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HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
Attachment of the PSGL-1 cytoplasmic domain to the actin cytoskeleton
is essential for leukocyte rolling on P-selectin
Karen R. Snapp, Christine E. Heitzig, and Geoffrey S. Kansas
P-selectin glycoprotein ligand–1 (PSGL-1)
serves as the leukocyte ligand for Pselectin, 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 PSGL1⌬cyto), were analyzed. Transfectants expressing full-length PSGL-1 rolled well on
P-selectin. In contrast, rolling was almost
completely absent in cells transfected
with PSGL-1⌬cyto, even at low shear.
Importantly, cells expressing truncated
PSGL-1 were able to bind soluble Pselectin and to bind COS cells overexpressing P-selectin, demonstrating that
the P-selectin binding site on the PSGL1⌬cyto transfectants was intact and was
capable of recognizing P-selectin. Impaired rolling by PSGL-1⌬cyto transfectants was not due to alterations in subcellular localization because both wild-type
and truncated PSGL-1 had similar surface
distributions on K562 transfectants. Treatment of cells expressing native PSGL-1
with actin cytoskeletal toxins inhibited
adhesion in a dose-dependent way.
PSGL-1 was associated with the actin
cytoskeleton, and this interaction was
greatly impaired in PSGL-1⌬cyto–
expressing cells. The PSGL-1 cytoplasmic domain interacted selectively with
the ezrin/radixin/moesin (ERM) protein
moesin, but not with other ERM proteins
or several other cytoskeletal linker proteins. Pharmacologic disruption of interactions between moesin and F-actin in
cells expressing PSGL-1 resulted in a
dose-dependent inhibition of rolling on
P-selectin. Thus, attachment of PSGL-1 to
the leukocyte cortical cytoskeleton is essential for leukocyte rolling on P-selectin.
(Blood. 2002;99:4494-4502)
© 2002 by The American Society of Hematology
Introduction
Leukocyte migration is mediated by several groups of adhesion
molecules, including the selectin family. Expression of Lselectin 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 receptorligand interactions, receptor internalization, redistribution, shedding, endocytic sorting, and signal transduction. Among leukocyte
adhesion molecules, leukocyte function-associated antigen–1 (LFA1), 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 ␣-actinin,
paxillin, vinculin, and talin.8-12 In contrast, linkage between CD44,
CD43, ICAM-1, or ICAM-2 and the cytoskeleton is mediated
through the ezrin/radixin/moesin (ERM) family of proteins.13-15
The ERM proteins are part of the band 4.1 superfamily of proteins
and are closely related structurally (⬃75% identity at the amino
acid level, rising to ⬃85% in their amino terminal half). Although
they are often coexpressed, these proteins differ in tissue and
cellular distribution and functional properties.16,17 ERM proteins
have both plasma membrane– and actin filament–binding domains
that allow them to function as linker proteins between the
membrane and the actin cytoskeleton.18 Although these associations have been biochemically documented, their functional consequences are not well understood.
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.
From the Department of Microbiology/Immunology, Northwestern University
Medical School, Chicago, IL.
Reprints: Karen R. Snapp, Northwestern University Medical School,
Department of Microbiology/Immunology, Tarry 6-728, 303 E Superior Ave,
Chicago, IL 60611; e-mail: [email protected].
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.
4494
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.
© 2002 by The American Society of Hematology
BLOOD, 15 JUNE 2002 䡠 VOLUME 99, NUMBER 12
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BLOOD, 15 JUNE 2002 䡠 VOLUME 99, NUMBER 12
THE PSGL-1 CYTOPLASMIC DOMAIN AND CELL ADHESION
4495
Parallel-plate flow assay
Materials and methods
Generation of PSGL-1 complementary DNA truncated in the
cytoplasmic domain
Introduction of a stop codon at position 1053 (residue 348) of the human
PSGL-1 complementary DNA (cDNA) by polymerase chain reaction
(PCR)–based site directed mutagenesis truncated the 69–amino acid
cytoplasmic domain to 4 residues, RLSR. Amplification used an antisense
oligonucleotide 5⬘ TT GGT ACC GGG GTA CAT GTG TCA CTT GCG
GGA GAG 3⬘, which inserted a stop codon at position 348 and maintained a
unique KpnI site (underlined) just downstream from the newly generated
stop codon, and a sense oligonucleotide 5⬘ AAT TTG TCC GTC AAC TAC
CCA GTG 3⬘, which incorporated a unique HincII site (underlined) just
upstream from the cytoplasmic tail. The 156-bp PCR-generated fragment
was gel purified, ligated into the HincII/KpnI site of the pBS.KS/PSGL-1
vector19 and sequenced to confirm the fidelity of the mutagenesis. The
mutant cDNA was subcloned into a modified SP65/RSV vector containing a
puromycin-resistant cassette and used to transfect K562/FucT-VII and
BJAB-FucT-VII cells.
