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J. Biol. Chem., Vol. 277, Issue 39, 36233-36243, September 27, 2002
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From the The binding and signaling properties of
neuronal NTS2 neurotensin (NT) receptors were examined in cultured rat
cerebellar granule cells. As shown by reverse transcription-PCR,
receptor autoradiography, and confocal microscopic localization of
fluorescent NT, these cells selectively express the NTS2 receptor
subtype. Accordingly, a single apparent class of
125I-NT-binding sites, with an affinity of 3.1 nM, was detected in cerebellar granule cell cultures. This
binding was competed for with high affinity (IC50 = 5.7 nM) by the NTS2 ligand levocabastine and with low affinity
(IC50 = 203 nM) by the NTS1 antagonist SR48692. Hypertonic acid stripping of surface-bound ligand and hyperosmolar sucrose treatment revealed that 64% of specifically bound
125I-NT was internalized at equilibrium via a
clathrin-dependent pathway. In cells loaded with the
Ca2+-sensitive fluorescent dye Fluo4, SR48692, but neither
NT nor levocabastine, triggered a marked increase in cytosolic
[Ca2+]i. By contrast, both NT and levocabastine,
but not SR48692, induced a sustained (>60 min) activation of the
mitogen-activated protein kinases, p42/p44, indicating functional
coupling of NTS2 receptors. Complementary experiments carried out on
synaptosomes from adult rat cerebellum demonstrated the presence of
presynaptic NTS2 receptors. However, in contrast to perikaryal NTS2
sites, these presynaptic receptors did not internalize in response to NT stimulation. Taken together, the present results demonstrate that
NTS2 receptors are present both presynaptically and postsynaptically in
central neurons and that NT and levocabastine act as agonists on these receptors.
Pharmacology and Functional Properties of NTS2 Neurotensin
Receptors in Cerebellar Granule Cells*
§,
,,,§§
Department of Neurology and
Neurosurgery, Montreal Neurological Institute, McGill University,
Montreal, Quebec H3A 2B4, Canada, the ¶ Service of Endocrinology,
Department of Medicine, Sherbrooke University, Quebec J1H 5N4,
Canada, and the ** Department of Physiology and Biophysics,
Faculty of Medicine, Sherbrooke University,
Quebec J1H 5N4, Canada
*
This work was supported by Canadian Institutes of Health
Research Grants MT-7366 (to A. B.) and MT-13679 (to M. D. P. and N. G. P.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
Recipient of a studentship from Fonds de la Recherche en
Santé du Québec.
Recipient of a studentship from the Ministère de
l'Education du Québec.
§§
To whom correspondence should be addressed: Dept. of Neurology
and Neurosurgery, Montreal Neurological Institute, 3801 University St.,
Montreal, PQ H3A 2B4, Canada. Tel.: 514-398-1913; Fax:
514-398-5871; E-mail: alain.beaudet@mcgill.ca.
Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
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