G2Cdb::Gene report

Gene id
Gene symbol
Homo sapiens
guanine nucleotide binding protein (G protein), alpha 13
G00001111 (Mus musculus)

Databases (7)

ENSG00000120063 (Ensembl human gene)
10672 (Entrez Gene)
356 (G2Cdb plasticity & disease)
GNA13 (GeneCards)
604406 (OMIM)
Marker Symbol
HGNC:4381 (HGNC)
Protein Sequence
Q14344 (UniProt)

Synonyms (2)

  • G13
  • MGC46138

Literature (67)

Pubmed - other

  • G protein subunit Galpha13 binds to integrin alphaIIbbeta3 and mediates integrin "outside-in" signaling.

    Gong H, Shen B, Flevaris P, Chow C, Lam SC, Voyno-Yasenetskaya TA, Kozasa T and Du X

    Department of Pharmacology, University of Illinois at Chicago, 835 South Wolcott Avenue, Room E403, Chicago, IL 60612, USA.

    Integrins mediate cell adhesion to the extracellular matrix and transmit signals within the cell that stimulate cell spreading, retraction, migration, and proliferation. The mechanism of integrin outside-in signaling has been unclear. We found that the heterotrimeric guanine nucleotide-binding protein (G protein) Galpha13 directly bound to the integrin beta3 cytoplasmic domain and that Galpha13-integrin interaction was promoted by ligand binding to the integrin alphaIIbbeta3 and by guanosine triphosphate (GTP) loading of Galpha13. Interference of Galpha13 expression or a myristoylated fragment of Galpha13 that inhibited interaction of alphaIIbbeta3 with Galpha13 diminished activation of protein kinase c-Src and stimulated the small guanosine triphosphatase RhoA, consequently inhibiting cell spreading and accelerating cell retraction. We conclude that integrins are noncanonical Galpha13-coupled receptors that provide a mechanism for dynamic regulation of RhoA.

    Funded by: NHLBI NIH HHS: HL062350, HL068819, HL080264, R01 HL062350, R01 HL062350-09, R01 HL068819, R01 HL068819-08, R01 HL080264, R01 HL080264-04; NIGMS NIH HHS: GM061454, GM074001, R01 GM061454, R01 GM061454-09, R01 GM074001, R01 GM074001-02

    Science (New York, N.Y.) 2010;327;5963;340-3

  • Involvement of aquaporin in thromboxane A2 receptor-mediated, G 12/13/RhoA/NHE-sensitive cell swelling in 1321N1 human astrocytoma cells.

    Saito M, Tanaka H, Sasaki M, Kurose H and Nakahata N

    Department of Cellular Signaling, Graduate School of Pharmaceutical Sciences, Tohoku University, Aoba 6-3, Aramaki, Aoba-ku, Sendai 980-8578, Japan.

    The physiological role of the thromboxane A(2) (TXA(2)) receptor expressed on glial cells remains unclear. We previously reported that 1321N1 human astrocytoma cells pretreated with dibutyryl cyclic AMP (dbcAMP) became swollen in response to U46619, a TXA(2) analogue. In the present study, we examined the detailed mechanisms of TXA(2) receptor-mediated cell swelling in 1321N1 cells. The cell swelling caused by U46619 was suppressed by expression of p115-RGS, an inhibitory peptide of G alpha(12/13) pathway and C3 toxin, an inhibitory protein for RhoA. The swelling was also inhibited by treatment with Y27632, a Rho kinase inhibitor and 5-(ethyl-N-isopropyl)amiloride (EIPA), a Na(+)/H(+)-exchanger inhibitor. Furthermore, cell swelling was suppressed by the pretreatment with aquaporin inhibitors mercury chloride or phloretin in a concentration-dependent manner, suggesting that aquaporins are involved in U46619-induced 1321N1 cell swelling. In fact, U46619 caused [(3)H]H(2)O influx into the cells, which was inhibited by p115-RGS, C3 toxin, EIPA, mercury chloride and phloretin. This is the first report that the TXA(2) receptor mediates water influx through aquaporins in astrocytoma cells via TXA(2) receptor-mediated activation of G alpha(12/13), Rho A, Rho kinase and Na(+)/H(+)-exchanger.

    Cellular signalling 2010;22;1;41-6

  • Suppression of tumor angiogenesis by Galpha(13) haploinsufficiency.

    Chen L, Zhang JJ, Rafii S and Huang XY

    Department of Physiology, Weill Medical College of Cornell University, New York, New York 10065, USA.

    Heterotrimeric G proteins are critical transducers of cellular signaling. Of the four families of G proteins, the physiological function of Galpha(13) is less well understood. Galpha(13) gene-deleted mice die at embryonic day approximately 9.5. Here, we show that heterozygous Galpha(13)(+/-) mice display defects in adult angiogenesis. Female Galpha(13)(+/-) mice showed a higher number of immature follicles and a lower density of blood vessels in the mature corpus luteum compared with Galpha(13)(+/+) mice. Furthermore, implanted tumors grew slower in Galpha(13)(+/-) host mice. These tumor tissues had many fewer blood vessels compared with those from Galpha(13)(+/+) host mice. Moreover, bone marrow-derived progenitor cells from Galpha(13)(+/+) mice rescued the failed growth of allografted tumors when reconstituted into irradiated Galpha(13)(+/-) mice. Hence, Galpha(13) is haploinsufficient for adult angiogenesis in both the female reproductive system and tumor angiogenesis.

    Funded by: NHLBI NIH HHS: HL91525, R01 HL091525, R01 HL091525-02

    The Journal of biological chemistry 2009;284;40;27409-15

  • Defining the human deubiquitinating enzyme interaction landscape.

    Sowa ME, Bennett EJ, Gygi SP and Harper JW

    Department of Pathology, Harvard Medical School, Boston, MA 02115, USA.

    Deubiquitinating enzymes (Dubs) function to remove covalently attached ubiquitin from proteins, thereby controlling substrate activity and/or abundance. For most Dubs, their functions, targets, and regulation are poorly understood. To systematically investigate Dub function, we initiated a global proteomic analysis of Dubs and their associated protein complexes. This was accomplished through the development of a software platform called CompPASS, which uses unbiased metrics to assign confidence measurements to interactions from parallel nonreciprocal proteomic data sets. We identified 774 candidate interacting proteins associated with 75 Dubs. Using Gene Ontology, interactome topology classification, subcellular localization, and functional studies, we link Dubs to diverse processes, including protein turnover, transcription, RNA processing, DNA damage, and endoplasmic reticulum-associated degradation. This work provides the first glimpse into the Dub interaction landscape, places previously unstudied Dubs within putative biological pathways, and identifies previously unknown interactions and protein complexes involved in this increasingly important arm of the ubiquitin-proteasome pathway.

    Funded by: NIA NIH HHS: AG085011, R01 AG011085, R01 AG011085-16; NIGMS NIH HHS: GM054137, GM67945, R01 GM054137, R01 GM054137-14, R01 GM067945

    Cell 2009;138;2;389-403

  • Activation of leukemia-associated RhoGEF by Galpha13 with significant conformational rearrangements in the interface.

    Suzuki N, Tsumoto K, Hajicek N, Daigo K, Tokita R, Minami S, Kodama T, Hamakubo T and Kozasa T

    Laboratory of Systems Biology and Medicine, Research Center for Advanced Science and Technology, The University of Tokyo, Tokyo 153-8904, Japan.

    The transient protein-protein interactions induced by guanine nucleotide-dependent conformational changes of G proteins play central roles in G protein-coupled receptor-mediated signaling systems. Leukemia-associated RhoGEF (LARG), a guanine nucleotide exchange factor for Rho, contains an RGS homology (RH) domain and Dbl homology/pleckstrin homology (DH/PH) domains and acts both as a GTPase-activating protein (GAP) and an effector for Galpha(13). However, the molecular mechanism of LARG activation upon Galpha(13) binding is not yet well understood. In this study, we analyzed the Galpha(13)-LARG interaction using cellular and biochemical methods, including a surface plasmon resonance (SPR) analysis. The results obtained using various LARG fragments demonstrated that active Galpha(13) interacts with LARG through the RH domain, DH/PH domains, and C-terminal region. However, an alanine substitution at the RH domain contact position in Galpha(13) resulted in a large decrease in affinity. Thermodynamic analysis revealed that binding of Galpha(13) proceeds with a large negative heat capacity change (DeltaCp degrees ), accompanied by a positive entropy change (DeltaS degrees ). These results likely indicate that the binding of Galpha(13) with the RH domain triggers conformational rearrangements between Galpha(13) and LARG burying an exposed hydrophobic surface to create a large complementary interface, which facilitates complex formation through both GAP and effector interfaces, and activates the RhoGEF. We propose that LARG activation is regulated by an induced-fit mechanism through the GAP interface of Galpha(13).

    Funded by: NIGMS NIH HHS: GM61454

    The Journal of biological chemistry 2009;284;8;5000-9

  • Galpha13 regulates MEF2-dependent gene transcription in endothelial cells: role in angiogenesis.

    Liu G, Han J, Profirovic J, Strekalova E and Voyno-Yasenetskaya TA

    Department of Pharmacology, University of Illinois, 835 S. Wolcott Ave., Chicago, IL 60612, USA.

    The alpha subunit of heterotrimeric G13 protein is required for the embryonic angiogenesis (Offermanns et al., Science 275:533-536, 1997). However, the molecular mechanism of Galpha13-dependent angiogenesis is not understood. Here, we show that myocyte-specific enhancer factor-2 (MEF2) mediates Galpha13-dependent angiogenesis. Our data showed that constitutively activated Galpha13Q226L stimulated MEF2-dependent gene transcription. In addition, downregulation of endogenous Galpha13 inhibited thrombin-stimulated MEF2-dependent gene transcription in endothelial cells. Both Ca(2+)/calmodulin-dependent kinase IV (CaMKIV) and histone deacetylase 5 (HDAC5) were involved in Galpha13-mediated MEF2-dependent gene transcription. Galpha13Q226L also increased Ca(2+)/calmodulin-independent CaMKIV activity, while dominant negative mutant of CaMKIV inhibited MEF2-dependent gene transcription induced by Galpha13Q226L. Furthermore, Galpha13Q226L was able to derepress HDAC5-mediated repression of gene transcription and induce the translocation of HDAC5 from nucleus to cytoplasm. Finally, downregulation of endogenous Galpha13 and MEF2 proteins in endothelial cells reduced cell proliferation and capillary tube formation. Decrease of endothelial cell proliferation that was caused by the Galpha13 downregulation was partially restored by the constitutively active MEF2-VP16. Our studies suggest that MEF2 proteins are an important component in Galpha13-mediated angiogenesis.

    Funded by: NHLBI NIH HHS: HL 06078, P01 HL077806, P01 HL077806-050001; NIGMS NIH HHS: GM 56159, R01 GM056159, R55 GM056159

    Angiogenesis 2009;12;1;1-15

  • RGS22, a novel testis-specific regulator of G-protein signaling involved in human and mouse spermiogenesis along with GNA12/13 subunits.

    Hu Y, Xing J, Chen L, Guo X, Du Y, Zhao C, Zhu Y, Lin M, Zhou Z and Sha J

    Laboratory of Reproductive Medicine, Department of Histology and Embryology, Nanjing Medical University, Nanjing 210029, China.