Generation and verification of stable transfectants in
K562/FucT-VII cells
The generation of both K562 and BJAB cells stably expressing FucT-VII
has been described.19,20 Wild-type and PSGL-1⌬cyto cDNAs were used to
stably transfect K562/FucT-VII and BJAB/FucT-VII by electroporation.
Bulk transfectants were drug selected in media containing 2.5 ␮g/mL
puromycin, screened by flow cytometry with the anti–PSGL-1 monoclonal
antibody (mAb) KPL1,21 and cloned by limiting dilution. Individual clones
were screened by flow cytometry with KPL1, and with HECA-452, which
recognizes a reporter epitope associated with FucT-VII enzymatic activity.20,22 FACS analysis was performed on a Becton Dickinson FACSCalibur,
and data were analyzed with CellQuest software (Mountain View, CA). Clones
that had equivalent surface staining of both these mAbs were tested by
semiquantitative reverse transcription-PCR (RT-PCR) analysis7,20 for core-2 ␤1,6
N-acetyglucosaminyltransferase-I (C2GlcNAcT-I) and ␣1,3-fucosyltransferaseVII (FucT-VII) messenger RNA (mRNA) expression to ensure that the selected
clones had equivalent levels of FucT-VII and C2GlcNAcT-I mRNA. Western
blotting of 1% Triton X-100 whole-cell lysates (WCLs, see below) were
generated from each transfectant, run on 6% sodium dodecyl sulfate–
polyacrylamide gel electrophoresis (SDS-PAGE) gels, transferred to nitrocellulose, probed with KPL1 followed by goat antimouse–horseradish peroxidase
(Biosource International, Camarillon, CA), and developed as described below.
Data in this report are representative of multiple experiments with numerous
clones from multiple independent transfections.
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 diethylaminoethyldextran 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.
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
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4496
BLOOD, 15 JUNE 2002 䡠 VOLUME 99, NUMBER 12
SNAPP et al
Immunoprecipitation
Statistics
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
␮L 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.
For statistical analysis of leukocyte rolling assays, the Student t test was
used. Differences were considered statistically significant with P ⬍ .05.
Scanning immunoelectron microscopy
The 106 transfectants expressing either PSGL-1 or PSGL-1⌬cyto were
resuspended in 100 ␮L PBS plus 5% goat serum containing KPL1,
incubated on ice for 15 minutes, rinsed twice, and resuspended in 100
␮L 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 ␮L
PBS plus 0.2% BSA. Approximately 50 ␮L 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 PSGL1⌬cyto distribution using a Hitachi S-900 LVSEM equipped with a
stereoviewer for enhanced spatial resolution.
Results
Generation and characterization of stably transfected
K562/FucT-VII cells expressing PSGL-1 or PSGL-1⌬cyto
Stable transfectants expressing either wild-type PSGL-1 or PSGL1⌬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 PSGL1⌬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⌬cyto, Western blotting also showed a less heterogeneous
banding pattern for both the monomeric and dimeric forms of
Figure 1. Flow cytometric analysis and Western
blotting of PSGL-1 and PSGL-1⌬cyto transfectants.
(A) HL60 cells (top panels) or K562/FucT-VII cells transfected with either PSGL-1 (middle panels) or PSGL1⌬cyto (bottom panels) were stained with either an
isotype-matched negative control (open histograms), the
anti-PSGL-1 mAb, KPL1 (left panels, filled histograms),
or HECA-452, an antibody that recognizes a reporter
epitope associated with FucT-VII activity (right panels,
filled histograms). (B) Western blotting of WCLs made
from K562/FucT-VII transfectants expressing either
PSGL-1 (lane 1) or PSGL-1⌬cyto (lane 2). Bands of the
appropriate molecular weight were seen in lysates from
PSGL-1 (lane 1), and a slight increase in electrophoretic
mobility was associated with truncation of 65 amino acids
(lane 2).
