G2Cdb::Gene report

Gene id
Gene symbol
Homo sapiens
guanine nucleotide binding protein (G protein), gamma 2
G00001092 (Mus musculus)

Databases (9)

Curated Gene
OTTHUMG00000029633 (Vega human gene)
ENSG00000186469 (Ensembl human gene)
54331 (Entrez Gene)
361 (G2Cdb plasticity & disease)
GNG2 (GeneCards)
606981 (OMIM)
Marker Symbol
HGNC:4404 (HGNC)
Protein Expression
3534 (human protein atlas)
Protein Sequence
P59768 (UniProt)

Literature (45)

Pubmed - other

  • 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

  • Toward a confocal subcellular atlas of the human proteome.

    Barbe L, Lundberg E, Oksvold P, Stenius A, Lewin E, Björling E, Asplund A, Pontén F, Brismar H, Uhlén M and Andersson-Svahn H

    Department of Biotechnology, AlbaNova University Center, Royal Institute of Technology, SE-106 91 Stockholm, Sweden.

    Information on protein localization on the subcellular level is important to map and characterize the proteome and to better understand cellular functions of proteins. Here we report on a pilot study of 466 proteins in three human cell lines aimed to allow large scale confocal microscopy analysis using protein-specific antibodies. Approximately 3000 high resolution images were generated, and more than 80% of the analyzed proteins could be classified in one or multiple subcellular compartment(s). The localizations of the proteins showed, in many cases, good agreement with the Gene Ontology localization prediction model. This is the first large scale antibody-based study to localize proteins into subcellular compartments using antibodies and confocal microscopy. The results suggest that this approach might be a valuable tool in conjunction with predictive models for protein localization.

    Molecular & cellular proteomics : MCP 2008;7;3;499-508

  • Heterotrimeric G protein betagamma subunits stimulate FLJ00018, a guanine nucleotide exchange factor for Rac1 and Cdc42.

    Ueda H, Nagae R, Kozawa M, Morishita R, Kimura S, Nagase T, Ohara O, Yoshida S and Asano T

    Department of Biomolecular Science, Faculty of Engineering, Gifu University, Yanagido 1-1, Gifu 501-1193, Japan. hueda@gifu-u.ac.jp

    We previously reported that Gbetagamma signaling regulates cell spreading or cell shape change through activation of a Rho family small GTPase, suggesting the existence of a Gbetagamma-regulated Rho guanine-nucleotide exchange factor (RhoGEF). In this study we examined various RhoGEF clones, found FLJ00018 to beaGbetagamma-activated RhoGEF, and investigated the molecular mechanism of Gbetagamma-induced activation of Rho family GTPases. Co-expression of the genes for FLJ00018 and Gbetagamma enhanced serum response element-mediated gene transcription in HEK-293 cells. Combined expression of Gbetagamma and FLJ00018 significantly induced activation of Rac and Cdc42 but not RhoA. FLJ00018 also enhanced gene transcription induced by carbachol-stimulated m2 muscarinic acetylcholine receptor, and this enhancement was blocked by pertussis toxin. Furthermore, we demonstrated Gbetagamma to interact directly with the N-terminal region of FLJ00018 and the N-terminal fragment of this molecule to inhibit serum response element-dependent transcription induced by Gbetagamma/FLJ00018 and carbachol. In NIH3T3 cells, FLJ00018 enhanced lysophosphatidic acid-induced cell spreading, which was also blocked by the N-terminal fragment of FLJ00018. These results provide evidence for a signaling pathway by which G(i)-coupled receptor specifically induces Rac and Cdc42 activation through direct interaction of Gbetagamma with FLJ00018.

    The Journal of biological chemistry 2008;283;4;1946-53

  • Phospholipase C beta3 is a key component in the Gbetagamma/PKCeta/PKD-mediated regulation of trans-Golgi network to plasma membrane transport.

    Díaz Añel AM

    Center for Molecular Genetics, University of California at San Diego, San Diego, CA, USA. adiazanel@immf.uncor.edu

    The requirement of DAG (diacylglycerol) to recruit PKD (protein kinase D) to the TGN (trans-Golgi network) for the targeting of transport carriers to the cell surface, has led us to a search for new components involved in this regulatory pathway. Previous findings reveal that the heterotrimeric Gbetagamma (GTP-binding protein betagamma subunits) act as PKD activators, leading to fission of transport vesicles at the TGN. We have recently shown that PKCeta (protein kinase Ceta) functions as an intermediate member in the vesicle generating pathway. DAG is capable of activating this kinase at the TGN, and at the same time is able to recruit PKD to this organelle in order to interact with PKCeta, allowing phosphorylation of PKD's activation loop. The most qualified candidates for the production of DAG at the TGN are PI-PLCs (phosphatidylinositol-specific phospholipases C), since some members of this family can be directly activated by Gbetagamma, utilizing PtdIns(4,5)P2 as a substrate, to produce the second messengers DAG and InsP3. In the present study we show that betagamma-dependent Golgi fragmentation, PKD1 activation and TGN to plasma membrane transport were affected by a specific PI-PLC inhibitor, U73122 [1-(6-{[17-3-methoxyestra-1,3,5(10)-trien-17-yl]amino}hexyl)-1H-pyrrole-2,5-dione]. In addition, a recently described PI-PLC activator, m-3M3FBS [2,4,6-trimethyl-N-(m-3-trifluoromethylphenyl)benzenesulfonamide], induced vesiculation of the Golgi apparatus as well as PKD1 phosphorylation at its activation loop. Finally, using siRNA (small interfering RNA) to block several PI-PLCs, we were able to identify PLCbeta3 as the sole member of this family involved in the regulation of the formation of transport carriers at the TGN. In conclusion, we demonstrate that fission of transport carriers at the TGN is dependent on PI-PLCs, specifically PLCbeta3, which is necessary to activate PKCeta and PKD in that Golgi compartment, via DAG production.

    Funded by: NIGMS NIH HHS: GM46224, GM53747, R01 GM046224, R01 GM053747

    The Biochemical journal 2007;406;1;157-65

  • 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

  • The G12 family of G proteins as a reporter of thromboxane A2 receptor activity.

    Zhang L, DiLizio C, Kim D, Smyth EM and Manning DR

    Department of Pharmacology, University of Pennsylvania School of Medicine, 3620 Hamilton Walk, Philadelphia, PA 19104-6084, USA.

    Despite advances in the understanding of pathways regulated by the G12 family of heterotrimeric G proteins, much regarding the engagement of this family by receptors remains unclear. We explore here, using the thromboxane A2 receptor TPalpha, the ability of G12 and G13 to report differences in the potency and efficacy of receptor ligands. We were interested especially in the potential of the isoprostane 8-iso-prostaglandin F (8-iso-PGF2alpha), among other ligands examined, to activate G12 and G13 through TPalpha explicitly. We were also interested in the functionality of TPalpha-Galpha fusion proteins germane to G12 and G13. Using fusion proteins in Spodoptera frugiperda (Sf9) cells and independently expressed proteins in human embryonic kidney 293 cells, and using guanosine 5'-O-(3-[35S]thio)triphosphate binding to evaluate Galpha activation directly, we found for Galpha that no ligand tested, including 8-iso-prostaglandin F (8-iso-PGF2alpha and a purported antagonist (pinane thromboxane A2), was silent. The activity of agonists was especially pronounced when evaluated for TPalpha-Galpha13 and in the context of receptor reserve. Agonist activity for 8-iso-PGF2 was diminished and that for pinane thromboxane A nonexistent when Galpha12 was the reporter. These data establish that G12 and G13 can report differentially potency and efficacy and underscore the relevance of receptor and G protein context.

