Internalized Receptors Reconstitute Catecholamine-stimulated Adenylate Cyclase Activity*

The adenylate cyclase-coupled &-adrenergic receptor of the frog erythrocyte has served as a useful model system for elucidating the mechanisms of catecholamine-induced desensitization. In this system, it has been previously demonstrated that agonist-induced refractoriness is associated with sequestration of the @adrenergic receptors in vesicles away from the cell surface and from their effector unit, the adenylate cyclase system (Stadel, J. M., Strulovici, B., Nambi, P., Lavin, T. N., Briggs, M. M., Caron, M. G., and Lefkowitz, R. J. (1983) J. Biol. Chern. 258, 30323038). These internalized @-adrenergic receptors appear to be structurally intact as assessed by photoaffinity labeling, but their functional status has previously been unknown. In the present studies, we sought to assess the functionality of the sequestered vesicular receptors by fusing them to Xenopus laevis erythrocytes. This cell is suitable for such studies, since it has almost no detectable &adrenergic receptor or catecholamine-sensitive adenylate cyclase, but contains prostaglandin El-stimulable adenylate cyclase. Fusion of &adrenergic receptor-containing vesicles from desensitized frog erythrocytes with X. laevis erythrocytes results in a 30-fold stimulation of the hybrid adenylate cyclase by the &adrenergic agonist isoproterenol. This effect was entirely blocked by the &antagonist propranolol. The catecholamine-sensitive adenylate cyclase activity established in the vesicle-Xenopus hybrids showed the characteristic agonist potency series of the donor frog erythrocyte &-adrenergic receptor. Fusion of vesicles from desensitized frog erythrocytes in which the 8adrenergic receptors had been inactivated with the group specific reagent dicyclohexylcarbodiimide, or of vesicles derived from control frog erythrocytes, which contain low amounts of @-adrenergic receptor, did not establish catecholamine-sensitive adenylate cyclase activity in the hybrids. These data demonstrate that &adrenergic receptors internalized during desensitization retain their functionality when recoupled to an adenylate cyclase system from a different source. The functional uncoupling of these receptors during desensitization is thus more likely due to their sequestration away from the other components of the adenylate cyclase than to any alterations in the receptors themselves.