    The heterotrimeric G-protein pathway controls numerous cellular processes, including proliferation, differentiation, migration, membrane trafficking, and embryonic development. Regulator of G-protein signaling (RGS) proteins are known to function at the G-protein level. Here, the functional role of a novel RGS protein, regulator of G-protein signaling 22 (RGS22), in the testis was investigated at the mRNA and protein levels. Our results demonstrate that RGS22 is a testis-specific gene. However, significantly decreased expression of RGS22 was found in the testes of patients with azoospermia. RGS22 was translated or posttranslationally modified into multiple proteins of different molecular sizes in prokaryocytes as well as in the testes. Its protein (NP_056483) was localized in spermatogenic cells and Leydig cells and could interact with guanine nucleotide binding protein, alpha 12, 13, and 11 (GNA12, GNA13, and GNA11). Fragmental GFP-fusion protein tracking revealed that the N-terminal of RGS22 was localized in the nucleus. RGS22 and GNA13 were localized in the nucleus from the elongated spermatid stage onward. Indirect immunofluorescence studies revealed defective expression of GNA13 in macrocephalic and global nucleus spermatozoa. These findings suggest that their functions in this subcellular compartment are likely related to the postmeiotic developmental phase, spermiogenesis. RGS22 may also play a role in GNA13 translocation from the cytoplasm to the nucleus during spermiogenesis.

    Biology of reproduction 2008;79;6;1021-9

  • Protein kinase C-related kinase and ROCK are required for thrombin-induced endothelial cell permeability downstream from Galpha12/13 and Galpha11/q.

    Gavard J and Gutkind JS

    Oral and Pharyngeal Cancer Branch, NIDCR, National Institutes of Health, Bethesda, Maryland 20892, USA. julie.gavard@inserm.fr

    Increase in vascular permeability occurs under many physiological conditions such as wound repair, inflammation, and thrombotic reactions and is central in diverse human pathologies, including tumor-induced angiogenesis, ocular diseases, and septic shock. Thrombin is a pro-coagulant serine protease, which causes the local loss of endothelial barrier integrity thereby enabling the rapid extravasation of plasma proteins and the local formation of fibrin-containing clots. Available information suggests that thrombin induces endothelial permeability by promoting actomyosin contractility through the Rho/ROCK signaling pathway. Here we took advantage of pharmacological inhibitors, knockdown approaches, and the emerging knowledge on how permeability factors affect endothelial junctions to investigate in detail the mechanism underlying thrombin-induced endothelial permeability. We show that thrombin signals through PAR-1 and its coupled G proteins Galpha(12/13) and Galpha(11/q) to induce RhoA activation and intracellular calcium elevation, and that these events are interrelated. In turn, this leads to the stimulation of ROCK, which causes actin stress-fiber formation. However, this alone is not sufficient to account for thrombin-induced permeability. Instead, we found that protein kinase C-related kinase, a Rho-dependent serine/threonine kinase, is activated in endothelial cells upon thrombin stimulation and that its expression is required for endothelial permeability and the remodeling of cell-extracellular matrix and cell-cell adhesions. Our results demonstrate that the signal initiated by thrombin bifurcates at the level of RhoA to promote changes in the cytoskeletal architecture through ROCK, and the remodeling of focal adhesion components through protein kinase C-related kinase. Ultimately, both pathways converge to cause cell-cell junction disruption and provoke vascular leakage.

    Funded by: Intramural NIH HHS

    The Journal of biological chemistry 2008;283;44;29888-96

  • Activated G(alpha)13 impairs cell invasiveness through p190RhoGAP-mediated inhibition of RhoA activity.

    Bartolomé RA, Wright N, Molina-Ortiz I, Sánchez-Luque FJ and Teixidó J

    Department of Cellular and Molecular Physiopathology, Centro de Investigaciones Biológicas, CSIC, Madrid, Spain.

    The GTPase RhoA is a downstream target of heterotrimeric G(13) proteins and plays key roles in cell migration and invasion. Here, we show that expression in human melanoma cells of a constitutively active, GTPase-deficient Galpha(13) form (G(alpha)(13)QL) or lysophosphatidylcholine (LPC)-promoted signaling through G(alpha)(13)-coupled receptors led to a blockade of chemokine-stimulated RhoA activation and cell invasion that was rescued by active RhoA. Melanoma cells expressing G(alpha)(13)QL or cells stimulated with LPC displayed an increase in p190RhoGAP activation, and defects in RhoA activation and invasion were recovered by knocking down p190RhoGAP expression, thus identifying this GTPase-activating protein (GAP) protein as a downstream G(alpha)(13) target that is responsible for these inhibitory responses. In addition, defective stress fiber assembly and reduced migration speed underlay inefficient invasion of G(alpha)(13)QL melanoma cells. Importantly, G(alpha)(13)QL expression in melanoma cells led to impairment in lung metastasis associated with prolonged survival in SCID mice. The data indicate that G(alpha)(13)-dependent downstream effects on RhoA activation and invasion tightly depend on cell type-specific GAP activities and that G(alpha)(13)-p190RhoGAP signaling might represent a potential target for intervention in melanoma metastasis.

    Cancer research 2008;68;20;8221-30

  • Regulation of cAMP responses by the G12/13 pathway converges on adenylyl cyclase VII.

    Jiang LI, Collins J, Davis R, Fraser ID and Sternweis PC

    Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA. lily.jiang@utsouthwestern.edu

    Regulation of intracellular cAMP by multiple pathways enables differential function of this ubiquitous second messenger in a context-dependent manner. Modulation of G(s)-stimulated intracellular cAMP has long been known to be modulated by the G(i) and G(q)/Ca(2+) pathways. Recently, the G(13) pathway was also shown to facilitate cAMP responses in murine macrophage cells. We report here that this synergistic regulation of cAMP synthesis by the G(s) and G(13) pathways is mediated by a specific isoform of adenylyl cyclase, AC7. Furthermore, this signaling paradigm exists in several hematopoietic lineages and can be recapitulated by exogenous expression of AC7 in HEK 293 cells. Mechanistic characterization of this synergistic interaction indicates that it occurs downstream of receptor activation and it can be mediated by the alpha subunit of either G(12) or G(13). Our results demonstrate that AC7 is a specific downstream effector of the G(12/13) pathway.

    Funded by: Intramural NIH HHS: ZIA AI001106-01, ZIA AI001107-01; NIGMS NIH HHS: GM31954, GM62114

    The Journal of biological chemistry 2008;283;34;23429-39

  • Reversible translocation of p115-RhoGEF by G(12/13)-coupled receptors.

    Meyer BH, Freuler F, Guerini D and Siehler S

    Center for Proteomic Chemistry, Novartis Institutes for BioMedical Research Basel, Novartis Pharma AG, 4002 Basel, Switzerland.

    G protein-coupled receptors (GPCRs) are important targets for medicinal agents. Four different G protein families, G(s), G(i), G(q), and G(12), engage in their linkage to activation of receptor-specific signal transduction pathways. G(12) proteins were more recently studied, and upon activation by GPCRs they mediate activation of RhoGTPase guanine nucleotide exchange factors (RhoGEFs), which in turn activate the small GTPase RhoA. RhoA is involved in many cellular and physiological aspects, and a dysfunction of the G(12/13)-Rho pathway can lead to hypertension, cardiovascular diseases, stroke, impaired wound healing and immune cell functions, cancer progression and metastasis, or asthma. In this study, regulator of G protein signaling (RGS) domain-containing RhoGEFs were tagged with enhanced green fluorescent protein (EGFP) to detect their subcellular localization and translocation upon receptor activation. Constitutively active Galpha(12) and Galpha(13) mutants induced redistribution of these RhoGEFs from the cytosol to the plasma membrane. Furthermore, a pronounced and rapid translocation of p115-RhoGEF from the cytosol to the plasma membrane was observed upon activation of several G(12/13)-coupled GPCRs in a cell type-independent fashion. Plasma membrane translocation of p115-RhoGEF stimulated by a GPCR agonist could be completely and rapidly reversed by subsequent application of an antagonist for the respective GPCR, that is, p115-RhoGEF relocated back to the cytosol. The translocation of RhoGEF by G(12/13)-linked GPCRs can be quantified and therefore used for pharmacological studies of the pathway, and to discover active compounds in a G(12/13)-related disease context.

    Journal of cellular biochemistry 2008;104;5;1660-70

  • 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

  • Regulation of apoptosis signal-regulating kinase 1 degradation by G alpha13.

    Kutuzov MA, Andreeva AV and Voyno-Yasenetskaya TA

    Department of Pharmacology, University of Illinois, 909 S. Wolcott Ave., Chicago, IL 60612, USA.

    Apoptosis signal-regulating kinase (ASK1) is a mitogen-activated protein kinase (MAPK) that transduces apoptotic signals from a variety of stresses. We have shown previously that alpha subunits of heterotrimeric G12 and G13 proteins stimulate ASK1 kinase activity and ASK1-dependent apoptosis. Here, we report a novel mechanism of G-protein-dependent regulation of ASK1. We demonstrated that G alpha13 forms a complex with ASK1 in an activation-independent manner. Both N- and C-terminal regulatory domains of ASK1 were essential for the efficient interaction, while its kinase domain was not required. Formation of the G alpha13-ASK1 complex was enhanced by JNK-interacting leucine zipper protein, JLP. Constitutively activated G alpha13Q226L increased ASK1 expression. Short-term activation of a serotonin 5-HT4 receptor that is coupled to G alpha13 also increased ASK1 expression. Importantly, prolonged activation of 5-HT4 receptor in COS-7 cells or prolonged treatment of human umbilical vein endothelial cells with thrombin concomitantly down-regulated both G alpha13 and ASK1. Data showed that G alpha13Q226L reduced the rate of ASK1 degradation, decreased ASK1 ubiquitination, and reduced association of ASK1 with an E3 ubiquitin ligase CHIP, previously shown to mediate ASK1 degradation. Our findings indicate that ASK1 expression levels can be regulated by G alpha13, at least in part via control of ASK1 ubiquitination and degradation.

    Funded by: NHLBI NIH HHS: HL-06078; NIGMS NIH HHS: GM-56159, GM-65160

    FASEB journal : official publication of the Federation of American Societies for Experimental Biology 2007;21;13;3727-36

  • Selective activation of human atrial Galpha12 and Galpha13 by Galphaq-coupled angiotensin and endothelin receptors.

    Kilts JD, Lin SS, Lowe JE and Kwatra MM

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

    Galphaq-coupled receptors such as alpha1-adrenergic, angiotensin, and endothelin receptors, play key roles in cardiac physiology. These receptors have also been shown to couple to G proteins of the G12 family, including Galpha12 and Galpha13. In this report, we determined whether these G proteins interact with endothelin, angiotensin, and alpha1-adrenergic receptors in the human heart. We find that these receptors activate cardiac Galpha12 and Galpha13 differentially; endothelin receptors activate only Galpha12 (to 218 +/- 22% of unstimulated levels), angiotensin receptors activate only Galpha13 (to 236 +/- 49% of unstimulated levels), and alpha1-adrenergic receptors activate neither Galpha12 (123 +/- 18% of unstimulated levels) nor Galpha13 (113 +/- 12% of unstimulated levels). Consistent with these data, translocation of guanine nucleotide exchange factor p115RhoGEF, which responds to Galpha13, occurs only after stimulation of angiotensin receptors (shifting from 73 +/- 12% to 41 +/- 10% cytosolic). These differences in the activation of Galpha12 and Galpha13 by Galphaq-coupled receptors may underlie reported differences in the functions of these receptors.

    Funded by: NIA NIH HHS: AG00029, AG15817

    Journal of cardiovascular pharmacology 2007;50;3;299-303

  • Distinct regions of Galpha13 participate in its regulatory interactions with RGS homology domain-containing RhoGEFs.

    Kreutz B, Hajicek N, Yau DM, Nakamura S and Kozasa T

    Department of Pharmacology (M/C 868), University of Illinois College of Medicine, 835 South Wolcott Ave., Chicago, IL 60612, USA.

