Platelet-activating factor-mediated endosome formation causes membrane translocation of p67phox and p40phox that requires recruitment and activation of p38 MAPK, Rab5a, and phosphatidylinositol 3-kinase in human neutrophils.
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 muM 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.
Bonfils Blood Center, University of Colorado Denver School of Medicine, Children's Hospital, Denver, CO 80230, USA.
Nathan J D
NJ
McLaughlin
Anirban
A
Banerjee
Samina Y
SY
Khan
Janet L
JL
Lieber
Marguerite R
MR
Kelher
Fabia
F
Gamboni-Robertson
Forest R
FR
Sheppard
Ernest E
EE
Moore
Gary W
GW
Mierau
David J
DJ
Elzi
Christopher C
CC
Silliman
D002462:Cell Membrane / Q000201:enzymology / Q000378:metabolism*; D034285:Dynamin II / Q000378:metabolism; D011992:Endosomes / Q000201:enzymology / Q000378:metabolism*; D004789:Enzyme Activation / Q000502:physiology; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D008024:Ligands; D020935:MAP Kinase Signaling System / Q000502:physiology; D009504:Neutrophils / Q000201:enzymology / Q000378:metabolism*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism*; D010972:Platelet Activating Factor / Q000378:metabolism / Q000502:physiology*; D010980:Platelet Membrane Glycoproteins / Q000378:metabolism; D021381:Protein Transport / Q000502:physiology; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism; D033921:Vesicular Transport Proteins / Q000378:metabolism; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism* / Q000502:physiology; D020696:rab5 GTP-Binding Proteins / Q000378:metabolism*
D008024:Ligands; D010750:Phosphoproteins; D010972:Platelet Activating Factor; D010980:Platelet Membrane Glycoproteins; D043562:Receptors, G-Protein-Coupled; D033921:Vesicular Transport Proteins; C093846:early endosome antigen 1; C085072:neutrophil cytosol factor 40K; C079789:neutrophil cytosol factor 67K; C039351:platelet activating factor receptor; D019869:Phosphatidylinositol 3-Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D020696:rab5 GTP-Binding Proteins; D034285:Dynamin II