that can be used to help prioritise interologs for further analysis. We show the application of our interolog prediction method to the genomic interactome of the fruit fly, Drosophila melanogaster. We analyse the resulting interaction networks and show that the method proposes new interactome members and interactions that are candidates for future experimental investigation. Our interolog prediction tool employs the Ensembl Perl API and PSICQUIC enabled protein interaction data sources to generate up to date interologs 'on-the-fly'. This represents a significant advance on previous methods for interolog prediction as it allows the use of the latest orthology and protein interaction data for all of the genomes in Ensembl. The module outputs simple text files, making it easy to customise the results by post-processing, allowing the

  5. Identification of putative QTLs for seedling stage phosphorus starvation response in finger millet (Eleusine coracana L. Gaertn.) by association mapping and cross species synteny analysis

    PubMed Central

    Ramakrishnan, M.; Ceasar, S. Antony; Vinod, K. K.; Duraipandiyan, V.; Ajeesh Krishna, T. P.; Upadhyaya, Hari D.; Al-Dhabi, N. A.

    2017-01-01

    A germplasm assembly of 128 finger millet genotypes from 18 countries was evaluated for seedling-stage phosphorus (P) responses by growing them in P sufficient (Psuf) and P deficient (Pdef) treatments. Majority of the genotypes showed adaptive responses to low P condition. Based on phenotype behaviour using the best linear unbiased predictors for each trait, genotypes were classified into, P responsive, low P tolerant and P non-responsive types. Based on the overall phenotype performance under Pdef, 10 genotypes were identified as low P tolerants. The low P tolerant genotypes were characterised by increased shoot and root length and increased root hair induction with longer root hairs under Pdef, than under Psuf. Association mapping of P response traits using mixed linear models revealed four quantitative trait loci (QTLs). Two QTLs (qLRDW.1 and qLRDW.2) for low P response affecting root dry weight explained over 10% phenotypic variation. In silico synteny analysis across grass genomes for these QTLs identified putative candidate genes such as Ser-Thr kinase and transcription factors such as WRKY and basic helix-loop-helix (bHLH). The QTLs for response under Psuf were mapped for traits such as shoot dry weight (qHSDW.1) and root length (qHRL.1). Putative associations of these QTLs over the syntenous regions on the grass genomes revealed proximity to cytochrome P450, phosphate transporter and pectin methylesterase inhibitor (PMEI) genes. This is the first report of the extent of phenotypic variability for P response in finger millet genotypes during seedling-stage, along with the QTLs and putative candidate genes associated with P starvation tolerance. PMID:28820887

  6. 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.

  7. 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

  8. 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

  9. 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.

  10. 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

  11. 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.

  12. 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.

  13. Structure and function of heterotrimeric G proteins in plants.

    PubMed

    Fujisawa, Y; Kato, H; Iwasaki, Y

    2001-08-01

    Heterotrimeric G proteins are mediators that transmit the external signals via receptor molecules to effector molecules. The G proteins consist of three different subunits: alpha, beta, and gamma subunits. The cDNAs or genes for all the alpha, beta, and gamma subunits have been isolated from many plant species, which has contributed to great progress in the study of the structure and function of the G proteins in plants. In addition, rice plants lacking the alpha subunit were generated by the antisense method and a rice mutant, Daikoku d1, was found to have mutation in the alpha-subunit gene. Both plants show abnormal morphology such as dwarfism, dark green leaf, and small round seed. The findings revealed that the G proteins are functional molecules regulating some body plans in plants. There is evidence that the plant G proteins participate at least in signaling of gibberellin at low concentrations. In this review, we summarize the currently known information on the structure of plant heterotrimeric G proteins and discuss the possible functions of the G proteins in plants.

  14. G protein-membrane interactions II: Effect of G protein-linked lipids on membrane structure and G protein-membrane interactions.

    PubMed

    Casas, Jesús; Ibarguren, Maitane; Álvarez, Rafael; Terés, Silvia; Lladó, Victoria; Piotto, Stefano P; Concilio, Simona; Busquets, Xavier; López, David J; Escribá, Pablo V

    2017-09-01

    G proteins often bear myristoyl, palmitoyl and isoprenyl moieties, which favor their association with the membrane and their accumulation in G Protein Coupled Receptor-rich microdomains. These lipids influence the biophysical properties of membranes and thereby modulate G protein binding to bilayers. In this context, we showed here that geranylgeraniol, but neither myristate nor palmitate, increased the inverted hexagonal (HII) phase propensity of phosphatidylethanolamine-containing membranes. While myristate and palmitate preferentially associated with phosphatidylcholine membranes, geranylgeraniol favored nonlamellar-prone membranes. In addition, Gαi1 monomers had a higher affinity for lamellar phases, while Gβγ and Gαβγ showed a marked preference for nonlamellar prone membranes. Moreover, geranylgeraniol enhanced the binding of G protein dimers and trimers to phosphatidylethanolamine-containing membranes, yet it decreased that of monomers. By contrast, both myristate and palmitate increased the Gαi1 preference for lamellar membranes. Palmitoylation reinforced the binding of the monomer to PC membranes and myristoylation decreased its binding to PE-enriched bilayer. Finally, binding of dimers and trimers to lamellar-prone membranes was decreased by palmitate and myristate, but it was increased in nonlamellar-prone bilayers. These results demonstrate that co/post-translational G protein lipid modifications regulate the membrane lipid structure and that they influence the physico-chemical properties of membranes, which in part explains why G protein subunits sort to different plasma membrane domains. This article is part of a Special Issue entitled: Membrane Lipid Therapy: Drugs Targeting Biomembranes edited by Pablo V. Escribá. Copyright © 2017 Elsevier B.V. All rights reserved.

  15. GPCR – G protein complexes – the fundamental signaling assembly

    PubMed Central

    Jastrzebska, Beata

    2013-01-01

    G protein coupled receptors (GPCR) constitute the largest group of cell surface receptors that transmit various signals across biological membranes through the binding and activation of heterotrimeric G proteins, which amplify the signal and activate downstream effectors leading to the biological responses. Thus, the first critical step in this signaling cascade is the interaction between receptor and its cognate G protein. Understanding this critical event at the molecular level is of high importance because abnormal function of GPCRs is associated with many diseases. Thus, these receptors are targets for drug development. PMID:24052187

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

    PubMed

    Port, Jesse A; Parker, Micaela S; Kodner, Robin B; Wallace, James C; Armbrust, E Virginia; Faustman, Elaine M

    2013-07-24

    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. 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. 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 evidence for some GPCR pathway

  17. 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

  18. 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.

  19. Isolation and fine mapping of 16 novel human zinc finger-encoding cDNAs identify putative candidate genes for developmental and malignant disorders

    SciTech Connect

    Tommerup, N.; Vissing, H.

    1995-05-20

    The authors have isolated and chromosomally fine-mapped 16 novel genes belonging to the human zinc finger Krueppel family (ZNF131-140, 142, 143, 148, 151, 154, and 155), including 1 of the GLI type (ZNF143) and 3 containing a KRAB (Krueppel-associated box) segment (ZNF133, 136, and 140). Based on their map position, several of these ZNF genes are putative candidate genes for both developmental and malignant disorders: ZNF138, ZNF139, and ZNF143 were localized to 7q11.2, 7q21.3-q22.1, and 11p15.3-p15.4, regions involved in deletions and/or translocations associated with Williams syndrome, split hand and foot disease (SHFD1), and Beckwith-Wiedemann syndrome, respectively. ZNF133 was localized to 20p11.2, close to, but probably distinct from, the region deleted in Alagille syndrome. Zinc finger genes mapping to regions commonly deleted in solid tumors included ZNF132, 134, 135, 137, 154, and 155, all located on 19q13 (thyroid adenoma), and ZNF151, at 1p36.1-p36.2 (neuroblastoma, colon cancer, and other tumors). In addition, several of the ZNFs mapped to regions implicated in recurrent chromosomal rearrangements in hematological malignancies (ZNF139, 7q21.3-q22.1; ZNF148, 3q21-q22; ZNF151, 1p36.1-p36.2). The study indicates that the number of ZNF genes in human is large and that systematic isolation and mapping of ZNF genes is a straightforward approach for the identification of novel candidate disease genes. 47 refs., 2 figs., 1 tab.

  20. Isolation and fine mapping of 16 novel human zinc finger-encoding cDNAs identify putative candidate genes for developmental and malignant disorders.

    PubMed

    Tommerup, N; Vissing, H

    1995-05-20

    We have isolated and chromosomally fine-mapped 16 novel genes belonging to the human zinc finger Krüppel family (ZNF131-140, 142, 143, 148, 151, 154, and 155), including 1 of the GLI type (ZNF143) and 3 containing a KRAB (Krüppel-associated box) segment (ZNF133, 136, and 140). Based on their map position, several of these ZNF genes are putative candidate genes for both developmental and malignant disorders: ZNF138, ZNF139, and ZNF143 were localized to 7q11.2, 7q21.3-q22.1, and 11p15.3-p15.4, regions involved in deletions and/or translocations associated with Williams syndrome, split hand and foot disease (SHFD1), and Beckwith-Wiedemann syndrome, respectively. ZNF133 was localized to 20p11.2, close to, but probably distinct from, the region deleted in Alagille syndrome. Zinc finger genes mapping to regions commonly deleted in solid tumors included ZNF132, 134, 135, 137, 154, and 155, all located on 19q13 (thyroid adenoma), and ZNF151, at 1p36.1-p36.2 (neuroblastoma, colon cancer, and other tumors). In addition, several of the ZNFs mapped to regions implicated in recurrent chromosomal rearrangements in hematological malignancies (ZNF139, 7q21.3-q22.1; ZNF148, 3q21-q22; ZNF151, 1p36.1-p36.2). The study indicates that the number of ZNF genes in human is large and that systematic isolation and mapping of ZNF genes is a straightforward approach for the identification of novel candidate disease genes.

  1. 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

  2. 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.

  3. 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.

  4. 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

  5. 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.

  6. Multiple functions of G protein-coupled receptor kinases.

    PubMed

    Watari, Kenji; Nakaya, Michio; Kurose, Hitoshi

    2014-03-06

    Desensitization is a physiological feedback mechanism that blocks detrimental effects of persistent stimulation. G protein-coupled receptor kinase 2 (GRK2) was originally identified as the kinase that mediates G protein-coupled receptor (GPCR) desensitization. Subsequent studies revealed that GRK is a family composed of seven isoforms (GRK1-GRK7). Each GRK shows a differential expression pattern. GRK1, GRK4, and GRK7 are expressed in limited tissues. In contrast, GRK2, GRK3, GRK5, and GRK6 are ubiquitously expressed throughout the body. The roles of GRKs in GPCR desensitization are well established. When GPCRs are activated by their agonists, GRKs phosphorylate serine/threonine residues in the intracellular loops and the carboxyl-termini of GPCRs. Phosphorylation promotes translocation of β-arrestins to the receptors and inhibits further G protein activation by interrupting receptor-G protein coupling. The binding of β-arrestins to the receptors also helps to promote receptor internalization by clathrin-coated pits. Thus, the GRK-catalyzed phosphorylation and subsequent binding of β-arrestin to GPCRs are believed to be the common mechanism of GPCR desensitization and internalization. Recent studies have revealed that GRKs are also involved in the β-arrestin-mediated signaling pathway. The GRK-mediated phosphorylation of the receptors plays opposite roles in conventional G protein- and β-arrestin-mediated signaling. The GRK-catalyzed phosphorylation of the receptors results in decreased G protein-mediated signaling, but it is necessary for β-arrestin-mediated signaling. Agonists that selectively activate GRK/β-arrestin-dependent signaling without affecting G protein signaling are known as β-arrestin-biased agonists. Biased agonists are expected to have potential therapeutic benefits for various diseases due to their selective activation of favorable physiological responses or avoidance of the side effects of drugs. Furthermore, GRKs are recognized as

  7. 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.

  8. G protein signaling in the parasite Entamoeba histolytica

    PubMed Central

    Bosch, Dustin E; Siderovski, David P

    2013-01-01

    The parasite Entamoeba histolytica causes amebic colitis and systemic amebiasis. Among the known amebic factors contributing to pathogenesis are signaling pathways involving heterotrimeric and Ras superfamily G proteins. Here, we review the current knowledge of the roles of heterotrimeric G protein subunits, Ras, Rho and Rab GTPase families in E. histolytica pathogenesis, as well as of their downstream signaling effectors and nucleotide cycle regulators. Heterotrimeric G protein signaling likely modulates amebic motility and attachment to and killing of host cells, in part through activation of an RGS-RhoGEF (regulator of G protein signaling–Rho guanine nucleotide exchange factor) effector. Rho family GTPases, as well as RhoGEFs and Rho effectors (formins and p21-activated kinases) regulate the dynamic actin cytoskeleton of E. histolytica and associated pathogenesis-related cellular processes, such as migration, invasion, phagocytosis and evasion of the host immune response by surface receptor capping. A remarkably large family of 91 Rab GTPases has multiple roles in a complex amebic vesicular trafficking system required for phagocytosis and pinocytosis and secretion of known virulence factors, such as amebapores and cysteine proteases. Although much remains to be discovered, recent studies of G protein signaling in E. histolytica have enhanced our understanding of parasitic pathogenesis and have also highlighted possible targets for pharmacological manipulation. PMID:23519208

  9. 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.

  10. 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.

  11. 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

  12. Heterotrimeric G protein signalling in the plant kingdom

    PubMed Central

    Urano, Daisuke; Chen, Jin-Gui; Botella, José Ramón; Jones, Alan M.

    2013-01-01

    In animals, heterotrimeric G proteins, comprising α-, β-and γ-subunits, perceive extracellular stimuli through cell surface receptors, and transmit signals to ion channels, enzymes and other effector proteins to affect numerous cellular behaviours. In plants, G proteins have structural similarities to the corresponding molecules in animals but transmit signals by atypical mechanisms and effector proteins to control growth, cell proliferation, defence, stomate movements, channel regulation, sugar sensing and some hormonal responses. In this review, we summarize the current knowledge on the molecular regulation of plant G proteins, their effectors and the physiological functions studied mainly in two model organisms: Arabidopsis thaliana and rice (Oryza sativa). We also look at recent progress on structural analyses, systems biology and evolutionary studies. PMID:23536550

  13. Heterotrimeric G protein signalling in the plant kingdom.

    PubMed

    Urano, Daisuke; Chen, Jin-Gui; Botella, José Ramón; Jones, Alan M

    2013-03-27

    In animals, heterotrimeric G proteins, comprising α-, β-and γ-subunits, perceive extracellular stimuli through cell surface receptors, and transmit signals to ion channels, enzymes and other effector proteins to affect numerous cellular behaviours. In plants, G proteins have structural similarities to the corresponding molecules in animals but transmit signals by atypical mechanisms and effector proteins to control growth, cell proliferation, defence, stomate movements, channel regulation, sugar sensing and some hormonal responses. In this review, we summarize the current knowledge on the molecular regulation of plant G proteins, their effectors and the physiological functions studied mainly in two model organisms: Arabidopsis thaliana and rice (Oryza sativa). We also look at recent progress on structural analyses, systems biology and evolutionary studies.

  14. 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.

  15. G protein diversity and the regulation of signaling pathways.

    PubMed

    Reed, R R

    1990-11-01

    The subunits of the heterotrimeric guanine nucleotide-binding (G) proteins mediate the transfer of information from receptor to effector molecules. Biochemical studies and recent molecular cloning efforts have revealed a rich but largely unexpected diversity in G protein subunit structure and function. Extreme specificity in signaling pathways could be accommodated by the combinatorial association of individual subunits. Alternatively, this wealth of diversity may allow for the simultaneous activation of related G alpha subunits and the modulation of several effector systems. In tissues where the subunits are expressed at different levels, activation of receptors could stimulate distinct effector pathways. Mutations in individual subunits as well as alterations in their level of expression can lead to profound physiological changes. The mechanisms that underlie these changes are being elucidated and will provide insight into the complex regulatory processes associated with the large G protein subunit family.

  16. Mutations of signal-transducing G proteins in human disease.

    PubMed

    Schnabel, P; Böhm, M

    1995-05-01

    Heterotrimeric guanine nucleotide binding proteins (G proteins) couple a large number of cell surface receptors to their intracellular effector molecules, such as enzymes or ion channels. Mutations of G proteins can lead to either activation or inactivation of the corresponding signal transduction pathway and thus cause clinical symptoms. Mutations of heterotrimeric G proteins have been found in a number of endocrine tumors, the McCune-Albright syndrome, Albright's hereditary osteodystrophy, and a combination of precocious puberty and pseudohypoparathyroidism Ia. The identification of the molecular defects underlying the above disorders and the investigation of their functional consequences for metabolism and growth regulation have been the subject of many studies over the past few years. A close understanding of these pathophysiologic mechanisms is crucial for the development of therapeutic strategies.

  17. Nanobody stabilization of G protein coupled receptor conformational states

    PubMed Central

    Steyaert, Jan; K Kobilka, Brian

    2011-01-01

    Remarkable progress has been made in the field of G protein coupled receptor (GPCR) structural biology during the past four years. Several obstacles to generating diffraction quality crystals of GPCRs have been overcome by combining innovative methods ranging from protein engineering to lipid-based screens and microdiffraction technology. The initial GPCR structures represent energetically stable inactive-state conformations. However, GPCRs signal through different G protein isoforms or G protein-independent effectors upon ligand binding suggesting the existence of multiple ligand-specific active states. These active-state conformations are unstable in the absence of specific cytosolic signaling partners representing new challenges for structural biology. Camelid single chain antibody fragments (nanobodies) show promise for stabilizing active GPCR conformations and as chaperones for crystallogenesis. PMID:21782416

  18. 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

  19. 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.

  20. 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

  1. Acupuncture upregulates G protein coupled activity in SAMP8 mice.

    PubMed

    Luo, Benhua; Zhao, Lan; Zhang, Xuezhu; Kan, Bohong; Liu, Yunhe; Jia, Yujie; Han, Jingxian; Yu, Jianchun

    2017-08-01

    Transmembrane and intracellular signal transduction of G protein is closely related to the pathophysiology of Alzheimer's disease (AD). To explore the effects of Sanjiao acupuncture on G protein signal transduction pathways in the pathogenesis of AD. 36 senescence-accelerated (SAM) prone 8 mice were divided into three groups that remained untreated (SAMP8, n=12) or received Sanjiao acupuncture (SAMP8+SA, n=12) or control acupuncture (SAMP8+CA, n=12). An additional control group of SAM resistant 1 mice was included (SAMR1 group, n=12). Morris water maze tests were used to investigate learning and memory abilities. Immunoprecipitation and Western blotting were used to study expression of G protein subunits and their activities in the cortex/hippocampus. Behavioural analysis showed that acupuncture attenuated the severe cognitive deficits observed in untreated/CA-treated SAMP8 mice. The findings of the G protein activation assays via immunoprecipitation and Western blots were that the physiologically coupled activation rate (PCAR) and maximal coupled activation rate (MCAR) of Gαs and Gαi were decreased in the cortex of SAMP8 vs SAMR1 mice. Sanjiao acupuncture induced an upregulation in the PCAR of Gαs and Gαi. In the hippocampus of untreated SAMP8 mice, the PCAR of Gαs and MCAR of both Gαs and Gαi declined, and Sanjiao acupuncture was associated with an upregulation in the MCAR of Gαs and Gαi. There were no significant differences in Gαs and Gαi expression between the groups. Sanjiao acupuncture attenuates cognitive deficits in a mouse model of AD via upregulation of G protein activity and stabilisation of the cellular signal. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.

  2. 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

  3. 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.

  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. 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.

  6. Extra-Large G Proteins Expand the Repertoire of Subunits in Arabidopsis Heterotrimeric G Protein Signaling1[OPEN

    PubMed Central

    Chakravorty, David; Gookin, Timothy E.; Milner, Matthew J.; Yu, Yunqing; Assmann, Sarah M.

    2015-01-01

    Heterotrimeric G proteins, consisting of Gα, Gβ, and Gγ subunits, are a conserved signal transduction mechanism in eukaryotes. However, G protein subunit numbers in diploid plant genomes are greatly reduced as compared with animals and do not correlate with the diversity of functions and phenotypes in which heterotrimeric G proteins have been implicated. In addition to GPA1, the sole canonical Arabidopsis (Arabidopsis thaliana) Gα subunit, Arabidopsis has three related proteins: the extra-large GTP-binding proteins XLG1, XLG2, and XLG3. We demonstrate that the XLGs can bind Gβγ dimers (AGB1 plus a Gγ subunit: AGG1, AGG2, or AGG3) with differing specificity in yeast (Saccharomyces cerevisiae) three-hybrid assays. Our in silico structural analysis shows that XLG3 aligns closely to the crystal structure of GPA1, and XLG3 also competes with GPA1 for Gβγ binding in yeast. We observed interaction of the XLGs with all three Gβγ dimers at the plasma membrane in planta by bimolecular fluorescence complementation. Bioinformatic and localization studies identified and confirmed nuclear localization signals in XLG2 and XLG3 and a nuclear export signal in XLG3, which may facilitate intracellular shuttling. We found that tunicamycin, salt, and glucose hypersensitivity and increased stomatal density are agb1-specific phenotypes that are not observed in gpa1 mutants but are recapitulated in xlg mutants. Thus, XLG-Gβγ heterotrimers provide additional signaling modalities for tuning plant G protein responses and increase the repertoire of G protein heterotrimer combinations from three to 12. The potential for signal partitioning and competition between the XLGs and GPA1 is a new paradigm for plant-specific cell signaling. PMID:26157115

  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. 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.

  9. [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.

  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. 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

  12. 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.

  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. Using melanopsin to study G protein signaling in cortical neurons.

    PubMed

    McGregor, K M; Bécamel, C; Marin, P; Andrade, R

    2016-09-01

    Our understanding of G protein-coupled receptors (GPCRs) in the central nervous system (CNS) has been hampered by the limited availability of tools allowing for the study of their signaling with precise temporal control. To overcome this, we tested the utility of the bistable mammalian opsin melanopsin to examine G protein signaling in CNS neurons. Specifically, we used biolistic (gene gun) approaches to transfect melanopsin into cortical pyramidal cells maintained in organotypic slice culture. Whole cell recordings from transfected neurons indicated that application of blue light effectively activated the transfected melanopsin to elicit the canonical biphasic modulation of membrane excitability previously associated with the activation of GPCRs coupling to Gαq-11 Remarkably, full mimicry of exogenous agonist concentration could be obtained with pulses as short as a few milliseconds, suggesting that their triggering required a single melanopsin activation-deactivation cycle. The resulting temporal control over melanopsin activation allowed us to compare the activation kinetics of different components of the electrophysiological response. We also replaced the intracellular loops of melanopsin with those of the 5-HT2A receptor to create a light-activated GPCR capable of interacting with the 5-HT2A receptor interacting proteins. The resulting chimera expressed weak activity but validated the potential usefulness of melanopsin as a tool for the study of G protein signaling in CNS neurons. Copyright © 2016 the American Physiological Society.

  15. 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

  16. 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.

  17. 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

  18. G Protein Activation Stimulates Phospholipase D Signaling in Plants.

    PubMed Central

    Munnik, T.; Arisz, S. A.; De Vrije, T.; Musgrave, A.

