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J. Biol. Chem., Vol. 279, Issue 43, 45057-45067, October 22, 2004

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Dynamic Confinement of NK2 Receptors in the Plasma Membrane

IMPROVED FRAP ANALYSIS AND BIOLOGICAL RELEVANCE^*

Laurence Cézanne, Sandra Lecat¶||, Bernard Lagane, Claire Millot, Jean-Yves Vollmer¶||**, Hans Matthes¶, Jean-Luc Galzi¶, and André Lopez

From the Institut de Pharmacologie et de Biologie Structurale/CNRS, 205 route de Narbonne, 31062 Toulouse Cedex, France and ^¶CNRS UPR9050, Récepteurs et Protéines membranaires, Ecole Supérieure de Biotechnologie de Strasbourg, Boulevard Sébastien Brandt, 67400 Illkirch, France

A functional fluorescent neurokinin NK2 receptor, EGFP-NK2, was previously used to follow, by fluorescence resonance energy transfer measurements in living cells, the binding of its fluorescently labeled agonist, bodipy-neurokinin A (NKA). Local agonist application suggested that the activation and desensitization of the NK2 receptors were compartmentalized at the level of the plasma membrane. In this study, fluorescence recovery after photobleaching experiments are carried out at variable observation radius (vrFRAP) to probe EGFP-NK2 receptor mobility and confinement. Experiments are carried out at 20 °C to maintain the number of receptors constant at the cell surface during recordings. In the absence of agonist, 35% EGFP-NK2 receptors diffuse within domains of 420 ± 80 nm in radius with the remaining 65% of receptors able to diffuse with a long range lateral diffusion coefficient between the domains. When cells are incubated with a saturating concentration of NKA, 30% EGFP-NK2 receptors become immobilized in small domains characterized by a radius equal to 170 ± 50 nm. Biochemical experiments show that the confinement of EGFP-NK2 receptor is not due to its association with rafts at any given time. Colocalization of the receptor with -arrestin and transferrin supports that the small domains, containing 30% of activated EGFP-NK2, correspond to clathrin-coated pre-pits. The similar amount of confined EGFP-NK2 receptors found before and after activation (30–35%) is discussed in term of putative transient interactions of the receptors with preexisting scaffolds of signaling molecules.

Received for publication, April 30, 2004 , and in revised form, July 8, 2004.

^* 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 fellowships from the Association pour la Recherche sur le Cancer.

^** Present address: Institut de Pathologie, Rue du Bugnon 25, 1011 Lausanne, Switzerland.

To whom correspondence should be addressed: IPBS/CNRS, 205, route de Narbonne, 31062 Toulouse Cedex, France. E-mail: laurence.cezanne{at}ipbs.fr.

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