α-Thrombin-mediated Phosphatidylinositol 3-Kinase Activation through Release of Gβγ Dimers from Gαq and Gα i2

Abstract

Chinese hamster embryonic fibroblasts (IIC9 cells) express the G α subunits Gαs, Gαi2, Gαi3, Gαo, Gαq/11, and Gα13. Consistent with reports in other cell types, α-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 Gα peptides, which block coupling of G-protein receptors to selective G proteins, as well as dominant negative xanthine nucleotide-binding Gα 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 a-thrombin in IIC9 cells is blocked by the expression of dominant negative Ras and β-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 β-arrestin1. Interestingly, inhibition of Gi2 and Gq activation blocks Ras activation and β-arrestin1 membrane translocation, respectively. Furthermore, expression of the Gβγ sequestrant, α-transducin, inhibits both Ras activation and membrane translocation of β-arrestin1, suggesting that Gβγ dimers from Gαi2 and Gαq activate different effectors to coordinately regulate the PI 3-kinase/Akt pathway.