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J. Biol. Chem., Vol. 280, Issue 11, 10219-10227, March 18, 2005

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Identification and Functional Characterization of a 5-Transmembrane Domain Variant Isoform of the NTS2 Neurotensin Receptor in Rat Central Nervous System^*

Amélie Perron, Philippe Sarret, Louis Gendron¶, Thomas Stroh, and Alain Beaudet||

From the Montreal Neurological Institute, Department of Neurology and Neurosurgery, McGill University, Montreal, Quebec H3A 2B4, Canada

The present study demonstrated that alternative splicing of the rat nts2 receptor gene generates a 5-transmembrane domain variant isoform (vNTS2) that is co-expressed with the full-length NTS2 receptor throughout the brain and spinal cord, as evidenced by reverse transcription-PCR. The vNTS2 polypeptide is 281 amino acids in length, which is 135 amino acids shorter than the full-length isoform. Immunohistochemical and radioligand binding studies revealed that the HA-tagged recombinant vNTS2 receptor is poorly targeted to plasma membranes in transfected COS-7 cells. Binding studies also showed that the truncated receptor displayed a 5000-fold lower affinity for neurotensin (NT) than its full-length counterpart (IC₅₀ of 10 µM and 2 nM, respectively). Yet NT binding induced efficient internalization of receptor-ligand complexes in vNTS2-transfected cells. Furthermore, it produced a rapid (<5 min) activation of the mitogen-activated protein kinases (ERK1/2) pathway, indicating functional coupling of the variant receptor. This activation is sustained (>1 h) and is also produced by the NTS2 agonist levocabastine. Western blotting experiments suggested that vNTS2 is not expressed in monomeric form in the rat central nervous system. However, it does appear to form a variety of multimeric complexes, including homodimers and heterodimers, with the full-length NTS2. Indeed, co-immunoprecipitation studies in dually transfected cells demonstrated that the two receptor isoforms can form stable associations. Taken together, the present results indicated that the rat vNTS2 is a functional receptor that may play a role in NT signaling in mammalian central nervous system.

Received for publication, September 14, 2004 , and in revised form, December 22, 2004.

^* This work was supported in part by a grant from the Canadian Institutes for Health Research. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Supported by a fellowship from the Fonds de la Recherche en Santé du Québec.

Present address: Dept. of Physiology and Biophysics, Faculty of Medicine, University of Sherbrooke, Sherbrooke, Quebec J1H 5N4, Canada.

^¶ Supported by a fellowship from the Canadian Institutes for Health Research.

^|| To whom correspondence should be addressed: Montreal Neurological Institute, Dept. of Neurology and Neurosurgery, Rm. 896, 3801 University St., Montreal, Quebec H3A 2B4, Canada. Tel.: 514-398-1913; Fax: 514-398-5871; E-mail: alain.beaudet{at}mcgill.ca.

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