    1995-01-01

    We provide direct evidence for phospholipase D (PLD) signaling in plants by showing that this enzyme is stimulated by the G protein activators mastoparan, ethanol, and cholera toxin. An in vivo assay for PLD activity in plant cells was developed based on the use of a "reporter alcohol" rather than water as a transphosphatidylation substrate. The product was a phosphatidyl alcohol, which, in contrast to the normal product phosphatidic acid, is a specific measure of PLD activity. When 32P-labeled cells were treated with 0.1% n-butanol, 32P-phosphatidyl butanol (32P-PtdBut) was formed in a time-dependent manner. In cells treated with any of the three G protein activators, the production of 32P-PtdBut was increased in a dose-dependent manner. The G protein involved was pertussis toxin insensitive. Ethanol could activate PLD but was itself consumed by PLD as transphosphatidylation substrate. In contrast, secondary alcohols (e.g., sec-butyl alcohol) activated PLD but did not function as substrate, whereas tertiary alcohols did neither. Although most of the experiments were performed with the green alga Chlamydomonas eugametos, the relevance for higher plants was demonstrated by showing that PLD in carnation petals could also be activated by mastoparan. The results indicate that PLD activation must be considered as a potential signal transduction mechanism in plants, just as in animals. PMID:12242371

  19. Two distinct light regulated G-proteins in octopus photoreceptors.

    PubMed

    Tsuda, M; Tsuda, T

    1990-04-09

    Two distinct light-regulated G-proteins were found in octopus photoreceptors. Gip, a 41 kDa protein from washed microvilli, was ADP ribosylated by pertussis toxin in the presence of GDP in the dark. Light and GTP analogues were inhibitory as with transducin (Gt; G-protein in vertebrate photoreceptors). G34, a 34 kDa protein from fresh octopus retina, was ADP ribosylated by both cholera and pertussis toxin in the dark. Light inhibited labeling of the 34 kDa protein by both toxins. Unlike Gip, G34 is soluble and is very labile to heat, freezing and thawing. Prolonged incubation of octopus retina with cholera toxin and labeled NAD produced an additional radioactive band at 46 kDa. Labeling of the 46 kDa protein, Gsp, was greatly enhanced by GTP analogues, but inhibited by a GDP analogue as with Gs in hormone-sensitive adenylate cyclase. In contrast to Gip and G34, labeling of the 46 kDa protein (Gsp) was not influenced by light. The two distinct light-regulated G-proteins, Gip and G34, found in octopus photoreceptors might be involved in either phototransduction or photoadaptation. The function of Gsp is not known.

  20. 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.

  1. 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.

  2. 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

  3. 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

  4. 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. Copyright © 2011 Elsevier Ltd. All rights reserved.

  5. The structure and function of G-protein-coupled receptors.

    PubMed

    Rosenbaum, Daniel M; Rasmussen, Søren G F; Kobilka, Brian K

    2009-05-21

    G-protein-coupled receptors (GPCRs) mediate most of our physiological responses to hormones, neurotransmitters and environmental stimulants, and so have great potential as therapeutic targets for a broad spectrum of diseases. They are also fascinating molecules from the perspective of membrane-protein structure and biology. Great progress has been made over the past three decades in understanding diverse GPCRs, from pharmacology to functional characterization in vivo. Recent high-resolution structural studies have provided insights into the molecular mechanisms of GPCR activation and constitutive activity.

  6. Fabrication and application of G protein-coupled receptor microarrays.

    PubMed

    Fang, Ye; Webb, Brian; Hong, Yulong; Ferrie, Ann; Lai, Fang; Frutos, Anthony G; Lahiri, Joydeep

    2004-01-01

    The increased number of drug targets and compounds demands novel high-throughput screening technologies that could be used for parallel analysis of many genes and proteins. Protein microarrays are evolving promising technologies for the parallel analysis of many proteins with respect to their abundance, location, modifications, and interactions with other biological and chemical molecules. This chapter specifically describes the fabrication of G protein-coupled receptor (GPCR) microarrays, a unique subset of protein microarrays, using contact-pin printing technology. The bioassays and potential applications of GPCR microarrays for the determination of compound affinities and potencies are also included.

  7. Aspergillus Oxylipin Signaling and Quorum Sensing Pathways Depend on G Protein-Coupled Receptors

    PubMed Central

    Affeldt, Katharyn J.; Brodhagen, Marion; Keller, Nancy P.

    2012-01-01

    Oxylipins regulate Aspergillus development and mycotoxin production and are also involved in Aspergillus quorum sensing mechanisms. Despite extensive knowledge of how these oxylipins are synthesized and what processes they regulate, nothing is known about how these signals are detected and transmitted by the fungus. G protein-coupled receptors (GPCR) have been speculated to be involved as they are known oxylipin receptors in mammals, and many putative GPCRs have been identified in the Aspergilli. Here, we present evidence that oxylipins stimulate a burst in cAMP in A. nidulans, and that loss of an A. nidulans GPCR, gprD, prevents this cAMP accumulation. A. flavus undergoes an oxylipin-mediated developmental shift when grown at different densities, and this regulates spore, sclerotial and aflatoxin production. A. flavus encodes two putative GprD homologs, GprC and GprD, and we demonstrate here that they are required to transition to a high-density development state, as well as to respond to spent medium of a high-density culture. The finding of GPCRs that regulate production of survival structures (sclerotia), inoculum (spores) and aflatoxin holds promise for future development of anti-fungal therapeutics. PMID:23105976

  8. Suppression of the heterotrimeric G protein causes abnormal morphology, including dwarfism, in rice.

    PubMed

    Fujisawa, Y; Kato, T; Ohki, S; Ishikawa, A; Kitano, H; Sasaki, T; Asahi, T; Iwasaki, Y

    1999-06-22

    Transgenic rice containing an antisense cDNA for the alpha subunit of rice heterotrimeric G protein produced little or no mRNA for the subunit and exhibited abnormal morphology, including dwarf traits and the setting of small seeds. In normal rice, the mRNA for the alpha subunit was abundant in the internodes and florets, the tissues closely related to abnormality in the dwarf transformants. The position of the alpha-subunit gene was mapped on rice chromosome 5 by mapping with the restriction fragment length polymorphism. The position was closely linked to the locus of a rice dwarf mutant, Daikoku dwarf (d-1), which is known to exhibit abnormal phenotypes similar to those of the transformants that suppressed the endogenous mRNA for the alpha subunit by antisense technology. Analysis of the cDNAs for the alpha subunits of five alleles of Daikoku dwarf (d-1), ID-1, DK22, DKT-1, DKT-2, and CM1361-1, showed that these dwarf mutants had mutated in the coding region of the alpha-subunit gene. These results show that the G protein functions in the formation of normal internodes and seeds in rice.

  9. 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

  10. 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.

  11. 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

  12. 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.

  13. 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.

  14. 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.

  15. 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

  16. Oncotargeting G proteins: The Hippo in the room.

    PubMed

    Feng, Xiaodong; Chen, Qianming; Gutkind, J Silvio

    2014-11-30

    The core components of the Hippo pathway are conserved from flies to mammals. In humans, these include a kinase cascade initiated by the Hippo kinase MST1/2 associated with the adaptor protein WW45/SAV1, and LATS1/2 in complex with MOB1, which in turn, phosphorylates and inhibits the mammalian transcription co-activator YAP and its related protein TAZ. YAP plays a critical role in organ size control during development, and its persistent nuclear localization and activation contributes to multiple human malignancies. The mechanisms driving YAP activation in most cancers, however, are often not clearly understood. In recent studies, we and Guan's team found that YAP activation represents a key molecular event contributing to uveal melanoma, the most frequent ocular malignancy in adults. Uveal melanoma growth is driven by gain-of-function mutations in GNAQ or GNA11 oncogenes, encoding persistently active G protein α subunits of the Gq family. As the signaling capacity of G proteins and their coupled receptors (GPCRs) has been extensively investigated, these findings provided an opportunity to identify cancer-associated mechanisms resulting in YAP activation, and to explore whether YAP represents a suitable oncotarget for cancer treatment.

  17. Identification of independent association signals and putative functional variants for breast cancer risk through fine-scale mapping of the 12p11 locus.

    PubMed

    Zeng, Chenjie; Guo, Xingyi; Long, Jirong; Kuchenbaecker, Karoline B; Droit, Arnaud; Michailidou, Kyriaki; Ghoussaini, Maya; Kar, Siddhartha; Freeman, Adam; Hopper, John L; Milne, Roger L; Bolla, Manjeet K; Wang, Qin; Dennis, Joe; Agata, Simona; Ahmed, Shahana; Aittomäki, Kristiina; Andrulis, Irene L; Anton-Culver, Hoda; Antonenkova, Natalia N; Arason, Adalgeir; Arndt, Volker; Arun, Banu K; Arver, Brita; Bacot, Francois; Barrowdale, Daniel; Baynes, Caroline; Beeghly-Fadiel, Alicia; Benitez, Javier; Bermisheva, Marina; Blomqvist, Carl; Blot, William J; Bogdanova, Natalia V; Bojesen, Stig E; Bonanni, Bernardo; Borresen-Dale, Anne-Lise; Brand, Judith S; Brauch, Hiltrud; Brennan, Paul; Brenner, Hermann; Broeks, Annegien; Brüning, Thomas; Burwinkel, Barbara; Buys, Saundra S; Cai, Qiuyin; Caldes, Trinidad; Campbell, Ian; Carpenter, Jane; Chang-Claude, Jenny; Choi, Ji-Yeob; Claes, Kathleen B M; Clarke, Christine; Cox, Angela; Cross, Simon S; Czene, Kamila; Daly, Mary B; de la Hoya, Miguel; De Leeneer, Kim; Devilee, Peter; Diez, Orland; Domchek, Susan M; Doody, Michele; Dorfling, Cecilia M; Dörk, Thilo; Dos-Santos-Silva, Isabel; Dumont, Martine; Dwek, Miriam; Dworniczak, Bernd; Egan, Kathleen; Eilber, Ursula; Einbeigi, Zakaria; Ejlertsen, Bent; Ellis, Steve; Frost, Debra; Lalloo, Fiona; Fasching, Peter A; Figueroa, Jonine; Flyger, Henrik; Friedlander, Michael; Friedman, Eitan; Gambino, Gaetana; Gao, Yu-Tang; Garber, Judy; García-Closas, Montserrat; Gehrig, Andrea; Damiola, Francesca; Lesueur, Fabienne; Mazoyer, Sylvie; Stoppa-Lyonnet, Dominique; Giles, Graham G; Godwin, Andrew K; Goldgar, David E; González-Neira, Anna; Greene, Mark H; Guénel, Pascal; Haeberle, Lothar; Haiman, Christopher A; Hallberg, Emily; Hamann, Ute; Hansen, Thomas V O; Hart, Steven; Hartikainen, Jaana M; Hartman, Mikael; Hassan, Norhashimah; Healey, Sue; Hogervorst, Frans B L; Verhoef, Senno; Hendricks, Carolyn B; Hillemanns, Peter; Hollestelle, Antoinette; Hulick, Peter J; Hunter, David J; Imyanitov, Evgeny N; Isaacs, Claudine; Ito, Hidemi; Jakubowska, Anna; Janavicius, Ramunas; Jaworska-Bieniek, Katarzyna; Jensen, Uffe Birk; John, Esther M; Joly Beauparlant, Charles; Jones, Michael; Kabisch, Maria; Kang, Daehee; Karlan, Beth Y; Kauppila, Saila; Kerin, Michael J; Khan, Sofia; Khusnutdinova, Elza; Knight, Julia A; Konstantopoulou, Irene; Kraft, Peter; Kwong, Ava; Laitman, Yael; Lambrechts, Diether; Lazaro, Conxi; Le Marchand, Loic; Lee, Chuen Neng; Lee, Min Hyuk; Lester, Jenny; Li, Jingmei; Liljegren, Annelie; Lindblom, Annika; Lophatananon, Artitaya; Lubinski, Jan; Mai, Phuong L; Mannermaa, Arto; Manoukian, Siranoush; Margolin, Sara; Marme, Frederik; Matsuo, Keitaro; McGuffog, Lesley; Meindl, Alfons; Menegaux, Florence; Montagna, Marco; Muir, Kenneth; Mulligan, Anna Marie; Nathanson, Katherine L; Neuhausen, Susan L; Nevanlinna, Heli; Newcomb, Polly A; Nord, Silje; Nussbaum, Robert L; Offit, Kenneth; Olah, Edith; Olopade, Olufunmilayo I; Olswold, Curtis; Osorio, Ana; Papi, Laura; Park-Simon, Tjoung-Won; Paulsson-Karlsson, Ylva; Peeters, Stephanie; Peissel, Bernard; Peterlongo, Paolo; Peto, Julian; Pfeiler, Georg; Phelan, Catherine M; Presneau, Nadege; Radice, Paolo; Rahman, Nazneen; Ramus, Susan J; Rashid, Muhammad Usman; Rennert, Gad; Rhiem, Kerstin; Rudolph, Anja; Salani, Ritu; Sangrajrang, Suleeporn; Sawyer, Elinor J; Schmidt, Marjanka K; Schmutzler, Rita K; Schoemaker, Minouk J; Schürmann, Peter; Seynaeve, Caroline; Shen, Chen-Yang; Shrubsole, Martha J; Shu, Xiao-Ou; Sigurdson, Alice; Singer, Christian F; Slager, Susan; Soucy, Penny; Southey, Melissa; Steinemann, Doris; Swerdlow, Anthony; Szabo, Csilla I; Tchatchou, Sandrine; Teixeira, Manuel R; Teo, Soo H; Terry, Mary Beth; Tessier, Daniel C; Teulé, Alex; Thomassen, Mads; Tihomirova, Laima; Tischkowitz, Marc; Toland, Amanda E; Tung, Nadine; Turnbull, Clare; van den Ouweland, Ans M W; van Rensburg, Elizabeth J; Ven den Berg, David; Vijai, Joseph; Wang-Gohrke, Shan; Weitzel, Jeffrey N; Whittemore, Alice S; Winqvist, Robert; Wong, Tien Y; Wu, Anna H; Yannoukakos, Drakoulis; Yu, Jyh-Cherng; Pharoah, Paul D P; Hall, Per; Chenevix-Trench, Georgia; Dunning, Alison M; Simard, Jacques; Couch, Fergus J; Antoniou, Antonis C; Easton, Douglas F; Zheng, Wei

    2016-06-21

    Multiple recent genome-wide association studies (GWAS) have identified a single nucleotide polymorphism (SNP), rs10771399, at 12p11 that is associated with breast cancer risk. We performed a fine-scale mapping study of a 700 kb region including 441 genotyped and more than 1300 imputed genetic variants in 48,155 cases and 43,612 controls of European descent, 6269 cases and 6624 controls of East Asian descent and 1116 cases and 932 controls of African descent in the Breast Cancer Association Consortium (BCAC; http://bcac.ccge.medschl.cam.ac.uk/ ), and in 15,252 BRCA1 mutation carriers in the Consortium of Investigators of Modifiers of BRCA1/2 (CIMBA). Stepwise regression analyses were performed to identify independent association signals. Data from the Encyclopedia of DNA Elements project (ENCODE) and the Cancer Genome Atlas (TCGA) were used for functional annotation. Analysis of data from European descendants found evidence for four independent association signals at 12p11, represented by rs7297051 (odds ratio (OR) = 1.09, 95 % confidence interval (CI) = 1.06-1.12; P = 3 × 10(-9)), rs805510 (OR = 1.08, 95 % CI = 1.04-1.12, P = 2 × 10(-5)), and rs1871152 (OR = 1.04, 95 % CI = 1.02-1.06; P = 2 × 10(-4)) identified in the general populations, and rs113824616 (P = 7 × 10(-5)) identified in the meta-analysis of BCAC ER-negative cases and BRCA1 mutation carriers. SNPs rs7297051, rs805510 and rs113824616 were also associated with breast cancer risk at P < 0.05 in East Asians, but none of the associations were statistically significant in African descendants. Multiple candidate functional variants are located in putative enhancer sequences. Chromatin interaction data suggested that PTHLH was the likely target gene of these enhancers. Of the six variants with the strongest evidence of potential functionality, rs11049453 was statistically significantly associated with the expression of PTHLH and its nearby

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

    PubMed

    Bondar, Alexey; Lazar, Josef

    2017-06-09

    The Gi/o protein family transduces signals from a diverse group of G protein-coupled receptors (GPCRs). The observed specificity of Gi/o-GPCR coupling and the high rate of Gi/o signal transduction have been hypothesized to be enabled by the existence of stable associates between Gi/o proteins and their cognate GPCRs in the inactive state (Gi/o-GPCR preassembly). To test this hypothesis, we applied the recently developed technique of two-photon polarization microscopy (2PPM) to Gαi1 subunits labeled with fluorescent proteins and four GPCRs: the α2A-adrenergic receptor, GABAB, cannabinoid receptor type 1 (CB1R), and dopamine receptor type 2. Our experiments with non-dissociating mutants of fluorescently labeled Gαi1 subunits (exhibiting impaired dissociation from activated GPCRs) showed that 2PPM is capable of detecting GPCR-G protein interactions. 2PPM experiments with non-mutated fluorescently labeled Gαi1 subunits and α2A-adrenergic receptor, GABAB, or dopamine receptor type 2 receptors did not reveal any interaction between the Gi1 protein and the non-stimulated GPCRs. In contrast, non-stimulated CB1R exhibited an interaction with the Gi1 protein. Further experiments revealed that this interaction is caused solely by CB1R basal activity; no preassembly between CB1R and the Gi1 protein could be observed. Our results demonstrate that four diverse GPCRs do not preassemble with non-active Gi1 However, we also show that basal GPCR activity allows interactions between non-stimulated GPCRs and Gi1 (basal coupling). These findings suggest that Gi1 interacts only with active GPCRs and that the well known high speed of GPCR signal transduction does not require preassembly between G proteins and GPCRs. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  19. 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 (

  20. Colloidal Aggregation Causes Inhibition of G Protein-Coupled Receptors

    PubMed Central

    2013-01-01

    Colloidal aggregation is the dominant mechanism for artifactual inhibition of soluble proteins, and controls against it are now widely deployed. Conversely, investigating this mechanism for membrane-bound receptors has proven difficult. Here we investigate the activity of four well-characterized aggregators against three G protein-coupled receptors (GPCRs) recognizing peptide and protein ligands. Each of the aggregators was active at micromolar concentrations against the three GPCRs in cell-based assays. This activity could be attenuated by either centrifugation of the inhibitor stock solution or by addition of Tween-80 detergent. In the absence of agonist, the aggregators acted as inverse agonists, consistent with a direct receptor interaction. Meanwhile, several literature GPCR ligands that resemble aggregators themselves formed colloids, by both physical and enzymological tests. These observations suggest that some GPCRs may be artifactually antagonized by colloidal aggregates, an effect that merits the attention of investigators in this field. PMID:23437772

  1. The G Protein-Coupled Receptor Rhodopsin: A Historical Perspective

    PubMed Central

    Hofmann, Lukas; Palczewski, Krzysztof

    2015-01-01

    Rhodopsin is a key light-sensitive protein expressed exclusively in rod photoreceptor cells of the retina. Failure to express this transmembrane protein causes a lack of rod outer segment formation and progressive retinal degeneration, including the loss of cone photoreceptor cells. Molecular studies of rhodopsin have paved the way to understanding a large family of cell-surface membrane proteins called G protein-coupled receptors (GPCRs). Work started on rhodopsin over 100 years ago still continues today with substantial progress made every year. These activities underscore the importance of rhodopsin as a prototypical GPCR and receptor required for visual perception—the fundamental process of translating light energy into a biochemical cascade of events culminating in vision. PMID:25697513

  2. 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

  3. 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

  4. 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

  5. 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

  6. 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

  7. 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.

  8. 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.

  9. 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.

  10. 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.

  11. 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.

  12. 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

  13. 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.

  14. 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.

  15. 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.

  16. 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.

  17. 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.

  18. [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.

  19. A G protein-coupled receptor for UDP-glucose.

    PubMed

    Chambers, J K; Macdonald, L E; Sarau, H M; Ames, R S; Freeman, K; Foley, J J; Zhu, Y; McLaughlin, M M; Murdock, P; McMillan, L; Trill, J; Swift, A; Aiyar, N; Taylor, P; Vawter, L; Naheed, S; Szekeres, P; Hervieu, G; Scott, C; Watson, J M; Murphy, A J; Duzic, E; Klein, C; Bergsma, D J; Wilson, S; Livi, G P

    2000-04-14

    Uridine 5'-diphosphoglucose (UDP-glucose) has a well established biochemical role as a glycosyl donor in the enzymatic biosynthesis of carbohydrates. It is less well known that UDP-glucose may possess pharmacological activity, suggesting that a receptor for this molecule may exist. Here, we show that UDP-glucose, and some closely related molecules, potently activate the orphan G protein-coupled receptor KIAA0001 heterologously expressed in yeast or mammalian cells. Nucleotides known to activate P2Y receptors were inactive, indicating the distinctly novel pharmacology of this receptor. The receptor is expressed in a wide variety of human tissues, including many regions of the brain. These data suggest that some sugar-nucleotides may serve important physiological roles as extracellular signaling molecules in addition to their familiar role in intermediary metabolism.

  20. 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.

  1. 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

  2. Meeting report - Arf and Rab family G proteins.

    PubMed

    Casanova, James E; Hsu, Victor W; Jackson, Catherine L; Kahn, Richard A; Roy, Craig R; Stow, Jennifer L; Wandinger-Ness, Angela; Sztul, Elizabeth

    2013-12-01

    A FASEB Summer Research Conference entitled 'Arf and Rab family G proteins' was held in July 2013 at Snowmass Village, Snowmass, Colorado. Arfs and Rabs are two families of GTPases that control membrane trafficking in eukaryotic cells, and increasing evidence indicates that their functions are tightly coordinated. Because many workers in this field have focused on only one family, this meeting was designed to integrate our understanding of the two families. The conference was organized by Elizabeth Sztul (University of Alabama, Birmingham, USA) and Jim Casanova (University of Virginia, Charlottesville, USA), and provided an opportunity for approximately 90 scientists to communicate their work and discuss future directions for the field. The talks highlighted the structural, functional and regulatory properties of Arf and Rab GTPases and the need to develop coordinated approaches to investigate them. Here, we present the major themes that emerged from the meeting.

  3. 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.

  4. 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

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

    PubMed

    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.

  6. 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

  7. Human G protein-coupled receptor studies in Saccharomyces cerevisiae.

    PubMed

    Liu, Rongfang; Wong, Winsy; IJzerman, Adriaan P

    2016-08-15

    G protein-coupled receptors (GPCRs) are one of the largest families of membrane proteins, with approximately 800 different GPCRs in the human genome. Signaling via GPCRs regulates many biological processes, such as cell proliferation, differentiation, and development. In addition, many receptors have a pivotal role in immunophysiology. Many hormones and neurotransmitters are ligands for these receptors, and hence it is not surprising that many drugs, either mimicking or blocking the action of the bodily substances, have been developed. It is estimated that 30-40% of current drugs on the market target GPCRs. Further identifying and elucidating the functions of GPCRs will provide opportunities for novel drug discovery, including for immunotherapy. The budding yeast Saccharomyces cerevisiae (S. cerevisiae) is a very important and useful platform in this respect. There are many advantages of using a yeast assay system, as it is cheap, safe and stable; it is also convenient for rapid feasibility and optimization studies. Moreover, it offers a "null" background when studying human GPCRs. New developments regarding human GPCRs expressed in a yeast platform are providing insight into GPCR activation and signaling, and facilitate agonist and antagonist identification. In this review we summarize the latest findings regarding human G-protein-coupled receptors in studies using S. cerevisiae, ever since the year 2005 when we last published a review on this topic. We describe 11 families of GPCRs in detail, while including the principles and developments of each yeast system applied to these different GPCRs and highlight and generalize the experimental findings of GPCR function in these systems. Copyright © 2016 Elsevier Inc. All rights reserved.

