Signal-dependent Slow Leukocyte Rolling Does Not Require Cytoskeletal Anchorage of P-selectin Glycoprotein Ligand-1 (PSGL-1) or Integrin αLβ2

Bojing Shao,Tadayuki Yago,Phillip A Coghill,Arkadiusz G Klopocki,Padmaja Mehta-D'Souza,David W Schmidtke,William Rodgers,Rodger P Mcever

doi:10.1074/jbc.m112.361519

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.

Highlights

The cytoskeleton contributes to receptor-initiated signaling and has been linked to force-regulated integrin activation
Neutrophils roll on P- or E-selectin, engage P-selectin glycoprotein ligand-1 (PSGL-1), and signal extension of integrin ␣L␤2 in a low affinity state to slow rolling on intercellular adhesion molecule-1 (ICAM-1)
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 ␤2 integrin-dependent slow rolling on P-selectin and ICAM-1

Summary

Background

The cytoskeleton contributes to receptor-initiated signaling and has been linked to force-regulated integrin activation. Binding of “primed” ␣L␤2 to immobilized, but not fluid-phase, ICAM-1 triggers energy-dependent conversion of ␣L␤2 to its high affinity state [33] These data support a model in which the cytoskeleton exerts lateral force on talin and/or kindlins to fully separate the integrin ␣ and ␤ tails and convert the extended ectodomain to the high affinity state, if the primed integrin binds to immobilized ligand [34]. If ␣L␤2 does not attach to the cytoskeleton, forces are likely to be applied axially but not laterally during rolling on ICAM-1 We tested these concepts by manipulating cytoskeletal anchorage or dimerization of PSGL-1 and by depolymerizing actin filaments or decreasing actomyosin tension. Additional chemokine signals, required actin filaments and actomyosin tension to arrest rolling cells on ICAM-1 or on a mAb to an ␣L␤2 conformation associated with high affinity for ligand

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION