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Platelet-Activating Factor-Mediated Endosome Formation Causes Membrane Translocation of p67^phox and p40^phox That Requires Recruitment and Activation of p38 MAPK, Rab5a, and Phosphatidylinositol 3-Kinase in Human Neutrophils¹

Nathan J. D. McLaughlin^*,, Anirban Banerjee, Samina Y. Khan^*,, Janet L. Lieber, Marguerite R. Kelher^*,, Fabia Gamboni-Robertson, Forest R. Sheppard, Ernest E. Moore, Gary W. Mierau, David J. Elzi^*, and Christopher C. Silliman^2,*,,

^* Bonfils Blood Center and Department of Pediatrics and Surgery, University of Colorado Denver School of Medicine, and Department of Pathology, Children’s Hospital, Denver, CO 80230

Neutrophils (polymorphonuclear leukocytes, PMNs) are vital to innate immunity and receive proinflammatory signals that activate G protein-coupled receptors (GPCRs). Because GPCRs transduce signals through clathrin-mediated endocytosis (CME), we hypothesized that platelet-activating factor (PAF), an effective chemoattractant that primes the PMN oxidase, would signal through CME, specifically via dynamin-2 activation and endosomal formation resulting in membrane translocation of cytosolic phagocyte oxidase (phox) proteins. PMNs were incubated with buffer or 2 µM PAF for 1–3 min, and in some cases activated with PMA, and O₂^– was measured, whole-cell lysates and subcellular fractions were prepared, or the PMNs were fixed onto slides for digital or electron microscopy. PAF caused activation of dynamin-2, resulting in endosomal formation that required PI3K and contained early endosomal Ag-1 (EEA-1) and Rab5a. The apoptosis signal-regulating kinase-1/MAPK kinase-3/p38 MAPK signalosome assembled on Rab5a and phosphorylated EEA-1 and Rab GDP dissociation inhibitor, with the latter causing Rab5a activation. Electron microscopy demonstrated that PAF caused two distinct sites for activation of p38 MAPK. EEA-1 provided a scaffold for recruitment of the p40^phox-p67^phox complex and PI3K-dependent Akt1 phosphorylation of these two phox proteins. PAF induced membrane translocation of p40^phox-p67^phox localizing to gp91^phox, which was PI3K-, but not p47^phox-, dependent. In conclusion, PAF transduces signals through CME, and such GPCR signaling may allow for pharmacological manipulation of these cells to decrease PMN-mediated acute organ injury.

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 Grants HL59355 and GM49222 from the National Heart, Lung, and Blood Institute and National Institute of General Medical Sciences, National Institutes of Health, and Bonfils Blood Center.

² Address correspondence and reprint requests to Dr. Christopher C. Silliman, Bonfils Blood Center, 717 Yosemite Street, Denver, CO 80230. E-mail address: christopher.silliman{at}uchsc.edu

³ Abbreviations used in this paper: CME, clathrin-mediated endocytosis; GPCR, G protein-coupled receptors; CCV, clathrin-coated vesicle; SNARE, soluble NSF attachment protein receptor; PMN, polymorphonuclear leukocyte; EEA-1, early endosome Ag-1; PAF, platelet-activating factor; PAFR, platelet-activating factor receptor; p67^phox, 67-kDa phagocyte oxidase protein; p40^phox, 40-kDa phagocyte oxidase protein; MKK3, mitogen-activated kinase kinase 3; RabGDI, Rab GDP dissociation inhibitor; PS, phosphoserine; PY, phosphotyrosine; FRET, fluorescent resonance energy transfer; FRET^C, FRET channel; a.l.u.f.i., arbitrary linear units of fluorescent intensity; PIP3, phosphatidylinositol 3,4,5-triphosphate; PIP2, phosphatidylinositol 4,5-diphosphate.

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