Ric-8 enhances G protein βγ-dependent signaling in response to βγ-binding peptides in intact cells

Peptides derived from a random-peptide phage display screen with purified G beta(1)gamma(2) subunits as the target promote the dissociation of G protein heterotrimers in vitro and activate G protein signaling in intact cells. In vitro, one of these peptides (SIRKALNILGYPDYD; SIRK) promotes subunit dissociation by binding directly to G beta gamma subunits and accelerating the dissociation of G alpha GDP without catalyzing nucleotide exchange. The experiments described here were designed to test whether the mechanism of SIRK action in vitro is in fact the mechanism of action in intact cells. We created a mutant of G beta(1) subunits (beta(1)W332A) that does not bind SIRK in vitro. Transfection of G beta(1)W332A mutant into Chinese hamster ovary cells blocked peptide-mediated activation of extracellular signal-regulated kinase (ERK), but it did not affect receptor-mediated G beta gamma subunit-dependent ERK activation, indicating that G beta gamma subunits are in fact the direct target in cells responsible for ERK activation. To determine whether free G alpha subunits were released from G protein heterotrimers upon peptide treatment, cells were transfected with Ric-8A, a guanine nucleotide exchange factor for free G alpha GDP, but not heterotrimeric G proteins. Ric-8A-transfected cells displayed enhanced myristoyl-SIRKALNILGYPDYD (mSIRK)-dependent inositol phosphate (IP) release and ERK activation. Ric-8A also enhanced ERK activation by the G(i)-linked G protein coupled receptor agonist lysophosphatidic acid. Inhibitors of G beta gamma subunit function blocked Ric-8-enhanced activation of ERK and IP release. These results suggest that one potential function of Ric-8 in cells is to enhance G protein G beta gamma subunit signaling. Overall, these experiments provide further support for the hypothesis that mSIRK promotes G protein subunit dissociation to release free beta gamma subunits in intact cells.