title: Selective localization of recognition complexes for leukotriene B4 and formyl-Met-Leu-Phe within lipid raft microdomains of human polymorphonuclear neutrophils.
abstract: 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, co-isolating with the lipid raft marker, flotillin-1. By contrast, formyl peptide receptor (FPR), the primary fMLP receptor, partitioned to high density fractions, co-isolating 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-beta-cyclodextrin, a lipid raft-disrupting agent, suppressed intracellular Ca(2+) 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.
authors.affiliation: Pulmonary and Critical Care Medicine Division, Department of Internal Medicine, University of Michigan, 1150 West Medical Drive, Ann Arbor, MI 48109, USA. rsitrin@umich.edu
authors.forename: Robert G
authors.initials: RG
authors.lastname: Sitrin
authors.forename: Sarah L
authors.initials: SL
authors.lastname: Emery
authors.forename: Timothy M
authors.initials: TM
authors.lastname: Sassanella
authors.forename: R Alexander
authors.initials: RA
authors.lastname: Blackwood
authors.forename: Howard R
authors.initials: HR
authors.lastname: Petty
mesh_terms: D002118:Calcium / Q000378:metabolism; D004789:Enzyme Activation / Q000276:immunology; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D007975:Leukotriene B4 / Q000378:metabolism*; D021962:Membrane Microdomains / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000378:metabolism*; D009504:Neutrophils / Q000166:cytology / Q000276:immunology / Q000378:metabolism*; D044042:Receptors, Formyl Peptide / Q000378:metabolism; D018102:Receptors, Leukotriene B4 / Q000378:metabolism; D015398:Signal Transduction / Q000276:immunology*; D020764:cdc42 GTP-Binding Protein / Q000378:metabolism
chemical_list: C513240:LTB4R protein, human; D008565:Membrane Proteins; D044042:Receptors, Formyl Peptide; D018102:Receptors, Leukotriene B4; C106354:flotillins; D007975:Leukotriene B4; D009240:N-Formylmethionine Leucyl-Phenylalanine; D048049:Extracellular Signal-Regulated MAP Kinases; D020764:cdc42 GTP-Binding Protein; D002118:Calcium