Reciprocal regulation of G(sα) by palmitate and the βγ subunit

Hormonal activation of G(s), the stimulatory regulator of adenylyl cyclase, promotes dissociation of α(s) from Gβγ, accelerates removal of covalently attached palmitate from the Gα subunit, and triggers release of a fraction of α(s) from the plasma membrane into the cytosol. To elucidate relations among these three events, we assessed biochemical effects in vitro of attached palmitate on recombinant α(s) prepared from Sf9 cells. In comparison to the unpalmitoylated protein (obtained from cytosol of Sf9 cells, treated with a palmitoyl esterase, or expressed as a mutant protein lacking the site for palmitoylation), palmitoylated α(s) (from Sf9 membranes, 50% palmitoylated) was more hydrophobic, as indicated by partitioning into TX-114, and bound βγ with 5-fold higher affinity. βγ protected GDP-bound α(s), but not α(s)· GTP[γS], from depalmitoylation by a recombinant esterase. We conclude that βγ binding and palmitoylation reciprocally potentiate each other in promoting membrane attachment of α(s) and that dissociation of α(s)·GTP from βγ is likely to mediate receptor-induced α(s) depalmitoylation and translocation of the protein to cytosol in intact cells.