    Galpha12 and Galpha13 transduce signals from G protein-coupled receptors to RhoA through RhoGEFs containing an RGS homology (RH) domain, such as p115 RhoGEF or leukemia-associated RhoGEF (LARG). The RH domain of p115 RhoGEF or LARG binds with high affinity to active forms of Galpha12 and Galpha13 and confers specific GTPase-activating protein (GAP) activity, with faster GAP responses detected in Galpha13 than in Galpha12. At the same time, Galpha13, but not Galpha12, directly stimulates the RhoGEF activity of p115 RhoGEF or nonphosphorylated LARG in reconstitution assays. In order to better understand the molecular mechanism by which Galpha13 regulates RhoGEF activity through interaction with RH-RhoGEFs, we sought to identify the region(s) of Galpha13 involved in either the GAP response or RhoGEF activation. For this purpose, we generated chimeras between Galpha12 and Galpha13 subunits and characterized their biochemical activities. In both cell-based and reconstitution assays of RhoA activation, we found that replacing the carboxyl-terminal region of Galpha12 (residues 267-379) with that of Galpha13 (residues 264-377) conferred gain-of-function to the resulting chimeric subunit, Galpha12C13. The inverse chimera, Galpha13C12, exhibited basal RhoA activation which was similar to Galpha12. In contrast to GEF assays, GAP assays showed that Galpha12C13 or Galpha13C12 chimeras responded to the GAP activity of p115 RhoGEF or LARG in a manner similar to Galpha12 or Galpha13, respectively. We conclude from these results that the carboxyl-terminal region of Galpha13 (residues 264-377) is essential for its RhoGEF stimulating activity, whereas the amino-terminal alpha helical and switch regions of Galpha12 and Galpha13 are responsible for their differential GAP responses to the RH domain.

    Funded by: NIGMS NIH HHS: GM61454; NINDS NIH HHS: NS41441

    Cellular signalling 2007;19;8;1681-9

  • Lysophosphatidic acid regulates trafficking of beta2-adrenergic receptors: the Galpha13/p115RhoGEF/JNK pathway stimulates receptor internalization.

    Shumay E, Tao J, Wang HY and Malbon CC

    Department of Pharmacology, Diabetes and Metabolic Diseases Research Program, School of Medicine, State University of New York, Stony Brook, New York 11794-8661, USA.

    Lysophosphatidic acid is an important lipid ligand regulating many aspects of cell function, including proliferation and migration. Operating via heterotrimeric G proteins to downstream effectors, lysophosphatidic acid was shown to regulate the function and trafficking of the G protein-coupled beta(2)-adrenergic receptor. C3 exotoxin, expression of dominant negative RhoA, and inhibition of c-Jun N-terminal kinase blocked the ability of lysophosphatidic acid to sequester the beta(2)-adrenergic receptor, whereas expression of constitutively active Galpha(13), p115RhoGEF, or RhoA mimicked lysophosphatidic acid (LPA) action, stimulating the internalization of the Galpha(s)-coupled beta(2)-adrenergic receptor. This study revealed a novel cross-talk exerted from the LPA/Galpha(13)/p115RhoGEF/RhoA pathway to the beta(2)-adrenergic receptor/Galpha(s)/adenylyl cyclase pathway, attenuating the ability of beta-adrenergic agonists to act following stimulation of cells by LPA as may occur during beta-adrenergic therapy of an inflammatory response.

    Funded by: NIDDK NIH HHS: DK42510

    The Journal of biological chemistry 2007;282;29;21529-41

  • G alpha 12/13 basally regulates p53 through Mdm4 expression.

    Kim MS, Lee SM, Kim WD, Ki SH, Moon A, Lee CH and Kim SG

    College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University, Seoul, Korea.

    G alpha(12/13), which belongs to the G alpha(12) family, participates in the regulation of diverse physiologic processes. In view of the control of G alpha(12/13) in cell proliferation, this study investigated the role of G alpha(12/13) in the regulation of p53 and mdm4. Immunoblotting and immunocytochemistry revealed that p53 was expressed in control embryonic fibroblasts and was largely localized in the nuclei. G alpha(12) deficiency decreased p53 levels and its DNA binding activity, accompanying p21 repression with Bcl(2) induction, whereas G alpha(13) deficiency exerted weak effects. G alpha(12) or G alpha(13) deficiency did not change p53 mRNA expression. ERK1/2 or Akt was not responsible for p53 repression due to G alpha(12) deficiency. Mdm4, a p53-stabilizing protein, was repressed by G alpha(12) deficiency and to a lesser extent by G alpha(13) deficiency, whereas mdm2, PTEN, beta-catenin, ATM, and Chk2 were unaffected. p53 accumulation by proteasomal inhibition during G alpha(12) deficiency suggested the role of G alpha(12) in p53 stabilization. Constitutively active G alpha(12) (G alpha(12)QL) or G alpha(13) (G alpha(13)QL) promoted p53 accumulation with mdm4 induction in MCF10A cells. p53 accumulation by mdm4 overexpression, but no mdm4 induction by p53 overexpression, and small interfering RNA knockdown verified the regulatory role of mdm4 for p53 downstream of G alpha(12/13). In control or G alpha(12)/G alpha(13)-deficient cells, genotoxic stress led to p53 accumulation. At concentrations increasing the flow cytometric pre-G(1) phase, doxorubicin or etoposide treatment caused serine phosphorylations in G alpha(12)-/- or G alpha(12/13)-/- cells, but did not induce mdm4. G alpha(12/13)QL transfection failed to phosphorylate p53 at serines. Our results indicate that G alpha(12/13) regulate basal p53 levels via mdm4, which constitutes a cell signaling pathway distinct from p53 phosphorylations elicited by genotoxic stress.

    Molecular cancer research : MCR 2007;5;5;473-84

  • The Galpha13-Rho signaling axis is required for SDF-1-induced migration through CXCR4.

    Tan W, Martin D and Gutkind JS

    Oral and Pharyngeal Cancer Branch, NIDCR, National Institutes of Health, Department of Health and Human Services, Bethesda, Maryland 20892-4330, USA.

    The CXC chemokine stromal cell-derived factor-1alpha (SDF-1) binds to CXCR4, a seven-transmembrane G protein-coupled receptor that plays a critical role in many physiological processes that involve cell migration and cell fate decisions, ranging from stem cell homing, angiogenesis, and neuronal development to immune cell trafficking. CXCR4 is also implicated in various pathological conditions, including metastatic spread and human immunodeficiency virus infection. Although SDF-1-induced cell migration in CXCR4-expressing cells is sensitive to pertussis toxin treatment, hence involving heterotrimeric G proteins of the G(i) family, whether other G proteins participate in the chemotactic response to SDF-1 is still unknown. In this study, we took advantage of the potent chemotactic activity of SDF-1 in Jurkat T-cells to examine the nature of the heterotrimeric G protein subunits contributing to CXCR4-mediated cell migration. We observed that whereas G(i) and Gbetagamma subunits are involved in SDF-1-induced Rac activation and cell migration, CXCR4 can also stimulate Rho potently leading to the phosphorylation of myosin light chain through the Rho effector, Rho kinase, but independently of G(i). Furthermore, we found that Galpha(13) mediates the activation of Rho by CXCR4 and that the functional activity of both Galpha(13) and Rho is required for directional cell migration in response to SDF-1. Collectively, our data indicate that signaling by CXCR4 to Rho through Galpha(13) contributes to cell migration when stimulated by SDF-1, thus identifying the Galpha(13)-Rho signaling axis as a potential pharmacological target in many human diseases that involve the aberrant function of CXCR4.

    Funded by: Intramural NIH HHS

    The Journal of biological chemistry 2006;281;51;39542-9

  • Proteomic and bioinformatic characterization of the biogenesis and function of melanosomes.

    Chi A, Valencia JC, Hu ZZ, Watabe H, Yamaguchi H, Mangini NJ, Huang H, Canfield VA, Cheng KC, Yang F, Abe R, Yamagishi S, Shabanowitz J, Hearing VJ, Wu C, Appella E and Hunt DF

    Department of Chemistry, University of Virginia, Charlottesville, Virginia 22904, USA.

    Melanin, which is responsible for virtually all visible skin, hair, and eye pigmentation in humans, is synthesized, deposited, and distributed in subcellular organelles termed melanosomes. A comprehensive determination of the protein composition of this organelle has been obstructed by the melanin present. Here, we report a novel method of removing melanin that includes in-solution digestion and immobilized metal affinity chromatography (IMAC). Together with in-gel digestion, this method has allowed us to characterize melanosome proteomes at various developmental stages by tandem mass spectrometry. Comparative profiling and functional characterization of the melanosome proteomes identified approximately 1500 proteins in melanosomes of all stages, with approximately 600 in any given stage. These proteins include 16 homologous to mouse coat color genes and many associated with human pigmentary diseases. Approximately 100 proteins shared by melanosomes from pigmented and nonpigmented melanocytes define the essential melanosome proteome. Proteins validated by confirming their intracellular localization include PEDF (pigment-epithelium derived factor) and SLC24A5 (sodium/potassium/calcium exchanger 5, NCKX5). The sharing of proteins between melanosomes and other lysosome-related organelles suggests a common evolutionary origin. This work represents a model for the study of the biogenesis of lysosome-related organelles.

    Funded by: NCRR NIH HHS: RR01744; NHGRI NIH HHS: U01-HG02712; NICHD NIH HHS: HD40179; NIGMS NIH HHS: GM 37537

    Journal of proteome research 2006;5;11;3135-44

  • A role for the G12 family of heterotrimeric G proteins in prostate cancer invasion.

    Kelly P, Stemmle LN, Madden JF, Fields TA, Daaka Y and Casey PJ

    Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina 27710, USA.

    Many studies have suggested a role for the members of the G12 family of heterotrimeric G proteins (Galpha12 and Galpha13) in oncogenesis and tumor cell growth. However, few studies have examined G12 signaling in actual human cancers. In this study, we examined the role of G12 signaling in prostate cancer. We found that expression of the G12 proteins is significantly elevated in prostate cancer. Interestingly, expression of the activated forms of Galpha12 or Galpha13 in the PC3 and DU145 prostate cancer cell lines did not promote cancer cell growth. Instead, expression of the activated forms of Galpha12 or Galpha13 in these cell lines induced cell invasion through the activation of the RhoA family of G proteins. Furthermore, inhibition of G12 signaling by expression of the RGS domain of the p115-Rho-specific guanine nucleotide exchange factor (p115-RGS) in the PC3 and DU145 cell lines did not reduce cancer cell growth. However, inhibition of G12 signaling with p115-RGS in these cell lines blocked thrombin- and thromboxane A2-stimulated cell invasion. These observations identify the G12 family proteins as important regulators of prostate cancer invasion and suggest that these proteins may be targeted to limit invasion- and metastasis-induced prostate cancer patient mortality.

    Funded by: NCI NIH HHS: CA100869; NIA NIH HHS: AG17952; NIDDK NIH HHS: DK60917

    The Journal of biological chemistry 2006;281;36;26483-90

  • Estrogen receptor alpha interacts with Galpha13 to drive actin remodeling and endothelial cell migration via the RhoA/Rho kinase/moesin pathway.

    Simoncini T, Scorticati C, Mannella P, Fadiel A, Giretti MS, Fu XD, Baldacci C, Garibaldi S, Caruso A, Fornari L, Naftolin F and Genazzani AR

    Molecular and Cellular Gynecological Endocrinology Laboratory, Department of Reproductive Medicine and Child Development, University of Pisa, Italy. t.simoncini@obgyn.med.unipi.it

    Sex steroids control cell movement and tissue organization; however, little is known of the involved mechanisms. This report describes the ongoing dynamic regulation by estrogen of the actin cytoskeleton and cell movement in human vascular endothelial cells that depends on rapid activation of the actin-regulatory protein moesin. Moesin activation is triggered by the interaction of the C-terminal portion of cell membrane estrogen receptor alpha with the G protein Galpha(13), leading to activation of the small GTPase RhoA and of the downstream effector Rho-associated kinase. The resulting phosphorylation of moesin on Thr(558) is the means of moesin's binding to actin and the remodeling of the actin cytoskeleton. This cascade of events ensues within minutes of estradiol administration and results in changes in cell morphology and to the development of specialized cell membrane structures such as ruffles and pseudopodia that are necessary for cell movement. These findings expand our knowledge of the basis of estrogen's effects on human cells, including the regulation of actin assembly, cell movement and migration. They highlight novel pathways of signal transduction of estrogen receptor alpha through nontranscriptional mechanisms. Furthermore, exposure of this estrogen receptor-dependent, nongenomic action of estrogen on human vascular endothelial cells is especially relevant to the present interest in the role of estrogen in cardiovascular protection.