  8. Saltational evolution of the heterotrimeric G protein signaling mechanisms in the plant kingdom.

    PubMed

    Urano, Daisuke; Maruta, Natsumi; Trusov, Yuri; Stoian, Richard; Wu, Qingyu; Liang, Ying; Jaiswal, Dinesh Kumar; Thung, Leena; Jackson, David; Botella, José Ramón; Jones, Alan M

    2016-09-20

    Signaling proteins evolved diverse interactions to provide specificity for distinct stimuli. Signaling complexity in the G protein (heterotrimeric guanosine triphosphate-binding protein) network was achieved in animals through subunit duplication and incremental evolution. By combining comprehensive and quantitative phenotypic profiles of Arabidopsis thaliana with protein evolution informatics, we found that plant heterotrimeric G protein machinery evolved by a saltational (jumping) process. Sequence similarity scores mapped onto tertiary structures, and biochemical validation showed that the extra-large Gα (XLG) subunit evolved extensively in the charophycean algae (an aquatic green plant) by gene duplication and gene fusion. In terrestrial plants, further evolution uncoupled XLG from its negative regulator, regulator of G protein signaling, but preserved an α-helix region that enables interaction with its partner Gβγ. The ancestral gene evolved slowly due to the molecular constraints imposed by the need for the protein to maintain interactions with various partners, whereas the genes encoding XLG proteins evolved rapidly to produce three highly divergent members. Analysis of A. thaliana mutants indicated that these Gα and XLG proteins all function with Gβγ and evolved to operate both independently and cooperatively. The XLG-Gβγ machinery specialized in environmental stress responses, whereas the canonical Gα-Gβγ retained developmental roles. Some developmental processes, such as shoot development, involve both Gα and XLG acting cooperatively or antagonistically. These extensive and rapid evolutionary changes in XLG structure compared to those of the canonical Gα subunit contrast with the accepted notion of how pathway diversification occurs through gene duplication with subsequent incremental coevolution of residues among interacting proteins. Copyright © 2016, American Association for the Advancement of Science.

  9. Do Plants Contain G Protein-Coupled Receptors?1[C][W][OPEN

    PubMed Central

    Taddese, Bruck; Upton, Graham J.G.; Bailey, Gregory R.; Jordan, Siân R.D.; Abdulla, Nuradin Y.; Reeves, Philip J.; Reynolds, Christopher A.

    2014-01-01

    Whether G protein-coupled receptors (GPCRs) exist in plants is a fundamental biological question. Interest in deorphanizing new GPCRs arises because of their importance in signaling. Within plants, this is controversial, as genome analysis has identified 56 putative GPCRs, including G protein-coupled receptor1 (GCR1), which is reportedly a remote homolog to class A, B, and E GPCRs. Of these, GCR2 is not a GPCR; more recently, it has been proposed that none are, not even GCR1. We have addressed this disparity between genome analysis and biological evidence through a structural bioinformatics study, involving fold recognition methods, from which only GCR1 emerges as a strong candidate. To further probe GCR1, we have developed a novel helix-alignment method, which has been benchmarked against the class A-class B-class F GPCR alignments. In addition, we have presented a mutually consistent set of alignments of GCR1 homologs to class A, class B, and class F GPCRs and shown that GCR1 is closer to class A and/or class B GPCRs than class A, class B, or class F GPCRs are to each other. To further probe GCR1, we have aligned transmembrane helix 3 of GCR1 to each of the six GPCR classes. Variability comparisons provide additional evidence that GCR1 homologs have the GPCR fold. From the alignments and a GCR1 comparative model, we have identified motifs that are common to GCR1, class A, B, and E GPCRs. We discuss the possibilities that emerge from this controversial evidence that GCR1 has a GPCR fold. PMID:24246381

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

    USDA-ARS?s Scientific Manuscript database

    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...

  11. 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").

  12. 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.

  13. 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

  14. 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.

  15. 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.

  16. 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. © 2013 Elsevier Inc. All rights reserved.

  17. 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. Copyright © 2012 Wiley Periodicals, Inc.

  18. 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.

  19. 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.

  20. 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

  1. 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

  2. Modeling G Protein-Coupled Receptors: a Concrete Possibility.

    PubMed

    Costanzi, Stefano

    2010-01-01

    G protein-coupled receptors (GPCRs) are a large superfamily of membrane bound signaling proteins that are involved in the regulation of a wide range of physiological functions and constitute the most common target for therapeutic intervention. Due to the paucity of crystal structures, homology modeling has become a widespread technique for the construction of GPCR models, which have been applied to the study of their structure-function relationships and to the identification of lead ligands through virtual screening. Rhodopsin has been for years the only available template. However, recent breakthroughs in GPCR crystallography have led to the solution of the structures of a few additional receptors. In light of these newly elucidated crystal structures, we have been able to produce a substantial amount of data to demonstrate that accurate models of GPCRs in complex with their ligands can be constructed through homology modeling followed by fully flexible molecular docking. These results have been confirmed by our success in the first blind assessment of GPCR modeling and docking, organized in coordination with the solution of the X-ray structure of the adenosine A(2A) receptor. Taken together, these data indicate that: a) the transmembrane helical bundle can be modeled with considerable accuracy; b) predicting the binding mode of a ligand, although doable, is challenging; c) modeling of the extracellular and intracellular loops is still problematic.

  3. Homology Modeling of Class A G Protein-Coupled Receptors

    PubMed Central

    Costanzi, Stefano

    2012-01-01

    G protein-coupled receptors (GPCRs) are a large superfamily of membrane bound signaling proteins that hold great pharmaceutical interest. Since experimentally elucidated structures are available only for a very limited number of receptors, homology modeling has become a widespread technique for the construction of GPCR models intended to study the structure-function relationships of the receptors and aid the discovery and development of ligands capable of modulating their activity. Through this chapter, various aspects involved in the constructions of homology models of the serpentine domain of the largest class of GPCRs, known as class A or rhodopsin family, are illustrated. In particular, the chapter provides suggestions, guidelines and critical thoughts on some of the most crucial aspect of GPCR modeling, including: collection of candidate templates and a structure-based alignment of their sequences; identification and alignment of the transmembrane helices of the query receptor to the corresponding domains of the candidate templates; selection of one or more templates receptor; election of homology or de novo modeling for the construction of specific extracellular and intracellular domains; construction of the three-dimensional models, with special consideration to extracellular regions, disulfide bridges, and interhelical cavity; validation of the models through controlled virtual screening experiments. PMID:22323225

  4. Portraying G Protein-Coupled Receptors with Fluorescent Ligands

    PubMed Central

    2015-01-01

    The thermodynamics of ligand–receptor interactions at the surface of living cells represents a fundamental aspect of G protein-coupled receptor (GPCR) biology; thus, its detailed elucidation constitutes a challenge for modern pharmacology. Interestingly, fluorescent ligands have been developed for a variety of GPCRs in order to monitor ligand–receptor binding in living cells. Accordingly, new methodological strategies derived from noninvasive fluorescence-based approaches, especially fluorescence resonance energy transfer (FRET), have been successfully developed to characterize ligand–receptor interactions. Importantly, these technologies are supplanting more hazardous and expensive radioactive binding assays. In addition, FRET-based tools have also become extremely powerful approaches for visualizing receptor–receptor interactions (i.e., GPCR oligomerization) in living cells. Thus, by means of the synthesis of compatible fluorescent ligands these novel techniques can be implemented to demonstrate the existence of GPCR oligomerization not only in heterologous systems but also in native tissues. Finally, there is no doubt that these methodologies would also be relevant in drug discovery in order to develop new high-throughput screening approaches or to identify new therapeutic targets. Overall, herein, we provide a thorough assessment of all technical and biological aspects, including strengths and weaknesses, of these fluorescence-based methodologies when applied to the study of GPCR biology at the plasma membrane of living cells. PMID:25010291

  5. 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

  6. 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

  7. 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.

  8. 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

  9. 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

  10. G-protein from Medicago sativa: functional association to photoreceptors.

    PubMed Central

    Muschietti, J P; Martinetto, H E; Coso, O A; Farber, M D; Torres, H N; Flawia, M M

    1993-01-01

    G-protein subunits were characterized from Medicago sativa (alfalfa) seedlings. Crude membranes and GTP-Sepharose-purified fractions were electrophoresed on SDS/polyacrylamide gels and analysed by Western blotting with 9193 (anti-alpha common) and AS/7 (anti-alpha t, anti-alpha i1 and anti-alpha i2) polyclonal antibodies. These procedures led to the identification of a specific polypeptide band of about 43 kDa. Another polypeptide reacting with the SW/1 (anti-beta) antibody, of about 37 kDa, was also detected. The 43 kDa polypeptide bound specifically [alpha-32P]GTP by a photoaffinity reaction and was ADP-ribosylated by activated cholera toxin, but not by pertussis toxin. Irradiation of etiolated Medicago sativa protoplast preparations at 660 nm for 1 min produced a maximal increase in the guanosine 5'-[gamma-thio]triphosphate (GTP[35S])-binding rate. After this period of irradiation, the binding rate tended to decrease. The effect of a red-light (660 nm) pulse on the binding rate was reversed when it was immediately followed by a period of far-red (> 730 nm) illumination. These results may suggest that activation of GTP[S]-binding rate was a consequence of conversion of phytochrome Pr into the Ptr form. Images Figure 1 Figure 2 Figure 3 PMID:8484719

  11. 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

  12. Chemogenomics approaches to G-protein coupled receptor lead finding.

    PubMed

    Klabunde, T; Jäger, R

    2006-01-01

    G-protein coupled receptors (GPCRs) are promising targets for the discovery of novel drugs. In order to identify novel chemical series, high-throughput screening (HTS) is often complemented by rational chemogenomics lead finding approaches. We have compiled a GPCR directed screening set by ligand-based virtual screening of our corporate compound database. This set of compounds is supplemented with novel libraries synthesized around proprietary scaffolds. These target-directed libraries are designed using the knowledge of privileged fragments and pharmacophores to address specific GPCR subfamilies (e.g., purinergic or chemokine-binding GPCRs). Experimental testing of the GPCR collection has provided novel chemical series for several GPCR targets including the adenosine A1, the P2Y12, and the chemokine CCR1 receptor. In addition, GPCR sequence motifs linked to the recognition of GPCR ligands (termed chemoprints) are identified using homology modeling, molecular docking, and experimental profiling. These chemoprints can support the design and synthesis of compound libraries tailor-made for a novel GPCR target.

  13. 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.

  14. 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.

  15. Erythrocyte G Protein as a Novel Target for Malarial Chemotherapy

    PubMed Central

    Murphy, Sean C; Harrison, Travis; Hamm, Heidi E; Lomasney, Jon W; Mohandas, Narla; Haldar, Kasturi

    2006-01-01

    Background Malaria remains a serious health problem because resistance develops to all currently used drugs when their parasite targets mutate. Novel antimalarial drug targets are urgently needed to reduce global morbidity and mortality. Our prior results suggested that inhibiting erythrocyte Gs signaling blocked invasion by the human malaria parasite Plasmodium falciparum. Methods and Findings We investigated the erythrocyte guanine nucleotide regulatory protein Gs as a novel antimalarial target. Erythrocyte “ghosts” loaded with a Gs peptide designed to block Gs interaction with its receptors, were blocked in β-adrenergic agonist-induced signaling. This finding directly demonstrates that erythrocyte Gs is functional and that propranolol, an antagonist of G protein–coupled β-adrenergic receptors, dampens Gs activity in erythrocytes. We subsequently used the ghost system to directly link inhibition of host Gs to parasite entry. In addition, we discovered that ghosts loaded with the peptide were inhibited in intracellular parasite maturation. Propranolol also inhibited blood-stage parasite growth, as did other β2-antagonists. β-blocker growth inhibition appeared to be due to delay in the terminal schizont stage. When used in combination with existing antimalarials in cell culture, propranolol reduced the 50% and 90% inhibitory concentrations for existing drugs against P. falciparum by 5- to 10-fold and was also effective in reducing drug dose in animal models of infection. Conclusions Together these data establish that, in addition to invasion, erythrocyte G protein signaling is needed for intracellular parasite proliferation and thus may present a novel antimalarial target. The results provide proof of the concept that erythrocyte Gs antagonism offers a novel strategy to fight infection and that it has potential to be used to develop combination therapies with existing antimalarials. PMID:17194200

  16. 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.

  17. 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

  18. 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

  19. Evolutionary trace of G protein-coupled receptors reveals clusters of residues that determine global and class-specific functions.

    PubMed

    Madabushi, Srinivasan; Gross, Alecia K; Philippi, Anne; Meng, Elaine C; Wensel, Theodore G; Lichtarge, Olivier

    2004-02-27

    G protein-coupled receptor (GPCR) activation mediated by ligand-induced structural reorganization of its helices is poorly understood. To determine the universal elements of this conformational switch, we used evolutionary tracing (ET) to identify residue positions commonly important in diverse GPCRs. When mapped onto the rhodopsin structure, these trace residues cluster into a network of contacts from the retinal binding site to the G protein-coupling loops. Their roles in a generic transduction mechanism were verified by 211 of 239 published mutations that caused functional defects. When grouped according to the nature of the defects, these residues sub-divided into three striking sub-clusters: a trigger region, where mutations mostly affect ligand binding, a coupling region near the cytoplasmic interface to the G protein, where mutations affect G protein activation, and a linking core in between where mutations cause constitutive activity and other defects. Differential ET analysis of the opsin family revealed an additional set of opsin-specific residues, several of which form part of the retinal binding pocket, and are known to cause functional defects upon mutation. To test the predictive power of ET, we introduced novel mutations in bovine rhodopsin at a globally important position, Leu-79, and at an opsin-specific position, Trp-175. Both were functionally critical, causing constitutive G protein activation of the mutants and rapid loss of regeneration after photobleaching. These results define in GPCRs a canonical signal transduction mechanism where ligand binding induces conformational changes propagated through adjacent trigger, linking core, and coupling regions.

  20. Shuttling of G protein subunits between the plasma membrane and intracellular membranes.

    PubMed

    Chisari, Mariangela; Saini, Deepak Kumar; Kalyanaraman, Vani; Gautam, Narasimhan

    2007-08-17

    Heterotrimeric G proteins (alphabetagamma) mediate the majority of signaling pathways in mammalian cells. It is long held that G protein function is localized to the plasma membrane. Here we examined the spatiotemporal dynamics of G protein localization using fluorescence recovery after photobleaching, fluorescence loss in photobleaching, and a photoswitchable fluorescent protein, Dronpa. Unexpectedly, G protein subunits shuttle rapidly (t1/2 < 1 min) between the plasma membrane and intracellular membranes. We show that consistent with such shuttling, G proteins constitutively reside in endomembranes. Furthermore, we show that shuttling is inhibited by 2-bromopalmitate. Thus, contrary to present thought, G proteins do not reside permanently on the plasma membrane but are constantly testing the cytoplasmic surfaces of the plasma membrane and endomembranes to maintain G protein pools in intracellular membranes to establish direct communication between receptors and endomembranes.

  1. 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

  2. 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.

  3. Two putative MAP kinase genes, ZrHOG1 and ZrHOG2, cloned from the salt-tolerant yeast Zygosaccharomyces rouxii are functionally homologous to the Saccharomyces cerevisiae HOG1 gene.

    PubMed

    Iwaki, T; Tamai, Y; Watanabe, Y

    1999-01-01

    The salt-tolerant yeast Zygosaccharomyces rouxii can adjust its osmotic balance when responding to osmotic shock by accumulating glycerol as the compatible osmolyte. However, the mechanism of glycerol production in Z. rouxii cells and its genetic regulation remain to be elucidated. Two putative mitogen-activated protein (MAP) kinase genes, ZrHOG1 and ZrHOG2, were cloned from Z. rouxii by their homology with HOG1 from Saccharomyces cerevisiae. The deduced amino acid sequences of ZrHog1p and ZrHog2p indicated close homology to that of Hog1p and contained a TGY motif for phosphorylation by MAP kinase kinase. When ZrHOG1 or ZrHOG2 was expressed in an S. cerevisiae hog1delta null mutant, the salt tolerance and osmotic tolerance characteristics of wild-type S. cerevisiae were restored. In addition, the aberrant cell morphology and low glycerol content of the hog1delta null mutant were corrected, indicating that ZrHog1p and ZrHog2p have functions similar to Hog1p. While the transcription of the glycerol-3-phosphate dehydrogenase gene (GPD1) of the ZrHOG1-harbouring S. cerevisiae mutant was similar to that of wild-type S. cerevisiae, the ZrHOG2-harbouring strain showed prolonged GPD1 transcription. Both Zrhog1delta and Zrhog2delta Z. rouxii null mutants showed a decrease in salt tolerance compared to the wild-type strain. The present study suggested the presence of a high-osmolarity glycerol response (HOG) pathway in Z. rouxii similar to that elucidated in S. cerevisiae. Two putative MAP kinase genes in Z. rouxii appeared to be significant in either osmotic regulation or ion homeostasis.

  4. 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.

  5. GIPC participates in G protein signaling downstream of insulin-like growth factor 1 receptor.

    PubMed

    Booth, Ronald A; Cummings, Cathy; Tiberi, Mario; Liu, X Johné

    2002-02-22

    Several recent studies have demonstrated that insulin-like growth factor (IGF)-1-induced mitogen-activated protein kinase (MAP kinase) activation is abolished by pertussis toxin, suggesting that trimeric G proteins of the G(i) class are novel cellular targets of the IGF-1 signaling pathway. We report here that the intracellular domain of the Xenopus IGF-1 receptor is capable of binding to the Xenopus homolog of mammalian GIPC, a PDZ domain-containing protein previously identified as a binding partner of G(i)-specific GAP (RGS-GAIP). Binding of xGIPC to xIGF-1 receptor is independent of the kinase activity of the receptor and appears to require the PDZ domain of xGIPC. Injection of two C-terminal truncation mutants that retained the PDZ domain blocked IGF-1-induced Xenopus MAP kinase activation and oocyte maturation. While full-length xGIPC injection did not significantly alter insulin response, it greatly enhanced human RGS-GAIP in stimulating the insulin response in frog oocytes. This represents the first demonstration that GIPC x RGS-GAIP complex acts positively in IGF-1 receptor signal transduction.

  6. 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.

  7. 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

  8. 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.

  9. 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

  10. A gene-rich linkage map in the dioecious species Actinidia chinensis (kiwifruit) reveals putative X/Y sex-determining chromosomes.

    PubMed

    Fraser, Lena G; Tsang, Gianna K; Datson, Paul M; De Silva, H Nihal; Harvey, Catherine F; Gill, Geoffrey P; Crowhurst, Ross N; McNeilage, Mark A

    2009-03-10

    The genus Actinidia (kiwifruit) consists of woody, scrambling vines, native to China, and only recently propagated as a commercial crop. All species described are dioecious, but the genetic mechanism for sex-determination is unknown, as is the genetic basis for many of the cluster of characteristics making up the unique fruit. It is, however, an important crop in the New Zealand economy, and a classical breeding program would benefit greatly by knowledge of the trait alleles carried by both female and male parents. The application of marker assisted selection (MAS) in seedling populations would also aid the accurate and efficient development of novel fruit types for the market. Gene-rich female, male and consensus linkage maps of the diploid species A. chinensis have been constructed with 644 microsatellite markers. The maps consist of twenty-nine linkage groups corresponding to the haploid number n = 29. We found that sex-linked sequence characterized amplified region (SCAR) markers and the 'Flower-sex' phenotype consistently mapped to a single linkage group, in a subtelomeric region, in a section of inconsistent marker order. The region also contained markers of expressed genes, some of unknown function. Recombination, assessed by allelic distribution and marker order stability, was, in the remainder of the linkage group, in accordance with other linkage groups. Fully informative markers to other genes in this linkage group identified the comparative linkage group in the female map, where recombination ratios determining marker order were similar to the autosomes. We have created genetic linkage maps that define the 29 linkage groups of the haploid genome, and have revealed the position and extent of the sex-determining locus in A. chinensis. As all Actinidia species are dioecious, we suggest that the sex-determining loci of other Actinidia species will be similar to that region defined in our maps. As the extent of the non-recombining region is limited, our

  11. A gene-rich linkage map in the dioecious species Actinidia chinensis (kiwifruit) reveals putative X/Y sex-determining chromosomes

    PubMed Central

    Fraser, Lena G; Tsang, Gianna K; Datson, Paul M; De Silva, H Nihal; Harvey, Catherine F; Gill, Geoffrey P; Crowhurst, Ross N; McNeilage, Mark A

    2009-01-01

    Background The genus Actinidia (kiwifruit) consists of woody, scrambling vines, native to China, and only recently propagated as a commercial crop. All species described are dioecious, but the genetic mechanism for sex-determination is unknown, as is the genetic basis for many of the cluster of characteristics making up the unique fruit. It is, however, an important crop in the New Zealand economy, and a classical breeding program would benefit greatly by knowledge of the trait alleles carried by both female and male parents. The application of marker assisted selection (MAS) in seedling populations would also aid the accurate and efficient development of novel fruit types for the market. Results Gene-rich female, male and consensus linkage maps of the diploid species A. chinensis have been constructed with 644 microsatellite markers. The maps consist of twenty-nine linkage groups corresponding to the haploid number n = 29. We found that sex-linked sequence characterized amplified region (SCAR) markers and the 'Flower-sex' phenotype consistently mapped to a single linkage group, in a subtelomeric region, in a section of inconsistent marker order. The region also contained markers of expressed genes, some of unknown function. Recombination, assessed by allelic distribution and marker order stability, was, in the remainder of the linkage group, in accordance with other linkage groups. Fully informative markers to other genes in this linkage group identified the comparative linkage group in the female map, where recombination ratios determining marker order were similar to the autosomes. Conclusion We have created genetic linkage maps that define the 29 linkage groups of the haploid genome, and have revealed the position and extent of the sex-determining locus in A. chinensis. As all Actinidia species are dioecious, we suggest that the sex-determining loci of other Actinidia species will be similar to that region defined in our maps. As the extent of the non

  12. A Monte Carlo study of the dynamics of G-protein activation.

    PubMed Central

    Mahama, P A; Linderman, J J

    1994-01-01

    To link quantitatively the cell surface binding of ligand to receptor with the production of cellular responses, it may be necessary to explore early events in signal transduction such as G-protein activation. Two different model frameworks relating receptor/ligand binding to G-protein activation are examined. In the first framework, a simple ordinary differential equation model is used to describe receptor/ligand binding and G-protein activation. In the second framework, the events leading to G-protein activation are simulated using a dynamic Monte Carlo model. In both models, reactions between ligand-bound receptors and G-proteins are assumed to be diffusion-limited. The Monte Carlo model predicts two regimes of G-protein activation, depending upon whether the lifetime of a receptor/ligand complex is long or short compared with the time needed for diffusional encounters of complexes and G-proteins. When the lifetime of a complex is relatively short compared with the diffusion time, the movement of ligand among free receptors by binding and unbinding ("switching") significantly enhances G-protein activation. Receptor antagonists dramatically reduce G-protein activation and, thus, signal transduction in this case, and significant clustering of active G-proteins near receptor/ligand complexes results. The simple ordinary differential equation model poorly predicts G-protein activation for this situation. In the alternative case, when diffusion is relatively fast, ligand movement among receptors is less important and the simple ordinary differential equation model and Monte Carlo model results are similar. In this case, there is little clustering of active G-proteins near receptor/ligand complexes. Results also indicate that as the GTPase activity of the alpha-subunit decreases, the steady-state level of alpha-GTP increases, although temporal sensitivity is compromised. PMID:7811949

  13. Comprehensive analysis of heterotrimeric G-protein complex diversity and their interactions with GPCRs in solution

    PubMed Central

    Hillenbrand, Matthias; Schori, Christian; Schöppe, Jendrik; Plückthun, Andreas

    2015-01-01

    Agonist binding to G-protein–coupled receptors (GPCRs) triggers signal transduction cascades involving heterotrimeric G proteins as key players. A major obstacle for drug design is the limited knowledge of conformational changes upon agonist binding, the details of interaction with the different G proteins, and the transmission to movements within the G protein. Although a variety of different GPCR/G protein complex structures would be needed, the transient nature of this complex and the intrinsic instability against dissociation make this endeavor very challenging. We have previously evolved GPCR mutants that display higher stability and retain their interaction with G proteins. We aimed at finding all G-protein combinations that preferentially interact with neurotensin receptor 1 (NTR1) and our stabilized mutants. We first systematically analyzed by coimmunoprecipitation the capability of 120 different G-protein combinations consisting of αi1 or αsL and all possible βγ-dimers to form a heterotrimeric complex. This analysis revealed a surprisingly unrestricted ability of the G-protein subunits to form heterotrimeric complexes, including βγ-dimers previously thought to be nonexistent, except for combinations containing β5. A second screen on coupling preference of all G-protein heterotrimers to NTR1 wild type and a stabilized mutant indicated a preference for those Gαi1βγ combinations containing γ1 and γ11. Heterotrimeric G proteins, including combinations believed to be nonexistent, were purified, and complexes with the GPCR were prepared. Our results shed new light on the combinatorial diversity of G proteins and their coupling to GPCRs and open new approaches to improve the stability of GPCR/G-protein complexes. PMID:25733868

  14. 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

  15. 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

  16. Identification, purification, and characterization of GRK5, a member of the family of G protein-coupled receptor kinases.

