Direct evidence that Gi-coupled receptor stimulation of mitogen-activated protein kinase is mediated by Gβγ activation of p21ras

Abstract

Stimulation of Gi-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 p21ras (Ras). Which G-protein subunit(s) (Gα or the Gβγ complex) primarily is responsible for triggering this signaling pathway, however, is unclear. We have demonstrated previously that the carboxyl terminus of the β-adrenergic receptor kinase, containing its Gβγ-binding domain, is a cellular Gβγ antagonist capable of specifically distinguishing Gα-and Gβγ-mediated processes. Using this Gβγ inhibitor, we studied Ras and MAP kinase activation through endogenous Gi-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 Gβγ antagonist. Likewise in COS-7 cells transfected with plasmids encoding Gi-coupled receptors (α2-adrenergic and M2 muscarinic), the activation of Ras and MAP kinase was significantly reduced in the presence of the coexpressed Gβγ antagonist. Ras-MAP kinase activation mediated through a Gq-coupled receptor (α1-adrenergic) or the tyrosine kinase epidermal growth factor receptor was unaltered by this Gβγ antagonist. These results identify Gβγ as the primary mediator of Ras activation and subsequent signaling via MAP kinase in response to stimulation of Gi-coupled receptors.