G2Cdb::Gene report

Gene id
G00001457
Gene symbol
GNAO1 (HGNC)
Species
Homo sapiens
Description
guanine nucleotide binding protein (G protein), alpha activating activity polypeptide O
Orthologue
G00000208 (Mus musculus)

Databases (8)

Curated Gene
OTTHUMG00000073092 (Vega human gene)
Gene
ENSG00000087258 (Ensembl human gene)
2775 (Entrez Gene)
353 (G2Cdb plasticity & disease)
GNAO1 (GeneCards)
Literature
139311 (OMIM)
Marker Symbol
HGNC:4389 (HGNC)
Protein Sequence
P09471 (UniProt)

Synonyms (1)

  • G-ALPHA-o

Literature (59)

Pubmed - other

  • Altered G-protein coupling in an mGluR6 point mutant associated with congenital stationary night blindness.

    Beqollari D, Betzenhauser MJ and Kammermeier PJ

    Department of Pharmacology and Physiology, University of Rochester School of Medicine and Dentistry, Rochester, New York, USA.

    The highly specialized metabotropic glutamate receptor type 6 (mGluR6) is postsynaptically localized and expressed only in the dendrites of ON bipolar cells. Upon activation of mGluR6 by glutamate released from photoreceptors, a nonselective cation channel is inhibited, causing these cells to hyperpolarize. Mutations in this gene have been implicated in the development of congenital stationary night blindness type 1 (CSNB1). We investigated five known mGluR6 point mutants that lead to CSNB1 to determine the molecular mechanism of each phenotype. In agreement with other studies, four mutants demonstrated trafficking impairment. However, mGluR6 E775K (E781K in humans) suggested no trafficking or signaling deficiencies measured by our initial assays. Most importantly, our results indicate a switch in G-protein coupling, in which E775K loses G(o) coupling but retains coupling to G(i), which may explain the phenotype.

    Molecular pharmacology 2009;76;5;992-7

  • RGS11 interacts preferentially with R7BP over Galpha(oa)--characterization of Gbeta5-free RGS11.

    Saleem Y and Kim KS

    Center for Neural Science, Korea Institute of Science and Technology (KIST), P.O. Box 131, Cheongryang, Seoul 130-650, Republic of Korea.

    Regulator of G protein signaling 11 (RGS11) is the least characterized member of the R7 family of Ggamma-like GGL domain-containing RGS proteins. All R7-RGS proteins of a variety of cell types are found in Gbeta5-containing complexes that exhibit a number of unique functional properties. However, presence of Gbeta5 reduced the affinity of R7-RGS7 for Galpha subunits, also only RGS7 bound to Muscarinic M3-Receptor, but the Gbeta5-RGS7 dimer did not, making it difficult to study differential interaction of R7-RGS proteins. Here, we report the successful purification of functionally intact, Gbeta5-free recombinant RGS11 (rRGS11), obtained by expressing N- and C-terminally truncated form of RGS11 in Escherichia coli BL 21 (DE3), that differentially interact with R7BP and Galpha(oa). rRGS11 was capable of interacting with Galpha(oa) and R7BP (RGS7 family binding protein) with equilibrium dissociation constants (K(D)) of 904 (+/- 208) nM, and 308 (+/- 97) nM, respectively. It also induced several-fold increase in the GTPase activity of Galpha(oa). The binding of rRGS11 was differential with a binding preference for R7BP over Galpha(oa) implying extended roles of R7BP. In addition, we identified a novel interaction between Galpha(oa) and R7BP with a K(D) of 592 (+/- 150) nM. The production of stable and functional rRGS11 would provide chances to discover more functions of RGS11 yet to be identified.

    Biochemical and biophysical research communications 2009;386;1;65-70

  • Reviews in molecular biology and biotechnology: transmembrane signaling by G protein-coupled receptors.

    Luttrell LM

    Division of Endocrinology, Diabetes and Medical Genetics, Department of Medicine, Medical University of South Carolina, 96 Jonathan Lucas Street, 816 CSB, P.O. Box 250624, Charleston, SC 29425, USA. luttrell@musc.edu

    As the most diverse type of cell surface receptor, the importance heptahelical G protein-coupled receptors (GPCRs) to clinical medicine cannot be overestimated. Visual, olfactory and gustatory sensation, intermediary metabolism, cell growth and differentiation are all influenced by GPCR signals. The basic receptor-G protein-effector mechanism of GPCR signaling is tuned by a complex interplay of positive and negative regulatory events that amplify the effect of a hormone binding the receptor or that dampen cellular responsiveness. The association of heptahelical receptors with a variety of intracellular partners other than G proteins has led to the discovery of potential mechanisms of GPCR signaling that extend beyond the classical paradigms. While the physiologic relevance of many of these novel mechanisms of GPCR signaling remains to be established, their existence suggests that the mechanisms of GPCR signaling are even more diverse than previously imagined.

    Molecular biotechnology 2008;39;3;239-64

  • Galphao/i-stimulated proteosomal degradation of RGS20: a mechanism for temporal integration of Gs and Gi pathways.

    Pagano M, Jordan JD, Neves SR, Nguyen T and Iyengar R

    Department of Pharmacology and Biological Chemistry, Mount Sinai School of Medicine, New York, NY 10029, United States.

    The G(s) and G(i) pathways interact to control the levels of intracellular cAMP. Although coincident signaling through G(s) and G(i)-coupled receptors can attenuate G(s)-stimulated cAMP levels, it is not known if prior activation of the G(i) pathway can affect signaling by G(s)-coupled receptors. We have found that activated Galpha(o/i) interact with RGS20, a GTPase activating protein for members of the Galpha(omicron/i) family. Interaction between Galpha(o/i) and RGS20 results in decreased cellular levels of RGS20. This decrease was induced by activated Galpha(o) and Galpha(i2) but not by Galpha(q), Galpha(i1) or Galpha(i3.) The Galpha(o/i)-induced decrease in RGS20 can be blocked by proteasomal inhibitors lactacystin or MG132. Activated Galpha(o) stimulates the ubiquitination of RGS20. The serotonin-1A receptor that couples to G(o/i) reduces the levels of RGS20 and this effect is blocked by lactacystin, suggesting that G(o/i) promotes the degradation of RGS20. Expression of RGS20 attenuates the inhibition of beta-adrenergic receptor-induced cAMP levels mediated by the serotonin-1A receptor. Prior activation of the serotonin-1A receptor results in loss of the RGS20-mediated attenuation, and the loss of attenuation is blocked when lactacystin is included during the prior treatment. These observations suggest that G(o/i)-coupled receptors, by stimulating the degradation of RGS20, can regulate how subsequent activation of the G(s) and G(i) pathways controls cellular cAMP levels, thus allowing for signal integration.

    Funded by: NHLBI NIH HHS: HL-007824, T32 HL007824; NIDA NIH HHS: DA-05798, F30 DA005798; NIDDK NIH HHS: DK038761-10, DK38761, R01 DK038761, R01 DK038761-10; NIGMS NIH HHS: F31 GM065065, GM-65065, R01 GM054508

    Cellular signalling 2008;20;6;1190-7

  • The alpha subunit of Go interacts with promyelocytic leukemia zinc finger protein and modulates its functions.

    Won JH, Park JS, Ju HH, Kim S, Suh-Kim H and Ghil SH

    Department of Life Science, Kyonggi University, Suwon 443-760, South Korea.

    Heterotrimeric GTP-binding proteins (G proteins) mediate signal transduction generated by neurotransmitters and hormones. Go, a member of the Go/Gi family, is the most abundant heterotrimeric G protein in the brain. Most mechanistic analyses on Go activation demonstrate that its action is mediated by the Gbetagamma dimer; downstream effectors for its alpha subunit (Goalpha) have not been clearly defined. Here, we employ the yeast two-hybrid system to screen for Goalpha-interacting partners in a cDNA library from human fetal brain. The transcription factor promyelocytic leukemia zinc finger protein (PLZF) specifically bound to Goalpha. Interactions between PLZF and Goalpha were confirmed using in vitro and in vivo affinity binding assays. Activated Goalpha interacted directly with PLZF, and enhanced its function as a transcriptional and cell growth suppressor. Notably, PLZF activity was additionally promoted by the Go/ialpha-coupled cannabinoid receptor (CB) in HL60 cells endogenously expressing CB and PLZF. These results collectively suggest that Goalpha modulates the function of PLZF via direct interactions. Our novel findings provide insights into the diverse cellular roles of Goalpha and its coupled receptor.

    Cellular signalling 2008;20;5;884-91

  • Mechanism of the receptor-catalyzed activation of heterotrimeric G proteins.

    Oldham WM, Van Eps N, Preininger AM, Hubbell WL and Hamm HE

    Department of Pharmacology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-6600, USA.

    Heptahelical receptors activate intracellular signaling pathways by catalyzing GTP for GDP exchange on the heterotrimeric G protein alpha subunit (G alpha). Despite the crucial role of this process in cell signaling, little is known about the mechanism of G protein activation. Here we explore the structural basis for receptor-mediated GDP release using electron paramagnetic resonance spectroscopy. Binding to the activated receptor (R*) causes an apparent rigid-body movement of the alpha5 helix of G alpha that would perturb GDP binding at the beta6-alpha5 loop. This movement was not observed when a flexible loop was inserted between the alpha5 helix and the R*-binding C terminus, which uncouples R* binding from nucleotide exchange, suggesting that this movement is necessary for GDP release. These data provide the first direct observation of R*-mediated conformational changes in G proteins and define the structural basis for GDP release from G alpha.

    Nature structural & molecular biology 2006;13;9;772-7

  • Diversification of transcriptional modulation: large-scale identification and characterization of putative alternative promoters of human genes.

    Kimura K, Wakamatsu A, Suzuki Y, Ota T, Nishikawa T, Yamashita R, Yamamoto J, Sekine M, Tsuritani K, Wakaguri H, Ishii S, Sugiyama T, Saito K, Isono Y, Irie R, Kushida N, Yoneyama T, Otsuka R, Kanda K, Yokoi T, Kondo H, Wagatsuma M, Murakawa K, Ishida S, Ishibashi T, Takahashi-Fujii A, Tanase T, Nagai K, Kikuchi H, Nakai K, Isogai T and Sugano S

    Life Science Research Laboratory, Central Research Laboratory, Hitachi, Ltd., Kokubunji, Tokyo, 185-8601, Japan.

    By analyzing 1,780,295 5'-end sequences of human full-length cDNAs derived from 164 kinds of oligo-cap cDNA libraries, we identified 269,774 independent positions of transcriptional start sites (TSSs) for 14,628 human RefSeq genes. These TSSs were clustered into 30,964 clusters that were separated from each other by more than 500 bp and thus are very likely to constitute mutually distinct alternative promoters. To our surprise, at least 7674 (52%) human RefSeq genes were subject to regulation by putative alternative promoters (PAPs). On average, there were 3.1 PAPs per gene, with the composition of one CpG-island-containing promoter per 2.6 CpG-less promoters. In 17% of the PAP-containing loci, tissue-specific use of the PAPs was observed. The richest tissue sources of the tissue-specific PAPs were testis and brain. It was also intriguing that the PAP-containing promoters were enriched in the genes encoding signal transduction-related proteins and were rarer in the genes encoding extracellular proteins, possibly reflecting the varied functional requirement for and the restricted expression of those categories of genes, respectively. The patterns of the first exons were highly diverse as well. On average, there were 7.7 different splicing types of first exons per locus partly produced by the PAPs, suggesting that a wide variety of transcripts can be achieved by this mechanism. Our findings suggest that use of alternate promoters and consequent alternative use of first exons should play a pivotal role in generating the complexity required for the highly elaborated molecular systems in humans.

    Genome research 2006;16;1;55-65

  • Towards a proteome-scale map of the human protein-protein interaction network.

    Rual JF, Venkatesan K, Hao T, Hirozane-Kishikawa T, Dricot A, Li N, Berriz GF, Gibbons FD, Dreze M, Ayivi-Guedehoussou N, Klitgord N, Simon C, Boxem M, Milstein S, Rosenberg J, Goldberg DS, Zhang LV, Wong SL, Franklin G, Li S, Albala JS, Lim J, Fraughton C, Llamosas E, Cevik S, Bex C, Lamesch P, Sikorski RS, Vandenhaute J, Zoghbi HY, Smolyar A, Bosak S, Sequerra R, Doucette-Stamm L, Cusick ME, Hill DE, Roth FP and Vidal M

    Center for Cancer Systems Biology and Department of Cancer Biology, Dana-Farber Cancer Institute, Harvard Medical School, 44 Binney Street, Boston, Massachusetts 02115, USA.