    Funded by: NICHD NIH HHS: HD 047003

    Molecular endocrinology (Baltimore, Md.) 2006;20;8;1756-71

  • 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

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

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

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

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

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

    Nature 2005;437;7062;1173-8

  • A novel mechanism of G protein-dependent phosphorylation of vasodilator-stimulated phosphoprotein.

    Profirovic J, Gorovoy M, Niu J, Pavlovic S and Voyno-Yasenetskaya T

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

    Vasodilator-stimulated phosphoprotein (VASP) is a major substrate of protein kinase A (PKA). Here we described the novel mechanism of VASP phosphorylation via cAMP-independent PKA activation. We showed that in human umbilical vein endothelial cells (HUVECs) alpha-thrombin induced phosphorylation of VASP. Specific inhibition of Galpha13 protein by the RGS domain of a guanine nucleotide exchange factor, p115RhoGEF, inhibited thrombin-dependent phosphorylation of VASP. More importantly, Galpha13-induced VASP phosphorylation was dependent on activation of RhoA and mitogen-activated protein kinase kinase kinase, MEKK1, leading to the stimulation of the NF-kappaB signaling pathway. alpha-Thrombin-dependent VASP phosphorylation was inhibited by small interfering RNA-mediated knockdown of RhoA, whereas Galpha13-dependent VASP phosphorylation was inhibited by a specific RhoA inhibitor botulinum toxin C3 and by a dominant negative mutant of MEKK1. We determined that Galpha13-dependent VASP phosphorylation was also inhibited by specific PKA inhibitors, PKI and H-89. In addition, the expression of phosphorylation-deficient IkappaB and pretreatment with the proteasome inhibitor MG-132 abolished Galpha13- and alpha-thrombin-induced VASP phosphorylation. In summary, we have described a novel pathway of Galpha13-induced VASP phosphorylation that involves activation of RhoA and MEKK1, phosphorylation and degradation of IkappaB, release of PKA catalytic subunit from the complex with IkappaB and NF-kappaB, and subsequent phosphorylation of VASP.

    Funded by: NHLBI NIH HHS: HL06078; NIGMS NIH HHS: GM56159, GM65160

    The Journal of biological chemistry 2005;280;38;32866-76

  • Coupling interaction between thromboxane A2 receptor and alpha-13 subunit of guanine nucleotide-binding protein.

    Chou KC

    Gordon Life Science Institute, 13784 Torrey Del Mar, San Diego, CA 92130, USA. kchou@san.rr.com

    G protein-coupled receptors (GPCRs) form a large superfamily of membrane proteins that play an essential role in modulating many vital physiological events, such as cell communication, neurotransmission, sensory perception, and chemotaxis. Understanding of the 3D (dimensional) structures of these receptors and their binding interactions with G proteins will help in the design of drugs for the treatment of GPCR-related diseases. By means of the approach of structural bioinformatics, the 3D structures of human alpha-13 subunit of guanine nucleotide-binding protein (G alpha 13) and human thromboxane A2 (TXA2) receptor were developed. The former plays an important role in the control of cell growth that may serve as a prototypical G protein; the latter is a target for nitric oxide-mediated desensitization that may serve as a prototypical GPCR. On the basis of the 3D models, their coupling interactions were investigated via docking studies. It has been found that the two proteins are coupled with each other mainly through the interaction between the minigene of G alpha 13 and the 3rd intracellular loop of the TXA2 receptor, consistent with the existing deduction in the literatures. However, it has also been observed via a close view that some residues of the TXA2 receptor that are sequentially far away but spatially quite close to the loop region are also involved in forming hydrogen bonds with the minigene of G alpha 13. These findings may provide useful information for conducting mutagenesis and reveal the molecular mechanism how the human TXA2 receptor interact with G alpha 13 to activate intracellular signaling. The findings may also provide useful insights for stimulating new therapeutic approaches by manipulating the interaction of the receptor with the relevant G proteins.

    Journal of proteome research 2005;4;5;1681-6

  • Functional consequences of G alpha 13 mutations that disrupt interaction with p115RhoGEF.

    Grabocka E and Wedegaertner PB

    Department of Microbiology and Immunology, Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, PA 19107, USA.

    The G-protein alpha subunit, alpha(13), regulates cell growth and differentiation through the monomeric Rho GTPase. Alpha(13) activates Rho through direct stimulation of the guanine nucleotide exchange factor p115RhoGEF, which contains a regulator of G-protein signaling homology domain (RH) in its N-terminus. Through its RH domain, p115RhoGEF also functions as a GAP for G alpha(13). The mechanism for the G alpha(13)/p115RhoGEF interaction is not well understood. Here, we determined specific alpha(13) residues important for its interaction with p115RhoGEF. GST-pulldowns and co-immunoprecipitation assays revealed that individually mutating alpha(13) residues Lys204, Glu229, or Arg232 to opposite charge residues disrupts the interaction of activated alpha(13) with the RH domain of p115RhoGEF or full-length p115RhoGEF. We further demonstrate that mutation of Glu229, and to a lesser extent Lys204 or Arg232, disrupts the ability of activated alpha(13) to induce the recruitment of p115RhoGEF to the plasma membrane (PM) and to activate Rho-mediated serum response element-luciferase gene transcription. Interestingly, an alpha(13) mutant where a conserved Gly was mutated to a Ser (G205S) retained its ability to bind to p115RhoGEF, induce p115RhoGEF recruitment to the PM, and activate Rho-dependent signaling, even though identical Gly to Ser mutations in other alpha disrupt their interaction with regulator of G-protein signaling (RGS) proteins. These results demonstrate that, whereas several features of a typical alpha/RGS interaction are preserved in the alpha(13)/p115RhoGEF interaction, there are also significant differences.

    Funded by: NIGMS NIH HHS: GM62884, R01 GM062884

    Oncogene 2005;24;13;2155-65

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

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

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

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

    Funded by: NIGMS NIH HHS: P50 GM-62413

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

  • 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

  • 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

  • RGS16 inhibits signalling through the G alpha 13-Rho axis.

    Johnson EN, Seasholtz TM, Waheed AA, Kreutz B, Suzuki N, Kozasa T, Jones TL, Brown JH and Druey KM

    Molecular Signal Transduction Section, Laboratory of Allergic Diseases, National Institute of Allergy and Infectious Diseases/National Institute of Health, Rockville, MD 20852, USA.

    G alpha 13 stimulates the guanine nucleotide exchange factors (GEFs) for Rho, such as p115Rho-GEF. Activated Rho induces numerous cellular responses, including actin polymerization, serum response element (SRE)-dependent gene transcription and transformation. p115Rho-GEF contains a Regulator of G protein Signalling domain (RGS box) that confers GTPase activating protein (GAP) activity towards G alpha 12 and G alpha 13 (ref. 3). In contrast, classical RGS proteins (such as RGS16 and RGS4) exhibit RGS domain-dependent GAP activity on G alpha i and G alpha q, but not G alpha 12 or G alpha 13 (ref 4). Here, we show that RGS16 inhibits G alpha 13-mediated, RhoA-dependent reversal of stellation and SRE activation. The RGS16 amino terminus binds G alpha 13 directly, resulting in translocation of G alpha 13 to detergent-resistant membranes (DRMs) and reduced p115Rho-GEF binding. RGS4 does not bind G alpha 13 or attenuate G alpha 13-dependent responses, and neither RGS16 nor RGS4 affects G alpha 12-mediated signalling. These results elucidate a new mechanism whereby a classical RGS protein regulates G alpha 13-mediated signal transduction independently of the RGS box.

    Funded by: NIGMS NIH HHS: GM36927

    Nature cell biology 2003;5;12;1095-103

  • Use of a nasogastric catheter to prevent soft tissue entanglement of the externally ported distractor arm.

    Kyi CS, Key SJ and Lloyd TW

    Department of Oral and Maxillofacial Surgery, UCLH NHS Trust, Mortimer Market, London WC1E 6AU, UK.

    A simple method for the protection of the surrounding soft tissue during activation of a percutaneously ported distractor arm is presented. This reduces tissue damage, enhances patient compliance and could improve the overall success rate of the technique.

    International journal of oral and maxillofacial surgery 2003;32;3;337-8

  • N-terminal short sequences of alpha subunits of the G12 family determine selective coupling to receptors.

    Yamaguchi Y, Katoh H and Negishi M

    Laboratory of Molecular Neurobiology, Graduate School of Biostudies, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan.

    The Galpha subunits of the G(12) family of heterotrimeric G proteins, defined by Galpha(12) and Galpha(13), have many cellular functions in common, such as stress fiber formation and neurite retraction. However, a variety of G protein-coupled receptors appear to couple selectively to Galpha(12) and Galpha(13). For example, thrombin and lysophosphatidic acid (LPA) have been shown to induce stress fiber formation via Galpha(12) and Galpha(13), respectively. We recently showed that active forms of Galpha(12) and Galpha(13) interact with Ser/Thr phosphatase type 5 through its tetratricopeptide repeat domain. Here we developed a novel assay to measure the activities of Galpha(12) and Galpha(13) by using glutathione S-transferase-fused tetratricopeptide repeat domain of Ser/Thr phosphatase type 5, taking advantage of the property that tetratricopeptide repeat domain strongly interacts with active forms of Galpha(12) and Galpha(13). By using this assay, we identified that thrombin and LPA selectively activate Galpha(12) and Galpha(13), respectively. Galpha(12) and Galpha(13) show a high amino acid sequence homology except for their N-terminal short sequences. Then we generated chimeric G proteins Galpha(12N/13C) and Galpha(13N/12C), in which the N-terminal short sequences are replaced by each other, and showed that thrombin and LPA selectively activate Galpha(12N/13C) and Galpha(13N/12C), respectively. Moreover, thrombin and LPA stimulate RhoA activity through Galpha(12) and Galpha(13), respectively, in a Galpha(12) family N-terminal sequence-dependent manner. Thus, N-terminal short sequences of the G(12) family determine the selective couplings of thrombin and LPA receptors to the Galpha(12) family.

    The Journal of biological chemistry 2003;278;17;14936-9

  • Mutation of an N-terminal acidic-rich region of p115-RhoGEF dissociates alpha13 binding and alpha13-promoted plasma membrane recruitment.

    Bhattacharyya R and Wedegaertner PB

    Department of Microbiology and Immunology and Kimmel Cancer Center, Thomas Jefferson University, 233 S 10th St, 839 BLSB, Philadelphia, PA 19107, USA.

    The Ras homology (Rho) guanine nucleotide exchange factor p115-RhoGEF couples the alpha(13) heterotrimeric guanine nucleotide binding protein (G protein) subunit to Rho GTPase. Alpha(13) binds to a regulator of G protein signaling (RGS) domain in p115-RhoGEF, but the mechanism of alpha(13) activation of p115-RhoGEF is poorly understood. In this report, we demonstrate in cell-based assays that the acidic-rich N-terminus, adjacent to the RGS domain, is required for binding to activated alpha(13), and refine the importance of this region by showing that mutation of glutamic acids 27 and 29 in full-length p115-RhoGEF is sufficient to prevent interaction with activated alpha(13). However, alpha(13)-interacting deficient N-terminal mutants of p115-RhoGEF retain alpha(13)-dependent plasma membrane recruitment. Overall, these findings demonstrate a critical role for the N-terminal extension of p115-RhoGEF in mediating binding to alpha(13) and dissociate two activities of p115-RhoGEF: binding to activated alpha(13) and translocation to the PM in response to activated alpha(13).

