title: Analysis of selective binding epitopes for the kappa-opioid receptor antagonist nor-binaltorphimine.
abstract: The structural determinants for the selective binding of the nonpeptide opioid receptor antagonist nor-binaltorphimine (nor-BNI) to the kappa-opioid receptor were characterized using a systematic series of chimeras between the kappa receptor and the homologous mu-opioid receptor. All 10 chimeric constructs bound the nonselective antagonists (-)-naloxone and diprenorphine with similar affinities, as did the two wild-type receptors. Introduction of amino-terminal segments of increasing length, extending to and including transmembrane segment VI, from the mu receptor into the kappa receptor did not impair the high affinity binding of nor-BNI, and neither did introduction of the intracellular carboxyl-terminal extension of the mu receptor. In contrast, nor-BNI binding was impaired > or = 600-fold in constructs in which extracellular loop 3 and transmembrane segment VII originated from the mu receptor. The exchange of a single residue within this region, Glu297, for lysine, the corresponding residue from the mu receptor, reduced the binding affinity of nor-BNI 142-fold, without affecting the binding the nonselective compounds (-)-naloxone and diprenorphine. It is concluded that the selective binding of nor-BNI to the kappa-opioid receptor is determined by nonconserved residues located in extracellular loop 3 and transmembrane segment VII and that Glu297, located just outside transmembrane segment VI, plays a major role in the kappa-selective binding characteristics of nor-BNI.
authors.affiliation: Department of Clinical Biochemistry, Rigshospitalet, Copenhagen, Denmark.
authors.forename: S A
authors.initials: SA
authors.lastname: Hjorth
authors.forename: K
authors.initials: K
authors.lastname: Thirstrup
authors.forename: D K
authors.initials: DK
authors.lastname: Grandy
authors.forename: T W
authors.initials: TW
authors.lastname: Schwartz
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000939:Epitopes / Q000032:analysis*; D005973:Glutamine / Q000235:genetics; D008969:Molecular Sequence Data; D009271:Naltrexone / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D051381:Rats; D017473:Receptors, Opioid, kappa / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D017450:Receptors, Opioid, mu / Q000235:genetics; D011993:Recombinant Fusion Proteins
chemical_list: D000939:Epitopes; D017473:Receptors, Opioid, kappa; D017450:Receptors, Opioid, mu; D011993:Recombinant Fusion Proteins; D005973:Glutamine; C051844:norbinaltorphimine; D009271:Naltrexone