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J. Biol. Chem., Vol. 280, Issue 6, 4880-4887, February 11, 2005

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Phosphorylation-independent -Arrestin Translocation and Internalization of Leukotriene B₄ Receptors^*

Venkatakrishna R. Jala, Wen-Hai Shao, and Bodduluri Haribabu

From the James Graham Brown Cancer Center and The Department of Microbiology & Immunology, University of Louisville Health Sciences Center, Louisville, Kentucky 40202

Leukotriene B₄ (LTB₄) activates the G-protein-coupled receptor leukotriene B₄ receptor 1 (BLT1) to mediate a diverse array of cellular responses in leukocytes including chemotaxis, calcium mobilization, degranulation, and gene expression. To determine the role of phosphorylation in BLT1 regulation, we generated mutants of BLT1 in which all of the serine/threonine residues in the C-tail are converted to alanine or to aspartate/glutamate. These mutants expressed in rat basophilic leukemia RBL-2H3 cells bound LTB₄ with similar affinity and activated all of the known functional activities of BLT1, albeit at different levels. The conversion of phosphorylation sites to alanine resulted in enhanced G-protein-mediated activities, whereas conversion to aspartate/glutamate resulted in reduced responses and a right shift in dose response, indicating that receptor phosphorylation is a critical regulator of G-protein-mediated pathways. Surprisingly, translocation of -arrestin and receptor internalization was completely independent of BLT1 phosphorylation. Real-time analysis of -arrestin translocation and receptor internalization using digital fluorescence video microscopy in cells expressing a red fluorescent protein labeled BLT1 and a green fluorescent protein-tagged -arrestin confirmed phosphorylation-independent -arrestin translocation and internalization of BLT1. In -arrestin-deficient mouse embryo fibroblasts, the BLT1 receptors failed to display endosomal localization upon stimulation. In these cells, co-expression of -arrestin-green fluorescent protein with BLT1-red fluorescent protein resulted in co-localization of BLT1 and -arrestin upon activation. Thus, receptor phosphorylation-dependent mechanisms regulate G-protein-mediated pathways; however, phosphorylation-independent mechanisms regulate -arrestin association and internalization of BLT1.

Received for publication, August 26, 2004 , and in revised form, November 10, 2004.

^* This work was supported by National Institutes of Health Grants AI-43184 and AI-52381 and a research grant from Kentucky Lung Cancer Research Program. 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 material.

To whom correspondence should be addressed: James Graham Brown Cancer Center, 580 S. Preston St., Rm. 119 B, Delia Baxter Bldg., Louisville, KY 40202. Tel.: 502-852-7503; Fax: 502-852-2123; E-mail: H0bodd01{at}gwise.louisville.edu.

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