Independent Regulation of Rap1 and Mitogen-Activated Protein Kinase by the α Chain of Go

Abstract

Receptors coupled to G_i/o proteins stimulate the mitogen-activated protein kinase (MAPK) cascade. The intracellular pathways linking the α chains of these G proteins to MAPK activation are not completely understood. One of the signaling molecules which has been suggested to act downstream of Gα_i/o is the small G protein Rap1. We investigated the role of Rap1 in MAPK stimulation by Gα_o in Chinese hamster ovary (CHO) cells. Our previous results have shown that in this cell system activated Gα_o strongly potentiates the MAPK response to the epidermal growth factor (EGF) receptor. Rap1 regulation was examined in cells transfected with Rap1 and wild-type Gα_o or the activated mutant Gα_o-Q205L. Immunocytochemical analysis detected both Rap1 and the Gα_o subunit at the plasma membrane as well as on perinuclear cytoplasmic vesicles. Expression of wild-type Gα_o had no significant effect on the levels of activated Rap1. In contrast, Gα_o-Q205L virtually abolished the activation of Rap1 induced by EGF. Further experiments showed that MAPK stimulation by EGF was greatly inhibited by expression of activated Rap1, suggesting that Rap1 inhibition could mediate the effect of Gα_o on the MAPK cascade. However, Gα_o-Q205L efficiently inhibited the activation of Rap1 induced by fibroblast growth factor (FGF). We have previously found that the ability of FGF to activate MAPK is not modified by Gα_o. In addition, expression of the GAP protein RAP1GAPII blocked Rap1 activation without affecting EGF- or FGF-dependent MAPK stimulation. These findings provide evidence for independent regulation of Rap1 and MAPK by the G_oα chain.