G2Cdb::Gene report

Gene id
Gene symbol
Homo sapiens
guanine nucleotide binding protein (G protein), beta polypeptide 1
G00000210 (Mus musculus)

Databases (8)

Curated Gene
OTTHUMG00000000940 (Vega human gene)
ENSG00000078369 (Ensembl human gene)
2782 (Entrez Gene)
25 (G2Cdb plasticity & disease)
GNB1 (GeneCards)
139380 (OMIM)
Marker Symbol
HGNC:4396 (HGNC)
Protein Sequence
P62873 (UniProt)

Diseases (1)

Disease Nervous effect Mutations Found Literature Mutations Type Genetic association?
D00000225: Retinitis pigmentosa (autosomal dominant) Y Y (17167406) Polymorphism (P) N


  • A screen for mutations in the transducin gene GNB1 in patients with autosomal dominant retinitis pigmentosa.

    Mylvaganam GH, McGee TL, Berson EL and Dryja TP

    Ocular Molecular Genetics Institute and the Berman-Gund Laboratory for the Study of Retinal Degenerations, Harvard Medical School, Massachusetts Eye and Ear Infirmary, Boston, MA 02114, USA.

    Purpose: To search for mutations in the GNB1 gene (coding for the transducin beta1-subunit protein) in patients with autosomal dominant retinitis pigmentosa.

    Methods: We screened 185 unrelated patients with autosomal dominant retinitis pigmentosa (ADRP) using direct genomic sequencing of the three non-coding exons and 9 coding exons, along with immediately flanking intron DNA.

    Results: We found 2 polymorphisms, one in intron 1 with a minor allele frequency of 24%, and one in intron 6 with a minor allele frequency of 12% among the 185 patients. Two rare variants (minor allele frequency <1%) were found in the 3' untranslated region of exon 12. No changes were found in the open reading frame (exons 3-11) or in the noncoding exons 1 and 2.

    Conclusions: No likely pathogenic GNB1 mutations have been found in any of 185 unrelated patients with ADRP. This result would be expected if hemizygosity for GNB1 does not result in ADRP or is a rare cause of ADRP.

    Funded by: NEI NIH HHS: EY00169, EY08683, P30 EY014104

    Molecular vision 2006;12;1496-8

Literature (72)

Pubmed - other

  • The G protein betagamma subunit mediates reannealing of adherens junctions to reverse endothelial permeability increase by thrombin.

    Knezevic N, Tauseef M, Thennes T and Mehta D

    Center for Lung and Vascular Biology, Department of Pharmacology, University of Illinois, Chicago, IL 60612, USA.

    The inflammatory mediator thrombin proteolytically activates protease-activated receptor (PAR1) eliciting a transient, but reversible increase in vascular permeability. PAR1-induced dissociation of Galpha subunit from heterotrimeric Gq and G12/G13 proteins is known to signal the increase in endothelial permeability. However, the role of released Gbetagamma is unknown. We now show that impairment of Gbetagamma function does not affect the permeability increase induced by PAR1, but prevents reannealing of adherens junctions (AJ), thereby persistently elevating endothelial permeability. We observed that in the naive endothelium Gbeta1, the predominant Gbeta isoform is sequestered by receptor for activated C kinase 1 (RACK1). Thrombin induced dissociation of Gbeta1 from RACK1, resulting in Gbeta1 interaction with Fyn and focal adhesion kinase (FAK) required for FAK activation. RACK1 depletion triggered Gbeta1 activation of FAK and endothelial barrier recovery, whereas Fyn knockdown interrupted with Gbeta1-induced barrier recovery indicating RACK1 negatively regulates Gbeta1-Fyn signaling. Activated FAK associated with AJ and stimulated AJ reassembly in a Fyn-dependent manner. Fyn deletion prevented FAK activation and augmented lung vascular permeability increase induced by PAR1 agonist. Rescuing FAK activation in fyn(-/-) mice attenuated the rise in lung vascular permeability. Our results demonstrate that Gbeta1-mediated Fyn activation integrates FAK with AJ, preventing persistent endothelial barrier leakiness.

    Funded by: NHLBI NIH HHS: HL71794, HL84153, R01 HL071794, R01 HL084153

    The Journal of experimental medicine 2009;206;12;2761-77

  • Gene expression in the mouse eye: an online resource for genetics using 103 strains of mice.

    Geisert EE, Lu L, Freeman-Anderson NE, Templeton JP, Nassr M, Wang X, Gu W, Jiao Y and Williams RW

    Department of Ophthalmology and Center for Vision Research, Memphis, TN 38163, USA. egeisert@utmem.edu

    Purpose: Individual differences in patterns of gene expression account for much of the diversity of ocular phenotypes and variation in disease risk. We examined the causes of expression differences, and in their linkage to sequence variants, functional differences, and ocular pathophysiology.

    Methods: mRNAs from young adult eyes were hybridized to oligomer microarrays (Affymetrix M430v2). Data were embedded in GeneNetwork with millions of single nucleotide polymorphisms, custom array annotation, and information on complementary cellular, functional, and behavioral traits. The data include male and female samples from 28 common strains, 68 BXD recombinant inbred lines, as well as several mutants and knockouts.

    Results: We provide a fully integrated resource to map, graph, analyze, and test causes and correlations of differences in gene expression in the eye. Covariance in mRNA expression can be used to infer gene function, extract signatures for different cells or tissues, to define molecular networks, and to map quantitative trait loci that produce expression differences. These data can also be used to connect disease phenotypes with sequence variants. We demonstrate that variation in rhodopsin expression efficiently predicts candidate genes for eight uncloned retinal diseases, including WDR17 for the human RP29 locus.

    Conclusions: The high level of strain variation in gene expression is a powerful tool that can be used to explore and test molecular networks underlying variation in structure, function, and disease susceptibility. The integration of these data into GeneNetwork provides users with a workbench to test linkages between sequence differences and eye structure and function.

    Funded by: NCRR NIH HHS: U24 RR021760; NEI NIH HHS: P30 EY013080, P30 EY013080-109002, R01 EY017841, R01 EY017841-03, R01EY017841; NIAAA NIH HHS: U01 AA013499, U01 AA014425, U01AA014425, U01AA13499; NIDA NIH HHS: P20 DA021131, P20-DA 21131; PHS HHS: U24 RP021760

    Molecular vision 2009;15;1730-63

  • Proteomic analysis of dorsolateral prefrontal cortex indicates the involvement of cytoskeleton, oligodendrocyte, energy metabolism and new potential markers in schizophrenia.

    Martins-de-Souza D, Gattaz WF, Schmitt A, Maccarrone G, Hunyadi-Gulyás E, Eberlin MN, Souza GH, Marangoni S, Novello JC, Turck CW and Dias-Neto E

    Laboratório de Neurociências, Instituto de Psiquiatria, Faculdade de Medicina da USP, Rua Dr. Ovídio Pires de Campos, SP, Brazil. martins@mpipsykl.mpg.de

    Schizophrenia is likely to be a consequence of serial alterations in a number of genes that, together with environmental factors, will lead to the establishment of the illness. The dorsolateral prefrontal cortex (Brodmann's Area 46) is implicated in schizophrenia and executes high functions such as working memory, differentiation of conflicting thoughts, determination of right and wrong concepts, correct social behavior and personality expression. We performed a comparative proteome analysis using two-dimensional gel electrophoresis of pools from 9 schizophrenia and 7 healthy control patients' dorsolateral prefrontal cortex aiming to identify, by mass spectrometry, alterations in protein expression that could be related to the disease. In schizophrenia-derived samples, our analysis revealed 10 downregulated and 14 upregulated proteins. These included alterations previously implicated in schizophrenia, such as oligodendrocyte-related proteins (myelin basic protein and transferrin), as well as malate dehydrogenase, aconitase, ATP synthase subunits and cytoskeleton-related proteins. Also, six new putative disease markers were identified, including energy metabolism, cytoskeleton and cell signaling proteins. Our data not only reinforces the involvement of proteins previously implicated in schizophrenia, but also suggests new markers, providing further information to foster the comprehension of this important disease.

