Chloroethylclonidine Binds Irreversibly to Exposed Cysteines in the Fifth Membrane-Spanning Domain of the Human α2A-Adrenergic Receptor
- Anne Marjamäki1,
- Marjo Pihlavisto1,
- Victor Cockcroft1,3,
- Petri Heinonen2,
- Juha-Matti Savola1,3 and
- Mika Scheinin1
- Departments of 1Pharmacology and Clinical Pharmacology (A.M., M.P., M.S.) and 2Chemistry (P.H.), University of Turku, FIN-20500, Turku, Finland, and 3Orion Corporation (V.C., J.-M.S.), Orion-Pharma, FIN-20101, Turku, Finland
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 (α2A, α2B, α2C) 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 α2A-AR. Based on a three-dimensional receptor model, we systematically mutated residues 197–201 and 204 in the fifth transmembrane domain of the human α2A-AR to cysteine. Chloroethylclonidine, an alkylating derivative of the α2-adrenergic agonist clonidine, binds irreversibly to α2A-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.
Footnotes
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Send reprint requests to: Dr. Anne Marjamäki, MediCity Research Laboratory, City of Turku, Tykistökatu 6 A, FIN-20520, Turku, Finland. E-mail: anmarja{at}utu.fi
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↵1 Current affiliation: Juvantia Pharma, Tykistökatu 6 A, FIN-20520 Turku, Finland.
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This work was supported by the Academy of Finland and Technology Development Centre of Finland.
- Abbreviations:
- AR
- adrenergic receptor
- CEC
- chloroethylclonidine
- CHO
- Chinese hamster ovary
- GPCR
- G protein-coupled receptor
- SH
- sulfhydryl
- TM
- transmembrane domain
- Hα2A
- human α2A-adrenergic receptor
- Hα2B
- human α2B-adrenergic receptor
- Hα2C
- human α2C-adrenergic receptor
- Mα2A
- mouse α2A-adrenergic receptor
- WT
- wild-type
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- Received July 21, 1997.
- Accepted October 8, 1997.
- The American Society for Pharmacology and Experimental Therapeutics
