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J Biol Chem, Vol. 274, Issue 53, 37915-37922, December 31, 1999

Subcellular Compartmentalization of Activation and Desensitization of Responses Mediated by NK2 Neurokinin Receptors^*

Jean-Yves Vollmer^§, Philippe Alix^¶, André Chollet, Kenneth Takeda^**, and Jean-Luc Galzi^§§

From the  Département Récepteurs et Protéines Membranaires, CNRS UPR 9050, Ecole Supérieure de Biotechnologie de Strasbourg, Boulevard Sébastien Brant, 67400 Illkirch, France,  Serono Pharmaceutical Research Institute, 14 chemin des Aulx, CH-1228 Plan-les-Ouates/Genève, Switzerland, and ^** Pharmacologie et Physicochimie des Interactions Cellulaires et Moléculaires, CNRS UMR 7034, Université Louis Pasteur de Strasbourg, Faculté de Pharmacie, B.P. 24, 67401 Illkirch, France

A functional fluorescent neurokinin NK2 receptor was constructed by joining enhanced green fluorescent protein to the amino-terminal end of the rat NK2 receptor and was expressed in human embryonic kidney cells. On cell suspensions, the binding of fluorescent Bodipy-labeled neurokinin A results in a saturatable and reversible decrease of NK2 receptor fluorescence via fluorescence resonance energy transfer. This can be quantified for nM to µM agonist concentrations and monitored in parallel with intracellular calcium responses. On single cells, receptor site occupancy and local agonist concentration can be determined in real time from the decrease in receptor fluorescence. Simultaneous measurement of intracellular calcium responses and agonist binding reveals that partial receptor site occupancy is sufficient to desensitize cellular response to a second agonist application to the same membrane area. Subsequent stimulation of a distal membrane area leads to a second response to agonist, provided that it had not been exposed to agonist during the first application. Together with persistent translocation of fluorescent protein kinase C to the membrane area exposed to agonist, the present data support that not only homologous desensitization but also heterologous desensitization of NK2 receptors is compartmentalized to discrete membrane domains.

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^* This work was supported by the Centre National de la Recherche Scientifique, the Association pour la Recherche sur le Cancer, the Fondation pour le Recherche Médicale, the Ligue Nationale Contre le Cancer (Comité du Haut-Rhin), the Communauté Urbaine de Strasbourg, and the Université Louis Pasteur de Strasbourg.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 fellowship from the Agence Nationale pour la Recherche sur le SIDA.

^¶ Present address: Laboratoire de Neurophysiologie (RCIM), Université d'Angers, rue Haute de Reculée, 49045 Angers Cedex, France.

^§§ To whom correspondence should be addressed: Tel.: (33) 3 88 65 52 93; Fax: (33) 3 88 65 52 98; E-mail: galzi@esbs.u-strasbg.fr.

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Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.

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