title: Identification in the mu-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 mu-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 mu 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 (IC50 = 20 microM) did not modify antagonists binding but decreased agonist affinity almost 10-fold. Modification of the less reactive site (IC50 = 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 mu-opioid receptor.
authors.affiliation: Institut de Pharmacologie et de Biologie Structurale, Centre National de la Recherche Scientifique, Unité Propre de Recherches No. 9062, Toulouse, France.
authors.forename: G
authors.initials: G
authors.lastname: Gaibelet
authors.forename: R
authors.initials: R
authors.lastname: Capeyrou
authors.forename: G
authors.initials: G
authors.lastname: Dietrich
authors.forename: L J
authors.initials: LJ
authors.lastname: Emorine
mesh_terms: D000477:Alkylating Agents / Q000378:metabolism / Q000494:pharmacology; D000478:Alkylation; D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D003001:Cloning, Molecular; D003545:Cysteine / Q000378:metabolism*; D004174:Diprenorphine / Q000378:metabolism; D004220:Disulfides / Q000737:chemistry / Q000378:metabolism; D004229:Dithiothreitol / Q000378:metabolism / Q000494:pharmacology*; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; D004745:Enkephalins / Q000378:metabolism; D005033:Ethylmaleimide / Q000378:metabolism / Q000494:pharmacology*; D006801:Humans; D008024:Ligands; D016297:Mutagenesis, Site-Directed; D009292:Narcotic Antagonists / Q000378:metabolism; D011485:Protein Binding; D017450:Receptors, Opioid, mu / Q000819:agonists / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D019163:Reducing Agents / Q000378:metabolism / Q000494:pharmacology; D013439:Sulfhydryl Reagents / Q000378:metabolism / Q000494:pharmacology*; D014162:Transfection
chemical_list: D000477:Alkylating Agents; D004220:Disulfides; D004745:Enkephalins; D008024:Ligands; D009292:Narcotic Antagonists; D017450:Receptors, Opioid, mu; D019163:Reducing Agents; D013439:Sulfhydryl Reagents; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; D004174:Diprenorphine; D003545:Cysteine; D005033:Ethylmaleimide; D004229:Dithiothreitol