DISTINCT PATHWAYS OF G(I)-MEDIATED AND G(Q)-MEDIATED MITOGEN-ACTIVATED PROTEIN-KINASE ACTIVATION

Receptors that couple to the heterotrimeric G proteins, G(1) or G(q), can stimulate phosphoinositide (PI) hydrolysis and mitogen-activated protein kinase (MAPK) activation. PI hydrolysis produces inositol 1,4,5-trisphosphate and diacylglycerol, leading to activation of protein kinase C (PKC), which can stimulate increased MAPK activity. However, the relationship between PI hydrolysis and MAPK activation in G(i) and G(q) signaling has not been clearly defined and is the subject study. The effects of several signaling inhibitors assessed including expression of a peptide derived from the carboxyl terminus of the beta adrenergic receptor kinase 1 (beta ARKct), which specifically blocks signaling mediated by the beta gamma subunits of G proteins (G beta gamma), expression of dominant negative mutants of p21(ras) (RasN17) and p74(raf-1) (N Delta Raf), protein-tyrosine kinase (PTK) inhibitors and cellular depletion of PRC. The G(1)-coupled alpha 2A adrenergic receptor (AR) stimulates MAPK activation which is blocked by expression of beta ARKct, RasN17, or N Delta Raf, or by PTK inhibitors, but unaffected by cellular depletion of PKC. In contrast, MAPK activation stimulated by the G(q) coupled, alpha 1B AR or M1 muscarinic cholinergic receptor is unaffected by expression of beta ARKct or RasN17 expression or by PTK inhibitors, but is blocked by expression of N Delta Raf or by PKC depletion. These data demonstrate that G(i)- and G(q)-coupled receptors stimulate MAPK activation via distinct signaling pathways. G beta gamma is responsible for mediating G(i)-coupled receptor-stimulated MAPK activation through a mechanism utilizing p21ras and p74raf independent of PKC. In contrast, G alpha mediates G(q)-coupled receptor-stimulated MAPK activation using a p21(ras)-independent mechanism employing PKC and p74(raf). To define the role of G beta gamma in G(i)-coupled receptor mediated PI hydrolysis and MAPK activation, direct stimulation with G beta gamma was used. Expression of G beta gamma resulted in MAPK activation that was sensitive to inhibition by expression of beta ARKct, RasN17, or N Delta Raf or by PTK inhibitors, but insensitive to PKC deletion. By comparison, G beta gamma-mediated PI hydrolysis not affected by beta ARKct, RasN17, or N Delta Raf expression or by PTK inhibitors. Together, these results demonstrate that G beta gamma mediates MAPK activation and PI hydrolysis via independent signaling pathways.