    Funded by: NHLBI NIH HHS: HL066233, R01 HL066233; NIGMS NIH HHS: GM066892

    Molecular pharmacology 2006;69;4;1433-40

  • A docking site for G protein βγ subunits on the parathyroid hormone 1 receptor supports signaling through multiple pathways.

    Mahon MJ, Bonacci TM, Divieti P and Smrcka AV

    Endocrine Unit, Massachusetts General Hospital, Boston, Massachuesetts 02114, USA. mahon@helix.mgh.harvard.edu

    The G protein-coupled receptor for PTH and PTH-related protein (PTH1R) signals via many intracellular pathways. The purpose of this work is to investigate a G protein binding site on an intracellular domain of the PTH1R. The carboxy-terminal, cytoplasmic tail of the PTH1R fused to glutathione-S-transferase interacts with Gi/o proteins in vitro. All three subunits of the heterotrimer interact with the receptor C-tail. Activation of the heterotrimeric complex with GTPgammaS has no effect on Gbetagamma interactions, but markedly disrupts binding of the Galphai/o subunits to the receptor tail, suggesting that direct Gbetagamma binding indirectly links Galpha subunits to this region of the receptor. Gbetagamma subunits alone bind the C-tail with an affinity that is comparable to the heterotrimeric G protein complex. G protein complexes consisting of Galphashis6-beta1gamma2 and Galphaqhis6-beta1gamma2 also interact with the PTH1R tail in vitro. The Gbetagamma interaction domain is located on the juxta-membrane region of the tail between amino acids 468 and 491. Mutations that disrupt Gbetagamma interactions block PTH signaling via phospholipase Cbeta/[Ca2+]i and MAPK and markedly reduce signaling via adenylyl cyclase/cAMP. Herein, we define a domain on the PTH1R that is capable of binding G protein heterotrimeric complexes via direct Gbetagamma interactions.

    Funded by: NHLBI NIH HHS: HLT3207949; NIDDK NIH HHS: K01 DK59900-01; NIGMS NIH HHS: GM60286

    Molecular endocrinology (Baltimore, Md.) 2006;20;1;136-46

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

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

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

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

    Genome research 2006;16;1;55-65

  • Protease-activated receptors in hemostasis, thrombosis and vascular biology.

    Coughlin SR

    Cardiovascular Research Institute, Department of Medicine, University of California, San Francisco, CA 94143-0130, USA. coughlin@cvrimail.ucsf.edu

    The coagulation cascade and protease-activated receptors (PARs) together provide an elegant mechanism that links mechanical information in the form of tissue injury to cellular responses. These receptors appear to largely account for the cellular effects of thrombin and can mediate signaling to other trypsin-like proteases. An important role for PARs in hemostasis and thrombosis is established in animal models, and studies in knockout mice and nonhuman primates raise the question of whether PAR inhibition might offer an appealing new approach to the prevention and treatment of thrombosis. PARs may also trigger inflammatory responses to tissue injury. For example, PAR activation on endothelial cells and perhaps sensory afferents can trigger local accumulation of leukocytes and platelets and transudation of plasma. However, panoply of signaling systems and cell types orchestrates inflammatory responses, and efforts to define the relative importance and roles of PARs in various inflammatory processes are just beginning. Lastly, roles for PARs in blood vessel formation and other processes during embryonic development are emerging, and whether these reflect new roles for the coagulation cascade and/or PAR signaling to other proteases remains to be explored.

    Journal of thrombosis and haemostasis : JTH 2005;3;8;1800-14

  • Phosducin-like protein acts as a molecular chaperone for G protein betagamma dimer assembly.

    Lukov GL, Hu T, McLaughlin JN, Hamm HE and Willardson BM

    Department of Chemistry and Biochemistry, Brigham Young University, Provo, UT 84602, USA.

    Phosducin-like protein (PhLP) is a widely expressed binding partner of the G protein betagamma subunit dimer (Gbetagamma). However, its physiological role is poorly understood. To investigate PhLP function, its cellular expression was blocked using RNA interference, resulting in inhibition of Gbetagamma expression and G protein signaling. This inhibition was caused by an inability of nascent Gbetagamma to form dimers. Phosphorylation of PhLP at serines 18-20 by protein kinase CK2 was required for Gbetagamma formation, while a high-affinity interaction of PhLP with the cytosolic chaperonin complex appeared unnecessary. PhLP bound nascent Gbeta in the absence of Ggamma, and S18-20 phosphorylation was required for Ggamma to associate with the PhLP-Gbeta complex. Once Ggamma bound, PhLP was released. These results suggest a mechanism for Gbetagamma assembly in which PhLP stabilizes the nascent Gbeta polypeptide until Ggamma can associate, resulting in membrane binding of Gbetagamma and release of PhLP to catalyze another round of assembly.

    Funded by: NEI NIH HHS: EY12287, R01 EY012287

    The EMBO journal 2005;24;11;1965-75

  • Regulatory interactions between the amino terminus of G-protein betagamma subunits and the catalytic domain of phospholipase Cbeta2.

    Bonacci TM, Ghosh M, Malik S and Smrcka AV

    Department of Pharmacology and Physiology, University of Rochester School of Medicine and Dentistry, 601 Elmwood Ave., Rochester, New York 14642, USA,

    We previously identified a 10-amino acid region from the Y domain of phospholipase Cbeta2 (PLCbeta2) that associates with G-protein betagamma subunits (Sankaran, B., Osterhout, J., Wu, D., and Smrcka, A. V. (1998) J. Biol. Chem. 273, 7148-7154). We mapped the site for cross-linking of a synthetic peptide (N20K) corresponding to this Y domain region to Cys(25) within the amino-terminal coiled-coil domain of Gbetagamma (Yoshikawa, D. M., Bresciano, K., Hatwar, M., and Smrcka, A. V. (2001) J. Biol. Chem. 276, 11246-11251). Here, further experiments with a series of variable length cross-linking agents refined the site of N20K binding to within 4.4-6.7 angstroms of Cys(25). A mutant within the amino terminus of the Gbeta subunit, Gbeta(1)(23-27)gamma(2), activated PLCbeta2 more effectively than wild type, with no significant change in the EC(50), indicating that this region is directly involved in the catalytic regulation of PLCbeta2. This mutant was deficient in cross-linking to N20K, suggesting that a binding site for the peptide had been eliminated. Surprisingly, N20K could still inhibit Gbeta(1)(23-27)gamma(2)-dependent activation of PLC, suggesting a second N20K binding site. Competition analysis with a peptide that binds to the Galpha subunit switch II binding surface of Gbetagamma indicates a second N20K binding site at this surface. Furthermore, mutations to the N20K region within the Y-domain of full-length PLCbeta2 inhibited Gbetagamma-dependent regulation of the enzyme, providing further evidence for aGbetagamma binding site within the catalytic domain of PLCbeta2. The data support a model with two modes of PLC binding to Gbetagamma through the catalytic domain, where interactions with the amino-terminal coiled-coil domain are inhibitory, and interactions with the Galpha subunit switch II binding surface are stimulatory.

    Funded by: NHLBI NIH HHS: HL/T3207949; NIGMS NIH HHS: GM60286

    The Journal of biological chemistry 2005;280;11;10174-81

  • High-throughput mapping of a dynamic signaling network in mammalian cells.

    Barrios-Rodiles M, Brown KR, Ozdamar B, Bose R, Liu Z, Donovan RS, Shinjo F, Liu Y, Dembowy J, Taylor IW, Luga V, Przulj N, Robinson M, Suzuki H, Hayashizaki Y, Jurisica I and Wrana JL

    Program in Molecular Biology and Cancer, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada, M5G 1X5.

