G2Cdb::Gene report

Gene id
G00002221
Gene symbol
GNAI3 (HGNC)
Species
Homo sapiens
Description
guanine nucleotide binding protein (G protein), alpha inhibiting activity polypeptide 3
Orthologue
G00000972 (Mus musculus)

Databases (8)

Curated Gene
OTTHUMG00000011648 (Vega human gene)
Gene
ENSG00000065135 (Ensembl human gene)
2773 (Entrez Gene)
359 (G2Cdb plasticity & disease)
GNAI3 (GeneCards)
Literature
139370 (OMIM)
Marker Symbol
HGNC:4387 (HGNC)
Protein Sequence
P08754 (UniProt)

Synonyms (1)

  • 87U6

Literature (65)

Pubmed - other

  • Genome-wide association study of recurrent early-onset major depressive disorder.

    Shi J, Potash JB, Knowles JA, Weissman MM, Coryell W, Scheftner WA, Lawson WB, DePaulo JR, Gejman PV, Sanders AR, Johnson JK, Adams P, Chaudhury S, Jancic D, Evgrafov O, Zvinyatskovskiy A, Ertman N, Gladis M, Neimanas K, Goodell M, Hale N, Ney N, Verma R, Mirel D, Holmans P and Levinson DF

    Department of Psychiatry, Stanford University, Stanford, CA, USA.

    A genome-wide association study was carried out in 1020 case subjects with recurrent early-onset major depressive disorder (MDD) (onset before age 31) and 1636 control subjects screened to exclude lifetime MDD. Subjects were genotyped with the Affymetrix 6.0 platform. After extensive quality control procedures, 671 424 autosomal single nucleotide polymorphisms (SNPs) and 25 068 X chromosome SNPs with minor allele frequency greater than 1% were available for analysis. An additional 1 892 186 HapMap II SNPs were analyzed based on imputed genotypic data. Single-SNP logistic regression trend tests were computed, with correction for ancestry-informative principal component scores. No genome-wide significant evidence for association was observed, assuming that nominal P<5 × 10(-8) approximates a 5% genome-wide significance threshold. The strongest evidence for association was observed on chromosome 18q22.1 (rs17077540, P=1.83 × 10(-7)) in a region that has produced some evidence for linkage to bipolar-I or -II disorder in several studies, within an mRNA detected in human brain tissue (BC053410) and approximately 75 kb upstream of DSEL. Comparing these results with those of a meta-analysis of three MDD GWAS data sets reported in a companion article, we note that among the strongest signals observed in the GenRED sample, the meta-analysis provided the greatest support (although not at a genome-wide significant level) for association of MDD to SNPs within SP4, a brain-specific transcription factor. Larger samples will be required to confirm the hypothesis of association between MDD (and particularly the recurrent early-onset subtype) and common SNPs.

    Funded by: NCRR NIH HHS: U54 RR020278; NIMH NIH HHS: R01 MH061686

    Molecular psychiatry 2011;16;2;193-201

  • Insulin-like growth factor-binding protein-5 stimulates growth of human intestinal muscle cells by activation of G{alpha}i3.

    Flynn RS, Mahavadi S, Murthy KS, Kellum JM and Kuemmerle JF

    Department of Medicine, Virginia Commonwealth University, Richmond, USA.

    In human intestinal smooth muscle cells, endogenous insulin-like growth factor-I (IGF-I) regulates growth and IGF-binding protein-5 (IGFBP-5) expression. The effects of IGF-I are facilitated by IGFBP-5. We previously showed that IGFBP-5 acts independently of IGF-I in human intestinal muscle to stimulate proliferation and upregulate IGF-I production by activation of Erk1/2 and p38 MAPK. Thus a positive feedback loop exists between IGF-I and IGFBP-5, whereby both stimulate muscle growth and production of the other factor. In Crohn's disease, IGF-I and IGFBP-5 expression are increased and contribute to stricture formation through this effect on muscle growth. To determine the signaling pathways coupling IGFBP-5 to MAPK activation and growth, smooth muscle cells were isolated from muscularis propria of human intestine and placed into primary culture. Erk1/2 and p38 MAPK activation and type I collagen production were measured by immunoblot. Proliferation was measured by [(3)H]thymidine incorporation. Activation of specific G proteins was measured by ELISA. AG1024, an IGF-I receptor tyrosine kinase inhibitor, was used to isolate the IGF-I-independent effects of IGFBP-5. IGFBP-5-induced phosphorylation of Erk1/2 and p38 MAPK and proliferation were abolished by pertussis toxin, implying the participation of Gi. IGFBP-5 specifically activated Gi3 but not other G proteins. Transfection of an inhibitory Galphai minigene specifically inhibited MAPK activation, proliferation, and both collagen-I and IGF-I production. Our results indicate that endogenous IGFBP-5 activates Gi3 and regulates smooth muscle growth, IGF-I production, and collagen production via the alpha-subunit of Gi3, independently of IGF-I, in normal human intestinal muscle cells.

    Funded by: NIDDK NIH HHS: DK-49691

    American journal of physiology. Gastrointestinal and liver physiology 2009;297;6;G1232-8

  • Activation of Galphai3 triggers cell migration via regulation of GIV.

    Ghosh P, Garcia-Marcos M, Bornheimer SJ and Farquhar MG

    Department of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, CA 92093, USA.

    During migration, cells must couple direction sensing to signal transduction and actin remodeling. We previously identified GIV/Girdin as a Galphai3 binding partner. We demonstrate that in mammalian cells Galphai3 controls the functions of GIV during cell migration. We find that Galphai3 preferentially localizes to the leading edge and that cells lacking Galphai3 fail to polarize or migrate. A conformational change induced by association of GIV with Galphai3 promotes Akt-mediated phosphorylation of GIV, resulting in its redistribution to the plasma membrane. Activation of Galphai3 serves as a molecular switch that triggers dissociation of Gbetagamma and GIV from the Gi3-GIV complex, thereby promoting cell migration by enhancing Akt signaling and actin remodeling. Galphai3-GIV coupling is essential for cell migration during wound healing, macrophage chemotaxis, and tumor cell migration, indicating that the Galphai3-GIV switch serves to link direction sensing from different families of chemotactic receptors to formation of the leading edge during cell migration.

    Funded by: NCI NIH HHS: CA100768, R01 CA100768; NIDDK NIH HHS: DK17780, T32 DK007202, T32 DK07202

    The Journal of cell biology 2008;182;2;381-93

  • Neither replication nor simulation supports a role for the axon guidance pathway in the genetics of Parkinson's disease.

    Li Y, Rowland C, Xiromerisiou G, Lagier RJ, Schrodi SJ, Dradiotis E, Ross D, Bui N, Catanese J, Aggelakis K, Grupe A and Hadjigeorgiou G

    Celera, Alameda, California, United States of America. yonghong.li@celera.com

    Susceptibility to sporadic Parkinson's disease (PD) is thought to be influenced by both genetic and environmental factors and their interaction with each other. Statistical models including multiple variants in axon guidance pathway genes have recently been purported to be capable of predicting PD risk, survival free of the disease and age at disease onset; however the specific models have not undergone independent validation. Here we tested the best proposed risk panel of 23 single nucleotide polymorphisms (SNPs) in two PD sample sets, with a total of 525 cases and 518 controls. By single marker analysis, only one marker was significantly associated with PD risk in one of our sample sets (rs6692804: P = 0.03). Multi-marker analysis using the reported model found a mild association in one sample set (two sided P = 0.049, odds ratio for each score change = 1.07) but no significance in the other (two sided P = 0.98, odds ratio = 1), a stark contrast to the reported strong association with PD risk (P = 4.64x10(-38), odds ratio as high as 90.8). Following a procedure similar to that used to build the reported model, simulated multi-marker models containing SNPs from randomly chosen genes in a genome wide PD dataset produced P-values that were highly significant and indistinguishable from similar models where disease status was permuted (3.13x10(-23) to 4.90x10(-64)), demonstrating the potential for overfitting in the model building process. Together, these results challenge the robustness of the reported panel of genetic markers to predict PD risk in particular and a role of the axon guidance pathway in PD genetics in general.

    PloS one 2008;3;7;e2707

  • 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

  • Dissociation of heterotrimeric g proteins in cells.

    Lambert NA

    Department of Pharmacology and Toxicology, Medical College of Georgia, Augusta, GA 30912-2300, USA. nlambert@mcg.edu

    Heterotrimeric G proteins dissociate into their component Galpha and Gbetagamma subunits when these proteins are activated in solution. Until recently, it has not been known if subunit dissociation also occurs in cells. The development of optical methods to study G protein activation in live cells has made it possible to demonstrate heterotrimer dissociation at the plasma membrane. However, subunit dissociation is far from complete, and many active [guanosine triphosphate (GTP)-bound] heterotrimers are intact in a steady state. This unexpectedly reluctant dissociation calls for inclusion of a GTP-bound heterotrimeric state in models of the G protein cycle and places renewed emphasis on the relation between subunit dissociation and effector activation.

    Science signaling 2008;1;25;re5

  • Purification and identification of G protein-coupled receptor protein complexes under native conditions.

    Daulat AM, Maurice P, Froment C, Guillaume JL, Broussard C, Monsarrat B, Delagrange P and Jockers R

    Department of Cell Biology, Institut Cochin, INSERM U567, CNRS UMR 8104, Université Paris Descartes, France.

    G protein-coupled receptors (GPCRs) constitute the largest family of membrane receptors and are of major therapeutic importance. The identification of GPCR-associated proteins is an important step toward a better understanding of these receptors. However, current methods are not satisfying as only isolated receptor domains (intracellular loops or carboxyl-terminal tails) can be used as "bait." We report here a method based on tandem affinity purification coupled to mass spectrometry that overcomes these limitations as the entire receptor is used to identify protein complexes formed in living mammalian cells. The human MT(1) and MT(2) melatonin receptors were chosen as model GPCRs. Both receptors were tagged with the tandem affinity purification tag at their carboxyl-terminal tails and expressed in human embryonic kidney 293 cells. Receptor solubilization and purification conditions were optimized. The method was validated by the co-purification of G(i) proteins, which are well known GPCR interaction partners but which are difficult to identify with current protein-protein interaction assays. Several new and functionally relevant MT(1)- and MT(2)-associated proteins were identified; some of them were common to both receptors, and others were specific for each subtype. Taken together, our protocol allowed for the first time the purification of GPCR-associated proteins under native conditions in quantities suitable for mass spectrometry analysis.