    Systematic mapping of protein-protein interactions, or 'interactome' mapping, was initiated in model organisms, starting with defined biological processes and then expanding to the scale of the proteome. Although far from complete, such maps have revealed global topological and dynamic features of interactome networks that relate to known biological properties, suggesting that a human interactome map will provide insight into development and disease mechanisms at a systems level. Here we describe an initial version of a proteome-scale map of human binary protein-protein interactions. Using a stringent, high-throughput yeast two-hybrid system, we tested pairwise interactions among the products of approximately 8,100 currently available Gateway-cloned open reading frames and detected approximately 2,800 interactions. This data set, called CCSB-HI1, has a verification rate of approximately 78% as revealed by an independent co-affinity purification assay, and correlates significantly with other biological attributes. The CCSB-HI1 data set increases by approximately 70% the set of available binary interactions within the tested space and reveals more than 300 new connections to over 100 disease-associated proteins. This work represents an important step towards a systematic and comprehensive human interactome project.

    Funded by: NCI NIH HHS: R33 CA132073; NHGRI NIH HHS: P50 HG004233, R01 HG001715, RC4 HG006066, U01 HG001715; NHLBI NIH HHS: U01 HL098166

    Nature 2005;437;7062;1173-8

  • A human protein-protein interaction network: a resource for annotating the proteome.

    Stelzl U, Worm U, Lalowski M, Haenig C, Brembeck FH, Goehler H, Stroedicke M, Zenkner M, Schoenherr A, Koeppen S, Timm J, Mintzlaff S, Abraham C, Bock N, Kietzmann S, Goedde A, Toksöz E, Droege A, Krobitsch S, Korn B, Birchmeier W, Lehrach H and Wanker EE

    Max Delbrueck Center for Molecular Medicine, 13092 Berlin-Buch, Germany.

    Protein-protein interaction maps provide a valuable framework for a better understanding of the functional organization of the proteome. To detect interacting pairs of human proteins systematically, a protein matrix of 4456 baits and 5632 preys was screened by automated yeast two-hybrid (Y2H) interaction mating. We identified 3186 mostly novel interactions among 1705 proteins, resulting in a large, highly connected network. Independent pull-down and co-immunoprecipitation assays validated the overall quality of the Y2H interactions. Using topological and GO criteria, a scoring system was developed to define 911 high-confidence interactions among 401 proteins. Furthermore, the network was searched for interactions linking uncharacterized gene products and human disease proteins to regulatory cellular pathways. Two novel Axin-1 interactions were validated experimentally, characterizing ANP32A and CRMP1 as modulators of Wnt signaling. Systematic human protein interaction screens can lead to a more comprehensive understanding of protein function and cellular processes.

    Cell 2005;122;6;957-68

  • CCL3, acting via the chemokine receptor CCR5, leads to independent activation of Janus kinase 2 (JAK2) and Gi proteins.

    Mueller A and Strange PG

    School of Animal and Microbial Sciences, University of Reading, Whiteknights, Reading RG6 6AJ, UK.

    The interaction of the chemokine receptor, CCR5, expressed in recombinant cells, with different G proteins was investigated and CCR5 was found to interact with Gi, Go and Gq species. Interaction with Gi leads to G protein activation, whereas Gq does not seem to be activated. Additionally, CCR5 activation also leads to phosphorylation of Janus kinase 2 (JAK2). Activation of JAK2 is independent of Gi or Gq activation. Gi protein activation was not prevented by inhibition of JAK, showing that heterotrimeric G protein activation and activation of the JAK/signal transducer and activator of transcription (STAT) pathway are independent of each other.

    FEBS letters 2004;570;1-3;126-32

  • A retinal-specific regulator of G-protein signaling interacts with Galpha(o) and accelerates an expressed metabotropic glutamate receptor 6 cascade.

    Dhingra A, Faurobert E, Dascal N, Sterling P and Vardi N

    Department of Neuroscience, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6058, USA. annu@retina.anatomy.upenn.edu

    G(o) is the most abundant G-protein in the brain, but its regulators are essentially unknown. In retina, Galpha(o1) is obligatory in mediating the metabotropic glutamate receptor 6 (mGluR6)-initiated ON response. To identify the interactors of G(o), we conducted a yeast two-hybrid screen with constituitively active Galpha(o) as a bait. The screen frequently identified a regulator of G-protein signaling (RGS), Ret-RGS1, the interaction of which we confirmed by coimmunoprecipitation with Galpha(o) in transfected cells and in retina. Ret-RGS1 localized to the dendritic tips of ON bipolar neurons, along with mGluR6 and Galpha(o1). When Ret-RGS1 was coexpressed in Xenopus oocytes with mGluR6, Galpha(o1), and a GIRK (G-protein-gated inwardly rectifying K+) channel, it accelerated the deactivation of the channel response to glutamate in a concentration-dependent manner. Because light onset suppresses glutamate release from photoreceptors onto the ON bipolar dendrites, Ret-RGS1 should accelerate the rising phase of the light response of the ON bipolar cell. This would tend to match its kinetics to that of the OFF bipolar that arises directly from ligand-gated channels.

    Funded by: NEI NIH HHS: EY 00128, EY 11105; NIGMS NIH HHS: GM068493

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2004;24;25;5684-93

  • Complete sequencing and characterization of 21,243 full-length human cDNAs.

    Ota T, Suzuki Y, Nishikawa T, Otsuki T, Sugiyama T, Irie R, Wakamatsu A, Hayashi K, Sato H, Nagai K, Kimura K, Makita H, Sekine M, Obayashi M, Nishi T, Shibahara T, Tanaka T, Ishii S, Yamamoto J, Saito K, Kawai Y, Isono Y, Nakamura Y, Nagahari K, Murakami K, Yasuda T, Iwayanagi T, Wagatsuma M, Shiratori A, Sudo H, Hosoiri T, Kaku Y, Kodaira H, Kondo H, Sugawara M, Takahashi M, Kanda K, Yokoi T, Furuya T, Kikkawa E, Omura Y, Abe K, Kamihara K, Katsuta N, Sato K, Tanikawa M, Yamazaki M, Ninomiya K, Ishibashi T, Yamashita H, Murakawa K, Fujimori K, Tanai H, Kimata M, Watanabe M, Hiraoka S, Chiba Y, Ishida S, Ono Y, Takiguchi S, Watanabe S, Yosida M, Hotuta T, Kusano J, Kanehori K, Takahashi-Fujii A, Hara H, Tanase TO, Nomura Y, Togiya S, Komai F, Hara R, Takeuchi K, Arita M, Imose N, Musashino K, Yuuki H, Oshima A, Sasaki N, Aotsuka S, Yoshikawa Y, Matsunawa H, Ichihara T, Shiohata N, Sano S, Moriya S, Momiyama H, Satoh N, Takami S, Terashima Y, Suzuki O, Nakagawa S, Senoh A, Mizoguchi H, Goto Y, Shimizu F, Wakebe H, Hishigaki H, Watanabe T, Sugiyama A, Takemoto M, Kawakami B, Yamazaki M, Watanabe K, Kumagai A, Itakura S, Fukuzumi Y, Fujimori Y, Komiyama M, Tashiro H, Tanigami A, Fujiwara T, Ono T, Yamada K, Fujii Y, Ozaki K, Hirao M, Ohmori Y, Kawabata A, Hikiji T, Kobatake N, Inagaki H, Ikema Y, Okamoto S, Okitani R, Kawakami T, Noguchi S, Itoh T, Shigeta K, Senba T, Matsumura K, Nakajima Y, Mizuno T, Morinaga M, Sasaki M, Togashi T, Oyama M, Hata H, Watanabe M, Komatsu T, Mizushima-Sugano J, Satoh T, Shirai Y, Takahashi Y, Nakagawa K, Okumura K, Nagase T, Nomura N, Kikuchi H, Masuho Y, Yamashita R, Nakai K, Yada T, Nakamura Y, Ohara O, Isogai T and Sugano S

    Helix Research Institute, 1532-3 Yana, Kisarazu, Chiba 292-0812, Japan.

    As a base for human transcriptome and functional genomics, we created the "full-length long Japan" (FLJ) collection of sequenced human cDNAs. We determined the entire sequence of 21,243 selected clones and found that 14,490 cDNAs (10,897 clusters) were unique to the FLJ collection. About half of them (5,416) seemed to be protein-coding. Of those, 1,999 clusters had not been predicted by computational methods. The distribution of GC content of nonpredicted cDNAs had a peak at approximately 58% compared with a peak at approximately 42%for predicted cDNAs. Thus, there seems to be a slight bias against GC-rich transcripts in current gene prediction procedures. The rest of the cDNAs unique to the FLJ collection (5,481) contained no obvious open reading frames (ORFs) and thus are candidate noncoding RNAs. About one-fourth of them (1,378) showed a clear pattern of splicing. The distribution of GC content of noncoding cDNAs was narrow and had a peak at approximately 42%, relatively low compared with that of protein-coding cDNAs.

    Nature genetics 2004;36;1;40-5

  • Oxidized human neuroglobin acts as a heterotrimeric Galpha protein guanine nucleotide dissociation inhibitor.

    Wakasugi K, Nakano T and Morishima I

    Department of Molecular Engineering, Graduate School of Engineering, Kyoto University, Japan. kei@wakasugi.mbox.media.kyoto-u.ac.jp

    Neuroglobin (Ngb) is a newly discovered vertebrate heme protein that is expressed in the brain and can reversibly bind oxygen. It has been reported that Ngb expression levels increase in response to oxygen deprivation and that it protects neurons from hypoxia in vitro and in vivo. However, the mechanism of this neuroprotection remains unclear. In the present study, we tried to clarify the neuroprotective role of Ngb under oxidative stress in vitro. By surface plasmon resonance, we found that ferric Ngb, which is generated spontaneously as a result of the rapid autoxidation, binds exclusively to the GDP-bound form of the alpha subunit of heterotrimeric G protein (Galphai). In GDP dissociation assays or guanosine 5'-O-(3-thio)triphosphate binding assays, ferric Ngb behaved as a guanine nucleotide dissociation inhibitor (GDI), inhibiting the rate of exchange of GDP for GTP. The interaction of GDP-bound Galphai with ferric Ngb will liberate Gbetagamma, leading to protection against neuronal death. In contrast, ferrous ligand-bound Ngb under normoxia did not have GDI activities. Taken together, we propose that human Ngb may be a novel oxidative stress-responsive sensor for signal transduction in the brain.

    The Journal of biological chemistry 2003;278;38;36505-12

  • A novel function of Goalpha: mediation of extracellular signal-regulated kinase activation by opioid receptors in neural cells.

    Zhang WB, Zhang Z, Ni YX, Wu YL and Pei G

    Laboratory of Molecular Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 320 Yue Yang Road, Shanghai 200031, China.

    Go is the most abundant G protein expressed in brain but its function is less known. Here we show a novel function of Goalpha as a mediator of opioid receptor-induced extracellular signal-regulated kinase activation in neural cells. The current study found that, in neuroblastoma x glioma NG108-15 hybrid cells, activation of extracellular signal-regulated kinase through delta opioid receptors was mediated by pertussis toxin-sensitive G protein and independent of Gbetagamma subunits, PI3 kinase and receptor internalization. Overexpression of a dominant negative form of Goalpha1, but not Gialpha2, completely blocked delta opioid receptor-induced extracellular signal-regulated kinase activity. Decreasing Goalpha expression by RNA interference greatly reduced delta opioid receptor-induced extracellular signal-regulated kinase activity and extracellular signal-regulated kinase-dependent gene expression, while knocking down Gialpha2 did not. By taking advantage of differences between human and mouse Goalpha gene sequences, we simultaneously knocked down endogenous Goalpha expression and expressed exogenous human Goalpha subunits. We found that both human Goalpha1 and Goalpha2 could mediate delta opioid receptor-induced extracellular signal-regulated kinase activation. This study suggests that one of the functions of Goalpha in the brain is to mediate extracellular signal-regulated kinase activation by G protein-coupled receptors.

    Journal of neurochemistry 2003;86;5;1213-22

  • Subcellular localization of LGN during mitosis: evidence for its cortical localization in mitotic cell culture systems and its requirement for normal cell cycle progression.

    Kaushik R, Yu F, Chia W, Yang X and Bahri S

    Institute of Molecular and Cell Biology, Singapore 117609.

    Mammalian LGN/AGS3 proteins and their Drosophila Pins orthologue are cytoplasmic regulators of G-protein signaling. In Drosophila, Pins localizes to the lateral cortex of polarized epithelial cells and to the apical cortex of neuroblasts where it plays important roles in their asymmetric division. Using overexpression studies in different cell line systems, we demonstrate here that, like Drosophila Pins, LGN can exhibit enriched localization at the cell cortex, depending on the cell cycle and the culture system used. We find that in WISH, PC12, and NRK but not COS cells, LGN is largely directed to the cell cortex during mitosis. Overexpression of truncated protein domains further identified the Galpha-binding C-terminal portion of LGN as a sufficient domain for cortical localization in cell culture. In mitotic COS cells that normally do not exhibit cortical LGN localization, LGN is redirected to the cell cortex upon overexpression of Galpha subunits of heterotrimeric G-proteins. The results also show that the cortical localization of LGN is dependent on microfilaments and that interfering with LGN function in cultured cell lines causes early disruption to cell cycle progression.

