G(I2)-MEDIATED ACTIVATION OF THE MAP KINASE CASCADE

The G(i) class of heterotrimeric G proteins has been implicated in tra nsmitting mitogenic signals from a variety of seven-transmembrane doma in receptors. In addition, the alpha subunit of G(i2) (alpha(i2)) is o ncogenic when mutated to a constitutively active form (gip2). The mech anism by which G(i2) stimulates cellular proliferation is unknown, but is believed to involve activation of the mitogen-activated protein ki nase (MAPK) signaling cascade. To study G(i2) activation of the cascad e, we transiently expressed a mutant, pertussis toxin (PTX)-resistant alpha(i2) in Chinese hamster ovary cells. After PTX treatment of these cells, G(i)-coupled receptors specifically activated PTX-resistant G( i2) without activating other G(i) proteins. Receptor-mediated activati on of G(i2) led to activation of MAPK and its upstream activator, MAPK /ERK-activating kinase (MEK). Activation of MAPK and MEK by G(i2) was blocked by expression of a dominant-negative mutant of Ras. G(i2) acti vation did not, however, detectably increase the proportion of Ras pro tein in the GTP-bound form. Additional experiments suggest that G(i2) stimulates the MAPK pathway, at least in part, by mechanisms that invo lve release of its beta gamma subunit, as well as activation of phosph atidylinositol-3 kinase.