HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Sitrin, R. G. Articles by Petty, H. R. Search for Related Content PubMed PubMed Citation Articles by Sitrin, R. G. Articles by Petty, H. R.

Selective Localization of Recognition Complexes for Leukotriene B4 and Formyl-Met-Leu-Phe within Lipid Raft Microdomains of Human Polymorphonuclear Neutrophils¹

Robert G. Sitrin^2,*, Sarah L. Emery^*, Timothy M. Sassanella^*, R. Alexander Blackwood and Howard R. Petty

^* Pulmonary and Critical Care Medicine Division, Department of Internal Medicine, Department of Pediatrics and Communicable Diseases, and Department of Ophthalmology, University of Michigan, Ann Arbor, MI 48109

Neutrophilic polymorphonuclear leukocytes contain glycosphingolipid- and cholesterol-enriched lipid raft microdomains within the plasma membrane. Although there is evidence that lipid rafts function as signaling platforms for CXCR chemokine receptors, their role in recognition systems for other chemotaxins such as leukotriene B4 (LTB4) and fMLP is unknown. To address this question, human neutrophils were extracted with 1% Brij-58 and fractionated on sucrose gradients. B leukotriene receptor-1 (BLT-1), the primary LTB4 receptor, partitioned to low density fractions, coisolating with the lipid raft marker, flotillin-1. By contrast, formyl peptide receptor (FPR), the primary fMLP receptor, partitioned to high density fractions, coisolating with a non-raft marker, Cdc42. This pattern was preserved after the cells were stimulated with LTB4 or fMLP. Fluorescence resonance energy transfer (FRET) was performed to confirm the proximity of BLT-1 and FPR with these markers. FRET was detected between BLT1 and flotillin-1 but not Cdc42, whereas FRET was detected between FPR and Cdc42, but not flotillin-1. Pretreating neutrophils with methyl--cyclodextrin, a lipid raft-disrupting agent, suppressed intracellular Ca²⁺ mobilization and ERK1/2 phosphorylation in response to LTB4 but had no effect on either of these responses to fMLP. We conclude that BLT-1 is physically located within lipid raft microdomains of human neutrophils and that disrupting lipid raft integrity suppresses LTB4-induced activation. By contrast, FPR is not associated with lipid rafts, and fMLP-induced signaling does not require lipid raft integrity. These findings highlight the complexity of chemotaxin signaling pathways and offer one mechanism by which neutrophils may spatially organize chemotaxin signaling within the plasma membrane.

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.

¹ This work was supported by National Institutes of Health Grants HL58283, AI060983, and AI51789.

² Address correspondence and reprint requests to Dr. Robert G. Sitrin, 6301 MSRB III, BOX 0642, 1150 West Medical Center Drive, Ann Arbor, MI 48109-0642. E-mail address: rsitrin{at}umich.edu

³ Abbreviations used in this paper: Lo, liquid ordered; LTB4, leukotriene B4; FRET, fluorescence resonance energy transfer; [Ca²⁺]_i, intracellular calcium concentration; MCD, methyl--cyclodextrin; -CD, -cyclodextrin; BLT-1, B leukotriene receptor-1; FPR, formyl peptide receptor.