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 ␣1B-or ␣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 ␣2A-AR is effectively attenuated by pretreatment with pertussis toxin or by coexpression of a G␤␥ subunit complex sequestrant peptide (␤ARK1ct) and dominantnegative mutants of Ras (N17-Ras), mSOS1 (SOS-Pro), and Raf (⌬N-Raf). Erk1/2 phosphorylation in response to ␣1B-AR stimulation is also attenuated by coexpression of N17-Ras, SOS-Pro, or ⌬N-Raf, but not by coexpression of ␤ARK1ct or by pretreatment with pertussis toxin. The ␣1B-and ␣2A-AR signals are both blocked by phospholipase C inhibition, intracellular Ca 2؉ 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 ␣1B-AR-and ␣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 mitogenactivated protein kinase phosphorylation after both ␣1B-and ␣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␤␥ subunit-mediated ␣2A-AR-and the G␣ q/11 -mediated ␣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.

Glucagon receptor activates extracellular signal-regulated protein kinase 1/2 via cAMP-dependent protein kinase

“…[Ca 2ϩ ] i has been shown to stimulate MAP kinase activation through different signaling molecules, which converge to activate a Ras-dependent Raf͞MEK͞MAPK kinase cascade (27,28). Thapsigargin and A23187 treatments induce a strong increase in [Ca 2ϩ ] i , which may stimulate Raf to activate MAP kinase.…”

The proliferative and antiapoptotic effects of substance P are facilitated by formation of a β-arrestin-dependent scaffolding complex

“…GPCRs activate ERK1͞2 by several mechanisms (18)(19)(20), including transactivation of receptor tyrosine kinases (20)(21)(22) and activation of proline rich tyrosine kinase 2 (PYK2), a tyrosine kinase that leads to shc phosphorylation and rasactivation (19,(23)(24)(25). To determine the mechanism used by NK1R, we examined the sensitivity of SP-stimulated ERK activation to GF103209X, a nonspecific protein kinase C inhibitor, genistein, a nonspecific tyrosine kinase inhibitor, and tyrphostin 25, an inhibitor of receptor tyrosine kinases.…”

Cross-talk between Cytosolic Phospholipase A2α (cPLA2α) and Secretory Phospholipase A2 (sPLA2) in Hydrogen Peroxide-induced Arachidonic Acid Release in Murine Mesangial Cells

“…Because BAPTA-AM has been shown to affect MAPK pathways in other cells, we evaluated whether ERK1/2 phosphorylation was affected by BAPTA-AM treatment (62)(63)(64) , and Western blot analysis was performed. SDS-PAGE was performed using 15 g of total cellular protein for each sample.…”