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ICln, a Novel Integrin
␣
IIb

3
-Associated Protein, Functionally
Regulates Platelet Activation*
Received for publication, February 26, 2004, and in revised form, April 8, 2004
Published, JBC Papers in Press, April 9, 2004, DOI 10.1074/jbc.M402159200
Deirdre Larkin‡, Derek Murphy§, Dermot F. Reilly‡, Martha Cahill‡, Ellen Sattler‡,
Pat Harriott¶, Dolores J. Cahill§, and Niamh Moran‡储
From the ‡Department of Clinical Pharmacology and the §Centre for Human Proteomics, Royal College of Surgeons
in Ireland, Dublin 2 and the ¶Medical Biology Centre, Queens University Belfast, Belfast BT9 7BL, Ireland
A critical role for the conserved
␣
-integrin cytoplas-
mic motif, KVGFFKR, is recognized in the regulation of
activation of the platelet integrin
␣
IIb

3
. To understand
the molecular mechanisms of this regulation, we sought
to determine the nature of the protein interactions with
this cytoplasmic motif. We used a tagged synthetic pep-
tide, biotin-KVGFFKR, to probe a high density protein
expression array (37,200 recombinant human proteins)
for high affinity interactions. A number of potential in-
tegrin-binding proteins were identified. One such pro-
tein, a chloride channel regulatory protein, ICln, was
characterized further because its affinity for the inte-
grin peptide was highest as was its expression in plate-
lets. We verified the presence of ICln in human platelets
by PCR, Western blots, immunohistochemistry, and its
co-association with
␣
IIb

3
by surface plasmon reso-
nance. The affinity of this interaction was 82.2 ⴞ24.4 nM
in a cell free assay. ICln co-immunoprecipitates with
␣
IIb

3
in platelet lysates demonstrating that this inter-
action is physiologically relevant. Furthermore, immo-
bilized KVGFFKR peptides, but not control KAAAAAR
peptides, specifically extract ICln from platelet lysates.
Acyclovir (100
Mto5mM), a pharmacological inhibitor
of the ICln chloride channel, specifically inhibits inte-
grin activation (PAC-1 expression) and platelet aggrega-
tion without affecting CD62 P expression confirming a
specific role for ICln in integrin activation. In parallel, a
cell-permeable peptide corresponding to the potential
integrin-recognition domain on ICln (AKFEEE, 10 –100
M) also inhibits platelet function. Thus, we have iden-
tified, verified, and characterized a novel functional in-
teraction between the platelet integrin and ICln, in the
platelet membrane.
Integrins are heterodimeric cell adhesion molecules com-
posed of
␣
- and

-subunits that mediate cell-cell and cell-
matrix adhesion and coordinate bi-directional signaling events
across a cell membrane. The platelet-specific integrin
␣
IIb

3
is
the most studied of all integrins and serves as a model for
exploring integrin activation and ligand interactions. Integrin
ligands bind preferentially to the activated integrin conforma-
tion. However, an understanding of integrin activation mech-
anisms has only recently been elaborated.
Activation of
␣
IIb

3
in response to cellular stimulation in-
volves a conformational change in the large extracellular com-
ponents of the integrin, possibly coordinated by the endogenous
thiol-isomerase activity of the

-subunit (1). However, the role
of the short integrin cytoplasmic domains has been the focus of
much research into the molecular mechanisms of this integrin
activation. Deletion or mutation of the highly conserved, mem-
brane-adjacent KXGFFKR motif in
␣
-integrins results in con-
stitutive integrin activation (2, 3). Furthermore, we and others
have shown that addition of exogenous cell-permeable peptides
corresponding to this sequence, KVGFFKR or KLGFFKR, di-
rectly activates integrins
␣
IIb

3
or
␣
2

1
, respectively (4, 5).
Thus this sequence plays a critical role in regulation of the
activation state of the integrin. Similarly, removal or mutation
of the corresponding sequence on the integrin

-cytoplasmic
tail can modulate integrin activation state (6, 7). It has been
demonstrated that the membrane-adjacent cytoplasmic regions
of the integrin
␣
- and

-subunits form a salt bridge that facil-
itates their interaction in resting integrins (8). Cellular activa-
tion alters the dynamics of this interaction and results in a
dissociation of the two tails. This is probably mediated by a
talin wedge that physically separates the two tails (9). How-
ever, the nature of the cytosolic stimulus that initiates integrin
activation following cellular activation is not understood. Be-
cause the integrin tails have no intrinsic enzymatic activity,
they are believed to dynamically recruit signaling molecules to
regulate their activation state. Many integrin-binding cytosolic
proteins have been identified and can be broadly divided into
structural, cytoskeletal proteins such as talin (10) or
␣
-actinin
(11), cytosolic proteins such as CIB (12), or

3
-endonexin (13)
and membrane proteins such as CD9 (14) and CD153 (15). New
integrin-binding proteins are continuously being identified
(16), but the precise molecular mechanisms by which they
regulate integrin activation remain unknown.
Although not generally associated with the regulation of ion
channels, integrins have recently been identified as playing a
role in osmoregulation and regulation of ion movement across
cell membranes. A number of groups have demonstrated that
integrins regulate potassium, calcium, or chloride fluxes either
indirectly (17–22) or directly (19, 21, 23, 26 –28). Vitronectin,
but not fibrinonectin or laminin, selectively affects potassium
currents in an Arg-Gly-Asp sequence-dependent manner in
developing mouse hippocampal neurons (29). Similarly, ligand
binding to integrins or integrin clustering modulates potas-
sium currents, leading to hyperpolarization of monocytes and
enhanced Ca
2⫹
influx (30). Integrin co-localization with ion
channels has been observed in LOX tumor cells where

