| |
|
|
|
|
|
|
|||
|
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
From the Department of Microbiology/Immunology,
Northwestern University Medical School, Chicago, IL.
P-selectin glycoprotein ligand-1 (PSGL-1) serves as the leukocyte
ligand for P-selectin, and many of the structural features of its
ectodomain required for interactions with P-selectin have been
uncovered. In contrast, the function of the highly conserved PSGL-1
cytoplasmic domain has not been explored. Stable transfectants expressing similar levels of either wild-type PSGL-1 or truncated PSGL-1 in which only 4 cytoplasmic residues were retained (designated PSGL-1 Leukocyte migration is mediated by several
groups of adhesion molecules, including the selectin family. Expression
of L-selectin on leukocytes is constitutive, whereas expression of
E-selectin on endothelium, and P-selectin on endothelium and platelets,
requires specific activators for surface expression.1 The
selectins recognize a heterogeneous array of glycoprotein ligands
including the homodimeric mucin P-selectin glycoprotein ligand-1
(PSGL-1).2 PSGL-1 interacts with both P-selectin and
L-selectin, and thus mediates leukocyte adhesion to endothelium,
platelets, and other leukocytes.3,4 Recognition of
selectins requires that specific ectodomain residues of PSGL-1 be
enzymatically modified,5,6 and dimerization through a
single extracellular cysteine residue is also required for leukocyte
rolling on P-selectin.7 To date, most research has
concentrated on essential modifications of the extracellular portions
of PSGL-1, but the role of the highly conserved PSGL-1 cytoplasmic tail
in leukocyte rolling has not been investigated.
The cytoplasmic domains of many adhesion molecules are known to play an
essential role in adhesive events by serving as sites for structural
and functional linkages between cell surface molecules and cytoskeletal
components. These interactions are involved in cell-cell and
cell-matrix adhesion, as well as receptor-ligand interactions, receptor
internalization, redistribution, shedding, endocytic sorting, and
signal transduction. Among leukocyte adhesion molecules, leukocyte
function-associated antigen-1 (LFA-1), very late activation-4
(VLA-4), intercellular adhesion molecule-1 (ICAM-1), L-selectin, and E-selectin have been shown to interact with the cytoskeleton, and these interactions are mediated through a series of
cytoplasmic linker proteins, including In this study, we investigated the role of the highly conserved
cytoplasmic tail of PSGL-1. The results indicate that interaction of
the PSGL-1 cytoplasmic domain with the actin cytoskeleton is essential
for rolling on P-selectin, and thereby suggest a novel paradigm for
adhesion receptors that mediate leukocyte rolling under flow.
Generation of PSGL-1 complementary DNA truncated in the
cytoplasmic domain
Generation and verification of stable transfectants in
K562/FucT-VII cells
Soluble P-selectin binding HL60 cells or transfectants were incubated with purified mouse P-selectin-human IgG fusion proteins (referred to as P-RIg; BD Pharmingen, San Diego, CA) for 20 minutes at 4°C, washed, and incubated with goat antihuman IgG fluorescein isothiocyanate (FITC; Biosource International) for an additional 20 minutes. Samples were analyzed on a Becton Dickinson FACSCalibur as described above.Low-shear COS cell adhesion assay The COS cells were transfected with P-selectin by the diethylaminoethyl-dextran method in 100-mm tissue culture-grade Petri dishes, replated on 35-mm dishes (assay plates), and allowed to readhere overnight. The next day, each assay plate was washed 3 times and either 2 × 106 HL60 cells or transfectants (in a final volume of 0.6 mL) were gently added. Assay plates with added cells were incubated on a constantly rocking platform for 15 minutes at 4°C, plates were washed 5 times followed by fixation with cold 0.37% formaldehyde. Mean number of cells bound per COS cell was determined by counting approximately 100 COS cells in multiple 40 × fields using a standard inverted light microscope.Parallel-plate flow assay Rolling of HL60 cells and transfectants on monolayers of stably transfected Chinese hamster ovary (CHO) cells expressing human P-selectin (referred to as CHO/P) was analyzed.22 CHO/P cells were grown to confluence in 35-mm culture dishes and positioned on a 0.0254-cm-thick gasket of a flow chamber (Glycotech, Rockville, MD), with the CHO/P monolayer serving as the bottom floor of the flow chamber. A constant flow level was maintained by drawing media containing cells at 0.5 × 106 cells/mL through the chamber using a PHD 2000 programmable syringe pump (Harvard Apparatus, Holliston, MA). The flow chamber was mounted on an inverted Eclipse TE300 microscope (Nikon, Melville, NY) with Hoffman interference contrast objectives (Modulation Optics, Greenvale, NY) and rolling data were collected with a video camera. Data were analyzed with Celltrak software (Compix, Cranberry Township, PA). Sequential images of tracked cells were digitized and matched on the basis of trajectories and morphology every 2 