    PubMed

    Premont, R T; Koch, W J; Inglese, J; Lefkowitz, R J

    1994-03-04

    A novel member of the family of G protein-coupled receptor kinases (GRKs), named GRK5, has been cloned from bovine taste epithelium. The cDNA sequence predicts a 590-amino acid protein with high overall similarity to rhodopsin kinase. GRK5 mRNA is found most abundantly in lung, heart, retina, and lingual epithelium, but is expressed very little in brain, liver, kidney, or testis. GRK5 expressed in Sf9 cells was purified to apparent homogeneity. GRK5 major autophosphorylation sites were mapped to Ser484 and Thr485. Purified GRK5 phosphorylates rhodopsin in a light-dependent manner and beta 2-adrenergic receptor in an agonist-dependent manner and phosphorylates the C-terminal tail regions of both receptor proteins. GRK5 possesses neither a CAAX motif specifying protein prenylation like rhodopsin kinase nor similarity to the G protein beta gamma-subunit binding domain of beta-adrenergic receptor kinases. GRK5 phosphorylation of rhodopsin or beta 2-adrenergic receptor is not stimulated by G protein beta gamma-subunits. The GRK5 protein does not undergo agonist-dependent translocation from cytosol to membranes as do beta-adrenergic receptor kinase and rhodopsin kinase, but rather appears to associate with membranes constitutively. GRK5 thus appears functionally similar to other characterized GRKs, but has distinct regulatory properties which may be important for its cellular function.

  17. 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

  18. A putative vulnerability locus to multiple sclerosis maps to 5p14-p12 in a region syntenic to the murine locus Eae2.

    PubMed

    Kuokkanen, S; Sundvall, M; Terwilliger, J D; Tienari, P J; Wikström, J; Holmdahl, R; Pettersson, U; Peltonen, L

    1996-08-01

    Multiple sclerosis (MS) is a chronic inflammatory disorder characterized by multifocal damage of myelin in the central nervous system (CNS). The prevalence of this putative autoimmune disease is 0.1% in individuals of northern European origin. Family, adoption and twin studies implicate genetic factors in the aetiology. MS is widely speculated to be a multifactorial disorder with a complex mode of inheritance. Despite many studies of candidate genes, only an association with HLA-DR2-DQ6 has been generally detected, and the number of susceptibility genes remains unknown. The chronic variant of experimental allergic encephalomyelitis (EAE), a T-cell mediated autoimmune disease in rodents, represents a relevant animal model for MS given the chronic relapsing disease course and inflammatory changes of CNS observed in these demyelinating disorders. Susceptibility to EAE is also influenced by the major histocompatibility complex (MHC). Human syntenic regions to murine loci predisposing to EAE were tested as candidate regions for genetic susceptibility of MS. Three chromosomal regions (1p22-q23, 5p14-p12 and Xq13.2-q22) were screened in 21 Finnish multiplex MS families most originating from a high risk region in western Finland. Several markers yielded positive lod scores on 5p14-p12, syntenic to the murine locus Eae2. Our data provide evidence for a predisposing locus for MS on 5p14-p12.

  19. 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.

  20. Evidence for a G protein-coupled diadenosine-5',5'''-P1,P4-tetraphosphate (Ap4A) receptor binding site in lung membranes from rat.

    PubMed

    Laubinger, W; Reiser, G

    1999-01-29

    Nucleotide receptors are of considerable importance in the treatment of lung diseases, such as cystic fibrosis. Because diadenosine polyphosphates may also be of significance as signalling molecules in lung, as they are in a variety of tissues, in the present work we investigated the binding sites for [3H]diadenosine-5',5'''-P1,P4-tetraphosphate (Ap4A) in plasma membranes from rat lung and studied their possible coupling to G proteins. We present evidence for a single high-affinity binding site for [3H]Ap4A with similar affinity for other diadenosine polyphosphates ApnA (n = 2 to 6). Displacement studies with different nucleotides revealed that the [3H]Ap4A binding site was different from P2X and P2Y2 receptor binding sites. Pretreatment of lung membranes with GTPgammaS or GTP in the presence of Mg2+ increased the Ki for Ap4A from 91 nM to 5.1 microM, which is indicative of G protein coupling. The putative coupling to G proteins was further confirmed by the enhancement of [35S]GTPgammaS binding (to Galpha proteins) to lung membranes by Ap4A (63% increase over basal) in a concentration-dependent manner. Therefore, our data for the first time provide evidence of a G protein-coupled Ap4A binding site in lung membranes.

  1. The beta-subunit of the Arabidopsis G protein negatively regulates auxin-induced cell division and affects multiple developmental processes.

    PubMed

    Ullah, Hemayet; Chen, Jin-Gui; Temple, Brenda; Boyes, Douglas C; Alonso, José M; Davis, Keith R; Ecker, Joseph R; Jones, Alan M

    2003-02-01

    Plant cells respond to low concentrations of auxin by cell expansion, and at a slightly higher concentration, these cells divide. Previous work revealed that null mutants of the alpha-subunit of a putative heterotrimeric G protein (GPA1) have reduced cell division. Here, we show that this prototypical G protein complex acts mechanistically by controlling auxin sensitivity toward cell division. Loss-of-function G protein mutants have altered auxin-mediated cell division throughout development, especially during the auxin-induced formation of lateral and adventitious root primordia. Ectopic expression of the wild-type Galpha-subunit phenocopies the Gbeta mutants (auxin hypersensitivity), probably by sequestering the Gbetagamma-subunits, whereas overexpression of Gbeta reduces auxin sensitivity and a constitutively active (Q222L) mutant Galpha behaves like the wild type. These data are consistent with a model in which Gbetagamma acts as a negative regulator of auxin-induced cell division. Accordingly, basal repression of approximately one-third of the identified auxin-regulated genes (47 of 150 upregulated genes among 8300 quantitated) is lost in the Gbeta transcript-null mutant. Included among these are genes that encode proteins proposed to control cell division in root primordia formation as well as several novel genes. These results suggest that although auxin-regulated cell division is not coupled directly by a G protein, the Gbeta-subunit attenuates this auxin pathway upstream of the control of mRNA steady state levels.

  2. 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

  3. 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

  4. 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.

  5. 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

  6. 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.

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

    SciTech Connect

    Klopffleisch, Karsten; Phan, Nguyen; Chen, Jay; Panstruga, Ralph; Uhrig, Joachim; Jones, Alan M

    2011-01-01

    The heterotrimeric G-protein complex is minimally composed of G{alpha}, G{beta}, and G{gamma} 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.

  8. Structural basis of GDP release and gating in G protein coupled Fe[superscript 2+] transport

    SciTech Connect

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

    2009-09-29

    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 Fe{sup 2+} 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.

  9. The beta-subunit of G proteins is a substrate of protein histidine phosphatase.

    PubMed

    Mäurer, Anette; Wieland, Thomas; Meissl, Florian; Niroomand, Feraydoon; Mehringer, Rebecca; Krieglstein, Josef; Klumpp, Susanne

    2005-09-09

    Increasing evidence suggests that reversible phosphorylation of histidine residues in proteins is important for signaling cascades in eukaryotic cells. Recently, the first eukaryotic protein histidine phosphatase (PHP) was identified. The beta1-subunit of heterotrimeric G proteins (Gbeta) undergoes phosphorylation on His266 which is apparently involved in receptor-independent G protein activation. We studied whether phosphorylated Gbeta-subunits are substrates of PHP. Phosphorylated Gbetagamma dimers of the retinal G protein transducin and Gbeta in membrane preparations of H10 cells (neonatal rat cardiomyocytes) were dephosphorylated by PHP. Overexpression of PHP in H10 cells showed that PHP and Gbeta also interfere within cells. In membranes of cells overexpressing PHP, the amount of phosphorylated Gbeta was largely reduced. Both our in vitro and cell studies indicate that phosphorylated Gbeta-subunits of heterotrimeric G proteins are substrates of PHP. Therefore, PHP might play a role in the regulation of signal transduction via heterotrimeric G proteins.

  10. 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

  11. Surface Density of the Hendra G Protein Modulates Hendra F Protein-Promoted Membrane Fusion: Role for Hendra G Protein Trafficking and Degradation

    PubMed Central

    Whitman, Shannon D.; Dutch, Rebecca Ellis

    2007-01-01

    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. PMID:17328935

  12. 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.

  13. 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

  14. Specific subunits of heterotrimeric G proteins play important roles during nodulation in soybean.

    PubMed

    Choudhury, Swarup Roy; Pandey, Sona

    2013-05-01

    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.

  15. 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.

  16. Neurogenetics and clinical evidence for the putative activation of the brain reward circuitry by a neuroadaptagen: proposing an addiction candidate gene panel map.

    PubMed

    Chen, Thomas J H; Blum, Kenneth; Chen, Amanda L C; Bowirrat, Abdalla; Downs, William B; Madigan, Margret A; Waite, Roger L; Bailey, John A; Kerner, Mallory; Yeldandi, Swetha; Majmundar, Neil; Giordano, John; Morse, Siohban; Miller, David; Fornari, Frank; Braverman, Eric R

    2011-01-01

    This document presents evidence supporting the role of the KB220/KB220Z neuroadaptagens consisting of amino-acid neurotransmitter precursors and enkephalinase-catecholamine-methyl-transferase (COMT) inhibition therapy called Neuroadaptagen Amino Acid Therapy (NAAT) in brain reward function. It is becoming increasingly clear that this novel formulation is the first neuroadaptagen known to activate the brain reward circuitry. Ongoing research repeatedly confirms the numerous clinical effects that ultimately result in significant benefits for victims having genetic antecedents for all addictive, compulsive and impulsive behaviors. These behaviors are correctly classified under the rubric of"Reward Deficiency Syndrome" (RDS). We are proposing a novel addiction candidate gene map. We present preliminary findings in the United States using qEGG and in China using Functional Magnetic Resonance Imaging (fMRI) regarding the effects of oral NAAT on the activation of brain reward circuitry in victims of SUD. In unpublished data utilizing an fMRI 2X2 design at resting state, NAAT in comparison to placebo shows activation of the caudate brain region and potentially a smoothing out of heroin-induced putamen (a site for emotionality) abnormal connectivity. Although awaiting final analysis, if confirmed by ongoing studies in China coupled with published qEEG results in America, showing an increase in alpha and low beta, NAAT may be shown to impact treatment outcomes.

  17. Priming GPCR signaling through the synergistic effect of two G proteins.

    PubMed

    Gupte, Tejas M; Malik, Rabia U; Sommese, Ruth F; Ritt, Michael; Sivaramakrishnan, Sivaraj

    2017-04-04

    Although individual G-protein-coupled receptors (GPCRs) are known to activate one or more G proteins, the GPCR-G-protein interaction is viewed as a bimolecular event involving the formation of a ternary ligand-GPCR-G-protein complex. Here, we present evidence that individual GPCR-G-protein interactions can reinforce each other to enhance signaling through canonical downstream second messengers, a phenomenon we term "GPCR priming." Specifically, we find that the presence of noncognate Gq protein enhances cAMP stimulated by two Gs-coupled receptors, β2-adrenergic receptor (β2-AR) and D1 dopamine receptor (D1-R). Reciprocally, Gs enhances IP1 through vasopressin receptor (V1A-R) but not α1 adrenergic receptor (α1-AR), suggesting that GPCR priming is a receptor-specific phenomenon. The C terminus of either the Gαs or Gαq subunit is sufficient to enhance Gα subunit activation and cAMP levels. Interaction of Gαs or Gαq C termini with the GPCR increases signaling potency, suggesting an altered GPCR conformation as the underlying basis for GPCR priming. We propose three parallel mechanisms involving (i) sequential G-protein interactions at the cognate site, (ii) G-protein interactions at distinct allosteric and cognate sites on the GPCR, and (iii) asymmetric GPCR dimers. GPCR priming suggests another layer of regulation in the classic GPCR ternary-complex model, with broad implications for the multiplicity inherent in signaling networks.

  18. 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.

  19. 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.

  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. 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.

  2. 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

  3. Cloning and mapping of a human RBP56 gene encoding a putative RNA binding protein similar to FUS/TLS and EWS proteins.

    PubMed

    Morohoshi, F; Arai, K; Takahashi, E I; Tanigami, A; Ohki, M

    1996-11-15

    The EWS gene was found at the chromosome breakpoints in Ewing sarcoma, and the FUS/TLS gene was found at the breakpoints of myxoid liposarcoma and acute myeloid leukemia. These genes encode proteins that carry a highly homologous RNA binding domain. Fusion proteins made of the N-terminal half of EWS or FUS/TLS and transcriptional regulatory proteins, also derived from genes located at breakpoints, have been suggested to be involved in the pathogenesis of tumors. By PCR amplification of human Namalwa cell cDNA using degenerate primers made from the conserved amino acid sequences in the RNA binding domain of EWS and FUS/TLS, we obtained a cDNA fragment (RBP56 cDNA), the predicted amino acid sequences of which were similar but not identical to those of EWS and FUS/TLS. Using this fragment as a probe, we obtained two isoforms of cDNAs consisting of 2144 and 2153 bp, respectively, which encode proteins consisting of 589 and 592 amino acid residues, respectively. The predicted amino acid sequences of RBP56 protein have a serine-, tyrosine-, glutamine-, and glycine-rich region in the N-terminal region, an RNA binding domain and a C2C2 finger motif in the central region, and degenerate repeats of DR(S)GG(G)-YGG sequences in the C-terminal region. The expression of RBP56 mRNA was observed in all of the human fetal and adult tissues examined, as was the expression of EWS and FUS/TLS mRNAs. The RBP56 gene was mapped to chromosome 17q11.2 to q12.

  4. G protein-coupled receptor-type G proteins are required for light-dependent seedling growth and fertility in Arabidopsis.

    PubMed

    Jaffé, Felix W; Freschet, Gian-Enrico C; Valdes, Billy M; Runions, John; Terry, Matthew J; Williams, Lorraine E

    2012-09-01

    G protein-coupled receptor-type G proteins (GTGs) are highly conserved membrane proteins in plants, animals, and fungi that have eight to nine predicted transmembrane domains. They have been classified as G protein-coupled receptor-type G proteins that function as abscisic acid (ABA) receptors in Arabidopsis thaliana. We cloned Arabidopsis GTG1 and GTG2 and isolated new T-DNA insertion alleles of GTG1 and GTG2 in both Wassilewskija and Columbia backgrounds. These gtg1 gtg2 double mutants show defects in fertility, hypocotyl and root growth, and responses to light and sugars. Histological studies of shoot tissue reveal cellular distortions that are particularly evident in the epidermal layer. Stable expression of GTG1(pro):GTG1-GFP (for green fluorescent protein) in Arabidopsis and transient expression in tobacco (Nicotiana tabacum) indicate that GTG1 is localized primarily to Golgi bodies and to the endoplasmic reticulum. Microarray analysis comparing gene expression profiles in the wild type and double mutant revealed differences in expression of genes important for cell wall function, hormone response, and amino acid metabolism. The double mutants isolated here respond normally to ABA in seed germination assays, root growth inhibition, and gene expression analysis. These results are inconsistent with their proposed role as ABA receptors but demonstrate that GTGs are fundamentally important for plant growth and development.

  5. 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

  6. 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

  7. 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

  8. 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.

  9. 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).

  10. Rice transgenic plants with suppressed expression of the β subunit of the heterotrimeric G protein.

    PubMed

    Utsunomiya, Yuzuko; Samejima, Chihiro; Fujisawa, Yukiko; Kato, Hisaharu; Iwasaki, Yukimoto

    2012-04-01

    The deficient mutant for the rice heterotrimeric G protein α subunit gene (RGA1), d1, showed dwarfism and set small seed due to a reduced cell number. Mutants for the rice heterotrimeric G protein β subunit gene (RGB1) have not been isolated. To determine the functions of RGB1, transgenic rice plants with suppressed expression of RGB1 were studied using the RNAi method. RGB1 knock-down lines showed browning of the lamina joint regions and nodes and reduced fertility, but these abnormality were not observed in d1. Transgenic plants in which the G protein β subunit was greatly decreased were not obtained, suggesting that the complete suppression of RGB1 mRNA may be lethal. In contrast, the d1 mutants, with complete loss of the G protein α subunit, were fertile and half the size of the WT. These studies suggest that RGB1 has different functions than RGA1.

  11. Rice transgenic plants with suppressed expression of the β subunit of the heterotrimeric G protein

    PubMed Central

    Utsunomiya, Yuzuko; Samejima, Chihiro; Fujisawa, Yukiko; Kato, Hisaharu; Iwasaki, Yukimoto

    2012-01-01

    The deficient mutant for the rice heterotrimeric G protein α subunit gene (RGA1), d1, showed dwarfism and set small seed due to a reduced cell number. Mutants for the rice heterotrimeric G protein β subunit gene (RGB1) have not been isolated. To determine the functions of RGB1, transgenic rice plants with suppressed expression of RGB1 were studied using the RNAi method. RGB1 knock-down lines showed browning of the lamina joint regions and nodes and reduced fertility, but these abnormality were not observed in d1. Transgenic plants in which the G protein β subunit was greatly decreased were not obtained, suggesting that the complete suppression of RGB1 mRNA may be lethal. In contrast, the d1 mutants, with complete loss of the G protein α subunit, were fertile and half the size of the WT. These studies suggest that RGB1 has different functions than RGA1. PMID:22499179

  12. 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.

  13. A novel photointermediate of octopus rhodopsin activates its G-protein.

    PubMed

    Nakagawa, M; Kikkawa, S; Tominaga, K; Tsugi, N; Tsuda, M

    1998-10-02

    The photointermediate of octopus rhodopsin responsible for G-protein activation was examined by a GTPgammaS-binding assay in a reconstituted system with purified rhodopsin and photoreceptor G-protein. When octopus rhodopsin alone was incubated in the dark after illumination, its ability to stimulate GTPgammaS-binding by the G-protein decreased in a time-dependent manner. We associate this decay with the decay of a novel photointermediate, transient acid metarhodopsin, which lies between mesorhodopsin and acid metarhodopsin. Spectroscopic evidence for its existence was suggested by its effects on the turbidity of the vesicles. These results suggest that the transient acid metarhodopsin, not the stable final photoproduct, acid metarhodopsin, activates a G-protein in octopus photoreceptors.

  14. 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

  15. Competition between internal AlF(4)(-) and receptor-mediated stimulation of dorsal raphe neuron G-proteins coupled to calcium current inhibition.

    PubMed

    Chen, Y; Penington, N J

    2000-03-01

    Intracellular aluminum fluoride (AlF(4)(-)), placed in a patch pipette, activated a G-protein, resulting in a "tonic" inhibition of the Ca(2+) current of isolated serotonergic neurons of the rat dorsal raphe nucleus. Serotonin (5-HT) also inhibits the Ca(2+) current of these cells. After external bath application and quick removal of 5-HT to an AlF(4)(-) containing cell, there was a reversal or transient disinhibition (TD) of the inhibitory effect of AlF(4)(-) on Ca(2+) current. A short predepolarization of the membrane potential to +70 mV, a condition that is known to reverse G-protein-mediated inhibition, reversed the inhibitory effect of AlF(4)(-) on Ca(2+) current and brought the Ca(2+) current to the same level as that seen at the peak of the TD current. With AlF(4)(-) in the pipette, the TD phenomenon could be eliminated by lowering pipette MgATP, or by totally chelating pipette Al(3+). In the presence of AlF(4)(-), but with either lowered MgATP or extreme efforts to eliminate pipette Al(3+), the rate of recovery from 5-HT on wash was slowed, a condition opposite to that where a TD occurred. The putative complex of AlF(4)(-)-bound G-protein (Galpha.GDP. AlF(4)(-)) appeared to free G-betagamma-subunits, mimicking the effect on Ca(2+) channels of the G.GTP complex. The ON-rate of the inhibition of Ca(2+) current, after a depolarizing pulse, by betagamma-subunits released by AlF(4)(-) in the pipette was significantly slower than that of the agonist-activated G-protein. The OFF-rate of the AlF(4)(-)-mediated inhibition in response to a depolarizing pulse, a measure of the affinity of the free G-betagamma-subunit for the Ca(2+) channel, was slightly slower than that of the agonist stimulated G-protein. In summary, AlF(4)(-) modified the OFF-rate kinetics of G-protein activation by agonists, but had little effect on the kinetics of the interaction of the betagamma-subunit with Ca(2+) channels. Agonist application temporarily reversed the effects of AlF(4

  16. G-protein diseases furnish a model for the turn-on switch

    NASA Astrophysics Data System (ADS)

    Iiri, Taroh; Farfel, Zvi; Bourne, Henry R.

