Association of Ezrin with Intercellular Adhesion Molecule-1
and -2 (ICAM-1 and ICAM-2)
REGULATION BY PHOSPHATIDYLINOSITOL 4,5-BISPHOSPHATE*
Leena
Heiska
,
Kaija
Alfthan§,
Mikaela
Grönholm
,
Pekka
Vilja¶,
Antti
Vaheri
, and
Olli
Carpén
**
From the Departments of
Pathology and
Virology, University of Helsinki, Haartman Institute, 00014 Helsinki, § VTT Biotechnology and Food Research,
FIN-02044 VTT (Espoo), and ¶ Medical School, University of
Tampere, 33101 Tampere, Finland
 |
ABSTRACT |
Ezrin is a cytoplasmic linker molecule
between plasma membrane components and the actin-containing
cytoskeleton. We studied whether ezrin is associated with intercellular
adhesion molecule (ICAM)-1, -2, and -3. In transfected cells, ICAM-1
and ICAM-2 colocalized with ezrin in microvillar projections, whereas
an ICAM-1 construct attached to cell membrane via a
glycophosphatidylinositol anchor was uniformly distributed on the
cell surface. An interaction of ICAM-2 and ezrin was seen by affinity
precipitation, microtiter binding assay, coimmunoprecipitation, and
surface plasmon resonance methods. The calculated
KD value was 3.3 × 10
7
M. Phosphatidylinositol 4,5-bisphosphate
(PtdIns(4,5)P2) induced an interaction of ezrin and ICAM-1
and enhanced the interaction of ezrin and ICAM-2, but ICAM-3 did not
bind ezrin even in the presence of PtdIns(4,5)P2.
PtdIns(4,5)P2 was shown to bind to cytoplasmic tails of
ICAM-1 and ICAM-2, which are the first adhesion proteins demonstrated
to interact with PtdIns(4,5)P2. The results indicate an
interaction of ezrin with ICAM-1 and ICAM-2 and suggest a regulatory
role of phosphoinositide signaling pathways in regulation of ICAM-ezrin
interaction.
 |
INTRODUCTION |
Ezrin is a member of the
ERM1 (ezrin/radixin/moesin)
family of proteins, which are involved in linking the actin-containing cytoskeleton to the plasma membrane (1, 2). Most epithelial and
lymphoid cells express ezrin (3-5). It is localized subcellularly underneath the plasma membrane in microvilli, at the leading edge and
at sites of cell-cell contacts (3, 4). The protein consists of an
NH2-terminal domain with sequence similarity to the protein 4.1 superfamily, an
-helical region, and an actin-binding COOH terminus (6-9). The COOH-terminal and NH2-terminal domains
self-associate by head-to-tail joining. The F-actin binding domain is
masked in the native ezrin molecule, possibly by the amino-terminal
domain (10), which enables regulatory control of ezrin's cytoskeletal associations. Ezrin can directly bind the cell surface glycoprotein CD44 (11). The NH2-terminal part of ezrin binds
phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) (12),
and PtdIns(4,5)P2 enhances the interaction between ezrin
and CD44, possibly through the regulation by the small GTP-binding
protein Rho (13).
Intercellular adhesion molecules (ICAMs) mediate leukocyte and
endothelial binding to
2-integrins (CD11/18). ICAM-1 is
widely expressed at low levels (14) and is strongly up-regulated by various inflammatory cytokines and on lymphoblasts (15). ICAM-2 is
expressed constitutively on lymphocytes, monocytes, platelets, and most
endothelial cells (16-19) and may be important for leukocyte recirculation in normal uninflamed tissues (20). ICAM-3 is expressed on
resting leukocytes and in tumors and has a role in the initiation of
the immune response (21-24).
The function of ICAMs is modulated by the association of cytoplasmic
domains with the actin-containing cytoskeleton. We have shown that
ICAM-1 and ICAM-2 interact with the cytoskeletal protein
-actinin
(25, 26). Moreover, ezrin is involved in regulation of the subcellular
distribution and adhesive function of ICAM-2 (27), but the biochemical
basis of this interaction is unclear. Here, we have studied whether
ezrin is directly associated with ICAM-1, ICAM-2, or ICAM-3. As
PtdIns(4,5)P2 regulates cytoskeletal assembly and linkage
to plasma membrane via interactions with many actin-binding proteins
(28, 29), we also studied how PtdIns(4,5)P2 affects these
interactions.
 |
EXPERIMENTAL PROCEDURES |
Antibodies and Lipids--
MAb UHP-9 (30) was used to detect
ICAM-1, and ICAM-2 was detected with mAb from Alexis, San Diego, CA.
Anti-ezrin polyclonal antibody (4), and mAb 3C12 (31) have been
described. Rabbit polyclonal anti-LexA antibody was a kind gift of Dr.
Erica Golemis, Fox Chase Cancer Center, Philadelphia, PA (32), and mAb
12CA5 specific for the hemagglutinin antigen (HA) epitope tag was from Boehringer Mannheim GmbH, Mannheim, Germany. Mouse X63 mAb and preimmune rabbit sera were used as controls. PtdIns(4,5)P2
(Sigma) was sonicated with a probe sonicator to a stock solution of 1 mg/ml in HE (20 mM HEPES, pH 7.4, 0.2 mM EGTA)
buffer. Polyphosphoinositide stocks were frozen in liquid nitrogen,
kept at
70 °C, and used within 2 days after thawing. Before use,
the lipids were further sonicated in a bath sonicator for 30 min.