seconds. Interactive "events," which correspond to rolling cells, were collected for 50 sequential paired images. In each experiment, each cell line or transfectant was analyzed twice. HL60 cells were included as a control in all experiments because they roll very well on P-selectin. In some experiments, cells were preincubated for 20 minutes with various concentrations of purified KPL1, washed, resuspended, and analyzed for rolling on CHO/P monolayers. In other experiments cells were pretreated for 30 to 60 minutes at 37°C with indicated doses of either latrunculin B (Calbiochem, La Jolla, CA), cytochalasin B (Sigma, St. Louis, MO), or staurosporine (Sigma), washed, resuspended, and analyzed for rolling on P-selectin.FACS analysis for cytoskeletal linkage of transmembrane receptors Minor modifications were made to previously published protocols.23-25 Cells were added to FACS tubes precoated with 1% bovine serum albumin (BSA) in phosphate-buffered saline (PBS) and incubated for 15 minutes with either purified KPL1 or an isotype-matched control mAb. Cells were washed and incubated with goat antimouse IgG FITC (Biosource) for an additional 15 minutes, pelleted, and the pellet was gently resuspended in buffer consisting of 0.5% Triton X-100, 13 mM Tris-HCl (pH 8.0), 50 mM NaCl, 2 mM MgCl2, 0.2 mM EGTA, 2% fetal bovine serum (FBS), and 1 mM each phenylmethylsulfonyl fluoride (PMSF), leupeptin, pepstatin A, and aprotinin, incubated at room temperature for 20 minutes, and washed with 3 mL of the above buffer without Triton X-100. Pellets (containing the insoluble fraction including the actin cytoskeleton and proteins linked to it) were gently resuspended in PBS/1% formaldehyde and analyzed by flow cytometry. Cells not treated with detergent were included as a control for the setting of data collection gates.Affinity capture assay and Western blotting The GST fusion proteins containing all 69 cytoplasmic tail residues of PSGL-1 (GST/PSGL-1) or GST alone (GST only) were generated using pGEX-2T by standard methods, coupled to glutathione-Sepharose, and used as an affinity matrix to capture cytoplasmic proteins from HL60 WCLs. HL60 cells were washed twice in PBS and resuspended at 1 × 107 cells/mL in lysis buffer (1% Triton X-100, 150 mM NaCl, 10 mM Tris-HCl (pH 8.0), and 1 mM each CaCl2, MgC12, aprotinin, leupeptin, pepstatin A, and PMSF). Glutathione-Sepharose beads with the indicated fusion proteins were incubated with WCLs for 2 hours at 4°C, washed with lysis buffer containing 0.1% Triton X-100, eluted with SDS-PAGE sample buffer, run on a 10% polyacrylamide gel, and transferred to nitrocellulose. Individual blots were probed with a mAb or pAb to several cytoskeletal proteins, including -actinin (Sigma), vinculin (Sigma), talin
(Sigma), moesin (mAb from Transduction Laboratories, San Diego,
CA and pAb provided by Dr A. Bretscher, Cornell University,
Ithaca, NY) and ezrin (pAb to ezrin provided by Dr A. Bretscher,
Cornell University). Blocking, incubations, washes, development, and
visualization by enhanced chemiluminescence (ECL) was carried out as
previously described.7,19
Immunoprecipitation The PSGL-1 and PSGL-1 cyto transfectants were
resuspended at 1 × 107 cells/mL in lysis buffer (0.5%
Triton X-100, 50 mM Hepes, 500 mM NaCl, and 1 mM each aprotinin,
leupeptin, pepstatin A, and PMSF) and incubated on ice for 45 minutes.
Extracts were clarified by centrifugation at 13 000g for 20 minutes at 4°C, and precleared with 50 µL of a 50% solution
(wt/vol) of Affigel coupled to an irrelevant isotype-matched control
mAb for 60 minutes with rotation at 4°C. Precleared lysates were
incubated with KPL1-coupled Affigel as described above.
Immunoprecipitates were washed twice in lysis buffer, boiled in
SDS-PAGE sample buffer, run on a 7.5% polyacrylamide gel, and
transferred to nitrocellulose. Blots were probed with either a mAb to
moesin, vinculin, talin, ezrin, -actinin or PSGL-1, or HECA-452
culture supernatants.
Scanning immunoelectron microscopy The 106 transfectants expressing either PSGL-1 or PSGL-1 cyto were resuspended in 100 µL PBS plus 5% goat serum
containing KPL1, incubated on ice for 15 minutes, rinsed twice, and
resuspended in 100 µL PBS plus 5% goat serum containing goat
antimouse IgG conjugated to 12 nm gold (Jackson Immunoresearch
Laboratory, West Grove, PA). Following a 15-minute incubation
on ice, cells were rinsed twice in PBS plus 0.2% BSA (BSA Fraction V,
Sigma), and resuspended in 100 µL PBS plus 0.2% BSA. Approximately
50 µL of the cell suspension was applied to degreased glass chips
precoated with 0.1% poly-L-lysine (Sigma), the cells were allowed to
settle on the chips for 10 minutes at room temperature, and the entire chip was transferred to a Petri dish containing 3% electron
microscopic grade glutaraldehyde (Electron Microscopy Services, Fort
Washington, PA) in 0.1 M sodium cacodylate (Sigma) containing
7.5% sucrose for overnight fixation. Samples were assigned a code
number and analyzed for either PSGL-1 or PSGL-1 cyto distribution
using a Hitachi S-900 LVSEM equipped with a stereoviewer for enhanced spatial resolution.
