[H-3]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

The binding of [H-3]SR 48692, a new potent and specific non-peptide 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 [H-3]SR 48692 was specific, time dependent, reversible, and saturable. Scatchard analysis of saturation experiments indicated that [H-3]SR 48692 bound to a single population of sites, with a K-d of 3.4 nM and a B-max value that was 30-40% greater than that observed in saturation experiments with [I-125]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 [H-3]SR 48692. Unlabeled NT inhibited [H-3]SR 48692 binding in a complex manner that was best analyzed with a three-site model, with high (K-i = 0.22 nM) and low (K-i = 57 nM) affinity NT binding sites and a site insensitive to unlabeled NT (up to 10 mu M), which represented 60, 20, and 20%, respectively, of the total number of [H-3]SR 48692 binding sites. Digitonin (10 mu g/ml) markedly reduced the proportion of NT-insensitive sites without affecting [H-3]SR 48692 binding. Na+ and guanosine-5'-(gamma-thio)triphosphate differentially modulated [H-3]SR 48692 and [I-125]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 [H-3]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 [H-3]SR 48692 represents a new, potent, nonpeptide antagonist radio-ligand 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.