1
-
integrins associate laterally with potassium channels during
adhesion contributing to the regulation of integrin function in
these cells (31). Co-localization of

1
-integrins with the
␣
-sub-
units of Na,K-ATPase, the epithelial sodium channel, and the
voltage-activated calcium channel has also been shown (23).
* The costs of publication of this article were defrayed in part by the
payment of page charges. This article must therefore be hereby marked
“advertisement” in accordance with 18 U.S.C. Section 1734 solely to
indicate this fact.
储To whom correspondence should be addressed. Tel.: 353-1-402-2153;
Fax: 353-1-402-2453; E-mail: nmoran@rcsi.ie.
THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 279, No. 26, Issue of June 25, pp. 27286–27293, 2004
© 2004 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.
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However, indirect evidence for integrin modulation of specific
chloride ion currents comes from studies that demonstrate that
conformational change of

2
-integrins in polymorphonuclear
leukocytes is inhibited by hypertonic saline (32) and is depend-
ent on altered chloride fluxes in these cells (33). In addition,
CLCA (chloride channel, calcium activatable) protein has been
identified as ligands for

4
-integrins facilitating tumor metas-
tasis. This interaction involves transcellular contacts of extra-
cellular sites on both proteins, and it is not clear if binding
activates the channel function (34). Thus a substantial amount
of recent evidence supports an active role for integrins in the
regulation of numerous ion channel proteins in active cells.
ICln is a 42-kDa multifunctional protein that is essential for
cell volume regulation.
1
It is believed to form a homodimeric
chloride channel (36). However, hydrophobicity analysis indi-
cates that ICln lacks the transmembrane helices necessary for
channel-pore formation by most vertebrates. When expressed
in Xenopus oocytes, it mediates a nucleotide-sensitive out-
wardly rectifying chloride channel closely resembling the bio-
physical properties of swelling-dependent chloride channels
involved in regulation of cell volume decrease after cell swelling
(36, 37). It is found in the cytosol in cells-at-rest but is associ-
ated with the cell membrane following a hypotonic challenge.
Thus, physiological cell swelling stimulates cytosol to mem-
brane transposition of ICln (21). However, controversy arises
over this hypothesis (38). Furthermore, reconstitution of re-
combinant ICln into lipid bilayers yields a cationic permeabil-
ity (39). Thus its exact role as a chloride channel is somewhat
controversial (40). It may, however, act as a channel facilitator
or regulator (41).
In this study, we have searched for novel integrin cytoplas-
mic binding partners by screening a large human recombinant
expression library for specific interactions with a tagged syn-
thetic peptide, biotin-KVGFFKR, corresponding to an integrin
motif known to critically regulate the integrin conformational
state (42). Our results highlighted an interaction with the
chloride channel protein ICln. We confirm the presence of ICln
in human platelets and verify an association between ICln and
␣
IIb

3
. Pharmacological modulators of ICln function specifi-
cally to modify integrin-mediated platelet responses, verifying
a physiological role for ICln in platelet activation events. Be-
cause ICln is thought to have many functional roles, we pro-
pose that one such role is in the regulation of the specific
platelet integrin
␣
IIb