    Molecular & cellular proteomics : MCP 2007;6;5;835-44

  • 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

  • G-protein alpha subunit interaction and guanine nucleotide dissociation inhibitor activity of the dual GoLoco motif protein PCP-2 (Purkinje cell protein-2).

    Willard FS, McCudden CR and Siderovski DP

    Department of Pharmacology, CB# 7365, 1106 Mary Ellen Jones Building, University of North Carolina, Chapel Hill, NC 27599-7365, USA. fwillard@med.unc.edu

    Purkinje cell protein-2 (PCP-2; L7/GPSM4) is a GoLoco motif-containing protein that is specifically expressed in Purkinje and retinal ON bipolar cells. An alternative splice variant of PCP-2 has recently been isolated which contains two GoLoco motifs. Although the second GoLoco motif (GL2) of PCP-2 has been reported to interact with Galpha-subunits, a complete biochemical analysis of each individual motif of PCP-2 has not been performed. We demonstrate that the first GoLoco motif (GL1) of PCP-2 is equipotent as a guanine nucleotide dissociation inhibitor (GDI) towards Galphai1 and Galphai2, while it has sevenfold lower GDI activity for Galphai3 and greater than 20-fold lower GDI activity against Galphao. In contrast we found PCP-2 GL2 to be essentially equipotent as a GDI for all Galphai subunits, but it had negligible activity toward Galphao. Using co-immunoprecipitation from COS-7 cells, we found that PCP-2 was only able to interact with Galphai1 but not Galphao nor Galpha-subunits from other families (Galphas, Galphaq, or Galpha12). Mutational analysis of a non-canonical residue (glycine 24) in human PCP-2 GL1 provided evidence for heterogeneity in mechanisms of Galphai interactions with GoLoco motifs. Collectively, the data demonstrate that PCP-2 is a comparatively weak GoLoco motif protein that exhibits highest affinity interactions and GDI activity toward Galphai1, Galphai2, and Galphai3 subunits.

    Funded by: NIGMS NIH HHS: P01 GM065533, R01 GM062338

    Cellular signalling 2006;18;8;1226-34

  • Expression, purification, and preliminary X-ray crystallographic analysis of the complex of G(alphai3)-RGS5 from human with GDP/Mg2+)/AlF4-.

    Rhee KH, Nam KH, Lee WH, Ko YG, Kim EE and Hwang KY

    Life Sciences Division, Biomedical Research Center, Korea Institute of Science and Technology, Division of Biotechnology, Korea University, Seoul 136-701, South Korea. chahong@korea.ac.kr.

    Regulator of G-protein signaling 5 (RGS5), an inhibitor of Gq and Gi activation, is a member of the small RGS protein subfamily. However, despite significant process in the investigation of RGS5, no structure is yet available. In order to elucidate the mechanism of the RGS5 in G protein signaling pathway, we have overexpressed the RGS5 and Galphai(3) from human in Escherichia coli and crystallized the complex of RGS5 and Galphai(3) proteins with GDP/Mg(2+)/AlF(4)(-) at 3.0 A resolution using a synchrotron radiation source. The complex crystals belong to the tetragonal space group P4(1)2(1)2 or P4(3)2(1)2, with unit cell parameters a=b=95.9 A, and c=138.8 A. Assuming one complex protein in the crystallographic asymmetric unit, the calculated Matthews parameter (V(M)) is 2.57 A(3)/Da and solvent content is 52.2 %.

    Protein and peptide letters 2006;13;9;945-9

  • Crystallization and preliminary X-ray crystallographic analysis of human RGS10 complexed with Galphai3.

    Lee HK, Rhee KH, Kim CW, Hwang KY and Kim EE

    Life Sciences Division, Korea Institute of Science and Technology, Seoul 130-650, South Korea.

    G-protein-coupled receptors, which are major targets for drug discovery, play a major role in diverse physiological processes by relating changes in the extracellular environment to intracellular functions via activation of heterotrimeric G-proteins. However, G-protein activity is also modulated by a family of proteins called regulators of G-protein signalling (RGS), which are classified into six subfamilies. RGS10 belongs to the subgroup D/R12 and is known to act specifically on activated forms of three Galpha proteins (Galphai3, Galphaz and Galphao but not Galphas). It is abundantly expressed in brain and immune tissues and has been implicated in the pathophysiology of schizophrenia. The RGS domain of RGS10 was cloned, purified, complexed with human Galphai3 and crystallized. The crystals containing both RGS and Galphai3 belong to space group P4(3)2(1)2 (or P4(1)2(1)2), with unit-cell parameters a = 99.88, b = 99.88, c = 144.59 A, alpha = beta = gamma = 90 degrees . A full set of diffraction data were collected to 2.5 A resolution at 100 K using synchrotron radiation at Pohang beamline 4A.

    Acta crystallographica. Section F, Structural biology and crystallization communications 2005;61;Pt 9;831-3

  • The status, quality, and expansion of the NIH full-length cDNA project: the Mammalian Gene Collection (MGC).

    Gerhard DS, Wagner L, Feingold EA, Shenmen CM, Grouse LH, Schuler G, Klein SL, Old S, Rasooly R, Good P, Guyer M, Peck AM, Derge JG, Lipman D, Collins FS, Jang W, Sherry S, Feolo M, Misquitta L, Lee E, Rotmistrovsky K, Greenhut SF, Schaefer CF, Buetow K, Bonner TI, Haussler D, Kent J, Kiekhaus M, Furey T, Brent M, Prange C, Schreiber K, Shapiro N, Bhat NK, Hopkins RF, Hsie F, Driscoll T, Soares MB, Casavant TL, Scheetz TE, Brown-stein MJ, Usdin TB, Toshiyuki S, Carninci P, Piao Y, Dudekula DB, Ko MS, Kawakami K, Suzuki Y, Sugano S, Gruber CE, Smith MR, Simmons B, Moore T, Waterman R, Johnson SL, Ruan Y, Wei CL, Mathavan S, Gunaratne PH, Wu J, Garcia AM, Hulyk SW, Fuh E, Yuan Y, Sneed A, Kowis C, Hodgson A, Muzny DM, McPherson J, Gibbs RA, Fahey J, Helton E, Ketteman M, Madan A, Rodrigues S, Sanchez A, Whiting M, Madari A, Young AC, Wetherby KD, Granite SJ, Kwong PN, Brinkley CP, Pearson RL, Bouffard GG, Blakesly RW, Green ED, Dickson MC, Rodriguez AC, Grimwood J, Schmutz J, Myers RM, Butterfield YS, Griffith M, Griffith OL, Krzywinski MI, Liao N, Morin R, Morrin R, Palmquist D, Petrescu AS, Skalska U, Smailus DE, Stott JM, Schnerch A, Schein JE, Jones SJ, Holt RA, Baross A, Marra MA, Clifton S, Makowski KA, Bosak S, Malek J and MGC Project Team

    The National Institutes of Health's Mammalian Gene Collection (MGC) project was designed to generate and sequence a publicly accessible cDNA resource containing a complete open reading frame (ORF) for every human and mouse gene. The project initially used a random strategy to select clones from a large number of cDNA libraries from diverse tissues. Candidate clones were chosen based on 5'-EST sequences, and then fully sequenced to high accuracy and analyzed by algorithms developed for this project. Currently, more than 11,000 human and 10,000 mouse genes are represented in MGC by at least one clone with a full ORF. The random selection approach is now reaching a saturation point, and a transition to protocols targeted at the missing transcripts is now required to complete the mouse and human collections. Comparison of the sequence of the MGC clones to reference genome sequences reveals that most cDNA clones are of very high sequence quality, although it is likely that some cDNAs may carry missense variants as a consequence of experimental artifact, such as PCR, cloning, or reverse transcriptase errors. Recently, a rat cDNA component was added to the project, and ongoing frog (Xenopus) and zebrafish (Danio) cDNA projects were expanded to take advantage of the high-throughput MGC pipeline.

    Funded by: PHS HHS: N01-C0-12400

    Genome research 2004;14;10B;2121-7

  • 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

  • Analysis of RGSZ1 protein interaction with Galphai subunits.

    Wang Y and Young KH

    Neuroscience Discovery Research, Wyeth Research, Princeton, New Jersey 08543, USA.

    RGSZ1 has been reported to interact with G-protein subunits of the Galphai family and function as a GTPase-accelerating protein on intrinsic Galphai GTPase activity. This article describes several experimental approaches and assays used to investigate the effect of RGSZ1 on Galphai subunits. The formats described here include physical and functional interaction assays by which the association of RGSZ1 with Galphai is explored both in vitro and in vivo. The methods analyzing physical interaction include pull-down and coimmunoprecipitation assays. We also apply yeast two-hybrid techniques to detect RGSZ1 protein interaction with Galpha subunits. Additionally, we developed several functional assay systems to identify the functional relationship between RGSZ1 and Galphai, such as the single turnover GTPase assay, yeast pheromone response assay, mitogen-activated protein kinase assay, and serum response element reporter assay.

    Methods in enzymology 2004;390;31-52

  • 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

  • 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

  • Promotion of G alpha i3 subunit down-regulation by GIPN, a putative E3 ubiquitin ligase that interacts with RGS-GAIP.

    Fischer T, De Vries L, Meerloo T and Farquhar MG

    Department of Cellular and Molecular Medicine, University of California at San Diego, La Jolla, CA 92093, USA.

    We have isolated an RGS-GAIP interacting protein that links RGS proteins to protein degradation. GIPN (GAIP interacting protein N terminus) is a 38-kDa protein with an N-terminal leucine-rich region, a central RING finger-like domain, and a putative C-terminal transmembrane domain. GIPN binds exclusively to RGS proteins of subfamily A, RGS-GAIP, RGSZ1, and RGSZ2. The N-terminal leucine-rich region of GIPN interacts with the cysteine-rich motif of RGS-GAIP. GIPN mRNA is ubiquitously expressed, and GIPN is found on the plasma membrane of transfected HEK293 cells. Endogenous GIPN is concentrated along the basolateral plasma membrane of proximal and distal tubules in rat kidney, where many G protein-coupled receptors and some G proteins are also located. Two immunoreactive species are found in rat kidney, a 38-kDa cytosolic form and an approximately 94-kDa membrane form. GIPN shows Zn2+- and E1/E2-dependent autoubiquitination in vitro, suggesting that it has E3 ubiquitin ligase activity. Overexpression of GIPN stimulates proteasome-dependent reduction of endogenous G alpha i3 in HEK293 cells and reduces the half-life of overexpressed G alpha i3-YFP. Thus, our findings suggest that GIPN is involved in the degradation of G alpha i3 subunits via the proteasome pathway. RGS-GAIP functions as a bifunctional adaptor that binds to G alpha subunits through its RGS domain and to GIPN through its cysteine string motif.