Statistics For statistical analysis of leukocyte rolling assays, the Student t test was used. Differences were considered statistically significant with P < .05.
Generation and characterization of stably transfected K562/FucT-VII
cells expressing PSGL-1 or PSGL-1 cyto were generated in K562/FucT-VII cells, a cell line that
we have previously shown does not express PSGL-1, but does express all
the glycosyltransferases required for appropriate enzymatic
modification of PSGL-1.7,19,20 Wild-type PSGL-1 and
PSGL-1 cyto were expressed at similar levels on the surface of the
transfected cells, although wild-type PSGL-1 was expressed approximately 2-fold higher compared to PSGL-1 cyto expression (Figure 1A, left panels). Both
transfectants stained equivalently with HECA-452 (Figure 1A, right
panels). HL60 cells were included as a positive control because they
endogenously express both PSGL-1 and HECA-452 and roll extremely well
on P-selectin even though they express approximately 10-fold lower
levels of PSGL-1 compared to both K562 transfectants. Western blotting
of WCLs generated from either native PSGL-1 or PSGL-1 cyto
transfectants showed the expected increase in mobility consistent with
the loss of 65 amino acids (Figure 1B). Individual clones were also
analyzed by semiquantitative RT-PCR using PCR cycles titered to be
below plateau phase,7,20 and showed equivalent levels of
FucT-VII and C2GlcNAcT-I mRNA (data not shown).
In addition to the decrease in the apparent molecular mass of
PSGL-1
To confirm that the functionally relevant modifications of the PSGL-1
ectodomain were not affected by truncation of the cytoplasmic domain,
the ability of recombinant soluble P-RIg fusion proteins to recognize
PSGL-1 Rolling of PSGL-1 or PSGL-1 cyto cells
rolled at greatly reduced levels in all experiments (7.3% ± 6.9%
of control PSGL-1 transfectants, n = 7, P < .05). To
confirm that this dramatic loss of rolling was not cell type specific,
the PSGL-1 or PSGL-1 cyto cDNAs were stably transfected into the
BJAB/FucT-VII cell line, another PSGL-1 cell line
containing all of the enzymes required for proper modification of
PSGL-1. Transfectants were generated, cloned, and characterized as
described above (data not shown). Rolling behavior of the BJAB cell
lines on CHO/P was quite similar to the K562 transfectants for cells
transfected with PSGL-1 cyto compared to BJAB cells transfected with
wild-type PSGL-1 (11.7% ± 10% of control PSGL-1 transfectants,
n = 7, P < .05; Figure 3B). Thus, truncation of the
PSGL-1 cytoplasmic tail blocks nearly all rolling on
P-selectin.
To confirm that the low residual rolling observed in PSGL-1 To determine if the remaining low rolling activity seen with
PSGL-1 In addition, PSGL-1 To further characterize interactions between PSGL-1 Interactions between the actin cytoskeleton and the cytoplasmic tail of PSGL-1 Because interactions between the cytoplasmic domains of many adhesion molecules and the actin cytoskeleton are essential for cell adhesion, we sought to determine if an intact actin cytoskeleton was required for PSGL-1 interactions with P-selectin. PSGL-1 transfectants were preincubated with increasing doses of either latrunculin B or cytochalasin B and washed; rolling was analyzed in the parallel-plate flow chamber. Rolling events were decreased in a dose-dependent manner (Figure 4A). Low doses sufficient to prevent de novo actin polymerization had only modest effects on rolling, whereas high doses capable of disrupting the pre-existing actin cytoskeleton sharply reduced rolling (Figure 4A). Importantly, surface expression of the PSGL-1 glycoprotein was only modestly reduced by the cytoskeletal inhibitors (Figure 4B), indicating that disruption of the actin cytoskeleton does not induce significant shedding or internalization of PSGL-1.
Because the pharmacologic disruption of F-actin gave similar rolling
results to that of PSGL-1
Identification of a cytoskeletal linker protein that interacts with PSGL-1 The above observation documents a functional link between PSGL-1 and the actin cytoskeleton, but does not identify the molecular basis for this interaction. To search for potential cytoplasmic mediators of this interaction, a GST fusion protein incorporating the entire PSGL-1 cytoplasmic domain was used as an affinity capture matrix to isolate cytoplasmic proteins from WCLs capable of interacting with the PSGL-1 cytoplasmic tail. Bound material was eluted, electrophoresed on SDS-PAGE gels, transferred to nitrocellulose, and probed with antibodies to -actinin, vinculin, talin, moesin, or ezrin. Under these conditions, specific interactions between the cytoplasmic domain
of PSGL-1 and moesin but not these other proteins were detected (Figure
6A and data not shown). To confirm these
results, coimmunoprecipitation studies with PSGL-1 and PSGL-1 cyto
transfectants were carried out. Immunoprecipitations were performed
with the anti-PSGL-1 mAb KPL1 coupled to Affigel followed by Western
blotting with an antimoesin mAb. Under these conditions, interactions
with moesin were detected in WCLs from PSGL-1 transfectants but not PSGL-1 cyto transfectants (Figure 6B). Controls showed that
equivalent amounts of PSGL-1 were immunoprecipitated (Figure 6B, right
panel). These findings confirm and extend previous observations in
which the cytoplasmic domain of PSGL-1 was shown to interact with the N-terminal domain of either recombinant or in vitro-translated moesin
(residues 1-310), or moesin in HL60 lysates.26 Thus, the
cytoplasmic domain of PSGL-1 interacts specifically and selectively with the ERM protein moesin, at least in vitro under the condition tested, suggesting an important role for moesin in leukocyte adhesion to P-selectin.
