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J. Biol. Chem., Vol. 279, Issue 11, 10338-10345, March 12, 2004

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Structural Determinants Regulating Expression of the High Affinity Leukotriene B₄ Receptor

INVOLVEMENT OF DILEUCINE MOTIFS AND -HELIX VIII^*

Rémi Gaudreau, Marie-Eve Beaulieu, Zhangguo Chen, Christian Le Gouill, Pierre Lavigne, Jana Staková, and Marek Rola-Pleszczynski

From the Immunology Division, Departments of Pediatrics and Pharmacology, Faculty of Medicine, Université de Sherbrooke, Sherbrooke, Quebec J1H 5N4, Canada

Mutational analysis of determinants located in the C-terminal (C) tail of the high affinity leukotriene (LT) B₄ receptor, BLT1, was performed to assess their significance in BLT1 trafficking. When expressed in COS-7 cells, a BLT1 deletion mutant lacking the C-tail (G291stop) displayed higher numbers of binding sites and increased signal transduction compared with wild-type (WT) BLT1. Addition of the C-tail from either the platelet-activating factor receptor or the LTD₄ receptor, CysLT1, did not restore WT phenotype. Moreover, the number of LTB₄ binding sites was higher in the chimeras than in the WT BLT1, suggesting the requirement for specific structural determinants within the BLT1 C-tail. Elimination of a distal C-tail dileucine motif (Leu³⁰⁴-Leu³⁰⁵), but not the proximal (Leu²⁹²-Leu²⁹³) motif, altered BLT1 pharmacological characteristics and caused a moderate constitutive receptor activation. Surprisingly, all mutant receptors were efficiently delivered to the plasma membrane, but not to a greater extent than WT BLT1, as assessed by flow cytometry. Furthermore, substitution of Leu³⁰⁴-Leu³⁰⁵ prevented LTB₄-induced BLT1 internalization. Molecular modeling of BLT1 on the bovine rhodopsin receptor scaffold strongly suggested the involvement of the distal dileucine motif (Leu³⁰⁴-Leu³⁰⁵) in a hydrophobic core, including intrahelical interactions within -helix VIII and interhelical interactions with residues of helix I. Disruption of this hydrophobic core is proposed to increase the population of receptors in the active form, to restrain their trafficking and to facilitate the activation of BLT1 as indicated by the increased maximal level of binding of the ligand and constitutive activation of the receptor.

Received for publication, August 19, 2003 , and in revised form, December 16, 2003.

^* This work was supported by a grant from the Canadian Institutes of Health Research and by a Studentship (to R. G.) from the Fonds pour les Chercheurs et l'Aide à la Recherche (FCAR). 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.

To whom correspondence should be addressed: Immunology Division, Dept. of Pediatrics, Faculty of Medicine, Université de Sherbrooke, 3001 N. 12th Avenue, Sherbrooke, QC J1H 5N4, Canada. Tel.: 819-346-1110 (ext. 14851); Fax: 819-564-5215; E-mail: marek.rola-pleszczynski{at}usherbrooke.ca.

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