    1998-07-01

    How does a trimeric G protein on the inside of a cell membrane respond to activation by a transmembrane receptor? G-protein mutations in patients with hypertension and inherited endocrine disorders enhance or block signals from stimulated receptors. In combination with three-dimensional crystal structures and results from biochemical experiments, the phenotypes produced by these mutations suggest a model for the molecular activation mechanism that relays hormonal and sensory signals transmitted by many transmembrane receptors.

  17. Eukaryotic G Protein Signaling Evolved to Require G Protein–Coupled Receptors for Activation

    PubMed Central

    Bradford, William; Buckholz, Adam; Morton, John; Price, Collin; Jones, Alan M.; Urano, Daisuke

    2016-01-01

    Although bioinformatic analysis of the increasing numbers of diverse genome sequences and amount of functional data has provided insight into the evolution of signaling networks, bioinformatics approaches have limited application for understanding the evolution of highly divergent protein families. We used biochemical analyses to determine the in vitro properties of selected divergent components of the heterotrimeric guanine nucleotide–binding protein (G protein) signaling network to investigate signaling network evolution. In animals, G proteins are activated by cell-surface seven-transmembrane (7TM) receptors, which are named G protein–coupled receptors (GPCRs) and function as guanine nucleotide exchange factors (GEFs). In contrast, the plant G protein is intrinsically active, and a 7TM protein terminates G protein activity by functioning as a guanosine triphosphatase–activating protein (GAP). We showed that ancient regulation of the G protein active state is GPCR-independent and “self-activating,” a property that is maintained in Bikonts, one of the two fundamental evolutionary clades containing eukaryotes, whereas G proteins of the other clade, the Unikonts, evolved from being GEF-independent to being GEF-dependent. Self-activating G proteins near the base of the Eukaryota are controlled by 7TM-GAPs, suggesting that the ancestral regulator of G protein activation was a GAP-functioning receptor, not a GEF-functioning GPCR. Our findings indicate that the GPCR paradigm describes a recently evolved network architecture found in a relatively small group of Eukaryota and suggest that the evolution of signaling network architecture is constrained by the availability of molecules that control the activation state of nexus proteins. PMID:23695163

  18. Automated large-scale purification of a recombinant g-protein-coupled neurotensin receptor.

    PubMed

    White, Jim F; Grisshammer, Reinhard

    2007-02-01

    Structure determination of G-protein-coupled receptors and other applications, such as nuclear magnetic resonance (NMR) studies, require milligram quantities of purified, functional receptor protein on a regular basis. This unit presents a step-by-step procedure for the automated two-column purification at the 10-milligram scale of a G protein-coupled receptor for neurotensin, expressed in functional form in Escherichia coli.

  19. Receptor-dependent G-protein activation in lipidic cubic phase.

    PubMed

    Navarro, Javier; Landau, Ehud M; Fahmy, Karim

    2002-01-01

    The primary step in cellular signaling by G-protein-coupled receptors (GPCRs) is the interaction of the agonist-activated transmembrane receptor with an intracellular G-protein. Understanding the underlying molecular mechanisms requires the structural determination of receptor G-protein complexes that are not yet achieved. The crystal structure of the bovine photoreceptor rhodopsin, a prototypical GPCR, was solved recently and the structures of different states of engineered G-proteins were reported. Posttranslational hydrophobic modifications of G-proteins are in most cases removed for crystallization but play functional roles for interactions among G-protein subunits with receptors, as well as membranes. Bovine rhodopsin is reconstituted into lipidic cubic phases to assess their potential for crystallization of receptor G-protein complexes under conditions that may preserve the structural and functional roles of hydrophobic protein modifications. Three-dimensional bilayers of a bicontinuous lipidic cubic phase are successfully employed for crystallization of membrane and soluble proteins. UV-visible absorption and attenuated total reflection Fourier transform IR difference spectroscopy reveal that light activation of cubic phase reconstituted rhodopsin results in the generation of a metarhodopsin II-like state. Via diffusion along aqueous channels, transducin couples efficiently to this photoproduct as evidenced by the nucleotide-dependent increase of transducin fluorescence. Thus, rhodopsin transducin interactions do not crucially depend on the presence of sn1 and sn2 acyl chains, phospholipid head groups, or membrane planarity. Because lipidic cubic phases preserve the essential functional and structural properties of native rhodopsin and transducin, they appear suitable for the detergent-free crystallization of receptor G-protein complexes carrying a normal pattern of hydrophobic modifications. Copyright 2002 Wiley Periodicals, Inc. Biopolymers

  20. 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

  1. 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

  2. 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.

  3. 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

  4. 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

  5. Mapping.

    ERIC Educational Resources Information Center

    Kinney, Douglas M.; McIntosh, Willard L.

    1979-01-01

    The area of geological mapping in the United States in 1978 increased greatly over that reported in 1977; state geological maps were added for California, Idaho, Nevada, and Alaska last year. (Author/BB)

  6. Cardiac hyporesponsiveness in severe sepsis is associated with nitric oxide-dependent activation of G protein receptor kinase.

    PubMed

    Dal-Secco, Daniela; DalBó, Silvia; Lautherbach, Natalia E S; Gava, Fábio N; Celes, Mara R N; Benedet, Patricia O; Souza, Adriana H; Akinaga, Juliana; Lima, Vanessa; Silva, Katiussia P; Kiguti, Luiz Ricardo A; Rossi, Marcos A; Kettelhut, Isis C; Pupo, André S; Cunha, Fernando Q; Assreuy, Jamil

    2017-07-01

    G protein-coupled receptor kinase isoform 2 (GRK2) has a critical role in physiological and pharmacological responses to endogenous and exogenous substances. Sepsis causes an important cardiovascular dysfunction in which nitric oxide (NO) has a relevant role. The present study aimed to assess the putative effect of inducible NO synthase (NOS2)-derived NO on the activity of GRK2 in the context of septic cardiac dysfunction. C57BL/6 mice were submitted to severe septic injury by cecal ligation and puncture (CLP). Heart function was assessed by isolated and perfused heart, echocardiography, and β-adrenergic receptor binding. GRK2 was determined by immunofluorescence and Western blot analysis in the heart and isolated cardiac myocytes. Sepsis increased NOS2 expression in the heart, increased plasma nitrite + nitrate levels, and reduced isoproterenol-induced isolated ventricle contraction, whole heart tension development, and β-adrenergic receptor density. Treatment with 1400W or with GRK2 inhibitor prevented CLP-induced cardiac hyporesponsiveness 12 and 24 h after CLP. Increased labeling of total and phosphorylated GRK2 was detected in hearts after CLP. With treatment of 1400W or in hearts taken from septic NOS2 knockout mice, the activation of GRK2 was reduced. 1400W or GRK2 inhibitor reduced mortality, improved echocardiographic cardiac parameters, and prevented organ damage. Therefore, during sepsis, NOS2-derived NO increases GRK2, which leads to a reduction in β-adrenergic receptor density, contributing to the heart dysfunction. Isolated cardiac myocyte data indicate that NO acts through the soluble guanylyl cyclase/cGMP/PKG pathway. GRK2 inhibition may be a potential therapeutic target in sepsis-induced cardiac dysfunction.NEW & NOTEWORTHY The main novelty presented here is to show that septic shock induces cardiac hyporesponsiveness to isoproterenol by a mechanism dependent on nitric oxide and mediated by G protein-coupled receptor kinase isoform 2. Therefore

  7. The G protein β subunit controls virulence and multiple growth- and development-related traits in Verticillium dahliae.

    PubMed

    Tzima, Aliki K; Paplomatas, Epaminondas J; Tsitsigiannis, Dimitrios I; Kang, Seogchan

    2012-04-01

    To gain insight into the role of G protein-mediated signaling in virulence and development of the soilborne, wilt causing fungus Verticillium dahliae, the G protein β subunit gene (named as VGB) was disrupted in tomato race 1 strain of V. dahliae. A resulting mutant strain, 70ΔGb15, displayed drastic reduction in virulence, increased microsclerotia formation and conidiation, and decreased ethylene production compared to the corresponding wild type (wt) strain 70wt-r1. Moreover, 70ΔGb15 exhibited an elongated rather than radial growth pattern on agar media. A transformant of 70ΔGb15 (named as 70ΔGbPKAC1) that carries an extra copy of VdPKAC1, a V. dahliae gene encoding the catalytic subunit of the cAMP-dependent protein kinase A, exhibited wt growth pattern and conidiation, was unable to form microsclerotia, produced high amounts of ethylene, and exhibited virulence between that of 70ΔGb15 and 70wt-r1 on tomato plants. Phenotypical changes observed in 70ΔGb15 and 70ΔGbPKAC1 correlated with transcriptional changes in several genes involved in signaling (MAP kinase VMK1) and development (hydrophobin VDH1 and ACC synthase ACS1) of V. dahliae. Results from the present work suggest a linkage between VGB and VdPKAC1 signaling pathways in regulating virulence, hormone production and development in V. dahliae. Copyright © 2012 Elsevier Inc. All rights reserved.

  8. 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.

  9. 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

  10. Membrane-Localized Extra-Large G Proteins and Gβγ of the Heterotrimeric G Proteins Form Functional Complexes Engaged in Plant Immunity in Arabidopsis1[OPEN

    PubMed Central

    Maruta, Natsumi; Trusov, Yuri; Brenya, Eric; Parekh, Urvi

    2015-01-01

    In animals, heterotrimeric G proteins, comprising Gα, Gβ, and Gγ subunits, are molecular switches whose function tightly depends on Gα and Gβγ interaction. Intriguingly, in Arabidopsis (Arabidopsis thaliana), multiple defense responses involve Gβγ, but not Gα. We report here that the Gβγ dimer directly partners with extra-large G proteins (XLGs) to mediate plant immunity. Arabidopsis mutants deficient in XLGs, Gβ, and Gγ are similarly compromised in several pathogen defense responses, including disease development and production of reactive oxygen species. Genetic analysis of double, triple, and quadruple mutants confirmed that XLGs and Gβγ functionally interact in the same defense signaling pathways. In addition, mutations in XLG2 suppressed the seedling lethal and cell death phenotypes of BRASSINOSTEROID INSENSITIVE1-associated receptor kinase1-interacting receptor-like kinase1 mutants in an identical way as reported for Arabidopsis Gβ-deficient mutants. Yeast (Saccharomyces cerevisiae) three-hybrid and bimolecular fluorescent complementation assays revealed that XLG2 physically interacts with all three possible Gβγ dimers at the plasma membrane. Phylogenetic analysis indicated a close relationship between XLGs and plant Gα subunits, placing the divergence point at the dawn of land plant evolution. Based on these findings, we conclude that XLGs form functional complexes with Gβγ dimers, although the mechanism of action of these complexes, including activation/deactivation, must be radically different form the one used by the canonical Gα subunit and are not likely to share the same receptors. Accordingly, XLGs expand the repertoire of heterotrimeric G proteins in plants and reveal a higher level of diversity in heterotrimeric G protein signaling. PMID:25588736

  11. 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

  12. 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

  13. 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

  14. 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

  15. Prediction and identification of the effectors of heterotrimeric G proteins in rice (Oryza sativa L.).

    PubMed

    Li, Kuan; Xu, Chaoqun; Huang, Jian; Liu, Wei; Zhang, Lina; Wan, Weifeng; Tao, Huan; Li, Ling; Lin, Shoukai; Harrison, Andrew; He, Huaqin

    2017-03-01

    Heterotrimeric G protein signaling cascades are one of the primary metazoan sensing mechanisms linking a cell to environment. However, the number of experimentally identified effectors of G protein in plant is limited. We have therefore studied which tools are best suited for predicting G protein effectors in rice. Here, we compared the predicting performance of four classifiers with eight different encoding schemes on the effectors of G proteins by using 10-fold cross-validation. Four methods were evaluated: random forest, naive Bayes, K-nearest neighbors and support vector machine. We applied these methods to experimentally identified effectors of G proteins and randomly selected non-effector proteins, and tested their sensitivity and specificity. The result showed that random forest classifier with composition of K-spaced amino acid pairs and composition of motif or domain (CKSAAP_PROSITE_200) combination method yielded the best performance, with accuracy and the Mathew's correlation coefficient reaching 74.62% and 0.49, respectively. We have developed G-Effector, an online predictor, which outperforms BLAST, PSI-BLAST and HMMER on predicting the effectors of G proteins. This provided valuable guidance for the researchers to select classifiers combined with different feature selection encoding schemes. We used G-Effector to screen the effectors of G protein in rice, and confirmed the candidate effectors by gene co-expression data. Interestingly, one of the top 15 candidates, which did not appear in the training data set, was validated in a previous research work. Therefore, the candidate effectors list in this article provides both a clue for researchers as to their function and a framework of validation for future experimental work. It is accessible at http://bioinformatics.fafu.edu.cn/geffector. © The Author 2016. Published by Oxford University Press. For Permissions, please email: journals.permissions@oup.com.

  16. Neurotransmitter signaling through heterotrimeric G proteins: insights from studies in C. elegans

    PubMed Central

    Koelle, Michael R.

    2016-01-01

    Neurotransmitters signal via G protein coupled receptors (GPCRs) to modulate activity of neurons and muscles. C. elegans has ~150 G protein coupled neuropeptide receptor homologs and 28 additional GPCRs for small-molecule neurotransmitters. Genetic studies in C. elegans demonstrate that neurotransmitters diffuse far from their release sites to activate GPCRs on distant cells. Individual receptor types are expressed on limited numbers of cells and thus can provide very specific regulation of an individual neural circuit and behavior. G protein coupled neurotransmitter receptors signal principally via the three types of heterotrimeric G proteins defined by the G alpha subunits Gαo, Gαq, and Gαs. Each of these G alpha proteins is found in all neurons plus some muscles. Gαo and Gαq signaling inhibit and activate neurotransmitter release, respectively. Gαs signaling, like Gαq signaling, promotes neurotransmitter release. Many details of the signaling mechanisms downstream of Gαq and Gαs have been delineated and are consistent with those of their mammalian orthologs. The details of the signaling mechanism downstream of Gαo remain a mystery. Forward genetic screens in C. elegans have identified new molecular components of neural G protein signaling mechanisms, including Regulators of G protein Signaling (RGS proteins) that inhibit signaling, a new Gαq effector (the Trio RhoGEF domain), and the RIC-8 protein that is required for neuronal Gα signaling. A model is presented in which G proteins sum up the variety of neuromodulator signals that impinge on a neuron to calculate its appropriate output level. PMID:26937633

  17. 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

  18. G-protein-coupled receptor inactivation by an allosteric inverse-agonist antibody.

    PubMed

    Hino, Tomoya; Arakawa, Takatoshi; Iwanari, Hiroko; Yurugi-Kobayashi, Takami; Ikeda-Suno, Chiyo; Nakada-Nakura, Yoshiko; Kusano-Arai, Osamu; Weyand, Simone; Shimamura, Tatsuro; Nomura, Norimichi; Cameron, Alexander D; Kobayashi, Takuya; Hamakubo, Takao; Iwata, So; Murata, Takeshi

    2012-01-29

    G-protein-coupled receptors are the largest class of cell-surface receptors, and these membrane proteins exist in equilibrium between inactive and active states. Conformational changes induced by extracellular ligands binding to G-protein-coupled receptors result in a cellular response through the activation of G proteins. The A(2A) adenosine receptor (A(2A)AR) is responsible for regulating blood flow to the cardiac muscle and is important in the regulation of glutamate and dopamine release in the brain. Here we report the raising of a mouse monoclonal antibody against human A(2A)AR that prevents agonist but not antagonist binding to the extracellular ligand-binding pocket, and describe the structure of A(2A)AR in complex with the antibody Fab fragment (Fab2838). This structure reveals that Fab2838 recognizes the intracellular surface of A(2A)AR and that its complementarity-determining region, CDR-H3, penetrates into the receptor. CDR-H3 is located in a similar position to the G-protein carboxy-terminal fragment in the active opsin structure and to CDR-3 of the nanobody in the active β(2)-adrenergic receptor structure, but locks A(2A)AR in an inactive conformation. These results suggest a new strategy to modulate the activity of G-protein-coupled receptors.

  19. 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.

  20. 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

  1. 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

  2. Deprotonation of the Schiff base of rhodopsin is obligate in the activation of the G protein.

    PubMed Central

    Longstaff, C; Calhoon, R D; Rando, R R

    1986-01-01

    Photolysis of rhodopsin leads to the formation of an activated intermediate that activates a G protein, thus beginning the visual cascade. This activated form of rhodopsin appears coincident in time with the spectroscopically defined intermediate, metarhodopsin II. Metarhodopsin I, the precursor of metarhodopsin II, contains a protonated Schiff base, whereas metarhodopsin II does not. The question of whether the deprotonation of the protonated Schiff base is obligate in the formation of activated rhodopsin was addressed by monomethylating the active-site lysine of permethylated rhodopsin and determining whether this pigment can activate the G protein upon photolysis. The photolysis of the new pigment, which absorbs at 520 nm, led to the formation of a relatively stable metarhodopsin I-like intermediate with a lambda max of approximately equal to 485 nm, with no apparent formation of either metarhodopsin II- or metarhodopsin III-like intermediates. The only probe available to detect formation of the active form of rhodopsin is G protein activation. Photolysis of the pigment in the presence of the G protein did not lead to measurable activation of the GTPase activity of the latter. These studies establish a functional link between Schiff base deprotonation and activation of the G protein. It is concluded that proton transfer from the protonated Schiff base of rhodopsin is obligate for the initiation of visual transduction. PMID:3012559

  3. G protein-coupled receptor for nicotinic acid in mouse macrophages.

    PubMed

    Lorenzen, Anna; Stannek, Christina; Burmeister, Anja; Kalvinsh, Ivars; Schwabe, Ulrich

    2002-08-15

    The use of the HDL-elevating drug nicotinic acid in the treatment and prevention of atherosclerotic disease is limited by the frequent induction of skin flushing. The therapeutic effects of nicotinic acid are attributed to inhibition of lipolysis in adipose tissue via a G protein-coupled receptor, whereas the mechanism of flush induction by release of prostaglandin D(2) from macrophages is not understood. In this study, we investigated if macrophages contain nicotinic acid receptors. Specific guanine nucleotide sensitive binding sites for [(3)H]nicotinic acid were detected in membranes from mouse RAW 264.7 macrophages. Nicotinic acid and related heterocycles stimulated activation of pertussis toxin-sensitive G proteins. The rank orders of potency in macrophage membranes were identical for inhibition of [(3)H]nicotinic acid binding and G protein activation, and were pharmacologically indistinguishable from that of the G protein-coupled nicotinic acid receptor in spleen membranes. These results indicate that the effects of nicotinic acid on macrophages, spleen and probably adipocytes are mediated via an identical, unique G protein-coupled receptor.

  4. Activation of G-proteins by receptor-stimulated nucleoside diphosphate kinase in Dictyostelium.

    PubMed Central

    Bominaar, A A; Molijn, A C; Pestel, M; Veron, M; Van Haastert, P J

    1993-01-01

    Recently, interest in the enzyme nucleoside diphosphate kinase (EC2.7.4.6) has increased as a result of its possible involvement in cell proliferation and development. Since NDP kinase is one of the major sources of GTP in cells, it has been suggested that the effects of an altered NDP kinase activity on cellular processes might be the result of altered transmembrane signal transduction via guanine nucleotide-binding proteins (G-proteins). In the cellular slime mould Dictyostelium discoideum, extracellular cAMP induces an increase of phospholipase C activity via a surface cAMP receptor and G-proteins. In this paper it is demonstrated that part of the cellular NDP kinase is associated with the membrane and stimulated by cell surface cAMP receptors. The GTP produced by the action of NDP kinase is capable of activating G-proteins as monitored by altered G-protein-receptor interaction and the activation of the effector enzyme phospholipase C. Furthermore, specific monoclonal antibodies inhibit the effect of NDP kinase on G-protein activation. These results suggest that receptor-stimulated NDP kinase contributes to the mediation of hormone action by producing GTP for the activation of GTP-binding proteins. Images PMID:8389692

  5. 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.

  6. 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

  7. Discovery of G Protein-Biased D2 Dopamine Receptor Partial Agonists.

    PubMed

    Chen, Xin; McCorvy, John D; Fischer, Matthew G; Butler, Kyle V; Shen, Yudao; Roth, Bryan L; Jin, Jian

    2016-12-08

    Biased ligands (also known as functionally selective ligands) of G protein-coupled receptors are valuable tools for dissecting the roles of G protein-dependent and independent signaling pathways in health and disease. Biased ligands have also been increasingly pursued by the biomedical community as promising therapeutics with improved efficacy and reduced side effects compared with unbiased ligands. We previously discovered first-in-class β-arrestin-biased agonists of dopamine D2 receptor (D2R) by extensively exploring multiple regions of aripiprazole, a balanced D2R agonist. In our continuing efforts to identify biased agonists of D2R, we unexpectedly discovered a G protein-biased agonist of D2R, compound 1, which is the first G protein-biased D2R agonist from the aripiprazole scaffold. We designed and synthesized novel analogues to explore two regions of 1 and conducted structure-functional selectivity relationship (SFSR) studies. Here we report the discovery of 1, findings from our SFSR studies, and characterization of novel G protein-biased D2R agonists.

  8. 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

  9. 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

  10. 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.

  11. Heterotrimeric G proteins regulate reproductive trait plasticity in response to water availability.

    PubMed

    Nilson, Sarah E; Assmann, Sarah M

    2010-02-01

    Phenotypic plasticity is the ability of one genotype to display different phenotypes under different environmental conditions. Although variation for phenotypic plasticity has been documented in numerous species, little is known about the genetic mechanisms underlying phenotypic plasticity. Given their widespread roles in hormonal and environmental signaling, we examined whether genes which encode heterotrimeric G proteins are plasticity genes. We grew multiple alleles of heterotrimeric G-protein mutants, together with wild-type Arabidopsis thaliana, under different watering regimes to determine the contributions of G-protein genes to phenotypic plasticity for a number of developmental and reproduction-related traits. G-protein mutations did not affect significantly the amount of phenotypic variation within an environment for any trait, but did affect significantly the amount of phenotypic plasticity for certain traits. AGB1, which encodes the beta subunit of the heterotrimeric G protein in Arabidopsis, is a plasticity gene and regulates reproductive trait plasticity in response to water availability, resulting in increased fitness (defined as seed production) under drought stress.

  12. 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.

  13. A novel human gene encoding a G-protein-coupled receptor (GPR15) is located on chromosome 3

    SciTech Connect

    Heiber, M.; Marchese, A.; O`Dowd, B.F.

    1996-03-05

    We used sequence similarities among G-protein-coupled receptor genes to discover a novel receptor gene. Using primers based on conserved regions of the opioid-related receptors, we isolated a PCR product that was used to locate the full-length coding region of a novel human receptor gene, which we have named GPR15. A comparison of the amino acid sequence of the receptor gene, which we have named GPR15. A comparison of the amino acid sequence of the receptor encoded by GPR15 with other receptors revealed that it shared sequence identity with the angiotensin II AT1 and AT2 receptors, the interleukin 8b receptor, and the orphan receptors GPR1 and AGTL1. GPR15 was mapped to human chromosome 3q11.2-q13.1. 12 refs., 2 figs.

  14. 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.

  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. 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.

  17. 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.

