Reconstitution of a hormone-sensitive adenylate cyclase system. The pure beta-adrenergic receptor and guanine nucleotide regulatory protein confer hormone responsiveness on the resolved catalytic unit.
A hormone responsive adenylate cyclase has been reconstituted in phosphatidylcholine vesicles from its isolated protein components. The proteins used were the affinity chromatography purified (500-2000-fold) or pure Mr = 64,000 beta-adrenergic receptors (beta AR) isolated from hamster and guinea pig lung membranes, the pure heterotrimeric (Mr: alpha = 42,000; beta = 35,000; gamma approximately equal to 5,000) guanine nucleotide regulatory protein (Ns) isolated from human erythrocyte membranes; and the catalytic unit of the adenylate cyclase (C) solubilized from bovine brain caudate nucleus and resolved from beta AR and Ns by gel filtration. Adenylate cyclase activity in vesicles containing C alone was stimulated by forskolin but not by guanine nucleotides or by the beta-adrenergic agonist isoproterenol. Reconstitution of Ns and C interactions in the lipid vesicles resulted in guanine nucleotide but not beta-adrenergic agonist sensitivity. When beta AR was inserted together with Ns and C into lipid vesicles, the catalytic unit became responsive to beta-adrenergic agonists as well and this stimulation was blocked in a stereoselective manner by the beta-adrenergic antagonist alprenolol. Regulation of adenylate cyclase activity in the reconstituted system by beta-adrenergic agonists, guanine nucleotides, and Mg2+ showed properties similar to those observed in native membranes. The interactions of the various protein components in the reconstituted system were also monitored by GTPase activity. Such activity was observed to occur primarily as a result of receptor-Ns interactions. The results described in this report document the feasibility of studying hormone-responsive adenylate cyclase in a totally reconstituted system which retains the major regulatory properties of the enzyme in its native membrane-bound environment.
Cerione, RA; Sibley, DR; Codina, J; Benovic, JL; Winslow, J; Neer, EJ; Birnbaumer, L; Caron, MG; Lefkowitz, RJ
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