3
through the KVGFFKR motif. In addi-
tion, a novel synthetic peptide, identified as a potential inte-
grin-recognition domain on ICln, specifically inhibits platelet
function. Thus we have identified and verified a novel platelet
integrin-binding protein that plays a role in the regulation of
integrin activation and platelet function.
EXPERIMENTAL PROCEDURES
High Density Arrays of Escherichia coli-expressed Proteins—Protein
arrays were generated from the hEx1 library, a redundant human fetal
brain protein expression cDNA library subcloned into pQE-30NST (ac-
cession no. AF074376) in E. coli strain SCS-1, which permits IPTG
2
-
inducible expression of His
6
-tagged fusion proteins directly on a mem-
brane (43, 44). The arrays consist of 2 PVDF membranes (22.2 ⫻22.2
cm, Millipore) containing a total of 37,200 clones arrayed in duplicate in
a5⫻5 pattern. The membranes are incubated overnight on 2YT agar
(supplemented with 100
g/ml ampicillin, 15
g/ml kanamycin, and 2%
glucose), and recombinant protein expression is induced on 2YT agar
containing 1 mMIPTG for 3–4 h. Next, the membranes are transferred
onto pre-soaked blotting paper (Whatman 3MM) with denaturing solu-
tion (0.5 MNaOH, 1.5 MNaCl) for 10 min to lyse the bacterial cells,
followed by 5-min incubation in neutralizing solution (1 MTris-HCl, pH
7.5, 1.5 MNaCl) and 15 min in 2⫻SSC. The filters are air-dried and
stored at room temperature.
Peptide Screening of Protein Arrays—Prior to screening, the dried
colonies are removed from the membranes with tissue paper and TB-
STT (20 mMTris-HCl, pH 7.4, 0.5 MNaCl, 0.05% Tween 20, 0.5% Triton
X-100). The protein array filters were incubated with 100
Mbiotin-
labeled KVGFFKR or KAAAAAR peptide in TBST (20 mMTris-HCl, pH
7.4, 0.5 MNaCl, 0.2% Tween 20) for 3 h, followed by three 5-min washes
in TBST at 4 °C. Bound peptide was detected using a Cy3-labeled
anti-biotin antibody (Amersham Biosciences) and visualized on a Ty-
phoon 9600 imager. Clones bound by the peptide were identified using
VisualGrid software (GPC Biotech) and sequenced to confirm their
identity.
Protein Expression and Purification—The E. coli clones expressing
the recombinant His-tagged proteins were grown in liquid cultures in
the presence of 1 mMIPTG. Bacteria were lysed by a freeze-thaw cycle
and incubated in 1 ml of lysis buffer (50 mMTris, 300 mMNaCl, pH 8.0,
10 mMimidazole, 1 mMphenylmethylsulfonyl fluoride, 0.6 mg/ml ly-
sozyme 0.6 mg/ml RNase, and 0.6 mg/ml DNase) per gram of wet
weight, overnight at 4 °C. The proteins were then purified using nickel-
nitrilotriacetic acid-agarose (Qiagen) according to the manufacturer’s
instructions (100
l/ml lysate). The purification of the proteins was
verified by SDS-PAGE.
Dot Blot Verification of Protein-Peptide Interactions—The 19 pro-
teins were dot-blotted in four serial dilutions corresponding to 0.1–5
pmol per spot. Specific binding of Biotin-KVGFFKR, and a control
peptide Biotin-KAAAAAR, was determined as above with an
␣
-biotin
secondary antibody (1:25,000). CIB (gift of Dr. Nelly Kieffer, Luxem-
bourg) was used as a positive control, to ascertain the efficiency of the
peptide interactions. Antibody detection was analyzed with SuperSig-
nal according to the manufacturer’s instructions (Pierce).
RNA Isolation and Purification from Platelets—1 ml of Tri reagent
was added to 10
8
platelets from a highly purified platelet preparation
(45) and allowed to lyse on ice for 15 min to 1 h. 1-Bromo-3-chloropro-
pane (Sigma) was then added (0.1 ml/ml Tri reagent), and samples were
shaken vigorously for 15 s then allowed to stand for 15 min. Platelets
were then centrifuged for 15 min at 12,000 ⫻gat 4 °C. The aqueous
phase was removed, avoiding the middle phase containing any genomic
or mitochondrial DNA. 0.5 ml of isopropanol was added per milliliter of
Tri reagent, and samples were mixed and allowed to stand for 15 min.
After centrifugation at 12,000 ⫻gfor 10 min at 4 °C, the supernatant
was carefully removed and the pellet was washed with 1 ml of 75%
ethanol per milliliter of Tri reagent. RNA was pelleted at 12,000 ⫻gfor
5 min at 4 °C. The pellet was allowed to air dry then was resuspended
in PCR-grade water (Sigma), and concentration and quality were as-
sessed using a spectrophotometer where the 260 nm/280 nm absorbance
ratio was 1.9:2.0.
ICln RT-PCR from Platelet RNA—The purification of the platelet
RNA was verified as previously described (45). Platelet RNA (1
g)
was denatured at 65 °C for 10 min and incubated at 37 °C overnight
in the presence of 1⫻reverse transcription buffer, 1
l of Moloney
murine leukemia virus reverse transcriptase, 25
Mdeoxycholate
triphosphates (dNTPs), and 1
l of random hexamers in a final
volume of 8
l. The reaction was stopped by heating at 95 °C for 5
min. A final assay volume of 50
l containing 1.5 mMMgCl
2
, 250
M
dNTPs, 10 pmol/
l of each sense ICln (5⬘-GAAGACAGTGATGAT-
GATGTTGAACC-3⬘) and antisense primer ICln (5⬘-TTCCACAT-
CATATTCTTCTCCATCGTA-3⬘), 2 units of TaqDNA polymerase (Pro-
mega), and 4
l of cDNA. The reaction cycles were as follows:
denaturation at 94 °C for 1 min, annealing at 62 °C for 1 min, and
extension at 72 °C for 1 min for 40 cycles. The PCR products were
separated on a 1.2% agarose gel containing 0.5
g/ml ethidium bro-
mide and visualized on an ultraviolet transilluminator.
Surface Plasmon Resonance—Real-time binding and kinetic analy-
ses were performed at the University of Reading on a BIAcore 3000
biosensor system (Amersham Biosciences Biosensor AB) using surface
plasmon resonance measurements. Purified
␣
IIb

3
(1
M, Calbiochem)
was immobilized on carboxymethylated sensor chips (type CM5) in 10
mMsodium acetate at pH 4.0 as described by the supplier. Control flow
cells were activated and blocked in the absence of integrin. Binding was
evaluated over a range of recombinant ICln concentrations (0.625
Mto
10
M) in 150 mMNaCl, 100 mMHEPES (pH 7.4), 0.05% Surfactant p20,
1.8 mMCaCl
2
, and 0.15 mg/ml bovine serum albumin under a contin-
uous flow of 5
l/min at 25 °C. 30
l of ICln-containing solutions were
1
BD Transduction Laboratories, pICln data sheet, available at www.
bdbioscience.com (2001).
2
The abbreviations used are: IPTG, isopropyl-1-thio-

