Signal-dependent slow leukocyte rolling does not require cytoskeletal anchorage of P-selectin glycoprotein ligand-1 (PSGL-1) or integrin αLβ2

J Biol Chem. 2012 Jun 1;287(23):19585-98. doi: 10.1074/jbc.M112.361519. Epub 2012 Apr 16.

Abstract

In inflamed venules, neutrophils roll on P- or E-selectin, engage P-selectin glycoprotein ligand-1 (PSGL-1), and signal extension of integrin α(L)β(2) in a low affinity state to slow rolling on intercellular adhesion molecule-1 (ICAM-1). Cytoskeleton-dependent receptor clustering often triggers signaling, and it has been hypothesized that the cytoplasmic domain links PSGL-1 to the cytoskeleton. Chemokines cause rolling neutrophils to fully activate α(L)β(2), leading to arrest on ICAM-1. Cytoskeletal anchorage of α(L)β(2) has been linked to chemokine-triggered extension and force-regulated conversion to the high affinity state. We asked whether PSGL-1 must interact with the cytoskeleton to initiate signaling and whether α(L)β(2) must interact with the cytoskeleton to extend. Fluorescence recovery after photobleaching of transfected cells documented cytoskeletal restraint of PSGL-1. The lateral mobility of PSGL-1 similarly increased by depolymerizing actin filaments with latrunculin B or by mutating the cytoplasmic tail to impair binding to the cytoskeleton. Converting dimeric PSGL-1 to a monomer by replacing its transmembrane domain did not alter its mobility. By transducing retroviruses expressing WT or mutant PSGL-1 into bone marrow-derived macrophages from PSGL-1-deficient mice, we show that PSGL-1 required neither dimerization nor cytoskeletal anchorage to signal β(2) integrin-dependent slow rolling on P-selectin and ICAM-1. Depolymerizing actin filaments or decreasing actomyosin tension in neutrophils did not impair PSGL-1- or chemokine-mediated integrin extension. Unlike chemokines, PSGL-1 did not signal cytoskeleton-dependent swing out of the β(2)-hybrid domain associated with the high affinity state. The cytoskeletal independence of PSGL-1-initiated, α(L)β(2)-mediated slow rolling differs markedly from the cytoskeletal dependence of chemokine-initiated, α(L)β(2)-mediated arrest.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Actin Cytoskeleton / genetics
  • Actin Cytoskeleton / metabolism
  • Actomyosin / genetics
  • Actomyosin / metabolism
  • Animals
  • Bridged Bicyclo Compounds, Heterocyclic / pharmacology
  • CHO Cells
  • Chemokines / genetics
  • Chemokines / metabolism
  • Cricetinae
  • Cricetulus
  • Humans
  • Intercellular Adhesion Molecule-1 / genetics
  • Intercellular Adhesion Molecule-1 / metabolism
  • Leukocyte Rolling / physiology*
  • Lymphocyte Function-Associated Antigen-1 / genetics
  • Lymphocyte Function-Associated Antigen-1 / metabolism*
  • Membrane Glycoproteins / genetics
  • Membrane Glycoproteins / metabolism*
  • Mice
  • Mice, Knockout
  • Neutrophils / cytology
  • Neutrophils / metabolism*
  • Protein Multimerization / drug effects
  • Protein Multimerization / physiology
  • Protein Structure, Tertiary
  • Signal Transduction / drug effects
  • Signal Transduction / physiology*
  • Thiazolidines / pharmacology

Substances

  • Bridged Bicyclo Compounds, Heterocyclic
  • Chemokines
  • Icam1 protein, mouse
  • Lymphocyte Function-Associated Antigen-1
  • Membrane Glycoproteins
  • P-selectin ligand protein
  • Thiazolidines
  • Intercellular Adhesion Molecule-1
  • Actomyosin
  • latrunculin B