    Signaling pathways transmit information through protein interaction networks that are dynamically regulated by complex extracellular cues. We developed LUMIER (for luminescence-based mammalian interactome mapping), an automated high-throughput technology, to map protein-protein interaction networks systematically in mammalian cells and applied it to the transforming growth factor-beta (TGFbeta) pathway. Analysis using self-organizing maps and k-means clustering identified links of the TGFbeta pathway to the p21-activated kinase (PAK) network, to the polarity complex, and to Occludin, a structural component of tight junctions. We show that Occludin regulates TGFbeta type I receptor localization for efficient TGFbeta-dependent dissolution of tight junctions during epithelial-to-mesenchymal transitions.

    Funded by: NIGMS NIH HHS: P50 GM-62413

    Science (New York, N.Y.) 2005;307;5715;1621-5

  • Real-time monitoring of receptor and G-protein interactions in living cells.

    Galés C, Rebois RV, Hogue M, Trieu P, Breit A, Hébert TE and Bouvier M

    Department of Biochemistry, Université de Montréal, P.O. Box 6128 Down-town station, Montréal, H3C 3J7, Canada.

    G protein-coupled receptors (GPCRs) represent the largest family of proteins involved in signal transduction. Here we present a bioluminescence resonance energy transfer (BRET) assay that directly monitors in real time the early interactions between human GPCRs and their cognate G-protein subunits in living human cells. In addition to detecting basal precoupling of the receptors to Galpha-, Gbeta- and Ggamma-subunits, BRET measured very rapid ligand-induced increases in the interaction between receptor and Galphabetagamma-complexes (t(1/2) approximately 300 ms) followed by a slower (several minutes) decrease, reflecting receptor desensitization. The agonist-promoted increase in GPCR-Gbetagamma interaction was highly dependent on the identity of the Galpha-subunit present in the complex. Therefore, this G protein-activity biosensor provides a novel tool to directly probe the dynamics and selectivity of receptor-mediated, G-protein activation-deactivation cycles that could be advantageously used to identify ligands for orphan GPCRs.

    Nature methods 2005;2;3;177-84

  • Crystallographic analysis of CaaX prenyltransferases complexed with substrates defines rules of protein substrate selectivity.

    Reid TS, Terry KL, Casey PJ and Beese LS

    Department of Biochemistry, Duke University Medical Center, Durham, NC 27710, USA.

    Post-translational modifications are essential for the proper function of many proteins in the cell. The attachment of an isoprenoid lipid (a process termed prenylation) by protein farnesyltransferase (FTase) or geranylgeranyltransferase type I (GGTase-I) is essential for the function of many signal transduction proteins involved in growth, differentiation, and oncogenesis. FTase and GGTase-I (also called the CaaX prenyltransferases) recognize protein substrates with a C-terminal tetrapeptide recognition motif called the Ca1a2X box. These enzymes possess distinct but overlapping protein substrate specificity that is determined primarily by the sequence identity of the Ca1a2X motif. To determine how the identity of the Ca1a2X motif residues and sequence upstream of this motif affect substrate binding, we have solved crystal structures of FTase and GGTase-I complexed with a total of eight cognate and cross-reactive substrate peptides, including those derived from the C termini of the oncoproteins K-Ras4B, H-Ras and TC21. These structures suggest that all peptide substrates adopt a common binding mode in the FTase and GGTase-I active site. Unexpectedly, while the X residue of the Ca1a2X motif binds in the same location for all GGTase-I substrates, the X residue of FTase substrates can bind in one of two different sites. Together, these structures outline a series of rules that govern substrate peptide selectivity; these rules were utilized to classify known protein substrates of CaaX prenyltransferases and to generate a list of hypothetical substrates within the human genome.

    Funded by: NCRR NIH HHS: RR07707; NIGMS NIH HHS: GM52382

    Journal of molecular biology 2004;343;2;417-33

  • 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

  • A single Gbeta subunit locus controls cross-talk between protein kinase C and G protein regulation of N-type calcium channels.

    Doering CJ, Kisilevsky AE, Feng ZP, Arnot MI, Peloquin J, Hamid J, Barr W, Nirdosh A, Simms B, Winkfein RJ and Zamponi GW

    Department of Physiology and Biophysics, Cellular and Molecular Neurobiology Research Group, University of Calgary, Calgary, Alberta T2N 4N1, Canada.

    The modulation of N-type calcium channels is a key factor in the control of neurotransmitter release. Whereas N-type channels are inhibited by Gbetagamma subunits in a G protein beta-isoform-dependent manner, channel activity is typically stimulated by activation of protein kinase C (PKC). In addition, there is cross-talk among these pathways, such that PKC-dependent phosphorylation of the Gbetagamma target site on the N-type channel antagonizes subsequent G protein inhibition, albeit only for Gbeta(1)-mediated responses. The molecular mechanisms that control this G protein beta subunit subtype-specific regulation have not been described. Here, we show that G protein inhibition of N-type calcium channels is critically dependent on two separate but adjacent approximately 20-amino acid regions of the Gbeta subunit, plus a highly conserved Asn-Tyr-Val motif. These regions are distinct from those implicated previously in Gbetagamma signaling to other effectors such as G protein-coupled inward rectifier potassium channels, phospholipase beta(2), and adenylyl cyclase, thus raising the possibility that the specificity for G protein signaling to calcium channels might rely on unique G protein structural determinants. In addition, we identify a highly specific locus on the Gbeta(1) subunit that serves as a molecular detector of PKC-dependent phosphorylation of the G protein target site on the N-type channel alpha(1) subunit, thus providing for a molecular basis for G protein-PKC cross-talk. Overall, our results significantly advance our understanding of the molecular details underlying the integration of G protein and PKC signaling pathways at the level of the N-type calcium channel alpha(1) subunit.

    The Journal of biological chemistry 2004;279;28;29709-17

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

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

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

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

    Nature genetics 2004;36;1;40-5

  • G Protein betagamma subunits stimulate p114RhoGEF, a guanine nucleotide exchange factor for RhoA and Rac1: regulation of cell shape and reactive oxygen species production.

    Niu J, Profirovic J, Pan H, Vaiskunaite R and Voyno-Yasenetskaya T

    Department of Pharmacology, College of Medicine, University of Illinois at Chicago, Chicago, Ill 60612, USA.

    Rho GTPases integrate the intracellular signaling in a wide range of cellular processes. Activation of these G proteins is tightly controlled by a number of guanine nucleotide exchange factors (GEFs). In this study, we addressed the functional role of the recently identified p114RhoGEF in in vivo experiments. Activation of endogenous G protein-coupled receptors with lysophosphatidic acid resulted in activation of a transcription factor, serum response element (SRE), that was enhanced by p114RhoGEF. This stimulation was inhibited by the functional scavenger of Gbetagamma subunits, transducin. We have determined that Gbetagamma subunits but not Galpha subunits of heterotrimeric G proteins stimulated p114RhoGEF-dependent SRE activity. Using coimmunoprecipitation assay, we have determined that Gbetagamma subunits interacted with full-length and DH/PH domain of p114RhoGEF. Similarly, Gbetagamma subunits stimulated SRE activity induced by full-length and DH/PH domain of p114RhoGEF. Using in vivo pull-down assays and dominant-negative mutants of Rho GTPases, we have determined that p114RhoGEF activated RhoA and Rac1 but not Cdc42 proteins. Functional significance of RhoA activation was established by the ability of p114RhoGEF to induce actin stress fibers and cell rounding. Functional significance of Rac1 activation was established by the ability of p114RhoGEF to induce production of reactive oxygen species (ROS) followed by activation of NADPH oxidase enzyme complex. In summary, our data showed that the novel guanine nucleotide exchange factor p114RhoGEF regulates the activity of RhoA and Rac1, and that Gbetagamma subunits of heterotrimeric G proteins are activators of p114RhoGEF under physiological conditions. The findings help to explain the integrated effects of LPA and other G-protein receptor-coupled agonists on actin stress fiber formation, cell shape change, and ROS production.

