G beta gamma subunits Src-dependent phosphorylation of the epidermal growth factor receptor - A scaffold for G protein-coupled receptor-mediated Ras activation

In many cells, stimulation of mitogen-activated protein kinases by both receptor tyrosine kinases and receptors that couple to pertussis toxin-sensitive heterotrimeric G proteins proceed via convergent signaling pathways. Both signals are sensitive to inhibitors of tyrosine protein kinases and require Res activation via phosphotyrosine-dependent recruitment of Ras guanine nucleotide exchange factors. Receptor tyrosine kinase stimulation mediates ligand-induced receptor autophosphorylation, which creates the initial binding sites for SH2 domain-containing docking proteins. However, the mechanism whereby G protein-coupled receptors mediate the phosphotyrosine-dependent assembly of a mitogenic signaling complex is poorly understood. We have studied the role of Src family nonreceptor tyrosine kinases in G protein-coupled receptor-mediated tyrosine phosphorylation in a transiently transfected COS-7 cell system. Stimulation of G(i)-coupled lysophosphatidic acid and alpha 2A adrenergic receptors or overexpression of G beta 1 gamma 2 subunits leads to tyrosine phosphorylation of the She adapter protein, which then associates with tyrosine phosphoproteins of approximately 130 and 180 kDa, as well as Grb2. The 180-kDa She-associated tyrosine phosphoprotein band contains both epidermal growth factor (EGF) receptor and p185(neu). 3-5-fold increases in EGF receptor but not p185(neu) tyrosine phosphorylation occur following G(i)-coupled receptor stimulation. Inhibition of endogenous Src family kinase activity by cellular expression of a dominant negative kinase-inactive mutant of c-Src inhibits G beta 1 gamma 2 subunit-mediated and G(i)-coupled receptor-mediated phosphorylation of both EGF receptor and She. Expression of Csk, which inactivates Src family kinases by phosphorylating the regulatory carboxyl-terminal tyrosine residue, has the same effect. The G(i)-coupled receptor-mediated increase in EGF receptor phosphorylation does not reflect increased EGF receptor autophosphorylation, assayed using an autophosphorylation-specific EGF receptor monoclonal antibody. Lysophosphatidic acid stimulates binding of EGF receptor to a GST fusion protein containing the c-Src SH2 domain, and this too is blocked by Csk expression. These data suggest that G beta gamma subunit-mediated activation of Src family nonreceptor tyrosine kinases can account for the G(i)-coupled receptor-mediated tyrosine phosphorylation events that direct recruitment of the She and Grb2 adapter proteins to the membrane.