    Funded by: NIGMS NIH HHS: GM62884

    FEBS letters 2003;540;1-3;211-6

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

    Tall GG, Krumins AM and Gilman AG

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

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

    Funded by: NIGMS NIH HHS: GM34497

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

  • Galpha12- and Galpha13-protein subunit linkage of D5 dopamine receptors in the nephron.

    Zheng S, Yu P, Zeng C, Wang Z, Yang Z, Andrews PM, Felder RA and Jose PA

    Department of Pediatrics, Georgetown University Medical Center, 3800 Reservoir Road, NW, Washington, DC 20007, USA.

    The roles of the G-protein alpha-subunits, Gs, Gi, and Gq/11, in the signal transduction of the D1-like dopamine receptors, D1 and D5, have been deciphered. Galpha12 and Galpha13, members of the 4th family of G protein subunits, are not linked with D1 receptors, and their linkage to D5 receptors is not known. Therefore, we studied the expression of Galpha12 and Galpha13 and interaction with D5 dopamine receptors in the kidney from normotensive Wistar-Kyoto (WKY) rats and D5 receptor-transfected HEK293 cells. Galpha12 and Galpha13 were found in the proximal tubule, distal convoluted tubule, and artery and vein in the WKY rat kidney. Whereas Galpha12 was expressed in the ascending limb of Henle, Galpha13 was expressed in the collecting duct and juxtaglomerular cells. In renal proximal tubules, Galpha12 and Galpha13, as with D5 receptors, were expressed in brush border membranes. Laser confocal microscopy revealed the colocalization of D5 receptors with Galpha12 and Galpha13 in rat renal brush border membranes, immortalized rat renal proximal tubule cells, and D5 receptor-transfected HEK293 cells. In these cells, a D1-like agonist, fenoldopam, increased the association of Galpha12 and Galpha13 with D5 receptors, results that were corroborated by immunoprecipitation experiments. We conclude that although both D1 and D5 receptors are linked to Galphas, they are differentially linked to Galpha12 and Galpha13. The consequences of the differential G-protein subunit linkage on D1- and D5-mediated sodium transport remains to be determined.

    Funded by: NHLBI NIH HHS: HL 23081, HL 62211, HL68686; NIDDK NIH HHS: DK 39308, DK52612

    Hypertension (Dallas, Tex. : 1979) 2003;41;3;604-10

  • Galpha 12 activates Rho GTPase through tyrosine-phosphorylated leukemia-associated RhoGEF.

    Suzuki N, Nakamura S, Mano H and Kozasa T

    Department of Pharmacology, University of Illinois, Chicago, IL 60612, USA.

    Heterotrimeric G proteins, G12 and G13, have been shown to transduce signals from G protein-coupled receptors to activate Rho GTPase in cells. Recently, we identified p115RhoGEF, one of the guanine nucleotide exchange factors (GEFs) for Rho, as a direct link between Galpha13 and Rho [Kozasa, T., et al. (1998) Science 280, 2109-2111; Hart, M. J., et al. (1998) Science 280, 2112-2114]. Activated Galpha13 stimulated the RhoGEF activity of p115 through interaction with the N-terminal RGS domain. However, Galpha12 could not activate Rho through p115, although it interacted with the RGS domain of p115. The biochemical mechanism from Galpha12 to Rho activation remained unknown. In this study, we analyzed the interaction of leukemia-associated RhoGEF (LARG), which also contains RGS domain, with Galpha12 and Galpha13. RGS domain of LARG demonstrated Galpha12- and Galpha13-specific GAP activity. LARG synergistically stimulated SRF activation by Galpha12 and Galpha13 in HeLa cells, and the SRF activation by Galpha12-LARG was further stimulated by coexpression of Tec tyrosine kinase. It was also found that LARG is phosphorylated on tyrosine by Tec. In reconstitution assays, the RhoGEF activity of nonphosphorylated LARG was stimulated by Galpha13 but not Galpha12. However, when LARG was phosphorylated by Tec, Galpha12 effectively stimulated the RhoGEF activity of LARG. These results demonstrate the biochemical mechanism of Rho activation through Galpha12 and that the regulation of RhoGEFs by heterotrimeric G proteins G1213 is further modulated by tyrosine phosphorylation.

    Proceedings of the National Academy of Sciences of the United States of America 2003;100;2;733-8

  • Protein kinase A-mediated phosphorylation of the Galpha13 switch I region alters the Galphabetagamma13-G protein-coupled receptor complex and inhibits Rho activation.

    Manganello JM, Huang JS, Kozasa T, Voyno-Yasenetskaya TA and Le Breton GC

    Department of Pharmacology, University of Illinois, Chicago, Illinois 60612, USA.

    The present studies mapped the protein kinase A (PKA) phosphorylation site of Galpha(13) and studied the consequences of its phosphorylation. Initial experiments using purified human Galpha(13) and the PKA catalytic subunit established that PKA directly phosphorylates Galpha(13). The location of this phosphorylation site was next investigated with a new synthetic peptide (G(13)SRI(pep)) containing the PKA consensus sequence (Arg-Arg-Pro-Thr(203)) within the switch I region of Galpha(13). G(13)SRI(pep) produced a dose-dependent inhibition of PKA-mediated Galpha(13) phosphorylation. On the other hand, the Thr-phosphorylated derivative of G(13)SRI(pep) possessed no inhibitory activity, suggesting that Galpha(13) Thr(203) may represent the phosphorylation site. Confirmation of this notion was obtained by showing that the Galpha(13)-T203A mutant (in COS-7 cells) could not be phosphorylated by PKA. Additional studies using co-elution affinity chromatography and co-immunoprecipitation demonstrated that Galpha(13) phosphorylation stabilized coupling of Galpha(13) with platelet thromboxane A(2) receptors but destabilized coupling of Galpha(13) to its betagamma subunits. In order to determine the functional consequences of this phosphorylation on Galpha(13) signaling, activation of the Rho pathway was investigated. Specifically, Chinese hamster ovary cells overexpressing human Galpha(13) wild type (Galpha(13)-WT) or Galpha(13)-T203A mutant were generated and assayed for Rho activation. It was found that 8-bromo-cyclic AMP caused a significant decrease (50%; p < 0.002) of Rho activation in Galpha(13) wild type cells but produced no change of basal Rho activation levels in the mutant (p > 0.4). These results therefore suggest that PKA blocks Rho activation by phosphorylation of Galpha(13) Thr(203).

    Funded by: NHLBI NIH HHS: HL-24530; NIGMS NIH HHS: GM 54159, GM59427, GM65160

    The Journal of biological chemistry 2003;278;1;124-30

  • Coordinated signaling through both G12/13 and G(i) pathways is sufficient to activate GPIIb/IIIa in human platelets.

    Dorsam RT, Kim S, Jin J and Kunapuli SP

    Department of Pharmacology, Temple University School of Medicine, Philadelphia, Pennsylvania 19140, USA.

    Activation of GPIIb/IIIa is known to require agonist-induced inside-out signaling through G(q), G(i), and G(z). Although activated by several platelet agonists, including thrombin and thromboxane A(2), the contribution of the G(12/13) signaling pathway to GPIIb/IIIa activation has not been investigated. In this study, we used selective stimulation of G protein pathways to investigate the contribution of G(12/13) activation to platelet fibrinogen receptor activation. YFLLRNP is a PAR-1-specific partial agonist that, at low concentrations (60 microm), selectively activates the G(12/13) signaling cascade resulting in platelet shape change without stimulating the G(q) or G(i) signaling pathways. YFLLRNP-mediated shape change was completely inhibited by the p160(ROCK) inhibitor, Y-27632. At this low concentration, YFLLRNP-mediated G(12/13) signaling caused platelet aggregation and enhanced PAC-1 binding when combined with selective G(i) or G(z) signaling, via selective stimulation of the P2Y(12) receptor or alpha(2A)-adrenergic receptor, respectively. Similar data were obtained when using low dose (10 nm), a thromboxane A(2) mimetic, to activate G(12/13) in the presence of G(i) signaling. These results suggest that selective activation of G(12/13) causes platelet GPIIb/IIIa activation when combined with G(i) signaling. Unlike either G(12/13) or G(i) activation alone, co-activation of both G(12/13) and G(i) resulted in a small increase in intracellular calcium. Chelation of intracellular calcium with dimethyl BAPTA dramatically blocked G(12/13) and G(i)-mediated platelet aggregation. No significant effect on aggregation was seen when using selective inhibitors for p160(ROCK), PKC, or MEKK1. PI 3-kinase inhibition lead to near abolishment of platelet aggregation induced by co-stimulation of G(q) and G(i) pathways, but not by G(12/13) and G(i) pathways. These data demonstrate that co-stimulation of G(12/13) and G(i) pathways is sufficient to activate GPIIb/IIIa in human platelets in a mechanism that involves intracellular calcium, and that PI 3-kinase is an important signaling molecule downstream of G(q) but not downstream of G(12/13) pathway.

    Funded by: NHLBI NIH HHS: HL60683, HL64943, T32 HL07777

    The Journal of biological chemistry 2002;277;49;47588-95

  • Hsp90 interactions and acylation target the G protein Galpha 12 but not Galpha 13 to lipid rafts.

    Waheed AA and Jones TL

    Metabolic Diseases Branch, NIDDK, National Institutes of Health, Bethesda, Maryland 20892, USA.

    The heterotrimeric G proteins, G(12) and G(13), are closely related in their sequences, signaling partners, and cellular effects such as oncogenic transformation and cytoskeletal reorganization. Yet G(12) and G(13) can act through different pathways, bind different proteins, and show opposing actions on some effectors. We investigated the compartmentalization of G(12) and G(13) at the membrane because other G proteins reside in lipid rafts, membrane microdomains enriched in cholesterol and sphingolipids. Lipid rafts were isolated after cold, nonionic detergent extraction of cells and gradient centrifugation. Galpha(12) was in the lipid raft fractions, whereas Galpha(13) was not associated with lipid rafts. Mutation of Cys-11 on Galpha(12), which prevents its palmitoylation, partially shifted Galpha(12) from the lipid rafts. Geldanamycin treatment, which specifically inhibits Hsp90, caused a partial loss of wild-type Galpha(12) and a complete loss of the Cys-11 mutant from the lipid rafts and the appearance of a higher molecular weight form of Galpha(12) in the soluble fractions. These results indicate that acylation and Hsp90 interactions localized Galpha(12) to lipid rafts. Hsp90 may act as both a scaffold and chaperone to maintain a functional Galpha(12) only in discrete membrane domains and thereby explain some of the nonoverlapping functions of G(12) and G(13) and control of these potent cell regulators.

    The Journal of biological chemistry 2002;277;36;32409-12

  • Galpha(12) and Galpha(13) interact with Ser/Thr protein phosphatase type 5 and stimulate its phosphatase activity.

    Yamaguchi Y, Katoh H, Mori K and Negishi M

    Laboratory of Molecular Neurobiology, Graduate School of Biostudies, Kyoto University, Sakyo-ku, 606-8502, Kyoto, Japan.

