title: Platelet-activating factor-induced clathrin-mediated endocytosis requires beta-arrestin-1 recruitment and activation of the p38 MAPK signalosome at the plasma membrane for actin bundle formation.
abstract: Clathrin-mediated endocytosis (CME) is a common pathway used by G protein-linked receptors to transduce extracellular signals. We hypothesize that platelet-activating factor (PAF) receptor (PAFR) ligation requires CME and causes engagement of beta-arrestin-1 and recruitment of a p38 MAPK signalosome that elicits distinct actin rearrangement at the receptor before endosomal scission. Polymorphonuclear neutrophils were stimulated with buffer or 2 microM PAF (1 min), and whole cell lysates or subcellular fractions were immunoprecipitated or slides prepared for colocalization and fluorescent resonance energy transfer analysis. In select experiments, beta-arrestin-1 or dynamin-2 were neutralized by intracellular introduction of specific Abs. PAFR ligation caused 1) coprecipitation of the PAFR and clathrin with beta-arrestin-1, 2) fluorescent resonance energy transfer-positive interactions among the PAFR, beta-arrestin-1, and clathrin, 3) recruitment and activation of the apoptosis signal-regulating kinase-1/MAPK kinase-3/p38 MAPK (ASK1/MKK3/p38 MAPK) signalosome, 4) cell polarization, and 5) distinct actin bundle formation at the PAFR. Neutralization of beta-arrestin-1 inhibited all of these cellular events, including PAFR internalization; conversely, dynamin-2 inhibition only affected receptor internalization. Selective p38 MAPK inhibition globally abrogated actin rearrangement; however, inhibition of MAPK-activated protein kinase-2 and its downstream kinase leukocyte-specific protein-1 inhibited only actin bundle formation and PAFR internalization. In addition, ASK1/MKK3/p38 MAPK signalosome assembly appears to occur in a novel manner such that the ASK1/p38 MAPK heterodimer is recruited to a beta-arrestin-1 bound MKK3. In polymorphonuclear neutrophils, leukocyte-specific protein-1 may play a role similar to fascin for actin bundle formation. We conclude that PAF signaling requires CME, beta-arrestin-1 recruitment of a p38 MAPK signalosome, and specific actin bundle formation at the PAFR for transduction before endosomal scission.
authors.affiliation: Department of Pediatrics, University of Colorado School of Medicine, Denver, CO 80262, USA.
authors.forename: Nathan J D
authors.initials: NJ
authors.lastname: McLaughlin
authors.forename: Anirban
authors.initials: A
authors.lastname: Banerjee
authors.forename: Marguerite R
authors.initials: MR
authors.lastname: Kelher
authors.forename: Fabia
authors.initials: F
authors.lastname: Gamboni-Robertson
authors.forename: Christine
authors.initials: C
authors.lastname: Hamiel
authors.forename: Forest R
authors.initials: FR
authors.lastname: Sheppard
authors.forename: Ernest E
authors.initials: EE
authors.lastname: Moore
authors.forename: Christopher C
authors.initials: CC
authors.lastname: Silliman
mesh_terms: D000199:Actins / Q000378:metabolism*; D019390:Arrestins / Q000378:metabolism* / Q000502:physiology; D002118:Calcium / Q000378:metabolism; D002462:Cell Membrane / Q000201:enzymology* / Q000378:metabolism; D002966:Clathrin / Q000378:metabolism / Q000502:physiology*; D034285:Dynamin II / Q000502:physiology; D004705:Endocytosis / Q000502:physiology*; D011992:Endosomes / Q000201:enzymology / Q000378:metabolism; D004789:Enzyme Activation / Q000502:physiology; D006801:Humans; D048848:MAP Kinase Kinase Kinase 5 / Q000302:isolation & purification / Q000378:metabolism; D020935:MAP Kinase Signaling System / Q000502:physiology*; D008840:Microfilament Proteins / Q000378:metabolism / Q000502:physiology; D009504:Neutrophils / Q000166:cytology / Q000201:enzymology / Q000378:metabolism; D010972:Platelet Activating Factor / Q000378:metabolism / Q000502:physiology*; D010980:Platelet Membrane Glycoproteins / Q000378:metabolism / Q000502:physiology; D021381:Protein Transport / Q000502:physiology; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism / Q000502:physiology; D013347:Subcellular Fractions / Q000201:enzymology; D000071559:beta-Arrestin 1; D000071557:beta-Arrestins; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism / Q000502:physiology*
chemical_list: C000605253:ARRB1 protein, human; D000199:Actins; D019390:Arrestins; D002966:Clathrin; C489183:LSP1 protein, human; D008840:Microfilament Proteins; D010972:Platelet Activating Factor; D010980:Platelet Membrane Glycoproteins; D043562:Receptors, G-Protein-Coupled; D000071559:beta-Arrestin 1; D000071557:beta-Arrestins; C039351:platelet activating factor receptor; D048051:p38 Mitogen-Activated Protein Kinases; D048848:MAP Kinase Kinase Kinase 5; D034285:Dynamin II; D002118:Calcium