Rolling on P-selectin is blocked after disruption of interactions between moesin and F-actin Moesin exists in both an active and inactive state within cells, with inactivation the result of intramolecular associations between the C- and N-terminal halves of the molecule.27-29 The active cross-linking form of moesin is generated and maintained by the phosphorylation of Thr558 by Rho kinase.30,31 Phosphorylation at this site can be prevented by treatment of cells with staurosporine, and this treatment prevents moesin from interacting with F-actin.32,33 Therefore, to determine the importance of interactions between PSGL-1, moesin, and F-actin, HL60 cells were incubated with increasing concentrations of staurosporine and then analyzed in the parallel plate flow assay for rolling on P-selectin (Figure 7). Rolling was significantly compromised at staurosporine concentrations as low as 0.01 µM (608 ± 101 events versus 1190 ± 220 in vehicle-treated control HL60 cells) and nearly completely eliminated at concentrations of 10 µM (16 ± 9 events). Thus, pharmacologic disruption of interactions between moesin and F-actin resulted in decreased rolling on P-selectin, indicating that interactions between PSGL-1, moesin, and F-actin are essential for rolling on P-selectin.
Subcellular localization of PSGL-1 and PSGL-1 cyto transfectants by scanning immunoelectron microscopy. Unlike freshly isolated human neutrophils, PSGL-1 on K562 transfectants showed a random pattern of distribution, with large numbers of PSGL-1 molecules on both the microvilli and the
planar surface of the cell (Figure 8,
left panel), as we showed previously.7 K562 cells
transfected with PSGL-1 cyto also showed this uniform distribution of
PSGL-1 on both the microvilli and the cell body (Figure 8, right
panel). Therefore, functional differences between PSGL-1 and
PSGL-1 cyto cells cannot be explained by altered subcellular
localization of the mutant.
Much research has been directed at the structure and function of the extracellular domain of PSGL-1. In contrast, considerably less is known about the function of the PSGL-1 cytoplasmic tail. The strong conservation between mouse35 and human36 sequences in this region, which exceeds that of the extracellular region, implies an important functional role for this domain. Other features of PSGL-1, including localization to microvilli, activation-induced surface redistribution, tyrosine phosphorylation of cytoplasmic proteins after ligand engagement, secretion of specific cytokines following adhesion to P-selectin, and suppression of proliferation by hematopoietic progenitors after ligation of PSGL-1, also suggest an essential role for the PSGL-1 cytoplasmic domain.34,37-40 In this report, we present evidence indicating that attachment of the PSGL-1 cytoplasmic domain to the actin cytoskeleton is crucial for rolling on P-selectin. Multiple observations support this hypothesis. Transfectants expressing
PSGL-1 Interactions between PSGL-1 and the actin cytoskeleton have previously been implicated in receptor localization or redistribution.37 PSGL-1 undergoes rapid surface redistribution to the uropod of activated, surface-bound neutrophils, and this redistribution of PSGL-1 is associated with decreased adhesion to P-selectin.37 Both the redistribution of PSGL-1 and the associated reduction in adhesion were prevented by a brief pretreatment of cells with 2 µM cytochalasin D, indicating a requirement for de novo actin polymerization in these processes.37 In contrast, we observed no significant decrease in rolling on P-selectin after preincubation with low-dose (10 µM) cytochalasin B and only modest effects with low dose (5 µM) latrunculin B, indicating that de novo actin polymerization is not essential for rolling of PSGL-1 transfectants. However, progressively higher doses of either actin cytoskeleton inhibitor dramatically reduced rolling on P-selectin in a dose-dependent fashion, implying that disruption of the pre-existing actin cytoskeletal network impaired PSGL-1-mediated rolling (Figure 4A). Importantly, these high doses did not cause significant decreases in surface expression of PSGL-1, indicating that treatment with cytoskeletal inhibitors did not cause shedding or internalization of the molecule (Figure 4B). That structurally dissimilar compounds with distinct mechanisms of action on actin29,41,42 exhibited very similar, dose-dependent inhibition of rolling on P-selectin argues against any nonspecific activity by these drugs. Thus, inhibition of de novo actin polymerization by low doses of inhibitors had little or no effect on PSGL-1-mediated rolling, whereas disruption of the intact actin cytoskeleton and inhibition of its repolymerization by high concentrations of actin inhibitors dramatically decreased rolling. These data suggest that a pre-existing actin cytoskeletal network, and linkages between this network and PSGL-1, are required for rolling on P-selectin. Treatment of L-selectin transfectants with high doses of cytochalasin B