-D-galactopyr-
anoside; MGC, a hypothetical protein; ICln, Homo sapiens nucleotide-
sensitive chloride channel (CLNS1a, P54105); CIB, calcium integrin-
binding protein; CD62P, P-selectin; PVDF, polyvinylidene difluoride;
RT, reverse transcription;
ICln Associates Platelet Integrin
␣
IIb

3
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flowed over the surface of the chip for 2 min. Binding of ICln to
␣
IIb

3
-immobilized flow cells was corrected for binding to control flow
cells. Flow cells were regenerated with running buffer, following re-
moval of ICln from
␣
IIb

3
using glycine pH 1.5. Binding data were fitted
using BIAevaluation version 3.1 software (BIAcore).
Peptide Synthesis—Peptides were synthesized on an Applied Biosys-
tems automated peptide synthesizer (model 432A, Norwalk, CT) using
a standard solid-phase Fmoc (n-(9-fluorenyl)methoxycarbonyl) proce-
dure, purified by high performance liquid chromatography, and masses
were verified by matrix-assisted laser desorption ionization time-of-
flight (Bruker Reflex III). Biotinylated KVGFFKR peptide and control
biotin-KAAAAAR peptides were synthesized with two biotin moieties
attached to each motif separated by a hexanoic acid spacer. Cell-per-
meable palmitoylated peptides (-AKFEEE, -LEFEEE, -ELFNDG, and
-KVGFFKR) were synthesized in an identical manner, except no spacer
was used.
Platelet Function—Gel-filtered platelets were prepared as previously
described (1, 4). Platelet aggregation assays were performed as described
using 0.1 unit/ml thrombin or 10
MU46619, a thromboxane mimetic.
Immunoprecipitations and KVGFFKR Pull-down—Platelet lysates
were prepared as described previously (1, 4) from gel-filtered platelets
using a mild detergent lysis buffer (20 mMTris, pH 7.4, 50 mMNaCl, 2
mMCaCl
2
,2mMMgCl
2
,1mMNa
3
VO
4
, 1% Brij-35, protease inhibitor
mixture (Calbiochem, 500
M4-(2-aminoethyl)benzenesulfonylfluoride
hydrochloride, 500
MEDTA, 1
ME-64, 1
Mleupeptin, 1
g/ml
aprotinin)). Aliquots of platelet lysates were incubated with a mono-
clonal antibody to ICln (1
g, Clone 32, BD Transduction laboratories)
or a control IgG overnight, then immunoprecipitated with blocked pro-
tein-G-Sepharose beads for 3 h, washed extensively in ice-cold buffers.
Proteins binding to the beads were removed by boiling in 1⫻(2% SDS,
50
Mdithiothreitol, 125 mMTris, pH 6.8, 20% glycerol, 0.0004% brom-
phenol blue) sample buffer, and separated on 7.5% SDS-PAGE before
being transferred to PVDF membranes and Western-blotted with
SZ22, an antibody detecting
␣
IIb
. Peptide pull-down assays were
performed in an identical manner using a biotinylated KVGFFKR
peptide (350
M), a control biotinylated KAAAAAR peptide (350
M),
and 40
l of NeutrAvidin beads (Pierce). All steps and buffers were
carried out at 4 °C.
Flow Cytometry of PAC-1 and CD62P—Gel-filtered platelet aliquots
of 50 ⫻10
3
/
l were pre-treated with acyclovir (up to 5 mM)orMe
2
SO
vehicle control (up to 0.5%) for 1 min before activation with 1 unit/ml
thrombin, or 10
MU46619 for 3 min at 37 °C. Platelets were then
incubated for 10 min in the presence of fluorescein isothiocyanate-
PAC-1 or fluorescein isothiocyanate-CD62P (10
l, Molecular Probes)
and fixed in 0.1% formaldehyde. A methanol vehicle control was used
for the AKFEEE and ELFNDG peptides. Platelet-associated fluores-
cence was estimated on a FACSCalibur flow cytometer.
Immunohistochemistry—Gel-filtered platelets (50 ⫻10
3
/
l) were ad-
hered to fibrinogen (20
g/ml)-coated slides at 37 °C for the indicated
times. Slides were fixed and permeabilized in 1% (v/v) formaldehyde
and 1% (v/v) Triton X-100. Using the Zenon Technology kit (Molecular
Probes) for monoclonal antibodies, the ICln (BD Transduction Labora-
tories) and the SZ22 (Immunotech) antibodies were directly labeled
prior to use. The platelets were probed simultaneously for ICln and
␣
IIb

