Catecholamine-stimulated GTPase cycle. Multiple sites of regulation by beta-adrenergic receptor and Mg2+ studied in reconstituted receptor-Gs vesicles.

Abstract

The regulation of the intermediary steps of the catecholamine-stimulated GTPase cycle by beta-adrenergic agonists and Mg2+ was investigated using unilamellar phosphatidylethanolamine-phosphatidylserine vesicles that contained purified beta-adrenergic receptor and the stimulatory GTP-binding protein of the adenylate cyclase system, Gs. The steady-state turnover number of the agonist-stimulated GTPase, normalized according to the receptor-responsive pool of Gs, was 0.8 min-1 for untreated vesicles and 1.7 min-1 for vesicles that had been treated with dithiothreitol to activate the receptors. The binding and release of [alpha-32P]GTP, [3H] GTP, and [gamma-32P]GTP were used to measure the binding and hydrolysis of GTP and the release of GDP. Agonist-liganded receptor stimulated both the binding of GTP and the release of the GDP product, and GDP release per se did not appear to be the mechanism by which receptor stimulated the binding of GTP. Both processes displayed apparent first order rate constants of about 0.5 min-1 for untreated vesicles and both rates increased about 5-fold after dithiothreitol treatment. Both processes were formally catalytic with respect to receptor, in that several (up to 8) molecules of Gs were stimulated per molecule of receptor. The hydrolysis of Gs X GTP to Gs X GDP was unaltered by agonist and occurred with a rate constant of about 4 min-1. The rates of these partial reactions were consistent with the overall rate of steady-state hydrolysis and with the ability of the agonist-liganded receptor to promote the formation of sufficient Gs X GTP to fully stimulate adenylate cyclase in a native membrane. The Mg2+ dependence of agonist-stimulated, steady-state GTPase activity appeared to consist of at least two, distinct Mg2+-requiring processes. Very low concentrations of Mg2+ (approximately 20 nM) were required for hydrolysis of Gs X GTP, and 10 microM Mg2+ was required to maximize the initial rate of agonist-stimulated [alpha-32P] GTP binding.