title: Chloroethylclonidine binds irreversibly to exposed cysteines in the fifth membrane-spanning domain of the human alpha2A-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.
authors.affiliation: Department of Pharmacology and Clinical Pharmacology, University of Turku, FIN-20500, Turku, Finland. anmarja@utu.fi
authors.forename: A
authors.initials: A
authors.lastname: Marjamaki
authors.forename: M
authors.initials: M
authors.lastname: Pihlavisto
authors.forename: V
authors.initials: V
authors.lastname: Cockcroft
authors.forename: P
authors.initials: P
authors.lastname: Heinonen
authors.forename: J M
authors.initials: JM
authors.lastname: Savola
authors.forename: M
authors.initials: M
authors.lastname: Scheinin
mesh_terms: D000317:Adrenergic alpha-Antagonists / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D003000:Clonidine / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D003545:Cysteine / Q000378:metabolism*; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D051381:Rats; D018341:Receptors, Adrenergic, alpha-2 / Q000378:metabolism*; D013329:Structure-Activity Relationship; D014162:Transfection
chemical_list: D000317:Adrenergic alpha-Antagonists; D018341:Receptors, Adrenergic, alpha-2; C051731:chlorethylclonidine; D003545:Cysteine; D003000:Clonidine