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. 2012 Mar 23;287(13):10693-10702.
doi: 10.1074/jbc.M111.318022. Epub 2012 Feb 6.

Ezrin-radixin-moesin-binding sequence of PSGL-1 glycoprotein regulates leukocyte rolling on selectins and activation of extracellular signal-regulated kinases

Caroline Spertini  1 , Bénédicte Baïsse  1 , Olivier Spertini  2
Affiliations

Ezrin-radixin-moesin-binding sequence of PSGL-1 glycoprotein regulates leukocyte rolling on selectins and activation of extracellular signal-regulated kinases

Caroline Spertini et al. J Biol Chem. .

Abstract

P-selectin glycoprotein ligand-1 (PSGL-1) mediates the capture (tethering) of free-flowing leukocytes and subsequent rolling on selectins. PSGL-1 interactions with endothelial selectins activate Src kinases and spleen tyrosine kinase (Syk), leading to α(L)β(2) integrin-dependent leukocyte slow rolling, which promotes leukocyte recruitment into tissues. In addition, but through a distinct pathway, PSGL-1 engagement activates ERK. Because ezrin, radixin and moesin proteins (ERMs) link PSGL-1 to actin cytoskeleton and because they serve as adaptor molecules between PSGL-1 and Syk, we examined the role of PSGL-1 ERM-binding sequence (EBS) on cell capture, rolling, and signaling through Syk and MAPK pathways. We carried out mutational analysis and observed that deletion of EBS severely reduced 32D leukocyte tethering and rolling on L-, P-, and E-selectin and slightly increased rolling velocity. Alanine substitution of Arg-337 and Lys-338 showed that these residues play a key role in supporting leukocyte tethering and rolling on selectins. Importantly, EBS deletion or Arg-337 and Lys-338 mutations abrogated PSGL-1-induced ERK activation, whereas they did not prevent Syk phosphorylation or E-selectin-induced leukocyte slow rolling. These studies demonstrate that PSGL-1 EBS plays a critical role in recruiting leukocytes on selectins and in activating the MAPK pathway, whereas it is dispensable to phosphorylate Syk and to lead to α(L)β(2)-dependent leukocyte slow rolling.