    Funded by: NCI NIH HHS: CA100768, CA58689, R01 CA100768; NIDDK NIH HHS: DK17780, R01 DK017780

    Proceedings of the National Academy of Sciences of the United States of America 2003;100;14;8270-5

  • 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

  • Regulator of G protein signaling Z1 (RGSZ1) interacts with Galpha i subunits and regulates Galpha i-mediated cell signaling.

    Wang Y, Ho G, Zhang JJ, Nieuwenhuijsen B, Edris W, Chanda PK and Young KH

    Neuroscience Discovery Research, Wyeth Research, Princeton, New Jersey 08543-8000, USA. wangy4@wyeth.com

    Regulator of G protein signaling (RGS) proteins constitute a family of over 20 proteins that negatively regulate heterotrimeric G protein-coupled receptor signaling pathways by enhancing endogenous GTPase activities of G protein alpha subunits. RGSZ1, one of the RGS proteins specifically localized to the brain, has been cloned previously and described as a selective GTPase accelerating protein for Galpha(z) subunit. Here, we employed several methods to provide new evidence that RGSZ1 interacts not only with Galpha(z,) but also with Galpha(i), as supported by in vitro binding assays and functional studies. Using glutathione S-transferase fusion protein pull-down assays, glutathione S-transferase-RGSZ1 protein was shown to bind (35)S-labeled Galpha(i1) protein in an AlF(4)(-)dependent manner. The interaction between RGSZ1 and Galpha(i) was confirmed further by co-immunoprecipitation studies and yeast two-hybrid experiments using a quantitative luciferase reporter gene. Extending these observations to functional studies, RGSZ1 accelerated endogenous GTPase activity of Galpha(i1) in single-turnover GTPase assays. Human RGSZ1 functionally regulated GPA1 (a yeast Galpha(i)-like protein)-mediated yeast pheromone response when expressed in a SST2 (yeast RGS protein) knockout strain. In PC12 cells, transfected RGSZ1 blocked mitogen-activated protein kinase activity induced by UK14304, an alpha(2)-adrenergic receptor agonist. Furthermore, RGSZ1 attenuated D2 dopamine receptor agonist-induced serum response element reporter gene activity in Chinese hamster ovary cells. In summary, these data suggest that RGSZ1 serves as a GTPase accelerating protein for Galpha(i) and regulates Galpha(i)-mediated signaling, thus expanding the potential role of RGSZ1 in G protein-mediated cellular activities.

    The Journal of biological chemistry 2002;277;50;48325-32

  • Cloning and characterization of a novel regulator of G protein signalling in human platelets.

    Gagnon AW, Murray DL and Leadley RJ

    Cardiovascular Drug Discovery, Aventis Pharmaceuticals, 500 Arcola Road, Collegeville, PA 19426, USA.

    In an effort to understand the modulation of G protein-coupled receptor (GPCR)-mediated signalling in platelets, we sought to identify which regulators of G protein signalling proteins (RGSs) are present in human platelets. Using degenerate oligonucleotides, we performed RT-PCR with human platelet and megakaryocytic cell line RNA. In addition to confirming the presence of several known RGS transcripts, we found a novel RGS domain-containing transcript in platelet RNA. Northern blot analysis of multiple human tissues indicates that this transcript is most abundantly expressed in platelets compared to other tissues examined. Full-length cloning of this novel RGS, which we now term RGS18, demonstrates that this transcript is predicted to encode a 235-amino acid protein that is most closely related to RGS5 (46% identity) and that has approximately 30-40% identity to other RGS proteins. RGS18 is expressed in platelet, leukocyte, and megakaryocyte cell lines and binds to endogenous Galphai1, Galphai2, Galphai3, and Galphaq but not Galphaz, Galphas or Galpha12 in vitro.

    Cellular signalling 2002;14;7;595-606

  • P2Y(13): identification and characterization of a novel Galphai-coupled ADP receptor from human and mouse.

    Zhang FL, Luo L, Gustafson E, Palmer K, Qiao X, Fan X, Yang S, Laz TM, Bayne M and Monsma F

    Human Genome Research, Schering-Plough Research Institute, Kenilworth, New Jersey 07033, USA. fang.zhang@spcorp.com

    We have identified an orphan G protein-coupled receptor, SP174, that shares a high degree of homology with the recently described ADP receptor P2Y(12). mRNA for SP174 is abundant in the brain and in cells of the immune system. In the present study, we demonstrate that SP174 is also a receptor for ADP, which is coupled to Galphai. ADP potently stimulates SP174 with an EC(50) of 60 nM, and other related nucleotides are active as well, with a rank order of potency 2-methylthio-ADP tetrasodium = adenosine 5'-O-2-(thio)diphosphate = 2-methylthio-ATP tetrasodium > ADP > AP3A >ATP > IDP. This pharmacological profile is similar to that for P2Y(12). We have also identified the murine homolog of SP174, which exhibits 75% homology to the human receptor. ADP is also a potent agonist at the murine receptor, and its pharmacological profile is similar to its human counterpart, but ADP and related nucleotides are more potent at the murine receptor than the human receptor. In keeping with the general nomenclature for the purinergic receptors, we propose designating this novel receptor P2Y(13).

    The Journal of pharmacology and experimental therapeutics 2002;301;2;705-13

  • Galpha i3 binding to calnuc on Golgi membranes in living cells monitored by fluorescence resonance energy transfer of green fluorescent protein fusion proteins.

    Weiss TS, Chamberlain CE, Takeda T, Lin P, Hahn KM and Farquhar MG

    Department of Cellular and Molecular Medicine, University of California at San Diego, La Jolla, CA 92093, USA.

    Galphai3 is found both on the plasma membrane and on Golgi membranes. Calnuc, an EF hand protein, binds both Galphai3 and Ca(2+) and is found both in the Golgi lumen and in the cytoplasm. To investigate whether Galphai3 binds calnuc in living cells and where this interaction takes place we performed fluorescence resonance energy transfer (FRET) analysis between Galphai3 and calnuc in COS-7 cells expressing Galphai3-yellow fluorescent protein (YFP) and calnuc-cyan fluorescent protein (CFP). The tagged proteins have the same localization as the endogenous, nontagged proteins. When Galphai3-YFP and calnuc-CFP are coexpressed, a FRET signal is detected in the Golgi region, but no FRET signal is detected on the plasma membrane. FRET is also seen within the Golgi region when Galphai3 is coexpressed with cytosolic calnuc(DeltaN2-25)-CFP lacking its signal sequence. No FRET signal is detected when Galphai3(DeltaC12)-YFP lacking the calnuc-binding region is coexpressed with calnuc-CFP or when Galphai3-YFP and calnuc(DeltaEF-1,2)-CFP, which is unable to bind Galphai3, are coexpressed. Galphai3(G2AC3A)-YFP lacking its lipid anchors is localized in the cytoplasm, and no FRET signal is detected when it is coexpressed with wild-type calnuc-CFP. These results indicate that cytosolic calnuc binds to Galphai3 on Golgi membranes in living cells and that Galphai3 must be anchored to the cytosolic surface of Golgi membranes via lipid anchors for the interaction to occur. Calnuc has the properties of a Ca(2+) sensor protein capable of binding to and potentially regulating interactions of Galphai3 on Golgi membranes.

    Funded by: NCI NIH HHS: CA58689; NIDDK NIH HHS: DK17780, R01 DK017780

    Proceedings of the National Academy of Sciences of the United States of America 2001;98;26;14961-6

  • RGS18 is a myeloerythroid lineage-specific regulator of G-protein-signalling molecule highly expressed in megakaryocytes.

    Yowe D, Weich N, Prabhudas M, Poisson L, Errada P, Kapeller R, Yu K, Faron L, Shen M, Cleary J, Wilkie TM, Gutierrez-Ramos C and Hodge MR

    Millennium Pharmaceuticals, 75 Sidney Street, Cambridge, MA 02139, USA. yowe@mpi.com

    Myelopoiesis and lymphopoiesis are controlled by haematopoietic growth factors, including cytokines, and chemokines that bind to G-protein-coupled receptors (GPCRs). Regulators of G-protein signalling (RGSs) are a protein family that can act as GTPase-activating proteins for G(alphai)- and G(alphaq)-class proteins. We have identified a new member of the R4 subfamily of RGS proteins, RGS18. RGS18 contains clusters of hydrophobic and basic residues, which are characteristic of an amphipathic helix within its first 33 amino acids. RGS18 mRNA was most highly abundant in megakaryocytes, and was also detected specifically in haematopoietic progenitor and myeloerythroid lineage cells. RGS18 mRNA was not detected in cells of the lymphoid lineage. RGS18 was also highly expressed in mouse embryonic 15-day livers, livers being the principal organ for haematopoiesis at this stage of fetal development. RGS1, RGS2 and RGS16, other members of the R4 subfamily, were expressed in distinct progenitor and mature myeloerythroid and lymphoid lineage blood cells. RGS18 was shown to interact specifically with the G(alphai-3) subunit in membranes from K562 cells. Furthermore, overexpression of RGS18 inhibited mitogen-activated-protein kinase activation in HEK-293/chemokine receptor 2 cells treated with monocyte chemotactic protein-1. In yeast cells, RGS18 overexpression complemented a pheromone-sensitive phenotype caused by mutations in the endogeneous yeast RGS gene, SST2. These data demonstrated that RGS18 was expressed most highly in megakaryocytes, and can modulate GPCR pathways in both mammalian and yeast cells in vitro. Hence RGS18 might have an important role in the regulation of megakaryocyte differentiation and chemotaxis.

    The Biochemical journal 2001;359;Pt 1;109-18

  • Phosphorylation and nuclear translocation of a regulator of G protein signaling (RGS10).