    Journal of psychiatric research 2009;43;11;978-86

  • beta-Arrestin1 interacts with the G-protein subunits beta1gamma2 and promotes beta1gamma2-dependent Akt signalling for NF-kappaB activation.

    Yang M, He RL, Benovic JL and Ye RD

    Department of Pharmacology, College of Medicine, University of Illinois, Chicago, IL 60612, USA. ming.yang@cvt.com

    beta-Arrestins are known to regulate G-protein signalling through interactions with their downstream effectors. In the present study, we report that beta-arrestin1 associates with the G-protein beta1gamma2 subunits in transfected cells, and purified beta-arrestin1 interacts with G(beta1gamma2) derived from in vitro translation. Deletion mutagenesis of beta-arrestin1 led to the identification of a region, comprising amino acids 181-280, as being responsible for its interaction with G(beta1gamma2). Overexpression of beta-arrestin1 facilitates G(beta1gamma2)-mediated Akt phosphorylation, and inhibition of endogenous beta-arrestin1 expression by siRNA (small interfering RNA) diminishes this effect. Through investigation of NF-kappaB (nuclear factor kappaB), a transcription factor regulated by Akt signalling, we have found that overexpression of beta-arrestin1 significantly enhances G(beta1gamma2)-mediated nuclear translocation of NF-kappaB proteins and expression of a NF-kappaB-directed luciferase reporter. Overexpression of beta-arrestin1 also promotes bradykinin-induced, G(betagamma)-mediated NF-kappaB luciferase-reporter expression, which is reverted by silencing the endogenous beta-arrestin1 with a specific siRNA. These results identify novel functions of beta-arrestin1 in binding to the beta1gamma2 subunits of heterotrimeric G-proteins and promoting G(betagamma)-mediated Akt signalling for NF-kappaB activation.

    Funded by: NIAID NIH HHS: AI040176, R01 AI040176, R01 AI040176-11, R56 AI040176; NIGMS NIH HHS: GM066182, R01 GM047417, R01 GM066182, R01 GM066182-04, R37 GM047417

    The Biochemical journal 2009;417;1;287-96

  • RACK1 regulates directional cell migration by acting on G betagamma at the interface with its effectors PLC beta and PI3K gamma.

    Chen S, Lin F, Shin ME, Wang F, Shen L and Hamm HE

    Department of Pharmacology, Vanderbilt University, Nashville, TN 37232, USA. songhai.chen@Vanderbilt.edu

    Migration of cells up the chemoattractant gradients is mediated by the binding of chemoattractants to G protein-coupled receptors and activation of a network of coordinated excitatory and inhibitory signals. Although the excitatory process has been well studied, the molecular nature of the inhibitory signals remains largely elusive. Here we report that the receptor for activated C kinase 1 (RACK1), a novel binding protein of heterotrimeric G protein betagamma (G betagamma) subunits, acts as a negative regulator of directed cell migration. After chemoattractant-induced polarization of Jurkat and neutrophil-like differentiated HL60 (dHL60) cells, RACK1 interacts with G betagamma and is recruited to the leading edge. Down-regulation of RACK1 dramatically enhances chemotaxis of cells, whereas overexpression of RACK1 or a fragment of RACK1 that retains G betagamma-binding capacity inhibits cell migration. Further studies reveal that RACK1 does not modulate cell migration through binding to other known interacting proteins such as PKC beta and Src. Rather, RACK1 selectively inhibits G betagamma-stimulated phosphatidylinositol 3-kinase gamma (PI3K gamma) and phospholipase C (PLC) beta activity, due to the competitive binding of RACK1, PI3K gamma, and PLC beta to G betagamma. Taken together, these findings provide a novel mechanism of regulating cell migration, i.e., RACK1-mediated interference with G betagamma-dependent activation of key effectors critical for chemotaxis.

    Funded by: NEI NIH HHS: EY010291, R01 EY010291

    Molecular biology of the cell 2008;19;9;3909-22

  • 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

  • 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

  • G Protein betagamma subunits augment UVB-induced apoptosis by stimulating the release of soluble heparin-binding epidermal growth factor from human keratinocytes.

    Seo M, Lee MJ, Heo JH, Lee YI, Kim Y, Kim SY, Lee ES and Juhnn YS

    Department of Biochemistry and Molecular Biology and Cancer Research Institute, Seoul National University College of Medicine, Seoul 110-779.

    UV radiation induces various cellular responses by regulating the activity of many UV-responsive enzymes, including MAPKs. The betagamma subunit of the heterotrimeric GTP-binding protein (Gbetagamma) was found to mediate UV-induced p38 activation via epidermal growth factor receptor (EGFR). However, it is not known how Gbetagamma mediates the UVB-induced activation of EGFR, and thus we undertook this study to elucidate the mechanism. Treatment of HaCaT-immortalized human keratinocytes with conditioned medium obtained from UVB-irradiated cells induced the phosphorylations of EGFR, p38, and ERK but not that of JNK. Blockade of heparin-binding EGF-like growth factor (HB-EGF) by neutralizing antibody or CRM197 toxin inhibited the UVB-induced activations of EGFR, p38, and ERK in normal human epidermal keratinocytes and in HaCaT cells. Treatment with HB-EGF also activated EGFR, p38, and ERK. UVB radiation stimulated the processing of pro-HB-EGF and increased the secretion of soluble HB-EGF in medium, which was quantified by immunoblotting and protein staining. In addition, treatment with CRM179 toxin blocked UV-induced apoptosis, but HB-EGF augmented this apoptosis. Moreover, UVB-induced apoptosis was reduced by inhibiting EGFR or p38. The overexpression of Gbeta(1)gamma(2) increased EGFR-activating activity and soluble HB-EGF content in conditioned medium, but the sequestration of Gbetagamma by the carboxyl terminus of G protein-coupled receptor kinase 2 (GRK2ct) produced the opposite effect. The activation of Src increased UVB-induced, Gbetagamma-mediated HB-EGF secretion, but the inhibition of Src blocked that. Overexpression of Gbetagamma increased UVB-induced apoptosis, and the overexpression of GRK2ct decreased this apoptosis. We conclude that Gbetagamma mediates UVB-induced human keratinocyte apoptosis by augmenting the ectodomain shedding of HB-EGF, which sequentially activates EGFR and p38.

    The Journal of biological chemistry 2007;282;34;24720-30

  • 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

  • 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

  • Association of the Asn306Ser variant of the SP4 transcription factor and an intronic variant in the beta-subunit of transducin with digenic disease.

    Gao YQ, Danciger M, Ozgul RK, Gribanova Y, Jacobson S and Farber DB

    Jules Stein Eye Institute, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095, USA.

    Purpose: SP4 is a transcription factor abundantly expressed in retina that binds to the GC promoter region of photoreceptor signal transduction genes. We have previously shown that SP4 may be involved in the transcriptional activation of these genes alone or together with other transcription factors such as SP1, neural retina leucine zipper protein (NRL), and cone-rod homeobox gene (CRX). Since mutations in NRL and CRX are involved in inherited retinal degenerations, SP4 was considered a good candidate for mutation screening in patients with this type of diseases. The purpose of this work, therefore, was to investigate possible mutations in SP4 in a cohort of patients affected with different forms of retinal degenerations.