    Funded by: NIGMS NIH HHS: GM56159, GM65160

    Circulation research 2003;93;9;848-56

  • Dose-dependent transcriptome changes by metal ores on a human acute lymphoblastic leukemia cell line.

    Sun NN, Fastje CD, Wong SS, Sheppard PR, Macdonald SJ, Ridenour G, Hyde JD and Witten ML

    Southwest Environmental Science Center and Department of Pediatrics, University of Arizona College of Medicine, Tucson, USA.

    The increased morbidity of childhood leukemia in Fallon, Nevada and Sierra Vista, Arizona has prompted great health concern. The main characteristic that these two towns share is the environmental pollution attributed to metal ore from abandoned mining operations. Consequently, we have investigated the transcriptome effects of metal ores from these endemic areas using a human T-cell acute lymphoblastic leukemia cell line (T-ALL). Metal ore from Fallon significantly increased cell growth after 24, 48 and 72 h of incubation at 1.5 microg/mL concentration, as measured by trypan-blue. Sierra Vista ore significantly increased cell growth with 0.15 and 1.5 microg/mL following 72 h of incubation. From human cDNA microarray, results indicate that in total, eight genes, mostly metallothionein (MT) genes, were up-regulated and 10 genes were down-regulated following treatment of the T-ALL cells with 0.15 and 1.5 microg/mL of metal ores at 72 h, in comparison with untreated cells. Twenty-eight metals of both ores were quantified and their presence may be associated with the cell growth rate and dose-dependent activation of transcriptomes in immature T-cells.

    Toxicology and industrial health 2003;19;7-10;157-63

  • T-type calcium channel regulation by specific G-protein betagamma subunits.

    Wolfe JT, Wang H, Howard J, Garrison JC and Barrett PQ

    Department of Pharmacology, University of Virginia, Charlottesville, Virginia 22908, USA.

    Low-voltage-activated (LVA) T-type calcium channels have a wide tissue distribution and have well-documented roles in the control of action potential burst generation and hormone secretion. In neurons of the central nervous system and secretory cells of the adrenal and pituitary, LVA channels are inhibited by activation of G-protein-coupled receptors that generate membrane-delimited signals, yet these signals have not been identified. Here we show that the inhibition of alpha1H (Ca(v)3.2), but not alpha(1G) (Ca(v)3.1) LVA Ca2+ channels is mediated selectively by beta2gamma2 subunits that bind to the intracellular loop connecting channel transmembrane domains II and III. This region of the alpha1H channel is crucial for inhibition, because its replacement abrogates inhibition and its transfer to non-modulated alpha1G channels confers beta2gamma2-dependent inhibition. betagamma reduces channel activity independent of voltage, a mechanism distinct from the established betagamma-dependent inhibition of non-L-type high-voltage-activated channels of the Ca(v)2 family. These studies identify the alpha1H channel as a new effector for G-protein betagamma subunits, and highlight the selective signalling roles available for particular betagamma combinations.

    Nature 2003;424;6945;209-13

  • Glucagon and regulation of glucose metabolism.

    Jiang G and Zhang BB

    Department of Metabolic Disorders and Molecular Endocrinology, Merck Research Laboratory, Rahway, New Jersey 07065, USA.

    As a counterregulatory hormone for insulin, glucagon plays a critical role in maintaining glucose homeostasis in vivo in both animals and humans. To increase blood glucose, glucagon promotes hepatic glucose output by increasing glycogenolysis and gluconeogenesis and by decreasing glycogenesis and glycolysis in a concerted fashion via multiple mechanisms. Compared with healthy subjects, diabetic patients and animals have abnormal secretion of not only insulin but also glucagon. Hyperglucagonemia and altered insulin-to-glucagon ratios play important roles in initiating and maintaining pathological hyperglycemic states. Not surprisingly, glucagon and glucagon receptor have been pursued extensively in recent years as potential targets for the therapeutic treatment of diabetes.

    American journal of physiology. Endocrinology and metabolism 2003;284;4;E671-8

  • Activation of heterotrimeric G proteins by a high energy phosphate transfer via nucleoside diphosphate kinase (NDPK) B and Gbeta subunits. Complex formation of NDPK B with Gbeta gamma dimers and phosphorylation of His-266 IN Gbeta.

    Cuello F, Schulze RA, Heemeyer F, Meyer HE, Lutz S, Jakobs KH, Niroomand F and Wieland T

    Institut für Pharmakologie und Toxikologie, Fakultät für Klinische Medizin Mannheim, Universität Heidelberg, Maybachstrasse 14-16, D-68169 Mannheim, Germany.

    G protein betagamma dimers can be phosphorylated in membranes from various tissues by GTP at a histidine residue in the beta subunit. The phosphate is high energetic and can be transferred onto GDP leading to formation of GTP. Purified Gbetagamma dimers do not display autophosphorylation, indicating the involvement of a separate protein kinase. We therefore enriched the Gbeta-phosphorylating activity present in preparations of the retinal G protein transducin and in partially purified G(i/o) proteins from bovine brain. Immunoblots, autophosphorylation, and enzymatic activity measurements demonstrated enriched nucleoside diphosphate kinase (NDPK) B in both preparations, together with residual Gbetagamma dimers. In the retinal NDPK B-enriched fractions, a Gbeta-specific antiserum co-precipitated phosphorylated NDPK B, and an antiserum against the human NDPK co-precipitated phosphorylated Gbetagamma. In addition, the NDPK-containing fractions from bovine brain reconstituted the phosphorylation of purified Gbetagamma. For identification of the phosphorylated histidine residue, bovine brain Gbetagamma and G(t)betagamma were thiophosphorylated with guanosine 5'-O-(3-[(35)S]thio)triphosphate, followed by digestion with endoproteinase Glu-C and trypsin, separation of the resulting peptides by gel electrophoresis and high pressure liquid chromatography, respectively, and sequencing of the radioactive peptides. The sequence information produced by both methods identified specific labeled fragments of bovine Gbeta(1) that overlapped in the heptapeptide, Leu-Met-Thr-Tyr-Ser-His-Asp (amino acids 261-267). We conclude that NDPK B forms complexes with Gbetagamma dimers and contributes to G protein activation by increasing the high energetic phosphate transfer onto GDP via intermediately phosphorylated His-266 in Gbeta(1) subunits.

    The Journal of biological chemistry 2003;278;9;7220-6

  • The familial hemiplegic migraine mutation R192Q reduces G-protein-mediated inhibition of P/Q-type (Ca(V)2.1) calcium channels expressed in human embryonic kidney cells.

    Melliti K, Grabner M and Seabrook GR

    Merck Sharp and Dohme Research Laboratories, The Neuroscience Research Centre, Terlings Park, Eastwick Road, Harlow, Essex CM20 2QR, UK. karim_melliti@merck.com

    Familial hemiplegic migraine is associated with at least 13 different missense mutations in the alpha1A Ca(2+) channel subunit. Some of these mutations have been shown to affect the biophysical properties of alpha1A currents. To date, no study has examined the influence of such mutations on the G-protein regulation of channel function. Because G-proteins inhibit movement of the voltage sensor, we examined the effects of the R192Q mutation, which neutralizes a positive charge in the first S4 segment. Human wild-type (WT) or R192Q mutant channels were expressed in human embryonic kidney tsA-201 cells along with dopamine D2 receptors. Application of quinpirole induced fast (approximately 1 s), pertussis toxin-sensitive inhibition of alpha1A(WT) and alpha1A(R192Q) Ca(2+) currents, consistent with the activation of a membrane-delimited pathway. alpha1A(WT) Ca(2+) currents were inhibited by 62.9 +/- 0.9 % (n = 27), whereas alpha1A(R192Q) Ca(2+) currents were inhibited by only 47.9 +/- 1.8 % (n = 35; P < 0.001). Concentration-response analysis showed that only the extent of inhibition was affected, with no change in agonist potency (EC(50) = 1 nM). Prepulse facilitation, which is a characteristic of voltage-dependent inhibition, was also reduced by the R192Q mutation. However, the kinetics of facilitation and slow activation were not affected, suggesting that G-protein-Ca(2+) channel affinity was unchanged. These results show that the R192Q mutation reduces the G-protein inhibition of P/Q-type Ca(2+) channels, probably by altering mechanisms by which Gbetagamma subunit binding induces a change in channel gating. Altered G-protein modulation and the consequent reduced presynaptic inhibition may contribute to migraine attacks by favouring a persistent state of hyperexcitability.