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BLOOD, 15 JUNE 2002 䡠 VOLUME 99, NUMBER 12
THE PSGL-1 CYTOPLASMIC DOMAIN AND CELL ADHESION
4497
HECA-452 (Figure 2A). Both PSGL-1 and PSGL-1⌬cyto were
recognized by HECA-452, suggesting that the glycosylation of
PSGL-1⌬cyto by FucT-VII is not altered by truncation of the
cytoplasmic tail.
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⌬cyto transfectants was examined. K562 transfectants were incubated with P-RIg and analyzed
by flow cytometry (Figure 2B). Under these conditions, both
wild-type and truncated PSGL-1 bound P-RIg, with PSGL-1⌬cyto
transfectants actually exhibiting slightly higher levels of P-RIg
binding. These data show that, as expected, the P-selectin binding
site is intact on PSGL-1⌬cyto.
Rolling of PSGL-1 or PSGL-1⌬cyto transfectants on P-selectin
Figure 2. PSGL-1 and PSGL-1⌬cyto transfectants are appropriately posttranslationally modified and can recognize soluble P-selectin. (A) WCLs were
generated from PSGL-1 (lane 1) or PSGL-1⌬cyto (lane 2) transfectants and
immunoprecipitated with the anti-PSGL-1 mAb KPL1 coupled to Affigel followed by
Western blotting with HECA-452. Arrows on right indicate dimeric (top arrow) and
monomeric (lower arrow) forms of PSGL-1. Both full-length and truncated PSGL-1
were recognized by HECA-452. (B) PSGL-1 and PSGL-1⌬cyto transfectants were
incubated with P-RIg (filled histograms) or second stage only (open histograms) and
analyzed by flow cytometry as described in “Materials and methods.” Both transfectants were capable of binding soluble P-selectin.
PSGL-1⌬cyto compared to wild-type (Figure 1B), suggesting
possible differences in glycosylation. To further investigate the
glycosylation of these molecules, WCLs generated from K562/FucTVII transfectants expressing either PSGL-1 or PSGL-1⌬cyto were
immunoprecipitated with KPL1 followed by Western blotting with
Figure 3. Rolling and adhesion of PSGL-1⌬cyto transfectants on P-selectin is significantly reduced. Total
rolling events in 50 sequential paired images were
acquired, recorded, and analyzed as described in “Materials and methods” for K562/FucT-VII cells (A, 1 of 7
experiments) or BJAB/FucT-VII cells (B, 1 of 7 experiments) expressing either full-length or truncated PSGL-1.
High numbers of rolling events were recorded for HL60
cells and PSGL-1 transfectants, but rolling was greatly
reduced in both PSGL-1⌬cyto transfectants. *P ⬍ .05
versus PSGL-1 transfectants. (C) Total rolling events
were collected as described for panels A and B, but
rolling was analyzed at 3 different shear stresses: 1.5,
1.0, and 0.5 dynes/cm2. Total rolling events in 50 fields
(1 of 3 experiments) were recorded for HL60 cells (open
boxes), K562/FucT-VII/PSGL-1 transfectants (closed triangles), and K562/FucT-VII/PSGL-1⌬cyto transfectants
(closed circles). High numbers of rolling events were
recorded for HL60 cells and PSGL-1 transfectants. In
contrast, rolling events were greatly reduced for PSGL1⌬cyto transfectants at the tested shear rates. (D) HL60
(8.8 ⫾ 2.9 cells/COS cell) and PSGL-1 transfectants
(7.4 ⫾ 2.32 cells/COS cell) bound at similar levels to
COS cells transfected with P-selectin, whereas PSGL1⌬cyto transfectants bound at lower levels (1.7 ⫾ 0.9
cells/COS cell).
Cells were analyzed in a parallel-plate flow system for rolling on
P-selectin. At a shear stress of 1.9 dynes/cm2, cells expressing PSGL-1
rolled extremely well on CHO/P monolayers, comparable with HL60
cells, with more than 1000 rolling events (Figure 3A). In contrast,
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 PSGL1⌬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.
