Distinct pathways of Gi- and Gq-mediated mitogen-activated protein kinase activation

Abstract

Receptors that couple to the heterotrimeric G proteins, Gi or Gq, 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 Gi and Gq signaling has not been clearly defined and is the subject of this study. The effects of several signaling inhibitors are assessed including expression of a peptide derived from the carboxyl terminus of the β adrenergic receptor kinase 1 (βARKct), which specifically blocks signaling mediated by the βγ subunits of G proteins (Gβγ), expression of dominant negative mutants of p21ras (RasN17) and p74raf-1 (NΔRaf), protein-tyrosine kinase (PTK) inhibitors and cellular depletion of PKC. The Gi-coupled α2A adrenergic receptor (AR) stimulates MAPK activation which is blocked by expression of βARKct, RasN17, or NΔRaf, or by PTK inhibitors, but unaffected by cellular depletion of PKC. In contrast, MAPK activation stimulated by the Gq-coupled α1B AR or M1 muscarinic cholinergic receptor is unaffected by expression of βARKct or RasN17 expression or by PTK inhibitors, but is blocked by expression of NΔRaf or by PKC depletion. These data demonstrate that Gi- and Gq-coupled receptors stimulate MAPK activation via distinct signaling pathways. Gβγ is responsible for mediating Gi-coupled receptor-stimulated MAPK activation through a mechanism utilizing p21ras and p74raf independent of PKC. In contrast, Gα mediates Gq-coupled receptor-stimulated MAPK activation using a p21ras-independent mechanism employing PKC and p74raf. To define the role of Gβγ in Gi-coupled receptor-mediated PI hydrolysis and MAPK activation, direct stimulation with Gβγ was used. Expression of Gβγ resulted in MAPK activation that was sensitive to inhibition by expression of βARKct, RasN17, or NΔRaf or by PTK inhibitors, but insensitive to PKC depletion. By comparison, Gβγ-mediated PI hydrolysis was not affected by βARKct, RasN17, or NΔRaf expression or by PTK inhibitors. Together, these results demonstrate that Gβγ mediates MAPK activation and PI hydrolysis via independent signaling pathways.