    The Journal of physiology 2003;546;Pt 2;337-47

  • Roles of G beta gamma in membrane recruitment and activation of p110 gamma/p101 phosphoinositide 3-kinase gamma.

    Brock C, Schaefer M, Reusch HP, Czupalla C, Michalke M, Spicher K, Schultz G and Nürnberg B

    Institut für Physiologische Chemie II, Klinikum der Heinrich-Heine-Universität, 40225 Düsseldorf, Germany.

    Receptor-regulated class I phosphoinositide 3-kinases (PI3K) phosphorylate the membrane lipid phosphatidylinositol (PtdIns)-4,5-P2 to PtdIns-3,4,5-P3. This, in turn, recruits and activates cytosolic effectors with PtdIns-3,4,5-P3-binding pleckstrin homology (PH) domains, thereby controlling important cellular functions such as proliferation, survival, or chemotaxis. The class IB p110 gamma/p101 PI3K gamma is activated by G beta gamma on stimulation of G protein-coupled receptors. It is currently unknown whether in living cells G beta gamma acts as a membrane anchor or an allosteric activator of PI3K gamma, and which role its noncatalytic p101 subunit plays in its activation by G beta gamma. Using GFP-tagged PI3K gamma subunits expressed in HEK cells, we show that G beta gamma recruits the enzyme from the cytosol to the membrane by interaction with its p101 subunit. Accordingly, p101 was found to be required for G protein-mediated activation of PI3K gamma in living cells, as assessed by use of GFP-tagged PtdIns-3,4,5-P3-binding PH domains. Furthermore, membrane-targeted p110 gamma displayed basal enzymatic activity, but was further stimulated by G beta gamma, even in the absence of p101. Therefore, we conclude that in vivo, G beta gamma activates PI3K gamma by a mechanism assigning specific roles for both PI3K gamma subunits, i.e., membrane recruitment is mediated via the noncatalytic p101 subunit, and direct stimulation of G beta gamma with p110 gamma contributes to activation of PI3K gamma.

    The Journal of cell biology 2003;160;1;89-99

  • G Protein beta gamma subunits act on the catalytic domain to stimulate Bruton's agammaglobulinemia tyrosine kinase.

    Lowry WE and Huang XY

    Department of Physiology, Weill Medical College of Cornell University, New York, New York 10021, USA.

    G proteins are critical cellular signal transducers for a variety of cell surface receptors. Both alpha and betagamma subunits of G proteins are able to transduce receptor signals. Several direct effect molecules for Gbetagamma subunits have been reported; yet the biochemical mechanism by which Gbetagamma executes its modulatory role is not well understood. We have shown that Gbetagamma could directly increase the kinase activity of Bruton's tyrosine kinase (Btk) whose defects are responsible for X chromosome-linked agammaglobulinemia in patients. The well characterized interaction of Gbetagamma with the PH (pleckstrin homology)/TH (Tec-homology) module of Btk was proposed to be the underlying activation mechanism. Here we show that Gbetagamma also interacts with the catalytic domain of Btk leading to increased kinase activity. Furthermore, we showed that the PH/TH module is required for Gbetagamma-induced membrane translocation of Btk. The membrane anchorage is also dependent on the interaction of Btk with phosphatidylinositol 3,4,5-trisphosphate, the product of phosphoinositide 3-kinase. These data support a dual role for Gbetagamma in the activation of Btk signaling function, namely membrane translocation and direct regulation of Btk catalytic activity.

    The Journal of biological chemistry 2002;277;2;1488-92

  • Gene expression profiling in human fetal liver and identification of tissue- and developmental-stage-specific genes through compiled expression profiles and efficient cloning of full-length cDNAs.

    Yu Y, Zhang C, Zhou G, Wu S, Qu X, Wei H, Xing G, Dong C, Zhai Y, Wan J, Ouyang S, Li L, Zhang S, Zhou K, Zhang Y, Wu C and He F

    Department of Genomics and Proteomics, Beijing Institute of Radiation Medicine, Chinese National Human Genome Center at Beijing, Beijing 100850, China.

    Fetal liver intriguingly consists of hepatic parenchymal cells and hematopoietic stem/progenitor cells. Human fetal liver aged 22 wk of gestation (HFL22w) corresponds to the turning point between immigration and emigration of the hematopoietic system. To gain further molecular insight into its developmental and functional characteristics, HFL22w was studied by generating expressed sequence tags (ESTs) and by analyzing the compiled expression profiles of liver at different developmental stages. A total of 13,077 ESTs were sequenced from a 3'-directed cDNA library of HFL22w, and classified as follows: 5819 (44.5%) matched to known genes; 5460 (41.8%) exhibited no significant homology to known genes; and the remaining 1798 (13.7%) were genomic sequences of unknown function, mitochondrial genomic sequences, or repetitive sequences. Integration of ESTs of known human genes generated a profile including 1660 genes that could be divided into 15 gene categories according to their functions. Genes related to general housekeeping, ESTs associated with hematopoiesis, and liver-specific genes were highly expressed. Genes for signal transduction and those associated with diseases, abnormalities, or transcription regulation were also noticeably active. By comparing the expression profiles, we identified six gene groups that were associated with different developmental stages of human fetal liver, tumorigenesis, different physiological functions of Itoh cells against the other types of hepatic cells, and fetal hematopoiesis. The gene expression profile therefore reflected the unique functional characteristics of HFL22w remarkably. Meanwhile, 110 full-length cDNAs of novel genes were cloned and sequenced. These novel genes might contribute to our understanding of the unique functional characteristics of the human fetal liver at 22 wk.

    Genome research 2001;11;8;1392-403

  • Gbeta gamma isoforms selectively rescue plasma membrane localization and palmitoylation of mutant Galphas and Galphaq.

    Evanko DS, Thiyagarajan MM, Siderovski DP and Wedegaertner PB

    Department of Microbiology and Immunology and Kimmel Cancer Institute, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.

    Mutation of Galpha(q) or Galpha(s) N-terminal contact sites for Gbetagamma resulted in alpha subunits that failed to localize at the plasma membrane or undergo palmitoylation when expressed in HEK293 cells. We now show that overexpression of specific betagamma subunits can recover plasma membrane localization and palmitoylation of the betagamma-binding-deficient mutants of alpha(s) or alpha(q). Thus, the betagamma-binding-defective alpha is completely dependent on co-expression of exogenous betagamma for proper membrane localization. In this report, we examined the ability of beta(1-5) in combination with gamma(2) or gamma(3) to promote proper localization and palmitoylation of mutant alpha(s) or alpha(q). Immunofluorescence localization, cellular fractionation, and palmitate labeling revealed distinct subtype-specific differences in betagamma interactions with alpha subunits. These studies demonstrate that 1) alpha and betagamma reciprocally promote the plasma membrane targeting of the other subunit; 2) beta(5), when co-expressed with gamma(2) or gamma(3), fails to localize to the plasma membrane or promote plasma membrane localization of mutant alpha(s) or alpha(q); 3) beta(3) is deficient in promoting plasma membrane localization of mutant alpha(s) and alpha(q), whereas beta(4) is deficient in promoting plasma membrane localization of mutant alpha(q); 4) both palmitoylation and interactions with betagamma are required for plasma membrane localization of alpha.

