Mammalian .beta.2-adrenergic receptor: reconstitution of functional interactions between pure receptor and pure stimulatory nucleotide binding protein of the adenylate cyclase system

Abstract

Pure .beta.-adrenergic receptors (.beta.-AR), isolated from guinea pig lung, and pure guanine nucleotide binding regulatory protein (NS) of adenylate cyclase, isolated from human erythrocytes, were inserted into phospholipid vesicles, resulting in the functional coupling of these 2 components. The reconstitution of receptor and NS interactions results in the establishment of a guanine nucleotide sensitive state of the receptor that binds agonists with high affinity. Competition curves of isoproterenol, for labeled antagonist binding to vesicles containing both .beta.-AR and NS, are biphasic and reveal 2 affinity states, one of high (.apprx. 2 nM) and the other of low (.apprx. 300 nM) affinity. In the presence of guanine nucleotides, the competition curves become monophasic; they are shifted to a single low-affinity state for the agonist, similar to the situation observed in membrane preparations. In addition, the interactions of the receptor and NS lead to the induction of a GTPase activity in NS. The GTPase activity can be stimulated by .beta.-adrenergic agonists such as isoproterenol (2- to 5-fold); it is completely blocked by antagonists, such as alprenolol, in a stereoselective manner. The established hormone responsive activity retains the .beta.2-adrenergic specificity conferred by the pure receptor, and similar extents of stimulation (up to 4-fold) are observed with pure receptor from frog erythrocytes, indicating a similar efficiency of coupling between receptors from different species and NS. These experiments demonstrate that no components, other than the pure .beta.-AR and NS, are necessary to elicit these activities; they now open the way for attempting to elucidate the molecular mechanisms by which transduction of receptor occupancy into NS activation leads to the stimulation of the rate of cAMP formation.