Ras-dependent mitogen-activated protein kinase activation by G protein-coupled receptors - Convergence of G(i)- and G(q)-mediated pathways on calcium/calmodulin, Pyk2, and Src kinase

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 effecters, In HEK-293 cells, stimulation of either alpha 1B- or alpha 2A-adrenergic receptors (ARs) leads to rapid 5-10-fold increases in Erk1/2 phosphorylation, Phosphorylation of Erk1/2 in response to stimulation of the alpha 2A-AR is effectively attenuated by pretreatment with pertussis toxin or by coexpression of a G beta gamma subunit complex sequestrant peptide (beta ARK1ct) and dominant-negative mutants of Ras (N17-Ras), mSOS1 (SOS-Pro), and Raf (Delta N-Raf), Erk1/2 phosphorylation in response to alpha 1B-AR stimulation is also attenuated by coexpression of N17-Ras, SOS-Pro, or Delta N-Raf, but not by coexpression of beta ARK1ct or by pretreatment with pertussis toxin. The alpha 1B- and alpha 2A-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 alpha 1B-AR- and alpha 2A-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 mitogen-activated protein kinase phosphorylation after both alpha 1B- and alpha 2A-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 G beta gamma subunit-mediated alpha 2A-AR- and the Ga-q/11-mediated alpha 1B-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.