    Methods: 270 unrelated probands with various forms of retinal degeneration including autosomal dominant and autosomal recessive retinitis pigmentosa (RP), autosomal dominant and autosomal recessive cone-rod dystrophy (CRD), and Leber's congenital amaurosis (LCA), were screened for mutations in the SP4 gene. Single strand conformation polymorphism (SSCP) analysis was performed on the six SP4 gene exons including flanking regions followed by direct sequencing of SSCP variants.

    Results: Nine different sequence variants were found in 29 patients, four in introns and five in exons. Many of the probands were previously screened for mutations in the genes encoding the alpha-, beta- and gamma-subunits of rod-specific cGMP phosphodiesterase (PDE6A, PDE6B, PDE6G), the beta-subunit of rod-specific transducin (GNB1), and peripherin/rds (RDS). One group of seven probands of Hispanic background that included five with arRP, one with RP of unknown inheritance (isolate) and 1 with arCRD carried an Asn306Ser mutation in SP4. Of the seven, the isolate case was homozygous and the other 6 heterozygous for the variant. Two arRP and the arCRD probands carried an additional intronic GNB1 variant. DNA from the family members of the arCRD proband could not be obtained, but for the other two families, all affected members and none of the unaffected carried both the SP4 Asn306Ser allele and the GNB1 intronic variant.

    Conclusions: If mutations in SP4 do cause retinal degenerative disease, their frequency would be low. While digenic disease with the SP4 Asn306Ser and the GNB1 intronic variant alleles has not been established, neither has it been ruled out. This leaves open the possibility of a cooperative involvement of SP4 and GNB1 in the normal function of the retina.

    Funded by: NEI NIH HHS: EY02651, R01 EY002651

    Molecular vision 2007;13;287-92

  • Conditional stimulation of type V and VI adenylyl cyclases by G protein betagamma subunits.

    Gao X, Sadana R, Dessauer CW and Patel TB

    Department of Pharmacology and Experimental Therapeutics, Loyola University Chicago, Stritch School of Medicine, Maywood, Illinois 60153, USA.

    In a yeast two-hybrid screen of mouse brain cDNA library, using the N-terminal region of human type V adenylyl cyclase (hACV) as bait, we identified G protein beta2 subunit as an interacting partner. Additional yeast two-hybrid assays showed that the Gbeta(1) subunit also interacts with the N-terminal segments of hACV and human type VI adenylyl cyclase (hACVI). In vitro adenylyl cyclase (AC) activity assays using membranes of Sf9 cells expressing hACV or hACVI showed that Gbetagamma subunits enhance the activity of these enzymes provided either Galpha(s) or forskolin is present. Deletion of residues 77-151, but not 1-76, in the N-terminal region of hACVI obliterated the ability of Gbetagamma subunits to conditionally stimulate the enzyme. Likewise, activities of the recombinant, engineered, soluble forms of ACV and ACVI, which lack the N termini, were not enhanced by Gbetagamma subunits. Transfection of the C terminus of G protein receptor kinase 2 to sequester endogenous Gbetagamma subunits attenuated the ability of isoproterenol to increase cAMP accumulation in COS-7 cells overexpressing hACVI even when G(i) was inactivated by pertussis toxin. Therefore, we conclude that the N termini of human hACV and hACVI are necessary for interactions with, and regulation by, Gbetagamma subunits both in vitro and in intact cells. Moreover, Gbetagamma subunits derived from a source(s) other than G(i) are necessary for the full activation of hACVI by isoproterenol in intact cells.

    Funded by: NHLBI NIH HHS: HL59679; NIGMS NIH HHS: GM060419, GM073181, R01 GM060419, R01 GM060419-07

    The Journal of biological chemistry 2007;282;1;294-302

  • A screen for mutations in the transducin gene GNB1 in patients with autosomal dominant retinitis pigmentosa.

    Mylvaganam GH, McGee TL, Berson EL and Dryja TP

    Ocular Molecular Genetics Institute and the Berman-Gund Laboratory for the Study of Retinal Degenerations, Harvard Medical School, Massachusetts Eye and Ear Infirmary, Boston, MA 02114, USA.

    Purpose: To search for mutations in the GNB1 gene (coding for the transducin beta1-subunit protein) in patients with autosomal dominant retinitis pigmentosa.

    Methods: We screened 185 unrelated patients with autosomal dominant retinitis pigmentosa (ADRP) using direct genomic sequencing of the three non-coding exons and 9 coding exons, along with immediately flanking intron DNA.

    Results: We found 2 polymorphisms, one in intron 1 with a minor allele frequency of 24%, and one in intron 6 with a minor allele frequency of 12% among the 185 patients. Two rare variants (minor allele frequency <1%) were found in the 3' untranslated region of exon 12. No changes were found in the open reading frame (exons 3-11) or in the noncoding exons 1 and 2.

    Conclusions: No likely pathogenic GNB1 mutations have been found in any of 185 unrelated patients with ADRP. This result would be expected if hemizygosity for GNB1 does not result in ADRP or is a rare cause of ADRP.

    Funded by: NEI NIH HHS: EY00169, EY08683, P30 EY014104

    Molecular vision 2006;12;1496-8

  • Identification of Gnr1p, a negative regulator of G alpha signalling in Schizosaccharomyces pombe, and its complementation by human G beta subunits.

    Goddard A, Ladds G, Forfar R and Davey J

    Department of Biological Sciences, University of Warwick, Coventry, UK. A.D.Goddard@warwick.ac.uk

    G protein-coupled receptors (GPCRs) are involved in the response of eukaryotic cells to a wide variety of stimuli, traditionally mediating their effects through heterotrimeric G proteins comprised of G alpha, G beta and G gamma subunits. The fission yeast Schizosaccharomyces pombe is an established tool for GPCR research, possessing two G alpha-dependent signalling cascades. A complete G alpha beta gamma complex has been characterised for the glucose-sensing pathway, but only the G alpha subunit, Gpa1p, has been identified in the pheromone-response pathway. Here, we report the use of the yeast two-hybrid system to identify a novel protein, Gnr1p, which interacts with Gpa1p. Gnr1p is predicted to contain seven WD repeats and to adopt a structure similar to typical G beta subunits. Disruption and overexpression studies reveal that Gnr1p negatively regulates the pheromone-response pathway but is not required for signalling. Human G beta subunits complement the loss of Gnr1p, functioning as negative regulators of G alpha signalling in fission yeast.

    Fungal genetics and biology : FG & B 2006;43;12;840-51

  • 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

  • A sperm component, HSD-3.8 (SPAG1), interacts with G-protein beta 1 subunit and activates extracellular signal-regulated kinases (ERK).

    Liu N, Qiao Y, Cai C, Lin W, Zhang J, Miao S, Zong S, Koide SS and Wang L

    National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, Peking Union Medical College, 5 Dong Dan San Tiao, Beijing, 100005, People Republic of China.

    HSD-3.8 cDNA (accession number AF311312) encodes a human sperm component. A 0.7 kb fragment (HSD-0.7) containing three immunological epitopes of HSD-3.8 cDNA was prepared and expressed in E. coli. Immunization of female rats with the recombinant HSD-0.7 proteins induced infertility. A cDNA fragment encoding the C-terminal 144 amino acids of human G-protein beta l subunit (Gbeta1-C144) was screened by yeast two-hybrid, when HSD-0.7 segment was used as a bait. Recombinant His6-tagged-Gbeta1-C144 protein was expressed in E. coli BL21 and Anti-Gbeta1 serum was raised with purified Gbeta1-C144. HA-tagged HSD-0.7 and FLAG-tagged Gbeta1 plasmids were constructed and co-transfected into human embryonal kidney 293 cells. Two proteins were localized at superimposable sites in the cytoplasm, and they formed a complex when 500 micromol/L GDP existed. Overexpression of HSD-0.7 activated the G-protein-mediated extracellular signal-regulated kinases (ERK1/2); however, the truncated fragments of HSD-0.7, which lacked either TPR domain or P-loop, lost the ability to activate the ERK1/2 pathway. Further study revealed that the activation of ERK1/2 was protein kinase C (PKC) rather than Ras dependent. These results provide evidence that HSD-3.8 present in spermatocytes and sperm may participate in spermatogenesis and fertilization process by activating the PKC-dependent ERK1/2 signal transduction pathway.