Phosphatidylcholine (PC) and phosphatidylserine (PS, Sigma) were
solutions in chloroform/methanol, and dried under nitrogen stream
before use. Mixed vesicles of PC and other phospholipids were made by
adding a solution of phosphoinositide into an Eppendorf tube lined with
dried PC, followed by extensive sonication. Large multilamellar
liposomes were prepared according to Ref. 12. All the incubations of
lipids with proteins were performed under nitrogen.
Cell Transfections and Indirect Immunofluorescence
Microscopy--
COS-1 cells were transfected with ICAM-1 cDNA
subcloned into CDM8 expression vector (33) or with a construct that
replaces the transmembrane and cytoplasmic domains with a
glycophosphatidylinositol (GPI) anchor (25). Transfections were
performed with the DEAE-dextran method as described (34). Chinese
hamster ovary cells stably expressing ICAM-2 (35) were transfected with
full-length ezrin cDNA expression vector (27) using LipofectAMINE
(Life Technologies, Inc.). The CDM8 expression vector alone was used as
a control for transfections. The transfected cells were grown on glass
coverslips for 48 h before fixation with 3.5% paraformaldehyde in
PBS at 4 °C for 10 min. The monoclonal antibodies were used at 10 µg/ml in PBS. After washes with PBS, the coverslips were reacted with fluorescein isothiocyanate-conjugated goat F(ab')2
anti-mouse IgG (Immunotech, Marseille, France). The cells were then
permeabilized with 0.1% Triton X-100 in PBS, stained with rabbit
antiserum against ezrin (1:50) or a control serum followed by staining
with tetramethylrhodamine isothiocyanate-conjugated goat
F(ab')2 anti-rabbit IgG (1:40) (Cappel, Durham, NC). The
coverslips were viewed with a Zeiss epifluorescence microscope (Carl
Zeiss, Thornwood, NY).
Peptide Synthesis and Immobilization--
Three peptides
of the following sequences were synthesized: IC1 peptide, RQRKI
KKYRLQQAQKGTPMKPNTQATPP; IC2 peptide, QHLRQQRMGTYGVRAAWRRLPQAFRP; and
IC3 peptide, REHQRSGSYHVREESTYLPLTSMQPTEAMGEEPSRAE, encompassing the predicted cytoplasmic domains of ICAM-1, ICAM-2, and ICAM-3, respectively. For microtiter well binding assays, the IC1 and IC3
peptide were biotinylated at the NH2 terminus via a
six-carbon spacer. In surface plasmon resonance (SPR) studies, IC2
peptide was replaced by the peptide ICK2, in which the
NH2-terminal glutamine of IC2 was deleted and histidine
replaced by lysine. The peptides were synthesized by Fmoc
(N-(9-fluorenyl)methoxycarbonyl) chemistry on a SMPS 350 multiple peptide synthesizer (Zinsser Analytic, Frankfurt, Germany).
The IC1, IC2, and IC3 peptides were coupled to CNBr-activated Sepharose
4B (Pharmacia Biotech, Uppsala, Sweden) at a concentration of 2 mg/ml
according to the protocol provided by the manufacturer. The coupling
efficiency was 93-99%.
Purification of Placental and Recombinant Proteins--
The
purification of placental ezrin was done essentially as described in
Ref. 36. The baculovirus ezrin expression construct was a kind gift of
P. Mangeat (Université Montpellier II, Montpellier, France) and
is described in Ref. 37. Recombinant ezrin was purified as described in
(13). The vinculin PtdIns(4,5)P2 binding glutathione S-transferase fusion construct, kindly provided by D. Critchley (University of Leicester, Leicester, United Kingdom), and its purification is described in Refs. 38 and 39. The glutathione S-transferase domain of recombinant vinculin was cleaved off
by thrombin.
Affinity Chromatography and Affinity Precipitation
Assays--
The ICAM-1 and ICAM-3 peptides were coupled to Sepharose
beads, and placental lysate was passed through the peptide-Sepharose column as described in detail in Ref. 25. For affinity precipitation, 2 µg of placental ezrin was incubated in the presence or absence of
lipids (50 µg/ml) in HKE buffer (20 mM HEPES, pH 7.4, 130 mM KCl, 0.2 mM EGTA) for 30 min at room
temperature. 30 µl of peptide-Sepharose 1:1 slurry was added, and the
mixture was shaken for 2 h. The beads were pelleted, the
supernatant removed and the beads washed in HKE buffer. Bound proteins
were eluted from the beads by boiling in Laemmli sample buffer and
analyzed in 8% SDS-PAGE gels stained with Coomassie Blue.
Enzyme-linked Immunosorbent Binding Assay--
Biotin-conjugated
ICAM-peptides were coated onto streptavidin-linked microtiter wells
(Labsystems, Helsinki, Finland) at a concentration of 0.01 mg/ml in
PBS. The wells were blocked with 2% bovine serum albumin in PBS for
2 h at 37 °C. Recombinant ezrin (500 ng) was incubated with
lipids (10 µg/ml) under nitrogen in HKE buffer at room temperature
for 30 min. The ezrin/lipid mixture was allowed to react with the
peptides in microtiter wells for 2 h at room temperature in a
plate shaker. The wells were washed extensively with HKT (20 mM HEPES, pH 7.4, 130 mM KCl, 0.05% Tween 20)
buffer and incubated with the primary antibody, mAb 3C12 at 1:6000
dilution in HKT, and the secondary antibody, rabbit horseradish peroxidase-conjugated anti-mouse (Dako, Copenhagen, Denmark), 1:3000
dilution, for 1 h each. After washes, the substrate reaction was
performed with o-phenylenediamine dihydrochloride (Sigma) and the absorbances were measured by Multiscan Mcc/340 (Labsystems) at
the wavelength of 492 nm. Specific values were calculated by subtracting the background absorbance values (<0.05 absorbance units)
from the total values. For inhibition assays, a dilution series of
soluble, non-biotinylated IC1 peptide was added to the reaction mixture
containing ezrin and PtdIns(4,5)P2.
