Ras-dependent mitogen-activated protein kinase activation by G protein- coupled receptors

Abstract

Many receptors that couple to heterotrimeric guanine-nucleotide binding proteins (G proteins) have been shown to mediate rapid activation of the mitogen-activated protein kinases Erk1 and Erk2. In different cell types, the signaling pathways employed appear to be a function of the available repertoire of receptors, G proteins, and effectors. In HEK-293 cells, stimulation of either αIB- or α2A-adrenergic receptors (ARs) leads to rapid 5-10-fold increases in Erk1/2 phosphorylation. Phosphorylation of Erk1/2 in response to stimulation of the α2A-AR is effectively attenuated by pretreatment with pertussis toxin or by coexpression of a Gβγ subunit complex sequestrant peptide (βARK1ct) and dominant-negative mutants of Ras (N17-Ras), mSOSl (SOS-Pro), and Raf (ΔN-Raf). Erk1/2 phosphorylation in response to α1B-AR stimulation is also attenuated by coexpression of N17- Ras, SOS-Pro, or ΔN-Raf, but not by coexpression of βARK1ct or by pretreatment with pertussis toxin. The α1B- and α2A-AR signals are both blocked by phospholipase C inhibition, intracellular Ca2+ chelation, and inhibitors of protein-tyrosine kinases. Overexpression of a dominant-negative mutant of c-Src or of the negative regulator of c-Src function, Csk, results in attenuation of the αlB-AR, and α2A-AR-mediated Erk1/2 signals. Chemical inhibitors of calmodulin, but not of PKC, and overexpression of a dominant- negative mutant of the protein-tyrosine kinase Pyk2 also attenuate mitogenactivated protein kinase phosphorylation after both αIB, and α2A-AR stimulation. Erk1/2 activation, then, proceeds via a common Ras-, calcium., and tyrosine kinase-dependent pathway for both G(i)- and G(q)/11/coupled receptors. These results indicate that in HEK-293 cells, the Gβγ subunit- mediated α3A-AR, and the Gα(q)/11-mediated α1B-AR-coupled Erk1/2 activation pathways converge at the level of phospholipase C. These data suggest that calcium-calmodulin plays a central role in the calcium-dependent regulation of tyrosine phosphorylation by G protein-coupled receptors in some systems.