    Funded by: NIGMS NIH HHS: GM56444

    The Journal of biological chemistry 2001;276;26;23945-53

  • 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

  • Cloning, characterization, and mapping of the gene encoding the human G protein gamma 2 subunit.

    Modarressi MH, Taylor KE and Wolfe J

    Department of Biology, University College London, United Kingdom.

    G proteins play vital roles in cellular responses to external signals. The specificity of G protein-receptor interaction is mediated mostly by the gamma-subunit and the individual members of the gamma-subunit multigene family would hence be expected to each have a particular expression profile. In an experiment designed to isolate genes expressed predominantly in human testis we identified a cDNA fragment corresponding to the gamma2 gene. Although the protein sequence of the gamma2 subunit has previously been published, the cDNA sequence, expression pattern, genomic structure, and localisation of the human GNG2 gene have not been described. We report the complete sequence of the GNG2 cDNA which is 1066 bp long and contains an open reading frame encoding a protein of 71 amino acids. This protein is 100% homologous to the bovine, mouse, and rat G protein gamma2 subunit. The gene structure is very similar to that of other Ggamma-subunit genes in that there are two introns, one located in the 5' UTR and the other within the ORF. We show that this gene is expressed in a range of foetal tissues as well as adult testis, adrenal gland, brain, white blood cells and lung but not in adult liver, muscle, sperm, prostate gland nor in the testes of two different infertile patients. There is evidence that GNG2 is expressed in malignant tissues. Using two independent methods, we have mapped the human GNG2 gene to chromosome 14q21.

    Biochemical and biophysical research communications 2000;272;2;610-5

  • Mutational analysis of Gbetagamma and phospholipid interaction with G protein-coupled receptor kinase 2.

    Carman CV, Barak LS, Chen C, Liu-Chen LY, Onorato JJ, Kennedy SP, Caron MG and Benovic JL

    Department of Microbiology & Immunology, Kimmel Cancer Institute, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.

    Agonist-dependent regulation of G protein-coupled receptors is dependent on their phosphorylation by G protein-coupled receptor kinases (GRKs). GRK2 and GRK3 are selectively regulated in vitro by free Gbetagamma subunits and negatively charged membrane phospholipids through their pleckstrin homology (PH) domains. However, the molecular binding determinants and physiological role for these ligands remain unclear. To address these issues, we generated an array of site-directed mutants within the GRK2 PH domain and characterized their interaction with Gbetagamma and phospholipids in vitro. Mutation of several residues in the loop 1 region of the PH domain, including Lys-567, Trp-576, Arg-578, and Arg-579, resulted in a loss of receptor phosphorylation, likely via disruption of phospholipid binding, that was reversed by Gbetagamma. Alternatively, mutation of residues distal to the C-terminal amphipathic alpha-helix, including Lys-663, Lys-665, Lys-667, and Arg-669, resulted in decreased responsiveness to Gbetagamma. Interestingly, mutation of Arg-587 in beta-sheet 3, a region not previously thought to interact with Gbetagamma, resulted in a specific and profound loss of Gbetagamma responsiveness. To further characterize these effects, two mutants (GRK2(K567E/R578E) and GRK2(R587Q)) were expressed in Sf9 cells and purified. Analysis of these mutants revealed that GRK2(K567E/R578E) was refractory to stimulation by negatively charged phospholipids but bound Gbetagamma similar to wild-type GRK2. In contrast, GRK2(R587Q) was stimulated by acidic phospholipids but failed to bind Gbetagamma. In order to examine the role of phospholipid and Gbetagamma interaction in cells, wild-type and mutant GRK2s were expressed with a beta(2)-adrenergic receptor (beta(2)AR) mutant that is responsive to GRK2 phosphorylation (beta(2)AR(Y326A)). In these cells, GRK2(K567E/R578E) and GRK2(R587Q) were largely defective in promoting agonist-dependent phosphorylation and internalization of beta(2)AR(Y326A). Similarly, wild-type GRK2 but not GRK2(K567E/R578E) or GRK2(R587Q) promoted morphinedependent phosphorylation of the mu-opioid receptor in cells. Thus, we have (i) identified several specific GRK2 binding determinants for Gbetagamma and phospholipids, and (ii) demonstrated that Gbetagamma binding is the limiting step for GRK2-dependent receptor phosphorylation in cells.

    Funded by: NCI NIH HHS: 5-T32-CA09662; NIGMS NIH HHS: GM44944

    The Journal of biological chemistry 2000;275;14;10443-52

  • Association of the proto-oncogene product dbl with G protein betagamma subunits.

    Nishida K, Kaziro Y and Satoh T

    Faculty of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama, Japan.

    The Rho family of GTP-binding proteins has been implicated in the regulation of various cellular functions including actin cytoskeleton-dependent morphological change. Its activity is directed by intracellular signals mediated by various types of receptors such as G protein-coupled receptors. However, the mechanisms underlying receptor-dependent regulation of Rho family members remain incompletely understood. The guanine nucleotide exchange factor (GEF) Dbl targets Rho family proteins thereby stimulating their GDP/GTP exchange, and thus is believed to be involved in receptor-mediated regulation of the proteins. Here, we show the association of Dbl with G protein betagamma subunits (Gbetagamma) in transient co-expression and cell-free systems. An amino-terminal portion conserved among a subset of Dbl family proteins is sufficient for the binding of Gbetagamma. In fact, Ost and Kalirin, which contain this Gbetagamma-binding motif, also associate with Gbetagamma. c-Jun N-terminal kinase was synergistically activated upon co-expression of Dbl and Gbeta in a dominant-negative Rho-sensitive manner. However, GEF activity of Dbl toward Rho as measured by in vitro GDP binding assays remained unaffected following Gbetagamma binding, suggesting that additional signals may be required for the regulation of Dbl.

    FEBS letters 1999;459;2;186-90

  • Gene structure and chromosome localization of the G gamma c subunit of human cone G-protein (GNGT2).

    Ong OC, Hu K, Rong H, Lee RH and Fung BK

    Jules Stein Eye Institute, Department of Molecular and Medical Pharmacology, University of California at Los Angeles School of Medicine 90095, USA.

    Phototransduction in the vertebrate rod and cone photoreceptors is regulated by structurally homologous and yet distinct groups of signaling proteins. We have previously identified in bovine retinas a cone-specific G-protein gamma subunit (G gamma c, previously named G gamma b), which may play a key role in coupling the cone visual pigment to phosphodiesterase (O. C. Ong et al., 1995, J. Biol. Chem. 270:8495-8500). We report here the characterization of human G gamma c and its gene structure. Human G gamma c subunit shares a high degree of sequence identity with the corresponding bovine G gamma c isoform (85%) and human rod G gamma 1 (63%). The protein is specifically localized in cones, as indicated by immunohistochemical staining using anti-G gamma c antibodies. Nucleotide sequence analysis of the G gamma c gene (GNGT2) reveals a structure consisting of three exons and two introns, with the intron splice sites similar to that of the rod G gamma 1 gene (GNGT1). By using fluorescence in situ hybridization, we have further localized the human GNGT2 gene to chromosome 17q21. The elucidation of the G gamma c gene structure would facilitate the identification of genetic defects associated with cone degeneration.

    Funded by: NEI NIH HHS: EY05895, EY07026, EY09936; ...