    Frontiers in bioscience : a journal and virtual library 2006;11;1679-89

  • 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

  • Gialpha and Gbeta subunits both define selectivity of G protein activation by alpha2-adrenergic receptors.

    Gibson SK and Gilman AG

    Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA. scott.gibson@utsouthwestern.edu

    Previous studies of the specificity of receptor interactions with G protein subunits in living cells have relied on measurements of second messengers or other downstream responses. We have examined the selectivity of interactions between alpha2-adrenergic receptors (alpha2R) and various combinations of Gialpha and Gbeta subunit isoforms by measuring changes in FRET between Gialpha-yellow fluorescent protein and cyan fluorescent protein-Gbeta chimeras in HeLa cells. All combinations of Gialpha1, -2, or -3 with Gbeta1, -2, or -4 were activated to some degree by endogenous alpha2Rs as judged by agonist-dependent decreases in FRET. The degree of G protein activation is determined by the combination of Gialpha and Gbeta subunits rather than by the identity of an individual subunit. RT-PCR analysis and small interfering RNA knockdown of alpha2R subtypes, followed by quantification of radiolabeled antagonist binding, demonstrated that HeLa cells express alpha2a- and alpha2b-adrenergic receptor isoforms in a 2:1 ratio. Increasing receptor number by overexpression of the alpha2aR subtype minimized the differences among coupling preferences for Gialpha and Gbeta isoforms. The molecular properties of each Gialpha, Gbeta, and alpha2-adrenergic receptor subtype influence signaling efficiency for the alpha2-adrenergic receptor-mediated signaling pathway.

    Funded by: NIGMS NIH HHS: GM34497, R01 GM034497, R37 GM034497

    Proceedings of the National Academy of Sciences of the United States of America 2006;103;1;212-7

  • 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

  • G betagamma binds histone deacetylase 5 (HDAC5) and inhibits its transcriptional co-repression activity.

    Spiegelberg BD and Hamm HE

    Department of Pharmacology, Vanderbilt University, Nashville, Tennessee 37232, USA.

    In a yeast two-hybrid screen designed to identify novel effectors of the G betagamma subunit of heterotrimeric G proteins, we found that G betagamma binds to histone deacetylase 5 (HDAC5), an enzyme involved in a pathway not previously recognized to be directly impacted by G proteins. Formation of the G beta1gamma2-HDAC5 complex in mammalian cells can be blocked by overexpression of G alpha(o), and this inhibition is relieved by activation of alpha2A-adrenergic receptor, suggesting that the interaction occurs in a signal-dependent manner. The C-terminal domain of HDAC5 binds directly to G betagamma through multiple motifs, and overexpression of this domain mimics the C terminus of G protein-coupled receptor kinase 2, a known G betagamma scavenger, in its ability to inhibit the G betagamma/HDAC5 interaction. The C terminus of HDAC4 shares significant similarity with that of HDAC5, and accordingly, HDAC4 is also able to form complexes with G beta1gamma2 in cultured cells, suggesting that the C-terminal domain of class II HDACs is a general G betagamma binding motif. Activation of a G(i/o)-coupled receptor results in a time-dependent activation of MEF2C, an HDAC5-regulated transcription factor, whereas inhibition of the interaction with a G betagamma scavenger inhibits MEF2C activity, suggesting a reduced potency of HDAC5-mediated inhibition. Taken together, these data imply that HDAC5 and possibly other class II HDACs can be added to the growing list of G betagamma effectors.

    Funded by: NEI NIH HHS: EY010291; NIMH NIH HHS: T32 MH65215-02

    The Journal of biological chemistry 2005;280;50;41769-76

  • Specific in vivo binding of activator of G protein signalling 1 to the Gbeta1 subunit.

    Hiskens R, Vatish M, Hill C, Davey J and Ladds G

    Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, UK.

    Activator of G protein signalling 1 (AGS1) is a Ras-like protein that affects signalling through heterotrimeric G proteins. Previous in vitro studies suggest that AGS1 can bind to G(alpha)-GDP subunits and promote nucleotide exchange, leading to activation of intracellular signalling pathways. This model is consistent with in vivo evidence demonstrating that AGS1 activates both G(alpha)- and G(betagamma)-dependent pathways in the absence of ligand. However, it does not easily explain how AGS1 blocks G(betagamma)-dependent, but not G(alpha)-dependent, signalling following receptor activation. We have used yeast two hybrid analysis and co-immunoprecipitation studies in mammalian cells to demonstrate a direct interaction between AGS1 and the G(beta1) subunit of heterotrimeric G proteins. The interaction is specific for G(beta1) and involves the cationic region of AGS1 and the C-terminal region of G(beta1). Possible implications of this novel interaction for the activity of AGS1 are discussed.

    Biochemical and biophysical research communications 2005;337;4;1038-46

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

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

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

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

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

    Nature 2005;437;7062;1173-8

  • 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

  • G protein-gated inhibitory module of N-type (ca(v)2.2) ca2+ channels.

    Agler HL, Evans J, Tay LH, Anderson MJ, Colecraft HM and Yue DT

    Department of Biomedical Engineering and Ca2+ Signals Laboratory, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.

    Voltage-dependent G protein (Gbetagamma) inhibition of N-type (CaV2.2) channels supports presynaptic inhibition and represents a central paradigm of channel modulation. Still controversial are the proposed determinants for such modulation, which reside on the principal alpha1B channel subunit. These include the interdomain I-II loop (I-II), the carboxy tail (CT), and the amino terminus (NT). Here, we probed these determinants and related mechanisms, utilizing compound-state analysis with yeast two-hybrid and mammalian cell FRET assays of binding among channel segments and G proteins. Chimeric channels confirmed the unique importance of NT. Binding assays revealed selective interaction between NT and I-II elements. Coexpressing NT peptide with Gbetagamma induced constitutive channel inhibition, suggesting that the NT domain constitutes a G protein-gated inhibitory module. Such inhibition was limited to NT regions interacting with I-II, and G-protein inhibition was abolished within alpha1B channels lacking these NT regions. Thus, an NT module, acting via interactions with the I-II loop, appears fundamental to such modulation.

    Neuron 2005;46;6;891-904

  • 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

  • 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

  • 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

  • Increased expression of Gi-coupled muscarinic acetylcholine receptor and Gi in atrium of elderly diabetic subjects.

    Richardson MD, Kilts JD and Kwatra MM

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

    In an ongoing investigation of the effects of age on G protein-coupled receptor signaling in human atrial tissue, we have found that the density of atrial muscarinic acetylcholine receptor (mAChR) increases with age but reaches statistical significance only in patients with diabetes. Moreover, we find that in elderly subjects of similar ages, those with diabetes have 1.7-fold higher levels of Galpha(i2) and twofold higher levels of Gbeta(1). Diabetes does not affect other atrial G proteins, including Galpha(i3,) Galpha(s), Galpha(o), and Gbeta(2). These data represent the first demonstration of an increase in a G(i)-coupled receptor, Galpha(i2), and Gbeta(1), in atrium of patients with diabetes. These findings suggest a molecular explanation for the increased risk of cardiac disease in patients with diabetes, because increased signaling through G(i) has been shown to lead to the development of dilated cardiomyopathy.

    Funded by: NIA NIH HHS: AG00029, AG15817

    Diabetes 2004;53;9;2392-6

  • 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

  • Mapping the Gbetagamma-binding sites in GIRK1 and GIRK2 subunits of the G protein-activated K+ channel.

