Reconstitution of Catecholamine-Stimulated Binding of Guanosine 5’ -O-(3-Thiotnphosphate) to the Stimulatory GTP-Binding Protein of Adenylate Cyclase

The stimulatory GTP-binding protein (Gs) of adenylate cyclase, purified from rabbit liver, and 0-adrenergic receptors, partially purified 1000-4000-fold from turkey erythrocyte plasma membranes, were coreconstituted into unilamellar phospholipid vesicles. The molar ratio of Gs to receptors in the vesicles varied from 3 to 10 in different preparations, as measured by guanosine 5′-O-(3-[35S]thiotriphosphate) ([35S]Gtpγs) binding to Gs and [125I]iodocyanopindolol binding to receptors. Activation of reconstituted Gs by Gtpγs was stimulated up to 10-fold by the addition of the β-adrenergic agonist (-)-isoproterenol. Activation was assayed functionally by reconstitution with the catalytic unit of adenylate cyclase. Because of the relative purity of this preparation, the quasi-irreversible binding of [35S]GTP7S could also be measured in the vesicles and was shown to parallel the functional activation of Gs under all conditions. Most of the assayable Gs in the vesicles could interact with the receptors and undergo agonist-stimulated activation. Agonist-stimulated activation and [35S]Gtpγs binding were complete in less than 3 min, even under suboptimal conditions, and could go to completion in 20 s under maximal stimulation. Agonist-stimulated binding did not require appreciable free Mg2+ (0.1 mM). Activation in the absence of agonist was stimulated by free Mg2+, but maximal activation took up to 10 min in the presence of 50 mM MgCl2. Reconstitution increased the stability of Gs to thermal denaturation. The addition of β-adrenergic agonist further stabilized Gs, presumably by the formation of a stable agonist-receptor-Gs complex. The apparent Kd for Gtpγs for agonist-stimulated binding was about 6 nM, almost 50-fold lower than that observed for Mg2+-stimulated binding in the vesicles and about 100-fold lower than that observed for soluble Gs. This variation in Ká can be qualitatively accounted for by the stabilization of Gs by reconstitution and by the interaction of Gs with the agonist-receptor complex. The ECS0 for (-)-isoproterenol for stimulation of GTP7S binding was 13 nM, far lower than its equilibrium Kd for binding to the receptor under similar conditions, 870 nM. This discrepancy, apparent spare receptors where Gs is in molar excess, reflects the ability of receptors to rapidly activate multiple Gs molecules.