3
or stained for polymerized actin using Alexa 488-phalloidin (Mo-
lecular Probes) in the platelet time course. Slides were viewed using a
Zeiss LSM501 confocal microscope.
RESULTS
KVGFFKR-specific Binding Proteins Identified from High
Density Protein Arrays of Human Recombinant Proteins—We
have previously established that the conserved amino acid
sequence KVGFFKR, immediately adjacent to the transmem-
brane domain of the platelet
␣
IIb
integrin subunit, plays a
critical role in the regulation of integrin activation (4, 42). To
identify the protein-protein interactions responsible for this
regulation, we synthesized a biotin-tagged peptide correspond-
ing to this sequence and used it as a probe to identify high
affinity protein interactions dependent on this motif. From a
high content, high density protein array derived from a redun-
dant human brain cDNA expression library (37,200 clones) (43,
44, 46), we identified a number of novel, potential integrin-
regulating proteins (Fig. 1A). Nineteen clones, corresponding to
thirteen different proteins, bound significantly and specifically
to the biotin-tagged KVGFFKR peptide on the array, but not to
a control peptide, biotin KAAAAAR. All clones that reacted
with biotin KVGFFKR were sequenced and identified (Fig. 1C).
His-tag purified proteins from these clones were dot-blotted
onto a PVDF membrane and re-probed to quantitate the inter-
actions. On this dot blot, many of the originally identified
proteins bound only weakly to biotin-KVGFFKR reflecting low
affinity or nonspecific binding to the bacterially expressed pro-
teins. The veracity of the technique was displayed by the spec-
ificity of the response observed on the proteins expressed more
than once. Thus, all four ferritin and both ribosomal L9 clones
identified on the array failed to register a sufficient interaction
with KVGFFKR when tested on the dot blot. Both myosin
light-chain clones maintained a weak interaction with the bi-
otin peptide, although this was considered too low to pursue.
Three of the clones, including two expressing a novel chloride
channel, ICln (clones 2 and 8), and one expressing a hypothet-
ical protein ‘‘MGC’’ (clone 6), demonstrated highly specific bind-
ing (Fig. 1B). Calcium-integrin-binding protein (CIB) identified
from a yeast-two-hybrid screen with the
␣
-cytoplasmic tail is
included as a positive control at equimolar concentrations and
binds KVGFFKR weakly in comparison to both ICln and MGC.
ICln Protein Is Present in Platelets—Although the KXG-
FFKR motif is highly conserved across all integrins, the specific
KVGFFKR sequence used to probe the array is found in 3
␣
-integrin subunits;
␣
IIb
, found exclusively in platelets, and
␣
L
and
␣
X
, found in neutrophils. Because the expression library on
FIG.1. Identification of clones that have specific binding to
KVGFFKR peptide. A, the hEx1 human recombinant protein array,
was screened with a biotinylated KVGFFKR peptide, and a control
biotinylated KAAAAAR peptide, to identify proteins that specifically
bind the KVGFFKR sequence. A small section of the PVDF array,
containing ⬃1,400 recombinant proteins, in duplicate in a distinct 5 ⫻
5 pattern around a guide dot, is shown. Four of the positive clones are
encircled on the array. No clear binding of any proteins was observed
with the control peptide. B, the 19 positive clones were sequenced, and
the proteins were expressed, purified, and dot-blotted onto PVDF mem-
branes before being probed again with the biotinylated peptides as
indicated. The proteins were loaded as equally as possible, in 5-pmol,
1-pmol, 0.5-pmol, and 100-fmol concentrations. Lb is the lysis buffer
control, and CIB (gift from Dr. Kieffer) was used as a positive control. C,
the identity of the positive clones, labeled 1–19, following sequencing
and BLAST searching.
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␣
IIb

3
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the protein array is derived from human fetal brain, the ob-
served interactions, though specific, might not be physiologi-
cally relevant to platelet biology. To address this, we therefore
examined the expression of ICln and MGC in platelets by PCR
analysis of purified platelet RNA. The preparation of pure
platelet RNA is not an insignificant task in platelets, because,
being non-nucleated, their RNA content is considered vestigial
and is consequently very low. Gel-filtered platelets were strin-
gently prepared by repeated differential centrifugation and
analyzed for white cell contamination by estimating T-cell re-
ceptor and CD45 (leucocyte common antigen) positivity by
RT-PCR assays (45). The presence of intact platelet RNA is
estimated by PCR analysis of mRNA for glycoprotein Ib
␣
. Only
platelet preparations that lacked T-cell receptor and CD45 and
expressed strong positive bands for GPIb
␣
were assayed for
ICln or MGC. The ICln message was present in platelet RNA,
but MGC was absent (Fig. 2A). We therefore chose to examine
the
␣
IIb

3
interaction with ICln in platelets.
Western blotting and immunofluorescent microscopy con-
firmed the presence of ICln in platelets using commercial
monoclonal antibodies (Clone 32) (Fig. 2, Band C) and poly-
clonal antibodies (gift from Dr. Paulmichl; data not shown).
Under reducing conditions, a 42-kDa monomer is observed in
platelet lysates identical to that observed in non-platelet ly-
sates such as the lymphoblastoma HUT-78 (Fig. 2B) or Chinese
hamster ovary cells (not shown). In contrast, however, under
non-reducing conditions, the platelet ICln-reactive band runs
as a high molecular mass complex of ⬎205 kDa. This complex
formation is unique to the platelet and is not observed in other
cell types (Fig. 2B).
ICln Protein Co-associates with Platelet Integrin
␣
IIb

3
—
Using immunofluorescent imaging, ICln shows even but low
intensity distribution in resting platelets. In the activated or
spread platelet, staining is most intense in the granulomere of
the spread platelet (Fig. 2C). However, it is difficult to ascer-
tain if there is co-localization with
␣
IIb

3
due to the low abun-
dance of ICln. To confirm the association of ICln with
␣
IIb

3
in
platelets, therefore, we needed to use more sensitive tech-
niques. Two types of co-precipitation studies were carried out.
First, immunoprecipitations using a monoclonal antibody to
ICln (Clone 32) demonstrated that
␣
IIb