    Ivanina T, Rishal I, Varon D, Mullner C, Frohnwieser-Steinecke B, Schreibmayer W, Dessauer CW and Dascal N

    Department of Physiology and Pharmacology, Sackler School of Medicine, Tel Aviv University, Ramat Aviv 69978, Israel.

    G protein-activated K+ channels (Kir3 or GIRK) are activated by direct binding of Gbetagamma. The binding sites of Gbetagamma in the ubiquitous GIRK1 (Kir3.1) subunit have not been unequivocally charted, and in the neuronal GIRK2 (Kir3.2) subunit the binding of Gbetagamma has not been studied. We verified and extended the map of Gbetagamma-binding sites in GIRK1 by using two approaches: direct binding of Gbetagamma to fragments of GIRK subunits (pull down), and competition of these fragments with the Galphai1 subunit for binding to Gbetagamma. We also mapped the Gbetagamma-binding sites in GIRK2. In both subunits, the N terminus binds Gbetagamma. In the C terminus, the Gbetagamma-binding sites in the two subunits are not identical; GIRK1, but not GIRK2, has a previously unrecognized Gbetagamma-interacting segments in the first half of the C terminus. The main C-terminal Gbetagamma-binding segment found in both subunits is located approximately between amino acids 320 and 409 (by GIRK1 count). Mutation of C-terminal leucines 262 or 333 in GIRK1, recognized previously as crucial for Gbetagamma regulation of the channel, and of the corresponding leucines 273 and 344 in GIRK2 dramatically altered the properties of K+ currents via GIRK1/GIRK2 channels expressed in Xenopus oocytes but did not appreciably reduce the binding of Gbetagamma to the corresponding fusion proteins, indicating that these residues are mainly important for the regulation of Gbetagamma-induced changes in channel gating rather than Gbetagamma binding.

    Funded by: NIGMS NIH HHS: GM60419, GM68493, R01 GM060419, R01 GM060419-04

    The Journal of biological chemistry 2003;278;31;29174-83

  • Directional sensing requires G beta gamma-mediated PAK1 and PIX alpha-dependent activation of Cdc42.

    Li Z, Hannigan M, Mo Z, Liu B, Lu W, Wu Y, Smrcka AV, Wu G, Li L, Liu M, Huang CK and Wu D

    Department of Genetics and Developmental Biology, University of Connecticut Health Center, Farmington, CT 06030, USA.

    Efficient chemotaxis requires directional sensing and cell polarization. We describe a signaling mechanism involving G beta gamma, PAK-associated guanine nucleotide exchange factor (PIX alpha), Cdc42, and p21-activated kinase (PAK) 1. This pathway is utilized by chemoattractants to regulate directional sensing and directional migration of myeloid cells. Our results suggest that G beta gamma binds PAK1 and, via PAK-associated PIX alpha, activates Cdc42, which in turn activates PAK1. Thus, in this pathway, PAK1 is not only an effector for Cdc42, but it also functions as a scaffold protein required for Cdc42 activation. This G beta gamma-PAK1/PIX alpha/Cdc42 pathway is essential for the localization of F-actin formation to the leading edge, the exclusion of PTEN from the leading edge, directional sensing, and the persistent directional migration of chemotactic leukocytes. Although ligand-induced production of PIP(3) is not required for activation of this pathway, PIP(3) appears to localize the activation of Cdc42 by the pathway.

    Cell 2003;114;2;215-27

  • 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

  • Activation of heterotrimeric G proteins by a high energy phosphate transfer via nucleoside diphosphate kinase (NDPK) B and Gbeta subunits. Specific activation of Gsalpha by an NDPK B.Gbetagamma complex in H10 cells.

    Hippe HJ, Lutz S, Cuello F, Knorr K, Vogt A, Jakobs KH, Wieland T and Niroomand F

    Innere Medizin III-Kardiologie, Universität Heidelberg, Bergheimer Strasse 58, D-69115 Heidelberg, Germany.

    Formation of GTP by nucleoside diphosphate kinase (NDPK) can contribute to G protein activation in vitro. To study the effect of NDPK on G protein activity in living cells, the NDPK isoforms A and B were stably expressed in H10 cells, a cell line derived from neonatal rat cardiomyocytes. Overexpression of either NDPK isoform had no effect on cellular GTP and ATP levels, basal cAMP levels, basal adenylyl cyclase activity, and the expression of G(s)alpha and G(i)alpha proteins. However, co-expression of G(s)alpha led to an increase in cAMP synthesis that was largely enhanced by the expression of NDPK B, but not NDPK A, and that was confirmed by direct measurement of adenylyl cyclase activity. Cells expressing an inactive NDPK B mutant (H118N) exhibited a decreased cAMP formation in response to G(s)alpha. Co-immunoprecipitation studies demonstrated a complex formation of the NDPK with Gbetagamma dimers. The overexpression of NDPK B, but not its inactive mutant or NDPK A, increased the phosphorylation of Gbeta subunits. In summary, our data demonstrate a specific NDPK B-mediated activation of a G protein in intact cells, which is apparently caused by formation of NDPK B.Gbetagamma complexes and which appears to contribute to the receptor-independent activation of heterotrimeric G proteins.

    The Journal of biological chemistry 2003;278;9;7227-33

  • 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

  • The betagamma subunit of heterotrimeric G proteins interacts with RACK1 and two other WD repeat proteins.

    Dell EJ, Connor J, Chen S, Stebbins EG, Skiba NP, Mochly-Rosen D and Hamm HE

    Institute for Neuroscience and Department of Molecular Pharmacology and Biological Chemistry, Northwestern University Medical School, Chicago, Illinois 60613, USA.

    A yeast two-hybrid approach was used to discern possible new effectors for the betagamma subunit of heterotrimeric G proteins. Three of the clones isolated are structurally similar to Gbeta, each exhibiting the WD40 repeat motif. Two of these proteins, the receptor for activated C kinase 1 (RACK1) and the dynein intermediate chain, co-immunoprecipitate with Gbetagamma using an anti-Gbeta antibody. The third protein, AAH20044, has no known function; however, sequence analysis indicates that it is a WD40 repeat protein. Further investigation with RACK1 shows that it not only interacts with Gbeta(1)gamma(1) but also unexpectedly with the transducin heterotrimer Galpha(t)beta(1)gamma(1). Galpha(t) alone does not interact, but it must contribute to the interaction because the apparent EC(50) value of RACK1 for Galpha(t)beta(1)gamma(1) is 3-fold greater than that for Gbeta(1)gamma(1) (0.1 versus 0.3 microm). RACK1 is a scaffold that interacts with several proteins, among which are activated betaIIPKC and dynamin-1 (1). betaIIPKC and dynamin-1 compete with Gbeta(1)gamma(1) and Galpha(t)beta(1)gamma(1) for interaction with RACK1. These findings have several implications: 1) that WD40 repeat proteins may interact with each other; 2) that Gbetagamma interacts differently with RACK1 than with its other known effectors; and/or 3) that the G protein-RACK1 complex may constitute a signaling scaffold important for intracellular responses.

    Funded by: NEI NIH HHS: EY 100291

    The Journal of biological chemistry 2002;277;51;49888-95

  • The role of G protein beta subunits in the release of ATP from human erythrocytes.

