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Vol. 53, Issue 3, 370-376, March 1998

Chloroethylclonidine Binds Irreversibly to Exposed Cysteines in the Fifth Membrane-Spanning Domain of the Human alpha 2A-Adrenergic Receptor

Anne Marjamäki, Marjo Pihlavisto, Victor Cockcroft,1 Petri Heinonen, Juha-Matti Savola,1 and Mika Scheinin

Departments of Pharmacology and Clinical Pharmacology (A.M., M.P., M.S.) and Chemistry (P.H.), University of Turku, FIN-20500, Turku, Finland, and Orion Corporation (V.C., J.-M.S.), Orion-Pharma, FIN-20101, Turku, Finland

The alpha 2-adrenergic receptors (alpha 2-ARs) mediate signals to intracellular second messengers via guanine nucleotide binding proteins. Three human genes encoding alpha 2-AR subtypes (alpha 2A, alpha 2B, alpha 2C) have been cloned. Several chemical compounds display subtype differences in their binding and/or functional activity. Site-directed mutagenesis and molecular modeling are new tools with which to investigate the subtype selectivity of ligands. In this study, we introduce a new approach to mapping of the binding site crevice of the human alpha 2A-AR. Based on a three-dimensional receptor model, we systematically mutated residues 197-201 and 204 in the fifth transmembrane domain of the human alpha 2A-AR to cysteine. Chloroethylclonidine, an alkylating derivative of the alpha 2-adrenergic agonist clonidine, binds irreversibly to alpha 2A-ARs by forming a covalent bond with the sulfhydryl side chain of a cysteine residue exposed in the binding cavity, leading to inactivation of the receptor. Irreversible binding of chloroethylclonidine was used as a criterion for identifying introduced cysteine residues as being exposed in the binding cavity. The results supported a receptor model in which the fifth transmembrane domain is alpha -helical, with residues Val197, Ser200, Cys201, and Ser204 exposed in the binding pocket. Residues Ile198, Ser199, Ile202, and Gly203 face the lipid bilayer of the plasma membrane. This approach emerges as a powerful tool for structural characterization of the alpha 2-ARs.

Copyright © 1998 by The American Society for Pharmacology and Experimental Therapeutics


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