(100 µM) eliminated adhesion to high endothelial venules and rolling
in vivo, indicating that L-selectin-mediated interactions are also
dependent on an intact actin cytoskeleton.43 Similar to
the data presented in this report for PSGL-1, truncation of the
L-selectin cytoplasmic tail also eliminated adhesion to high endothelial venules and rolling in vivo.43,44 L-selectin
also interacts with the actin cytoskeleton, but these interactions appear to be mediated by PSGL-1 is localized primarily to the tips of microvilli on resting
leukocytes, which is thought to be important for interactions with
selectins.34 Because ERM proteins are concentrated in
microvilli and available evidence suggests that ERM proteins may be
necessary for microvilli formation in leukocytes,45,46 it
is reasonable to hypothesize that interactions with moesin are
essential for both subcellular localization and PSGL-1 adhesive
functions. However, PSGL-1 displayed a random pattern of distribution
on K562 cells, being found on the microvilli, ruffles, and planar body
of this cell line7 (Figure 8A), suggesting that interaction
with moesin is not sufficient for preferential localization to
microvilli. An identical pattern of surface expression was present on
transfectants expressing PSGL-1 Our findings confirm and extend a previous report that the ERM protein
moesin appears to serve as a linker protein between PSGL-1 and the
actin cytoskeleton. Moesin and PSGL-1 colocalize to the uropods of
activated neutrophils,26 moesin in HL60 lysates selectively bound to GST fusion proteins containing the PSGL-1 cytoplasmic tail (Figure 6A),26 and moesin was
coimmunoprecipitated in transfectants expressing full-length PSGL-1 but
not PSGL-1 This is the first report describing an essential role for the highly conserved PSGL-1 cytoplasmic domain in rolling on P-selectin. Truncation of the PSGL-1 cytoplasmic domain abrogated rolling of cells on P-selectin and sharply reduced interactions with the actin cytoskeleton. This loss of rolling was recapitulated in cells expressing native PSGL-1 by treatment of cells with reagents that disrupt either the pre-existing actin cytoskeleton or interactions between moesin and F-actin. These data collectively indicate that attachment of PSGL-1 to the actin cytoskeleton is essential for cell adhesion and suggest that moesin participates in this interaction. It is likely that some level of interaction between PSGL-1 and the actin cytoskeleton is constitutive, and that these associations can be modulated by various factors such as chemokines or cytokines.
The authors gratefully acknowledge Dr Robert D. Nelson and Michael
Herron (Department of Dermatology, University of Minnesota, Minneapolis) for their excellent technical support in generating the
scanning electron photomicrographs of PSGL-1 and PSGL-1
Submitted August 10, 2001; accepted February 11, 2002.
Supported by the American Cancer Society, Illinois Chapter, grant 99-47 (to K.R.S.), the American Heart Association grant 003003N (to K.R.S.), and grant RPG-96-097-04-CSM from the National American Cancer Society (to G.S.K.). G.S.K. is an Established Investigator of the American Heart Association.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Karen R. Snapp, Northwestern University Medical School, Department of Microbiology/Immunology, Tarry 6-728, 303 E Superior Ave, Chicago, IL 60611; e-mail: krs133{at}northwestern.edu.
1.
Kansas GS.
Selectins and their ligands: current concepts and controversies.
Blood.
1996;88:3259-3286 2. Varki A. Selectin ligands: will the real ones please stand up? J Clin Invest. 1997;99:1-5[Medline] [Order article via Infotrieve]. 3. Geng JG, Bevilacqua MP, Moore KL, et al. Rapid neutrophil adhesion to activated endothelium mediated by GMP-140. Nature. 1990;343:757-760[CrossRef][Medline] [Order article via Infotrieve]. 4. Walchek B, Moore KL, McEver RP, Kishimoto TK. Neutrophil-neutrophil interactions under hydrodynamic shear stress involve L-selectin and PSGL-1. J Clin Invest. 1996;98:1081-1087[Medline] [Order article via Infotrieve]. 5. Sako D, Comess KM, Barone KM, Camphausen RT, Cumming DA, Shaw GD. A sulfated peptide segment at the amino terminus of PSGL-1 is critical for P-selectin binding. Cell. 1995;83:323-331[CrossRef][Medline] [Order article via Infotrieve].
6.
Li F, Wilkins PP, Crawley S, Weinstein J, Cummings RD, McEver RP.
Post-translational modifications of recombinant P-selectin glycoprotein ligand-1 required for binding to P- and E-selectin.
J Biol Chem.
1996;271:3255-3264
7.
Snapp KR, Craig R, Herron M, Nelson RD, Stoolman LM, Kansas GS.
Dimerization of P-selectin glycoprotein ligand-1 (PSGL-1) required for optimal recognition of P-selectin.
J Cell Biol.
1998;142:263-270
8.
Yoshida M, Westlin WF, Wang N, et al.
Leukocyte adhesion to vascular endothelium induces E-selectin linkage to the actin cytoskeleton.
J Cell Biol.
1996;133:445-455
9.
Otey CA, Pavalko FM, Burridge K.
An interaction between alpha-actinin and the beta 1 integrin subunit in vitro.
J Cell Biol.
1990;111:721-729 10. Pavalko FM, LaRoche SM. Activation of human neutrophils induces an interaction between the integrin beta 2-subunit (CD18) and the actin binding protein alpha-actinin. J Immunol. 1993;151:3795-3807[Abstract].
11.
Pavalko FM, Walker DM, Graham L, Goheen M, Doerschuk CM, Kansas GS.
The cytoplasmic domain of L-selectin interacts with cytoskeletal proteins via
12.
Carpen O, Pallai P, Staunton DE, Springer TA.
Association of intercellular adhesion molecule-1 (ICAM-1) with actin-containing cytoskeleton and alpha-actinin.
J Cell Biol.
1992;118:1223-1234
13.
Tsukita S, Oishi K, Sato N, Sagara J, Kawai A, Tsukita S.
ERM family members as molecular linkers between the cell surface glycoprotein CD44 and actin-based cytoskeletons.
J Cell Biol.
1994;126:391-401
14.
Serrador JM, Nieto M, Alonso-Lebrero JL, et al.
CD43 interacts with moesin and ezrin and regulates its redistribution to the uropods of T lymphocytes at the cell-cell contacts.
Blood.
1998;91:4632-4644
15.
Heiska L, Alfthan K, Gronholm M, Vilja P, Vaheri A, Carpen O.
Association of ezrin with intercellular adhesion molecule-1 and -2 (ICAM-1 and ICAM-2).
J Biol Chem.
1998;273:21893-21900 16. Rees DJG, Ades SE, Singer SJ, Hynes RO. Sequence and domain structure of talin. Nature. 1990;347:685-689[CrossRef][Medline] [Order article via Infotrieve].
17.
Sato N, Funayama N, Nagafuchi A, Yonemura S, Tsukita S, Tsukita S.
A gene family consisting of ezrin, radixin and moesin. Its specific localization at actin filament/plasma membrane association sites.
J Cell Sci.
1992;103:131-143
18.
Reczek D, Bretscher A.
The carboxyl-terminal region of EBP50 binds to a site in the amino-terminal domain of ezrin that is masked in the dormant molecule.
J Biol Chem.
1998;273:18452-18458
19.
Snapp KR, Wagers AJ, Craig R, Stoolman LM, Kansas GS.
P-selectin glycoprotein ligand-1 (PSGL-1) is essential for adhesion to P-selectin but not E-selectin in stably transfected hematopoietic cell lines.
Blood.
1996;89:896-901 20. Wagers AJ, Stoolman LM, Kannagi R, Craig R, Kansas GS. Expression of leukocyte fucosyltransferases regulates binding to E-selectin. Relationship to previously implicated carbohydrate epitopes. J Immunol. 1997;159:1917-1929[Abstract].
21.
Snapp KR, Ding H, Atkins K, Warnke R, Luscinskas FW, Kansas GS.
A novel P-selectin glycoprotein ligand-1 (PSGL-1) monoclonal antibody recognizes an epitope within the tyrosine sulfate motif of human PSGL-1 and blocks recognition of both P- and L-selectin.
Blood.
1998;91:154-164
22.
Knibbs RN, Craig RA, Natsuka S, et al.
The fucosyltransferase FucT-VII regulates E-selectin ligand synthesis in human T cells.
J Cell Biol.
1996;133:911-920 23. Albrecht DL, Noelle RJ. Membrane Ig-cytoskeletal interactions, I: flow cytofluorometric and biochemical analysis of membrane IgM-cytoskeletal interactions. J Immunol. 1988;141:3915-3921[Abstract]. 24. Geppert TD, Lipsky PE. Associations of various T cell-surface molecules with the cytoskeleton: effect of cross-linking and activation. J Immunol. 1991;146:3298-3307[Abstract].
25.
Evans SS, Bowman L, Schleider DM, Kansas GS, Black JD.
Dynamic association of L-selectin with the lymphocyte cortical cytoskeleton.
J Immunol.
1999;162:3615-3624
26.
Alonso-Lebrero J-L, Dominguez C, Serrador JM, et al.
Polarization of adhesion molecules and its interaction with ERM actin-binding proteins in chemoattractant-stimulated neutrophils.
Blood.
2000;95:2413-2419 27. Gary R, Bretscher A. Ezrin self-association involves binding of an N-terminal domain to a normally masked C-terminal domain that includes the F-actin binding site. Mol Biol Cell. 1995;6:1061-1075[Abstract].
28.
Magendantz M, Henry MD, Lander A, Solomon F.
Interdomain interactions of radixin in vitro.
J Biol Chem.