    Sprague RS, Bowles EA, Olearczyk JJ, Stephenson AH and Lonigro AJ

    Saint Louis University School of Medicine, MO 63104, USA. spraguer@slu.edu

    Previously, we demonstrated that adenosine triphosphate (ATP) is released from human erythrocytes in response to mechanical deformation and that this release requires activation of a signal-transduction pathway involving adenylyl cyclase and the heterotrimeric G protein, Gs. Here we investigate the role of heterotrimeric G proteins of the Gi subtype in the release of ATP from human erythrocytes. In addition, we determined the profile of heterotrimeric G protein beta subunits present in these erythrocyte membranes. The activity of Gi was stimulated by incubation of erythrocytes (20% hematocrit) with mastoparin (10 microM). ATP release was measured using the luciferin/luciferase assay. Heterotrimeric G protein beta subunits present in erythrocyte membranes were resolved using gel electrophoresis and subunit specific antibodies. Incubation of human erythrocytes with mastoparan (an activator of Gi/o) resulted in a 4.1 +/- 0.6-fold increase in ATP present in the medium (P<0.01). Human erythrocyte membranes stain positively for beta subunit types 1, 2, 3 and 4, all of which been reported to activate of some isoforms of adenylyl cyclase. Activation of the heterotrimeric G protein, Gi, results in ATP release from erythrocytes. This effect is may be related to the activity of beta subunits associated with this G protein in the human erythrocyte.

    Funded by: NHLBI NIH HHS: HL-39226, HL-51298, HL-52675

    Journal of physiology and pharmacology : an official journal of the Polish Physiological Society 2002;53;4 Pt 1;667-74

  • 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

  • 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

  • Genomic characterization of the human heterotrimeric G protein alpha, beta, and gamma subunit genes.

    Hurowitz EH, Melnyk JM, Chen YJ, Kouros-Mehr H, Simon MI and Shizuya H

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

    Heterotrimeric guanine nucleotide binding proteins (G proteins) transduce extracellular signals received by transmembrane receptors to effector proteins. Each subunit of the G protein complex is encoded by a member of one of three corresponding gene families. Currently, 16 different members of the alpha subunit family, 5 different members of the beta subunit family, and 11 different members of the gamma subunit family have been described in mammals. Here we have identified and characterized Bacterial Artificial Chromosomes (BACs) containing the human homologs of each of the alpha, beta, and gamma subunit genes as well as a G alpha11 pseudogene and a previously undiscovered G gamma5-like gene. The gene structure and chromosome location of each gene was determined, as were the orientations of paired genes. These results provide greater insight into the evolution and functional diversity of the mammalian G protein subunit genes.

    Funded by: NHGRI NIH HHS: HG01464; NIGMS NIH HHS: GM34236

    DNA research : an international journal for rapid publication of reports on genes and genomes 2000;7;2;111-20

  • 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

  • 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

  • Ggamma13 colocalizes with gustducin in taste receptor cells and mediates IP3 responses to bitter denatonium.

    Huang L, Shanker YG, Dubauskaite J, Zheng JZ, Yan W, Rosenzweig S, Spielman AI, Max M and Margolskee RF

    Howard Hughes Medical Institute, Mount Sinai School of Medicine of New York University, Box 1677, One Gustave L. Levy Place, New York, New York 10029, USA.

    Gustducin is a transducin-like G protein selectively expressed in taste receptor cells. The alpha subunit of gustducin (alpha-gustducin) is critical for transduction of responses to bitter or sweet compounds. We identified a G-protein gamma subunit (Ggamma13) that colocalized with alpha-gustducin in taste receptor cells. Of 19 alpha-gustducin/Ggamma13-positive taste receptor cells profiled, all expressed the G protein beta3 subunit (Gbeta3); approximately 80% also expressed Gbeta1. Gustducin heterotrimers (alpha-gustducin/Gbeta1/Ggamma13) were activated by taste cell membranes plus bitter denatonium. Antibodies against Ggamma13 blocked the denatonium-induced increase of inositol trisphosphate (IP3) in taste tissue. We conclude that gustducin heterotrimers transduce responses to bitter and sweet compounds via alpha-gustducin's regulation of phosphodiesterase (PDE) and Gbetagamma's activation of phospholipase C (PLC).

    Funded by: NIDCD NIH HHS: DC03155; NIDCR NIH HHS: DE10754; NIMH NIH HHS: MH57241; ...

    Nature neuroscience 1999;2;12;1055-62

  • 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

  • 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

  • Binding of the G protein betagamma subunit to multiple regions of G protein-gated inward-rectifying K+ channels.

    Huang CL, Jan YN and Jan LY

    Department of Physiology, the University of California at San Francisco, 94143-0724, USA. chuan1@mednet.swmed.edu

    We have previously shown that direct binding of the betagamma subunit of G protein (G betagamma) to both the N-terminal domain and the C-terminal domain of a cloned G protein-gated inward-rectifying K+ channel subunit, GIRK1, is important for channel activation. We have now further localized the G betagamma binding region in the N-terminal domain of GIRK1 to amino acids 34-86 and the G betagamma binding region in the C-terminal domain of GIRK1 to two separate fragments of amino acids 318-374 and amino acids 390-462. Of the four cloned mammalian GIRK subunits, GIRK1-4, GIRK1 and 4 form heteromeric K+ channels in the heart and similar channels in the brain include heteromultimers of GIRK1 and 2, and possibly other GIRK homomultimers and heteromultimers. We found that the N-terminal and the C-terminal domains of all four GIRKs bound G betagamma. The G betagamma binding activities for the C-terminal domains of GIRK2-4 were lower than that for the C-terminal domain of GIRK1. The higher G betagamma binding activity for the C-terminal domain of GIRK1 is due to amino acids 390-462 which are unique to GIRK1. We also found that the N-terminal and C-terminal domains of GIRKs interacted with each other, and the N-terminal domain of either GIRK1 or GIRK4 together with the C-terminal domain of GIRK1 exhibited much enhanced binding of G betagamma. These results are consistent with the idea that the N- and C-terminal domains of the cardiac G protein-gated K+ channel subunits may interact with each other to form higher affinity binding site(s) for G betagamma.

    FEBS letters 1997;405;3;291-8

  • Large-scale concatenation cDNA sequencing.

    Yu W, Andersson B, Worley KC, Muzny DM, Ding Y, Liu W, Ricafrente JY, Wentland MA, Lennon G and Gibbs RA

    A total of 100 kb of DNA derived from 69 individual human brain cDNA clones of 0.7-2.0 kb were sequenced by concatenated cDNA sequencing (CCS), whereby multiple individual DNA fragments are sequenced simultaneously in a single shotgun library. The method yielded accurate sequences and a similar efficiency compared with other shotgun libraries constructed from single DNA fragments (> 20 kb). Computer analyses were carried out on 65 cDNA clone sequences and their corresponding end sequences to examine both nucleic acid and amino acid sequence similarities in the databases. Thirty-seven clones revealed no DNA database matches, 12 clones generated exact matches (> or = 98% identity), and 16 clones generated nonexact matches (57%-97% identity) to either known human or other species genes. Of those 28 matched clones, 8 had corresponding end sequences that failed to identify similarities. In a protein similarity search, 27 clone sequences displayed significant matches, whereas only 20 of the end sequences had matches to known protein sequences. Our data indicate that full-length cDNA insert sequences provide significantly more nucleic acid and protein sequence similarity matches than expressed sequence tags (ESTs) for database searching.

    Funded by: NHGRI NIH HHS: 1F32 HG00169-01, F32 HG000169, F33 HG000210, P30 HG00210-05, R01 HG00823, U54 HG003273

    Genome research 1997;7;4;353-8

  • 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

  • Phosphorylation-dependent activation of the Ras-GRF/CDC25Mm exchange factor by muscarinic receptors and G-protein beta gamma subunits.

    Mattingly RR and Macara IG

    Department of Pathology, University of Vermont College of Medicine, Burlington 05405, USA.