    Molecular biology of the cell 2003;14;8;3144-55

  • Functional characterization of human receptors for short chain fatty acids and their role in polymorphonuclear cell activation.

    Le Poul E, Loison C, Struyf S, Springael JY, Lannoy V, Decobecq ME, Brezillon S, Dupriez V, Vassart G, Van Damme J, Parmentier M and Detheux M

    Euroscreen, rue Adrienne Bolland 47, 6041 Gosselies, Laboratory of Molecular Immunology, Rega Institute for Medical Research, Catholic University of Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium.

    Short chain fatty acids (SCFAs), including acetate, propionate, and butyrate, are produced at high concentration by bacteria in the gut and subsequently released in the bloodstream. Basal acetate concentrations in the blood (about 100 microm) can further increase to millimolar concentrations following alcohol intake. It was known previously that SCFAs can activate leukocytes, particularly neutrophils. In the present work, we have identified two previously orphan G protein-coupled receptors, GPR41 and GPR43, as receptors for SCFAs. Propionate was the most potent agonist for both GPR41 and GPR43. Acetate was more selective for GPR43, whereas butyrate and isobutyrate were more active on GPR41. The two receptors were coupled to inositol 1,4,5-trisphosphate formation, intracellular Ca2+ release, ERK1/2 activation, and inhibition of cAMP accumulation. They exhibited, however, a differential coupling to G proteins; GPR41 coupled exclusively though the Pertussis toxin-sensitive Gi/o family, whereas GPR43 displayed a dual coupling through Gi/o and Pertussis toxin-insensitive Gq protein families. The broad expression profile of GPR41 in a number of tissues does not allow us to infer clear hypotheses regarding its biological functions. In contrast, the highly selective expression of GPR43 in leukocytes, particularly polymorphonuclear cells, suggests a role in the recruitment of these cell populations toward sites of bacterial infection. The pharmacology of GPR43 matches indeed the effects of SCFAs on neutrophils, in terms of intracellular Ca2+ release and chemotaxis. Such a neutrophil-specific SCFA receptor is potentially involved in the development of a variety of diseases characterized by either excessive or inefficient neutrophil recruitment and activation, such as inflammatory bowel diseases or alcoholism-associated immune depression. GPR43 might therefore constitute a target allowing us to modulate immune responses in these pathological situations.

    The Journal of biological chemistry 2003;278;28;25481-9

  • Mammalian Ric-8A (synembryn) is a heterotrimeric Galpha protein guanine nucleotide exchange factor.

    Tall GG, Krumins AM and Gilman AG

    Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas 75390-9041, USA.

    The activation of heterotrimeric G proteins is accomplished primarily by the guanine nucleotide exchange activity of ligand-bound G protein-coupled receptors. The existence of nonreceptor guanine nucleotide exchange factors for G proteins has also been postulated. Yeast two-hybrid screens with Galpha(o) and Galpha(s) as baits were performed to identify binding partners of these proteins. Two mammalian homologs of the Caenorhabditis elegans protein Ric-8 were identified in these screens: Ric-8A (Ric-8/synembryn) and Ric-8B. Purification and biochemical characterization of recombinant Ric-8A revealed that it is a potent guanine nucleotide exchange factor for a subset of Galpha proteins including Galpha(q), Galpha(i1), and Galpha(o), but not Galpha(s). The mechanism of Ric-8A-mediated guanine nucleotide exchange was elucidated. Ric-8A interacts with GDP-bound Galpha proteins, stimulates release of GDP, and forms a stable nucleotide-free transition state complex with the Galpha protein; this complex dissociates upon binding of GTP to Galpha.

    Funded by: NIGMS NIH HHS: GM34497

    The Journal of biological chemistry 2003;278;10;8356-62

  • Thrombin receptors activate G(o) proteins in endothelial cells to regulate intracellular calcium and cell shape changes.

    Vanhauwe JF, Thomas TO, Minshall RD, Tiruppathi C, Li A, Gilchrist A, Yoon EJ, Malik AB and Hamm HE

    Institute for Neuroscience, Department of Molecular Pharmacology and Biological Chemistry, Northwestern University, Chicago, IL 60611, USA.

    Thrombin receptors couple to G(i/o), G(q), and G(12/13) proteins to regulate a variety of signal transduction pathways that underlie the physiological role of endothelial cells in wound healing or inflammation. Whereas the involvement of G(i), G(q), G(12), or G(13) proteins in thrombin signaling has been investigated extensively, the role of G(o) proteins has largely been ignored. To determine whether G(o) proteins could contribute to thrombin-mediated signaling in endothelial cells, we have developed minigenes that encode an 11-amino acid C-terminal peptide of G(o1) proteins. Previously, we have shown that use of the C-terminal minigenes can specifically block receptor activation of G protein families (). In this study, we demonstrate that G(o) proteins are present in human microvascular endothelial cells (HMECs). Moreover, we show that thrombin receptors can stimulate [(35)S]guanosine-5'-O-(3-thio)triphosphate binding to G(o) proteins when co-expressed in Sf9 membranes. The potential coupling of thrombin receptors to G(o) proteins was substantiated by transfection of the G(o1) minigene into HMECs, which led to a blockade of thrombin-stimulated release of [Ca(2+)](i) from intracellular stores. Transfection of the beta-adrenergic kinase C terminus blocked the [Ca(2+)](i) response to the same extent as with G(o1) minigene peptide, suggesting that this G(o)-mediated [Ca(2+)](i) transient was caused by Gbetagamma stimulation of PLCbeta. Transfection of a G(i1/2) minigene had no effect on thrombin-stimulated [Ca(2+)](i) signaling in HMEC, suggesting that Gbetagamma derived from G(o) but not G(i) could activate PLCbeta. The involvement of G(o) proteins on events downstream from calcium signaling was further evidenced by investigating the effect of G(o1) minigenes on thrombin-stimulated stress fiber formation and endothelial barrier permeability. Both of these effects were sensitive to pertussis toxin treatment and could be blocked by transfection of G(o1) minigenes but not G(i1/2) minigenes. We conclude that the G(o) proteins play a role in thrombin signaling distinct from G(i1/2) proteins, which are mediated through their Gbetagamma subunits and involve coupling to calcium signaling and cytoskeletal rearrangements.

    Funded by: NHLBI NIH HHS: HL 60678-01A1

    The Journal of biological chemistry 2002;277;37;34143-9

  • Serine 232 of the alpha(2A)-adrenergic receptor is a protein kinase C-sensitive effector coupling switch.

    Liang M, Freedman NJ, Theiss CT and Liggett SB

    Department of Medicine, University of Cincinnati College of Medicine, 231 Albert Sabin Way, ML 0564, Cincinnati, Ohio 45267-0564, USA.

    alpha(2)-adrenergic receptors (alpha(2)AR) couple to multiple effectors including adenylyl cyclase and phospholipase C. We hypothesized that signaling selectivity to these effectors is dynamically directed by kinase-sensitive domains within the third intracellular loop of the receptor. Substitution of Ala for Ser232, which is in the N-terminal region of this loop in the alpha(2A)AR, resulted in a receptor that was markedly uncoupled ( approximately 82% impairment) from stimulation of inositol phosphate accumulation while the capacity to inhibit adenylyl cyclase remained relatively intact. In S232A alpha(2A)AR transfected cell membranes, agonist-promoted [(35)S]GTPgammaS binding was reduced by approximately 50%. Coexpression of modified G proteins rendered insensitive to pertussis toxin revealed that the S232A receptor was uncoupled from both G(i) and G(o). S232 is a potential PKC phosphorylation site, and whole cell phosphorylation studies showed that the mutant had depressed phosphorylation compared to wild type (1.3- vs 2.1-fold/basal). Consistent with S232 directing coupling to phospholipase C, PMA exposure resulted in approximately 67% desensitization of agonist-promoted inositol phosphate accumulation without significantly affecting inhibition of adenylyl cyclase. The dominant effect of mutation or phosphorylation at this site on inositol phosphate as compared to cAMP signaling was found to most likely be due to the low efficiency of signal transduction via phospholipase C vs adenylyl cyclase. Taken together, these results indicate that S232 acts as a selective, PKC-sensitive, modulator of effector coupling of the alpha(2A)AR to inositol phosphate stimulation. This represents one mechanism by which cells route stimuli directed to multifunctional receptors to selected effectors so as to attain finely targeted signaling.

    Funded by: NHLBI NIH HHS: HL52318, HL53436

    Biochemistry 2001;40;49;15031-7

  • Multiplicity of mechanisms of serotonin receptor signal transduction.

    Raymond JR, Mukhin YV, Gelasco A, Turner J, Collinsworth G, Gettys TW, Grewal JS and Garnovskaya MN

    The Research Service of the Ralph H. Johnson Veterans Affairs Medical Center, Charleston, SC 29401, USA. raymondj@musc.edu

    The serotonin (5-hydroxytryptamine, 5-HT) receptors have been divided into 7 subfamilies by convention, 6 of which include 13 different genes for G-protein-coupled receptors. Those subfamilies have been characterized by overlapping pharmacological properties, amino acid sequences, gene organization, and second messenger coupling pathways. Post-genomic modifications, such as alternative mRNA splicing or mRNA editing, creates at least 20 more G-protein-coupled 5-HT receptors, such that there are at least 30 distinct 5-HT receptors that signal through G-proteins. This review will focus on what is known about the signaling linkages of the G-protein-linked 5-HT receptors, and will highlight some fascinating new insights into 5-HT receptor signaling.

    Funded by: NIDDK NIH HHS: DK02694, DK053981, DK52448, DK54720

    Pharmacology & therapeutics 2001;92;2-3;179-212

  • Binding of G alpha(o) N terminus is responsible for the voltage-resistant inhibition of alpha(1A) (P/Q-type, Ca(v)2.1) Ca(2+) channels.

    Kinoshita M, Nukada T, Asano T, Mori Y, Akaike A, Satoh M and Kaneko S

    Department of Neuropharmacology, Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan.

    G-protein-mediated inhibition of presynaptic voltage-dependent Ca(2+) channels is comprised of voltage-dependent and -resistant components. The former is caused by a direct interaction of Ca(2+) channel alpha(1) subunits with G beta gamma, whereas the latter has not been characterized well. Here, we show that the N terminus of G alpha(o) is critical for the interaction with the C terminus of the alpha(1A) channel subunit, and that the binding induces the voltage-resistant inhibition. An alpha(1A) C-terminal peptide, an antiserum raised against G alpha(o) N terminus, and a G alpha(o) N-terminal peptide all attenuated the voltage-resistant inhibition of alpha(1A) currents. Furthermore, the N terminus of G alpha(o) bound to the C terminus of alpha(1A) in vitro, which was prevented either by the alpha(1A) channel C-terminal or G alpha(o) N-terminal peptide. Although the C-terminal domain of the alpha(1B) channel showed similar ability in the binding with G alpha(o) N terminus, the above mentioned treatments were ineffective in the alpha(1B) channel current. These findings demonstrate that the voltage-resistant inhibition of the P/Q-type, alpha(1A) channel is caused by the interaction between the C-terminal domain of Ca(2+) channel alpha(1A) subunit and the N-terminal region of G alpha(o).

    The Journal of biological chemistry 2001;276;31;28731-8

  • Coincident signalling between the Gi/Go-coupled delta-opioid receptor and the Gq-coupled m3 muscarinic receptor at the level of intracellular free calcium in SH-SY5Y cells.

    Yeo A, Samways DS, Fowler CE, Gunn-Moore F and Henderson G

    Department of Pharmacology, University of Bristol, Bristol, UK.

    In SH-SY5Y cells, activation of delta-opioid receptors with [D-Pen(2,5)]-enkephalin (DPDPE; 1 microM) did not alter the intracellular free Ca(2+) concentration [Ca(2+)](i). However, when DPDPE was applied during concomitant Gq-coupled m3 muscarinic receptor stimulation by carbachol or oxotremorine-M, it produced an elevation of [Ca(2+)](i). The DPDPE-evoked increase in [Ca(2+)](i) was abolished when the carbachol-sensitive intracellular Ca(2+) store was emptied. There was a marked difference between the concentration-response relationship for the elevation of [Ca(2+)](i) by carbachol (EC(50) 13 microM, Hill slope 1) and the concentration-response relationship for carbachol's permissive action in revealing the delta-opioid receptor-mediated elevation of [Ca(2+)] (EC(50) 0.7 mM; Hill slope 1.8). Sequestration of free G protein beta gamma dimers by transient transfection of cells with a beta gamma binding protein (residues 495-689 of the C terminal tail of G protein-coupled receptor kinase 2) reduced the ability of delta opioid receptor activation to elevate [Ca(2+)](i). However, DPDPE did not elevate either basal or oxotremorine-M-evoked inositol phosphate production indicating that delta-opioid receptor activation did not stimulate phospholipase C. Furthermore, delta-opioid receptor activation did not result in the reversal of muscarinic receptor desensitization, membrane hyperpolarization or stimulation of sphingosine kinase. There was no coincident signalling between the delta-opioid receptor and the lysophosphatidic acid receptor which couples to elevation of [Ca(2+)](i) in SH-SY5Y cells by a PLC-independent mechanism. In SH-SY5Y cells the coincident signalling between the endogenously expressed delta-opioid and m3 muscarinic receptors appears to occur in the receptor activation-Ca(2+) release signalling pathway at a step after the activation of phospholipase C.