1995;270:25324-25327 29. Pearson MA, Reczek D, Bretscher A, Karplus PA. Structure of the ERM protein moesin reveals the FERM domain fold masked by an extended actin binding tail domain. Cell. 2000;101:259-270[CrossRef][Medline] [Order article via Infotrieve].
30.
Hirao M, Sato N, Kondo T, et al.
Regulation mechanism of ERM (ezrin/radixin/moesin) protein/plasma membrane association: possible involvement of phosphatidylinositol turnover and Rho-dependent signaling pathway.
J Cell Biol.
1996;135:37-51
31.
Mackay DJG, Esch F, Furthmayr H, Hall A.
Rho- and Rac-dependent assembly of focal adhesion complexes and actin filaments in permeabilized fibroblasts: an essential role for ezrin/radixin/moesin proteins.
J Cell Biol.
1997;138:927-938
32.
Nakamura F, Amieva MR, Furthmayr H.
Phosphorylation of threonine 558 in the carboxyl-terminal actin-binding domain of moesin by thrombin activation of human platelets.
J Biol Chem.
1995;270:31377-31385
33.
Nakamura F, Huang L, Pestonjamasp K, Luna EJ, Furthmayr H.
Regulation of F-actin binding to platelet moesin in vitro by both phosphorylation of threonine 558 and polyphosphatidylinositides.
Mol Biol Cell.
1999;10:2669-2685
34.
Moore KL, Patel KD, Breuhl RE, et al.
P-selectin glycoprotein ligand-1 mediates rolling of human neutrophils on P-selectin.
J Cell Biol.
1995;128:661-671
35.
Yang J, Galipeau J, Kozak CA, Furie BC, Furie B.
Mouse P-selectin glycoprotein ligand-1: molecular cloning, chromosomal localization and expression of a functional P-selectin receptor.
Blood.
1996;87:4176-4186 36. Sako D, Chang X-J, Barone KM, et al. Expression cloning of a functional glycoprotein ligand for P-selectin. Cell. 1993;75:1179-1186[CrossRef][Medline] [Order article via Infotrieve]. 37. Lorant DE, McEver RP, McIntyre TM, Moore KL, Prescott SM, Zimmerman GA. Activation of polymorphonuclear leukocytes reduces their adhesion to P-selectin and causes redistribution of ligands for P-selectin on their surfaces. J Clin Invest. 1995;96:171-182[Medline] [Order article via Infotrieve].
38.
Kazuya L-P, Hidari J, Weyrich AS, Zimmerman GA, McEver RP.
Engagement of P-selectin glycoprotein ligand-1 enhances tyrosine phosphorylation and activates mitogen-activated protein kinases in human neutrophils.
J Biol Chem.
1997;272:28750-28756 39. Weyrich AS, Elstad MR, McEver RP, et al. Activated platelets signal chemokine synthesis by human monocytes. J Clin Invest. 1996;97:1525-1534[Medline] [Order article via Infotrieve]. 40. Levesque JP, Zannettino AC, Pudney M, et al. PSGL-1-mediated adhesion of human hematopoietic progenitors to P-selectin results in suppression of hematopoiesis. Immunity. 1999;11:369-378[CrossRef][Medline] [Order article via Infotrieve].
41.
Spector I, Shochet NR, Kashman Y, Groweiss A.
Latrunculins: novel marine toxins that disrupt microfilament organization in cultured cells.
Science.
1983;219:493-495 42. MacLean-Fletcher S, Pollard T. Mechanism of action of cytochalasin B on actin. Cell. 1980;20:329-341[CrossRef][Medline] [Order article via Infotrieve].
43.
Kansas GS, Ley K, Munro JM, Tedder TF.
Regulation of leukocyte rolling and adhesion to HEV through the cytoplasmic domain of L-selectin.
J Exp Med.
1993;177:833-838
44.
Dwir O, Kansas GS, Alon R.
Cytoplasmic anchorage of L-selectin controls leukocyte capture and rolling by increasing the mechanical stability of the selectin tether.
J Cell Biol.
2001;155:145-156
45.
Yonemura S, Tsukita S, Tshkita S.
Direct involvement of ezrin/radixin/moesin (ERM)-binding membrane proteins in the organization of microvilli in collaboration with activated ERM proteins.
J Cell Biol.
1999;145:1497-1509
46.
Takeuchi K, Sato N, Kasahara H, et al.
Perturbation of cell adhesion and microvilli formation by antisense oligonucleotides to ERM family members.
J Cell Biol.
1994;125:1371-1384
47.
Martin M, Andreoli C, Sahuquet A, Montcourrier P, Algrain M, Mangeat P.
Ezrin NH2-terminal domain inhibits the cell extension activity of the COOH-terminal domain.
J Cell. Biol.
1995;128:1081-1093
48.
Huang L, Wong TYW, Lin RCC, Furthmayr H.
Replacement of threonine 558, a critical site of phosphorylation of moesin in vivo, with aspartate activates F-actin binding of moesin.
J Biol. Chem.
1999;274:12803-12810
© 2002 by The American Society of Hematology.