    The Galpha subunits of the G(12) family of heterotrimeric G proteins, defined by Galpha(12) and Galpha(13), are involved in many signaling pathways and diverse cellular functions. In an attempt to elucidate downstream effectors of Galpha(12) for cellular functions, we have performed a yeast two-hybrid screening of a rat brain cDNA library and revealed that Ser/Thr protein phosphatase type 5 (PP5) is a novel effector of Galpha(12) and Galpha(13). PP5 is a newly identified phosphatase and consists of a C-terminal catalytic domain and an N-terminal regulatory tetratricopeptide repeat (TPR) domain [2]. Arachidonic acid was recently shown to activate PP5 phosphatase activity by binding to its TPR domain, however the precise regulatory mechanism of PP5 phosphatase activity is not fully determined. In this study, we show that active forms of Galpha(12) and Galpha(13) specifically interact with PP5 through its TPR domain and activate its phosphatase activity about 2.5-fold. Active forms of Galpha(12) and Galpha(13) also enhance the arachidonic acid-stimulated PP5 phosphatase activity about 2.5-fold. Moreover, we demonstrate that the active form of Galpha(12) translocates PP5 to the cell periphery and colocalizes with PP5. These results propose a new signaling pathway of G(12) family G proteins.

    Current biology : CB 2002;12;15;1353-8

  • Galpha12 and Galpha13 negatively regulate the adhesive functions of cadherin.

    Meigs TE, Fedor-Chaiken M, Kaplan DD, Brackenbury R and Casey PJ

    Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina 27710, USA.

    Cadherins function to promote adhesion between adjacent cells and play critical roles in such cellular processes as development, tissue maintenance, and tumor suppression. We previously demonstrated that heterotrimeric G proteins of the G12 subfamily comprised of Galpha12 and Galpha13 interact with the cytoplasmic domain of cadherins and cause the release of the transcriptional activator beta-catenin (Meigs, T. E., Fields, T. A., McKee, D. D., and Casey, P. J. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 519-524). Because of the importance of beta-catenin in cadherin-mediated cell-cell adhesion, we examined whether G12 subfamily proteins could also regulate cadherin function. The introduction of mutationally activated G12 proteins into K562 cells expressing E-cadherin blocked cadherin-mediated cell adhesion in steady-state assays. Also, in breast cancer cells, the introduction of activated G12 proteins blocked E-cadherin function in a fast aggregation assay. Aggregation mediated by a mutant cadherin that lacks G12 binding ability was not affected by activated G12 proteins, indicating a requirement for direct G12-cadherin interaction. Furthermore, in wound-filling assays in which ectopic expression of E-cadherin inhibits cell migration, the expression of activated G12 proteins reversed the inhibition via a mechanism that was independent of G12-mediated Rho activation. These results validate the G12-cadherin interaction as a potentially important event in cell biology and suggest novel roles for G12 proteins in the regulation of cadherin-mediated developmental events and in the loss of cadherin function that is characteristic of metastatic tumor progression.

    Funded by: NCI NIH HHS: CA91159; NIAMS NIH HHS: AR44713; NIGMS NIH HHS: GM55717

    The Journal of biological chemistry 2002;277;27;24594-600

  • Plexin-B1 directly interacts with PDZ-RhoGEF/LARG to regulate RhoA and growth cone morphology.

    Swiercz JM, Kuner R, Behrens J and Offermanns S

    Institute of Pharmacology, University of Heidelberg, Im Neuenheimer Feld 366, Germany.

    Plexins are widely expressed transmembrane proteins that, in the nervous system, mediate repulsive signals of semaphorins. However, the molecular nature of plexin-mediated signal transduction remains poorly understood. Here, we demonstrate that plexin-B family members associate through their C termini with the Rho guanine nucleotide exchange factors PDZ-RhoGEF and LARG. Activation of plexin-B1 by semaphorin 4D regulates PDZ-RhoGEF/LARG activity leading to RhoA activation. In addition, a dominant-negative form of PDZ-RhoGEF blocks semaphorin 4D-induced growth cone collapse in primary hippocampal neurons. Our study indicates that the interaction of mammalian plexin-B family members with the multidomain proteins PDZ-RhoGEF and LARG represents an essential molecular link between plexin-B and localized, Rho-mediated downstream signaling events which underly various plexin-mediated cellular phenomena including axonal growth cone collapse.

    Neuron 2002;35;1;51-63

  • The chemokine stromal cell-derived factor-1 alpha modulates alpha 4 beta 7 integrin-mediated lymphocyte adhesion to mucosal addressin cell adhesion molecule-1 and fibronectin.

    Wright N, Hidalgo A, Rodríguez-Frade JM, Soriano SF, Mellado M, Parmo-Cabañas M, Briskin MJ and Teixidó J

    Department of Immunology, Centro de Investigaciones Biológicas, Velázquez 144, 28006 Madrid, Spain.

    The interaction between the integrin alpha(4)beta(7) and its ligand, mucosal addressin cell adhesion molecule-1, on high endothelial venules represents a key adhesion event during lymphocyte homing to secondary lymphoid tissue. Stromal cell-derived factor-1alpha (SDF-1alpha) is a chemokine that attracts T and B lymphocytes and has been hypothesized to be involved in lymphocyte homing. In this work we show that alpha(4)beta(7)-mediated adhesion of CD4(+) T lymphocytes and the RPMI 8866 cell line to mucosal addressin cell adhesion molecule-1 was up-regulated by SDF-1alpha in both static adhesion and cell detachment under shear stress assays. Both naive and memory phenotype CD4(+) T cells were targets of SDF-1alpha-triggered increased adhesion. In addition, SDF-1alpha augmented alpha(4)beta(7)-dependent adhesion of RPMI 8866 cells to connecting segment-1 of fibronectin. While pertussis toxin totally blocked chemotaxis of CD4(+) and RPMI 8866 cells to SDF-1alpha, enhanced alpha(4)beta(7)-dependent adhesion triggered by this chemokine was partially inhibited, indicating the participation of Galpha(i)-dependent as well as Galpha(i)-independent signaling. Accordingly, we show that SDF-1alpha induced a rapid and transient association between its receptor CXCR4 and Galpha(i), whereas association of pertussis toxin-insensitive Galpha(13) with CXCR4 was slower and of a lesser extent. SDF-1alpha also activated the small GTPases RhoA and Rac1, and inhibition of RhoA activation reduced the up-regulation of alpha(4)beta(7)-mediated lymphocyte adhesion in response to SDF-1alpha, suggesting that activation of RhoA could play an important role in the enhanced adhesion. These data indicate that up-regulation by SDF-1alpha of lymphocyte adhesion mediated by alpha(4)beta(7) could contribute to lymphocyte homing to secondary lymphoid tissues.

    Journal of immunology (Baltimore, Md. : 1950) 2002;168;10;5268-77

  • Galpha13 induces preproET-1 gene expression via JNK.

    Yamakaw K, Kitamura K, Nonoguchi H, Takasu N, Miller RT and Tomita K

    Third Department of Internal Medicine, Kumamoto University School of Medicine, Japan.

    The endothelin B receptor (ETBR) has been shown to mediate autoinduction of endothelin-1 (ET-1). We previously reported that the ET(B)R interacts with Galpha13, a member of the heterotrimeric GTP-binding protein family. In the present study, we examined whether Galpha13 induces preproET-1 (ppET-1) gene transcription, which could result in ET-1 autoinduction in a renal epithelial cell line. We generated a reporter gene construct under control of the ppET-1 promoter region. The construct was transiently expressed in COS-7 cells. Transient expression of ETBR increased the promoter activity of ppET-1 following treatment with 100 nmol/l of ET-1. Expression of Galpha13Q226L or Galpha9209L, constitutively active forms of Galpha13 and Galpha9, also activated the ppET-1 promoter. ETBR-stimulated ppET-1 promoter activity was partially diminished by the expression of dominant negative forms of c-Jun N-terminal kinase (JNK1APF) or MAPK/ERK kinase (MEKK97M). Expression of JNK1APF also inhibited Galpha13Q226L-induced ppET-1 promoter activation. These findings indicate that Galpha13 can induce ppET-1 gene expression through a JNK-mediated pathway. Our results also suggest that this Galpha13-coupled signaling pathway may play an important role in a sustained ET-1 autoinduction loop in various pathophysiological conditions.

    Funded by: NIDDK NIH HHS: DK41726

    Hypertension research : official journal of the Japanese Society of Hypertension 2002;25;3;427-32

  • Interaction of heterotrimeric G13 protein with an A-kinase-anchoring protein 110 (AKAP110) mediates cAMP-independent PKA activation.

    Niu J, Vaiskunaite R, Suzuki N, Kozasa T, Carr DW, Dulin N and Voyno-Yasenetskaya TA

    Department of Pharmacology, University of Illinois, Chicago, IL 60612, USA.

    Heterotrimeric G proteins and protein kinase A (PKA) are two important transmitters that transfer signals from a wide variety of cell surface receptors to generate physiological responses. The established mechanism of PKA activation involves the activation of the Gs-cAMP pathway. Binding of cAMP to the regulatory subunit of PKA (rPKA) leads to a release and subsequent activation of a catalytic subunit of PKA (cPKA). Here, we report a novel mechanism of PKA stimulation that does not require cAMP. Using yeast two-hybrid screening, we found that the alpha subunit of G13 protein interacted with a member of the PKA-anchoring protein family, AKAP110. Using in vitro binding and coimmunoprecipitation assays, we have shown that only activated G alpha 13 binds to AKAP110, suggesting a potential role for AKAP110 as a G alpha subunit effector protein. Importantly, G alpha 13, AKAP110, rPKA, and cPKA can form a complex, as shown by coimmunoprecipitation. By characterizing the functional significance of the G alpha 13-AKAP110 interaction, we have found that G alpha 13 induced release of the cPKA from the AKAP110-rPKA complex, resulting in a cAMP-independent PKA activation. Finally, AKAP110 significantly potentiated G alpha 13-induced activation of PKA. Thus, AKAP110 provides a link between heterotrimeric G proteins and cAMP-independent activation of PKA.

    Funded by: NICHD NIH HHS: HD36408; NIGMS NIH HHS: GM56159, GM59427

    Current biology : CB 2001;11;21;1686-90

  • Interaction of Galpha 12 and Galpha 13 with the cytoplasmic domain of cadherin provides a mechanism for beta -catenin release.

    Meigs TE, Fields TA, McKee DD and Casey PJ

    Departments of Pharmacology and Cancer Biology, and Pathology, Duke University Medical Center, Durham, NC 27710, USA.

    The G12 subfamily of heterotrimeric G proteins, comprised of the alpha-subunits Galpha12 and Galpha13, has been implicated as a signaling component in cellular processes ranging from cytoskeletal changes to cell growth and oncogenesis. In an attempt to elucidate specific roles of this subfamily in cell regulation, we sought to identify molecular targets of Galpha12. Here we show a specific interaction between the G12 subfamily and the cytoplasmic tails of several members of the cadherin family of cell-surface adhesion proteins. Galpha12 or Galpha13 binding causes dissociation of the transcriptional activator beta-catenin from cadherins. Furthermore, in cells lacking the adenomatous polyposis coli protein required for beta-catenin degradation, expression of mutationally activated Galpha12 or Galpha13 causes an increase in beta-catenin-mediated transcriptional activation. These findings provide a potential molecular mechanism for the previously reported cellular transforming ability of the G12 subfamily and reveal a link between heterotrimeric G proteins and cellular processes controlling growth and differentiation.

    Funded by: NIGMS NIH HHS: GM 55717, R01 GM055717

    Proceedings of the National Academy of Sciences of the United States of America 2001;98;2;519-24

  • Leukemia-associated Rho guanine nucleotide exchange factor (LARG) links heterotrimeric G proteins of the G(12) family to Rho.

    Fukuhara S, Chikumi H and Gutkind JS

    Oral and Pharyngeal Cancer Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, 9000 Rockville Pike, Building 30, Room 211, Bethesda, MD 20892-4330, USA.