  18. G protein-induced trafficking of voltage-dependent calcium channels.

    PubMed

    Tombler, Eugene; Cabanilla, Nory Jun; Carman, Paul; Permaul, Natasha; Hall, John J; Richman, Ryan W; Lee, Jessica; Rodriguez, Jennifer; Felsenfeld, Dan P; Hennigan, Robert F; Diversé-Pierluissi, María A

    2006-01-20

    Calcium channels are well known targets for inhibition by G protein-coupled receptors, and multiple forms of inhibition have been described. Here we report a novel mechanism for G protein-mediated modulation of neuronal voltage-dependent calcium channels that involves the destabilization and subsequent removal of calcium channels from the plasma membrane. Imaging experiments in living sensory neurons show that, within seconds of receptor activation, calcium channels are cleared from the membrane and sequestered in clathrin-coated vesicles. Disruption of the L1-CAM-ankyrin B complex with the calcium channel mimics transmitter-induced trafficking of the channels, reduces calcium influx, and decreases exocytosis. Our results suggest that G protein-induced removal of plasma membrane calcium channels is a consequence of disrupting channel-cytoskeleton interactions and might represent a novel mechanism of presynaptic inhibition.

  19. 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

  20. 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

  1. 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.

  2. 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.

  3. 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

  4. 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

  5. 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

  6. Recent insights into Pasteurella multocida toxin and other G-protein-modulating bacterial toxins.

    PubMed

    Wilson, Brenda A; Ho, Mengfei

    2010-08-01

    Over the past few decades, our understanding of the bacterial protein toxins that modulate G proteins has advanced tremendously through extensive biochemical and structural analyses. This article provides an updated survey of the various toxins that target G proteins, ending with a focus on recent mechanistic insights in our understanding of the deamidating toxin family. The dermonecrotic toxin from Pasteurella multocida (PMT) was recently added to the list of toxins that disrupt G-protein signal transduction through selective deamidation of their targets. The C3 deamidase domain of PMT has no sequence similarity to the deamidase domains of the dermonecrotic toxins from Escherichia coli (cytotoxic necrotizing factor [CNF]1-3), Yersinia (CNFY) and Bordetella (dermonecrotic toxin). The structure of PMT-C3 belongs to a family of transglutaminase-like proteins, with active site Cys-His-Asp catalytic triads distinct from E. coli CNF1.

  7. 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.

  8. 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

  9. 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.

  10. 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

  11. 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.

  12. 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.

  13. Synergistic activation of G protein-gated inwardly rectifying potassium channels by cholesterol and PI(4,5)P2.

    PubMed

    Bukiya, Anna N; Rosenhouse-Dantsker, Avia

    2017-07-01

    G-protein gated inwardly rectifying potassium (GIRK or Kir3) channels play a major role in the control of the heart rate, and require the membrane phospholipid phosphatidylinositol-bis-phosphate (PI(4,5)P2) for activation. Recently, we have shown that the activity of the heterotetrameric Kir3.1/Kir3.4 channel that underlies atrial KACh currents was enhanced by cholesterol. Similarly, the activities of both the Kir3.4 homomer and its active pore mutant Kir3.4* (Kir3.4_S143T) were also enhanced by cholesterol. Here we employ planar lipid bilayers to investigate the crosstalk between PI(4,5)P2 and cholesterol, and demonstrate that these two lipids act synergistically to activate Kir3.4* currents. Further studies using the Xenopus oocytes heterologous expression system suggest that PI(4,5)P2 and cholesterol act via distinct binding sites. Whereas PI(4,5)P2 binds to the cytosolic domain of the channel, the putative binding region of cholesterol is located at the center of the transmembrane domain overlapping the central glycine hinge region of the channel. Together, our data suggest that changes in the levels of two key membrane lipids - cholesterol and PI(4,5)P2 - could act in concert to provide fine-tuning of Kir3 channel function. Copyright © 2017 Elsevier B.V. All rights reserved.

  14. Metabolic Parameters and Emotionality Are Little Affected in G-Protein Coupled Receptor 12 (Gpr12) Mutant Mice

    PubMed Central

    Frank, Elisabeth; Wu, Yizhen; Piyaratna, Naomi; Body, William James; Snikeris, Peta; South, Timothy; Gerdin, Anna-Karin; Bjursell, Mikael; Bohlooly-Y, Mohammad; Storlien, Leonard; Huang, Xu-Feng

    2012-01-01

    Background G-protein coupled receptors (GPR) bear the potential to serve as yet unidentified drug targets for psychiatric and metabolic disorders. GPR12 is of major interest given its putative role in metabolic function and its unique brain distribution, which suggests a role in emotionality and affect. We tested Gpr12 deficient mice in a series of metabolic and behavioural tests and subjected them to a well-established high-fat diet feeding protocol. Methodology/Principal Findings Comparing the mutant mice with wild type littermates, no significant differences were seen in body weight, fatness or weight gain induced by a high-fat diet. The Gpr12 mutant mice displayed a modest but significant lowering of energy expenditure and a trend to lower food intake on a chow diet, but no other metabolic parameters, including respiratory rate, were altered. No emotionality-related behaviours (assessed by light-dark box, tail suspension, and open field tests) were affected by the Gpr12 gene mutation. Conclusions/Significance Studying metabolic and emotionality parameters in Gpr12 mutant mice did not reveal a major phenotypic impact of the gene mutation. Compared to previous results showing a metabolic phenotype in Gpr12 mice with a mixed 129 and C57Bl6 background, we suggest that a more pure C57Bl/6 background due to further backcrossing might have reduced the phenotypic penetrance. PMID:22879962

  15. Discovery and cardioprotective effects of the first non-Peptide agonists of the G protein-coupled prokineticin receptor-1.

    PubMed

    Gasser, Adeline; Brogi, Simone; 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.

  16. [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.

  17. 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.

  18. 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.

  19. Interaction of rhodopsin, G-protein and kinase in octopus photoreceptors.

    PubMed

    Tsuda, M; Hirata, H; Tsuda, T

    1992-12-01

    Light induced phosphorylation of octopus rhodopsin was greatly enhanced by guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S), suggesting that the kinases are involved in regulating interaction between rhodopsin and G-protein. We determined phosphorylated peptides of octopus rhodopsin in the presence or absence of GTP gamma S. Possible phosphorylation sites for octopus rhodopsin enhanced by GTP gamma S were Thr329, Thr330 and/or Thr336, which suggest that the G-protein associates with cytoplasmic loops including C-terminal peptide in the seventh helix of octopus rhodopsin.

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

    PubMed

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

    2015-08-01

    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 effects associated with these drugs. Variability in drug responses is due, in part, to individuals' diversity in alternative splicing of pain-relevant GPCRs. G protein-coupled receptor alternative splice variants often exhibit distinct tissue distribution patterns, drug-binding properties, and signaling characteristics that may impact disease pathology as well as the extent and direction of analgesic effects. We review the importance of GPCRs and their known splice variants to the management of pain.

  1. Fidelity of G protein β-subunit association by the G protein γ-subunit-like domains of RGS6, RGS7, and RGS11

    PubMed Central

    Snow, Bryan E.; Betts, Laurie; Mangion, Joan; Sondek, John; Siderovski, David P.

    1999-01-01

    Several regulators of G protein signaling (RGS) proteins contain a G protein γ-subunit-like (GGL) domain, which, as we have shown, binds to Gβ5 subunits. Here, we extend our original findings by describing another GGL-domain-containing RGS, human RGS6. When RGS6 is coexpressed with different Gβ subunits, only RGS6 and Gβ5 interact. The expression of mRNA for RGS6 and Gβ5 in human tissues overlaps. Predictions of α-helical and coiled-coil character within GGL domains, coupled with measurements of Gβ binding by GGL domain mutants, support the contention that Gγ-like regions within RGS proteins interact with Gβ5 subunits in a fashion comparable to conventional Gβ/Gγ pairings. Mutation of the highly conserved Phe-61 residue of Gγ2 to tryptophan, the residue present in all GGL domains, increases the stability of the Gβ5/Gγ2 heterodimer, highlighting the importance of this residue to GGL/Gβ5 association. PMID:10339615

  2. MAPS

    Atmospheric Science Data Center

    2014-07-03

    ... Measurement of Air Pollution from Satellites (MAPS) data were collected during Space Shuttle flights in 1981, ... Facts Correlative Data  - CDIAC - Spring & Fall 1994 - Field and Aircraft Campaigns SCAR-B Block:  ...

  3. Expression of G-Protein Subunit α-14 Is Increased in Human Placentas from Preeclamptic Pregnancies

    PubMed Central

    Zhao, Ying-Jie; Zou, Qing-Yun; Li, Yan; Li, Hui-Hui; Wu, Yan-Ming; Li, Xing-Fu; Wang, Kai

    2014-01-01

    G-proteins mediate cellular function upon interaction with G-protein coupled receptors. Of the 16 mammalian G-protein α subunits identified, G-protein subunit α-11 (GNA11) and -14 (GNA14) have been implicated in modulating hypertension and endothelial function. However, little is known about their expression and roles in human placentas. Here, we examined GNA11 and GNA14 protein expression in first trimester (FT), normal term (NT), and severe preeclamptic (sPE) human placentas as well as in NT human umbilical cords. We found that GNA11 and GNA14 were immunolocalized primarily in trophoblasts, villous stromal cells, and endothelial cells in placentas as well as in endothelial and/or smooth muscle cells of the umbilical cord artery and vein. Western blotting revealed that the GNA14, but not GNA11, protein levels were increased (2.5-2.9 fold; p<0.01) in sPE vs. NT placentas. GNA11 protein was detected only in NT, but not FT, placentas, whereas GNA14 protein levels were increased (7.7-10.6 fold; p<0.01) in NT vs. FT placentas. Thus, GNA11 and GNA14 may mediate the function of several cell types in placentas. Moreover, the high expression of GNA14 in sPE placentas may also imply its importance in sPE pregnancies as in the other hypertension-related disorders. PMID:24423937

  4. Expression of G-protein subunit α-14 is increased in human placentas from preeclamptic pregnancies.

    PubMed

    Zhao, Ying-Jie; Zou, Qing-Yun; Li, Yan; Li, Hui-Hui; Wu, Yan-Ming; Li, Xing-Fu; Wang, Kai; Zheng, Jing

    2014-05-01

    G-proteins mediate cellular function upon interaction with G-protein coupled receptors. Of the 16 mammalian G-protein α subunits identified, G-protein subunit α-11 (GNA11) and -14 (GNA14) have been implicated in modulating hypertension and endothelial function. However, little is known about their expression and roles in human placentas. Here, we examined GNA11 and GNA14 protein expression in first trimester (FT), normal term (NT), and severe preeclamptic (sPE) human placentas as well as in NT human umbilical cords. We found that GNA11 and GNA14 were immunolocalized primarily in trophoblasts, villous stromal cells, and endothelial cells in placentas as well as in endothelial and/or smooth muscle cells of the umbilical cord artery and vein. Western blotting revealed that the GNA14, but not GNA11, protein levels were increased (2.5-2.9 fold; p<0.01) in sPE vs. NT placentas. GNA11 protein was detected only in NT, but not FT, placentas, whereas GNA14 protein levels were increased (7.7-10.6 fold; p<0.01) in NT vs. FT placentas. Thus, GNA11 and GNA14 may mediate the function of several cell types in placentas. Moreover, the high expression of GNA14 in sPE placentas may also imply its importance in sPE pregnancies as in the other hypertension-related disorders.

  5. 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.

  6. 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.

  7. Discovery of Natural Phenols as G Protein-Coupled Receptor-35 (GPR35) Agonists.

    PubMed

    Deng, Huayun; Hu, Haibei; Ling, Shizhang; Ferrie, Ann M; Fang, Ye

    2012-02-09

    We report the discovery and characterization of natural phenols as G protein-coupled receptor-35 (GPR35) agonists. Pharmacological characterization using label-free dynamic mass redistribution and Tango β-arrestin translocation assays revealed that GPR35-active natural phenols are divergent in their biased agonism.

  8. Discovery of Natural Phenols as G Protein-Coupled Receptor-35 (GPR35) Agonists

    PubMed Central

    2012-01-01

    We report the discovery and characterization of natural phenols as G protein-coupled receptor-35 (GPR35) agonists. Pharmacological characterization using label-free dynamic mass redistribution and Tango β-arrestin translocation assays revealed that GPR35-active natural phenols are divergent in their biased agonism. PMID:24900447

  9. Crystal structure of a common GPCR-binding interface for G protein and arrestin

    PubMed Central

    Szczepek, Michal; Beyrière, Florent; Hofmann, Klaus Peter; Elgeti, Matthias; Kazmin, Roman; Rose, Alexander; Bartl, Franz J.; von Stetten, David; Heck, Martin; Sommer, Martha E.; Hildebrand, Peter W.; Scheerer, Patrick

    2014-01-01

    G-protein-coupled receptors (GPCRs) transmit extracellular signals to activate intracellular heterotrimeric G proteins (Gαβγ) and arrestins. For G protein signalling, the Gα C-terminus (GαCT) binds to a cytoplasmic crevice of the receptor that opens upon activation. A consensus motif is shared among GαCT from the Gi/Gt family and the ‘finger loop’ region (ArrFL1–4) of all four arrestins. Here we present a 2.75 Å crystal structure of ArrFL-1, a peptide analogue of the finger loop of rod photoreceptor arrestin, in complex with the prototypical GPCR rhodopsin. Functional binding of ArrFL to the receptor was confirmed by ultraviolet-visible absorption spectroscopy, competitive binding assays and Fourier transform infrared spectroscopy. For both GαCT and ArrFL, binding to the receptor crevice induces a similar reverse turn structure, although significant structural differences are seen at the rim of the binding crevice. Our results reflect both the common receptor-binding interface and the divergent biological functions of G proteins and arrestins. PMID:25205354

  10. 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.

  11. 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.

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

    USDA-ARS?s Scientific Manuscript database

    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...

  13. 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.

  14. Conformational dynamics of a G-protein α subunit is tightly regulated by nucleotide binding

    PubMed Central

    Goricanec, David; Stehle, Ralf; Egloff, Pascal; Grigoriu, Simina; Wagner, Gerhard; Hagn, Franz

    2016-01-01

    Heterotrimeric G proteins play a pivotal role in the signal-transduction pathways initiated by G-protein–coupled receptor (GPCR) activation. Agonist–receptor binding causes GDP-to-GTP exchange and dissociation of the Gα subunit from the heterotrimeric G protein, leading to downstream signaling. Here, we studied the internal mobility of a G-protein α subunit in its apo and nucleotide-bound forms and characterized their dynamical features at multiple time scales using solution NMR, small-angle X-ray scattering, and molecular dynamics simulations. We find that binding of GTP analogs leads to a rigid and closed arrangement of the Gα subdomain, whereas the apo and GDP-bound forms are considerably more open and dynamic. Furthermore, we were able to detect two conformational states of the Gα Ras domain in slow exchange whose populations are regulated by binding to nucleotides and a GPCR. One of these conformational states, the open state, binds to the GPCR; the second conformation, the closed state, shows no interaction with the receptor. Binding to the GPCR stabilizes the open state. This study provides an in-depth analysis of the conformational landscape and the switching function of a G-protein α subunit and the influence of a GPCR in that landscape. PMID:27298341

  15. 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

  16. 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.

  17. 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.

  18. 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

  19. 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

  20. Interaction between G proteins and tyrosine kinases upon T cell receptor.CD3-mediated signaling.

    PubMed

    Stanners, J; Kabouridis, P S; McGuire, K L; Tsoukas, C D

    1995-12-22

    Engagement of the T cell receptor (TCR).CD3 complex results in the induction of multiple intracellular events, with protein tyrosine kinases playing a pivotal role in their initiation. Biochemical studies also exist suggesting the involvement of heterotrimeric GTP-binding proteins (G proteins); however, the functional consequence of this participation in TCR.CD3-mediated signaling is unresolved. Here, we report TCR.CD3-mediated guanine nucleotide exchange among the 42-kDa G protein alpha subunits of the G alpha q/11 family, their physical association with CD3 epsilon, and the G alpha 11-dependent activation of phospholipase C beta. Protein tyrosine kinase inhibitors, however, abrogate TCR.CD3-mediated G protein activation. Quite interesting is the observation that cells transfected with a function-deficient mutant of G alpha 11 display diminished tyrosine phosphorylation of TCR.CD3 zeta and epsilon chains, as well as ZAP-70, upon anti-CD3 antibody triggering. These data indicate the involvement of the G alpha q/11 family in TCR.CD3 signaling at a step proximal to the receptor and suggest a reciprocal regulation between tyrosine kinases and G proteins in T cells.

  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. 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.

  3. Coordinate Regulation of G Protein Signaling via Dynamic Interactions of Receptor and GAP

    PubMed Central

    Turcotte, Marc; Tang, Wei; Ross, Elliott M.

    2008-01-01

    Signal output from receptor–G-protein–effector modules is a dynamic function of the nucleotide exchange activity of the receptor, the GTPase-accelerating activity of GTPase-activating proteins (GAPs), and their interactions. GAPs may inhibit steady-state signaling but may also accelerate deactivation upon removal of stimulus without significantly inhibiting output when the receptor is active. Further, some effectors (e.g., phospholipase C-β) are themselves GAPs, and it is unclear how such effectors can be stimulated by G proteins at the same time as they accelerate G protein deactivation. The multiple combinations of protein–protein associations and interacting regulatory effects that allow such complex behaviors in this system do not permit the usual simplifying assumptions of traditional enzyme kinetics and are uniquely subject to systems-level analysis. We developed a kinetic model for G protein signaling that permits analysis of both interactive and independent G protein binding and regulation by receptor and GAP. We evaluated parameters of the model (all forward and reverse rate constants) by global least-squares fitting to a diverse set of steady-state GTPase measurements in an m1 muscarinic receptor–Gq–phospholipase C-β1 module in which GTPase activities were varied by ∼104-fold. We provide multiple tests to validate the fitted parameter set, which is consistent with results from the few previous pre-steady-state kinetic measurements. Results indicate that (1) GAP potentiates the GDP/GTP exchange activity of the receptor, an activity never before reported; (2) exchange activity of the receptor is biased toward replacement of GDP by GTP; (3) receptor and GAP bind G protein with negative cooperativity when G protein is bound to either GTP or GDP, promoting rapid GAP binding and dissociation; (4) GAP indirectly stabilizes the continuous binding of receptor to G protein during steady-state GTPase hydrolysis, thus further enhancing receptor activity

  4. 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.

  5. A constitutively active Gα subunit provide insights into the mechanism of G protein activation

    PubMed Central

    Singh, Garima; Ramachandran, Sekar; Cerione, Richard A.

    2013-01-01

    The activation of Gα subunits of heterotrimeric G proteins by G protein-coupled receptors (GPCRs) is a critical event underlying a variety of biological responses. Understanding how G proteins are activated will require structural and biochemical analyses of GPCRs complexed to their G protein partners, together with structure-function studies of Gα mutants that shed light on the different steps in the activation pathway. Previously, we reported that the substitution of a glycine for a proline at position 56 within the linker region connecting the helical and GTP-binding domains of a Gα chimera, designated αT*, yields a more readily exchangeable state for guanine nucleotides. Here we show that GDP-GTP exchange on αT*(G56P), in the presence of the light-activated GPCR, rhodopsin (R*), is less sensitive to the β1γ1 subunit complex as compared to wild-type αT*. We solved the x-ray crystal structure for the αT*(G56P) mutant and found that the G56P substitution leads to concerted changes that are transmitted to the conformationally sensitive switch regions, the α4/β6 loop, and the β6 strand. The α4/β6 loop has been proposed to be a GPCR contact site that signals to the TCAT motif and weakens the binding of the guanine ring of GDP, whereas, the switch regions are the contact sites for the β1γ1 complex. Collectively, these biochemical and structural data lead us to suggest that αT*(G56P) may be adopting a conformation that is normally induced within Gα subunits by the combined actions of a GPCR and a Gβγ subunit complex during the G protein activation event. PMID:22448927

  6. 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.

  7. 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.

  8. 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.

  9. A G protein functions immediately downstream of Smoothened in Hedgehog signaling

    PubMed Central

    Ogden, Stacey K.; Fei, Dennis Liang; Schilling, Neal S.; Ahmed, Yashi F.; Hwa, John; Robbins, David J.

    2009-01-01

    The Hedgehog (Hh) signaling pathway plays an evolutionarily conserved role in patterning fields of cells during metazoan development, and is inappropriately activated in cancer1,2. Hh pathway activity is absolutely dependent upon signaling by the seven-transmembrane protein Smoothened (Smo), which is regulated by the Hh receptor Patched (Ptc). Smo signals to an intracellular multi-protein complex containing the Kinesin related protein Costal2 (Cos2), the protein kinase Fused (Fu) and the transcription factor Cubitus interruptus (Ci)3. In the absence of Hh, this complex regulates the cleavage of full length Ci to a truncated repressor protein, Ci75 in a process that is dependent upon the proteosome and priming phosphorylations by Protein Kinase A (PKA)4. Binding of Hh to Ptc blocks Ptc-mediated Smo inhibition, allowing Smo to signal to the intracellular components to attenuate Ci cleavage. Because of its homology with the Frizzled family of G protein coupled receptors (GPCR)5, a likely candidate for an immediate Smo effector would be a heterotrimeric G protein. However, the role G proteins may play in Hh signal transduction is unclear and quite controversial6-10, which has led to widespread speculation that Smo signals through a variety of novel G protein independent mechanisms. Here, we present in vitro and in vivo evidence that Smo activates a G protein to modulate intracellular cyclic AMP (cAMP) levels in response to Hh. Our results demonstrate that Smo functions as a canonical GPCR, which signals through Gαi to regulate Hh pathway activation. PMID:18987629

  10. 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

  11. Activator of G protein signaling 3 is a guanine dissociation inhibitor for Gαi subunits

    PubMed Central

    De Vries, Luc; Fischer, Thierry; Tronchère, Hélène; Brothers, Greg M.; Strockbine, Bentley; Siderovski, David P.; Farquhar, Marilyn Gist

    2000-01-01

    Activator of G protein signaling 3 (AGS3) is a newly identified protein shown to act at the level of the G protein itself. AGS3 belongs to the GoLoco family of proteins, sharing the 19-aa GoLoco motif that is a Gαi/o binding motif. AGS3 interacts only with members of the Gαi/o subfamily. By surface plasmon resonance, we found that AGS3 binds exclusively to the GDP-bound form of Gαi3. In GTPγS binding assays, AGS3 behaves as a guanine dissociation inhibitor (GDI), inhibiting the rate of exchange of GDP for GTP by Gαi3. AGS3 interacts with both Gαi3 and Gαo subunits, but has GDI activity only on Gαi3, not on Gαo. The fourth GoLoco motif of AGS3 is a major contributor to this activity. AGS3 stabilizes Gαi3 in its GDP-bound form, as it inhibits the increase in tryptophan fluorescence of the Gαi3-GDP subunit stimulated by AlF4−. AGS3 is widely expressed as it is detected by immunoblotting in brain, testis, liver, kidney, heart, pancreas, and in PC-12 cells. Several different sizes of the protein are detected. By Northern blotting, AGS3 shows 2.3-kb and 3.5-kb mRNAs in heart and brain, respectively, suggesting tissue-specific alternative splicing. Taken together, our results demonstrate that AGS3 is a GDI. To the best of our knowledge, no other GDI has been described for heterotrimeric G proteins. Inhibition of the Gα subunit and stimulation of heterotrimeric G protein signaling, presumably by stimulating Gβγ, extend the possibilities for modulating signal transduction through heterotrimeric G proteins. PMID:11121039

  12. 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.