    Burgon PG, Lee WL, Nixon AB, Peralta EG and Casey PJ

    Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts 02138, USA. burgon@fas.harvard.edu

    Heterotrimeric G proteins are involved in the transduction of hormonal and sensory signals across plasma membranes of eukaryotic cells. Hence, they are a critical point of control for a variety of agents that modulate cellular function. Activation of these proteins is dependent on GTP binding to their alpha (Galpha) subunits. Regulators of G protein signaling (RGS) bind specifically to activated Galpha proteins, potentiating the intrinsic GTPase activity of the Galpha proteins and thus expediting the termination of Galpha signaling. Although there are several points in most G protein controlled signaling pathways that are affected by reversible covalent modification, little evidence has been shown addressing whether or not the functions of RGS proteins are themselves regulated by such modifications. We report in this study the acute functional regulation of RGS10 thru the specific and inducible phosphorylation of RGS10 protein at serine 168 by cAMP-dependent kinase A. This phosphorylation nullifies the RGS10 activity at the plasma membrane, which controls the G protein-dependent activation of the inwardly rectifying potassium channel. Surprisingly, the phosphorylation-mediated attenuation of RGS10 activity was not manifested in an alteration of its ability to accelerate GTPase activity of Galpha. Rather, the phosphorylation event correlates with translocation of RGS10 from the plasma membrane and cytosol into the nucleus.

    Funded by: NCI NIH HHS: F32 CA76711; NIGMS NIH HHS: R01 GM36259, R01 GM55717

    The Journal of biological chemistry 2001;276;35;32828-34

  • RGS12 and RGS14 GoLoco motifs are G alpha(i) interaction sites with guanine nucleotide dissociation inhibitor Activity.

    Kimple RJ, De Vries L, Tronchère H, Behe CI, Morris RA, Gist Farquhar M and Siderovski DP

    Department of Pharmacology, Lineberger Comprehensive Cancer Center, University of North Caro 1f40 lina, Chapel Hill, North Carolina 27599-7365, USA.

    The regulators of G-protein signaling (RGS) proteins accelerate the intrinsic guanosine triphosphatase activity of heterotrimeric G-protein alpha subunits and are thus recognized as key modulators of G-protein-coupled receptor signaling. RGS12 and RGS14 contain not only the hallmark RGS box responsible for GTPase-accelerating activity but also a single G alpha(i/o)-Loco (GoLoco) motif predicted to represent a second G alpha interaction site. Here, we describe functional characterization of the GoLoco motif regions of RGS12 and RGS14. Both regions interact exclusively with G alpha(i1), G alpha(i2), and G alpha(i3) in their GDP-bound forms. In GTP gamma S binding assays, both regions exhibit guanine nucleotide dissociation inhibitor (GDI) activity, inhibiting the rate of exchange of GDP for GTP by G alpha(i1). Both regions also stabilize G alpha(i1) in its GDP-bound form, inhibiting the increase in intrinsic tryptophan fluorescence stimulated by AlF(4)(-). Our results indicate that both RGS12 and RGS14 harbor two distinctly different G alpha interaction sites: a previously recognized N-terminal RGS box possessing G alpha(i/o) GAP activity and a C-terminal GoLoco region exhibiting G alpha(i) GDI activity. The presence of two, independent G alpha interaction sites suggests that RGS12 and RGS14 participate in a complex coordination of G-protein signaling beyond simple G alpha GAP activity.

    Funded by: NCI NIH HHS: CA58689; NIDDK NIH HHS: DK07386, DK17780; NIGMS NIH HHS: GM07040, GM62338; NIMH NIH HHS: F30 MH064319, F30 MH064319-04

    The Journal of biological chemistry 2001;276;31;29275-81

  • 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

  • CB1 receptor-G protein association. Subtype selectivity is determined by distinct intracellular domains.

    Mukhopadhyay S and Howlett AC

    Department of Pharmacological and Physiological Science, Saint Louis University School of Medicine, St Louis, MO, USA.

    The CB1 cannabinoid receptor in N18TG2 neuroblastoma cells inhibits adenylate cyclase, and this response can be mimicked by a peptide corresponding to the juxtamembrane C-terminal domain (CB(1)401-417). Guanosine 5'-O-(3-thio)triphosphate binding to G proteins can be stimulated by both peptide CB(1)401-417 and peptides corresponding to the third intracellular loop [Howlett, A.C., Song, C., Berglund, B.A., Wilken, G.H. & Pigg, J.J. (1998) Mol. Pharmacol. 53, 504-510; Mukhopadhyay, S., Cowsik, S.M., Welsh, W.J. & Howlett, A.C. (1999) Biochemistry 38, 3447-3455]. In Chaps-solubilized N18TG2 membranes, the CB1 receptor coimmunoprecipitated with all three Gi subtypes. Pertussis toxin significantly reduced the CB(1) receptor-G alpha(i) association and attenuated the CB(1)401-417-induced inhibition of adenylate cyclase. CB(1)401-417 significantly reduced the CB(1) receptor association with G alpha(i3), but not with G alpha(i1) or G alpha(i2). In contrast, third intracellular loop peptides significantly reduced the CB(1) receptor association with G alpha(i1) and G alpha(i2), but not G alpha(i3). These interactions are specific for the CB(1) receptor because a peptide corresponding to the juxtamembrane C-terminal domain of the CB(2) receptor failed to compete for the association of the CB1 receptor with any of the Gi alpha subtypes, and was not able to activate Gi proteins to inhibit adenylate cyclase. These studies indicate that different domains of the CB(1) receptor direct the interaction with specific G protein subtypes.

    Funded by: NIDA NIH HHS: K05-DA00182, R01 DA003690, R01-DA03690

    European journal of biochemistry 2001;268;3;499-505

  • Identification of the platelet ADP receptor targeted by antithrombotic drugs.

    Hollopeter G, Jantzen HM, Vincent D, Li G, England L, Ramakrishnan V, Yang RB, Nurden P, Nurden A, Julius D and Conley PB

    Department of Cellular and Molecular Pharmacology, University of California, San Francisco 94143, USA.

    Platelets have a crucial role in the maintenance of normal haemostasis, and perturbations of this system can lead to pathological thrombus formation and vascular occlusion, resulting in stroke, myocardial infarction and unstable angina. ADP released from damaged vessels and red blood cells induces platelet aggregation through activation of the integrin GPIIb-IIIa and subsequent binding of fibrinogen. ADP is also secreted from platelets on activation, providing positive feedback that potentiates the actions of many platelet activators. ADP mediates platelet aggregation through its action on two G-protein-coupled receptor subtypes. The P2Y1 receptor couples to Gq and mobilizes intracellular calcium ions to mediate platelet shape change and aggregation. The second ADP receptor required for aggregation (variously called P2Y(ADP), P2Y(AC), P2Ycyc or P2T(AC)) is coupled to the inhibition of adenylyl cyclase through Gi. The molecular identity of the Gi-linked receptor is still elusive, even though it is the target of efficacious antithrombotic agents, such as ticlopidine and clopidogrel and AR-C66096 (ref. 9). Here we describe the cloning of this receptor, designated P2Y12, and provide evidence that a patient with a bleeding disorder has a defect in this gene. Cloning of the P2Y12 receptor should facilitate the development of better antiplatelet agents to treat cardiovascular diseases.

    Nature 2001;409;6817;202-7

  • Coupling of dopamine receptor subtypes to multiple and diverse G proteins.

    Sidhu A and Niznik HB

    Laboratory of Molecular Neurochemistry, Department of Pediatrics, Georgetown University Medical Center, Georgetown University, 3970 Reservoir Road, NW, Washington, DC 20007, USA. sidhua@odrge.odr.georgetown.edu

    The family of five dopamine receptors subtypes activate cellular effector systems through G proteins. Historically, dopamine receptors were thought to only stimulate or inhibit adenylyl cyclase, by coupling to either G(s)alpha or G(i)alpha, respectively. Recent studies in transfected cells, reviewed here, have shown that multiple and highly diverse signaling pathways are activated by specific dopamine receptor subtypes. This multiplicity of signaling responses occurs through selective coupling to distinct G proteins and each of the receptors can interact with more than one G protein. Although some of the multiple coupling of dopamine receptors to different G proteins occurs from within the same family of G proteins, these receptors can also couple to G proteins belonging to different families. Such multiple interactions between receptors and G proteins elicits functionally distinct physiological effects which acts to enhance and subsequently suppress the original receptor response, and to activate apparently distinct signaling pathways. In the brain, where coexpression of functionally distinct receptors in heterogeneous cells further adds to the complexity of dopamine signaling, minor alterations in receptor/G protein coupling states during either development or in adults, may underlie the imbalanced signaling seen in dopaminergic-linked diseases such as schizophrenia, Parkinson's disease and attention deficit hyperactivity disorder.

    Funded by: NINDS NIH HHS: NS-29685, NS-34914

    International journal of developmental neuroscience : the official journal of the International Society for Developmental Neuroscience 2000;18;7;669-77

  • Functional reconstitution of the angiotensin II type 2 receptor and G(i) activation.

    Hansen JL, Servant G, Baranski TJ, Fujita T, Iiri T and Sheikh SP

    Laboratory for Molecular Cardiology and the Department of Medicine B, University of Copenhagen, Denmark.

    On the basis of the patterns of conserved amino acid sequence, the angiotensin II type 2 (AT(2)) receptor belongs to the family of serpentine receptors, which relay signals from extracellular stimuli to heterotrimeric G proteins. However, the AT(2) receptor signal transduction mechanisms are poorly understood. We have measured AT(2)-triggered activation of purified heterotrimeric proteins in urea-extracted membranes from cultured COS-7 cells expressing the recombinant receptor. This procedure removes contaminating GTP-binding proteins without inactivating the serpentine receptor. Binding studies using [(125)I] angiotensin (Ang) II revealed a single binding site with a K(d)=0.45 and a capacity of 627 fmol/mg protein in the extracted membranes. The AT(2) receptor caused a rapid activation of alpha(i) and alpha(o) but not of alpha(q) and alpha(s), as measured by radioactive guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) binding. Activation required the presence of activated receptors, betagamma, and alpha subunits. As a first step aimed at developing an in vitro assay to examine AT(2) receptor pharmacology, we tested a battery of Ang II-related ligands for their ability to promote AT(1) or AT(2) receptor-catalyzed G(i) activation. Two proteolytic fragments of Ang II, Ang III and Ang1-7, also promoted activation of alpha(i) through the AT(2) receptor. Furthermore, we found that [Sar(1),Ala(8)]Ang II is an antagonist for both AT(1) and AT(2) receptors and that CPG42112 behaves as a partial agonist for the AT(2) receptor. In combination with previous observations, these results show that the AT(2) receptor is fully capable of activating G(i) and provides a new tool for exploring AT(2) receptor pharmacology and interactions with G-protein trimers.

