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[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

[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

C Labbé-Jullié, J M Botto, M V Mas, J Chabry, J Mazella, J P Vincent, D Gully, J P Maffrand, P Kitabgi

Mol Pharmacol. 1995 May;47(5):1050-6.

PMID: 7746272

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.

Chemicals Related in the Paper:

Catalog Number	Product Name	Structure	CAS Number	Price
AP146362701	SR 48692		146362-70-1	Price

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