Alpha-thrombin-mediated phosphatidylinositol 3-kinase activation through release of Gbetagamma dimers from Galphaq and Galphai2

• Published in: The Journal of biological chemistry 2004-02, Vol.279 (8), p.6701-6710
• Main Authors: Goel, Reema, Phillips-Mason, Polly J, Gardner, Alice, Raben, Daniel M, Baldassare, Joseph J
• Format: Article
• Language: English
• Subjects: Adenosine Diphosphate - chemistry; Adenosine Diphosphate - metabolism; Animals; Arrestins - metabolism; beta-Arrestins; Blotting, Western; Cell Membrane - metabolism; Cricetinae; Dimerization; DNA, Complementary - metabolism; Enzyme Activation; Fibroblasts - metabolism; Genes, Dominant; GTP-Binding Protein alpha Subunit, Gi2; GTP-Binding Protein alpha Subunits, Gi-Go - chemistry; GTP-Binding Protein alpha Subunits, Gq-G11 - chemistry; GTP-Binding Protein beta Subunits - chemistry; GTP-Binding Protein gamma Subunits - chemistry; Guanosine 5'-O-(3-Thiotriphosphate) - metabolism; Microscopy, Confocal; Models, Biological; Mutation; Peptides - chemistry; Pertussis Toxin - pharmacology; Phosphatidylinositol 3-Kinases - metabolism; Phosphorylation; Precipitin Tests; Protein Binding; Protein Structure, Tertiary; Protein Transport; Proto-Oncogene Proteins - chemistry; Ribose - chemistry; Signal Transduction; Thrombin - chemistry; Thrombin - metabolism; Transducin - metabolism; Transfection; Xanthine - metabolism
• ISSN: 0021-9258

Chinese hamster embryonic fibroblasts (IIC9 cells) express the Galpha subunits Galphas, Galphai2, Galphai3, Galphao, Galpha(q/11), and Galpha13. Consistent with reports in other cell types, alpha-thrombin stimulates a subset of the expressed G proteins in IIC9 cells, namely Gi2, G13, and Gq as measured by an in vitro membrane [35S]guanosine 5'-O-(3-thio)triphosphate binding assay. Using specific Galpha peptides, which block coupling of G-protein receptors to selective G proteins, as well as dominant negative xanthine nucleotide-binding Galpha mutants, we show that activation of the phosphatidylinositol 3-kinase/Akt pathway is dependent on Gq and Gi2. To examine the role of the two G proteins, we examined the events upstream of PI 3-kinase. The activation of the PI 3-kinase/Akt pathway by alpha-thrombin in IIC9 cells is blocked by the expression of dominant negative Ras and beta-arrestin1 (Phillips-Mason, P. J., Raben, D. M., and Baldassare, J. J. (2000) J. Biol. Chem. 275, 18046-18053, and Goel, R., Phillips-Mason, P. J., Raben, D. M., and Baldassare, J. J. (2002) J. Biol. Chem. 277, 18640-18648), indicating a role for Ras and beta-arrestin1. Interestingly, inhibition of Gi2 and Gq activation blocks Ras activation and beta-arrestin1 membrane translocation, respectively. Furthermore, expression of the Gbetagamma sequestrant, alpha-transducin, inhibits both Ras activation and membrane translocation of beta-arrestin1, suggesting that Gbetagamma dimers from Galphai2 and Galphaq activate different effectors to coordinately regulate the PI 3-kinase/Akt pathway.