3
co-precipitated with
ICln from platelet lysate (Fig. 3A). Second, to confirm the
specificity of interaction with the peptide motif, a biotin-pep-
tide pull-down assay was performed using immobilized KVG-
FFKR or control KAAAAAR peptides to extract platelet pro-
teins. Western blots of these precipitates for ICln showed a
selective interaction of ICln with the KVGFFKR peptide veri-
fying that the peptide-protein interaction observed on the cell-
free protein array is also valid in a platelet (Fig. 3B). The
observation that KVGFFKR specifically precipitates ICln is a
powerful verification of its co-association with this unique pep-
tide motif and confirms that the interaction between ICln and
␣
IIb

3
is physiological and occurs in platelets.
Finally, surface plasmon resonance analysis of the direct
interactions of ICln with purified preparations of
␣
IIb

3
con-
firmed a specific and saturable, high affinity interaction be-
tween the two proteins in a cell-free system (Fig. 3C). The
calculated affinity (K
d
) of this interaction was 82.2 ⫾24.4 nM.
Inhibitors of ICln Modulate Platelet Function and Integrin
Activation—We next needed to establish a functional role for
ICln in integrin activation. Acyclovir, the known antiviral
agent, is established as a specific inhibitor of the ICln chloride
channel with minimal effects on other chloride fluxes (39, 47,
48). We therefore examined the effects of acyclovir on platelet
responses induced by the potent platelet agonists, thrombin,
the PAR-1 agonist, and U46619, a thromboxane mimetic (Fig.
4A). Acyclovir causes a dose-dependent inhibition of platelet
aggregation by these agonists and, in parallel, inhibits activa-
FIG.2. Identification of the chlo-
ride channel ICln in human platelets.
A, ICln was amplified by RT-PCR from
human platelets (PLT) as a 168-bp prod-
uct in two separate donor samples along-
side a Chinese hamster ovary cell control.
B, ICln was identified in platelets by
Western blotting of platelet lysate in both
reducing (R Plt) and non-reducing (NR
Plt) conditions. On non-reducing gels the
protein runs as a single high molecular
weight band. When reduced, ICln is seen
at its predicted molecular mass of 42 kDa.
For comparison, lymphocytes (Lymph)
and HUT78 cells, under reducing (R
HUT) and non-reducing (NR HUT), con-
ditions were probed in parallel. Both cell
types have a single reactive band ob-
served at 42 kDa. C, distribution of ICln
(green) relative to
␣
IIb

3
(red) in human
platelets. Measurement bars represent 2
m. Gel-filtered platelets adhered to fi-
brinogen-coated glass slides (20
g/ml)
were examined by confocal microscopy on
a Zeiss LSM510 microscope using a 63⫻
oil immersion lens. High zoom images
(upper panel) showing a single platelet
or low zoom images (lower panel) show-
ing multiple platelets are presented.
Data represents a single dataset repre-
sentative of five similar independent
observations.
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tion of
␣
IIb

3
as determined by binding of the activation-de-
pendent antibody PAC-1 (Fig. 4B). The effect of acyclovir on
platelets appears to be integrin-selective, because it has no
effect on secretion measured as CD62P expression (Fig. 4B).
Thus specific inhibition of ICln prevents integrin activation
and inhibits platelet aggregation. This strongly suggests a
functional association between
␣
IIb

3
and ICln in the platelet.
Identification of an Integrin Recognition Motif on ICln—To
identify a potential integrin-recognizing motif in ICln, we
aligned its sequence with that of other putative integrin-bind-
ing proteins, including CIB (12), calreticulin (49), F-actin (50),
caveolin-1 (50, 51), CD9, CD63 (14), CD151 (15), Aup-1, and
CD98 (52) using pairwise alignment of sequences with Clust-
alW or T-coffee paradigms (53). No significant sequence homol-
ogy was identified between the proteins and ICln suggesting
that, if a consensus recognition sequence for binding to the
KVGFFKR motif exists, it is of a non-linear nature. However,
because it is also established that the conserved
␣
-integrin
motif binds specifically to the corresponding

-tail, we aligned
the ICln protein with the

-cytoplasmic tail using T-Coffee at
EMBnet (53). Interestingly, we identified a sequence of six
identical amino acids common to the two proteins (Fig. 5A). The
identified sequence
728
AKFEEE
733
is present in the

-cytoplas-
mic tail of
␣
IIb

3
, between the known
␣
-integrin-recognition
domain LITIHD and the tyrosine phosphorylation motifs 747
and 759.
84
AKFEEE
89
is also present in ICln as part of the
fourth predicted transmembrane domain of the channel pore
(36). This unique sequence is not found elsewhere in the human
genome.
Inhibition of Platelet Function by the Putative Integrin Rec-
ognition Motif on ICln—To ascertain if this sequence has a
functional significance, cell-permeable peptides corresponding
to this sequence, palmitoyl-AKFEEE, were tested in platelet
function assays. Specific, dose-dependent, and complete inhibi-
tion of platelet aggregation in response to thrombin (0.1 unit/
ml; not shown), thrombin receptor-activating peptide (5
M; not
shown) or the thromboxane mimetic, U46619 (10
M) was dem-
onstrated (Fig. 5, Band C). Control peptides have no effect (Fig.
5C). In addition Pal-AKFEEE inhibits PAC-1 binding but not
CD62 P expression on thrombin-activated platelets (data not
shown). Platelet spreading on immobilized fibrinogen is also
inhibited significantly in the presence of 100
MAKFEEE
compared with controls (Fig. 5D). Thus, the AKFEEE motif,
common only to ICln and the