Ezrin and ICAM Fusion Protein Constructs and the
Coimmunoprecipitation Assay of Yeast Lysates--
The cDNAs
encoding the cytoplasmic domains of ICAM-1 (amino acids 478-505) and
ICAM-2 (amino acids 229-254) or amino- and carboxyl-terminal domains
(amino acids 1-309 and 278-585, respectively) of ezrin were
introduced to the pEG202 yeast expression vector, which contains a LexA
binding site (40). The same ICAM-cDNAs and the full-length ezrin
cDNA were introduced to the pJG4-5 yeast expression vector, which
contains the HA epitope tag (41). The sequences of all constructs were
verified by sequencing. BOY yeast cells cotransformed with the EG202
fusion construct and the JG4-5 fusion construct were grown overnight
at 30 °C, washed once with PBS, and lysed by vortexing in the
presence of 1 ml of acid-washed glass beads (Sigma) in 0.5 ml of ELB
buffer (50 mM HEPES, pH 7.4, 150 mM NaCl, 5 mM EDTA) supplemented with 1% Nonidet P-40 and protease
inhibitors. The debris was removed by centrifugation, and the
supernatant diluted with ELB plus 0.1% Nonidet P-40 to a final Nonidet
P-40 concentration of 0.5%. The protein concentration was measured at
A280 nm. 250 µg of total protein lysate was
incubated in the presence or absence of 5 µg/ml
PtdIns(4,5)P2 for 20 min at room temperature. The 12CA5
(anti-HA) antibody was added at a dilution of 1:1000 and incubated for
30 min on ice, followed by a protein A-Sepharose bead incubation for
2 h. After washes with ELB and 0.1% Nonidet P-40, the bound
proteins were eluted by boiling in Laemmli sample buffer, and analyzed
by subsequent SDS-PAGE and immunoblotting.
Immunoblot Analysis--
The samples were separated by 10%
SDS-PAGE, blotted onto nitrocellulose sheets, and blocked overnight
using 5% nonfat milk powder in PBS plus 0.1% Tween 20. Primary
antibodies were incubated for 1 h. Anti-LexA antibody was diluted
1:5000 and 12CA5 mAb 1:1500 in PBS plus 0.1% Tween 20. As secondary
antibodies, either sheep peroxidase-conjugated anti-rabbit IgG or sheep
peroxidase-conjugated anti-mouse IgG (Boehringer Mannheim GmbH,
Mannheim, Germany) was used at a 1:1500 dilution for 30 min. The bound
antibodies were detected by enhanced chemiluminescence (Boehringer).
The comparison of the immunoprecipitates was done by densitometric
analysis option of NIH Image program.
SPR Assays--
Real-time analysis of the binding between ezrin
and ICAM-peptides was performed with a BIAcore 1000TM
(BIAcore AB, Uppsala, Sweden). Peptides were immobilized on a sensor
chip according to the manufacturer's protocol using the Amine Coupling
Kit (BIAcore AB). ICAM-1 and ICAM-2 peptides (IC1 and ICK2) were
diluted in 10 mM phosphate buffer (pH 7.0), and ICAM-3
peptide (IC3) was diluted in 10 mM acetate buffer (pH 4.0) to 200 µg/ml. The resonance units (RUs) for immobilized ICAM-1, ICAM-2, and ICAM-3 peptides were 2300, 2500, and 1800 RU, respectively. Ezrin was diluted in the running buffer (HKE) to 2.9 µM,
and the solution was run over the peptide surfaces in the absence and presence of 9 µM PtdIns(4,5)P2 micelles or
100 µg/ml mixed vesicles. Typically, the lipids were sonicated and
preincubated with ezrin for 30 min at room temperature. In some
experiments, the preincubation was omitted and
PtdIns(4,5)P2 (4.5 µM) was first injected
over the peptide surface, followed by an injection of ezrin, vinculin fragment, or bovine serum albumin (1.45 µM). All samples
were run over a control surface (activated and blocked surface). The measurements were performed at 25 °C under a constant flow of 5 µl/min. Kinetic constants for the interaction between the ICAM-2 peptide and ezrin were determined by immobilizing 1224 RU of the peptide on the sensor surface and injecting 2.9 µM ezrin
with the flow rate of 15 µl/min at 25 °C over the ICAM-2 peptide
surface. The apparent association rate (ka) and
dissociation rate (kd) constants for the interaction
were determined from three parallel runs, and the data were analyzed
according to a 1:1 interaction model using the BIAevaluation 2.1 software supplied by the manufacturer. The apparent dissociation
constant (KD) was calculated from the ratio
kd/ ka.
 |
RESULTS |
Colocalization of Ezrin, ICAM-1, and ICAM-2--
Earlier studies
have shown that transfection of ezrin into thymoma cells redistributes
ICAM-2 on the cell surface and renders them sensitive for killer cell
lysis (27). Ezrin also colocalizes with ICAM-2 in these cells,
suggesting an interaction between the proteins. To extend the
colocalization studies, we transfected wild type ICAM-1, and an
engineered ICAM-1 form in which the cytoplasmic and transmembrane
region is replaced with a GPI anchor, to COS-1 cells and compared their
distribution to ezrin by indirect immunofluorescence microscopy.
