DETERMINATION OF THE AMINO-ACID RESIDUE INVOLVED IN [H-13]BETA-FUNALTREXAMINE COVALENT BINDING IN THE CLONED RAT MU-OPIOID RECEPTOR

We previously demonstrated that [H-3]beta-funaltrexamine ([H-3]beta-FN A) labeled the rat mu opioid receptor expressed in Chinese hamster ova ry cells with high specificity, and [H-3] beta-FNA-Iabeled receptors m igrated as one broad band with a mass of 80 kDa. In this study, we det ermined 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-label ed receptors were solubilized and purified to similar to 10% purity by immunoaffinity chromatography with antibodies against a C-terminal do main peptide. The site of covalent bond formation was determined to be within Ala(206)-Met(243) by CNBr cleavage of partially purified label ed mu receptors and determinations of sizes of labeled receptor fragme nts. The amino acid residue of beta-FNA covalent incorporation was the n determined by site-directed mutagenesis studies within this region. Mutation of Lys(233) to Ala, Arg, His, and Leu completely eliminated c ovalent binding of [H-3]beta-FNA, although these mutants bound beta-FN A with high affinity. Mutations of other amino acid residues did not a ffect 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 ri gid molecule, the information will be very useful for molecular modeli ng of interaction between morphinans and the mu receptor.