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J. Biol. Chem., Vol. 283, Issue 44, 30225-30234, October 31, 2008

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Functional Adhesiveness of the CX3CL1 Chemokine Requires Its Aggregation

ROLE OF THE TRANSMEMBRANE DOMAIN^*

Patricia Hermand, Frédéric Pincet^¶, Stéphanie Carvalho¹, Hervé Ansanay^||, Eric Trinquet^||, Mehdi Daoudi², Christophe Combadière^**, and Philippe Deterre³

From the Laboratoire d'Immunologie Cellulaire, INSERM UMR-S 543, and the FacultédeMédecine Pitié-Salpêtrière, Université Pierre et Marie Curie-Paris 06, 91 boulevard de l'Hôpital, 75013 Paris, France, the ^¶Laboratoire de Physique Statistique, UMR CNRS 8550, École Normale Supérieure, rue Lhomond, 75231 Paris, France, ^||Homogeneous Time-resolved Fluorescence (HTRF) Research, Cisbio International, BP 84175, 30204 Bagnols-sur-Cèze, France, and the ^**Laboratoire d'Immunologie Cellulaire et Tissulaire, Assistance Publique-Hôpitaux de Paris, Pitié-Salpêtrière, 87 boulevard de l'Hôpital, 75013 Paris, France

In its native form, the chemokine CX3CL1 is a firmly adhesive molecule promoting leukocyte adhesion and migration and hence involved, along with its unique receptor CX3CR1, in various inflammatory processes. Here we investigated the role of molecular aggregation in the CX3CL1 adhesiveness. Assays of bioluminescence resonance energy transfer (BRET) and homogeneous time-resolved fluorescence (HTRF) in transfected cell lines and in primary cells showed specific signals indicative of CX3CL1 clustering. Truncation experiments showed that the transmembrane domain played a central role in this aggregation. A chimera with mutations of the 12 central transmembrane domain residues had significantly reduced BRET signals and characteristics of a non-clustering molecule. This mutant was weakly adhesive according to flow and dual pipette adhesion assays and was less glycosylated than CX3CL1, although, as we demonstrated, loss of glycosylation did not affect the CX3CL1 adhesive potency. We postulate that cell surfaces express CX3CL1 as a constitutive oligomer and that this oligomerization is essential for its adhesive potency. Inhibition of CX3CL1 self-assembly could limit the recruitment of CX3CR1-positive cells and may be a new pathway for anti-inflammatory therapies.

Received for publication, April 4, 2008 , and in revised form, August 19, 2008.

^* This work was supported by grants from the Association de Recherche contre le Cancer and the Institut National de la Santé et de la Recherche Médicale (Programme National De Recherche Sur Les Maladies Cardiovasculaires) and by European FP6 "INNOCHEM" Contract LSHB-CT-2005-518167 (to C. C.). 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.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Table S1.

¹ Present address: Laboratoire de Neurobiologie, CNRS UMR8544, Ecole Normale Supérieure, 46 rue d'Ulm, 75230 Paris Cedex 05, France.

² Present address: Laboratoire Récepteurs Nucléaires, Lipoprotéines et Athérosclérose, INSERM U545, blvd. du Professeur J. Leclerc, 59045 Lille Cedex, France.

³ To whom correspondence should be addressed: Laboratoire d'Immunologie Cellulaire, INSERM U543, UPMC-Université Paris 06, FacultédeMédecine Pitié-Salpêtrière, 91 blvd. de l'Hôpital, 75013 Paris, France. Fax: 33-1-40-77-97-34; E-mail: deterre{at}ccr.jussieu.fr.

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