Transfected ICAM-1-wt was concentrated in microvilli where it perfectly
colocalized with endogenous ezrin (Fig.
1A). In contrast, the
ICAM-1-GPI construct, which is attached to the cell membrane via a
lipid, showed no preferential localization but was uniformly
distributed on the cell surface (Fig. 1A). No apparent
codistribution with ezrin was detected. We also analyzed the
colocalization of transfected ezrin in adherent Chinese hamster ovary
cells that stably express human ICAM-2. The colocalization of these
proteins was high (Fig. 1B), concentrating at microvillar structures.

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Fig. 1.
Distribution of ICAM-1 or ICAM-2 and ezrin in
transfected cells. A, COS-1 cells were transfected
either with a wild-type ICAM-1 cDNA (upper
panel) or with a GPI-ICAM-1 cDNA construct that replaces
the transmembrane and cytoplasmic domains of ICAM-1 with a GPI anchor
(lower panel). The cells were fixed, and ICAM-1
was visualized with specific mAb and FITC-conjugated goat anti-mouse
IgG (left) and endogenous ezrin with a rabbit anti-ezrin
antibody and TRITC-conjugated goat anti-rabbit IgG (right).
Note the punctate microvillar staining of wild type ICAM-1 in the
upper panel and its colocalization with ezrin. In contrast,
the GPI-anchored ICAM-1 is uniformly distributed on the cell surface
and lacks colocalization with ezrin. Bar, 10 µm.
B, Chinese hamster ovary cells stably expressing transfected
ICAM-2 were cotransfected with an ezrin cDNA, fixed and
immunostained for ICAM-2 (right) and ezrin (left)
as described in A. ICAM-2, in analogy with ICAM-1, shows a
punctate microvillar staining and colocalization with ezrin.
Bar, 5 µm.
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Binding of Ezrin to ICAM Cytoplasmic Domain Peptides--
When
placental lysate was passed over a column containing ICAM-1 cytoplasmic
peptide as an affinity matrix, only a few polypeptides were retained to
the peptide beads. One of the most prominent proteins eluted from the
column migrated at a molecular mass of 75 kDa and reacted with an
ezrin-specific antiserum (Fig.
2A), indicating a linkage
between ICAM-1 and ezrin. The eluate did not contain several other
cytoskeletal components, talin, vinculin, and spectrin, which were
immunoblotted as a control (data not shown) (25). In a similar
experiment, in which ICAM-3 cytoplasmic peptide was used instead of
ICAM-1, the profile of the bound peptides was different (Fig.
2A, lane 4 versus lane 1)
and ezrin was not detected among the proteins eluted from the
column.

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Fig. 2.
Binding of ezrin to ICAM-1 or ICAM-3
cytoplasmic domain peptide by affinity precipitation and microtiter
assay. A, placental lysate was passed through an
immobilized ICAM-1 or ICAM-3 cytoplasmic peptide column. The bound
proteins were eluted by soluble peptide and separated in SDS-PAGE.
Lanes 1-3, eluted material of ICAM-1 peptide column,
visualized by silver staining (lane 1), immunoblotted with a
control antibody X63 (lane 2) or with an ezrin-specific mAb
3C12 (lane 3). Lanes 4-6, eluted material of
ICAM-3 peptide column, visualized by silver staining (lane
4), or immunoblotted with the ezrin mAb 3C12 (lane 6).
Lane 5 shows the input lysate of the ICAM-3 peptide column,
immunoblotted with 3C12. B, streptavidin-linked microtiter
wells were coated with biotinylated ICAM-1 or ICAM-3 cytoplasmic
peptides and incubated with ezrin in the presence of indicated lipids.
PIP2, PtdIns(4,5)P2). After washes,
bound ezrin was detected by an ezrin-specific antibody and a secondary,
horseradish peroxidase-conjugated antibody followed by a colorimetric
substrate reaction. C, inhibition of ezrin binding to ICAM-1
peptide-coated microtiter well in the presence of
PtdIns(4,5)P2 by soluble, non-biotinylated ICAM-1 peptide.
The soluble peptide was added to the ezrin/lipid mixture in indicated
amounts prior to reaction with the immobilized ICAM-1 peptide.
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To study whether a direct association exists between ezrin and ICAM-1,
we purified placental ezrin and analyzed its binding to
streptavidin-linked microtiter wells coated with biotinylated ICAM-1
peptide. ICAM-1 peptide bound a small amount of ezrin in the absence of
phospholipids (Fig. 2B). The binding was not affected when
ezrin had been preincubated with PC or PS, but was markedly increased
when ezrin had been preincubated with PtdIns(4,5)P2. The
binding in the presence of PtdIns(4,5)P2 was inhibited by soluble non-biotinylated ICAM-1 peptide in a
concentration-dependent manner (Fig. 2C). In
contrast, ezrin did not bind the ICAM-3 cytoplasmic peptide in the
presence or absence of phosphoinositides (Fig. 2B).
Another peptide-based approach was used to study the interaction of
ICAM-2 and ezrin and the effect of PtdIns(4,5)P2 on ezrin association to both ICAM-1 and ICAM-2. Synthetic peptides encompassing the cytoplasmic domains of ICAM-1, -2 and -3 were coupled to Sepharose beads and incubated with ezrin in the presence or absence of
PtdIns(4,5)P2 or PC. In these experiments (Fig.
