Inactivation of the purified bovine mu opioid receptor by sulfhydryl reagents.

Abstract

We have investigated the role of cysteine residues in a highly purified mu opioid receptor protein (muORP) by examining the effect of -SH reagents on the binding of opioid ligands. Treatment of muORP, which is devoid of additional proteins, eliminates complications that arise from reaction of -SH reagents with other components, such as G proteins. Reagents tested include N-ethylmaleimide, 5,5'-dithiobis(2-nitrobenzoic) acid, and two derivatives of methanethiosulfonate. Specific opioid binding was inactivated by micromolar concentrations of all -SH reagents tested. Agonist binding ([3H]DAMGO) was much more sensitive to inactivation than antagonist binding ([3H]bremazocine). Prebinding muORP with 100 nM naloxone protected antagonist and agonist binding from inactivation by -SH reagents. The results of these experiments strongly suggest that at least one, and possibly more, reactive cysteine residue(s) is present on the mu opioid receptor protein molecule, positioned near the ligand binding site and accessible to -SH reagents.