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The precise mechanism whereby epidermal growth factor (EGF) activates the serine-threonine kinase Akt and the mammalian target of rapamycin (mTOR) complex 1 (mTORC1) remains elusive. Here, we report that the α subunits of the heterotrimeric guanine nucleotide–binding proteins (G proteins) Gαi1 and Gαi3 are critical for this activation process. Both Gαi1 and Gαi3 formed complexes with growth factor receptor binding 2 (Grb2)–associated binding protein 1 (Gab1) and the EGF receptor (EGFR) and were required for the phosphorylation of Gab1 and its subsequent interaction with the p85 subunit of phosphatidylinositol 3-kinase in response to EGF. Loss of Gαi1 and Gαi3 severely impaired the activation of Akt and of p70 S6 kinase and 4E-BP1, downstream targets of mTORC1, in response to EGF, heparin-binding EGF-like growth factor, and transforming growth factor α, but not insulin, insulin-like growth factor, or platelet-derived growth factor. In addition, ablation of Gαi1 and Gαi3 largely inhibited EGF-induced cell growth, migration, and survival, and the accumulation of cyclin D1. Overall, this study suggests that Gαi1 and Gαi3 lie downstream of EGFR, but upstream of Gab1-mediated activation of Akt and mTORC1, thus revealing a role for Gαi proteins in mediating EGFR signaling.

VEGF binding to VEGFR2 leads to VEGFR2 endocytosis and downstream signaling activation to promote angiogenesis.Methods: Using genetic strategies, we tested the requirement of α subunits of heterotrimeric G proteins (Gαi1/3) in the process.Results: Gαi1/3 are located in the VEGFR2 endocytosis complex (VEGFR2-Ephrin-B2-Dab2-PAR-3), where they are required for VEGFR2 endocytosis and downstream signaling transduction. Gαi1/3 knockdown, knockout or dominant negative mutation inhibited VEGF-induced VEGFR2 endocytosis, and downstream Akt-mTOR and Erk-MAPK activation. Functional studies show that Gαi1/3 shRNA inhibited VEGF-induced proliferation, invasion, migration and vessel-like tube formation of HUVECs. In vivo, Gαi1/3 shRNA lentivirus inhibited alkali burn-induced neovascularization in mouse cornea. Further, oxygen-induced retinopathy (OIR)-induced retinal neovascularization was inhibited by intravitreal injection of Gαi1/3 shRNA lentivirus. Moreover, in vivo angiogenesis by alkali burn and OIR was significantly attenuated in Gαi1/3 double knockout mice. Significantly, Gαi1/3 proteins are upregulated in proliferative retinal tissues of proliferative diabetic retinopathy (PDR) patients.Conclusion: These results provide mechanistic insights into the critical role played by Gαi1/3 proteins in VEGF-induced VEGFR2 endocytosis, signaling and angiogenesis.