The stimulatory G protein (Gs) mediates the activation of hormone- and neurotransmitter-responsive adenylyl cyclase. Gs consists of an alpha subunit (Gs-alpha) which has an intrinsic GTPase activity, and beta and gamma subunits that are tightly associated. Extensive research has produced a well-established model describing the molecular events that are mediated by Gs during hormone or neurotransmitter stimulation. Activation is initiated when the agonist-receptor complex promotes the exchange of GDP bound to Gs-alpha for GTP. Nucleotide exchange in turn leads to dissociation of the Gs-alpha from the beta-gamma-subunit complex. Subunit dissociation is believed to be pivotal since G(beta- gamma) can inhibit the activity of Gs-alpha. Gs-alpha activates adenylyl cyclase until GTP is hydrolyzed, whereupon G(beta-gamma)reassociates with Gs-alpha and prevents further activation of adenylyl cyclase until another round of nucleotide exchange can trigger subunit dissociation. To investigate the role of subunit dissociation on Gs activation, the heterotrimeric protein was partially purified from bovine brain and rabbit liver. Complete and irreversible activation of Gs was achieved by incubating it with the nonhydrolyzable GTP analogue, GTP-gamma-S, in the presence of different MgCl2 concentrations. Activation was assessed by reconstitution of adenylyl cyclase activity in S49 cyc-membranes (which lack Gs-alpha), and by measuring [35S]GTP-gamma-S binding to Gs. Subunit dissociation was determined by immunoprecipitating Gs-alpha, and measuring the amount of beta-subunit that coprecipitated. By these assays, it was determined that high concentrations of MgCl2 caused subunit dissociation even in the absence of GTP-gamma-S. When Gs was activated with low MgCl2 concentrations, there was little or no subunit dissociation. It was also determined that the time course for Gs activation was more rapid than that of subunit dissociation. These data suggest that subunit dissociation may be unrelated to Gs activation, and indicate that the model for Gs activation may need revision.