  13. 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. © 2014 Xu, Jiang, et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

  14. 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

  15. 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

  16. 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

  17. 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.

  18. Expression, Purification and Crystallisation of the Adenosine A2A Receptor Bound to an Engineered Mini G Protein.

    PubMed

    Carpenter, Byron; Tate, Christopher G

    2017-04-20

    G protein-coupled receptors (GPCRs) promote cytoplasmic signalling by activating heterotrimeric G proteins in response to extracellular stimuli such as light, hormones and nucleosides. Structure determination of GPCR-G protein complexes is central to understanding the precise mechanism of signal transduction. However, these complexes are challenging targets for structural studies due to their conformationally dynamic and inherently transient nature. We recently developed an engineered G protein, mini-Gs, which addressed these problems and allowed the formation of a stable GPCR-G protein complex. Mini-Gs facilitated the structure determination of the human adenosine A2A receptor (A2AR) in its G protein-bound conformation at 3.4 Å resolution. Here, we describe a step by step protocol for the expression and purification of A2AR, and crystallisation of the A2AR-mini-Gs complex.

  19. 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

  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. Role of Membrane Integrity on G protein-coupled Receptors: Rhodopsin Stability and Function

    PubMed Central

    Jastrzebska, Beata; Debinski, Aleksander; Filipek, Slawomir; Palczewski, Krzysztof

    2011-01-01

    Rhodopsin is a prototypical G protein-coupled receptor (GPCR) – a member of the superfamily that shares a similar structural architecture consisting of seven-transmembrane helices and propagates various signals across biological membranes. Rhodopsin is embedded in the lipid bilayer of specialized disk membranes in the outer segments of retinal rod photoreceptor cells where it transmits a light-stimulated signal. Photoactivated rhodopsin then activates a visual signaling cascade through its cognate G protein, transducin or Gt, that results in a neuronal response in the brain. Interestingly, the lipid composition of ROS membranes not only differs from that of the photoreceptor plasma membrane but is critical for visual transduction. Specifically, lipids can modulate structural changes in rhodopsin that occur after photoactivation and influence binding of transducin. Thus, altering the lipid organization of ROS membranes may result in visual dysfunction and blindness. PMID:21435354

  2. 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

  3. 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.

  4. 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.

  5. 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

  6. 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

  7. Signaling within Allosteric Machines: Signal Transmission Pathways Inside G Protein-Coupled Receptors.

    PubMed

    Bartuzi, Damian; Kaczor, Agnieszka A; Matosiuk, Dariusz

    2017-07-15

    In recent years, our understanding of function of G protein-coupled receptors (GPCRs) has changed from a picture of simple signal relays, transmitting only a particular signal to a particular G protein heterotrimer, to versatile machines, capable of various responses to different stimuli and being modulated by various factors. Some recent reports provide not only the data on ligands/modulators and resultant signals induced by them, but also deeper insights into exact pathways of signal migration and mechanisms of signal transmission through receptor structure. Combination of these computational and experimental data sheds more light on underlying mechanisms of signal transmission and signaling bias in GPCRs. In this review we focus on available clues on allosteric pathways responsible for complex signal processing within GPCRs structures, with particular emphasis on linking compatible in silico- and in vitro-derived data on the most probable allosteric connections.

  8. 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

  9. Expression, Purification, and Analysis of G-Protein-Coupled Receptor Kinases

    PubMed Central

    Sterne-Marr, Rachel; Baillargeon, Alison I.; Michalski, Kevin R.; Tesmer, John J.G.

    2015-01-01

    G-protein-coupled receptor (GPCR) kinases (GRKs) were first identified based on their ability to specifically phosphorylate activated GPCRs. Although many soluble substrates have since been identified, the chief physiological role of GRKs still remains the uncoupling of GPCRs from heterotrimeric G-proteins by promoting β-arrestin binding through the phosphorylation of the receptor. It is expected that GRKs recognize activated GPCRs through a docking site that not only recognizes the active conformation of the transmembrane domain of the receptor but also stabilizes a more catalytically competent state of the kinase domain. Many of the recent gains in understanding GRK-receptor interactions have been gleaned through biochemical and structural analysis of recombinantly expressed GRKs. Described herein are current techniques and procedures being used to express, purify, and assay GRKs in both in vitro and living cells. PMID:23351749

  10. A G protein-coupled receptor phosphatase required for rhodopsin function.

    PubMed

    Vinós, J; Jalink, K; Hardy, R W; Britt, S G; Zuker, C S

    1997-08-01

    Heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors are phosphorylated by kinases that mediate agonist-dependent receptor deactivation. Although many receptor kinases have been isolated, the corresponding phosphatases, necessary for restoring the ground state of the receptor, have not been identified. Drosophila RDGC (retinal degeneration C) is a phosphatase required for rhodopsin dephosphorylation in vivo. Loss of RDGC caused severe defects in the termination of the light response as well as extensive light-dependent retinal degeneration. These phenotypes resulted from the hyperphosphorylation of rhodopsin because expression of a truncated rhodopsin lacking the phosphorylation sites restored normal photoreceptor function. These results suggest the existence of a family of receptor phosphatases involved in the regulation of G protein-coupled signaling cascades.

  11. 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.

  12. 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

  13. 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.

  14. [G-proteins, their role in the transduction of the hormonal message and their pathology].

    PubMed

    Homburger, V

    1991-01-01

    Heterotrimeric G proteins belong to the superfamily of proteins which bind and hydrolyse GTP. Each of these GTPases acts as a molecular switch whose "on" and "off" states are triggered by binding and hydrolysis of GTP. Heterotrimeric G proteins (alpha beta gamma complex) transduce hormonal and sensory stimuli across the plasma membrane. The alpha chain contains the site of GTP binding and hydrolysis and the domains of interactions with receptors, effectors and beta gamma complex. GTP binding site is composed of five distinct domains denoted G1-5. Specific aminoacidic substitution or alteration in G2 and G3 domains triggers a decrease of GTP hydolysis, trapping the alpha chain in its active state. Such alterations are detected during cholera disease and in some secreting pituitary tumors. A genetic deficiency of the Gs alpha subunit also occurs in most patients who have Albright hereditary osteodystrophy.

  15. 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

  16. 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

  17. 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

  18. 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

  19. Accessories to addiction: G protein regulators play a key role in cocaine seeking and neuroplasticity.

    PubMed

    Kelley, Ann E; Schiltz, Craig A

    2004-04-22

    The prefrontal cortex mediates many aspects of addiction. In this issue of Neuron, Bowers et al. demonstrate that an activator of G protein signaling (AGS3) is persistently upregulated in the prefrontal cortex after cessation of chronic cocaine treatment. Furthermore, they find that AGS3 is responsible for altered behavior, such as enhanced drug seeking, and altered neurotransmission in cocaine-treated rats, representing a novel therapeutic target.

  20. 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.

  1. 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.

  2. 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

  3. An allosteric modulator to control endogenous G protein-coupled receptors with light.

    PubMed

    Pittolo, Silvia; Gómez-Santacana, Xavier; Eckelt, Kay; Rovira, Xavier; Dalton, James; Goudet, Cyril; Pin, Jean-Philippe; Llobet, Artur; Giraldo, Jesús; Llebaria, Amadeu; Gorostiza, Pau

    2014-10-01

    Controlling drug activity with light offers the possibility of enhancing pharmacological selectivity with spatial and temporal regulation, thus enabling highly localized therapeutic effects and precise dosing patterns. Here we report on the development and characterization of what is to our knowledge the first photoswitchable allosteric modulator of a G protein-coupled receptor. Alloswitch-1 is selective for the metabotropic glutamate receptor mGlu5 and enables the optical control of endogenous mGlu5 receptors.

  4. 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

  5. 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.

  6. 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

  7. Arabidopsis heterotrimeric G-protein regulates cell wall defense and resistance to necrotrophic fungi.

    PubMed

    Delgado-Cerezo, Magdalena; Sánchez-Rodríguez, Clara; Escudero, Viviana; Miedes, Eva; Fernández, Paula Virginia; Jordá, Lucía; Hernández-Blanco, Camilo; Sánchez-Vallet, Andrea; Bednarek, Pawel; Schulze-Lefert, Paul; Somerville, Shauna; Estevez, José Manuel; Persson, Staffan; Molina, Antonio

    2012-01-01

    The Arabidopsis heterotrimeric G-protein controls defense responses to necrotrophic and vascular fungi. The agb1 mutant impaired in the Gβ subunit displays enhanced susceptibility to these pathogens. Gβ/AGB1 forms an obligate dimer with either one of the Arabidopsis Gγ subunits (γ1/AGG1 and γ2/AGG2). Accordingly, we now demonstrate that the agg1 agg2 double mutant is as susceptible as agb1 plants to the necrotrophic fungus Plectosphaerella cucumerina. To elucidate the molecular basis of heterotrimeric G-protein-mediated resistance, we performed a comparative transcriptomic analysis of agb1-1 mutant and wild-type plants upon inoculation with P. cucumerina. This analysis, together with metabolomic studies, demonstrated that G-protein-mediated resistance was independent of defensive pathways required for resistance to necrotrophic fungi, such as the salicylic acid, jasmonic acid, ethylene, abscisic acid, and tryptophan-derived metabolites signaling, as these pathways were not impaired in agb1 and agg1 agg2 mutants. Notably, many mis-regulated genes in agb1 plants were related with cell wall functions, which was also the case in agg1 agg2 mutant. Biochemical analyses and Fourier Transform InfraRed (FTIR) spectroscopy of cell walls from G-protein mutants revealed that the xylose content was lower in agb1 and agg1 agg2 mutants than in wild-type plants, and that mutant walls had similar FTIR spectratypes, which differed from that of wild-type plants. The data presented here suggest a canonical functionality of the Gβ and Gγ1/γ2 subunits in the control of Arabidopsis immune responses and the regulation of cell wall composition.

  8. 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.

  9. 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

  10. 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

  11. 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.

  12. Buried ionizable networks are an ancient hallmark of G protein-coupled receptor activation.

    PubMed

    Isom, Daniel G; Dohlman, Henrik G

    2015-05-05

    Seven-transmembrane receptors (7TMRs) have evolved in prokaryotes and eukaryotes over hundreds of millions of years. Comparative structural analysis suggests that these receptors may share a remote evolutionary origin, despite their lack of sequence similarity. Here we used structure-based computations to compare 221 7TMRs from all domains of life. Unexpectedly, we discovered that these receptors contain spatially conserved networks of buried ionizable groups. In microbial 7TMRs these networks are used to pump ions across the cell membrane in response to light. In animal 7TMRs, which include light- and ligand-activated G protein-coupled receptors (GPCRs), homologous networks were found to be characteristic of activated receptor conformations. These networks are likely relevant to receptor function because they connect the ligand-binding pocket of the receptor to the nucleotide-binding pocket of the G protein. We propose that agonist and G protein binding facilitate the formation of these electrostatic networks and promote important structural rearrangements such as the displacement of transmembrane helix-6. We anticipate that robust classification of activated GPCR structures will aid the identification of ligands that target activated GPCR structural states.

  13. 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.

  14. Different Properties of the Native and Reconstituted Heterotrimeric G Protein Transducin†

    PubMed Central

    Goc, Anna; Angel, Thomas E.; Jastrzebska, Beata; Wang, Benlian; Wintrode, Patrick L.; Palczewski, Krzysztof

    2008-01-01

    Visual signal transduction serves as one of the best understood G protein-coupled receptor signaling systems. Signaling is initiated when a photon strikes rhodopsin (Rho) causing a conformational change leading to productive interaction of this G protein-coupled receptor with the heterotrimeric G protein, transducin (Gt). Here we describe a new method for Gt purification from native bovine rod photoreceptor membranes without subunit dissociation caused by exposure to photoactivated rhodopsin (Rho*). Native electrophoresis followed by immunoblotting revealed that Gt purified by this method formed more stable heterotrimers and interacted more efficiently with membranes containing Rho* or its target, phosphodiesterase 6, than did Gt purified by a traditional method involving subunit dissociation and reconstitution in solution without membranes. Because these differences could result from selective extraction, we characterized the type and amount of posttranslational modifications on both purified native and reconstituted Gt preparations. Similar N-terminal acylation of the Gtα subunit was observed for both proteins as was farnesylation and methylation of the terminal Gtγ subunit Cys residue. However, hydrogen/deuterium exchange experiments revealed less incorporation of deuterium into the Gtα and Gtβ subunits of native Gt as compared to reconstituted Gt. These findings may indicate differences in conformation and heterotrimer complex formation between the two preparations or altered stability of the reconstituted Gt that assembles differently than the native protein. Therefore, Gt extracted and purified without subunit dissociation appears to be more appropriate for future studies. PMID:18975915

  15. 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

  16. From GTP and G proteins to TRPC channels: a personal account.

    PubMed

    Birnbaumer, Lutz

    2015-09-01

    By serendipity and good fortune, as a postdoctoral fellow in 1967, I landed at the right place at the right time, as I was allowed to investigate the mechanism by which hormones activate the enzyme adenylyl cyclase (then adenyl cyclase) in Martin Rodbell's Laboratory at the NIH in Bethesda, Maryland. The work uncovered first, the existence of receptors separate from the enzyme and then, the existence of transduction mechanisms requiring guanosine-5'-triphosphate (GTP) and Mg(2+). With my laboratory colleagues first and postdoctoral fellows after leaving NIH, I participated in the development of the field "signal transduction by G proteins," uncovered by molecular cloning several G-protein-coupled receptors (GPCRs) and became interested in both the molecular makeup of voltage-gated Ca channels and Ca2+ homeostasis downstream of activation of phospholipase C (PLC) by the Gq/11 signaling pathway. We were able to confirm the hypothesis that there would be mammalian homologues of the Drosophila "transient receptor potential" channel and discovered the existence of six of the seven mammalian genes, now called transient receptor potential canonical (TRPC) channels. In the present article, I summarize from a bird's eye view of what I feel were key findings along this path, not only from my laboratory but also from many others, that allowed for the present knowledge of cell signaling involving G proteins to evolve. Towards the end, I summarize roles of TRPC channels in health and disease.

  17. 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.

  18. 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.

  19. Allosteric control of an asymmetric transduction in a G protein-coupled receptor heterodimer.

    PubMed

    Liu, Junke; Zhang, Zongyong; Moreno-Delgado, David; Dalton, James Ar; Rovira, Xavier; Trapero, Ana; Goudet, Cyril; Llebaria, Amadeu; Giraldo, Jesús; Yuan, Qilin; Rondard, Philippe; Huang, Siluo; Liu, Jianfeng; Pin, Jean-Philippe

    2017-08-10

    GPCRs play critical roles in cell communication. Although GPCRs can form heteromers, their role in signaling remains elusive. Here we used rat metabotropic glutamate (mGlu) receptors as prototypical dimers to study the functional interaction between each subunit. mGluRs can form both constitutive homo- and heterodimers. Whereas both mGlu2 and mGlu4 couple to G proteins, G protein activation is mediated by mGlu4 heptahelical domain (HD) exclusively in mGlu2-4 heterodimers. Such asymmetric transduction results from the action of both the dimeric extracellular domain, and an allosteric activation by the partially-activated non-functional mGlu2 HD. G proteins activation by mGlu2 HD occurs if either the mGlu2 HD is occupied by a positive allosteric modulator or if mGlu4 HD is inhibited by a negative modulator. These data revealed an oriented asymmetry in mGlu heterodimers that can be controlled with allosteric modulators. They provide new insight on the allosteric interaction between subunits in a GPCR dimer.

  20. Activator of G Protein Signaling 3 Promotes Epithelial Cell Proliferation in PKD

    PubMed Central

    Nadella, Rama; Blumer, Joe B.; Jia, Guangfu; Kwon, Michelle; Akbulut, Talha; Qian, Feng; Sedlic, Filip; Wakatsuki, Tetsuro; Sweeney, William E.; Wilson, Patricia D.; Lanier, Stephen M.

    2010-01-01

    The activation of heterotrimeric G protein signaling is a key feature in the pathophysiology of polycystic kidney diseases (PKD). In this study, we report abnormal overexpression of activator of G protein signaling 3 (AGS3), a receptor-independent regulator of heterotrimeric G proteins, in rodents and humans with both autosomal recessive and autosomal dominant PKD. Increased AGS3 expression correlated with kidney size, which is an index of severity of cystic kidney disease. AGS3 expression localized exclusively to distal tubular segments in both normal and cystic kidneys. Short hairpin RNA–induced knockdown of endogenous AGS3 protein significantly reduced proliferation of cystic renal epithelial cells by 26 ± 2% (P < 0.001) compared with vehicle-treated and control short hairpin RNA–expressing epithelial cells. In summary, this study suggests a relationship between aberrantly increased AGS3 expression in renal tubular epithelia affected by PKD and epithelial cell proliferation. AGS3 may play a receptor-independent role to regulate Gα subunit function and control epithelial cell function in PKD. PMID:20488951

  1. UNC119 is required for G protein trafficking in sensory neurons

    PubMed Central

    Zhang, Houbin; Constantine, Ryan; Vorobiev, Sergey; Chen, Yang; Seetharaman, Jayaraman; Huang, Yuanpeng Janet; Xiao, Rong; Montelione, Gaetano T.; Gerstner, Cecilia D.; Davis, M. Wayne; Inana, George; Whitby, Frank G.; Jorgensen, Erik M.; Hill, Christopher P.; Tong, Liang; Baehr, Wolfgang

    2011-01-01

    SUMMARY UNC119 is widely expressed among vertebrates and invertebrates. Here we report that UNC119 recognized the acylated N-terminus of the rod photoreceptor transducin α-subunit (Tα) as well as C. elegans G proteins Odr-3 and Gpa-13. The crystal structure of human UNC119 at 1.95 Å resolution revealed an immunoglobulin-like β-sandwich fold. Pulldowns and isothermal titration calorimetry revealed a tight interaction between UNC119 and acylated Gα peptides. Co-crystallization of UNC119 with an acylated Tα N-terminal peptide at 2.0 Å revealed that the lipid chain is buried deeply into UNC119's hydrophobic cavity. UNC119 bound TαGTP inhibiting its GTPase activity, thereby providing a stable UNC119-TαGTP complex that is capable of diffusing from the inner segment back to the outer segment following light-induced translocation. UNC119 deletion in both mouse and C. elegans lead to G protein mislocalization. These results establish UNC119 as a novel Gα-subunit cofactor that is essential for G-protein trafficking in sensory cilia. PMID:21642972

  2. A New Molecular Mechanism To Engineer Protean Agonism at a G Protein-Coupled Receptor.

    PubMed

    De Min, Anna; Matera, Carlo; Bock, Andreas; Holze, Janine; Kloeckner, Jessica; Muth, Mathias; Traenkle, Christian; De Amici, Marco; Kenakin, Terry; Holzgrabe, Ulrike; Dallanoce, Clelia; Kostenis, Evi; Mohr, Klaus; Schrage, Ramona

    2017-04-01

    Protean agonists are of great pharmacological interest as their behavior may change in magnitude and direction depending on the constitutive activity of a receptor. Yet, this intriguing phenomenon has been poorly described and understood, due to the lack of stable experimental systems and design strategies. In this study, we overcome both limitations: First, we demonstrate that modulation of the ionic strength in a defined experimental set-up allows for analysis of G protein-coupled receptor activation in the absence and presence of a specific amount of spontaneous receptor activity using the muscarinic M2 acetylcholine receptor as a model. Second, we employ this assay system to show that a dualsteric design principle, that is, molecular probes, carrying two pharmacophores to simultaneously adopt orthosteric and allosteric topography within a G protein-coupled receptor, may represent a novel approach to achieve protean agonism. We pinpoint three molecular requirements within dualsteric compounds that elicit protean agonism at the muscarinic M2 acetylcholine receptor. Using radioligand-binding and functional assays, we posit that dynamic ligand binding may be the mechanism underlying protean agonism of dualsteric ligands. Our findings provide both new mechanistic insights into the still enigmatic phenomenon of protean agonism and a rationale for the design of such compounds for a G protein-coupled receptor. Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.

  3. 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.

  4. 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

  5. Ligand Binding Ensembles Determine Graded Agonist Efficacies at a G Protein-coupled Receptor*

    PubMed Central

    Bock, Andreas; Bermudez, Marcel; Krebs, Fabian; Matera, Carlo; Chirinda, Brian; Sydow, Dominique; Dallanoce, Clelia; Holzgrabe, Ulrike; De Amici, Marco; Lohse, Martin J.; Wolber, Gerhard; Mohr, Klaus

    2016-01-01

    G protein-coupled receptors constitute the largest family of membrane receptors and modulate almost every physiological process in humans. Binding of agonists to G protein-coupled receptors induces a shift from inactive to active receptor conformations. Biophysical studies of the dynamic equilibrium of receptors suggest that a portion of receptors can remain in inactive states even in the presence of saturating concentrations of agonist and G protein mimetic. However, the molecular details of agonist-bound inactive receptors are poorly understood. Here we use the model of bitopic orthosteric/allosteric (i.e. dualsteric) agonists for muscarinic M2 receptors to demonstrate the existence and function of such inactive agonist·receptor complexes on a molecular level. Using all-atom molecular dynamics simulations, dynophores (i.e. a combination of static three-dimensional pharmacophores and molecular dynamics-based conformational sampling), ligand design, and receptor mutagenesis, we show that inactive agonist·receptor complexes can result from agonist binding to the allosteric vestibule alone, whereas the dualsteric binding mode produces active receptors. Each agonist forms a distinct ligand binding ensemble, and different agonist efficacies depend on the fraction of purely allosteric (i.e. inactive) versus dualsteric (i.e. active) binding modes. We propose that this concept may explain why agonist·receptor complexes can be inactive and that adopting multiple binding modes may be generalized also to small agonists where binding modes will be only subtly different and confined to only one binding site. PMID:27298318

  6. 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.

  7. 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

  8. 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

  9. G-protein alpha subunits distribution in the cyprid of Balanus amphitrite (=Amphibalanus amphitrite) (Cirripedia, Crustacea).

    PubMed

    Gallus, Lorenzo; Ferrando, Sara; Gambardella, Chiara; Amaroli, Andrea; Faimali, Marco; Piazza, Veronica; Masini, Maria Angela

    2012-12-01

    The acorn barnacle Balanus amphitrite is a marine crustacean with six nauplius and one cyprid larval stages and a sessile adult, that represent one of the main constituents of sea biofouling. The cyprid is the last larval stage, specialized for settlement, and the study of its biology is interesting also in the frame of antifouling strategies. In this study, a novel approach to the neurobiology of B. amphitrite cyprid has undertaken, studying immunohistochemically the distribution of some G-protein α subunits (Gαs, Gαo Gαi, and Gαq) on B. amphitrite cyprid. Gαs-like immunoreactivity was observed in the intestinal mucosa, oral cone, epithelial cells along the outer face of the mantle and thorax; Gαo into the fibers of the neuropile of the central nervous system; Gαi in oil cells, epithelial cells, and limbs and thorax muscles; Gαq was not detected. The results suggest the involvement of the G-protein α subunits in different tissues and functions that seem to be in agreement with the distribution of the ones from the same class of G-proteins in vertebrates. Copyright © 2012 Wiley Periodicals, Inc.

