Abstract

The α₂-adrenergic receptors (α₂-ARs) mediate signals to intracellular second messengers via guanine nucleotide binding proteins. Three human genes encoding α₂-AR subtypes (α_2A, α_2B, α_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 α_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 α_2A-AR to cysteine. Chloroethylclonidine, an alkylating derivative of the α₂-adrenergic agonist clonidine, binds irreversibly to α_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 α-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 α₂-ARs.