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J. Biol. Chem., Vol. 279, Issue 43, 45175-45184, October 22, 2004

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N-Formyl Peptide Receptors Cluster in an Active Raft-associated State Prior to Phosphorylation^*

Mei Xue, Charlotte M. Vines¶, Tione Buranda||, Daniel F. Cimino, Teresa A. Bennett**, and Eric R. Prossnitz

From the Department of Cell Biology and Physiology and the ^||Department of Pathology and the University of New Mexico Cancer Research and Treatment Center, University of New Mexico Health Sciences Center, Albuquerque, New Mexico 87131

In response to ligand binding, G protein-coupled receptors undergo phosphorylation and activate cellular internalization machinery. An important component of this process is the concentration of receptors into clusters on the plasma membrane. Aside from organizing the receptor in anticipation of internalization, little is known of the function of ligand-mediated G protein-coupled receptor clustering, which has traditionally been thought of as being a phosphorylation-dependent event prior to receptor internalization. We now report that following receptor activation, the N-formyl peptide receptor (FPR) forms distinct membrane clusters prior to its association with arrestin. To determine whether this clustering is dependent upon receptor phosphorylation, we used a mutant form of the FPR, ST-FPR, which lacks all phosphorylation sites in the carboxyl-terminal domain. We found that activation of the signaling-competent ST-FPR resulted in rapid receptor clustering on the plasma membrane independent of G_i protein activation. This clustering required receptor activation since the D71A mutant receptor, which binds ligand but is incapable of transitioning to an active state, failed to induce receptor clustering. Furthermore we demonstrated that FPR-mediated clustering and signaling were cholesterol-dependent processes, suggesting that translocation of the active receptor to lipid rafts may be required for maximal signaling activity. Finally we showed that FPR stimulation in the absence of receptor phosphorylation resulted in translocation of FPR to GM1-rich clusters. Our results demonstrate for the first time that formation of a clustered activated receptor state precedes receptor phosphorylation, arrestin binding, and internalization.

Received for publication, June 23, 2004 , and in revised form, July 28, 2004.

^* This work was supported by National Institutes of Health Grants AI36357 (to E. R. P.) and K25AI60036 (to T. B.). Flow cytometry and confocal microscopy data were generated in the Flow Cytometry and Microscopy Facilities at the University of New Mexico Health Sciences Center, which received support from National Center for Research Resources (NCRR) Grant 1 S10 RR14668, National Science Foundation Grant MCB9982161, NCRR Grant P20 RR11830, and NCI, National Institutes of Health Grant R24 CA88339, the University of New Mexico Health Sciences Center, and the University of New Mexico Cancer Center. 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.

Both authors contributed equally to this work.

^¶ Supported by the University of New Mexico Cancer Research and Treatment Center and recipient of National Institutes of Health Post-doctoral Training Fellowship T32 AI007538.

^** Present address: Novasite Pharmaceuticals Inc., 11095 Flintkote Ave., San Diego, CA 92121.

To whom correspondence should be addressed. Tel.: 505-272-5647; Fax: 505-272-1421; E-mail: eprossnitz{at}salud.unm.edu.

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