3B) purified ezrin bound to ICAM-2, but not to ICAM-1 peptide beads in the absence of
phospholipids. PC did not affect the interaction, but incubation with
PtdIns(4,5)P2 induced ezrin binding to ICAM-1 and
remarkably increased the interaction with ICAM-2. ICAM-3 cytoplasmic
peptide did not bind ezrin even in the presence of
PtdIns(4,5)P2. As a control, we purified recombinant PtdIns(4,5)P2-binding fragment (amino acids 881-1066) of
vinculin (38, 39), a cytoskeletal component of focal adhesions with no
apparent cellular colocalization with ICAMs. The fragment was able to
bind to PtdIns(4,5)P2-containing large multilamellar
liposomes in a manner comparable with purified ezrin (Fig.
3A). However, the recombinant vinculin did not bind to
ICAM-peptide beads, and the presence of PtdIns(4,5)P2 did
not induce a significant binding to beads as compared with ezrin
binding (Fig. 3C).

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Fig. 3.
Binding of ezrin to ICAM-1, ICAM-2, and
ICAM-3 peptide-Sepharose. A, the upper panel
shows Coomassie staining of the purified placental ezrin and
recombinant vinculin fragment used in the experiments. The
arrows indicate ezrin and vinculin, respectively. In
lower panel, both proteins were incubated with large
multilamellar liposomes containing PC (left) or 4:1
PC:PtdIns(4,5)P2 (PIP2)
(right). The liposomes were pelleted, and the proteins
retaining in pellet (P) or supernatant (S) were
visualized by Coomassie staining of SDS gels. B, the ICAM-1,
ICAM-2, and ICAM-3 peptides (IC1, IC2, and
IC3, respectively) were coupled to Sepharose beads and
allowed to react with purified ezrin in the presence of indicated
lipids. The supernatant was collected, the beads washed, and bound
ezrin eluted by boiling in Laemmli buffer. Ezrin was detected by
Coomassie staining of SDS-polyacrylamide gels. , no lipids added;
PIP2, PtdIns(4,5)P2.
Supernatants from the peptide beads (representing unbound protein)
contain one fourth of the total material. C, the ICAM-1 and
ICAM-2 peptide Sepharose beads were reacted with vinculin
PtdIns(4,5)P2 binding fragment and bound protein detected
as in B. The right lane shows the intensity of
the input material in the gel.
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A real-time analysis of the binding between ezrin and ICAM peptides was
performed with the SPR-based method. The peptides were immobilized on
sensor chips, and ezrin or ezrin/PtdIns(4,5)P2 mixture was
run over the peptide-containing surfaces. Fig.
4 (A and B) shows
overlaid sensorgrams in the absence and presence of added phospholipid,
respectively. Analysis of the binding curves of several experiments
indicated an interaction between immobilized ICAM-2 peptide and ezrin,
whereas no binding to ICAM-1 and ICAM-3 peptides compared with the
control surface (activated and blocked sensor chip) was observed. When
ezrin was preincubated with micellar PtdIns(4,5)P2, it
bound both to ICAM-1 and ICAM-2 peptides, but not to ICAM-3. Ezrin
binding to ICAM-2 was enhanced by the presence of
PtdIns(4,5)P2. The apparent ka and
kd values determined for the interaction between the
ICAM-2 peptide and ezrin in the absence of phospholipids were 2.9 ± 0.63 × 103 M
1
s
1 and 9.5 ± 0.85 × 10
4
s
1, respectively. The apparent KD
calculated was 3.3 × 10
7 M. No
significant differences in the kinetic constants were observed, when
about 5-fold less of ICAM-2 peptide (237 RU) was immobilized on the
sensor surface.

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Fig. 4.
Binding of ezrin to ICAM-1 and ICAM-2
peptides studied by surface plasmon resonance. ICAM-1, ICAM-2, and
ICAM-3 cytoplasmic peptides (IC1, ICK2, and
IC3, respectively) were immobilized on a sensor chip, and
purified ezrin was injected on the peptide surfaces under a constant
flow. A, overlaid sensorgrams of ICAM-1, ICAM-2, and ICAM-3
peptide surfaces and a control surface (activated and blocked sensor
surface). Ezrin (2.9 µM) was injected in the absence of
PtdIns(4,5)P2. B, overlaid sensorgrams of
ICAM-1, ICAM-2, and ICAM-3 peptide surfaces and a control surface.
Ezrin was incubated with 9 µM PtdIns(4,5)P2
for 30 min at room temperature before the injections. C,
overlaid sensorgrams of ICAM-1 peptide surface and a control. Ezrin was
incubated for 30 min with lipid vesicles consisting of PC as a carrier
mixed with 8% (molar fraction) PS or PtdIns(4,5)P2
(PIP2) before injection over ICAM-1 peptide. In
control, ezrin preincubated with PC/PtdIns(4,5)P2 was
injected over control surface. The total lipid concentration was 100 µg/ml. D, overlaid sensorgrams of ICAM-2 peptide surface
as in C.
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To study the possibility that ezrin was associating to ICAM-1 and
ICAM-2 just via PtdIns(4,5)P2 micellar aggregates, the SPR assays were performed also by preincubating ezrin with vesicles consisting of a neutral phospholipid PC as a carrier, mixed with 8%
(molar fraction) of PS or PtdIns(4,5)P2. The interaction
inducing effect of PtdIns(4,5)P2 was retained under these
conditions, whereas the PC/PS vesicles had no effect on ICAM-1-ezrin
interaction (Fig. 4C). SPR results confirmed the role of
PtdIns(4,5)P2-containing vesicles also in enhancing ezrin
binding to ICAM-2 (Fig. 4D).