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
![]() |
A. Zarbock, H. Muller, Y. Kuwano, and K. Ley PSGL-1-dependent myeloid leukocyte activation J. Leukoc. Biol., November 1, 2009; 86(5): 1119 - 1124. [Abstract] [Full Text] [PDF] |
||||
![]() |
H. Oh, E. R. Mohler III, A. Tian, T. Baumgart, and S. L. Diamond Membrane Cholesterol Is a Biomechanical Regulator of Neutrophil Adhesion Arterioscler Thromb Vasc Biol, September 1, 2009; 29(9): 1290 - 1297. [Abstract] [Full Text] [PDF] |
||||
![]() |
J. M. van Gils, J. J. Zwaginga, and P. L. Hordijk Molecular and functional interactions among monocytes, platelets, and endothelial cells and their relevance for cardiovascular diseases J. Leukoc. Biol., February 1, 2009; 85(2): 195 - 204. [Abstract] [Full Text] [PDF] |
||||
![]() |
G. Xu and J.-Y. Shao Human neutrophil surface protrusion under a point load: location independence and viscoelasticity Am J Physiol Cell Physiol, November 1, 2008; 295(5): C1434 - C1444. [Abstract] [Full Text] [PDF] |
||||
![]() |
J. J. Miner, L. Xia, T. Yago, J. Kappelmayer, Z. Liu, A. G. Klopocki, B. Shao, J. M. McDaniel, H. Setiadi, D. W. Schmidtke, et al. Separable requirements for cytoplasmic domain of PSGL-1 in leukocyte rolling and signaling under flow Blood, September 1, 2008; 112(5): 2035 - 2045. [Abstract] [Full Text] [PDF] |
||||
![]() |
S. C. Kerr, C. B. Fieger, K. R. Snapp, and S. D. Rosen Endoglycan, a Member of the CD34 Family of Sialomucins, Is a Ligand for the Vascular Selectins J. Immunol., July 15, 2008; 181(2): 1480 - 1490. [Abstract] [Full Text] [PDF] |
||||
![]() |
A. Zarbock and K. Ley Mechanisms and Consequences of Neutrophil Interaction with the Endothelium Am. J. Pathol., January 1, 2008; 172(1): 1 - 7. [Abstract] [Full Text] [PDF] |
||||
![]() |
A. Urzainqui, G. Martinez del Hoyo, A. Lamana, H. de la Fuente, O. Barreiro, I. M. Olazabal, P. Martin, M. K. Wild, D. Vestweber, R. Gonzalez-Amaro, et al. Functional Role of P-Selectin Glycoprotein Ligand 1/P-Selectin Interaction in the Generation of Tolerogenic Dendritic Cells J. Immunol., December 1, 2007; 179(11): 7457 - 7465. [Abstract] [Full Text] [PDF] |
||||
![]() |
Y. Takai, K. Kitano, S.-i. Terawaki, R. Maesaki, and T. Hakoshima Structural basis of PSGL-1 binding to ERM proteins. Genes Cells, December 1, 2007; 12(12): 1329 - 1338. [Abstract] [Full Text] [PDF] |
||||
![]() |
O. Dwir, V. Grabovsky, R. Pasvolsky, E. Manevich, R. Shamri, P. Gutwein, S. W. Feigelson, P. Altevogt, and R. Alon Membranal Cholesterol Is Not Required for L-Selectin Adhesiveness in Primary Lymphocytes but Controls a Chemokine-Induced Destabilization of L-Selectin Rolling Adhesions J. Immunol., July 15, 2007; 179(2): 1030 - 1038. [Abstract] [Full Text] [PDF] |
||||
![]() |
C. Abbal, M. Lambelet, D. Bertaggia, C. Gerbex, M. Martinez, A. Arcaro, M. Schapira, and O. Spertini Lipid raft adhesion receptors and Syk regulate selectin-dependent rolling under flow conditions Blood, November 15, 2006; 108(10): 3352 - 3359. [Abstract] [Full Text] [PDF] |
||||
![]() |
S.-C. Chen, C.-C. Huang, C.-L. Chien, C.-J. Jeng, H.-T. Su, E. Chiang, M.-R. Liu, C. H. H. Wu, C.-N. Chang, and R.-H. Lin Cross-linking of P-selectin glycoprotein ligand-1 induces death of activated T cells Blood, November 15, 2004; 104(10): 3233 - 3242. [Abstract] [Full Text] [PDF] |
||||
![]() |
W. D. Hanley, D. Wirtz, and K. Konstantopoulos Distinct kinetic and mechanical properties govern selectin-leukocyte interactions J. Cell Sci., May 15, 2004; 117(12): 2503 - 2511. [Abstract] [Full Text] [PDF] |
||||
![]() |
H. Setiadi and R. P. McEver Signal-dependent distribution of cell surface P-selectin in clathrin-coated pits affects leukocyte rolling under flow J. Cell Biol., December 22, 2003; 163(6): 1385 - 1395. [Abstract] [Full Text] [PDF] |
||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| Copyright © 2002 by American Society of Hematology Online ISSN: 1528-0020 | |||||||||