Reciprocal regulation of Gsα 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. trimeric G proteins lipid modification membrane localization protein–protein interaction