    A putative guanine nucleotide exchange factor (GEF), termed leukemia-associated RhoGEF (LARG), was recently identified upon fusion to the coding sequence of the MLL gene in acute myeloid leukemia. Although the function of LARG is still unknown, it exhibits a number of structural domains suggestive of a role in signal transduction, including a PDZ domain, a LH/RGS domain, and a Dbl homology/pleckstrin homology domain. Here, we show that LARG can activate Rho in vivo. Furthermore, we present evidence that LARG is an integral component of a novel biochemical route whereby G protein-coupled receptors (GPCRs) and heterotrimeric G proteins of the G alpha(12) family stimulate Rho-dependent signaling pathways.

    FEBS letters 2000;485;2-3;183-8

  • Activation of RhoA by association of Galpha(13) with Dbl.

    Jin S and Exton JH

    Howard Hughes Medical Institute and Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee, 37232-0295, USA.

    RhoA is a small G protein that is implicated in the regulation of the actin cytoskeleton, gene expression, and cell cycle progression. It is activated by many agonists whose receptors are linked to heterotrimeric G proteins, but the mechanisms are incompletely understood. In this study, we show that the constitutively active alpha-subunit of the heterotrimeric G protein G(13) associated with the Rho family guanine nucleotide exchange factor Dbl in NIH 3T3 cells and that this resulted in activation of RhoA. This activation was not seen with wild-type Galpha(13) or if Dbl and active Galpha(13) were expressed separately and mixed. In contrast, coexpression of constitutively active Galpha(q) with Dbl did not lead to their association and caused a weak activation of RhoA that was no greater than that observed with wild-type Galpha(q). These findings illustrate that activated Galpha(13) and Dbl can associate in vivo and that this leads to Rho activation.

    Biochemical and biophysical research communications 2000;277;3;718-21

  • Conformational activation of radixin by G13 protein alpha subunit.

    Vaiskunaite R, Adarichev V, Furthmayr H, Kozasa T, Gudkov A and Voyno-Yasenetskaya TA

    Department of Pharmacology, University of Illinois, Chicago, Illinois 60612, USA.

    G(13) protein, one of the heterotrimeric guanine nucleotide-binding proteins (G proteins), regulates diverse and complex cellular responses by transducing signals from the cell surface presumably involving more than one pathway. Yeast two-hybrid screening of a mouse brain cDNA library identified radixin, a member of the ERM family of three closely related proteins (ezrin, radixin, and moesin), as a protein that interacted with Galpha(13). Interaction between radixin and Galpha(13) was confirmed by in vitro binding assay and by co-immunoprecipitation technique. Activated Galpha(13) induced conformational activation of radixin, as determined by binding of radixin to polymerized F-actin and by immunofluorescence in intact cells. Finally, two dominant negative mutants of radixin inhibited Galpha(13)-induced focus formation of Rat-1 fibroblasts but did not affect Ras-induced focus formation. Our results identifying a new signaling pathway for Galpha(13) indicate that ERM proteins can be activated by and serve as effectors of heterotrimeric G proteins.

    The Journal of biological chemistry 2000;275;34;26206-12

  • G13alpha-mediated PYK2 activation. PYK2 is a mediator of G13alpha -induced serum response element-dependent transcription.

    Shi CS, Sinnarajah S, Cho H, Kozasa T and Kehrl JH

    BCell Molecular Immunology Section, Laboratory of Immunoregulation, NIAID, National Institutes of Health, Bethesda, Maryland 20892-1876, USA.

    G(12)alpha/G(13)alpha transduces signals from G-protein-coupled receptors to stimulate growth-promoting pathways and the early response gene c-fos. Within the c-fos promoter lies a key regulatory site, the serum response element (SRE). Here we show a critical role for the tyrosine kinase PYK2 in muscarinic receptor type 1 and G(12)alpha/G(13)alpha signaling to an SRE reporter gene. A kinase-inactivate form of PYK2 (PYK2 KD) inhibits muscarinic receptor type 1 signaling to the SRE and PYK2 itself triggers SRE reporter gene activation through a RhoA-dependent pathway. Placing PYK2 downstream of G-protein activation but upstream of RhoA, the expression of PYK2 KD blocks the activation of an SRE reporter gene by GTPase-deficient forms of G(12)alpha or G(13)alpha but not by RhoA. The GTPase-deficient form of G(13)alpha triggers PYK2 kinase activity and PYK2 tyrosine phosphorylation, and co-expression of the RGS domain of p115 RhoGEF inhibits both responses. Finally, we show that in vivo G(13)alpha, although not G(12)alpha, readily associates with PYK2. Thus, G-protein-coupled receptors via G(13)alpha activation can use PYK2 to link to SRE-dependent gene expression.

    The Journal of biological chemistry 2000;275;32;24470-6

  • Acylation of Galpha(13) is important for its interaction with thrombin receptor, transforming activity and actin stress fiber formation.

    Ponimaskin E, Behn H, Adarichev V, Voyno-Yasenetskaya TA, Offermanns S and Schmidt MF

    Institut für Immunologie und Molekularbiologe, Freie Universität Berlin, Phillippstrasse 13, D-10115, Berlin, Germany.

    Palmitoylation of alpha-subunits in heterotrimeric G proteins has become a research object of growing attention. Following our recent report on the acylation of the mono-palmitoylated Galpha(12) [Ponimaskin et al., FEBS Lett. 429 (1998) 370-374], we report here on the identification of three palmitoylation sites in the second member of the G(12) family, Galpha(13), and on the biological significance of fatty acids on the particular sites. Using mutants of alpha(13) in which the potentially palmitoylated cysteine residues (Cys) were replaced by serine residues, we find that Cys-14, Cys-18 and Cys-37 all serve as palmitoylation sites, and that the mutants lacking fatty acids are functionally defective. The following biological functions of Galpha(13) were found to be inhibited: coupling to the PAR1 thrombin receptor, cell transformation and actin stress fiber formation. Results from established assays for the above functions with a series of mutants, including derivatives of the constitutively active mutant Galpha(13)Q226L, revealed a graded inhibitory response on the above mentioned parameters. As a rule, it appears that palmitoylation of the N-proximal sites (e.g. Cys-14 and Cys-18) contributes more effectively to biological function than of the acylation site located more internally (Cys-37). However, the mutant with Cys-37 replaced by serine is more severely inhibited in stress fiber formation (80%) than in cell transformation (50%), pointing to the possibility of a differential involvement of the three palmitoylation sites in Galpha(13).

    FEBS letters 2000;478;1-2;173-7

  • Galpha 13 requires palmitoylation for plasma membrane localization, Rho-dependent signaling, and promotion of p115-RhoGEF membrane binding.

    Bhattacharyya R and Wedegaertner PB

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

    Most heterotrimeric G protein alpha subunits are covalently modified by palmitate attached to one or more N-terminal cysteine residues. Although a wide variety of proteins undergo palmitoylation, the role of this fatty acid modification in G protein signaling is not well understood. Thus, we examined the role of palmitoylation of alpha(13), a G protein alpha subunit that regulates many pathways involved in cell growth. Both N-terminal cysteines at positions 14 and 18 were required for palmitoylation. Mutant alpha(13), in which both cysteines were changed to serines, failed to localize to plasma membranes in transfected cells and failed to activate Rho-dependent serum response factor-mediated transcription and actin stress fiber formation. However, nonpalmitoylated, cysteine to serine mutant alpha(13) retained the ability to co-immunoprecipitate with a direct effector, p115-RhoGEF. Finally, we report the novel observation that activated alpha(13) induces a redistribution of p115-RhoGEF from the cytoplasm to plasma membranes, but non-palmitoylated mutants of alpha(13) fail to cause p115-RhoGEF translocation. These findings identify palmitoylation of alpha(13) as critical for its proper membrane localization and signaling and provide insight into the mechanism of activation of Rho-dependent signaling pathways by alpha(13).

    Funded by: NIGMS NIH HHS: GM56444

    The Journal of biological chemistry 2000;275;20;14992-9

  • Activation of the small GTPases, rac and cdc42, after ligation of the platelet PAR-1 receptor.

    Azim AC, Barkalow K, Chou J and Hartwig JH

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

    Stimulation of platelet PAR-1 receptors results in the rapid (10 to 30 seconds) and extensive (30% to 40% of total) guanosine triphosphate (GTP) charging of endogenous platelet rac, previously identified as a possible key intermediate in the signal pathway between PAR-1 and actin filament barbed-end uncapping, leading to actin assembly. During PAR-1-mediated platelet activation, rac distributes from the cell interior to the cell periphery, and this reorganization is resistant to the inhibition of PI-3-kinase activity. Rac, in resting or activated platelets, is Triton X-100 soluble, suggesting that it does not form tight complexes with actin cytoskeletal proteins, though its retention in octyl-glucoside-treated platelets and ultrastructural observations of activated platelets implies that rac binds to plasma membranes, where it can interact with phosphoinositide kinases implicated in actin assembly reactions. PAR-1 stimulation also rapidly and extensively activates cdc42, though, in contrast to rac, some cdc42 associates with the actin cytoskeleton in resting platelets, and the bound fraction increases during stimulation. The differences in subcellular distribution and previous evidence showing quantitatively divergent effects of rac and cdc42 on actin nucleation in permeabilized platelets indicate different signaling roles for these GTPases.

    Funded by: NHLBI NIH HHS: HL56252, HL56949

    Blood 2000;95;3;959-64

  • The G protein subunit gene families.

    Downes GB and Gautam N

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

    Genomics 1999;62;3;544-52

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

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

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

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

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

  • Differential coupling of the sphingosine 1-phosphate receptors Edg-1, Edg-3, and H218/Edg-5 to the G(i), G(q), and G(12) families of heterotrimeric G proteins.

    Windh RT, Lee MJ, Hla T, An S, Barr AJ and Manning DR

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

    Sphingosine 1-phosphate (S1P) is one of several bioactive phospholipids that exert profound mitogenic and morphogenic actions. Originally characterized as a second messenger, S1P is now recognized to achieve many of its effects through cell surface, G protein-coupled receptors. We used a subunit-selective [(35)S]GTPgammaS binding assay to investigate whether the variety of actions exerted through Edg-1, a recently identified receptor for S1P, might be achieved through multiple G proteins. We found, employing both Sf9 and HEK293 cells, that Edg-1 activates only members of the G(i) family, and not G(s), G(q), G(12), or G(13). We additionally established that Edg-1 activates G(i) in response not only to S1P but also sphingosylphosphorylcholine; no effects of lysophosphatidic acid through Edg-1 were evident. Our assays further revealed a receptor(s) for S1P endogenous to HEK293 cells that mediates activation of G(13) as well as G(i). Because several of the biological actions of S1P are assumed to proceed through the G(12/13) family, we tested whether Edg-3 and H218/Edg-5, two other receptors for S1P, might have a broader coupling profile than Edg-1. Indeed, Edg-3 and H218/Edg-5 communicate not only with G(i) but also with G(q) and G(13). These studies represent the first characterization of S1P receptor activity through G proteins directly and establish fundamental differences in coupling.

    Funded by: NIGMS NIH HHS: GM51196

    The Journal of biological chemistry 1999;274;39;27351-8

  • Coupling of thromboxane A2 receptor isoforms to Galpha13: effects on ligand binding and signalling.

    Becker KP, Garnovskaya M, Gettys T and Halushka PV

    Department of Cell and Molecular Pharmacology and Experimental Therapeutics, Medical University of South Carolina, 171 Ashley Avenue, Charleston, SC 29425, USA.

