Molecular Pharmacology, Vol 17, 163-171, Copyright © 1980 by the American Society for Pharmacology and Experimental Therapeutics

Inactivation of -Adrenergic Receptors by N-Ethylmaleimide: Permissive Role of -Adrenergic Agents in Relation to Adenylate Cyclase Activation

¹ Biochemical Pathology, Institute Molecular Biology, Free University Brussels, V.U.B., 65, Paardenstraat, B-1640 St. Genesius Rode, Belgium
² Biochemical Pathology, Institute Molecular Biology, Free University Brussels, V.U.B., 65, Paardenstraat, B-1640 St. Genesius Rode, Belgium, and Molecular Immunology, Institute Molecular Biology, University Paris VII, Place Jussieu 2, Paris, France

The ₁-adrenergic receptors of turkey erythrocyte membranes have been identified by the specific binding of the radiolabeled antagonist (-)-[³H]dihydroalprenolol. Binding of -adrenergic agonists to these receptor sites sensitizes them to inactivation by the alkylating agent N-ethylmaleimide. A dose- and time-dependent decrease of 45 to 60% of the sites is commonly observed. Binding of (-)-³H-dihydroalprenolol and -adrenergic agonists to the remaining sites occurs with the same characteristics as for the untreated receptor population. Kinetic experiments reveal that the rate of inactivation is proportional to the concentration of N-ethylmaleimide (between 5.5 and 450 µM). In contrast, the rate of inactivation reaches a plateau value when increasing the concentration of the agonist. The rate of inactivation is half-maximal in presence of 1.3 µM (-)-epinephrine or 20 µM (+)-epinephrine. This marked stereospecificity, along with the close identity of the above concentrations with the equilibrium dissociation constant (K_D) of the epinephrine isomers for binding to the -receptor (i.e., 2.0 µM for (-)-epinephrine and 21 µM for (+)-epinephrine) indicate that N-ethylmaleimide inactivates the agonist-bound form of the receptor. The second-order rate constant (k₂) of the inactivation process, in the presence of 15 -adrenergic ligands, was found to correlate with their capability to stimulate the adenylate cyclase activity, i.e., "intrinsic activity." Since all tested ligands were able to cause a complete and dose-dependent displacement of bound (-)-[³H]dihydroalprenolol, it is likely that both the intrinsic activity and k₂ of each adrenergic ligand reflect an inherent property of the ligand-bound receptor. The proportionality between k₂ and the intrinsic activity further suggests that -adrenergic agonists "induce" or "favor" a conformational change of the receptor, resulting in adenylate cyclase activation and the uncovering of an alkylable group which becomes exposed to N-ethylmaleimide in the active conformation.

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