  10. GDP Release Preferentially Occurs on the Phosphate Side in Heterotrimeric G-proteins

    PubMed Central

    Louet, Maxime; Martinez, Jean; Floquet, Nicolas

    2012-01-01

    After extra-cellular stimulation of G-Protein Coupled Receptors (GPCRs), GDP/GTP exchange appears as the key, rate limiting step of the intracellular activation cycle of heterotrimeric G-proteins. Despite the availability of a large number of X-ray structures, the mechanism of GDP release out of heterotrimeric G-proteins still remains unknown at the molecular level. Starting from the available X-ray structure, extensive unconstrained/constrained molecular dynamics simulations were performed on the complete membrane-anchored Gi heterotrimer complexed to GDP, for a total simulation time overcoming 500 ns. By combining Targeted Molecular Dynamics (TMD) and free energy profiles reconstruction by umbrella sampling, our data suggest that the release of GDP was much more favored on its phosphate side. Interestingly, upon the forced extraction of GDP on this side, the whole protein encountered large, collective motions in perfect agreement with those we described previously including a domain to domain motion between the two ras-like and helical sub-domains of Gα. PMID:22829757

  11. 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.

  12. 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

  13. A purified agonist-activated G-protein coupled receptor: truncated octopus Acid Metarhodopsin.

    PubMed

    Ashida, Akemi; Matsumoto, Kumi; Ebrey, Thomas G; Tsuda, Motoyuki

    2004-03-01

    G-protein coupled receptors (GPCRs) mediate responses to many types of extracellular signals. So far, bovine rhodopsin, the inactive form of a GPCR, is the only member of the family whose three dimensional structure has been determined. It would be desirable to determine the structure of the active form of a GPCR. In this paper, we report the large scale preparation of a stable, homogenous species, truncated octopus rhodopsin (t-rhodopsin) in which proteolysis has removed the proline-rich C-terminal; this species retains the spectral properties and the ability for light-induced G-protein activation of unproteolyzed octopus rhodopsin. Moreover, starting from this species we can prepare a pure, active form of pigment, octopus t-Acid Metarhodopsin which has an all-trans-retinal as its agonist. Photoisomerization of t-Acid Metarhodopsin leads back to the inactive form, t-rhodopsin with the inverse agonist 11-cis-retinal. Octopus t-Acid Metarhodopsin can activate an endogenous octopus G-protein in the dark and this activity is reduced by irradiation with orange light which photoregenerates t-Acid Metarhodopsin back to the initial species, t-rhodopsin.

  14. 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.

  15. G protein-coupled receptor kinases: more than just kinases and not only for GPCRs

    PubMed Central

    Gurevich, Eugenia V.; Tesmer, John J. G.; Mushegian, Arcady; Gurevich, Vsevolod V.

    2011-01-01

    G protein-coupled receptor (GPCR) kinases (GRKs) are best known for their role in homologous desensitization of GPCRs. GRKs phosphorylate activated receptors and promote high affinity binding of arrestins, which precludes G protein coupling. GRKs have a multidomain structure, with the kinase domain inserted into a loop of a regulator of G protein signaling homology domain. Unlike many other kinases, GRKs do not need to be phosphorylated in their activation loop to achieve an activated state. Instead, they are directly activated by docking with active GPCRs. In this manner they are able to selectively phosphorylate Ser/Thr residues on only the activated form of the receptor, unlike related kinases such as protein kinase A. GRKs also phosphorylate a variety of non-GPCR substrates and regulate several signaling pathways via direct interactions with other proteins in a phosphorylation-independent manner. Multiple GRK subtypes are present in virtually every animal cell, with the highest expression levels found in neurons, with their extensive and complex signal regulation. Insufficient or excessive GRK activity was implicated in a variety of human disorders, ranging from heart failure to depression to Parkinson’s disease. As key regulators of GPCR-dependent and -independent signaling pathways, GRKs are emerging drug targets and promising molecular tools for therapy. Targeted modulation of expression and/or of activity of several GRK isoforms for therapeutic purposes was recently validated in cardiac disorders and Parkinson’s disease. PMID:21903131

  16. Increased G protein-coupled receptor kinase (GRK) expression in the anterior cingulate cortex in schizophrenia.

    PubMed

    Funk, Adam J; Haroutunian, Vahram; Meador-Woodruff, James H; McCullumsmith, Robert E

    2014-10-01

    Current pharmacological treatments for schizophrenia target G protein-coupled receptors (GPCRs), including dopamine receptors. Ligand-bound GPCRs are regulated by a family of G protein-coupled receptor kinases (GRKs), members of which uncouple the receptor from heterotrimeric G proteins, desensitize the receptor, and induce receptor internalization via the arrestin family of scaffolding and signaling molecules. GRKs initiate the activation of downstream signaling pathways, can regulate receptors and signaling molecules independent of GPCR phosphorylation, and modulate epigenetic regulators like histone deacetylases (HDACs). We hypothesize that the expression of GRK proteins is altered in schizophrenia, consistent with previous findings of alterations upstream and downstream from this family of molecules that facilitate intracellular signaling processes. In this study, we measured protein expression via Western blot analysis for GRKs 2, 3, 5, and 6 in the anterior cingulate cortex of patients with schizophrenia (n=36) and a comparison group (n=33). To control for antipsychotic treatment, we measured these same targets in haloperidol-treated vs. untreated rats (n=10 for both). We found increased levels of GRK5 in schizophrenia. No changes were detected in GRK protein expression in rats treated with haloperidol decanoate for 9 months. These data suggest that increased GRK5 expression may contribute to the pathophysiology of schizophrenia via abnormal regulation of the cytoskeleton, endocytosis, signaling, GPCRs, and histone modification. Copyright © 2014 Elsevier B.V. All rights reserved.

  17. 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.

  18. 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

  19. 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.

  20. 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

  1. 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.

  2. 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.

  3. Cholesterol up-regulates neuronal G protein-gated inwardly rectifying potassium (GIRK) channel activity in the hippocampus.

    PubMed

    Bukiya, Anna N; Durdagi, Serdar; Noskov, Sergei; Rosenhouse-Dantsker, Avia

    2017-04-14

    Hypercholesterolemia is a well known risk factor for the development of neurodegenerative disease. However, the underlying mechanisms are mostly unknown. In recent years, it has become increasingly evident that cholesterol-driven effects on physiology and pathophysiology derive from its ability to alter the function of a variety of membrane proteins including ion channels. Yet, the effect of cholesterol on G protein-gated inwardly rectifying potassium (GIRK) channels expressed in the brain is unknown. GIRK channels mediate the actions of inhibitory brain neurotransmitters. As a result, loss of GIRK function can enhance neuron excitability, whereas gain of GIRK function can reduce neuronal activity. Here we show that in rats on a high-cholesterol diet, cholesterol levels in hippocampal neurons are increased. We also demonstrate that cholesterol plays a critical role in modulating neuronal GIRK currents. Specifically, cholesterol enrichment of rat hippocampal neurons resulted in enhanced channel activity. In accordance, elevated currents upon cholesterol enrichment were also observed in Xenopus oocytes expressing GIRK2 channels, the primary GIRK subunit expressed in the brain. Furthermore, using planar lipid bilayers, we show that although cholesterol did not affect the unitary conductance of GIRK2, it significantly enhanced the frequency of channel openings. Last, combining computational and functional approaches, we identified two putative cholesterol-binding sites in the transmembrane domain of GIRK2. These findings establish that cholesterol plays a critical role in modulating GIRK activity in the brain. Because up-regulation of GIRK function can reduce neuronal activity, our findings may lead to novel approaches for prevention and therapy of cholesterol-driven neurodegenerative disease. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  4. The Visual Cycle in the Inner Retina of Chicken and the Involvement of Retinal G-Protein-Coupled Receptor (RGR).

    PubMed

    Díaz, Nicolás M; Morera, Luis P; Tempesti, Tomas; Guido, Mario E

    2017-05-01

    The vertebrate retina contains typical photoreceptor (PR) cones and rods responsible for day/night vision, respectively, and intrinsically photosensitive retinal ganglion cells (ipRGCs) involved in the regulation of non-image-forming tasks. Rhodopsin/cone opsin photopigments in visual PRs or melanopsin (Opn4) in ipRGCs utilizes retinaldehyde as a chromophore. The retinoid regeneration process denominated as "visual cycle" involves the retinal pigment epithelium (RPE) or Müller glial cells. Opn4, on the contrary, has been characterized as a bi/tristable photopigment, in which a photon of one wavelength isomerizes 11-cis to all-trans retinal (Ral), with a second photon re-isomerizing it back. However, it is unknown how the chromophore is further metabolized in the inner retina. Nor is it yet clear whether an alternative secondary cycle occurs involving players such as the retinal G-protein-coupled receptor (RGR), a putative photoisomerase of unidentified inner retinal activity. Here, we investigated the role of RGR in retinoid photoisomerization in Opn4x (Xenopus ortholog) (+) RGC primary cultures free of RPE and other cells from chicken embryonic retinas. Opn4x (+) RGCs display significant photic responses by calcium fluorescent imaging and photoisomerize exogenous all-trans to 11-cis Ral and other retinoids. RGR was found to be expressed in developing retina and in primary cultures; when its expression was knocked down, the levels of 11-cis, all-trans Ral, and all-trans retinol in cultures exposed to light were significantly higher and those in all-trans retinyl esters lower than in dark controls. The results support a novel role for RGR in ipRGCs to modulate retinaldehyde levels in light, keeping the balance of inner retinal retinoid pools.

  5. 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

  6. 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

  7. 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.

  8. 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.

  9. 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

  10. Characterization of structurally novel G protein biased CB1 agonists: Implications for drug development.

    PubMed

    Ford, Benjamin M; Franks, Lirit N; Tai, Sherrica; Fantegrossi, William E; Stahl, Edward L; Berquist, Michael D; Cabanlong, Christian V; Wilson, Catheryn D; Penthala, Narsimha R; Crooks, Peter A; Prather, Paul L

    2017-08-23

    The human cannabinoid subtype 1 receptor (hCB1R) is highly expressed in the CNS and serves as a therapeutic target for endogenous ligands as well as plant-derived and synthetic cannabinoids. Unfortunately, acute use of hCB1R agonists produces unwanted psychotropic effects and chronic administration results in development of tolerance and dependence, limiting the potential clinical use of these ligands. Studies in β-arrestin knockout mice suggest that interaction of certain GPCRs, including μ-, δ-, κ-opioid and hCB1Rs, with β-arrestins might be responsible for several adverse effects produced by agonists acting at these receptors. Indeed, agonists that bias opioid receptor activation toward G-protein, relative to β-arrestin signaling, produce less severe adverse effects. These observations indicate that therapeutic utility of agonists acting at hCB1Rs might be improved by development of G-protein biased hCB1R agonists. Our laboratory recently reported a novel class of indole quinulidinone (IQD) compounds that bind cannabinoid receptors with relatively high affinity and act with varying efficacy. The purpose of this study was to determine whether agonists in this novel cannabinoid class exhibit ligand bias at hCB1 receptors. Our studies found that a novel IQD-derived hCB1 receptor agonist PNR-4-20 elicits robust G protein-dependent signaling, with transduction ratios similar to the non-biased hCB1R agonist CP-55,940. In marked contrast to CP-55,940, PNR-4-20 produces little to no β-arrestin 2 recruitment. Quantitative calculation of bias factors indicates that PNR-4-20 exhibits from 5.4-fold to 29.5-fold bias for G protein, relative to β-arrestin 2 signaling (when compared to G protein activation or inhibition of forskolin-stimulated cAMP accumulation, respectively). Importantly, as expected due to reduced β-arrestin 2 recruitment, chronic exposure of cells to PNR-4-20 results in significantly less desensitization and down-regulation of hCB1Rs compared to

  11. 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.

  12. 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.

  13. 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.

  14. 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.

  15. 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

  16. 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.

  17. The complex G protein-coupled receptor kinase 2 (GRK2) interactome unveils new physiopathological targets

    PubMed Central

    Penela, Petronila; Murga, Cristina; Ribas, Catalina; Lafarga, Vanesa; Mayor, Federico

    2010-01-01

    GRK2 is a ubiquitous member of the G protein-coupled receptor kinase (GRK) family that appears to play a central, integrative role in signal transduction cascades. GRKs participate together with arrestins in the regulation of G protein-coupled receptors (GPCR), a family of hundreds of membrane proteins of key physiological and pharmacological importance, by triggering receptor desensitization from G proteins and GPCR internalization, and also by helping assemble macromolecular signalosomes in the receptor environment acting as agonist-regulated adaptor scaffolds, thus contributing to signal propagation. In addition, emerging evidence indicates that GRK2 can phosphorylate a growing number of non-GPCR substrates and associate with a variety of proteins related to signal transduction, thus suggesting that this kinase could also have diverse ‘effector’ functions. We discuss herein the increasing complexity of such GRK2 ‘interactome’, with emphasis on the recently reported roles of this kinase in cell migration and cell cycle progression and on the functional impact of the altered GRK2 levels observed in several relevant cardiovascular, inflammatory or tumour pathologies. Deciphering how the different networks of potential GRK2 functional interactions are orchestrated in a stimulus, cell type or context-specific way is critical to unveil the contribution of GRK2 to basic cellular processes, to understand how alterations in GRK2 levels or functionality may participate in the onset or development of several cardiovascular, tumour or inflammatory diseases, and to assess the feasibility of new therapeutic strategies based on the modulation of the activity, levels or specific interactions of GRK2. PMID:20590581

  18. 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.

  19. 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.

  20. Rho and Rab Small G Proteins Coordinately Reorganize Stress Fibers and Focal Adhesions in MDCK Cells

    PubMed Central

    Imamura, Hiroshi; Takaishi, Kenji; Nakano, Katsutoshi; Kodama, Atsuko; Oishi, Hideto; Shiozaki, Hitoshi; Monden, Morito; Sasaki, Takuya; Takai, Yoshimi

    1998-01-01

    The Rho subfamily of the Rho small G protein family (Rho) regulates formation of stress fibers and focal adhesions in many types of cultured cells. In moving cells, dynamic and coordinate disassembly and reassembly of stress fibers and focal adhesions are observed, but the precise mechanisms in the regulation of these processes are poorly understood. We previously showed that 12-O-tetradecanoylphorbol-13-acetate (TPA) first induced disassembly of stress fibers and focal adhesions followed by their reassembly in MDCK cells. The reassembled stress fibers showed radial-like morphology that was apparently different from the original. We analyzed here the mechanisms of these TPA-induced processes. Rho inactivation and activation were necessary for the TPA-induced disassembly and reassembly, respectively, of stress fibers and focal adhesions. Both inactivation and activation of the Rac subfamily of the Rho family (Rac) inhibited the TPA-induced reassembly of stress fibers and focal adhesions but not their TPA-induced disassembly. Moreover, microinjection or transient expression of Rab GDI, a regulator of all the Rab small G protein family members, inhibited the TPA-induced reassembly of stress fibers and focal adhesions but not their TPA-induced disassembly, indicating that, furthermore, activation of some Rab family members is necessary for their TPA-induced reassembly. Of the Rab family members, at least Rab5 activation was necessary for the TPA-induced reassembly of stress fibers and focal adhesions. The TPA-induced, small G protein-mediated reorganization of stress fibers and focal adhesions was closely related to the TPA-induced cell motility. These results indicate that the Rho and Rab family members coordinately regulate the TPA-induced reorganization of stress fibers and focal adhesions that may cause cell motility. PMID:9725912

  1. 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.

  2. 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.

  3. A peptide core motif for binding to heterotrimeric G protein alpha subunits.

    PubMed

    Ja, William W; Adhikari, Anirban; Austin, Ryan J; Sprang, Stephen R; Roberts, Richard W

    2005-09-16

    Recently, in vitro selection using mRNA display was used to identify a novel peptide sequence that binds with high affinity to Galpha(i1). The peptide was minimized to a 9-residue sequence (R6A-1) that retains high affinity and specificity for the GDP-bound state of Galpha(i1) and acts as a guanine nucleotide dissociation inhibitor (GDI). Here we demonstrate that the R6A-1 peptide interacts with Galpha subunits representing all four G protein classes, acting as a core motif for Galpha interaction. This contrasts with the consensus G protein regulatory(GPR) sequence, a 28-mer peptide GDI derived from the GoLoco (Galpha(i/0)-Loco interaction)/GPR motif that shares no homology with R6A-1 and binds only to Galpha(i1-3) in this assay. Binding of R6A-1 is generally specific to the GDP-bound state of the Galpha subunits and excludes association with Gbetagamma. R6A-Galpha(i1) complexes are resistant to trypsin digestion and exhibit distinct stability in the presence of Mg(2+), suggesting that the R6A and GPR peptides exert their activities using different mechanisms. Studies using Galpha(i1)/Galpha(s) chimeras identify two regions of Galpha(i1) (residues 1-35 and 57-88) as determinants for strong R6A-G(ialpha1) interaction. Residues flanking the R6A-1 peptide confer unique binding properties, indicating that the core motif could be used as a starting point for the development of peptides exhibiting novel activities and/or specificity for particular G protein subclasses or nucleotide-bound states.

  4. G-protein-coupled receptors for neurotransmitter amino acids: C-terminal tails, crowded signalosomes.

    PubMed Central

    El Far, Oussama; Betz, Heinrich

    2002-01-01

    G-protein-coupled receptors (GPCRs) represent a superfamily of highly diverse integral membrane proteins that transduce external signals to different subcellular compartments, including nuclei, via trimeric G-proteins. By differential activation of diffusible G(alpha) and membrane-bound G(beta)gamma subunits, GPCRs might act on both cytoplasmic/intracellular and plasma-membrane-bound effector systems. The coupling efficiency and the plasma membrane localization of GPCRs are regulated by a variety of interacting proteins. In this review, we discuss recently disclosed protein interactions found with the cytoplasmic C-terminal tail regions of two types of presynaptic neurotransmitter receptors, the group III metabotropic glutamate receptors and the gamma-aminobutyric acid type-B receptors (GABA(B)Rs). Calmodulin binding to mGluR7 and other group III mGluRs may provide a Ca(2+)-dependent switch for unidirectional (G(alpha)) versus bidirectional (G(alpha) and G(beta)gamma) signalling to downstream effector proteins. In addition, clustering of mGluR7 by PICK1 (protein interacting with C-kinase 1), a polyspecific PDZ (PSD-95/Dlg1/ZO-1) domain containing synaptic organizer protein, sheds light on how higher-order receptor complexes with regulatory enzymes (or 'signalosomes') could be formed. The interaction of GABA(B)Rs with the adaptor protein 14-3-3 and the transcription factor ATF4 (activating transcription factor 4) suggests novel regulatory pathways for G-protein signalling, cytoskeletal reorganization and nuclear gene expression: processes that may all contribute to synaptic plasticity. PMID:12006104

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

    PubMed

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

    2015-01-01

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

  6. 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

  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. 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

  10. The PIX-GIT complex: a G protein signaling cassette in control of cell shape.

    PubMed

    Frank, Scott R; Hansen, Steen H

    2008-06-01

    Arf and Rho GTP-binding proteins coordinately regulate membrane dynamics and cytoskeletal rearrangements. The Cdc42/Rac guanine nucleotide exchange factor PIX and the Arf GTPase-activating protein GIT form a stable complex in cells. The PIX-GIT complex functions to integrate signaling among Arf, Cdc42, and Rac proteins in response to cues emanating from integrins, heterotrimeric G proteins, receptor tyrosine kinases, and cell-cell interactions. A concept that emerges from the literature is that the PIX-GIT complex serves as a cassette to elicit changes in cell shape essential for polarized cell responses in a wide range of biological contexts.

  11. 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.

  12. 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.

  13. 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.

  14. Quantifying the allosteric interactions within a G-protein-coupled receptor heterodimer.

    PubMed

    Zhou, Bin; Giraldo, Jesús

    2017-07-27

    G-protein-coupled receptors are central to signal transduction and cell communication. The possibility that cells use receptor heteromerization to modulate individual receptor pathways is a surmise that cannot be precluded. Given the complexity of these processes, mathematical models contribute to understanding how receptors and their respective ligands regulate signaling. Here, a mathematical model is presented that quantifies the allosteric interactions within a receptor heterodimer. The model is based on the operational model of allosterism including constitutive receptor activity, which provides the pharmacological analysis of heteromerization with well-established and widely used modeling and fitting procedures. Copyright © 2017 Elsevier Ltd. All rights reserved.

  15. 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

  16. Kinetics and dynamics in the G protein-coupled receptor signaling cascade.

    PubMed

    Vilardaga, Jean-Pierre; Romero, Guillermo; Feinstein, Timothy N; Wehbi, Vanessa L

    2013-01-01

    We describe optical and microscopy methods based on Förster resonance energy transfer, fluorescence recovery after photobleaching, and imaging cross-correlation spectroscopy that permit to determine kinetic and dynamic properties of key reactions involved G protein-coupled receptor (GPCR) signaling from the initial ligand binding step to the generation of the second messenger, cAMP. Well suited to determine rate-limiting reactions taking place along a GPCR signaling cascade in live cells, these techniques have also uncovered new concepts in GPCR signaling as well as many interesting mechanistic subtleties by which GPCRs transmit neurotransmitter and hormone signals into cells. Copyright © 2013 Elsevier Inc. All rights reserved.

  17. 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

  18. 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

  19. 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.

  20. Lipid-mediated regulation of G protein-coupled receptor kinases 2 and 3.

    PubMed

    DebBurman, S K; Ptasienski, J; Boetticher, E; Lomasney, J W; Benovic, J L; Hosey, M M

    1995-03-17

    G protein-coupled receptor-mediated signaling is attenuated by a process referred to as desensitization, wherein agonist-dependent phosphorylation of receptors by G protein-coupled receptor kinases (GRKs) is proposed to be a key initial event. However, mechanisms that activate GRKs are not fully understood. In one scenario, beta gamma-subunits of G proteins (G beta gamma) activate certain GRKs (beta-adrenergic receptor kinases 1 and 2, or GRK2 and GRK3), via a pleckstrin homology domain in the COOH terminus. This interaction has been proposed to translocate cytosolic beta-adrenergic receptor kinases (beta ARKs) to the plasma membrane and facilitate interaction with receptor substrates. Here, we report a novel finding that membrane lipids modulate beta ARK activity in vitro in a manner that is analogous and competitive with G beta gamma. Several lipids, including phosphatidylserine (PS), stimulated, whereas phosphatidylinositol 4,5-bisphosphate inhibited, the ability of these GRKs to phosphorylate agonist-occupied m2 muscarinic acetylcholine receptors. Furthermore, both PS and phosphatidylinositol 4,5-bisphosphate specifically bound to beta ARK1, whereas phosphatidylcholine, a lipid that did not modulate beta ARK activity, did not bind to beta ARK1. The lipid regulation of beta ARKs did not occur via a modulation of its autophosphorylation state. PS- and G beta gamma-mediated stimulation of beta ARK1 was compared and found strikingly similar; moreover, their effects together were not additive (except at initial stages of reaction), which suggests that PS and G beta gamma employed a common interaction and activation mechanism with the kinase. The effects of these lipids were prevented by two well known G beta gamma-binding proteins, phosducin and GST-beta ARK-(466-689) fusion protein, suggesting that the G beta gamma-binding domain (possibly the plecks