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 bot h receptor tyrosine kinases and receptors that couple to pertussis tox in-sensitive heterotrimeric G proteins proceed via convergent signalin g pathways. Both signals are sensitive to inhibitors of tyrosine prote in kinases and require Res activation via phosphotyrosine-dependent re cruitment of Ras guanine nucleotide exchange factors. Receptor tyrosin e kinase stimulation mediates ligand-induced receptor autophosphorylat ion, which creates the initial binding sites for SH2 domain-containing docking proteins. However, the mechanism whereby G protein-coupled re ceptors mediate the phosphotyrosine-dependent assembly of a mitogenic signaling complex is poorly understood. We have studied the role of Sr c family nonreceptor tyrosine kinases in G protein-coupled receptor-me diated tyrosine phosphorylation in a transiently transfected COS-7 cel l 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 t hen associates with tyrosine phosphoproteins of approximately 130 and 180 kDa, as well as Grb2. The 180-kDa She-associated tyrosine phosphop rotein band contains both epidermal growth factor (EGF) receptor and p 185(neu). 3-5-fold increases in EGF receptor but not p185(neu) tyrosin e phosphorylation occur following G(i)-coupled receptor stimulation. I nhibition of endogenous Src family kinase activity by cellular express ion of a dominant negative kinase-inactive mutant of c-Src inhibits G beta 1 gamma 2 subunit-mediated and G(i)-coupled receptor-mediated pho sphorylation of both EGF receptor and She. Expression of Csk, which in activates Src family kinases by phosphorylating the regulatory carboxy l-terminal tyrosine residue, has the same effect. The G(i)-coupled rec eptor-mediated increase in EGF receptor phosphorylation does not refle ct increased EGF receptor autophosphorylation, assayed using an autoph osphorylation-specific EGF receptor monoclonal antibody. Lysophosphati dic acid stimulates binding of EGF receptor to a GST fusion protein co ntaining the c-Src SH2 domain, and this too is blocked by Csk expressi on. These data suggest that G beta gamma subunit-mediated activation o f Src family nonreceptor tyrosine kinases can account for the G(i)-cou pled receptor-mediated tyrosine phosphorylation events that direct rec ruitment of the She and Grb2 adapter proteins to the membrane.