In sequential analyses, where PtdIns(4,5)P2 was run over
ICAM-1 and ICAM-2 peptide surfaces prior to ezrin, a significant increase in the resonance units was induced on both surfaces by PtdIns(4,5)P2 alone (Fig.
5A). This result suggested an
interaction between the ICAMs and PtdIns(4,5)P2. In
contrast, ICAM-3 peptide surface did not bind PtdIns(4,5)P2
(data not shown). Binding of ezrin on the ICAM-1 or ICAM-2 peptide
surfaces pretreated with PtdIns(4,5)P2 was evident (Fig.
5A). As an additional test for the specificity of
PtdIns(4,5)P2 effect, the recombinant
PtdIns(4,5)P2-binding fragment of vinculin was injected
over the surfaces. The vinculin fragment induced only a minor shift in
resonance units when run over ICAM-1 (Fig. 5C) or ICAM-2
(data not shown) peptide surfaces after injection of micellar
PtdIns(4,5)P2. Bovine serum albumin bound neither surface
after the PtdIns(4,5)P2 treatment (data not shown).

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Fig. 5.
Binding of PtdIns(4,5)P2 to
ICAM-1 and ICAM-2 cytoplasmic domains and comparison of ezrin and
vinculin binding. A, overlaid sensorgrams of sequential
injections over ICAM-1, ICAM-2, and control surfaces. 4.5 µM PtdIns(4,5)P2 (PIP2)
was injected over sensorchip, followed by subsequent injection of 1.45 µM ezrin. B, overlaid sensorgrams of ICAM-1
surface injected with 4.5 µM PtdIns(4,5)P2,
followed by subsequent injection of 1.45 µM vinculin
fragment (lower curve) or purified ezrin (upper
curve). In control, PtdIns(4,5)P2 and vinculin
fragment were injected on a control surface.
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Coimmunoprecipitation of Ezrin and ICAM Cytoplasmic
Domains--
We made fusion protein constructs containing the
cytoplasmic domains of ICAM-1 or ICAM-2, and cotransfected them into
yeast together with constructs expressing full-length or
NH2-terminal or COOH-terminal fragments of ezrin (Fig.
6A). All the constructs were expressed at expected molecular sizes as analyzed by
immunoblotting (Fig. 6B). The full-length ezrin fusion
protein was immunoprecipitated and the presence of
coimmunoprecipitating ICAM fusion products detected. A weak band of
ICAM-1 and a stronger band of ICAM-2 fusion protein were detectable in
ezrin immunoprecipitates. The effect of PtdIns(4,5)P2 was
analyzed by incubating identical lysates with PtdIns(4,5)P2
before the coimmunoprecipitation. PtdIns(4,5)P2 treatment
resulted in a 40-fold increase in the amount of coimmunoprecipitated ICAM-1 and in an 8-fold increase of coimmunoprecipitated ICAM-2 as
analyzed densitometrically. A control fusion protein, which lacked ICAM
cytoplasmic domains, did not coimmunoprecipitate with ezrin constructs
under any conditions (data not shown). When ICAM fusion construct were
immunoprecipitated, the carboxyl-terminal ezrin fusion protein did not
coimmunoprecipitate either in the presence or absence of
PtdIns(4,5)P2. On the other hand, the amino-terminal fusion
construct coimmunoprecipitated with both ICAMs, and the presence of
PtdIns(4,5)P2 caused a 4-fold enhancement of binding to
ICAM-2, but did not affect binding to ICAM-1 (Fig. 6C).

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Fig. 6.
Coimmunoprecipitation of ICAM-1, ICAM-2, and
ezrin fusion proteins. A, a schematic picture of ezrin
molecule and the constructs used in this assay. The structure of ezrin
consists of a globular amino terminus (oval), an -helical
region (striped bar) and a COOH-terminal domain (white
bar) containing a charged actin-binding region (black
box). The fusion protein constructs contain either an HA epitope
tag or sites detected by LexA antibody. The numbers depict
the amino acid residues of ezrin, ICAM-1, and ICAM-2 contained in the
constructs. B, yeast cells were cotransfected with either an
ICAM-1 or ICAM-2 and an ezrin fusion protein cDNA construct. The
expression of fusion proteins was verified by immunoblotting of the
yeast cell lysates with anti-HA-tag or anti-LexA antibody. Lysate of
yeast cells transfected with HA-ezrin-wt (lane 1), HA-ICAM-1
(lane 2), HA-ICAM-2 (lane 3), LexA-ICAM-1
(lane 4), LexA-ICAM-2 (lane 5), LexA-ezrin N
(lane 6), or LexA-ezrin C (lane 7). Lanes
1-3 are blotted with an anti- HA antibody, and lanes
4-7 with an anti-LexA antibody. The positions of molecular
size markers (kDa) are indicated by lines and
numbers. C, the yeast cell lysate was incubated
in the absence or presence of added PtdIns(4,5)P2. In the
left panel, the fusion proteins were immunoprecipitated with
an anti-HA-tag antibody recognizing HA-ezrin-wt and coprecipitating
fusion proteins detected by immunoblotting with an anti-LexA antibody
recognizing LexA-ICAM-1 or LexA-ICAM-2. In the middle and
right panels, the the fusion proteins were
immunoprecipitated with an anti-HA-tag antibody recognizing HA-ICAM-1
or HA-ICAM-2, respectively, and coprecipitating fusion proteins
detected by immunoblotting with an anti-LexA antibody recognizing
LexA-ezrin-N or LexA-ezrin-C.
|
|
 |
DISCUSSION |
In this study, we report that the cytoplasmic domain of ICAM-2
interacts with ezrin. The interaction is highly facilitated by
PtdIns(4,5)P2, and PtdIns(4,5)P2 induces an
interaction between ezrin and ICAM-1 but not ICAM-3. An association was
first suggested by transfection studies, which showed a close
codistribution of wild-type ICAM-1 or ICAM-2 and endogenous or
cotransfected ezrin in adherent cells, and ICAM-2 and ezrin in thymoma
cells (27). An engineered ICAM-1 molecule linked to the membrane by a
glycophospholipid anchor showed a diffuse staining pattern without
microvillar colocalization, indicating the requirement of an intact
intracellular domain of ICAM-1 for the specific surface distribution
and codistribution with ezrin.