    Previous subtyping of thromboxane A2 (TXA2) receptors in platelets and vascular smooth muscle cells was based on pharmacological criteria. Two distinct carboxy-terminal splice variants for TXA2 receptors exist and they couple to several different G protein alpha subunits including Galpha13, but it has not been established whether either or both isoforms interact with and signal through it. We sought to determine: (1) which TXA2 receptor isoforms exist in vascular smooth muscle, (2) if Galpha13 is present in vascular smooth muscle and (3) if Galpha13 interacts with either or both of the two TXA2 receptor isoforms as determined by changes in ligand binding properties and generation of intracellular signals. Both TXA2 receptor isoforms and Galpha13 were found in vascular smooth muscle cells. Both the alpha and beta isoforms of the TXA2 receptors were transiently transfected with or without Galpha13 into COS-7 (radioligand binding assays) or CHO cells (agonist induced Na+/H+ exchange). Co-expression of each receptor isoform with Galpha13 significantly (P<0.05) increased the affinity of each receptor for the two agonists, I-BOP and ONO11113, and decreased the affinity of the receptor for the antagonists, SQ29,548 and L657,925. I-BOP stimulated Na+/H+ exchange in vascular smooth muscle cells. Co-expression of Galpha13 with each TXA2 receptor isoform in CHO cells resulted in a significant (P<0.04) agonist induced increase in Na+/H+ exchange compared to cells not transfected with Galpha13. The results support the possibility that the previous classification of TXA2 receptor subtypes based on pharmacological criteria reflect unique interactions with specific G protein alpha subunits.

    Funded by: NHLBI NIH HHS: HL36838

    Biochimica et biophysica acta 1999;1450;3;288-96

  • A novel PDZ domain containing guanine nucleotide exchange factor links heterotrimeric G proteins to Rho.

    Fukuhara S, Murga C, Zohar M, Igishi T and Gutkind JS

    Oral and Pharyngeal Cancer Branch, NIDCR, National Institutes of Health, Bethesda, Maryland 20892-4330, USA.

    Small GTP-binding proteins of the Rho family play a critical role in signal transduction. However, there is still very limited information on how they are activated by cell surface receptors. Here, we used a consensus sequence for Dbl domains of Rho guanine nucleotide exchange factors (GEFs) to search DNA data bases, and identified a novel human GEF for Rho-related GTPases harboring structural features indicative of its possible regulatory mechanism(s). This protein contained a tandem DH/PH domain closely related to those of Rho-specific GEFs, a PDZ domain, a proline-rich domain, and an area of homology to Lsc, p115-RhoGEF, and a Drosophila RhoGEF that was termed Lsc-homology (LH) domain. This novel molecule, designated PDZ-RhoGEF, activated biological and biochemical pathways specific for Rho, and activation of these pathways required an intact DH and PH domain. However, the PDZ domain was dispensable for these functions, and mutants lacking the LH domain were more active, suggesting a negative regulatory role for the LH domain. A search for additional molecules exhibiting an LH domain revealed a limited homology with the catalytic region of a newly identified GTPase-activating protein for heterotrimeric G proteins, RGS14. This prompted us to investigate whether PDZ-RhoGEF could interact with representative members of each G protein family. We found that PDZ-RhoGEF was able to form, in vivo, stable complexes with two members of the Galpha12 family, Galpha12 and Galpha13, and that this interaction was mediated by the LH domain. Furthermore, we obtained evidence to suggest that PDZ-RhoGEF mediates the activation of Rho by Galpha12 and Galpha13. Together, these findings suggest the existence of a novel mechanism whereby the large family of cell surface receptors that transmit signals through heterotrimeric G proteins activate Rho-dependent pathways: by stimulating the activity of members of the Galpha12 family which, in turn, activate an exchange factor acting on Rho.

    The Journal of biological chemistry 1999;274;9;5868-79

  • Direct stimulation of the guanine nucleotide exchange activity of p115 RhoGEF by Galpha13.

    Hart MJ, Jiang X, Kozasa T, Roscoe W, Singer WD, Gilman AG, Sternweis PC and Bollag G

    Onyx Pharmaceuticals, 3031 Research Drive, Richmond, CA 94806, USA.

    Signaling pathways that link extracellular factors to activation of the monomeric guanosine triphosphatase (GTPase) Rho control cytoskeletal rearrangements and cell growth. Heterotrimeric guanine nucleotide-binding proteins (G proteins) participate in several of these pathways, although their mechanisms are unclear. The GTPase activities of two G protein alpha subunits, Galpha12 and Galpha13, are stimulated by the Rho guanine nucleotide exchange factor p115 RhoGEF. Activated Galpha13 bound tightly to p115 RhoGEF and stimulated its capacity to catalyze nucleotide exchange on Rho. In contrast, activated Galpha12 inhibited stimulation by Galpha13. Thus, p115 RhoGEF can directly link heterotrimeric G protein alpha subunits to regulation of Rho.

    Funded by: NIGMS NIH HHS: GM 31954, GM34497

    Science (New York, N.Y.) 1998;280;5372;2112-4

  • G protein heterotrimer Galpha13beta1gamma3 couples the angiotensin AT1A receptor to increases in cytoplasmic Ca2+ in rat portal vein myocytes.

    Macrez-Leprêtre N, Kalkbrenner F, Morel JL, Schultz G and Mironneau J

    Laboratoire de Physiologie Cellulaire et Pharmacologie Moléculaire, CNRS ESA 5017, Université de Bordeaux II, 146 rue Léo Saignat, 33076 Bordeaux Cedex, France.

    The subunit composition of angiotensin AT1 receptor-activated G protein was identified by using antisense oligonucleotide injection into the nucleus of rat portal vein myocytes. In these cells, we have previously shown that increases in the cytoplasmic calcium concentration ([Ca2+]i) induced by activation of angiotensin AT1 receptors were dependent on extracellular Ca2+ entry by L-type Ca2+ channels and subsequent Ca2+-induced Ca2+ release from the intracellular stores. The angiotensin AT1 receptor-activated increases in [Ca2+]i were selectively inhibited by injection of antisense oligonucleotides directed against the mRNAs coding for the alpha13, beta1, and gamma3 subunits. A correlating reduction in Galpha13, Gbeta1, and Ggamma3 protein expression was confirmed by immunocytochemistry. In addition, anti-alpha13 antibody and synthetic peptide corresponding to the carboxyl terminus of the Galpha13 subunit inhibited, in a concentration-dependent manner, the angiotensin AT1 receptor-mediated Ca2+ response. Reverse transcription-polymerase chain reaction analysis showed that only the angiotensin AT1A receptor was expressed in rat portal vein smooth muscle. Furthermore, injection of anti-AT1A oligonucleotides selectively inhibited the angiotensin II-induced increase in [Ca2+]i. We conclude that the receptor-activated signal leading to increases in [Ca2+]i is transduced by the heterotrimeric G13 protein composed of alpha13/beta1/gamma3 subunits and that the carboxyl terminus of the Galpha13 subunit interacts with the angiotensin AT1A receptor.

    The Journal of biological chemistry 1997;272;15;10095-102

  • Vascular system defects and impaired cell chemokinesis as a result of Galpha13 deficiency.

    Offermanns S, Mancino V, Revel JP and Simon MI

    Division of Biology 147-75, California Institute of Technology, Pasadena, CA 91125, USA.

    Heterotrimeric GTP-binding proteins (G proteins) participate in cellular signaling and regulate a variety of physiological processes. Disruption of the gene encoding the G protein subunit alpha13 (Galpha13) in mice impaired the ability of endothelial cells to develop into an organized vascular system, resulting in intrauterine death. In addition, Galpha13 (-/-) embryonic fibroblasts showed greatly impaired migratory responses to thrombin. These results demonstrate that Galpha13 participates in the regulation of cell movement in response to specific ligands, as well as in developmental angiogenesis.

    Funded by: NIA NIH HHS: AG 12288; NIGMS NIH HHS: GM 34236

    Science (New York, N.Y.) 1997;275;5299;533-6

  • Galpha12 and galpha13 are phosphorylated during platelet activation.

    Offermanns S, Hu YH and Simon MI

    Division of Biology 147-75, California Institute of Technology, Pasadena, California 91125, USA.

    The ubiquitously expressed G-proteins G12 and G13 whose function is currently not clear have been shown to be activated in platelet membranes through receptors that stimulate platelet aggregation. We used intact human platelets to determine whether alpha subunits of both G-proteins can be phosphorylated under physiological conditions. Activation of human platelets by thrombin and the thromboxane A2 receptor agonist U46619 lead to phosphorylation of Galpha12 and Galpha13. Phosphorylation occurred rapidly after addition of thrombin and was not mediated by glycoprotein IIb/IIIa (integrin alphaIIbbeta3) activation. Phosphorylation of Galpha12 and Galpha13 could be mimicked by phorbol 12-myristate 13-acetate, and thrombin-induced phosphorylation was inhibited by the protein kinase C inhibitor calphostin C indicating an involvement of protein kinase C in Galpha12/13 phosphorylation induced by thrombin in human platelets. The phosphorylation of both G protein alpha subunits was reconstituted in COS-7 cells cotransfected with Galpha12 or Galpha13 and different protein kinase C isoforms. Among the protein knase C isoforms tested, protein kinase C beta, delta, and epsilon were most effective in promoting phosphorylation of Galpha12 and Galpha13 in a phorbol 12-myristate 13-acetate-dependent manner. These data demonstrate that Galpha12 and Galpha13 are phosphorylated under in vivo conditions and that this phosphorylation involves protein kinase C.

    The Journal of biological chemistry 1996;271;42;26044-8

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

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

    Institut für Pharmakologie, Freie Universität Berlin, Germany.

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

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

  • Expression of GTP-binding protein alpha subunits in human thymocytes.

    Kabouridis PS, Waters ST, Escobar S, Stanners J and Tsoukas CD

    Department of Biology, San Diego State University, CA 92182, USA.

    In this report, we investigate G protein alpha subunit diversity in human thymocytes, utilizing common properties shared by these genes and reverse transcription-polymerase chain reaction (RT-PCR). Sequence analysis of PCR amplified gene portions, indicate the presence of members from all four G-protein families that have been described thus far. The alpha subunit genes identified are: G alpha i1-3 and G alpha z but not G alpha o from the Gi family, G alpha s from the Gs family, G alpha 11, G alpha q, and G alpha 16 from the Gq family, and G alpha 12 and G alpha 13 from the G12 family. Also in this report we present the nucleotide and predicted amino acid sequences of the human G alpha 13 cloned from a thymocyte cDNA library. The sequence of the human G alpha 13 has not been previously reported. Comparison of this sequence with the reported murine G alpha 13 shows > 90% identity at the deduced amino acid sequence level. We conclude that thymocytes represent a useful experimental system for the study of G protein involvement in immune responses and lymphocyte development.

    Funded by: NIGMS NIH HHS: GM39518, GM45765

    Molecular and cellular biochemistry 1995;144;1;45-51

  • Mechanism of GTP hydrolysis by G-protein alpha subunits.

    Kleuss C, Raw AS, Lee E, Sprang SR and Gilman AG

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

    Hydrolysis of GTP by a variety of guanine nucleotide-binding proteins is a crucial step for regulation of these biological switches. Mutations that impair the GTPase activity of certain heterotrimeric signal-transducing G proteins or of p21ras cause tumors in man. A conserved glutamic residue in the alpha subunit of G proteins has been hypothesized to serve as a general base, thereby activating a water molecule for nucleophilic attack on GTP. The results of mutagenesis of this residue (Glu-207) in Gi alpha 1 refute this hypothesis. Based on the structure of the complex of Gi alpha 1 with GDP, Mg2+, and AlF-4, which appears to resemble the transition state for GTP hydrolysis, we believe that Gln-204 of Gi alpha 1, rather than Glu-207, supports catalysis of GTP hydrolysis by stabilization of the transition state.

    Funded by: NIGMS NIH HHS: GM34497

    Proceedings of the National Academy of Sciences of the United States of America 1994;91;21;9828-31

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

    Offermanns S, Laugwitz KL, Spicher K and Schultz G

    Institut für Pharmakologie, Freie Universität Berlin, Germany.

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

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

Gene lists (4)

Gene List Source Species Name Description Gene count
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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