    Circulation research 2000;87;9;753-9

  • Interaction of folate receptor with signaling molecules lyn and G(alpha)(i-3) in detergent-resistant complexes from the ovary carcinoma cell line IGROV1.

    Miotti S, Bagnoli M, Tomassetti A, Colnaghi MI and Canevari S

    Unit of Molecular Therapies, Department of Experimental Oncology, Istituto Nazionale per lo Studio e la Cura dei Tumori, Milan, Italy. miotti@istitutotumori.mi.it.

    Using as a model the ovary carcinoma cell line IGROV1, we analyzed the partitioning of the glycosyl-phosphatidylinositol-anchored folate receptor into lipid rafts based on its relative detergent insolubility, with a focus on physically and functionally associated signaling molecules. A variable amount (40-60%) of folate receptor was found in low-density Triton X-100 insoluble complexes together with subunits of heterotrimeric G-proteins and the src-family non-receptor tyrosine kinases p53-56 lyn. In the same fraction the structural component of caveolae, caveolin, was not detected at the protein level, although the corresponding mRNA was detected in trace amounts. Comodulation of folate receptor and signalling molecules was observed in the detergent-insoluble complexes during cell proliferation or induced by phosphatidylinositol-specific phospholipase C treatment or by interaction with anti-folate receptor monoclonal antibodies. Moreover, complexes of folate receptor, lyn and the G(&agr;)(i-3) subunit were immunoprecipitated using either anti-folate receptor or anti-lyn antibodies. In vitro kinase assay of the immunoprecipitates revealed stimulation of phosphorylation of common and specific proteins. In particular, the p53 form of lyn appeared to be enriched and phosphorylated in the anti-folate receptor MOv19 monoclonal antibody immunoprecipitate, whereas a 40 kDa band common to anti-folate receptor and anti-lyn immunoprecipitates was the phosphorylated form of the G(&agr;)(i-3) subunit. These findings point to the functional interaction between folate receptor and associated signaling molecules.

    Journal of cell science 2000;113 Pt 2;349-57

  • The G protein subunit gene families.

    Downes GB and Gautam N

    Department of Anesthesiology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.

    Genomics 1999;62;3;544-52

  • Dual signaling of human Mel1a melatonin receptors via G(i2), G(i3), and G(q/11) proteins.

    Brydon L, Roka F, Petit L, de Coppet P, Tissot M, Barrett P, Morgan PJ, Nanoff C, Strosberg AD and Jockers R

    CNRS-UPR 0415 and Université Paris VII, Institut Cochin de Génétique Moléculaire, Paris, France.

    Mel 1a melatonin receptors belong to the super-family of guanine nucleotide-binding regulatory protein (G protein)-coupled receptors. So far, interest in Mel 1a receptor signaling has focused mainly on the modulation of the adenylyl cyclase pathway via pertussis toxin (PTX)-sensitive G proteins. To further investigate signaling of the human Mel 1a receptor, we have developed an antibody directed against the C terminus of this receptor. This antibody detected the Mel 1a receptor as a protein with an apparent molecular mass of approximately 60 kDa in immunoblots after separation by SDS-PAGE. It also specifically precipitated the 2-[125I]iodomelatonin (125I-Mel)-labeled receptor from Mel 1a-transfected HEK 293 cells. Coprecipitation experiments showed that G(i2), G(i3), and G(q/11) proteins couple to the Mel 1a receptor in an agonist-dependent and guanine nucleotide-sensitive manner. Coupling was selective since other G proteins present in HEK 293 cells, (G(i1), G(o), G(s), G(z), and G12) were not detected in receptor complexes. Coupling of the Mel 1a receptor to G(i) and G(q) was confirmed by inhibition of high-affinity 125I-Mel binding to receptors with subtype-selective G protein alpha-subunit antibodies. G(i2) and/or G(i3) mediated adenylyl cyclase inhibition while G(q/11) induced a transient elevation in cytosolic calcium concentrations in HEK 293 cells stably expressing Mel 1a receptors. Melatonin-induced cytosolic calcium mobilization via PTX-insensitive G proteins was confirmed in primary cultures of ovine pars tuberalis cells endogenously expressing Mel 1a receptors. In conclusion, we report the development of the first antibody recognizing the cloned human Mel 1a melatonin receptor protein. We show that Mel 1a receptors functionally couple to both PTX-sensitive and PTX-insensitive G proteins. The previously unknown signaling of Mel 1a receptors through G(q/11) widens the spectrum of potential targets for melatonin.

    Molecular endocrinology (Baltimore, Md.) 1999;13;12;2025-38

  • Changes in expression of adenyl cyclase activity in human endometrium during hormone replacement therapy and ovarian stimulation.

    Bernardini L, Moretti-Rojas I, Brush M, Rojas FJ and Balmaceda JP

    Department of Obstetrics and Gynaecology, University of Genoa, Genoa, Italy.

    We have investigated membrane fractions prepared from human endometrium for activity of the signalling adenyl cyclase (AC). We characterized the AC guanine nucleotide-binding proteins (G proteins) and examined the changes in AC activity during evaluation cycles of oestrogen and progesterone replacement therapy as well during ovarian stimulation cycles. AC activity was determined by the conversion of substrate ATP into cyclic AMP under basal conditions and in the presence of guanine nucleotide or forskolin. G proteins were determined by Western Blot using specific polyclonal antibodies against Gsalpha, Gi1,2alpha and Gi3alpha. Our results indicate that endometrial AC was highly responsive to activation by both guanine nucleotide and forskolin and its rate of cyclic AMP production was highly pronounced. Mean activity reached 920 pmol/l/min/mg membrane protein in the presence of forskolin, a value approximately 5-fold higher than those detected in corpus luteum. Hormonal induction of AC activities increased Gsalpha protein, which couples with and stimulates the catalytic component of AC. We conclude that human endometrium is rich in AC and that enzyme activity is induced by oestrogen and progesterone treatment. These data strongly support the concept that the transmembrane signalling AC system and its messenger cyclic AMP are major regulators of endometrial function in the human.

    Molecular human reproduction 1999;5;10;955-60

  • 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

  • RGS7 and RGS8 differentially accelerate G protein-mediated modulation of K+ currents.

    Saitoh O, Kubo Y, Odagiri M, Ichikawa M, Yamagata K and Sekine T

    Department of Molecular and Cellular Neurobiology, Tokyo Metropolitan Institute for Neuroscience, 2-6 Musashidai, Fuchu-shi, Tokyo 183-8526, Japan. osaito@tmin.ac.jp

    The recently discovered family of RGS (regulators of G protein signaling) proteins acts as GTPase activating proteins which bind to alpha subunits of heterotrimeric G proteins. We previously showed that a brain-specific RGS, RGS8 speeds up the activation and deactivation kinetics of the G protein-coupled inward rectifier K+ channel (GIRK) upon receptor stimulation (Saitoh, O., Kubo, Y., Miyatani, Y., Asano, T., and Nakata, H. (1997) Nature 390, 525-529). Here we report the isolation of a full-length rat cDNA of another brain-specific RGS, RGS7. In situ hybridization study revealed that RGS7 mRNA is predominantly expressed in Golgi cells within granule cell layer of cerebellar cortex. We observed that RGS7 recombinant protein binds preferentially to Galphao, Galphai3, and Galphaz. When co-expressed with GIRK1/2 in Xenopus oocytes, RGS7 and RGS8 differentially accelerate G protein-mediated modulation of GIRK. RGS7 clearly accelerated activation of GIRK current similarly with RGS8 but the acceleration effect of deactivation was significantly weaker than that of RGS8. These acceleration properties of RGS proteins may play important roles in the rapid regulation of neuronal excitability and the cellular responses to short-lived stimulations.

    The Journal of biological chemistry 1999;274;14;9899-904

  • Regulators of G protein signaling exhibit distinct patterns of gene expression and target G protein specificity in human lymphocytes.

    Beadling C, Druey KM, Richter G, Kehrl JH and Smith KA

    Immunology Program, Cornell University Graduate School of Medical Sciences, Division of Immunology, Cornell University Medical College, New York, NY 10021, USA.

    The newly recognized regulators of G protein signaling (RGS) attenuate heterotrimeric G protein signaling pathways. We have cloned an IL-2-induced gene from human T cells, cytokine-responsive gene 1, which encodes a member of the RGS family, RGS16. The RGS16 protein binds Gialpha and Gqalpha proteins present in T cells, and inhibits Gi- and Gq-mediated signaling pathways. By comparison, the mitogen-induced RGS2 inhibits Gq but not Gi signaling. Moreover, the two RGS genes exhibit marked differences in expression patterns. The IL-2-induced expression of the RGS16 gene in T cells is suppressed by elevated cAMP, whereas the RGS2 gene shows a reciprocal pattern of regulation by these stimuli. Because the mitogen and cytokine receptors that trigger expression of RGS2 and RGS16 in T cells do not activate heterotrimeric G proteins, these RGS proteins and the G proteins that they regulate may play a heretofore unrecognized role in T cell functional responses to Ag and cytokine activation.

    Funded by: NIAID NIH HHS: R01-AI32031-22

    Journal of immunology (Baltimore, Md. : 1950) 1999;162;5;2677-82

  • RGS3 inhibits G protein-mediated signaling via translocation to the membrane and binding to Galpha11.

    Dulin NO, Sorokin A, Reed E, Elliott S, Kehrl JH and Dunn MJ

    Department of Medicine and Cardiovascular Research Center, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, USA.

