DIRECT EVIDENCE THAT G(I)-COUPLED RECEPTOR STIMULATION OF MITOGEN-ACTIVATED PROTEIN-KINASE IS MEDIATED BY G(BETA-GAMMA) ACTIVATION OF P21(RAS)

Stimulation of G(i)-coupled receptors leads to the activation of mitogen-activated protein kinases (MAP kinases). In several cell types, this appears to be dependent on the activation of p21(ras) (Ras). Which G-protein subunit(s) (G(alpha) or the G(beta gamma) complex) primarily is responsible for triggering this signaling pathway, however, is unclear. We have demonstrated previously that the carboxyl terminus of the beta-adrenergic receptor kinase, containing its G(beta gamma)-binding domain, is a cellular G(beta gamma) antagonist capable of specifically distinguishing G(alpha)- and G(beta gamma)-mediated processes. Using this G(beta gamma) inhibitor, we studied Ras and MAP kinase activation through endogenous G(i)-coupled receptors in Rat-1 fibroblasts and through receptors expressed by transiently transfected COS-7 cells. We report here that both Ras and MAP kinase activation in response to lysophosphatidic acid is markedly attenuated in Rat-1 cells stably transfected with a plasmid encoding this G(beta gamma) antagonist. Likewise in COS-7 cells transfected with plasmids encoding G(i)-coupled receptors (alpha(2)-adrenergic and M2 muscarinic), the activation of Ras and MAP kinase was significantly reduced in the presence of the coexpressed G(beta gamma) antagonist. Ras-MAP kinase activation mediated through a G(q)-coupled receptor (alpha(1)-adrenergic) or the tyrosine kinase epidermal growth factor receptor was unaltered by this G(beta gamma) antagonist. These results identify G(beta gamma) as the primary mediator of Ras activation and subsequent signaling via MAP kinase in response to stimulation of G(i)-coupled receptors.