Identification in the μ-opioid receptor of cysteine residues responsible for inactivation of ligand binding by thiol alkylating and reducing agents

Abstract

Inactivation by thiol reducing and alkylating agents of ligand binding to the human μ-opioid receptor was examined. Dithiothreitol reduced the number of [ 3H]diprenorphine binding sites. Replacement by seryl residues of either C142 or C219 in extracellular loops 1 and 2 of the μ receptor resulted in a complete loss of opioid binding. A disulfide bound linking C142 to C219 may thus be essential to maintain a functional conformation of the receptor. We also demonstrated that inactivation of ligand binding upon alkylation by N-ethylmaleimide occurred at two sites. Alteration of the more sensitive (IC 50=20 μM) did not modify antagonists binding but decreased agonist affinity almost 10-fold. Modification of the less reactive site (IC 50=2 mM) decreased the number of both agonist and antagonist binding sites. The alkylation site of higher sensitivity to N-ethylmaleimide was shown by mutagenesis experiments to be constituted of both C81 and C332 in transmembrane domains 1 and 7 of the μ-opioid receptor.