A direct interaction of purified ezrin and ICAM-2 was detected with
several different methods (Sepharose bead affinity precipitation, microtiter binding assay, coimmunoprecipitation, and surface plasmon resonance). The apparent dissociation constant of 3.3 × 10
7 M is in line with the affinities of other
membrane protein-cytoskeleton interactions. For instance,
-actinin
was reported to bind to
1-integrin cytoplasmic domain
peptide with KD of 1.6 × 10
8
M (42) and protein 4.1 binds to the cytoplasmic domain of
CD44 with KD value on the order of 10
7
M (43), whereas moesin binding to CD44 was too weak for
quantitation at physiological ionic strength (13).
In contrast to ICAM-2, only a very weak binding of ezrin to ICAM-1 was
seen in the microtiter and coimmunoprecipitation assays in the absence
of PtdIns(4,5)P2. When placental lysate was passed through
ICAM-1 peptide column, ezrin was one of the few proteins that were
retained. The lysate apparently contained PtdIns(4,5)P2. With all different methods used, the interaction of ICAM-1 or -2 and
ezrin was either induced or highly enhanced by
PtdIns(4,5)P2. This is in analogy with the interaction
between ezrin and the transmembrane adhesion protein CD44 (13). The
microtiter and SPR assays with phospholipid vesicles consisting of a
neutral carrier lipid PC mixed with a small portion of
PtdIns(4,5)P2, resembling the natural composition of
cellular membranes, showed that the binding is not just an aggregation
of PtdIns(4,5)P2 micelles to the peptides, but a specific
effect. Another cytoskeleton-associated protein, vinculin, is regulated
by PtdIns(4,5)P2 (39, 44, 45), but compared with ezrin the
phosphoinositide binding fragment of vinculin did not show significant
binding to ICAM peptide surfaces pretreated with
PtdIns(4,5)P2. This result further emphasizes the
specificity of ezrin binding to ICAM peptides in the presence of
PtdIns(4,5)P2.
ICAM-3 did not bind ezrin even in the presence of added phospholipids.
When T-cells produce cellular uropods in response to cytokines and
cAMP, ICAM-3 is redistributed to the uropod region together with
myosin, not with the actin-based cytoskeleton (46-48). Moesin has been
shown to interact with ICAM-3 in polarized T-cells, and stimulation of
T-cells redistributed ICAM-3 and moesin, but not ezrin to the formed
uropods (48). In these cells, the expression level of ICAM-1 is low. It
is possible that ICAM adhesion molecules and ERM proteins, with partly
overlapping and redundant functions, display variable combinations in
different physiological and cellular environments. In the present
study, we have not analyzed whether other ERM family members interact
with ICAM-1 and ICAM-2 in a fashion similar to ezrin. Considering the
high sequence homology and a similar binding ability to CD44 (13), it
is likely that radixin and moesin can also associate with ICAM-1 and
-2.
The SPR measurements demonstrated that PtdIns(4,5)P2
interacts with the cytoplasmic tails of ICAM-1 and ICAM-2. Previously, a variety of cytoplasmic polypeptides, particularly actin-binding proteins, have been shown to bind PtdIns(4,5)P2, and
PtdIns(4,5)P2 is an important modulator of the
actin-containing cytoskeleton (28, 29). However, ICAM-1 and ICAM-2 are
the first adhesion proteins reported to interact with
PtdIns(4,5)P2. The consensus binding sequences for
PtdIns(4,5)P2 binding are
(K/R)XXXXKX(K/R)(K/R) or
KXXXKXKK (49), although other binding
sequences exist. The binding of phosphoinositides is not simply
electrostatic, but also secondary structure and hydrophobic segments
are thought to be important (49-52). The cytoplasmic domains of ICAM-1
and ICAM-2 contain both basic and hydrophobic residues resembling the
described PtdIns(4,5)P2 binding sites, and in particular
the juxtamembrane sequence of ICAM-1 is highly homologous with the PtdIns(4,5)P2 binding consensus motif. ICAM-3 did not bind
PtdIns(4,5)P2 in SPR measurements, and its amino acid
sequence also differs from the typical PtdIns(4,5)P2
binding site pattern. The differences in sequences suggest that the
cytoplasmic interactions of ICAM-3 are possibly not regulated the same
way as the interactions of ICAM-1 and -2.
Phosphoinositides promote conformational changes of the peptides to
which they are bound (52, 53) and may increase or stabilize the
formation of higher form oligomers, as suggested in the dimerization of
the protein kinase Akt (54). PtdIns(4,5)P2 is implicated in
adhesion; it is needed for focal adhesion formation (45), and the
levels of PtdIns(4,5)P2 are increased by integrin clustering, which activates cytoskeleton-associated PtdInsP kinase locally within focal adhesion complex (55). It is tempting to speculate
that increased phophoinositide levels caused by preadhesion signaling
or by initial adhesion would modulate the adhesive functions and
intracellular interactions of ICAM-1 and ICAM-2, maybe by changing the
conformation of the short cytoplasmic tails or by oligomerization of
the molecules. Alternatively, local accumulation of
PtdIns(4,5)P2 in the plasma membrane could lead to bridging of ICAMs and ezrin.