    Genomics 1997;44;1;101-9

  • Direct interaction of gbetagamma with a C-terminal gbetagamma-binding domain of the Ca2+ channel alpha1 subunit is responsible for channel inhibition by G protein-coupled receptors.

    Qin N, Platano D, Olcese R, Stefani E and Birnbaumer L

    Department of Anesthesiology, University of California, Los Angeles, CA 90095, USA.

    Several classes of voltage-gated Ca2+ channels (VGCCs) are inhibited by G proteins activated by receptors for neurotransmitters and neuromodulatory peptides. Evidence has accumulated to indicate that for non-L-type Ca2+ channels the executing arm of the activated G protein is its betagamma dimer (Gbetagamma). We report below the existence of two Gbetagamma-binding sites on the A-, B-, and E-type alpha1 subunits that form non-L-type Ca2+ channels. One, reported previously, is in loop 1 connecting transmembrane domains I and II. The second is located approximately in the middle of the ca. 600-aa-long C-terminal tails. Both Gbetagamma-binding regions also bind the Ca2+ channel beta subunit (CCbeta), which, when overexpressed, interferes with inhibition by activated G proteins. Replacement in alpha1E of loop 1 with that of the G protein-insensitive and Gbetagamma-binding-negative loop 1 of alpha1C did not abolish inhibition by G proteins, but the exchange of the alpha1E C terminus with that of alpha1C did. This and properties of alpha1E C-terminal truncations indicated that the Gbetagamma-binding site mediating the inhibition of Ca2+ channel activity is the one in the C terminus. Binding of Gbetagamma to this site was inhibited by an alpha1-binding domain of CCbeta, thus providing an explanation for the functional antagonism existing between CCbeta and G protein inhibition. The data do not support proposals that Gbetagamma inhibits alpha1 function by interacting with the site located in the loop I-II linker. These results define the molecular mechanism by which presynaptic G protein-coupled receptors inhibit neurotransmission.

    Funded by: NIAMS NIH HHS: AR43411; NIDDK NIH HHS: DK19318; NIGMS NIH HHS: F32 GM017120; PHS HHS: 38970

    Proceedings of the National Academy of Sciences of the United States of America 1997;94;16;8866-71

  • The human thromboxane A2 receptor alpha isoform (TP alpha) functionally couples to the G proteins Gq and G11 in vivo and is activated by the isoprostane 8-epi prostaglandin F2 alpha.

    Kinsella BT, O'Mahony DJ and Fitzgerald GA

    Department of Biochemistry, University College Dublin, Ireland.

    To establish whether the thromboxane A2 (TXA2) receptor (TP) functionally couples to the Gq family of heterotrimeric G proteins in vivo, we have coexpressed the cDNAs coding for the human platelet/placental TP alpha isoform (TP alpha) and the alpha subunits of Gq or G11 in human embryonic kidney (HEK) 293 cells. TP activation in response to ligand stimulation was monitored by analyzing mobilization of intracellular calcium (Ca++i) in FURA2/AM-loaded transfected HEK 293 and in platelets. Second, we wished to examine the possible interaction of the isoprostane 8-epi prostaglandin F2 alpha with the TP alpha, in transfected HEK 293 cells and with the TPs expressed in platelets. Thus both the prostaglandin endoperoxide/TXA2 analog (U46619) and the 8-epi PGF2 alpha were utilized as ligand probes of TP alpha activation. The results demonstrate that each ligand induced elevations of Ca++i levels in HEK 293 cells, cotransfected with either the TP alpha and G alpha q or the TP alpha and G alpha 11, and also in platelets. Initial stimulation of these cells with U46619 or 8-epi PGF 2 alpha desensitized a subsequent rise in [Ca++]i in response to U46619 or 8-epi PGF 2 alpha, respectively. Moreover, prestimulation with U46619 desensitized a subsequent rise in Ca++i concentration in response to 8-epi PGF 2 alpha, and vice versa. These responses were blocked by the TP antagonist SQ29,548 in both cell types. In contrast, prestimulation of the transfected HEK 293 cells or platelets with thrombin did not desensitize a subsequent rise in [Ca++]i in response to U46619 or 8-epi PGF 2 alpha. After stimulation with either U46619 or 8-epi PGF 2 alpha, no significant rise in Ca++i levels was observed in HEK 293 cells transfected with the TP alpha receptor only or in control cells transfected with the vector pCMV5. These results demonstrate that the TP alpha isoform functionally couples with either Gq or G11 in vivo, whether activated by a PG/TXA2 analog or by the F2 isoprostane 8-epi PGF2 alpha.

    Funded by: Wellcome Trust

    The Journal of pharmacology and experimental therapeutics 1997;281;2;957-64

  • Molecular basis of receptor/G protein coupling selectivity studied by coexpression of wild type and mutant m2 muscarinic receptors with mutant G alpha(q) subunits.

    Kostenis E, Conklin BR and Wess J

    Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, Maryland 20892, USA.

    The molecular basis of receptor/G protein coupling selectivity was studied by using the m2 muscarinic receptor, a prototypical G(i/o)-coupled receptor as a model system. We could recently show that the m2 receptor can efficiently interact with mutant G protein alpha(q) subunits in which the last five amino acids were replaced with alpha(i2) or alpha(o) sequence [Liu, J., Conklin, B. R., Blin, N., Yun, J., & Wess, J. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 11642-11646]. Additional mutagenesis studies led to the identification of a four-amino-acid motif on the m2 receptor (Val385, Thr386, Ile389, and Leu390) that is predicted to functionally interact with the C-terminal portion of alpha(i/o) subunits. To further investigate the structural requirements for this interaction to occur, these four m2 receptor residues were replaced, either individually or in combination, with the corresponding residues present in the G(q/11)-coupled muscarinic receptors (m1, m3, and m5). The ability of the resulting mutant m2 receptors to interact with a mutant alpha(q) subunit (qo5) in which the last five amino acids were replaced with alpha(o) sequence was investigated in co-transfected COS-7 cells [studied biochemical response: stimulation of phosphatidyl inositol (PI) hydrolysis]. Our data suggest that the presence of three of the four targeted m2 receptor residues (Val385, Thr386, and Ile389) is essential for efficient recognition of C-terminal alpha(i/o) sequences. To study which specific amino acids within the C-terminal segment of alpha(i/o) subunits are critical for this interaction to occur, the wild type m2 receptor was co-expressed with a series of mutant alpha(q) subunits containing single or multiple alpha(q) --> alpha(i1,2) point mutations at their C-terminus. Remarkably, the wild type m2 receptor, while unable to efficiently stimulate wild type alpha(q), gained the ability to productively interact with three alpha(q) single-point mutants, providing the first example that the receptor coupling selectivity of G protein alpha subunits can be switched by single amino acid substitutions. Given the high degree of structural homology among different G protein-coupled receptors and among different classes of G protein alpha subunits, our results should be of broad general relevance.

    Biochemistry 1997;36;6;1487-95

  • Direct binding of G-protein betagamma complex to voltage-dependent calcium channels.

    De Waard M, Liu H, Walker D, Scott VE, Gurnett CA and Campbell KP

    Howard Hughes Medical Institute, University of Iowa College of Medicine, Iowa City 52242, USA.

    Voltage-dependent Ca2+ channels play a central role in controlling neurotransmitter release at the synapse. They can be inhibited by certain G-protein-coupled receptors, acting by a pathway intrinsic to the membrane. Here we show that this inhibition results from a direct interaction between the G-protein betagamma complex and the pore-forming alpha1 subunits of several types of these channels. The interaction is mediated by the cytoplasmic linker connecting the first and second transmembrane repeats. Within this linker, binding occurs both in the alpha1 interaction domain (AID), which also mediates the interaction between the alpha1 and beta subunits of the channel, and in a second downstream sequence. Further analysis of the binding site showed that several amino-terminal residues in the AID are critical for Gbetagamma binding, defining a site distinct from the carboxy-terminal residues shown to be essential for binding the beta-subunit of the Ca2+ channel. Mutation of an arginine residue within the N-terminal motif abolished betagamma binding and rendered the channel refractory to G-protein modulation when expressed in Xenopus oocytes, showing that the interaction is indeed responsible for G-protein-dependent modulation of Ca2+ channel activity.