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Figures

FIGURE 1.
FIGURE 1.
SRK mutations inhibit CHO-PSGL-1 transfectant rolling on P-selectin but do not affect P-selectin/μ chimera binding and cell rolling velocity. A, schematic representation of WT and mutant PSGL-1 used in this study. The dashed box represents the signal peptide and propeptide. TM, transmembrane domain. The EBS is boxed on WT PSGL1. Residues are numbered according to the PSGL-1 amino acid sequence reported by Sako et al. (42). B and C, flow cytometric histograms of CHO transfectants showing matched expression levels of WT PSGL-1 and AAA mutant detected by anti-PSGL-1 mAb PS5 (B) and P-selectin/μ chimera binding (gray histograms) (C). White histograms indicate immunostaining with isotype-matched control mAb (B) or complete inhibition of staining with 10 mm EDTA (C). Mean fluorescence intensity is indicated in each histogram. D, CHO cells expressing matched levels of WT PSGL-1 or AAA mutant were perfused at 1.5 dynes/cm2 on recombinant P-selectin. Cell recruitment was assessed by video microscopy, and results represent the mean ± S.E. (error bars) of 11 experiments. E, cumulative rolling velocity of CHO transfectants on P-selectin. The number of analyzed rolling cells (n) and the mrv are indicated in the bottom table (***, p < 0.001 compared with WT).
FIGURE 2.
FIGURE 2.
EBS deletion or mutations do not impair L- or P-selectin binding to 32D leukocytes. Flow cytometric histograms showing that 32D transfectants express matched levels of PSGL-1 (A) and are bound by l-selectin/μ (B) and P-selectin/μ chimera (C) similarly (gray histograms). Immunostaining with isotype-matched control mAb or inhibition of selectin binding with 10 mm EDTA is indicated by white histograms. The figure illustrates one of three representative experiments. Mean fluorescence intensity is indicated in each histogram.
FIGURE 3.
FIGURE 3.
EBS deletion impairs 32D leukocyte rolling on selectins and increases rolling velocity. Transfectants (0.5 × 106/ml) were perfused at 1.5 dynes/cm2 on L- or P-selectin/μ chimera and at 1.0 dyn/cm2 on E-selectin/μ chimera. A, leukocyte recruitment; B, cumulative rolling velocity. The number of analyzed rolling cells (n) and mrv are indicated in the tables. C, tethering of transfectants expressing WT PSGL-1 or ΔEBS mutant. Results represent the mean ± S.E. (error bars) of at least three independent experiments. ***, p < 0.001 compared with WT.
FIGURE 4.
FIGURE 4.
ERMs stabilize PSGL-1-dependent rolling on L-selectin. A, illustration of 1 of 20 representative 32D cells expressing WT PSGL-1 or ΔEBS or AAA mutants tracked each 40 ms for 2 s; mrv is indicated by a dashed line. B, distribution of distances traveled by 32D transfectants during successive 40 ms time lapses (mrd, mean rolling distance; ***, p < 0.001 compared with WT).
FIGURE 5.
FIGURE 5.
SRK or RK mutations inhibit leukocyte recruitment and tethering on L-, P-, and E-selectin but do not affect slow rolling on E-selectin plus ICAM-1. A, 32D cell recruitment; B, cumulative rolling velocity; C, tethering on L-, P-, and E-selectin. Transfectants expressing matched levels of WT PSGL-1 or SAA or AAA mutants (0.5–1 × 106 cells/ml) were perfused under constant shear stress on selectins. Results represent the mean ± S.E. (error bars) of 3–9 independent experiments (**, p < 0.01; ***, p < 0.001, compared with WT PSGL-1). D, mrv of 32D leukocytes expressing WT PSGL-1 or ΔEBS or SAA mutants on E-selectin or E-selectin plus ICAM-1 (***, p < 0.001, compared with mrv on E-selectin alone). Results represent the mean ± S.E. of at least three independent experiments.
FIGURE 6.
FIGURE 6.
PSGL-1 signaling on ERK is dependent on the EBS and on lipid raft integrity. A–C, 32D leukocytes expressing WT PSGL-1 or ΔEBM, SAA, or AAA mutants were incubated with anti-PSGL-1 mAb at 37 °C for the indicated times and then lysed. Equivalent amounts of lysates were analyzed by Western blotting with antibody directed against phospho-ERK (P-ERK) followed by ERK antibody. When indicated, transfectants expressing WT PSGL-1 were pretreated with vehicle or MEK inhibitor PD98059. D, THP1 cells exposed to mβCD or vehicle were incubated and immunoblotted as indicated in A–C. E, detergent-insoluble fractions of CHO cells expressing WT or AAA PSGL-1 were isolated by centrifugation on a sucrose gradient and analyzed by immunoblotting with anti-PSGL-1 PS5 mAb (bottom panels). Phospho-ERK or phospho-Syk expression ratios were calculated as described under “Experimental Procedures.” Lipid raft-containing fractions were identified with HRP-labeled cholera toxin (top panels). Data are representative of at least three independent experiments.
FIGURE 7.
FIGURE 7.
PSGL-1 binding to ERMs is not required to activate Syk. A and B, 32D leukocytes expressing WT PSGL-1, ΔEBS, SAA, or AAA mutants were incubated with anti-PSGL-1 mAb at 37 °C for the indicated times and then lysed. Equivalent amounts of lysates were immunoblotted with antibody directed against phospho-Syk (P-Syk) and then Syk. When indicated, leukocytes were incubated with the SFK inhibitor PP2 or its inactive analog PP3 before PSGL-1 cross-linking. Data are representative of 3–6 independent experiments. C, THP1 cells exposed to mβCD or vehicle were incubated and immunoblotted as indicated in A and B. Data are representative of at least three independent experiments.
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