    Journal of neurochemistry 2001;76;6;1688-700

  • Nrg-1 belongs to the endothelial differentiation gene family of G protein-coupled sphingosine-1-phosphate receptors.

    Malek RL, Toman RE, Edsall LC, Wong S, Chiu J, Letterle CA, Van Brocklyn JR, Milstien S, Spiegel S and Lee NH

    Department of Molecular and Cellular Biology, Institute for Genomic Research, Rockville, Maryland 20850, USA.

    The previously cloned rat nerve growth factor-regulated G protein-coupled receptor NRG-1 (Glickman, M., Malek, R. L., Kwitek-Black, A. E., Jacob, H. J., and Lee N. H. (1999) Mol. Cell. Neurosci. 14, 141-52), also known as EDG-8, binds sphingosine-1-phosphate (S1P) with high affinity and specificity. In this paper we examined the signal transduction pathways regulated by the binding of S1P to EDG-8. In Chinese hamster ovary cells heterologously expressing EDG-8, S1P inhibited forskolin-induced cAMP accumulation and activated c-Jun NH2-terminal kinase. Surprisingly, S1P inhibited serum-induced activation of extracellular regulated protein kinase 1 and 2 (ERK1/2). Treatment with pertussis toxin, which ADP-ribosylates and inactivates G(i), blocked S1P-mediated inhibition of cAMP accumulation, but had no effect on c-Jun NH2-terminal kinase activation or inhibition of ERK1/2. The inhibitory effect of S1P on ERK1/2 activity was abolished by treatment with orthovanadate, suggesting the involvement of a tyrosine phosphatase. A subunit selective [35S] guanosine 5'-3-O-(thio)triphosphate binding assay demonstrates that EDG-8 activated G(i/o) and G12 but not Gs and G(q/11) in response to S1P. In agreement, EDG-8 did not stimulate phosphoinositide turnover or cAMP accumulation. The ability of S1P to induce mitogenesis in cells expressing the EDG-1 subfamily of G protein-coupled receptors is well characterized. In contrast, S1P inhibited proliferation in Chinese hamster ovary cells expressing EDG-8 but not empty vector. The antiproliferative effect, like S1P-mediated ERK1/2 inhibition, was orthovanadate-sensitive and pertussis toxin-insensitive. Our results indicate that EDG-8, a member of the EDG-1 subfamily, couples to unique signaling pathways.

    Funded by: NCI NIH HHS: CA61774; NINDS NIH HHS: NS352321

    The Journal of biological chemistry 2001;276;8;5692-9

  • Characterization of RGS5 in regulation of G protein-coupled receptor signaling.

    Zhou J, Moroi K, Nishiyama M, Usui H, Seki N, Ishida J, Fukamizu A and Kimura S

    Department of Biochemistry and Molecular Pharmacology, Chiba University Graduate School of Medicine, Japan.

    RGS proteins (regulators of G protein signaling) serve as GTPase-activating proteins (GAPs) for G alpha subunits and negatively regulate G protein-coupled receptor signaling. In this study, we characterized biochemical properties of RGS5 and its N terminal (1-33)-deleted mutant (deltaN-RGS5). RGS5 bound to G alpha(i1), G alpha(i2), G alpha(i3), G alpha(o) and G alpha(q) but not to G alpha(s) and G alpha13 in the presence of GDP/AIF4-, and accelerated the catalytic rate of GTP hydrolysis of G alpha(i3) subunit. When expressed in 293T cells stably expressing angiotensin (Ang) AT1a receptors (AT1a-293T cells), RGS5 suppressed Ang II- and endothelin (ET)-1-induced intracellular Ca2+ transients. The effect of RGS5 was concentration-dependent, and the slope of the concentration-response relationship showed that a 10-fold increase in amounts of RGS5 induced about 20-25% reduction of the Ca2+ signaling. Furthermore, a comparison study of three sets of 293T cells with different expression levels of AT1a receptors showed that RGS5 inhibited Ang II-induced responses more effectively in 293T cells with the lower density of AT1a receptors, suggesting that the degree of inhibition by RGS proteins reflects the ratio of amounts of RGS proteins to those of activated G alpha subunits after receptor stimulation by agonists. When expressed in AT1a-293T cells, deltaN-RGS5 was localized almost exclusively in the cytosolic fraction, and exerted the inhibitory effects as potently as RGS5 which was present in both membrane and cytosolic fractions. Studies on relationship between subcellular localization and inhibitory effects of RGS5 and deltaN-RGS5 revealed that the N terminal (1-33) of RGS5 plays a role in targeting this protein to membranes, and that the N terminal region of RGS5 is not essential for exerting activities.

    Life sciences 2001;68;13;1457-69

  • The light response of ON bipolar neurons requires G[alpha]o.

    Dhingra A, Lyubarsky A, Jiang M, Pugh EN, Birnbaumer L, Sterling P and Vardi N

    Department of Neuroscience, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.

    ON bipolar neurons in retina detect the glutamate released by rods and cones via metabotropic glutamate receptor 6 (mGluR6), whose cascade is unknown. The trimeric G-protein G(o) might mediate this cascade because it colocalizes with mGluR6. To test this, we studied the retina in mice negative for the alpha subunit of G(o) (Galpha(o)-/-). Retinal layering, key cell types, synaptic structure, and mGluR6 expression were all normal, as was the a-wave of the electroretinogram, which represents the rod and cone photocurrents. However, the b-wave of the electroretinogram, both rod- and cone-driven components, was entirely missing. Because the b-wave represents the massed response of ON bipolar cells, its loss in the Galpha(o) null mouse establishes that the light response of the ON bipolar cell requires G(o). This represents the first function to be defined in vivo for the alpha subunit of the most abundant G-protein of the brain.

    Funded by: NEI NIH HHS: EY00828, EY02660, EY11105; ...

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2000;20;24;9053-8

  • Molecular cloning and characterization of a novel type of histamine receptor preferentially expressed in leukocytes.

    Oda T, Morikawa N, Saito Y, Masuho Y and Matsumoto S

    Helix Research Institute, Inc., 1532-3 Yana, Kisarazu-shi, Chiba, 292-0812, Japan.

    Recently cDNA encoding the histamine H3 receptor was isolated after 15 years of considerable research. However, several studies have proposed heterogeneity of the H3 receptor. We report here the molecular cloning and characterization of a novel type of histamine receptor. A novel orphan G-protein-coupled receptor named GPRv53 was obtained through a search of the human genomic DNA data base and analyzed by rapid amplification of cDNA ends (RACE). GPRv53 possessed the features of biologic amine 226 receptors and had the highest homology with H3 receptor among known G-protein-coupled receptors. Mammalian cells expressing GPRv53 were demonstrated to bind and respond to histamine in a concentration-dependent manner. In functional assays, not only an H3 receptor agonist, R-(alpha)-methylhistamine, but also a H3 receptor antagonist, clobenpropit, and a neuroleptic, clozapine, activated GPRv53-expressing cells. Tissue distribution analysis revealed that expression of GPRv53 is localized in the peripheral blood leukocytes, spleen, thymus, and c 1d12 olon, which was totally different from the H3 receptor, whose expression was restricted to the brain. The discovery of the GPRv53 receptor will open a new phase of research on the physiological role of histamine.

    The Journal of biological chemistry 2000;275;47;36781-6

  • RGS14 is a novel Rap effector that preferentially regulates the GTPase activity of galphao.

    Traver S, Bidot C, Spassky N, Baltauss T, De Tand MF, Thomas JL, Zalc B, Janoueix-Lerosey I and Gunzburg JD

    INSERM U-528, Institut Curie-Recherche, 26 rue d'Ulm, 75248 Paris Cedex 05, France.

    In an attempt to elucidate the physiological function(s) of the Ras-related Rap proteins, we used the yeast two-hybrid system and isolated a cDNA encoding a protein that interacts with both Rap1 and Rap2, but not with Ras; the use of Rap2 mutants showed that this interaction is characteristic of a potential Rap effector. This protein was identified as RGS14, a member of the recently discovered family of RGS ('regulators of G-protein signalling') proteins that stimulate the GTPase activity of the GTP-binding alpha subunit of heterotrimeric G-proteins (Galpha). Deletion analysis, as well as in vitro binding experiments, revealed that RGS14 binds Rap proteins through a domain distinct from that carrying the RGS identity, and that this domain shares sequence identity with the Ras/Rap binding domain of B-Raf and Raf-1 kinases. RGS14 is distinguished from other RGS proteins by its marked preference for Galpha(o) over other Galpha subunits: RGS14 binds preferentially to Galpha(o) in isolated brain membranes, and also interacts preferentially with Galpha(o) (as compared with Galpha(i1)) to stimulate its GTPase activity. In adult mice, RGS14 expression is restricted to spleen and brain. In situ hybridization studies showed that it is highly expressed only in certain areas of mouse brain (such as the CA1 and CA2 regions of the hippocampus), and that this pattern closely resembles that of Rap2, but not Rap1, expression. Double in situ hybridization experiments revealed that certain cells in the hippocampus express both RGS14 and Galpha(o), as well as both RGS14 and Rap2, showing that the interaction of RGS14 with Galpha(o) and Rap2 is physiologically possible. Taken together, these results suggest that RGS14 could constitute a bridging molecule that allows cross-regulation of signalling pathways downstream from G-protein-coupled receptors involving heterotrimeric proteins of the G(i/o) family and those involving the Ras-related GTPase Rap2.

    The Biochemical journal 2000;350 Pt 1;19-29

  • Interaction between HIV-1 NEF and G(o) proteins in transfected COS-7 cells.

    Guzzi F, Celano E, Levi G and Parenti M

    Department of Experimental and Environmental Medicine and Biotechnology, School of Medicine, University of Milano-Bicocca, Via Cadore 48, Monza, 20052, Italy.

    Nef protein of HIV/SIV lentiviruses affects G-protein-mediated signaling, and physically associates to Lck, a myristoylated and palmitoylated Src-like tyrosine kinase. To assess whether Nef interacts with alpha-subunits of heterotrimeric G proteins (Galpha), carrying the same lipidation motif as Lck, we transiently expressed Nef and G(o)alpha (wild-type or nonpalmitoylated C3S mutant), individually or in combination, in transfected COS-7 cells. Recombinant Nef was mostly recovered in particulate fractions, and a Nef-Green Fluorescent Protein chimera was localized at the plasmalemma by in vivo fluorescence imaging. Moreover, Nef and C3S were entirely solubilized by cold Triton X-100, and excluded from low buoyant density sucrose gradient fractions, containing caveolin-1, whereas wild-type G(o)alpha was partially resistant to Triton extraction, and colocalized with caveolin-1. After coexpression, Nef recruited soluble C3S to membranes, and the two proteins were coimmunoprecipitated by G(o)alpha and Nef antisera. We conclude that Nef interacts with nonpalmitoylated G(o)alpha, presumably outside caveolin-rich microdomains of the plasma membrane.

    Biochemical and biophysical research communications 2000;270;2;570-5

  • A novel spliced variant of the type 1 corticotropin-releasing hormone receptor with a deletion in the seventh transmembrane domain present in the human pregnant term myometrium and fetal membranes.

    Grammatopoulos DK, Dai Y, Randeva HS, Levine MA, Karteris E, Easton AJ and Hillhouse EW

    Sir Quinton Hazell Molecular Medicine Research Centre, Department of Biological Sciences, University of Warwick, Coventry, United Kingdom. chdg@dna.bio.warwick.ac.uk

    CRH exerts its actions via activation of specific G protein-coupled receptors, which exist in two types, CRH-R1 and CRH-R2, and arise from different genes with multiple spliced variants. RT-PCR amplification of CRH receptor sequences from human myometrium and fetal membranes yielded cDNAs that encode a novel CRH-R type 1 spliced variant. This variant (CRH-R1d) is present in the human pregnant myometrium at term only, which suggests a physiologically important role at the end of human pregnancy and labor. The amino acid sequence of CRH-R1d is identical to the CRH-R1alpha receptor except that it contains an exon deletion resulting in the absence of 14 amino acids in the predicted seventh transmembrane domain. Binding studies in HEK-293 cells stably expressing the CRH-R1d or CRH-R1alpha receptors revealed that the deletion does not change the binding characteristics of the variant receptor. In contrast, studies on the G protein activation demonstrated that CRH-R1d is not well coupled to the four subtypes of G proteins (G(s), G(i), G(o), G(q)) that CRH-R1alpha can activate. These data suggest that although the deleted segment is not important for CRH binding, it plays a crucial role in CRH receptor signal transduction. Second messenger studies of the variant receptor showed that CRH and CRH-like peptides can stimulate the adenylate cyclase system, with reduced sensitivity and potency by 10-fold compared with the CRH-R1alpha. Furthermore, CRH failed to stimulate inositol trisphosphate production. Coexpression studies between the CRH-R1d or CRH-R1alpha showed that this receptor does not play a role as a dominant negative receptor for CRH.

