Determination of the amino acid residue involved in [H-13]beta-funaltrexamine covalent binding in the cloned rat mu opioid receptor

作者：Chen CG; Yin YL; deRiel JK; DesJarlais RL; Raveglia LF; Zhu JM; LiuChen LY

来源：Journal of Biological Chemistry, 1996, 271(35): 21422-21429.

We previously demonstrated that [H-3]beta-funaltrexamine ([H-3]beta-FNA) labeled the rat mu opioid receptor expressed in Chinese hamster ovary cells with high specificity, and [H-3] beta-FNA-Iabeled 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 mu receptor involved in the covalent binding of [H-3]beta-FNA. [H-3]beta-FNA-labeled receptors were solubilized and purified to similar 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 Ala(206)-Met(243) by CNBr cleavage of partially purified labeled mu receptors and determinations of sizes of labeled receptor fragments. The amino acid residue of beta-FNA covalent incorporation was then determined by site-directed mutagenesis studies within this region. Mutation of Lys(233) to Ala, Arg, His, and Leu completely eliminated covalent binding of [H-3]beta-FNA, although these mutants bound beta-FNA with high affinity. Mutations of other amino acid residues did not affect covalent binding of [H-3]beta-FNA. These results indicate that [H-3]beta-FNA binds covalently to Lys(233). Since [H-3]beta-FNA is a rigid molecule, the information will be very useful for molecular modeling of interaction between morphinans and the mu receptor.

• 出版日期1996-8-30