3
integrin, represents an impor-
tant sequence in integrin activation and platelet function.
Whether this effect is mediated via the integrin or the chloride
channel or both is, as yet, unknown.
DISCUSSION
To identify potential integrin-binding proteins that regulate
functional platelet responses, we utilized a novel protein array
technology. Probing a high density array of 37,200 E. coli clones
FIG.3. Association of platelet integrin
␣
IIb

3
and the chloride
channel ICln. A, a specific association between the chloride channel
and
␣
IIb

3
is demonstrated by co-immunoprecipitation of ICln from a
platelet lysate. Anti-Icln-immunoprecipitates were Western-blotted
with an antibody specific to
␣
IIb
, SZ22. Lane 1 shows a commercial
monoclonal antibody (Clone 32) ICln, lane 2 uses beads only with no
primary antibody, and lane 3 is a positive control using SZ22 to precip-
itate
␣
IIb
. The proteins are separated on 7.5% SDS-PAGE and Western
blotted with SZ22 to detect
␣
IIb
. Data represents a single dataset rep-
resentative of three similar independent observations. B, peptide pull-
down assays were performed using biotinylated KVGFFKR (lane 2), or
a control biotin-KAAAAAR peptide (lane 3), or NeutrAvidin beads as a
negative control (lane 1). Samples were separated on 7.5% SDS gels and
transferred to PVDF membranes. The membrane was Western blotted
for ICln. The antibody detected a 42-kDa protein band only in the
KVGFFKR precipitate but not in the KAAAAAR precipitate. Data rep-
resents a single dataset representative of two similar independent
observations. C, the specific interaction between
␣
IIb

3
and ICln was
examined by surface plasmon resonance (BIAcore 3000). The recombi-
nant ICln protein binds in a dose-dependent manner (0.625
Mto 10
M
as indicated) to purified
␣
IIb

3
immobilized on a carboxymethylated
sensor CM5 chip. Data represent a single dataset representative of
three similar independent observations.
FIG.4. Effect of acyclovir on thrombin-activated platelets. A,
platelet aggregation to the thromboxane mimetic U46619 (10
M)is
determined in the absence (1) or presence of decreasing concentrations
of acyclovir, 5 mM(2),1mM(3), or 100
M(4), where platelet aggrega-
tion is observed as an increase in percent light transmission. Data
represent a single dataset representative of three similar independent
observations. B, PAC-1 binding to the integrin
␣
IIb

3
, in the presence of
acyclovir was examined. Resting platelets (light gray histograms)or
platelets treated with 1 unit/ml thrombin alone (black line)orinthe
presence of 5 mMacyclovir with 1 unit/ml thrombin (dark gray line)
were assessed for expression of PAC-1 antibody binding (upper panel)or
CD62P expression (lower panel) as indicated. Data represent a single
dataset representative of four similar independent observations.
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expressing recombinant human proteins with a tagged integrin
peptide probe, we identified 19 clones that represented poten-
tial integrin-binding proteins. Of these, two proteins (three
clones) emerged as novel potential integrin-associated proteins.
The KVGFFKR sequence from the platelet-specific
␣
IIb
cyto-
plasmic tail was used to probe these arrays. This sequence is
not unique to the platelet integrin. It is also present in the
␣
L
and
␣
X
subunits of the

2
-integrin family in polymorphonuclear
lymphocytes but not in
␣
M

2
, which has a KLGFFKR sequence.
Thus, this experiment has the potential to identify leukocyte
proteins that bind to the cytoplasmic tails of the
␣
L
and
␣
X
integrins, in addition to proteins that will bind to the platelet-
specific integrin
␣
IIb
. We therefore examined platelet expres-
sion of the two candidate proteins.
One, the protein ICln, was expressed both at RNA and at a
protein level. ICln, a 42-kDa transmembrane protein, is in-
volved in regulating cell volume (54, 55). It is ubiquitously
expressed in all cell types investigated, and deletion of this
chloride channel results in cell death (36, 55). The other, a
hypothetical protein, MGC, was not expressed in platelets as
determined by PCR analysis of platelet mRNA.
ICln co-immunoprecipitated with platelet
␣
IIb

3
from plate-
let lysates and interacted strongly and specifically with immo-
bilized KVGFFKR-peptide in a pull-down assay. Surface plas-
mon resonance confirmed a specific high affinity interaction
between
␣
IIb

3
and ICln in a cell-free system with a measura-
ble affinity of 82 ⫾24 nM. Functional verification of this inter-
action was achieved through the use of a specific inhibitor of
the ICln channel protein, acyclovir (47). Acyclovir showed dose-
dependent (100
Mto5mM) inhibition of platelet aggregation
and integrin activation, regardless of the agonists used, but
had no effect on integrin-independent indices of platelet acti-
vation such as CD62P expression. This result strongly supports
a functional role for the ICln chloride channel in platelet
aggregation.
A role for chloride channels in platelet responses has previ-
ously been identified. Thrombin receptors activate chloride
fluxes in megakaryocytes causing a rapid and immediate ion
efflux following cell stimulation (56). Menegazzi and colleagues
(33, 57) have further identified a chloride efflux in polymorpho-
nuclear lymphocytes that is directly and reversibly associated
with