    In the present study, we investigated the function and the mechanism of action of RGS3, a member of a family of proteins called regulators of G protein signaling (RGS). Polyclonal antibodies against RGS3 were produced and characterized. An 80-kDa protein was identified as RGS3 by immunoprecipitation and immunoblotting with anti-RGS3 antibodies in a human mesangial cell line (HMC) stably transfected with RGS3 cDNA. Coimmunoprecipitation experiments in RGS3-overexpressing cell lysates revealed that RGS3 bound to aluminum fluoride-activated Galpha11 and to a lesser extent to Galphai3 and that this binding was mediated by the RGS domain of RGS3. A role of RGS3 in postreceptor signaling was demonstrated by decreased calcium responses and mitogen-activated protein (MAP) kinase activity induced by endothelin-1 in HMC stably overexpressing RGS3. Moreover, depletion of endogenous RGS3 by transfection of antisense RGS3 cDNA in NIH 3T3 cells resulted in enhanced MAP kinase activation induced by endothelin-1. The study of intracellular distribution of RGS3 indicated its unique cytosolic localization. Activation of G proteins by AlF4-, NaF, or endothelin-1 resulted in redistribution of RGS3 from cytosol to the plasma membrane as determined by Western blotting of the cytosolic and particulate fractions with RGS3 antiserum as well as by immunofluorescence microscopy. Agonist-induced translocation of RGS3 occurred by a dual mechanism involving both C-terminal (RGS domain) and N-terminal regions of RGS3. Thus, coexpression of RGS3 with a constitutively active mutant of Galpha11 (Galpha11-QL) resulted in the binding of RGS3, but not of its N-terminal fragment, to the membrane fraction and in its interaction with Galpha11-QL in vitro without any stimuli. However, both full-length RGS3 and its N-terminal domain translocated to the plasma membrane upon stimulation of intact cells with endothelin-1 as assayed by immunofluorescence microscopy. The effect of endothelin-1 was also mimicked by calcium ionophore A23187, suggesting the importance of Ca2+ in the mechanism of redistribution of RGS3. These data indicate that RGS3 inhibits G protein-coupled receptor signaling by a complex mechanism involving its translocation to the membrane in addition to its established function as a GTPase-activating protein.

    Funded by: NHLBI NIH HHS: HL 22563, R01 HL022563; NIDDK NIH HHS: DK 41684, R01 DK041684

    Molecular and cellular biology 1999;19;1;714-23

  • 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

  • A single receptor encoded by vzg-1/lpA1/edg-2 couples to G proteins and mediates multiple cellular responses to lysophosphatidic acid.

    Fukushima N, Kimura Y and Chun J

    Department of Pharmacology, School of Medicine, University of California at San Diego, La Jolla, CA 92093-0636, USA.

    Extracellular lysophosphatidic acid (LPA) produces diverse cellular responses in many cell types. Recent reports of several molecularly distinct G protein-coupled receptors have raised the possibility that the responses to LPA stimulation could be mediated by the combination of several uni-functional receptors. To address this issue, we analyzed one receptor encoded by ventricular zone gene-1 (vzg-1) (also referred to as lpA1/edg-2) by using heterologous expression in a neuronal and nonneuronal cell line. VZG-1 expression was necessary and sufficient in mediating multiple effects of LPA: [3H]-LPA binding, G protein activation, stress fiber formation, neurite retraction, serum response element activation, and increased DNA synthesis. These results demonstrate that a single receptor, encoded by vzg-1, can activate multiple LPA-dependent responses in cells from distinct tissue lineages.

    Proceedings of the National Academy of Sciences of the United States of America 1998;95;11;6151-6

  • 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

  • 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

  • Leukotriene D4-induced activation and translocation of the G-protein alpha i3-subunit in human epithelial cells.

    Adolfsson JL, Ohd JF and Sjölander A

    Division of Experimental Pathology, Lund University, Wallenberg Laboratory, Malmö, Sweden.

    The present results show that stimulation of Intestine 407 epithelial cells with LTD4 (Leukotriene D4) triggers a rapid activation of the pertussis-toxin-sensitive Gi3-protein and a simultaneous translocation of its alpha-subunits to a crude cytoskeletal fraction. The activation of G alpha i3, which was measured as the GTP/ GDP exchange ratio, peaked about 15 s after the addition of LTD4. Western blot analyses of subcellular fractions showed that G alpha i3-subunits accumulated in the cytoskeletal fraction and decreased in the membrane fraction, and the decrease was most marked 15 s after the exposure to LTD4. None of the other pertussis-toxin-sensitive G-proteins (Gi3-Z and G alpha 0) were activated or translocated upon stimulation with LTD4. This agonist was also found to reduce the GTP/GDP exchange ratio of Gi-proteins without affecting the subcellular distribution of its alpha-subunits. These findings imply that the Gi3-protein is the pertussis-toxin-sensitive G-protein previously found to mediate several downstream LTD4-stimulated signalling events. Furthermore, the translocation of G alpha i3-subunits to the cytoskeleton and the simultaneous inhibition of G3-proteins indicate that the cytoskeleton might participate in the signalling process of human epithelial cells.

    Biochemical and biophysical research communications 1996;226;2;413-9

  • RGS10 is a selective activator of G alpha i GTPase activity.

    Hunt TW, Fields TA, Casey PJ and Peralta EG

    Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts 02138, USA.

    Polypeptides that define a protein family termed RGS (for regulators of G-protein signalling) are encoded by the SST2 gene of the yeast Saccharomyces cerevisiae, the EGL-10 gene of the nematode Caenorhabdatis elegans, and several related mammalian genes. Genetic studies in invertebrates and mammalian cell-transfection experiments indicate that RGS proteins negatively regulate signalling pathways involving seven transmembrane receptors and heterotrimeric G proteins. However, the biochemical mechanism by which RGS proteins control these pathways is unknown. Here we report the characterization of human RGS10, a member of this protein family. Co-immunoprecipitation studies demonstrate that RGS10 associates specifically with the activated forms of two related G-protein subunits, G alphai3, and G alphaz, but fails to interact with the structurally and functionally distinct G alphas subunit. In vitro assays with purified proteins indicate that RGS10 increases potently and selectively the GTP hydrolytic activity of several members of the G alphai family, including G alphai3, G alphaz, and G alpha0. These results demonstrate that RGS proteins can attenuate signalling pathways involving heterotrimeric G proteins by serving as GTPase-activating proteins for specific types of G alpha subunits.

    Nature 1996;383;6596;175-7

  • The AT2 receptor selectively associates with Gialpha2 and Gialpha3 in the rat fetus.

    Zhang J and Pratt RE

    Division of Cardiovascular Medicine, Falk Cardiovascular Research Center, Stanford University School of Medicine, Stanford, California 94305-5246, USA.

    The effects of angiotensin II are mediated by a family of seven transmembrane receptors. In the adult, the majority of the receptors are of the AT1 isoform, which is coupled to heterotrimeric G proteins (either Gqalpha or Gialpha). In contrast, the AT2 receptor is expressed at low 145f levels in the adult but is the major form expressed in the fetal and neonatal animal. Previous results have failed to show G protein coupling of the AT2 receptor in the fetus. We now provide evidence that the AT2 receptor is G protein-coupled. An antibody that binds several Galpha subunits immunoselected angiotensin II receptor-Galpha complexes. In addition, Gialpha1-3 antibody, which recognizes Gialpha1, Gialpha2 and Gialpha3, also co-immunoselect the AT2 receptor. Anti-Gialpha2 and anti-Gialpha3 antibodies were both able to co-immunoselected AT2 receptor-Gialpha complexes, but consistent with the lack of Gialpha1 in the fetal extracts, anti-Gialpha1 antibodies did not nor did any other G protein-directed antisera. The finding that AT2 receptor couples to both Gialpha2 and Gialpha3 raises the possibility that selective interactions between AT2 receptor and different G proteins may result in specific cellular effects mediated by AT2 stimulation.

    Funded by: NHLBI NIH HHS: HL 42663, HL 48638

    The Journal of biological chemistry 1996;271;25;15026-33

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

    Solomon KR, Rudd CE and Finberg RW

    Laboratory of Infectious Diseases, Dana-Farber Cancer Institute, Boston, MA 02115, USA.

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

    Funded by: NIAID NIH HHS: 2 R01-AI31628-04

    Proceedings of the National Academy of Sciences of the United States of America 1996;93;12;6053-8

  • The inducible G protein-coupled receptor edg-1 signals via the G(i)/mitogen-activated protein kinase pathway.

    Lee MJ, Evans M and Hla T

    Department of Molecular Biology, Holland Laboratory, American Red Cross, Rockville, Maryland 20855, USA.

    The edg-1 gene encodes an inducible G protein-coupled receptor (GPR) homologue that is induced during the in vitro differentiation of human endothelial cells. The aim of this study was to investigate the G protein-coupling and -signaling properties of the edg-1 polypeptide. The third cytosolic loop (i3) of edg-1 associates with G(i) alpha and G(o) alpha polypeptides in a guanosine 5'-O-(thiotriphosphate)-sensitive manner. Immunoprecipitation of the edg-1 polypeptide in transfected cells results in the co-precipitation of G(i) alpha 1 and G(i) alpha 3 polypeptides. These data strongly suggest that edg-1 is capable of coupling to the Gi pathway. Overexpression of the edg-1 GPR in human embryonic kidney 293 cells results in the sustained activation of the MAP kinase activity that is blocked by pertussis toxin treatment. Moreover, NIH3T3 cells permanently transfected with edg-1 exhibit enhanced MAP kinase and phospholipase A2 activities. These data suggest that the G(i)/mitogen-activated protein kinase pathway is a major signaling pathway regulated by the orphan receptor edg-1.

    Funded by: NHLBI NIH HHS: HL 49094; NIDDK NIH HHS: DK 45659

    The Journal of biological chemistry 1996;271;19;11272-9

  • 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

  • GAIP, a protein that specifically interacts with the trimeric G protein G alpha i3, is a member of a protein family with a highly conserved core domain.

