title: Radioligand-dependent discrepancy in agonist affinities enhanced by mutations in the kappa-opioid receptor.
abstract: A series of kappa/mu receptor chimeras and a number of kappa receptors substituted in the second transmembrane segment (TM-II) were investigated using as radioligands, respectively, the kappa-selective agonist [3H]C1977 and the nonselective opioid antagonist [3H]diprenorphine (DIP). All of the receptor constructs bound [3H]DIP with similar and high affinity, whereas the apparent affinity of the nonpeptide agonist C1977, when estimated in competition binding with the antagonist [3H]DIP, was impaired between 42- and > 500-fold in the kappa/mu chimeras and between 64- and 153-fold in three of the kappa receptor mutants that had been substituted in the TM-II segment. However, homologous competition binding experiments, using [3H]C1977 as radioligand, showed that the high affinity binding of this nonpeptide agonist was in fact not impaired in four of the kappa/mu chimeras and in three TM-II substituted kappa receptors compared with the wild-type kappa receptor. In all cases in which mutations decreased the apparent affinity of C1977 without affecting its actual affinity, as determined in homologous assays using [3H]C1977, the calculated number of receptor sites (Bmax) was decreased. In three of the kappa/mu constructs, binding of [3H]C1977 was undetectable, indicating that in these chimeras the affinity of the nonpeptide agonist had actually been affected. Also, for the kappa-selective peptide agonist dynorphin A(1-8), the measured affinity for the receptor mutants was strongly dependent on whether it was determined using the antagonist [3H]DIP or the agonist [3H]C1977 in that < or = 800-fold higher Ki values were determined in competition with the antagonist. It is concluded that mutations in the kappa-opioid receptor can cause large discrepancies between the affinity determined for agonists in homologous versus heterologous competition binding assays and that this pattern, which is compatible with a partial uncoupling of receptors, is observed in surprisingly many types of receptor mutations.
authors.affiliation: Laboratory for Molecular Pharmacology 6321, Rigshospitalet, Copenhagen, Denmark. siv@molpharm.dk
authors.forename: S A
authors.initials: SA
authors.lastname: Hjorth
authors.forename: K
authors.initials: K
authors.lastname: Thirstrup
authors.forename: T W
authors.initials: TW
authors.lastname: Schwartz
mesh_terms: D000818:Animals; D001572:Benzofurans / Q000378:metabolism / Q000494:pharmacology; D019556:COS Cells / Q000187:drug effects / Q000378:metabolism; D004174:Diprenorphine / Q000378:metabolism / Q000494:pharmacology; D004399:Dynorphins / Q000378:metabolism / Q000494:pharmacology; D007700:Kinetics; D009154:Mutation; D009292:Narcotic Antagonists / Q000378:metabolism / Q000494:pharmacology; D010446:Peptide Fragments / Q000378:metabolism / Q000494:pharmacology; D011759:Pyrrolidines / Q000378:metabolism / Q000494:pharmacology; D011869:Radioligand Assay; D017473:Receptors, Opioid, kappa / Q000819:agonists* / Q000235:genetics* / Q000378:metabolism; D017450:Receptors, Opioid, mu / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014316:Tritium
chemical_list: D001572:Benzofurans; D009292:Narcotic Antagonists; D010446:Peptide Fragments; D011759:Pyrrolidines; D017473:Receptors, Opioid, kappa; D017450:Receptors, Opioid, mu; D011993:Recombinant Fusion Proteins; D014316:Tritium; D004174:Diprenorphine; D004399:Dynorphins; C026770:dynorphin (1-8); C067110:enadoline