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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.

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.

Pubmed ID: 16709866


  • McLaughlin NJ
  • Banerjee A
  • Kelher MR
  • Gamboni-Robertson F
  • Hamiel C
  • Sheppard FR
  • Moore EE
  • Silliman CC


Journal of immunology (Baltimore, Md. : 1950)

Publication Data

June 1, 2006

Associated Grants

  • Agency: NIGMS NIH HHS, Id: GM-49222
  • Agency: NHLBI NIH HHS, Id: HL-59355-06

Mesh Terms

  • Actins
  • Arrestins
  • Calcium
  • Cell Membrane
  • Clathrin
  • Dynamin II
  • Endocytosis
  • Endosomes
  • Enzyme Activation
  • Humans
  • MAP Kinase Kinase Kinase 5
  • MAP Kinase Signaling System
  • Microfilament Proteins
  • Neutrophils
  • Platelet Activating Factor
  • Platelet Membrane Glycoproteins
  • Protein Transport
  • Receptors, G-Protein-Coupled
  • Subcellular Fractions
  • p38 Mitogen-Activated Protein Kinases