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4498
SNAPP et al
To confirm that the low residual rolling observed in PSGL1⌬cyto transfectants was due to interactions between PSGL-1 and
P-selectin, PSGL-1⌬cyto transfectants were preincubated with
either KPL1 or an isotype control mAb prior to rolling on CHO/P.
Preincubation of PSGL-1⌬cyto transfectants with KPL1 almost
completely inhibited this low level of rolling on P-selectin (126 ⫾ 46
events in the absence of KPL1 versus 3 ⫾ 1 in the presence of 2.5
␮g/mL KPL1).
To determine if the remaining low rolling activity seen with
PSGL-1⌬cyto cells was a function of shear stress, transfectants
were analyzed in a parallel-plate flow system as described above,
but the shear stress was adjusted to either 0.5, 1.0, or 1.5 dynes/cm2.
At all 3 shear rates, cells expressing full-length PSGL-1 rolled
extremely well on P-selectin, with total events slightly increasing at
the lowest shear rate (Figure 3C). In contrast, PSGL-1⌬cyto cells
rolled at greatly reduced levels even at the lowest shear of 0.5
dynes/cm2. Total events for PSGL-1⌬cyto cells at the lowest shear
level were an average of 572 events (⫾ 184) compared to 2343
events (⫾ 427) for transfectants expressing PSGL-1. Similar
results were obtained with BJAB/FucT-VII transfectants expressing either PSGL-1 or PSGL-1⌬cyto (data not shown).
In addition, PSGL-1⌬cyto transfectants rolled with faster
velocities than PSGL-1 transfectants. PSGL-1 transfectants rolled
with very low velocities at all 3 shears, with mean velocities
decreasing slightly as the shear stress decreased, as expected
(ranging from 2.68 ␮m/s at 0.5 dynes/cm2 to 4.21 ␮m/s at 1.5
dynes/cm2). In contrast, PSGL-1⌬cyto mean velocities were significantly higher than PSGL-1 transfectants. At a shear stress of 0.5
dynes/cm,2 PSGL-1⌬cyto transfectants rolled with a mean velocity
of 23.31 ␮m/s (⫾ 12.76). In addition to these large differences in
rolling velocities, the rolling patterns of these 2 cell lines were very
distinct. PSGL-1 transfectants attached to the monolayer and rolled
at a consistent low velocity across the entire filed of view. In
contrast, PSGL-1⌬cyto transfectants attached briefly, rolled with
one or 2 long “steps,” and detached. In additional experiments, the
shear stress was turned off and PSGL-1⌬cyto cells were allowed to
settle on the CHO/P monolayer. When the shear stress was turned
back on, the settled PSGL-1⌬cyto transfectants still rapidly
detached without rolling (data not shown). These data show that the
rolling defect associated with truncation of the PSGL-1 cytoplasmic tail is not a function of the level of shear stress (0.5-1.9
dynes/cm2), and cannot be overcome by eliminating the attachment step.
To further characterize interactions between PSGL-1⌬cyto and
P-selectin, binding of the transfectants to COS cells overexpressing
P-selectin was evaluated. Interactions between PSGL-1⌬cyto and
P-selectin were easily detectable in this low shear assay, but at
BLOOD, 15 JUNE 2002 䡠 VOLUME 99, NUMBER 12
levels approximately 4-fold lower compared to wild-type PSGL-1
(Figure 3D). These data recapitulate the low level of activity
observed at 0.5 dynes/cm2 in the flow assay (Figure 3C), and
indicate that the P-selectin binding domain of PSGL-1⌬cyto
transfectants can recognize P-selectin under low shear conditions,
but that these interactions are compromised to a greater degree
under the more physiologic conditions of the parallel-plate
flow assay.
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⌬cyto transfectants, we hypothesized that truncation of the cytoplasmic tail disrupted essential
interactions with the actin cytoskeleton. We used a combined
detergent fractionation and flow cytometric assay, which has
previously been used to characterize interactions between the
cytoskeleton and L-selectin, major histocompatibility complex
class II, and several other cell surface molecules,23-25 to determine
if PSGL-1 was physically associated with the actin cytoskeleton.