    Funded by: Wellcome Trust

    Molecular endocrinology (Baltimore, Md.) 1999;13;12;2189-202

  • Kinetics of ternary complex formation with fusion proteins composed of the A(1)-adenosine receptor and G protein alpha-subunits.

    Waldhoer M, Wise A, Milligan G, Freissmuth M and Nanoff C

    Institute of Pharmacology, University of Vienna, Währinger Strasse 13a, A-1090 Vienna, Austria.

    High affinity agonist binding to G protein-coupled receptors depends on the formation of a ternary complex between agonist, receptor, and G protein. This process is too slow to be accounted for by a simple diffusion-controlled mechanism. We have tested if the interaction between activated receptor and G protein is rate-limiting by fusing the coding sequence of the human A(1)-adenosine receptor to that of Galpha(i-1) (A(1)/Galpha(i-1)) and of Galpha(o) (A(1)/Galpha(o)). Fusion proteins of the expected molecular mass were detected following transfection of HEK293 cells. Ternary complex formation was monitored by determining the kinetics for binding of the high affinity agonist (-)-N(6)-3[(125)I](iodo-4-hydroxyphenylisopropyl)adenosine; these were similar in the wild-type receptor and the fusion proteins over the temperature range of 10 to 30 degrees C. Agonist dissociation may be limited by the stability of the ternary complex. This assumption was tested by creating fusion proteins in which the Cys(351) of Galpha(i-1) was replaced with glycine (A(1)/Galpha(i-1)C351G) or isoleucine (A(1)/Galpha(i-1)C351I) to lower the affinity of the receptor for the G protein. In these mutated fusion proteins, the dissociation rate of the ternary complex was accelerated; in contrast, the rate of the forward reaction was not affected. We therefore conclude that (i) receptor activation per se rather than its interaction with the G protein is rate-limiting in ternary complex formation; (ii) the stability of the ternary complex is determined by the dissociation rate of the G protein. These features provide for a kinetic proofreading mechanism that sustains the fidelity of receptor-G protein coupling.

    The Journal of biological chemistry 1999;274;43;30571-9

  • Structural basis for the selectivity of the RGS protein, GAIP, for Galphai family members. Identification of a single amino acid determinant for selective interaction of Galphai subunits with GAIP.

    Woulfe DS and Stadel JM

    Department of Pharmacology, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.

    GAIP is a regulator of G protein signaling (RGS) that accelerates the rate of GTP hydrolysis by some G protein alpha subunits. In the present studies, we have examined the structural basis for the ability of GAIP to discriminate among members of the Galphai family. Galphai1, Galphai3, and Galphao interacted strongly with GAIP, whereas Galphai2 interacted weakly and Galphas did not interact at all. A chimeric G protein composed of a Galphai2 N terminus and a Galphai1 C terminus interacted as strongly with GAIP as native Galphai1, whereas a chimeric N-terminal Galphai1 with a Galphai2 C terminus did not interact. These results suggest that the determinants responsible for GAIP selectivity between these two Galphais reside within the C-terminal GTPase domain of the G protein. To further localize residues contributing to G protein-GAIP selectivity, a panel of 15 site-directed Galphai1 and Galphai2 mutants were assayed. Of the Galphai1 mutants tested, only that containing a mutation at aspartate 229 located at the N terminus of Switch 3 did not interact with GAIP. Furthermore, the only Galphai2 variant that interacted strongly with GAIP contained a replacement of the corresponding Galphai2 Switch 3 residue (Ala230) with aspartate. To determine whether GAIP showed functional preferences for Galpha subunits that correlate with the binding data, the ability of GAIP to enhance the GTPase activity of purified alpha subunits was tested. GAIP catalyzed a 3-5-fold increase in the rate of GTP hydrolysis by Galphai1 and Galphai2(A230D) but no increase in the rate of Galphai2 and less than a 2-fold increase in the rate of Galphai1(D229A) under the same conditions. Thus, GAIP was able to discriminate between Galphai1 and Galphai2 in both binding and functional assays, and in both cases residue 229/230 played a critical role in selective recognition.

    The Journal of biological chemistry 1999;274;25;17718-24

  • Identification and expression of Go1 and Go2 alpha-subunit transcripts in human myometrium in relation to pregnancy.

    Duc-Goiran P, Bourgeois C, Mignot TM, Robert B, Tanguy G and Ferré F

    INSERM U. 361, Université René Descartes Paris V, Pavillon Baudelocque, 75014 Paris, France. u361@cochin.inserm.fr

    The 39-kDa Goalpha protein, the alpha subunit of a major heterotrimeric G protein of brain and neuroendocrine cells, was found to be present in human myometrium. Using three different antisera, we showed its strong expression in myometrium from pregnant patients as compared to nonpregnant ones. This is in agreement with the high expression level of its two isoforms (alphao1 and alphao2), previously described in late pregnancy. To better ascertain the nature of these immunoreactive isoforms, we investigated transcripts of the Goalpha gene in myometrium from pregnant and nonpregnant patients by reverse transcription-polymerase chain reaction (RT-PCR). In this tissue, the amplified cDNA product of a region common to both Go1alpha and Go2alpha mRNA variants was recognized as the Goalpha nucleotide sequence. Transcripts of Go1alpha and Go2alpha were identified by sequencing. A partial cDNA Go2alpha sequence was described, which differed from the Goalpha gene by two nucleotides in exon 8B. Levels of Go1alpha and Go2alpha transcripts analyzed by semi-quantitative RT-PCR were significantly higher in myometrium from pregnant than from nonpregnant patients. It is suggested that Goalpha gene expression in this tissue may contribute to modifications seen in the signaling pathways observed at the end of pregnancy.

    Biology of reproduction 1999;60;6;1528-35

  • Interaction of heterotrimeric G protein Galphao with Purkinje cell protein-2. Evidence for a novel nucleotide exchange factor.

    Luo Y and Denker BM

    Renal Division, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115, USA.

    The heterotrimeric G protein Galphao is ubiquitously expressed throughout the central nervous system, but many of its functions remain to be defined. To search for novel proteins that interact with Galphao, a mouse brain library was screened using the yeast two-hybrid interaction system. Pcp2 (Purkinje cell protein-2) was identified as a partner for Galphao in this system. Pcp2 is expressed in cerebellar Purkinje cells and retinal bipolar neurons, two locations where Galphao is also expressed. Pcp2 was first identified as a candidate gene to explain Purkinje cell degeneration in pcd mice (Nordquist, D. T., Kozak, C. A., and Orr, H. T. (1988) J. Neurosci. 8, 4780-4789), but its function remains unknown as Pcp2 knockout mice are normal (Mohn, A. R., Feddersen, R. M., Nguyen, M. S., and Koller, B. H. (1997) Mol. Cell. Neurosci. 9, 63-76). Galphao and Pcp2 binding was confirmed in vitro using glutathione S-transferase-Pcp2 fusion proteins and in vitro translated [35S]methionine-labeled Galphao. In addition, when Galphao and Pcp2 were cotransfected into COS cells, Galphao was detected in immunoprecipitates of Pcp2. To determine whether Pcp2 could modulate Galphao function, kinetic constants kcat and koff of bovine brain Galphao were determined in the presence and absence of Pcp2. Pcp2 stimulates GDP release from Galphao more than 5-fold without affecting kcat. These findings define a novel nucleotide exchange function for Pcp2 and suggest that the interaction between Pcp2 and Galphao is important to Purkinje cell function.

    The Journal of biological chemistry 1999;274;16;10685-8

  • Activation of Go-proteins by membrane depolarization traced by in situ photoaffinity labeling of galphao-proteins with [alpha32P]GTP-azidoanilide.

    Anis Y, Nürnberg B, Visochek L, Reiss N, Naor Z and Cohen-Armon M

    Department of Physiology and The Cardiac Research Institute, Sackler School of Medicine, Tel-Aviv University, 69978 Tel-Aviv, Israel.

    Evidence for depolarization-induced activation of G-proteins in membranes of rat brain synaptoneurosomes has been previously reported (Cohen-Armon, M., and Sokolovsky, M. (1991) J. Biol. Chem. 266, 2595-2605; Cohen-Armon, M., and Sokolovsky, M. (1993) J. Biol. Chem. 268, 9824-9838). In the present work we identify the activated G-proteins as Go-proteins by tracing their depolarization-induced in situ photoaffinity labeling with [alpha32P]GTP-azidoanilide (GTPAA). Labeled GTPAA was introduced into transiently permeabilized rat brain-stem synaptoneurosomes. The resealed synaptoneurosomes, while being UV-irradiated, were depolarized. Relative to synaptoneurosomes at resting potential, the covalent binding of [alpha32P]GTPAA to Galphao1- and Galphao3-proteins, but not to Galphao2- isoforms, was enhanced by 5- to 7-fold in depolarized synaptoneurosomes, thereby implying an accelerated exchange of GDP for [alpha32P]GTPAA. Their depolarization-induced photoaffinity labeling was independent of stimulation of Go-protein-coupled receptors and could be reversed by membrane repolarization, thus excluding induction by transmitters release. It was, however, dependent on depolarization-induced activation of the voltage-gated sodium channels (VGSC), regardless of Na+ current. The alpha subunit of VGSC was cross-linked and co-immunoprecipitated with Galphao-proteins in depolarized brain-stem and cortical synaptoneurosomes. VGSC alpha subunit most efficiently cross-linked with guanosine 5'-O-2-thiodiphosphate-bound rather than to guanosine 5'-O-(3-thiotriphosphate)-bound Galphao-proteins in isolated synaptoneurosomal membranes. These findings support a possible involvement of VGSC in depolarization-induced activation of Go-proteins.

    The Journal of biological chemistry 1999;274;11;7431-40

  • Expression of GTPase-deficient Gialpha2 results in translocation of cytoplasmic RGS4 to the plasma membrane.

    Druey KM, Sullivan BM, Brown D, Fischer ER, Watson N, Blumer KJ, Gerfen CR, Scheschonka A and Kehrl JH

    Laboratory of Immunoregulation, National Institutes of Health, Bethesda, Maryland 20892-1876, USA.

    The members of a recently identified protein family termed regulators of G-protein signaling (RGS) act as GTPase-activating proteins for certain Galpha subunits in vitro, but their physiological effects in cells are uncertain in the face of similar biochemical activity and overlapping patterns of tissue expression. Consistent with its activity in in vitro GTPase-activating protein assays, RGS4 interacts efficiently with endogenous proteins of the Gi and Gq subclasses of Galpha subunits but not with G12alpha or Gsalpha. Unlike other RGS proteins such as RGS9, RGS-GAIP, and Sst2p, which have been reported to be largely membrane-associated, a majority of cellular RGS4 is found as a soluble protein in the cytoplasm. However, the expression of a GTPase-deficient Gialpha subunit (Gialpha2-Q204L) resulted in the translocation of both wild type RGS4 and a non-Gialpha-binding mutant (L159F) to the plasma membrane. These data suggest that RGS4 may be recruited to the plasma membrane indirectly by G-protein activation and that multiple RGS proteins within a given cell might be differentially localized to determine a physiologic response to a G-protein-linked stimulus.

    Funded by: NIDDK NIH HHS: DK38452

    The Journal of biological chemistry 1998;273;29;18405-10

  • The mammalian calcium-binding protein, nucleobindin (CALNUC), is a Golgi resident protein.

    Lin P, Le-Niculescu H, Hofmeister R, McCaffery JM, Jin M, Hennemann H, McQuistan T, De Vries L and Farquhar MG

    Division of Cellular and Molecular Medicine and Department of Pathology, University of California, San Diego, La Jolla, California 92093-0651, USA.

    We have identified CALNUC, an EF-hand, Ca2+-binding protein, as a Golgi resident protein. CALNUC corresponds to a previously identified EF-hand/calcium-binding protein known as nucleobindin. CALNUC interacts with Galphai3 subunits in the yeast two-hybrid system and in GST-CALNUC pull-down assays. Analysis of deletion mutants demonstrated that the EF-hand and intervening acidic regions are the site of CALNUC's interaction with Galphai3. CALNUC is found in both cytosolic and membrane fractions. The membrane pool is tightly associated with the luminal surface of Golgi membranes. CALNUC is widely expressed, as it is detected by immunofluorescence in the Golgi region of all tissues and cell lines examined. By immunoelectron microscopy, CALNUC is localized to cis-Golgi cisternae and the cis-Golgi network (CGN). CALNUC is the major Ca2+-binding protein detected by 45Ca2+-binding assay on Golgi fractions. The properties of CALNUC and its high homology to calreticulin suggest that it may play a key role in calcium homeostasis in the CGN and cis-Golgi cisternae.

