title: [3H]SR 48692, the first nonpeptide neurotensin antagonist radioligand: characterization of binding properties and evidence for distinct agonist and antagonist binding domains on the rat neurotensin receptor.
abstract: The binding of [3H]SR 48692, a new potent and specific nonpeptide neurotensin (NT) receptor antagonist, was characterized in membranes from mouse fibroblast LTK- cells stably transfected with the G protein-coupled rat NT receptor. The binding of [3H]SR 48692 was specific, time dependent, reversible, and saturable. Scatchard analysis of saturation experiments indicated that [3H]SR 48692 bound to a single population of sites, with a Kd of 3.4 nM and a Bmax value that was 30-40% greater than that observed in saturation experiments with [125I]NT. Two SR 48692-related enantiomers, SR 48527 and SR 49711, were 10 and 1000 times less potent, respectively, than unlabeled SR 48692 in inhibiting [3H]SR 48692. Unlabeled NT inhibited [3H]SR 48692 binding in a complex manner that was best analyzed with a three-site model, with high (Ki = 0.22 nM) and low (Ki = 57 nM) affinity NT binding sites and a site insensitive to unlabeled NT (up to 10 microM), which represented 60, 20, and 20%, respectively, of the total number of [3h]SR 48692 binding sites. Digitonin (10 micrograms/ml) markedly reduced the proportion of NT-insensitive sites without affecting [3H]SR 48692 binding. Na+ and guanosine-5'-(gamma-thio)triphosphate differentially modulated [3H]SR 48692 and [125I]NT binding and inverted the proportions of the high and low affinity NT binding sites. A mutant rat NT receptor that contained a deletion in a region (amino acids 45-60) of the amino-terminal extracellular domain near the first transmembrane helix and was expressed in COS M6 cells retained the same affinity for [3H]SR 48692 and the same stereoselectivity for SR 48527 and SR 49711 as the wild-type receptor. In contrast, it bound NT with 3000-fold lower potency. In conclusion, the data indicate that [3H]SR 48692 represents a new, potent, nonpeptide antagonist radioligand of the NT receptor that differentiates between agonist- and antagonist-receptor interactions. Furthermore, the data demonstrate that the peptide agonist and the nonpeptide antagonist bind to distinct regions of the NT receptor.
authors.affiliation: Institut de Pharmacologie Moléculaire et Cellulaire du CNRS, Université de Nice-Sophia Antipolis, Valbonne, France.
authors.forename: C
authors.initials: C
authors.lastname: Labbé-Jullié
authors.forename: J M
authors.initials: JM
authors.lastname: Botto
authors.forename: M V
authors.initials: MV
authors.lastname: Mas
authors.forename: J
authors.initials: J
authors.lastname: Chabry
authors.forename: J
authors.initials: J
authors.lastname: Mazella
authors.forename: J P
authors.initials: JP
authors.lastname: Vincent
authors.forename: D
authors.initials: D
authors.lastname: Gully
authors.forename: J P
authors.initials: JP
authors.lastname: Maffrand
authors.forename: P
authors.initials: P
authors.lastname: Kitabgi
mesh_terms: D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D002460:Cell Line; D004072:Digitonin / Q000494:pharmacology; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D007700:Kinetics; D051379:Mice; D009154:Mutation; D011720:Pyrazoles / Q000378:metabolism* / Q000494:pharmacology*; D011804:Quinolines / Q000378:metabolism* / Q000494:pharmacology*; D011869:Radioligand Assay; D051381:Rats; D018028:Receptors, Neurotensin / Q000819:agonists / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D012964:Sodium / Q000494:pharmacology; D014162:Transfection
chemical_list: D011720:Pyrazoles; D011804:Quinolines; D018028:Receptors, Neurotensin; C086487:SR 48527; C079087:SR 48692; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D012964:Sodium; D004072:Digitonin