Platelet-activating factor-mediated endosome formation causes membrane translocation of p67phox and p40phox that requires recruitment and activation of p38 …

NJD McLaughlin, A Banerjee, SY Khan… - The Journal of …, 2008 - journals.aai.org
NJD McLaughlin, A Banerjee, SY Khan, JL Lieber, MR Kelher, F Gamboni-Robertson…
The Journal of Immunology, 2008journals.aai.org
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) …
Abstract
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 O 2− 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.
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