After exposure to detergent containing buffer, high levels of KPL1
staining were largely retained on cells transfected with PSGL-1,
demonstrating that the cytoplasmic tail of PSGL-1 interacts with
the actin cytoskeleton (Figure 5, left side). In contrast, sharply
reduced staining was observed for PSGL-1⌬cyto transfectants,
indicating that interactions with the actin cytoskeleton were greatly
compromised (Figure 5, right side). Thus, the PSGL-1 cytoplasmic
tail interacts with the actin cytoskeleton, and the elimination of
these interactions, either by truncation of the PSGL-1 cytoplasmic
domain or by pharmacologic disruption of the actin cytoskeleton,
dramatically reduced rolling on P-selectin.
Figure 4. Disruption of the actin cytoskeleton in
PSGL-1 transfectants blocks rolling on P-selectin.
(A) PSGL-1 transfectants were treated with either 10, 30,
or 100 ␮M cytochalasin B (open squares), or 5, 50, or 100
␮M latrunculin B (filled squares) and analyzed for rolling
on CHO/P. To facilitate comparison between experiments, rolling events were normalized to the control
group, which consisted of cells treated with vehicle alone
(dimethyl sulfoxide). One of 3 similar experiments.
(B) Expression of PSGL-1 on cells treated with 50 ␮M
latrunculin B (open histogram) and untreated (filled histogram) PSGL-1 transfectants. Similar results were obtained when cells were treated with 100 ␮M cytochalasin
B (data not shown).
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BLOOD, 15 JUNE 2002 䡠 VOLUME 99, NUMBER 12
THE PSGL-1 CYTOPLASMIC DOMAIN AND CELL ADHESION
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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
Figure 5. Association of the cytoplasmic domain of PSGL-1 with the actin
cytoskeleton is disrupted in PSGL-1⌬cyto transfectants. The detergent fractionation assay was performed as described in “Materials and methods.” High levels of
KPL1 staining remained in the detergent-insoluble pellet associated with PSGL-1
transfectants (left panels), but this staining was greatly reduced in PSGL-1⌬cyto cells
(right panels). Top, untreated cells; bottom, detergent-treated cells.
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
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 crosslinking 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 Factin.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 ␮M (608 ⫾ 101 events versus 1190 ⫾ 220 in
vehicle-treated control HL60 cells) and nearly completely eliminated at concentrations of 10 ␮M (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
It has been reported that PSGL-1 is localized to the microvilli of
normal human neutrophils.34 To determine if truncation of the
PSGL-1 cytoplasmic domain altered its distribution on K562 cells,
we examined PSGL-1 and PSGL-1⌬cyto transfectants by scanning
Figure 6. The PSGL-1 cytoplasmic tail interacts with moesin. (A) GST fusion proteins were generated, which incorporated the PSGL-1 cytoplasmic domain (lane 3), or GST
only (negative control, lane 2), and were incubated with HL60 WCLs. The blot was probed with a rabbit pAb to moesin, which also recognizes ezrin. GST fusion proteins
expressing the cytoplasmic tail of PSGL-1 (lane 3) affinity captured moesin, but not ezrin. (B) Coimmunoprecipitations of WCLs from PSGL-1 or PSGL-1⌬cyto transfectants
were performed with the anti-PSGL-1 mAb KPL1 coupled to Affigel followed by Western blotting with an antimoesin mAb (left panel) or KPL-1 (right panel). Interactions with
moesin were detected in either untreated WCLs (left panel, lane 1) or immunoprecipitates from PSGL-1 transfectants (left panel, lane 2), but not PSGL-1⌬cyto transfectants
(left panel, lane 3). Probing of either untreated WCLs (right panel, lane 1) or immunoprecipitates from PSGL-1 (right panel, lane 2) or PSGL-1⌬cyto (right panel, lane 3) with
KPL1 revealed either full-length (lane 1 and lane 2) or truncated (lane 3) PSGL-1.
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4500
SNAPP et al
Figure 7. Treatment of cells with staurosporine inhibits rolling on P-selectin.
HL60 cells were incubated with increasing concentrations of staurosporine as
described in “Materials and methods.” Total rolling events at 1.9 dynes/cm2 were
collected and analyzed as described for Figure 2. Staurosporine caused a dosedependent decrease in rolling on P-selectin (1 of 3 experiments).
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.