    Nature 1997;385;6615;446-50

  • Differential ability to form the G protein betagamma complex among members of the beta and gamma subunit families.

    Yan K, Kalyanaraman V and Gautam N

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

    We have determined the relative abilities of several members of the G protein beta and gamma subunit families to associate with each other using the yeast two-hybrid system. We show first that the mammalian beta1 and gamma3 fusion proteins form a complex in yeast and that formation of the complex activates the reporter gene for beta-galactosidase. Second, the magnitude of reporter activity stimulated by various combinations of beta and gamma subunit types varies widely. Third, the reporter activity evoked by a particular combination of beta and gamma subunit types is not correlated with the expression levels of these subunit types in the yeast cells. Finally, the reporter activity shows a direct relationship with the amount of hybrid betagamma complex formed in the cell as determined by immunoprecipitation. These results suggest that different beta and gamma subunit types interact with each other with widely varying abilities, and this in combination with the level of expression of a subunit type in a mammalian cell determines which G protein will be active in that cell. The strong preference of all gamma subunit types for the beta1 subunit type explains the preponderence of this subunit type in most G proteins.

    The Journal of biological chemistry 1996;271;12;7141-6

  • 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

  • Cloning and characterization of a G protein-activated human phosphoinositide-3 kinase.

    Stoyanov B, Volinia S, Hanck T, Rubio I, Loubtchenkov M, Malek D, Stoyanova S, Vanhaesebroeck B, Dhand R, Nürnberg B et al.

    Max Planck Research Unit in Growth Factor Signal Transduction, Medical Faculty, University of Jena, Germany.

    Phosphoinositide-3 kinase activity is implicated in diverse cellular responses triggered by mammalian cell surface receptors and in the regulation of protein sorting in yeast. Receptors with intrinsic and associated tyrosine kinase activity recruit heterodimeric phosphoinositide-3 kinases that consist of p110 catalytic subunits and p85 adaptor molecules containing Src homology 2 (SH2) domains. A phosphoinositide-3 kinase isotype, p110 gamma, was cloned and characterized. The p110 gamma enzyme was activated in vitro by both the alpha and beta gamma subunits of heterotrimeric guanosine triphosphate (GTP)-binding proteins (G proteins) and did not interact with p85. A potential pleckstrin homology domain is located near its amino terminus. The p110 gamma isotype may link signaling through G protein-coupled receptors to the generation of phosphoinositide second messengers phosphorylated in the D-3 position.

    Science (New York, N.Y.) 1995;269;5224;690-3

  • A direct interaction between G-protein beta gamma subunits and the Raf-1 protein kinase.

    Pumiglia KM, LeVine H, Haske T, Habib T, Jove R and Decker SJ

    Parke-Davis Pharmaceutical Research Division, Department of Signal Transduction, Ann Arbor, Michigan 48106, USA.

    Raf-1 is a serine/threonine protein kinase positioned downstream of Ras in the mitogen-activated protein kinase cascade. Using a yeast two-hybrid strategy to identify other proteins that interact with and potentially regulate Raf-1, we isolated a clone encoding the carboxyl-terminal half of the G beta 2 subunit of heterotrimeric G-proteins. In vitro, purified G beta gamma subunits specifically bound to a GST fusion protein encoding amino acids 1-330 of Raf-1 (Raf/330). Binding assays with truncation mutants of GST-Raf indicate that the region located between amino acids 136 and 239 is a primary determinant for interaction with G beta gamma. In competition experiments, the carboxyl terminus of beta-adrenergic receptor kinase (beta ARK) blocked the binding of G beta gamma to Raf/330; however, the Raf-1-binding proteins, Ras and 14-3-3, had no effect. Scatchard analysis of in vitro binding between Raf/330 and G beta gamma revealed an affinity of interaction (Kd = 163 +/- 36 nM), similar to that seen between G beta gamma and beta ARK (Kd = 87 +/- 24 nM). The formation of native heterotrimeric G alpha beta gamma complexes, as measured by pertussis toxin ADP-ribosylation of G alpha, could be disrupted by increasing amounts of Raf/330, with an EC50 of approximately 200 nM, in close agreement with the estimated binding affinity. In vivo complexes of Raf-1 and G beta gamma were isolated from human embryonic kidney 293-T cells transfected with epitope-tagged G beta 2. The identification and characterization of this novel interaction raises several possibilities for signaling cross-talk between growth factor receptors and those receptors coupled to heterotrimeric G-proteins.

    Funded by: NCI NIH HHS: CA55652

    The Journal of biological chemistry 1995;270;24;14251-4

  • Binding of beta gamma subunits of heterotrimeric G proteins to the PH domain of Bruton tyrosine kinase.

    Tsukada S, Simon MI, Witte ON and Katz A

    Department of Microbiology and Molecular Genetics, University of California, Los Angeles 90024-1662.

    Bruton tyrosine kinase (Btk) has been implicated as the defective gene in both human and murine B-cell deficiencies. The identification of molecules that interact with Btk may shed light on critical processes in lymphocyte development. The N-terminal unique region of Btk contains a pleckstrin homology domain. This domain is found in a broad array of signaling molecules and implicated to function in protein-protein interactions. By using an in vitro binding assay and an in vivo competition assay, the pleckstrin homology domain of Btk was shown to interact with the beta gamma dimer of heterotrimeric guanine nucleotide-binding proteins (G proteins). A highly conserved tryptophan residue in subdomain 6 of the pleckstrin homology domain was shown to play a critical role in the binding. The interaction of Btk with beta gamma suggests the existence of a unique connection between cytoplasmic tyrosine kinases and G proteins in cellular signal transduction.

    Funded by: NCI NIH HHS: CA12800; NIGMS NIH HHS: GM 34236

    Proceedings of the National Academy of Sciences of the United States of America 1994;91;23;11256-60

  • 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

  • G proteins of the G12 family are activated via thromboxane A2 and thrombin receptors in human platelets.

    Offermanns S, Laugwitz KL, Spicher K and Schultz G

    Institut für Pharmakologie, Freie Universität Berlin, Germany.

    Using subtype-specific antisera, we were able to identify the recently described alpha subunits of G12 and G13 in platelet membranes as 43-kDa proteins. Activation of the thromboxane A2 and the thrombin receptors in platelet membranes led to increased incorporation of the photoreactive GTP analogue [alpha-32P]GTP azidoanilide into immunoprecipitated alpha 12 and alpha 13, indicating that both receptors couple to G12 and G13. In addition, both activated receptors were demonstrated to couple to one or more members of the Gq family. In the absence of receptor agonists, incorporation of [alpha-32P]GTP azidoanilide into alpha 12 and alpha 13 was low over a long time period (up to 45 min) due to an obviously low basal nucleotide exchange rate, whereas an agonist-stimulated photolabeling of alpha 12 and alpha 13 could be observed after 4-8 min and reached a maximum after 30-45 min. Effective activation of G12 and G13 via the thromboxane A2 and the thrombin receptors was not dependent on the presence of GDP. Our results provide evidence that G12 and G13 play a functional role in transmembrane signal transduction and suggest that both proteins are involved in pathways leading to platelet activation.

    Proceedings of the National Academy of Sciences of the United States of America 1994;91;2;504-8

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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