Chloroethylclonidine Binds Irreversibly to Exposed Cysteines in the Fifth Membrane-Spanning Domain of the Human α_2A-Adrenergic Receptor

Abstract

The alpha2-adrenergic receptors (alpha2-ARs) mediate signals to intracellular second messengers via guanine nucleotide binding proteins. Three human genes encoding alpha2-AR subtypes (alpha2A, alpha2B, alpha2C) 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 alpha2A-AR. Based on a three-dimensional receptor model, we systematically mutated residues 197-201 and 204 in the fifth transmembrane domain of the human alpha2A-AR to cysteine. Chloroethylclonidine, an alkylating derivative of the alpha2-adrenergic agonist clonidine, binds irreversibly to alpha2A-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 alpha2-ARs.

Chloroethylclonidine and 2-Aminoethyl Methanethiosulfonate Recognize Two Different Conformations of the Human α2A-Adrenergic Receptor

“…In our previous study, TM5 of H␣2A was shown to be consistent with an ␣-helix with residues Val-197, Cys-201, and Ser-204, pointing into the binding-site crevice, residues Ile-198, Ser-199, Ile-202, and Gly-203, facing the lipid bilayer, and Ser-200, pointing partly toward TM4 (9). Surprisingly, the pattern of accessibility to MTSEA in TM5 of the dopamine D2 receptor was not consistent with a fixed ␣-helical structure for TM5 (8).…”

“…Mutagenesis and Expression Vectors-Site-directed mutagenesis was performed utilizing the Altered Sites II in vitro mutagenesis system (Promega, Madison, WI) as described previously (9). The wild type H␣2A and the mutated receptor cDNAs were subcloned into the KpnI/ BamHI sites of the expression vector pREP4 (In Vitrogen, NV Leek, The Netherlands).…”

“…Cell Culture and Transfections-Adherent CHO cells (American Type Culture Collection, Manassas, VA) were cultured as reported previously (9). The pREP4-based expression constructs were transfected into cells using the Lipofectin reagent kit (Life Technologies, Inc.) (9).…”

“…The pREP4-based expression constructs were transfected into cells using the Lipofectin reagent kit (Life Technologies, Inc.) (9). Hygromycin B (Roche Molecular Biochemicals)-resistant (550 g/ ml) cell cultures were examined for their ability to bind the ␣ 2 -AR antagonist [ 3 H]RX821002.…”

“…We recently used the agonist chloroethylclonidine (CEC) to map the structure of TM5 of the human ␣ 2A -AR with a modified substituted cysteine-accessibility method (9). The reactive aziridinium ion derivative of CEC forms a covalent bond with the sulfhydryl side chain of a cysteine exposed in the bindingsite crevice and accessible to CEC.…”