Glucagon receptor-mediated activation of G(s) is accompanied by subunit dissociation

The effect of the glucagon receptor on the activation of the stimulatory GTP-binding protein of adenylyl cyclase (G(s)) in the native rat liver membrane environment was studied. The activated state of G(s) was assessed by its ability to reconstitute the cyc^- S49 cell membrane adenylyl cyclase. The G(s) protein was activated by saturating concentrations of guanosine 5'-thiotriphosphate (GTPγS) or guanyl-5'-yl imidodiphosphate in a hormone-dependent manner at 0.4 mM Mg²⁺ in native membranes or in membranes that had been treated with 1 mM N-ethylmaleimide to eliminate the catalytic activity of adenylyl cyclase. At 50 mM Mg²⁺, G(s) was fully activated by GTPγS in the absence of hormone. The unactivated G(s) protein migrates around 4 S, whereas activated G(s) migrates around 2 S on sucrose density gradients. When pure G(s) is analyzed on sucrose density gradients, it is found that the unactivated protein migrates at 4.1 S. G(s) was activated by saturating concentrations of GTPγS and Mg²⁺, and the α subunit of G(s) was chromatographically purified. The resolved α subunit of G(s) that is capable of stimulating the cyc^- adenylyl cyclase migrates at 2.1 S. From these data, we conclude that activation of G(s) results in the dissociation of this protein in the membrane environment and that the hormone-occupied receptor promotes this dissociation process under conditions where Mg²⁺ ions are limiting.