    Funded by: NCI NIH HHS: CA 58689; NIDDK NIH HHS: DK 17780, R01 DK017780

    The Journal of cell biology 1998;141;7;1515-27

  • RGS7 attenuates signal transduction through the G(alpha q) family of heterotrimeric G proteins in mammalian cells.

    Shuey DJ, Betty M, Jones PG, Khawaja XZ and Cockett MI

    Department of CNS Disorders, Wyeth-Ayerst Research, Princeton, New Jersey 08543-8000, USA.

    The RGS proteins are a recently discovered family of G protein regulators that have been shown to act as GTPase-activating proteins (GAPs) on the G(alpha i) and G(alpha q) subfamilies of the heterotrimeric G proteins. Here, we demonstrate that RGS7 is a potent GAP in vitro on G(alpha i1), and G(alpha o) heterotrimeric proteins and that RGS7 acts to down-regulate G(alpha q)-mediated calcium mobilization in a whole-cell assay system using a transient expression protocol. This RGS protein and RGS4 are reported to be expressed predominantly in brain, and in situ hybridization studies have revealed similarities in the regional distribution of RGS and G(alpha q) mRNA expression. Our findings provide further evidence to support a functional role for RGS4 and RGS7 in G(alpha q)-mediated signaling in the CNS.

    Journal of neurochemistry 1998;70;5;1964-72

  • Analysis of the N-terminal binding domain of Go alpha.

    Busconi L and Denker BM

    Renal Division, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115, USA.

    Signalling from membrane receptors through heterotrimeric G-proteins (G alpha and G beta gamma) to intracellular effectors is a highly regulated process. Receptor activation causes exchange of GTP for GDP on G alpha and dissociation of G alpha from G beta gamma. Both subunits remain membrane-associated and interact with a series of other molecules throughout the cycle of activation. The N-terminal binding domain of G alpha subunits interacts with the membrane by several partially defined mechanisms: the anchoring of G alpha to the more hydrophobic G beta gamma subunits, the interaction of N-terminal lipids (palmitate and/or myristate) with the membrane, and attachment of amino acid regions to the membrane {amino acids 11-14 of Go alpha (D[11-14]); Busconi, Boutin and Denker (1997) Biochem. J. 323, 239-244}. We characterized N-terminal mutants of Go alpha with known G beta gamma-binding properties for the ability to interact with phospholipid vesicles and membranes prepared from cultured cells (acceptor membranes). In vitro analysis allows membrane interactions that are important to the activated and depalmitoylated state of G alpha to be characterized. Subcellular localization was also determined in transiently transfected COS cells. All of the mutant proteins are myristoylated, and differences in myristoylation do not account for changes in membrane binding. Disrupting the N-terminal alpha-helix of Go alpha with a proline point mutation at Arg-9 (R9P) does not affect interactions with G beta gamma on sucrose-density gradients but significantly reduces acceptor membrane binding. Deletion of amino acids 6-15 (D[6-15]; reduced G beta gamma binding) or deletion of amino acids 3-21 (D[3-21]); no detectable G beta gamma binding) further reduces acceptor membrane binding. When expressed in COS cells, R9P and D[6-15] are localized in the membrane similar to wild-type Go alpha as a result of the contribution from palmitoylation. In contrast, D[3-21] is completely soluble in COS cells, and no palmitoylation is detected. The binding of Go alpha and mutants translated in vitro to liposomes indicates that Go alpha preferentially binds to neutral phospholipids (phosphatidylcholine). R9P and D[11-14] bind to phosphatidylcholine liposomes like Go alpha, but D[6-15] exhibits no detectable binding. Taken together, these studies suggest that interactions of the N-terminus of G alpha subunits with the membrane may be affected by both membrane proteins and lipids. A detailed understanding of G alpha-membrane interactions may reveal unique mechanisms for regulating signal transduction.

    Funded by: NIDDK NIH HHS: DK02110

    The Biochemical journal 1997;328 ( Pt 1);23-31

  • Interaction of human retinal RGS with G-protein alpha-subunits.

    Natochin M, Lipkin VM and Artemyev NO

    Department of Physiology and Biophysics, University of Iowa College of Medicine, Iowa City 52242, USA.

    A novel family of RGS proteins negatively regulates signaling via heterotrimeric G-proteins by accelerating the GTPase activity of G-protein alpha subunits. We have investigated interaction of human retinal RGS protein (hRGSr) with in vitro translated G(alpha) subunits: G(t alpha), G(i alpha1), G(o alpha) and G(s alpha). hRGSr binds well to G(t alpha), G(i alpha1) and G(o alpha) in the presence of AIF4-, but does not interact with G(s alpha). The N- and C-terminally truncated G(alpha) subunits interact with hRGSr similarly to the intact G(alpha) polypeptides. Analysis of interaction between hRGSr and G(o alpha)/G(s alpha) chimeras suggests that a region of G(o alpha), G(o alpha)22-212, contains major structural determinants for binding to RGS proteins.

    Funded by: NEI NIH HHS: EY-10843; NIDDK NIH HHS: DK-25295

    FEBS letters 1997;411;2-3;179-82

  • Characterization of a novel mammalian RGS protein that binds to Galpha proteins and inhibits pheromone signaling in yeast.

    Chen C, Zheng B, Han J and Lin SC

    Regulatory Biology Laboratory, Institute of Molecular and Cell Biology, National University of Singapore, 10 Kent Ridge Crescent, Singapore 119260, Republic of Singapore.

    Genetic studies of molecules that negatively regulate G-coupled receptor functions have led to the identification of a large gene family with an evolutionarily conserved domain, termed the RGS domain. It is now understood that RGS proteins serve as GTPase-activating proteins for subfamilies of the heterotrimeric G-proteins. We have isolated from mouse pituitary a full-length cDNA clone encoding a novel member of the RGS protein family, termed RGS16, as well as the full-length cDNA of mRGS5 and mRGS2. Tissue distribution analysis shows that the novel RGS16 is predominantly expressed in liver and pituitary, and that RGS5 is preferentially expressed in heart and skeletal muscle. In contrast, RGS2 is widely expressed. Genetic analysis using the pheromone response halo assay and FUS1 gene induction assay show that overexpression of the RGS16 gene dramatically inhibits yeast response to alpha-factor, whereas neither RGS2 nor RGS5 has any discernible effect on pheromone sensitivity, pointing to a possible functional diversity among RGS proteins. In vitro binding assays reveal that RGS5 and RGS16 bind to Galphai and Galphao subunits of heterotrimeric G-proteins, but not to Galphas. Based on mutational analysis of the conserved residues in the RGS domain, we suggest that the G-protein binding and GTPase-activating protein activity may involve distinct functional structures of the RGS proteins, indicating that RGS proteins may exert a dual function in the attenuation of signaling via G-coupled receptors.

    The Journal of biological chemistry 1997;272;13;8679-85

  • G alpha(o) is necessary for muscarinic regulation of Ca2+ channels in mouse heart.

    Valenzuela D, Han X, Mende U, Fankhauser C, Mashimo H, Huang P, Pfeffer J, Neer EJ and Fishman MC

    Laboratory of Developmental Biology and Cardiovascular Research Center, Massachusetts General Hospital, Charlestown 02129, USA.

    Heterotrimeric G proteins, composed of G alpha and G betagamma subunits, transmit signals from cell surface receptors to cellular effector enzymes and ion channels. The G alpha(o) protein is the most abundant G alpha subtype in the nervous system, but it is also found in the heart. Its function is not completely known, although it is required for regulation of N-type Ca2+ channels in GH3 cells and also interacts with GAP43, a major protein in growth cones, suggesting a role in neuronal pathfinding. To analyze the function of G alpha(o), we have generated mice lacking both isoforms of G alpha(o) by homologous recombination. Surprisingly, the nervous system is grossly intact, despite the fact that G alpha(o) makes up 0.2-0.5% of brain particulate protein and 10% of the growth cone membrane. The G alpha(o)-/- mice do suffer tremors and occasional seizures, but there is no obvious histologic abnormality in the nervous system. In contrast, G alpha(o)-/- mice have a clear and specific defect in ion channel regulation in the heart. Normal muscarinic regulation of L-type calcium channels in ventricular myocytes is absent in the mutant mice. The L-type calcium channel responds normally to isoproterenol, but there is no evident muscarinic inhibition. Muscarinic regulation of atrial K+ channels is normal, as is the electrocardiogram. The levels of other G alpha subunits (G alpha(s), G alpha(q), and G alpha(i)) are unchanged in the hearts of G alpha(o)-/- mice, but the amount of G betagamma is decreased. Whichever subunit, G alpha(o) or G betagamma, carries the signal forward, these studies show that muscarinic inhibition of L-type Ca2+ channels requires coupling of the muscarinic receptor to G alpha(o). Other cardiac G alpha subunits cannot substitute.

    Funded by: NHLBI NIH HHS: HL52320, P50 HL052320; NIGMS NIH HHS: GM36359

    Proceedings of the National Academy of Sciences of the United States of America 1997;94;5;1727-32

  • GAIP is membrane-anchored by palmitoylation and interacts with the activated (GTP-bound) form of G alpha i subunits.

    De Vries L, Elenko E, Hubler L, Jones TL and Farquhar MG

    Division of Cellular and Molecular Medicine, University of California at San Diego, La Jolla 92093-0651, USA.

    GAIP (G Alpha Interacting Protein) is a member of the recently described RGS (Regulators of G-protein Signaling) family that was isolated by interaction cloning with the heterotrimeric G-protein G alpha i3 and was recently shown to be a GTPase-activating protein (GAP). In AtT-20 cells stably expressing GAIP, we found that GAIP is membrane-anchored and faces the cytoplasm, because it was not released by sodium carbonate treatment but was digested by proteinase K. When Cos cells were transiently transfected with GAIP and metabolically labeled with [35S]methionine, two pools of GAIP--a soluble and a membrane-anchored pool--were found. Since the N terminus of GAIP contains a cysteine string motif and cysteine string proteins are heavily palmitoylated, we investigated the possibility that membrane-anchored GAIP might be palmitoylated. We found that after labeling with [3H]palmitic acid, the membrane-anchored pool but not the soluble pool was palmitoylated. In the yeast two-hybrid system, GAIP was found to interact specifically with members of the G alpha i subfamily, G alpha i1, G alpha i2, G alpha i3, G alpha z, and G alpha o, but not with members of other G alpha subfamilies, G alpha s, G alpha q, and G alpha 12/13. The C terminus of G alpha i3 is important for binding because a 10-aa C-terminal truncation and a point mutant of G alpha i3 showed significantly diminished interaction. GAIP interacted preferentially with the activated (GTP) form of G alpha i3, which is in keeping with its GAP activity. We conclude that GAIP is a membrane-anchored GAP with a cysteine string motif. This motif, present in cysteine string proteins found on synaptic vesicles, pancreatic zymogen granules, and chromaffin granules, suggests GAIP's possible involvement in membrane trafficking.

    Funded by: NCI NIH HHS: CA58689, F32 CA066289; NIDDK NIH HHS: DK17780, R01 DK017780; NIGMS NIH HHS: GM07752, T32 GM007752

    Proceedings of the National Academy of Sciences of the United States of America 1996;93;26;15203-8

  • Immunoexpression of the alpha subunit of a guanine nucleotide-binding protein (Go) in pulmonary neuroendocrine cells and neoplasms.

    Kawano N, Ito T, Kitamura H, Shibagaki T, Kameda Y, Nakamura N and Kanisawa M

    Department of Pathology, Yokohama City University School of Medicine, Japan.

    The alpha subunit of a GTP-binding protein, Go, was investigated in pulmonary neuroendocrine neoplasms and fetal tissues of the lung by an immunohistochemical method. Positive immunostaining for the alpha subunit of Go (Go alpha) was found predominantly on the cell membrane and found occasionally in the cytoplasm. Typical carcinoids were all positively stained (9/9), and small cell carcinoma showed weaker and less frequent staining (5 positive cases in 10). Atypical carcinoids were variously stained (3/4). The tendency for obvious neuroendocrine differentiation to be immunohistochemically determined in typical carcinoids and not in small cell carcinoma is also true of staining for neuron specific enolase (NSE), chromogranin A (CG-A) and synaptophysin. In the lung, Go alpha-immunostaining was positive not only in nerve tissues but also in the airway epithelium. In the fetal lung, serial sections immunostained for NSE, CG-A and Go alpha confirmed that Go alpha-immunoreactive cells belong to the neuroendocrine cell population. The biological significance of Go alpha is unclear in normal and neoplastic lung tissues, but Go alpha is a useful marker of neuroendocrine cells and neoplasma of the lung.

    Pathology international 1996;46;6;393-8

  • The human thyrotropin receptor: a heptahelical receptor capable of stimulating members of all four G protein families.