    Muscarinic receptors activate Ras through a pathway distinct from that mediated through translocation of the exchange factor mSos1 by receptor tyrosine kinases. Here we report that muscarinic receptors can activate another Ras exchange factor, CDC25Mm, or p140Ras-GRF (refs 5,6). In NIH-3T3 cells expressing subtype 1 human muscarinic receptors (hm1), the agonist carbachol selectively increased the specific activity and phosphorylation state of epitope-tagged Ras-GRF. This stimulation was reversed by protein phosphatase 1 (PP1), and prevented by transducin alpha-subunits. Carbachol treatment of neonatal rat brain explants increasd Ras exchange factor activity and the phosphorylation state of endogenous Ras-GRF. In COS-7 cells, cotransfection of hm1 or hm2 receptors with Ras-GRF conferred carbachol-dependent increases in exchange-factor activity, whereas cotransfection with G-protein beta gamma subunits caused a constitutive activation that was sensitive to PP1. These results demonstrate a G-protein-coupled mechanism for Ras activation, mediated by p140 Ras-GRF.

    Nature 1996;382;6588;268-72

  • A "double adaptor" method for improved shotgun library construction.

    Andersson B, Wentland MA, Ricafrente JY, Liu W and Gibbs RA

    Department of Molecular and Human Genetics, Baylor College of Medicine, One Baylor Plaza, Houston, Texas, 77030, USA.

    The efficiency of shotgun DNA sequencing depends to a great extent on the quality of the random-subclone libraries used. We here describe a novel "double adaptor" strategy for efficient construction of high-quality shotgun libraries. In this method, randomly sheared and end-repaired fragments are ligated to oligonucleotide adaptors creating 12-base overhangs. Nonphosphorylated oligonucleotides are used, which prevents formation of adaptor dimers and ensures efficient ligation of insert to adaptor. The vector is prepared from a modified M13 vector, by KpnI/PstI digestion followed by ligation to oligonucleotides with ends complementary to the overhangs created in the digest. These adaptors create 5'-overhangs complementary to those on the inserts. Following annealing of insert to vector, the DNA is directly used for transformation without a ligation step. This protocol is robust and shows three- to fivefold higher yield of clones compared to previous protocols. No chimeric clones can be detected and the background of clones without an insert is <1%. The procedure is rapid and shows potential for automation.

    Funded by: NHGRI NIH HHS: R01 HG00823

    Analytical biochemistry 1996;236;1;107-13

  • 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

  • Crystal structure of a G-protein beta gamma dimer at 2.1A resolution.

    Sondek J, Bohm A, Lambright DG, Hamm HE and Sigler PB

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

    Many signalling cascades use seven-helical transmembrane receptors coupled to heterotrimeric G proteins (G alpha beta gamma) to convert extracellular signals into intracellular responses. Upon nucleotide exchange catalysed by activated receptors, heterotrimers dissociate into GTP-bound G alpha subunits and G beta gamma dimers, either of which can modulate many downstream effectors. Here we use multiwavelength anomalous diffraction data to solve the crystal structure of the beta gamma dimer of the G protein transducin. The beta-subunit is primarily a seven-bladed beta-propeller that is partially encircled by an extended gamma-subunit. The beta-propeller, which contains seven structurally similar WD repeats, defines the stereochemistry of the WD repeat and the probable architecture of all WD-repeat-containing domains. The structure details interactions between G protein beta- and gamma-subunits and highlights regions implicated in effector modulation for the conserved family of G protein beta gamma dimers.

    Nature 1996;379;6563;369-74

  • 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

  • Isolation of cDNA clones encoding eight different human G protein gamma subunits, including three novel forms designated the gamma 4, gamma 10, and gamma 11 subunits.

    Ray K, Kunsch C, Bonner LM and Robishaw JD

    Weis Center for Research, Geisinger Clinic, Danville, Pennsylvania 17822, USA.

    With the growing awareness that the G protein beta and gamma subunits directly regulate the activities of various enzymes and ion channels, the importance of identifying and characterizing these subunits is underscored. In this paper, we report the isolation of cDNA clones encoding eight different human gamma subunits, including three novel forms designated gamma 4, gamma 10, and gamma 11. The predicted protein sequence of gamma 4 shares the most identity (60-77%) with gamma 2, gamma 3, and gamma 7 and the least identity (38%) with gamma 1. The gamma 4 is modified by a geranylgeranyl group and is capable of interacting with both beta 1 and beta 2 but not with beta 3. The predicted protein sequence of gamma 10 shows only modest to low identity (35-53%) with the other known gamma subunits, with most of the differences concentrated in the N-terminal region, suggesting gamma 10 may interact with a unique subclass of alpha. The gamma 10 is modified by a geranylgeranyl group and is capable of interacting with beta 1 and beta 2 but not with beta 3. Finally, the predicted protein sequence of gamma 11 shows the most identity to gamma 1 (76% identity) and the least identity to the other known gamma (33-44%). Unlike most of the other known gamma subunits, gamma 11 is modified by a farnesyl group and is not capable of interacting with beta 2. The close resemblance of gamma 11 to gamma 1 raises intriguing questions regarding its function since the mRNA for gamma 11 is abundantly expressed in all tissues tested except for brain, whereas the mRNA for gamma 1 is expressed only in the retina where the protein functions in phototransduction.

    The Journal of biological chemistry 1995;270;37;21765-71

  • Mitogen-activated protein kinase activation requires two signal inputs from the human anaphylatoxin C5a receptor.

    Buhl AM, Osawa S and Johnson GL

    Department of Chemistry, Aarhus University, Denmark.

    The anaphylatoxin C5a receptor activates the Ras/Raf/mitogen-activated protein (MAP) kinase pathway in human neutrophils. The signal pathways involved in Ras/Raf/MAP kinase activation in response to C5a and other chemoattractant receptors is poorly understood. Stimulation of the C5a receptor expressed in HEK293 cells results in modest MAP kinase activation, which is inhibited by pertussis toxin-catalyzed ADP-ribosylation of G(i). Coexpression of the C5a receptor and the G16 alpha subunit (alpha 16) results in the G16-mediated activation of phospholipase C beta and a robust MAP kinase activation. Pertussis toxin treatment of C5a receptor/alpha 16-cotransfected cells inhibits C5a stimulation of MAP kinase activity approximately 60% relative to the control response. Similarly, the protein kinase C inhibitor, GF109203X inhibits activation of MAP kinase activation in C5a receptor/alpha 16-cotransfected cells by 60%; the protein kinase C inhibitor does not affect the modest C5a receptor response in the absence of alpha 16 expression. These results demonstrate that two independent signals are required for the maximal activation of MAP kinase by G protein-coupled receptors.

    Funded by: NIDDK NIH HHS: DK37871; NIGMS NIH HHS: GM30324

    The Journal of biological chemistry 1995;270;34;19828-32

  • 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

  • Purification of recombinant G proteins from Sf9 cells by hexahistidine tagging of associated subunits. Characterization of alpha 12 and inhibition of adenylyl cyclase by alpha z.

    Kozasa T and Gilman AG

    Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas 75235.

    A method is described for purification of G protein alpha and beta gamma subunits from Sf9 cells infected with recombinant baculoviruses. The subunit to be purified is coexpressed with an associated subunit bearing a hexahistidine tag. After adsorption of the oligomer to a Ni(2+)-containing column, the subunit to be purified is eluted specifically by promoting subunit dissociation with AIF4-. The alpha subunits of G12, Gq, Gz, and Gi1 and the beta 1 gamma 2 subunit complex were easily and efficiently purified by this method. Results was superior to established procedures in all cases. Purified alpha 12 was characterized for the first time. The protein has a slow rate of guanine nucleotide exchange (kon, GTP gamma S = 0.01 min-1) and a very slow kcat for hydrolysis of GTP (0.1-0.2 min-1). GTP gamma S (guanosine 5' -3-O- (thio)triphosphate) alpha 12 does not influence the activity of several adenylyl cyclases or phospholipases. Activated alpha z inhibits the activity of type I and type V adenylyl cyclases. It is a somewhat more potent inhibitor of type V adenylyl cyclase than is activated alpha i1.

