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Volume 271, Number 35, Issue of August 30, 1996 pp. 21422-21429
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.

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Determination of the Amino Acid Residue Involved in [³H]-Funaltrexamine Covalent Binding in the Cloned Rat µ Opioid Receptor

(Received for publication, March 1, 1996, and in revised form, June 6, 1996)

Chongguang Chen , Jinling Yin , J. Kim de Riel , Renee L. DesJarlais ^§ , Luca F. Raveglia ^¶ , Jinmin Zhu and Lee-Yuan Liu-Chen

From the Department of Pharmacology and  Fels Institute for Molecular Biology and Cancer Research, Temple University School of Medicine, Philadelphia, Pennsylvania 19140, ^§ Department of Physical and Structural Chemistry, SmithKline Beecham Pharmaceuticals, King of Prussia, Pennsylvania 19406, and ^¶ Department of Chemistry, SmithKline Beecham S.P.A., Via Zambeletti 20021, Baranzate, Milan, Italy

We previously demonstrated that [³H]-funaltrexamine ([³H]-FNA) labeled the rat µ opioid receptor expressed in Chinese hamster ovary cells with high specificity, and [³H]-FNA-labeled receptors migrated as one broad band with a mass of 80 kDa. In this study, we determined the region and then the amino acid residue of the µ receptor involved in the covalent binding of [³H]-FNA. [³H]-FNA-labeled receptors were solubilized and purified to ~10% purity by immunoaffinity chromatography with antibodies against a C-terminal domain peptide. The site of covalent bond formation was determined to be within Ala²⁰⁶-Met²⁴³ by CNBr cleavage of partially purified labeled µ receptors and determinations of sizes of labeled receptor fragments. The amino acid residue of -FNA covalent incorporation was then determined by site-directed mutagenesis studies within this region. Mutation of Lys²³³ to Ala, Arg, His, and Leu completely eliminated covalent binding of [³H]-FNA, although these mutants bound -FNA with high affinity. Mutations of other amino acid residues did not affect covalent binding of [³H]-FNA. These results indicate that [³H]-FNA binds covalently to Lys²³³. Since [³H]-FNA is a rigid molecule, the information will be very useful for molecular modeling of interaction between morphinans and the µ receptor.

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