    Laugwitz KL, Allgeier A, Offermanns S, Spicher K, Van Sande J, Dumont JE and Schultz G

    Institut für Pharmakologie, Freie Universität Berlin, Germany.

    Thyrotropin is the primary hormone that, via one heptahelical receptor, regulates thyroid cell functions such as secretion, specific gene expression, and growth. In human thyroid, thyrotropin receptor activation leads to stimulation of the adenylyl cyclase and phospholipase C cascades. However, the G proteins involved in thyrotropin receptor action have been only partially defined. In membranes of human thyroid gland, we immunologically identified alpha subunits of the G proteins Gs short, Gs long, Gi1, Gi2, Gi3, G(o) (Go2 and another form of Go, presumably Go1), Gq, G11, G12, and G13. Activation of the thyrotropin (TSH) receptor by bovine TSH led to increased incorporation of the photoreactive GTP analogue [alpha-32P]GTP azidoanilide into immunoprecipitated alpha subunits of all G proteins detected in thyroid membranes. This effect was receptor-dependent and not due to direct G protein stimulation because it was mimicked by TSH receptor-stimulating antibodies of patients suffering from Grave disease and was abolished by a receptor-blocking antiserum from a patient with autoimmune hypothyroidism. The TSH-induced activation of individual G proteins occurred with EC50 values of 5-50 milliunits/ml, indicating that the activated TSH receptor coupled with similar potency to different G proteins. When human thyroid slices were pretreated with pertussis toxin, the TSH receptor-mediated accumulation of cAMP increased by approximately 35% with TSH at 1 milliunits/ml, indicating that the TSH receptor coupled to Gs and G(i). Taken together, these findings show that, at least in human thyroid membranes, in which the protein is expressed at its physiological levels, the TSH receptor resembles a naturally occurring example of a general G protein-activating receptor.

    Proceedings of the National Academy of Sciences of the United States of America 1996;93;1;116-20

  • The structure of the G protein heterotrimer Gi alpha 1 beta 1 gamma 2.

    Wall MA, Coleman DE, Lee E, Iñiguez-Lluhi JA, Posner BA, Gilman AG and Sprang SR

    Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas 75235, USA.

    The crystallographic structure of the G protein heterotrimer Gi alpha 1(GDP)beta 1 gamma 2 (at 2.3 A) reveals two nonoverlapping regions of contact between alpha and beta, an extended interface between beta and nearly all of gamma, and limited interaction of alpha with gamma. The major alpha/beta interface covers switch II of alpha, and GTP-induced rearrangement of switch II causes subunit dissociation during signaling. Alterations in GDP binding in the heterotrimer (compared with alpha-GDP) explain stabilization of the inactive conformation of alpha by beta gamma. Repeated WD motifs in beta form a circularized sevenfold beta propeller. The conserved cores of these motifs are a scaffold for display of their more variable linkers on the exterior face of each propeller blade.

    Funded by: NIDDK NIH HHS: DK46371; NIGMS NIH HHS: GM34497

    Cell 1995;83;6;1047-58

  • Analysis of the relative interactions between the alpha 2C10 adrenoceptor and the guanine-nucleotide-binding proteins G(o)1 alpha and Gi 2 alpha following co-expression of these polypeptides in rat 1 fibroblasts.

    Grassie MA and Milligan G

    Molecular Pharmacology Group, University of Glasgow, Scotland, U.K.

    Rat 1 fibroblasts which had been transfected to express the human alpha 2C10 adrenoceptor (clone 1C) were further co-transfected with a plasmid containing the hygromycin-B-resistance gene and a plasmid containing a cDNA encoding the alpha-subunit of the rat pertussis-toxin-sensitive G-protein G(o)1. In clone 3 the receptor was expressed at some 2.2 pmol/mg of membrane protein, and G(o)1 alpha at approx. 100 pmol/mg of membrane protein. The interaction of these two polypeptides and that between the receptor and Gi2 alpha (endogenously expressed at some 50 pmol/mg of membrane protein) were studied. Agonist activation of G(o)1 alpha was observed in membranes of the alpha 2C10-adrenoceptor(+)-G(o)1 alpha+ cells (clone 3), but not in alpha 2C10-adrenoceptor(+)-G(o)alpha-cells (clone 1C), whereas similar agonist-dependent activation of Gi2 alpha was observed in both cell types. alpha 2C10-adrenoceptor activation of G(o)1 alpha and Gi2 alpha in clone-3 membranes was produced with similar agonist-dose-effect curves. These observations indicate that the receptor interacts with equivalent affinity with each of these G-proteins. Agonist-dependent cholera-toxin-catalysed [32P]ADP-ribosylation of G(o)1 alpha was terminated when the alpha 2-adrenoceptor antagonist yohimbine was added subsequent to agonist-induced initiation of the reaction and release of GDP, demonstrating the conformational requirement for this reaction to be the ternary complex of agonist-occupied receptor and guanine-nucleotide-denuded G-protein.

    The Biochemical journal 1995;306 ( Pt 2);525-30

  • Up-regulation in late pregnancy of both Go1 alpha and Go2 alpha isoforms in human myometrium.

    Zumbihl R, Breuiller-Fouché M, Carrette J, Dufour MN, Ferré F, Bockaert J and Rouot B

    INSERM U65, Université Montpellier II, France.

    The nature of the 39 kDa pertussis toxin substrate previously detected in human pregnant myometrium was investigated. Comparison of membranes from non-pregnant and from 39-40 week pregnant myometrium revealed a higher level at 39 kDa of pertussis toxin ADP-ribosylation and Go alpha immunoreactivity in late pregnancy. Furthermore, quantification of both Go alpha isoforms with specific anti-alpha o1- and alpha o2-antibodies revealed an increase in their expression in late pregnancy. At the same time, only limited changes in the levels of Gi1,2, Gi3 and Gq were observed. In rat myometrial membranes, neither pertussis toxin substrate nor Go alpha immunoreactivity could be detected at the 39 kDa level. These results demonstrate that, in the human myometrium in late pregnancy, there is an increase in the expression of both isoforms of the Go alpha subunits, suggesting a role for these proteins throughout gestation and/or near term for parturition.

    European journal of pharmacology 1994;288;1;9-15

  • Association of heterotrimeric GTP binding regulatory protein (Go) with mitosis.

    Wu HC and Lin CT

    Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan, Republic of China.

    Background: The heterotrimeric GTP-binding regulatory proteins (or G proteins) play an important role in regulation of membrane signal transduction. Previously, we have identified the beta-subunit of G proteins (G beta) to be closely associated with mitotic spindles. In the present study, we have conducted experiments to examine whether the G alpha subunit is also associated with mitotic spindles.

    We prepared polyclonal antibodies against synthetic oligopeptides from different G alpha subunits and used them to localize G alpha in a nasopharyngeal carcinoma cell line by both immunohistochemistry and immunoelectron microscopy, and to see whether any specific G alpha subunit was present in the mitotic spindle region. Subsequently, we used a colocalization method to identify the overlapping distributions of specific G alpha subunits and beta-tubulin. To be sure that the findings represented a general phenomenon, several other cell types were also examined. To identify whether the G alpha subunit in the mitotic spindle was present in a heterotrimeric form, we performed ADP ribosylation experiments.

    Results: (a) Only Go alpha subunit but not Gi alpha or Gs alpha was localized in the mitotic spindle. (b) Go alpha was colocalized with beta-tubulin in the mitotic spindle. (c) The precise site of localization of Go alpha on spindle microtubules was confirmed by immunoultrastructural study. (d) The association of Go alpha with the mitotic spindle could be found in both human and animal cell types. (e) The mitotic spindle preparation could be ADP ribosylated in the presence of pertussis toxin.

    Conclusions: The heterotrimeric Go protein is shown to be closely associated with the mitotic spindle. This finding suggests that Go protein may play some role in the regulation of mitotic spindles in addition to its regulation of signal transduction in neuronal cell membranes.

    Laboratory investigation; a journal of technical methods and pathology 1994;71;2;175-81

  • Structural determinants for activation of the alpha-subunit of a heterotrimeric G protein.

    Lambright DG, Noel JP, Hamm HE and Sigler PB

    Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06510.

    The 1.8 A crystal structure of transducin alpha.GDP, when compared to that of the activated complex with GTP-gamma S, reveals the nature of the conformational changes that occur on activation of a heterotrimeric G-protein alpha-subunit. Structural changes initiated by direct contacts with the terminal phosphate of GTP propagate to regions that have been implicated in effector activation. The changes are distinct from those observed in other members of the GTPase superfamily.

    Nature 1994;369;6482;621-8

  • Identification of GTP-binding proteins in human glomeruli.

    Nitta K, Uchida K, Kawashima A, Tsutsui T, Ozu H, Naito T, Yumura W and Nihei H

    Department of Medicine, Kidney Center, Tokyo Women's Medical College, Japan.

    The localization of GTP-binding proteins (G-proteins) in human glomeruli was examined using immunohistochemistry and immunoblotting. Immunohistochemical staining for G-protein subunits demonstrated the existence of Gs alpha, Gi alpha and Go alpha proteins in the glomeruli. Moreover, immunoblots further revealed Gs alpha (52 kD), Gi alpha 1/2 (40-41 kD), Gi alpha 3 (40 kD) and Go alpha (39 kD) in the glomerular membranes. The predominant subspecies of Gs was a 52-kD protein, and Go alpha was detectable in the smallest amounts of the G-protein subunits. However, immunoblots failed to demonstrate detectable amounts of G-proteins in cytosolic extracts. This is the first report that characterizes G-protein subunits in human glomeruli. Further study is required to determine the roles of G-proteins in signal transductions in human glomeruli.

    Nihon Jinzo Gakkai shi 1994;36;1;9-12

  • Direct measurements of in situ interactions of rat brain opioid receptors with the guanine nucleotide-binding protein Go.

    Georgoussi Z, Carr C and Milligan G

    Institute of Biology, National Center for Scientific Research, Demokritos, Athens, Greece.

    The interactions of rat brain cortical opioid receptors with the guanine nucleotide-binding protein (G protein) Go were probed in membranes by examining the ability of selective antipeptide anti-G protein antisera to disrupt receptor-G protein interactions. This was measured both by antibody-induced alterations in the characteristics of agonist binding to mu and delta receptor binding sites and by antibody attenuation of opioid stimulation of high affinity GTPase activity. Antisera to the amino-terminal 16 amino acids (ON1), amino acids 22-35 (IM1), and the carboxyl-terminal decapeptide (OC2) of forms of Go alpha were able to selectively immunoprecipitate Go from rat cortical membranes. Both antisera OC2 and ON1 were able to immunoprecipitate Go alpha quantitatively. Preincubation of rat cortical membranes with an IgG fraction isolated from antiserum OC2 was able to produce a marked reduction in the ability of the synthetic enkephalin [D-Ala2,D-Leu5] enkephalin (DADLE) (which interacts with delta and mu but not significantly with kappa receptors) to displace specific binding of [3H] diprenorphine (which binds to all of these sites), demonstrating a clear interaction of the mu and delta receptors with one or more variants of Go. An IgG fraction from antiserum ON1 was able to mimic this effect, suggesting that the amino-terminal region of G protein alpha subunits also plays a role in receptor-G protein interactions. In contrast, an IgG fraction from antiserum IM1 was unable to alter the characteristics of DADLE displacement of [3H] diprenorphine binding. Similarly, an antiserum (SG1) directed against the carboxyl-terminal decapeptide common to the alpha subunits of Gi1 and Gi2 was unable to reduce the affinity of DADLE binding to opioid receptors. Use of antiserum OC2 in experiments that allowed pharmacological examination of only the mu-opioid receptor provided independent evidence for the interaction of this receptor site with Go.

    Molecular pharmacology 1993;44;1;62-9

  • Factors determining the specificity of signal transduction by guanine nucleotide-binding protein-coupled receptors. II. Preferential coupling of the alpha 2C-adrenergic receptor to the guanine nucleotide-binding protein, Go.

    Coupry I, Duzic E and Lanier SM

    Cellular and Molecular Research Laboratory, Massachusetts General Hospital, Harvard Medical School, Boston 02114.

    Cell to cell communication by many hormones and neurotransmitters involves three major entities: receptor (R), G-protein (G), and effector molecule (E). Plasticity in this system is conferred by the existence of each entity as isoforms or closely related subtypes that are expressed in a tissue-specific and developmentally regulated manner. Factors that determine signal specificity in this system are poorly understood. Such factors include the relative affinity and stoichiometry of R-G or G-E and the possible colocalization of R-G-E in cellular microdomains. Utilizing the alpha 2-adrenergic receptor (alpha 2-AR) system as a representative subfamily of this class of signal transducers, we determined the relative importance of these factors. By analysis of R-G coupling in mammalian cells cotransfected with alpha 2-AR genes and G alpha cDNA, we demonstrate preferential coupling between an alpha 2-AR subtype and Go. Our data implicate R-G affinity as an important determinant of signal transduction specificity and indicate that a critical level of Go alpha is required for coupling. This report indicates the utility of R-G cotransfection in mammalian cells as a "natural environment model" to characterize events occurring at the R-G and G-E interface.