    De Vries L, Mousli M, Wurmser A and Farquhar MG

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

    Using the yeast two-hybrid system we have identified a human protein, GAIP (G Alpha Interacting Protein), that specifically interacts with the heterotrimeric GTP-binding protein G alpha i3. Interaction was verified by specific binding of in vitro-translated G alpha i3 with a GAIP-glutathione S-transferase fusion protein. GAIP is a small protein (217 amino acids, 24 kDa) that contains two potential phosphorylation sites for protein kinase C and seven for casein kinase 2. GAIP shows high homology to two previously identified human proteins, GOS8 and 1R20, two Caenorhabditis elegans proteins, CO5B5.7 and C29H12.3, and the FLBA gene product in Aspergillus nidulans--all of unknown function. Significant homology was also found to the SST2 gene product in Saccharomyces cerevisiae that is known to interact with a yeast G alpha subunit (Gpa1). A highly conserved core domain of 125 amino acids characterizes this family of proteins. Analysis of deletion mutants demonstrated that the core domain is the site of GAIP's interaction with G alpha i3. GAIP is likely to be an early inducible phosphoprotein, as its cDNA contains the TTTTGT sequence characteristic of early response genes in its 3'-untranslated region. By Northern analysis GAIP's 1.6-kb mRNA is most abundant in lung, heart, placenta, and liver and is very low in brain, skeletal muscle, pancreas, and kidney. GAIP appears to interact exclusively with G alpha i3, as it did not interact with G alpha i2 and G alpha q. The fact that GAIP and Sst2 interact with G alpha subunits and share a common domain suggests that other members of the GAIP family also interact with G alpha subunits through the 125-amino-acid core domain.

    Funded by: NCI NIH HHS: CA58689; NIDDK NIH HHS: DK17780

    Proceedings of the National Academy of Sciences of the United States of America 1995;92;25;11916-20

  • Differential coupling of m2 and m4 muscarinic receptors to inhibition of adenylyl cyclase by Gi alpha and G(o)alpha subunits.

    Migeon JC, Thomas SL and Nathanson NM

    Department of Pharmacology, University of Washington, Seattle 98195-7750, USA.

    We compared the G-protein requirements for coupling of human and chicken m2 and m4 muscarinic acetylcholine receptors (mAChRs) to inhibition of adenylyl cyclase, using a luciferase reporter gene under the transcriptional control of a cAMP response element as a sensitive monitor of intracellular cAMP levels. Previously, we used this system to demonstrate that the chick m4 receptor preferentially coupled to Gi alpha-2 and G(o)alpha over Gi alpha-1 and Gi alpha-3. We found that both the chick and human m2 mAChRs can couple to Gi alpha-1, Gi alpha-2, Gi alpha-3, and G(o)alpha, while the human m4 mAChR preferentially couples to Gi alpha-2 and G(o)alpha. Both the G(o)1 and G(o)2 forms of the G(o)alpha subunit were effective in reconstituting coupling of the m2 and m4 mAChRs to inhibit adenylyl cyclase activity. The m2 and m4 mAChRs thus couple to inhibition of adenylyl cyclase by overlapping but different sets of G-protein alpha subunits.

    Funded by: NIGMS NIH HHS: GM07108, GM07750; NINDS NIH HHS: NS07332

    The Journal of biological chemistry 1995;270;27;16070-4

  • Stabilization of C5a receptor--G-protein interactions through ligand binding.

    Wennogle LP, Conder L, Winter C, Braunwalder A, Vlattas S, Kramer R, Cioffi C and Hu SI

    Research Department, CIBA-GEIGY Pharmaceuticals Division, Summit, New Jersey 07901.

    Binding of biotin-C5a to the C5a receptor in membrane fragments followed by detergent solubilization and purification with streptavidin-agarose affinity chromatography resulted in the isolation of a receptor complex with associated G-proteins. In contrast, when receptor was detergent-solubilized in the absence of C5a and purified by affinity chromatography with Affigel-C5a, G-proteins did not copurify. Since the results indicate that receptor ligation stabilized the receptor--G-protein interaction to allow purification of the complex, the findings emphasize the dynamic nature of the C5a receptor-effector interactions. When biotin-C5a-ligated receptor was purified from a mouse cell line overexpressing recombinant human receptor, both Gialpha2 and Gialpha3 subunits copurified, confirming that multiple transducing systems are linked to the C5a receptor. The method of stabilization of receptor-transducer complexes offers the opportunity to further elaborate the interactions of the C5a receptor with diverse transducing elements and second messenger systems.

    Journal of cellular biochemistry 1994;55;3;380-8

  • 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

  • Cell-specific physical and functional coupling of human 5-HT1A receptors to inhibitory G protein alpha-subunits and lack of coupling to Gs alpha.

    Raymond JR, Olsen CL and Gettys TW

    Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710.

    We have studied the physical and functional linkages of heterologously expressed human 5-HT1A receptors to G protein alpha-subunits in HeLa and CHO-K1 cells. HeLa cells expressed immunoreactivity to G(i) proteins with an apparent rank order of G(i) alpha 3 (approximately 1 pmol/mg of protein) > G(i) alpha 1 (approximately 0.1 pmol/mg) > G(i) alpha 2 (< 0.02 pmol/mg), whereas CHO-K1 cells expressed immunoreactivity to G(i) alpha 2 (approximately 5 pmol/mg) > G(i) alpha 3 (approximately 0.7 pmol/mg), but not to G(i) alpha 1. Both cell lines expressed large and small forms of Gs alpha, but neither expressed detectable G(o) alpha. Agonist-promotable physical coupling of the 5-HT1A receptor to G proteins was examined with high-affinity agonist binding and with co-immunoprecipitation using rabbit anti-receptor IgG fractions. Agonist treatment induced coupling of the 5-HT1A receptors to G proteins with an apparent rank order of G(i) alpha 3 > G(i) alpha 1, G(i) alpha 2 in HeLa cells and G(i) alpha 3 > G(i) alpha 2 in CHO-K1 cells. Agonist-promotable functional coupling of the 5-HT1A receptors to inhibition of adenylylcyclase was measured in membranes derived from HeLa and CHO-K1 cells expressing approximately 2.5-3 pmol of receptors/mg of protein by preincubation with antisera raised against the carboxyl termini of the G(i) protein alpha-subunits. A noteworthy difference between the two cell types was that antisera against the predominant G protein (G(i) alpha 2) were substantially more efficacious than G(i) alpha 3 antisera at blocking functional coupling to adenylylcyclase inhibition in CHO-K1 cells, whereas in HeLa cells, antisera against nonpredominant G proteins (G(i) alpha 1/G(i) alpha 2) were equally as effective as those against the predominant G protein (G(i) alpha 3). No physical or functional coupling of the 5-HT1A receptor to Gs alpha isoforms was detected in either cell line. These findings suggest that the 5-HT1A receptor can physically couple to multiple distinct G(i) proteins in mammalian cell membranes and that functional coupling to adenylylcyclase inhibition may be mediated by G(i) alpha 1, G(i) alpha 2, and G(i) alpha 3. One factor influencing the relative importance of those G proteins for 5-HT1A receptor-inhibited adenylylcyclase activity appears to be their-relative levels of expression.(ABSTRACT TRUNCATED AT 250 WORDS)

    Funded by: NIDDK NIH HHS: DK42486; NINDS NIH HHS: NS30927

    Biochemistry 1993;32;41;11064-73

  • G protein-coupled signal transduction pathways for interleukin-8.

    Wu D, LaRosa GJ and Simon MI

    Division of Biology, California Institute of Technology, Pasadena 91125.

    Interleukin-8 (IL-8) is one of the major mediators of the inflammatory response. The pathways by which IL-8 activates inositide-specific phospholipase C (PLC) were investigated by co-expression of different components of the guanosine triphosphate binding protein (G protein) pathway in COS-7 cells. Two distinct IL-8 receptors reconstituted ligand-dependent activation of endogenous PLC when transfected together with the G protein alpha subunits G alpha 14, G alpha 15, or G alpha 16. However, reconstitution was not observed with cells that overexpressed G alpha q or G alpha 11. Furthermore, IL-8 receptors interacted with endogenous pertussis toxin-sensitive G proteins or with the recombinant G protein Gi to release free beta gamma subunits that could then specifically activate the beta 2 isoform of PLC. These findings suggest that IL-8 acts through signal-transducing pathways that are limited to specific heterotrimeric G proteins and effectors. These may provide suitable targets for the development of anti-inflammatory agents.

    Science (New York, N.Y.) 1993;261;5117;101-3

  • Identification and expression of G-proteins in human myometrium: up-regulation of G alpha s in pregnancy.

    Europe-Finner GN, Phaneuf S, Watson SP and López Bernal A

    University of Oxford, Nuffield Department of Obstetrics and Gynecology, John Radcliffe Hospital, Headington, United Kingdom.

    We report that human myometrium contains G alpha i1, G alpha i3, and G alpha q, and G alpha 11, which are expressed at similar levels in tissues from pregnant and nonpregnant women. G alpha i2 is also expressed, but at a slightly reduced level, in tissue taken from pregnant compared to nonpregnant donors. The major finding of this investigation is the substantial increase in G alpha s expression in pregnant myometrium. The increase in G alpha s levels may play a crucial role in maintaining relaxation of the uterus by favoring cAMP formation during pregnancy.

    Endocrinology 1993;132;6;2484-90

  • Guanine nucleotide-binding regulatory proteins in retinal pigment epithelial cells.

    Jiang M, Pandey S, Tran VT and Fong HK

    Department of Microbiology, University of Southern California School of Medicine, Los Angeles 90033.

    The expression of GTP-binding regulatory proteins (G proteins) in retinal pigment epithelial (RPE) cells was analyzed by RNA blot hybridization and cDNA amplification. Both adult and fetal human RPE cells contain mRNA for multiple G protein alpha subunits (G alpha) including Gs alpha, Gi-1 alpha, Gi-2 alpha, Gi-3 alpha, and Gz alpha (or Gx alpha), where Gs and Gi are proteins that stimulate or inhibit adenylyl cyclase, respectively, and Gz is a protein that may mediate pertussis toxin-insensitive events. Other G alpha-related mRNA transcripts were detected in fetal RPE cells by low-stringency hybridization to Gi-2 alpha and Gs alpha protein-coding cDNA probes. The diversity of G proteins in RPE cells was further studied by cDNA amplification with reverse transcriptase and the polymerase chain reaction. This approach revealed that, besides the above mentioned members of the G alpha gene family, at least two other G alpha subunits are expressed in RPE cells. Human retinal cDNA clones that encode one of the additional G alpha subunits were isolated and characterized. The results indicate that this G alpha subunit belongs to a separate subfamily of G proteins that may be insensitive to inhibition by pertussis toxin.

    Funded by: NEI NIH HHS: EY08364

    Proceedings of the National Academy of Sciences of the United States of America 1991;88;9;3907-11

  • Distinct guanine nucleotide binding and release properties of the three Gi proteins.

