α-thrombin-mediated phosphatidylinositol 3-kinase activation through release of Gβγ dimers from Gαq and Gαi2

Chinese hamster embryonic fibroblasts (IIC9 cells) express the Galpha subunits Galpha(s), Galpha(i2), Galpha(i3), Galpha(o), Galpha(q/11), and Galpha(13). Consistent with reports in other cell types, alpha-thrombin stimulates a subset of the expressed G proteins in IIC9 cells, namely G(i2), G(i3), and G(q) as measured by an in vitro membrane [S-35]guanosine 5'-O-(3-thio)triphosphate binding assay. Using specific Ga peptides, which block coupling of G-protein receptors to selective G proteins, as well as dominant negative xanthine nucleotide-binding Ga mutants, we show that activation of the phosphatidylinositol 3-kinase/Akt pathway is dependent on G(q) and G(i2). 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 G(i2) and G(q) 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 Galpha(i2) and Galpha(q) activate different effectors to coordinately regulate the PI 3-kinase/Akt pathway.