    Funded by: FIC NIH HHS: 3 FO5 TW4313; NINDS NIH HHS: NS-24821

    The Journal of biological chemistry 1992;267;14;9852-7

  • Mutagenesis of the amino terminus of the alpha subunit of the G protein Go. In vitro characterization of alpha o beta gamma interactions.

    Denker BM, Neer EJ and Schmidt CJ

    Department of Medicine, Brigham and Women's Hospital, Boston, Massachusetts.

    Heterotrimeric guanine nucleotide-binding proteins are composed of alpha and beta gamma subunits and couple a variety of cell-surface receptors to intracellular enzymes or ion channels. The heterotrimer dissociates into alpha and beta gamma subunits when the alpha subunit is activated by guanine nucleoside triphosphates. Several lines of evidence show that the amino terminus of the alpha subunit is important for the interaction with the beta gamma subunit (Neer, E. J., Pulsifer, L., and Wolf, L. G. (1988) J. Biol. Chem. 263, 8996-9000; Fung, B. K.-K., and Nash, C. R. (1983) J. Biol. Chem. 258, 10503-10510). We have mutagenized the amino terminus of alpha o to dissect the relative contributions of amino-terminal myristoylation and specific amino acid sequences to subunit interaction. Wild-type and mutant alpha o cDNAs were translated in vitro in a rabbit reticulocyte lysate. All proteins were able to bind guanosine 5'-(gamma-thio)triphosphate and to achieve the necessary conformation for protection from tryptic digestion. Two assays of alpha o beta gamma interactions were used: sucrose density gradients to look for stable heterotrimer formation and ADP-ribosylation by pertussis toxin to detect weak or transient alpha o beta gamma interactions. Our results indicate that myristoylation is essential for stable heterotrimer formation, but that nonmyristoylated proteins are also capable of interacting with the beta gamma subunit. Amino acids 7-10 have an important role in alpha o beta gamma interactions whether alpha o is myristoylated or not. Deletion of this region diminishes the ability of alpha o to interact with the beta gamma subunit, but substitutions at this position indicate that other amino acids can be tolerated without affecting subunit interaction.

    Funded by: NIGMS NIH HHS: GM12578-03, GM35417, GM36259

    The Journal of biological chemistry 1992;267;9;6272-7

  • Structure of the human gene and two rat cDNAs encoding the alpha chain of GTP-binding regulatory protein Go: two different mRNAs are generated by alternative splicing.

    Tsukamoto T, Toyama R, Itoh H, Kozasa T, Matsuoka M and Kaziro Y

    Institute of Medical Science, University of Tokyo, Japan.

    Go is a specific class ("other") of signal-transducing heterotrimeric GTP-binding proteins (G proteins) that is expressed in high levels in mammalian brain. We have cloned two different rat cDNAs encoding the alpha subunit of Go (Go alpha-1 and Go alpha-2) and a human Go alpha chromosomal gene. The human Go alpha gene spans more than 100 kilobases and contains 11 exons, including one noncoding exon in the 3' flanking region. The 5' flanking region is highly G + C-rich and contains five G.C boxes (Sp1 binding sites) but no TATA box. Exons 7 and 8 coding for amino acid residues 242-354 of Go alpha protein are duplicated (referred to as exons 7A, 7B, 8A, and 8B). It was found that exons 7A and 8A code for Go alpha-1, and 7B and 8B code for Go alpha-2. This indicates that two different Go alpha mRNAs may be generated by alternative splicing of a single Go alpha gene. The splice sites of the Go alpha-1 and Go alpha-2 genes are completely identical with those encoding human inhibitory G protein alpha subunits Gi2 alpha and Gi3 alpha [Itoh, H., Toyama, R., Kozasa, T., Tsukamoto, T., Matsuoka, M. & Kaziro, Y. (1988) J. Biol. Chem. 263, 6656-6664] and also transducin G protein alpha subunit Gt1 alpha [Raport, C. J., Dere, B. & Hurley, J. (1989) J. Biol. Chem. 264, 7122-7128]. Sequence homology and conservation of the exon-intron organization indicate that the genes coding for Go alpha, Gi2 alpha, Gi3 alpha, Gt1 alpha, and probably Gi1 alpha may be evolved from a common progenitor. Like Go alpha-1, Go alpha-2 is expressed mainly in brain.

    Proceedings of the National Academy of Sciences of the United States of America 1991;88;8;2974-8

  • Structural and functional studies of cross-linked Go protein subunits.

    Yi F, Denker BM and Neer EJ

    Department of Medicine, Brigham and Women's Hospital, Boston, MA 02115.

    The guanine nucleotide binding proteins (G proteins) that couple hormone and other receptors to a variety of intracellular effector enzymes and ion channels are heterotrimers of alpha, beta, and gamma subunits. One way to study the interfaces between subunits is to analyze the consequences of chemically cross-linking them. We have used 1,6-bismaleimidohexane (BMH), a homobifunctional cross-linking reagent that reacts with sulfhydryl groups, to cross-link alpha to beta subunits of Go and Gi-1. Two cross-linked products are formed from each G protein with apparent molecular masses of 140 and 122 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Both bands formed from Go reacted with anti-alpha o and anti-beta antibody. The mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis is anomalous since the undenatured, cross-linked proteins have the same Stokes radius as the native, uncross-linked alpha beta gamma heterotrimer. Therefore, each cross-linked product contains one alpha and one beta subunit. Activation of Go by guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) does not prevent cross-linking of alpha to beta gamma, consistent with an equilibrium between associated and dissociated subunits even in the presence of GTP gamma S. The same cross-linked products of Go are formed in brain membranes reacted with BMH as are formed in solution, indicating that the residues cross-linked by BMH in the pure protein are accessible when Go is membrane bound. Analysis of tryptic peptides formed from the cross-linked products indicates that the alpha subunit is cross-linked to the 26-kDa carboxyl-terminal portion of the beta subunit. The cross-linked G protein is functional, and its alpha subunit can change conformation upon binding GTP gamma S. GTP gamma S stabilizes alpha o to digestion by trypsin (Winslow, J.W., Van Amsterdam, J.R., and Neer, E.J. (1986) J. Biol. Chem. 261, 7571-7579) and also stabilizes the alpha subunit in the cross-linked product. Cross-linked G o can be ADP-ribosylated by pertussis toxin. This ADP-ribosylation is inhibited by GTP gamma S with a concentration dependence that is indistinguishable from that of the control, uncross-linked G o. These two kinds of experiments indicate that alpha o is able to change its conformation even though it cannot separate completely from beta gamma. Thus, although dissociation of the subunits accompanies activation of G o in solution, it is not obligatory for a conformational change to occur in the alpha subunit.

    Funded by: NIGMS NIH HHS: GM12578, GM36259

    The Journal of biological chemistry 1991;266;6;3900-6

  • Different forms of Go alpha mRNA arise by alternative splicing of transcripts from a single gene on human chromosome 16.

    Murtagh JJ, Eddy R, Shows TB, Moss J and Vaughan M

    Laboratory of Cellular Metabolism, National Heart, Lung, and Blood Institute, Bethesda, Maryland 20892.

    Go alpha, (gene symbol GNA01), a member of the signal-transducing guanine nucleotide-binding (G) protein family, has been implicated in ion channel regulation. Some tissues contain multiple Go alpha mRNAs of different sizes that differ in the 3' untranslated regions (UTRs). Using sequence-specific 48-base oligonucleotides, two complementary to the different 3' UTRs and one complementary to the coding region, we investigated the origin of the multiple Go alpha transcripts, the organization of the Go alpha gene, the interspecies conservation of 3' UTRs, and the chromosomal localization of Go alpha. Oligonucleotides labeled to high specific activity by using terminal deoxynucleotidyltransferase each hybridized with a single band of restriction enzyme-digested mouse and human DNAs. In three of four digests of human DNA, the two probes specific for the different 3' UTRs hybridized with the same restriction fragment. Thus, these nucleotide sequences are in close proximity in the human genome. The order of the UTRs in the bovine, human, and mouse genomes was confirmed directly by polymerase chain reaction (PCR) amplification and sequencing. Hybridization of bovine oligonucleotide sequence with mouse and human genomic DNA indicated a high degree of interspecies sequence conservation: conservation was confirmed by PCR amplification and sequencing. Bands detected by both UTR probes, as well as the predominant bands detected by a bovine Go alpha cDNA, segregated with human chromosome 16 on Southern blot analysis of human-mouse somatic cell hybrids. We conclude that Go alpha mRNAs with different 3' UTRs arise by alternative splicing of transcripts from a single gene. The UTRs, which exhibit a high degree of interspecies conservation, may play a role in regulation of Go alpha expression during differentiation or in specific tissues. The use of oligonucleotide probes of the type described here represents a new strategy, potentially widely applicable for mapping and elucidating structural features of genes.

    Molecular and cellular biology 1991;11;2;1146-55

  • Ultrastructural localization of the GTP-binding protein Go in neurons.

    Gabrion J, Brabet P, Nguyen Than Dao B, Homburger V, Dumuis A, Sebben M, Rouot B and Bockaert J

    Laboratoire de Neurobiologie Endocrinologique, UA 1197 CNRS, Université de Montpellier II, France.

    The ultrastructural localization of Go, a GTP-binding protein (G protein) highly expressed in nervous tissues, was performed in cultured fetal and adult murine neurons, using affinity-purified polyclonal antibodies against the alpha subunit of the Go protein (Go alpha). These antibodies recognized denatured Go alpha and both the native Go alpha-subunit and the Go alpha beta gamma heterotrimer. At the ultrastructural level, the positive immunoreactivity detected in cultured cells as well as in thin frozen sections, showed that Go was largely distributed in cell bodies and neuritic cytoplasm. Labelling was principally noted on the cytoplasmic face of the plasma membrane lining the cell body and the neurites, especially in 'cell-cell' contacts, but also in the cytoplasmic matrix, between endoplasmic reticulum and Golgi cisternae. No immunoreactivity was observed on the inner face of the pre- or postsynaptic membranes in both adult brain and in cultured neurons. This last finding strongly suggests that the Go protein is not involved in transducing chemical signals at the level of synapses, but more probably modulates the synaptic functions by controlling the activity of effectors localized outside of the synaptic densities.

    Cellular signalling 1989;1;1;107-23

  • Molecular cloning and DNA sequence analysis of the human guanine nucleotide-binding protein Go alpha.

    Lavu S, Clark J, Swarup R, Matsushima K, Paturu K, Moss J and Kung HF

    Biological Response Modifiers Program, National Cancer Institute-Frederick Cancer Research Facility, MD 21701.

    The nucleotide sequence of human Go alpha was determined from a partial human brain cDNA clone and the sequence of the first two 5' coding exons of a human genomic Go alpha clone. Comparison of this sequence with bovine and rat Go alpha shows greater than 90% homology at the nucleotide and deduced amino acid level. There is 100% identity at the amino acid level for the cholera and pertussis toxin-catalyzed ADP ribosylation sites, the putative guanine nucleotide binding, and the GTP hydrolysis sites.

    Biochemical and biophysical research communications 1988;150;2;811-5

Gene lists (10)

Gene List Source Species Name Description Gene count
L00000009 G2C Homo sapiens Human PSD Human orthologues of mouse PSD adapted from Collins et al (2006) 1080
L00000011 G2C Homo sapiens Human clathrin Human orthologues of mouse clathrin coated vesicle genes adapted from Collins et al (2006) 150
L00000012 G2C Homo sapiens Human Synaptosome Human orthologues of mouse synaptosome adapted from Collins et al (2006) 152
L00000013 G2C Homo sapiens Human mGluR5 Human orthologues of mouse mGluR5 complex adapted from Collins et al (2006) 52
L00000016 G2C Homo sapiens Human PSP Human orthologues of mouse PSP adapted from Collins et al (2006) 1121
L00000049 G2C Homo sapiens TAP-PSD-95-CORE TAP-PSD-95 pull-down core list (ortho) 120
L00000059 G2C Homo sapiens BAYES-COLLINS-HUMAN-PSD-CONSENSUS Human cortex PSD consensus 748
L00000061 G2C Homo sapiens BAYES-COLLINS-MOUSE-PSD-CONSENSUS Mouse cortex PSD consensus (ortho) 984
L00000069 G2C Homo sapiens BAYES-COLLINS-HUMAN-PSD-FULL Human cortex biopsy PSD full list 1461
L00000071 G2C Homo sapiens BAYES-COLLINS-MOUSE-PSD-FULL Mouse cortex PSD full list (ortho) 1556
© G2C 2014. The Genes to Cognition Programme received funding from The Wellcome Trust and the EU FP7 Framework Programmes:
EUROSPIN (FP7-HEALTH-241498), SynSys (FP7-HEALTH-242167) and GENCODYS (FP7-HEALTH-241995).

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