PtdIns(4,5)P2 acts as a precursor for the secondary
messenger molecules inositol trisphosphate and diacylglycerol and is
one of the substrates of PI 3-kinase, but also has independent
signaling properties. It can interact with a number of actin-binding
proteins and thereby regulate actin polymerization (28). Ezrin and
other ERM proteins are effector molecules of the Rho signaling pathway (56), which controls actin filament and focal adhesion assembly. Rho
regulates also phosphatidylinositol 4-phosphate 5-kinase (57), suggesting that Rho could activate ezrin through the controlled synthesis of PtdIns(4,5)P2. Our studies together with the
results from the binding of CD44 (13) indicate a role for
PtdIns(4,5)P2 in controlling the ability of ezrin to bind
transmembrane proteins. The amino-terminal domain of ezrin has an
exceptionally high affinity to the carboxyl-terminal part, and in an
inactive conformation apparently masks the actin binding domain by
intra- or intermolecular association (10, 58). The mechanism of ezrin
activation is unknown but is speculated to involve phosphorylation or
dimerization/oligomerization of the molecule (59, 60). A
PtdIns(4,5)P2 binding site is contained in the
NH2-terminal half of ezrin (12), and
PtdIns(4,5)P2 binding could be an alternative or additional
mechanism to modulate the conformation of ezrin through uncovering
binding sites for F-actin and other interacting molecules. This type of
regulation has been reported for vinculin, in which
PtdIns(4,5)P2 dissociates an intramolecular head-to-tail
joining (44) rendering the masked talin and actin binding sites
accessible, and exposing a phosphorylation site for protein kinase C
(39, 45). Our coimmunoprecipitation results support the concept that
the binding site for transmembrane proteins is in the amino-terminal
part of ezrin. The finding that PtdIns(4,5)P2 enhances
ICAM-2 binding also to the NH2-terminal fragment of ezrin
shows a complex role for PtdIns(4,5)P2, possibly modulating
ICAM-2 activity in addition to ezrin regulation.
Ezrin is a prominent constituent of microvilli and other cellular
protrusions and the leading edge. It is located at cell membrane
regions that primarily come into contact with other cells. Thus ezrin,
as well as other ERM-members, serve as ideal cytoplasmic binding
partners for adhesion molecules. The ability of ezrin to redistribute
ICAM-2 (27), and probably also ICAM-1, to microvilli or concentrated
patches on uropod-like structures increases the accessibility of these
molecules and could also increase their avidity by clustering (Fig.
7). Concentration of ICAMs would benefit the cells during conjugation between lymphoid cells and their target
cells, and during migration of lymphocytes through endothelium to
tissues. An interaction between ezrin and ICAMs also facilitates post-binding events, as demonstrated by experiments in which
relocalization of ICAM-2 to the uropod region after transfection of
ezrin rendered target cells susceptible for NK cell killing (27). A
similar mechanism may be involved in killing of viral infected cells, when ezrin redistributes from the cytoplasm into newly formed microvilli (61). The finding that PtdIns(4,5)P2 can be
sequestered into lateral domains in the membrane upon binding to
specific proteins (62) raises the possibility that the interplay of all three components, ezrin, ICAM-1 or ICAM-2 and
PtdIns(4,5)P2, is regulated by their local distribution. In
this manner, the versatile interactions between adhesion molecules and
the cytoskeleton could be linked to the signals that cells obtain from
their environment and from inside the cell.

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|
Fig. 7.
A model of the events related to ezrin/ICAM
interaction. A signal (e.g. growth factor) induces
transient accumulation of phosphoinositides and activation of ezrin.
Ezrin binds to ICAMs and F-actin. The changes induce cytoskeletal
rearrangement and local concentration of adhesion molecules to cellular
extensions, which strengthens adhesion by increasing the accessibility
and avidity of adhesion molecules via clustering.
|
|
 |
ACKNOWLEDGEMENTS |
We thank E. Golemis for anti-LexA mAb, R. Brent for plasmids, D. Critchley and P. Mangeat for the vinculin and
ezrin expression constructs, P. Janmey for fruitful discussions, F. Zhao and M. Sainio for help with computer programs, and T. Halmesvaara
and M.-L. Mäntylä for skillful technical assistance.
 |
Note Added in Proof |
While this article was under
review, another paper describing an interaction between ICAM-2 and
ezrin was published (63).
 |
FOOTNOTES |
*
This work was supported by the Academy of Finland, the
Finnish Cancer Society, the Sigrid Juselius Foundation, and the Ida Montin Foundation.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
**
To whom correspondence should be addressed: Haartman Institute,
University of Helsinki, P. O. Box 21 (Haartmanink. 3), FIN-00014 Helsinki, Finland. Tel.: 358-9-19126413; Fax: 358-9-19126700; E-mail:
olli.carpen{at}helsinki.fi.
The abbreviations used are:
ERM, ezrin/radixin/moesin; GPI, glycophosphatidylinositol; HA, hemagglutinin
antigen; ICAM, intercellular adhesion molecule; PC, phosphatidylcholine; PS, phosphatidylserine; PtdIns(4, 5)P2,
phosphatidylinositol 4,5-bisphosphateSPR, surface plasmon resonancemAb, monoclonal antibodyPAGE, polyacrylamide gel electrophoresisRU, resonance unit(s)PBS, phosphate-buffered saline.
 |
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