Stable Expression of the Cloned Rat Brain Neurotensin Receptor into Fibroblasts: Binding Properties, Photoaffinity Labeling, Transduction Mechanisms, and Internalization

Authors

  • Joëlle Chabry,

    Corresponding author
    1. Institut de Pharmacologie Moléculaire et Cellulaire du Centre National de la Recherche Scientifique, Université de Nice-Sophia Antipolis, Valbonne, France
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  • Catherine Labbé-Jullié,

    1. Institut de Pharmacologie Moléculaire et Cellulaire du Centre National de la Recherche Scientifique, Université de Nice-Sophia Antipolis, Valbonne, France
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  • Danielle Gully,

    1. Institut de Pharmacologie Moléculaire et Cellulaire du Centre National de la Recherche Scientifique, Université de Nice-Sophia Antipolis, Valbonne, France
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    • The present address of Dr. D. Gully is Sanofi Recherche, 195, Route d'Espagne, 31036 Toulouse Cédex, France.

  • Patrick Kitabgi,

    1. Institut de Pharmacologie Moléculaire et Cellulaire du Centre National de la Recherche Scientifique, Université de Nice-Sophia Antipolis, Valbonne, France
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  • Jean-Pierre Vincent,

    1. Institut de Pharmacologie Moléculaire et Cellulaire du Centre National de la Recherche Scientifique, Université de Nice-Sophia Antipolis, Valbonne, France
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  • Jean Mazella

    1. Institut de Pharmacologie Moléculaire et Cellulaire du Centre National de la Recherche Scientifique, Université de Nice-Sophia Antipolis, Valbonne, France
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Address correspondence and reprint requests to Dr. J. Chabry at Institut de Pharmacologie Moléculaire et Cellulaire du Centre National de la Recherche Scientifique, Université de Nice-Sophia Antipolis, Sophia Antipolis, 660, Route des Lucioles, 06560 Valbonne, France.

Abstract

Abstract: The study of the pharmacological, biochemical, and transduction properties of the cloned rat brain neurotensin receptor was carried out in thymidine kinase mutant fibroblasts stably transfected with the receptor cDNA. The interaction of neurotensin with transfected fibroblasts leads to a concentration-dependent stimulation of phosphatidylinositol hydrolysis and intracellular calcium. These effects are totally inhibited by the nonpeptide neurotensin antagonist SR48692. By contrast, this receptor remains unable to modulate intracellular levels of cyclic nucleotides. The transfected neurotensin receptor can be solubilized in an active form by digitonin with an identical pharmacological profile, whereas the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonic acid is unable to solubilize the binding activity. The binding of iodinated neurotensin to transfected fibroblasts bearing the cloned receptor remains partly undissociated even after an acid washing step, indicating that the transfected neurotensin receptor retains the capacity to be internalized according to a temperature-dependent mechanism. Indeed, the sequestration of the neurotensin-receptor complex can be blocked by phenylarsine oxide. Finally, photoaffinity labeling experiments reveal that the cloned rat brain neurotensin receptor is expressed under two forms with molecular masses of 50 and 60 kDa. Labeling and internalization of these two proteins are totally blocked by the neurotensin antagonist SR48692.

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