Molecular Pharmacology, Vol 18, 11-19, Copyright © 1980 by the American Society for Pharmacology and Experimental Therapeutics

Neurotensin Binding to Extraneural and Neural Receptors: Comparison with Biological Activity and Structure—Activity Relationships

¹ Institut National de la Santé et de la Recherche Médicale (INSERM) Groupe de Recherches sur les Hormones Polypeptidiques et la Physiopathologie Endocrinienne, U 145, et Laboratoire de Médecine Expérimentale, Faculté de Médecine, Université de Nice, Chemin de Vallombrose, 06034 Nice Cédex, France
² Laboratoire de Biochimie, Faculté de Médecine Nord, Boulevard Pierre Dramard, 13326 Marseille Cédex 3, France
³ Laboratories for Neuroendocrinology, The Salk Institute for Biological Studies, La Jolla, California 92037
⁴ Service de Biochimie, Centre d’Etudes Nucléaires de Saclay, B.P. No. 2, 91190 Gif-sur-Yvette, France

The binding of [³H]neurotensin to a cell line (HT 29) derived from a human colon carcinoma was characterized and compared with [³H]neurotensin binding to rat brain synaptic membranes. Both systems were used as radioreceptor assays for neurotensin and 18 neurotensin synthetic analogs, and the binding affinities thus derived were compared to the biological potencies obtained from the peptide abilities to contract isolated longitudinal smooth muscle strips of the guinea pig ileum. Tritiated neurotensin bound specifically and reversibly to HT 29 cells. The characteristics of [³H]neurotensin binding to cells at 24°C were those of a simple, bimolecular reaction involving one class of noncooperative binding sites. A K_d value of 1.5 nM was independently obtained from association kinetic and equilibrium experiments; total binding capacity was 37 fmol/10⁶ cells (22,000 neurotensin-binding sites/cell). Peptides structurally not related to neurotensin did not affect [³H]neurotensin binding. These binding characteristics were very similar to those observed for the binding of [³H]neurotensin to rat brain synaptic membranes. When the binding affinities of neurotensin and neurotensin analogs were compared in the extraneural (HT 29 cells) and neural (brain membranes) systems, a highly significant correlation between the two binding systems was observed. A highly significant correlation was also found when the biological potencies of neurotensin and neurotensin analogs were compared with their binding affinities in either the neural or the extraneural radioreceptor assay. The positive charge on both arginyl residues 8 and 9 and the L-configuration of Arg⁹ were important for binding and biological activity. An aromatic residue in the L-configuration was required in position 11 of the neurotensin molecule. The side-chain methyl groups of Ile¹² and carboxy-terminal residue Leu¹³, as well as the presence of Leu¹³ in the L-configuration, were required for activity.

Note:
ACKNOWLEDGMENTS We would like to thank Dr. A. Zweibaum for kindly supplying us with HT 29 cells. We also wish to thank G. Visciano for expert technical assistance and J. Duch for excellent secretarial assistance.

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