Discussion
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⌬cyto bound poorly to COS cells overexpressing P-selectin in low-shear assays and rolled poorly on P-selectin
under defined shear flow compared to PSGL-1 transfectants. This
impaired adhesion was not due to alterations in the PSGL-1
ectodomain, because soluble P-selectin and HECA-452 recognized
PSGL-1⌬cyto as well as full-length PSGL-1, indicating that the
P-selectin binding site was intact. Full-length PSGL-1 was physically associated with the actin cytoskeleton, and these interactions
were greatly reduced in PSGL-1⌬cyto transfectants. Both pharmacologic disruption of the pre-existing actin cytoskeleton and
pharmacologic inhibition of interactions between moesin and
F-actin disrupted the rolling of cells expressing wild-type PSGL-1
in a dose-dependent way. Collectively, these data indicate that the
BLOOD, 15 JUNE 2002 䡠 VOLUME 99, NUMBER 12
highly conserved PSGL-1 cytoplasmic tail interacts with the
pre-existing intact actin cytoskeleton, and that these associations
are essential for rolling on P-selectin.
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 ␮M
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 ␮M) cytochalasin B and only modest effects with low dose (5
␮M) 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 ␮M) 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 ␣-actinin, not ERM proteins.11 Thus, both L-selectin
Figure 8. Cell surface distribution of PSGL-1 and PSGL-1⌬cyto on transfected
K562 cells is indistinguishable. High-power (original magnification ⫻ 8000) photomicrographs of PSGL-1 transfectants (left panel) and PSGL-1⌬cyto transfectants
(right panel). Both transfectants showed a similar and uniform distribution of KPL1
immunogold particles on the microvilli and cell body. Scale bar is 860 nm.
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BLOOD, 15 JUNE 2002 䡠 VOLUME 99, NUMBER 12
THE PSGL-1 CYTOPLASMIC DOMAIN AND CELL ADHESION
and PSGL-1 require interactions between their cytoplasmic domains and an intact actin cytoskeleton, but not de novo actin
polymerization, for leukocyte rolling. Our findings therefore suggest a new paradigm for leukocyte migration: that the function of
leukocyte “rolling receptors” requires interactions with the preexisting actin cortical network, and therefore that ligand recognition is necessary but not sufficient for cell adhesion.
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⌬cyto (Figure 8B), and a similar random
pattern of distribution was also found with BJAB cells transfected
with either PSGL-1 or PSGL-1⌬cyto (data not shown). Thus, the
significant decreases in rolling by PSGL-1⌬cyto transfectants
cannot be attributed to improper cellular localization. Whether
localization of PSGL-1 to microvilli is essential for leukocyte
rolling has yet to be definitively determined.
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⌬cyto transfectants (Figure 6B). ERM proteins exist in
both active and inactive forms within cells, with inactivation
resulting from an intramolecular association of the N- and Cterminal halves of the protein.27,28,47 The generation and maintenance of the active cross-linking form of moesin, which is essential
for interactions with F-actin, requires phosphorylation of
Thr558.32,33,48 Phosphorylation at this site is inhibited by treatment
4501
with staurosporine, which converts moesin into its inactive,
unphosphorylated form and prevents it from interacting with
F-actin.33 We observed a dose-dependent decrease in rolling on
P-selectin after exposure to staurosporine, which supports a role for
moesin in PSGL-1–mediated adhesion (Figure 7). Staurosporine
also inhibits phosphorylation of other cellular proteins including
ezrin and radixin,32,33 so it is possible that staurosporine treatment
might be disrupting these interactions and therefore inhibits
PSGL-1–mediated adhesion. However, we were unable to detect
interactions between the PSGL-1 cytoplasmic tail and ezrin by
either GST binding assays or immunoprecipitation. In addition,
neutrophils express no detectable ezrin by Western blotting (data
not shown) and freshly isolated neutrophils roll extremely well on
P-selectin, suggesting that ezrin is not essential for PSGL-1–
mediated rolling on P-selectin. These data further support a role for
interactions between PSGL-1, moesin, and F-actin in rolling on
P-selectin, but do not rule out a role for other as yet unidentified
cytoplasmic proteins in leukocyte rolling.
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.
Acknowledgments
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⌬cyto distribution on K562/FucT-VII transfectants.
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2002 99: 4494-4502
doi:10.1182/blood.V99.12.4494
Attachment of the PSGL-1 cytoplasmic domain to the actin cytoskeleton is
essential for leukocyte rolling on P-selectin
Karen R. Snapp, Christine E. Heitzig and Geoffrey S. Kansas
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