    Carty DJ, Padrell E, Codina J, Birnbaumer L, Hildebrandt JD and Iyengar R

    Department of Pharmacology, Mount Sinai School of Medicine, City University, New York, New York 10029.

    The native pertussis toxin sensitive GTP-binding proteins (Gi proteins) were individually resolved, and their guanine nucleotide binding and release properties were studied. Gi2 and Gi3, the two major GTP-binding proteins of human erythrocytes, were purified to apparent homogeneity by fast protein liquid chromatography. Gi1 was purified from bovine brain. The three proteins bound 0.6-0.85 mol of guanosine 5'-O-(thio-triphosphate (GTP gamma S)/mol of protein with similar affinities (KD(app) = 50-100 nM). The rate of [35S]GTP gamma S binding to Gi2 was 5-8-fold faster than to Gi1 or Gi3 at 2 mm Mg2+. There were no observable differences in the binding characteristics between bovine brain Gi1 and human erythrocyte Gi3. At 50 mM Mg2+, all three Gi proteins exhibited fast binding, although Gi1 and Gi3 were marginally slower than Gi2. All three Gi proteins exhibited different rates of [32P]GDP release at 2 mM Mg2+. GDP release from Gi2 was severalfold faster than that from Gi1 or Gi3. GDP release rates from Gi1 and Gi3 were similar, although Gi3 was somewhat (60-80%) faster than Gi1. These data indicate that rates of GDP release and GTP binding may be independently regulated for these three proteins and that the relative proportions of Gi2/Gi1 or Gi2/Gi3 will be a crucial factor in determining the kinetics of signal transduction through Gi-coupled effectors.

    Funded by: NCI NIH HHS: CA 44998; NIDDK NIH HHS: DK 19318, DK 38761; ...

    The Journal of biological chemistry 1990;265;11;6268-73

  • Identification of cDNA encoding an additional alpha subunit of a human GTP-binding protein: expression of three alpha i subtypes in human tissues and cell lines.

    Kim SY, Ang SL, Bloch DB, Bloch KD, Kawahara Y, Tolman C, Lee R, Seidman JG and Neer EJ

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

    The guanine nucleotide-binding proteins (G proteins), which mediate hormonal regulation of many membrane functions, are composed of alpha, beta, and gamma subunits. We have cloned and characterized cDNA from a human T-cell library encoding a form of alpha i that is different from the human alpha i subtypes previously reported [Didsbury, J. R., Ho, Y.-S. & Snyderman, R. (1987) FEBS Lett. 211, 160-164 and Bray, P., Carter, A., Guo, V., Puckett, C., Kamholz, J., Spiegel, A. & Nirenberg, M. (1987) Proc. Natl. Acad. Sci. USA 84, 5115-5119]. alpha i is the alpha subunit of a class of G proteins that inhibits adenylate cyclase and regulates other enzymes and ion channels. This cDNA encodes a polypeptide of 354 amino acids and is assigned to encode the alpha i-3 subtype of G proteins on the basis of its similarity to other alpha i-like cDNAs and the presence of a predicted site for ADP ribosylation by pertussis toxin. We have determined the expression of mRNA for this and two other subtypes of human alpha i (alpha i-1 and alpha i-2) in a variety of human fetal tissues and in human cell lines. All three alpha i subtypes were present in the tissues tested. However, analysis of individual cell types reveals specificity of alpha i-1 expression. mRNA for alpha i-1 is absent in T cells, B cells, and monocytes but is present in other cell lines. The finding of differential expression of alpha i-1 genes may permit characterization of distinct physiological roles for this alpha i subunit. mRNA for alpha i-2 and alpha i-3 was found in all the primary and transformed cell lines tested. Thus, some cells contain all three alpha i subtypes. This observation raises the question of how cells prevent cross talk among receptors that are coupled to effectors through such similar alpha proteins.

    Funded by: NHLBI NIH HHS: HL01835; NIAID NIH HHS: AI 18436, AI 19148

    Proceedings of the National Academy of Sciences of the United States of America 1988;85;12;4153-7

  • Alpha i-3 cDNA encodes the alpha subunit of Gk, the stimulatory G protein of receptor-regulated K+ channels.

    Codina J, Olate J, Abramowitz J, Mattera R, Cook RG and Birnbaumer L

    Department of Cell Biology, Baylor College of Medicine, Houston, Texas 77030.

    cDNA cloning has identified the presence in the human genome of three genes encoding alpha subunits of pertussis toxin substrates, generically called "Gi." They are named alpha i-1, alpha i-2 and alpha i-3. However, none of these genes has been functionally identified with any of the alpha subunits of several possible G proteins, including pertussis toxin-sensitive Gp's, stimulatory to phospholipase C or A2, Gi, inhibitory to adenylyl cyclase, or Gk, stimulatory to a type of K+ channels. We now report the nucleotide sequence and the complete predicted amino acid sequence of human liver alpha i-3 and the partial amino acid sequence of proteolytic fragments of the alpha subunit of human erythrocyte Gk. The amino acid sequence of the proteolytic fragment is uniquely encoded by the cDNA of alpha i-3, thus identifying it as alpha k. The probable identity of alpha i-1 with alpha p and possible roles for alpha i-2, as well as additional roles for alpha i-1 and alpha i-3 (alpha k) are discussed.

    Funded by: NHLBI NIH HHS: HL-31163; NIDDK NIH HHS: DK-19318, DK-26785

    The Journal of biological chemistry 1988;263;14;6746-50

  • Presence of three distinct molecular species of Gi protein alpha subunit. Structure of rat cDNAs and human genomic DNAs.

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

    Institute of Medical Science, University of Tokyo, Japan.

    We have cloned a new species of rat Gi alpha (Gi3 alpha) cDNA and genomic DNAs for three distinct human Gi alpha proteins (Gi1 alpha, Gi2 alpha, and Gi3 alpha). Gi3 alpha cDNA codes for a protein of 354 amino acids (Mr 40,522) whose sequence is closely related but distinct from that of the previously isolated rat Gi alpha (Gi2 alpha). By screening the human genomic libraries with the two rat Gi alpha cDNAs as probes, clones encoding human Gi1 alpha, Gi2 alpha, and Gi3 alpha were isolated. The human Gi2 alpha and Gi3 alpha genes are composed of eight coding exons and seven introns and possess a completely identical exon-intron organization. Southern blot analysis indicates that a single copy of each Gi alpha gene is present per haploid human genome.

    The Journal of biological chemistry 1988;263;14;6656-64

  • A small multigene family encodes Gi signal-transduction proteins.

    Beals CR, Wilson CB and Perlmutter RM

    Howard Hughes Medical Institute, University of Washington, Seattle 98195.

    The guanine nucleotide-binding regulatory proteins known as G proteins are receptor-associated signal-transduction molecules that are implicated in the control of a variety of metabolic processes. Recent evidence suggests that G proteins may mediate B-lymphocyte responses to bacterial lipopolysaccharide and may also transduce signals from the T-cell antigen receptor. Since these receptors are uniquely expressed on lymphoid cells, we used molecular cloning strategies to ask whether lymphocytes contain specialized G-protein alpha subunits to assist in signal transduction. Comparison of our two deduced human alpha i amino acid sequences with those previously determined for bovine and rodent G proteins permits the identification of three closely related but distinct types of alpha i molecules that comprise a small multigene family. Using gene-specific probes, we found that both of our alpha i genes are expressed in most cell types but in differing ratios. Our data support the view that a modest repertoire of extremely closely related G proteins mediates the transduction of signals derived from multiple different receptor molecules.

    Funded by: NIGMS NIH HHS: GM07266

    Proceedings of the National Academy of Sciences of the United States of America 1987;84;22;7886-90

  • The human genome encodes at least three non-allellic G proteins with alpha i-type subunits.

    Suki WN, Abramowitz J, Mattera R, Codina J and Birnbaumer L

    The amino acid sequence and composition of alpha-subunits of signal transducing G proteins of the same kind appear to vary by no more than 2% from species to species. Here we isolated a human liver cDNA using an oligonucleotide complementary to the sequences encoding the pertussis toxin (PTX) ADP-ribosylation site of the alpha-subunit of the rat brain G protein called Gi. Its open reading frame characterizes it as an alpha i-type cDNA--as opposed to alpha o-type--but predicts an amino acid composition that differs by 7% and 14%, respectively, from two other human alpha i-type molecules. Together with human brain alpha i (type-1) and human monocyte alpha i (type-2), the new human liver alpha i cDNA (type-3) forms parts of a family of alpha i molecules. Type-3 alpha i cDNA hybridizes to a approximately 3.6 kilobase long mRNA and type-2 alpha i cDNA hybridizes to an mRNA species of approximately 2.7 kilobases. This indicates that the human genome has at least three non-allellic genes encoding non-alpha o-type PTX substrates and provides structural evidence for the hypothesis that distinct effector systems are regulated by similar but nevertheless distinct PTX substrates.

    Funded by: NHLBI NIH HHS: HL-31164; NICHD NIH HHS: HD-09581; NIDDK NIH HHS: DK-19318; ...

    FEBS letters 1987;220;1;187-92

  • Molecular cloning of a new human G protein. Evidence for two Gi alpha-like protein families.

    Didsbury JR and Snyderman R

    The amino acid sequence of a novel G protein alpha subunit (Gx alpha) has been deduced from the nucleotide sequence of a human cDNA clone isolated from a differentiated HL-60 cDNA library. The cDNA encodes a polypeptide of 354 amino acids (Mr 40,519) which is closely related to Gi alpha proteins. The amino acid sequence homology between Gx alpha and human myeloid Gi alpha is 86% with 15 nonconservative substitutions. Gx alpha also shares 86% homology with both rat brain and mouse macrophage Gi alpha but is more homologous (94%) to bovine brain Gi alpha with only 5 nonconservative amino acid differences. G proteins previously termed Gi alpha may fall into at least two distinct groups, with one including human myeloid Gi alpha, rat brain Gi alpha and mouse macrophage Gi alpha; and other Gx alpha and bovine brain Gi alpha. One group probably contains true Gi and the other a new class of G protein whose function remains to be determined.

    Funded by: NCI NIH HHS: CA29589; NCRR NIH HHS: 1 U41 RR-01685-03; NIDCR NIH HHS: DE03738

    FEBS letters 1987;219;1;259-63

Gene lists (3)

Gene List Source Species Name Description Gene count
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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