    The Journal of biological chemistry 1995;270;4;1734-41

  • 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 protein beta gamma subunits. Simplified purification and properties of novel isoforms.

    Ueda N, Iñiguez-Lluhi JA, Lee E, Smrcka AV, Robishaw JD and Gilman AG

    Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas 75235.

    The beta and gamma subunits of heterotrimeric guanine nucleotide-binding regulatory proteins (G proteins) form tightly associated complexes. To examine functional differences among the large number of possible combinations of unique beta and gamma subunits, we have synthesized and characterized beta gamma complexes containing gamma 5 and gamma 7, two widely distributed gamma subunits. When either gamma 5 or gamma 7 is expressed concurrently with beta 1 or beta 2 subunits in a baculovirus/Sf9 cell system, all four subunit complexes support pertussis toxin-catalyzed ADP-ribosylation of rGi alpha 1 (where "r" indicates recombinant), indicating formation of functional complexes. Each of the complexes was purified by subunit exchange chromatography, using the G203A mutant of rGi alpha 1 as the immobilized ligand. The purified preparations were compared with other recombinant beta gamma subunits, including beta 1 gamma 1 and beta 1 gamma 2, for their ability to modulate type I and II adenylyl cyclase activities; stimulate phosphoinositide-specific phospholipase C beta; support pertussis toxin-catalyzed ADP-ribosylation of rGi alpha 1 and Go alpha; and inhibit steady-state GTP hydrolysis catalyzed by Gs alpha, Go alpha, and myristoylated rGi alpha 2. The results emphasize the unique properties of beta 1 gamma 1. The properties of the complexes containing gamma 5 or gamma 7 were similar to each other and to those of beta 1 gamma 2.

    Funded by: NIGMS NIH HHS: GM31954, GM34497, GM39867; ...

    The Journal of biological chemistry 1994;269;6;4388-95

  • 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

  • Diversity among the beta subunits of heterotrimeric GTP-binding proteins: characterization of a novel beta-subunit cDNA.

    von Weizsäcker E, Strathmann MP and Simon MI

    Institut für Entwicklungsbiologie, Universität Köln, FRG.

    Heterotrimeric guanine nucleotide binding proteins transduce signals from cell surface receptors to intracellular effectors. The alpha subunit is believed to confer receptor and effector specificity on the G protein. This role is reflected in the diversity of genes that encode these subunits. The beta and gamma subunits are thought to have a more passive role in G protein function; biochemical data suggests that beta-gamma dimers are shared among the alpha subunits. However, there is growing evidence for active participation of beta-gamma dimers in some G protein mediated signaling systems. To further investigate this role, we examined the diversity of the beta subunit family in mouse. Using the polymerase chain reaction, we uncovered a new member of this family, G beta 4, which is expressed at widely varying levels in a variety of tissues. The predicted amino acid sequence of G beta 4 is 79% to 89% identical to the three previously known beta subunits. The diversity of beta gene products may be an important corollary to the functional diversity of G proteins.

    Funded by: NIGMS NIH HHS: GM34236

    Biochemical and biophysical research communications 1992;183;1;350-6

  • Chromosomal localization of the genes encoding two forms of the G protein beta polypeptide, beta 1 and beta 3, in man.

    Levine MA, Modi WS and O'Brien SJ

    Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.

    The signal-transducing G proteins are heterotrimers composed of three subunits, alpha, beta, and gamma. Multiple distinctive forms of the alpha, beta, and gamma subunits, each encoded by a distinct gene, have been described. To investigate further the structural diversity of the beta subunits, we recently cloned and characterized a novel cDNA encoding a third form of the G protein beta subunit, which we have termed beta 3. The protein corresponding to beta 3 has not yet been identified. The three forms of the beta subunit show 81-90% amino acid sequence identity. Previous studies had localized the human genes for the beta 1 and beta 2 subunits to chromosomes 1 and 7, respectively. The present studies were designed to determine whether the gene encoding beta 3 is linked to either the beta 1 or the beta 2 gene. Genomic DNA was isolated from a panel of rodent-human hybrid cell lines and analyzed by hybridization to cDNAs for beta 1 and beta 3. Discordancy analysis allowed assignment of the beta 3 gene to chromosome 12 and confirmed the previous assignment of the beta 1 gene to chromosome 1. These results were confirmed and extended by using in situ chromosome hybridization, which permitted the regional localization of the beta 1 gene to 1pter----p31.2 and the beta 3 gene to 12pter----p12.3. Digestion of human genomic DNA with 10 restriction enzymes failed to disclose a restriction fragment length polymorphism for the beta 3 gene. These data indicate that there is considerable diversity in the genomic organization of the beta subunit family.

    Genomics 1990;8;2;380-6

  • Beta-subunits of the human liver Gs/Gi signal-transducing proteins and those of bovine retinal rod cell transducin are identical.

    Codina J, Stengel D, Woo SL and Birnbaumer L

    The complete cDNA encoding the beta-subunit of the human liver signal transducing proteins Gs/Gi (beta G) has been cloned from a lambda gtll library using an oligonucleotide as a screening agent. The cDNA has 3088 nucleotides and an 11 nucleotide poly(A) tail, of which 280 nucleotides constitute the 5'-untranslated region, 1023 form the open reading frame (ORF) and its stop codon, and 1785 are the 3'-untranslated region with two AATAAA cleavage and polyadenylation signals separated by 1467 nucleotides. The ORF codes for a 340 amino acid polypeptide that is identical to that encoded by bovine retinal rod cell cDNA of the beta-subunit of transducin. Yet, it does so by using 87 different codons. Curiously, the 280 nucleotide 5' leader sequence obtained starts with an ATG that is part of another ORF encoding a putative peptide X of 75 amino acids (nucleotide 280 to 55). This work proves for the first time that the beta-subunits of all signal-transducing G-proteins, including transducin, are the same.

    Funded by: NIADDK NIH HHS: AM-19318, AM-27685; NICHD NIH HHS: HD-09581; ...

    FEBS letters 1986;207;2;187-92

Gene lists (10)

Gene List Source Species Name Description Gene count
L00000009 G2C Homo sapiens Human PSD Human orthologues of mouse PSD adapted from Collins et al (2006) 1080
L00000011 G2C Homo sapiens Human clathrin Human orthologues of mouse clathrin coated vesicle genes adapted from Collins et al (2006) 150
L00000012 G2C Homo sapiens Human Synaptosome Human orthologues of mouse synaptosome adapted from Collins et al (2006) 152
L00000015 G2C Homo sapiens Human NRC Human orthologues of mouse NRC adapted from Collins et al (2006) 186
L00000016 G2C Homo sapiens Human PSP Human orthologues of mouse PSP adapted from Collins et al (2006) 1121
L00000033 G2C Homo sapiens Pocklington H2 Human orthologues of cluster 2 (mouse) from Pocklington et al (2006) 13
L00000059 G2C Homo sapiens BAYES-COLLINS-HUMAN-PSD-CONSENSUS Human cortex PSD consensus 748
L00000061 G2C Homo sapiens BAYES-COLLINS-MOUSE-PSD-CONSENSUS Mouse cortex PSD consensus (ortho) 984
L00000069 G2C Homo sapiens BAYES-COLLINS-HUMAN-PSD-FULL Human cortex biopsy PSD full list 1461
L00000071 G2C Homo sapiens BAYES-COLLINS-MOUSE-PSD-FULL Mouse cortex PSD full list (ortho) 1556
© G2C 2014. The Genes to Cognition Programme received funding from The Wellcome Trust and the EU FP7 Framework Programmes:
EUROSPIN (FP7-HEALTH-241498), SynSys (FP7-HEALTH-242167) and GENCODYS (FP7-HEALTH-241995).

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