2
integrins and suggest a role for intracellular chloride
ions as a second messenger in these cells. Interestingly, poly-
morphonuclear lymphocytes have been demonstrated to re-
spond to activation with a fall in intracellular chloride concen-
trations and a corresponding integrin activation (33, 57).
Although the authors did not identify which precise chloride
channel was responsible for this alteration in chloride homeo-
stasis, they demonstrated that its effect was specific for
␣
L
and
␣
X
integrins (containing KVGFFKR), not the
␣
M
integrin (con-
tains KLGFFKR). This would support a specific interaction of
the common KVGFFKR motif with ICln and indicate that the
di-amino acid sequence prior to the absolutely conserved
GFFKR motif is critical in
␣
-integrin function, as we have
previously indicated (42).
A unique 6-amino acid peptide sequence (AKFEEE) was
identified as being common to the ICln protein and the integrin

3
cytoplasmic tail. This sequence represents a potential inte-
grin-binding domain. No other common linear sequence was
identified between ICln and any other putative integrin-bind-
FIG.5.Effect of AKFEEE on platelet
activation, through aggregation and
spreading. A, AKFEEE is a conserved
motif common only to the transmembrane
region of ICln (ICln) and the

cytoplas-
mic tail (Integrin

3), identified as the red
highlight from a T-Coffee alignment. B,
the effect of the cell-permeable palmitoy-
lated AKFEEE peptide was examined in
platelet aggregation assays in a four-
channel Bio-Data aggregometer. At point
aAKFEEE is added to channel 2 at 80
M, to channel 3 at 50
M, and to channel
4at10
Maggregations. Thrombin alone
(0.2 unit/ml) was added to channel 1 at
this time point. The AKFEEE (2–3)-
treated platelets are then further stimu-
lated with 0.2 unit/ml thrombin at point b
and the effect observed. AKFEEE has no
agonist effects on platelet aggregation but
showed dose-dependent inhibition throm-
bin-induced responses. C, a control palmi-
toylated LEFEEE peptide is added at
point a to channels 1 and 2, and a vehicle
control of 1% Me
2
SO added to channel 3.
All channels are then stimulated with 0.2
unit/ml thrombin at point b, and the effect
observed. D, to determine the effect of
palmitoyl AKFEEE peptide on the
spreading of platelets, a time course of
platelets spreading on fibrinogen-coated
slides (20
g/ml) was carried out on un-
treated platelets, vehicle-treated (meth-
anol 2.2%), and control peptide pal-
ELFNDG (100
M)- or AKFEEE (100
M)-treated platelets. The platelet mor-
phology is altered in the AKFEEE-
treated samples only. Measurement bars
are representative of 2
m. Data repre-
sent a single dataset representative of
three similar independent observations.
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3
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ing proteins identified in the literature, including CIB (12),
calreticulin (59), F-actin (50), caveolin-1 (50, 51), CD9, CD63
(14), CD151 (15), Aup-1, and CD98 (52). Furthermore, the
sequence AKFEEE was found only in ICln and integrin

3
within the entire human genome. If this sequence represents
the functional site of interaction of ICln with
␣
IIb

3
, then we
would expect to observe inhibition of integrin-mediated events
in the platelet if the peptide sequence was supplied in excess.
We have previous experience of delivering synthetic peptide to
the platelet cytoplasm by adding a cell-permeable lipid tail,
palmitate (4). Cell-permeable AKFEEE, but not control peptide
sequences (LEFEEE or ELFNDG), induced a specific, dose-de-
pendent inhibition of platelet aggregation, adhesion, and integrin
activation with no measurable effect on CD62 P expression, an
integrin-independent event. These data suggest that the chloride
channel provides some of the signaling mediated by
␣
IIb

3
, fol-
lowing complex formation between the two proteins.
ICln is not abundantly expressed in platelets. We calculate
that it is present at a ratio of ⬃1:70 relative to
␣
IIb

3
. This was
calculated from the knowledge that there are ⬃55,000
␣
IIb

3
integrin molecules per platelet (25). Using Western blots of
limiting dilutions of known concentrations of recombinant
ICln, and comparing the signal intensity with Western blots of
platelet lysates, we estimated that there are ⬃700 –800 ICln
molecules per platelet. This is entirely dependent on the ability
of antibodies to quantitatively identify protein in platelet ly-
sates (data not shown). However, it is likely that some portion
of the ICln protein may be detergent-insoluble under some
circumstances. Furthermore, the commercial antibody may not
recognize altered conformations of the ICln protein. From non-
reducing gels, we know that ICln can form high molecular
weight, thiol-dependent complexes not seen in other cells. It is
probable that a chloride channel, once activated, will pump
many ions out of the platelet. Because the platelet volume is
small, strict control over such a process would be necessary as
activation of a few ion channels could result in a very signifi-
cant fall in cell volume. The association of a single, or a dimeric,
ICln channel with an integrin cluster may be sufficient to
control the precise ion flux necessary for platelet activation and
spreading. This would be consistent with such a low ratio of
chloride channel proteins relative to the integrin cell adhesion
molecule. In addition surface plasmon resonance shows rapid
association and slow dissociation consistent with a prolonged
opening of a channel.
To address the question of how alterations in intra-platelet
chloride could regulate integrin function, very little informa-
tion is available. NMR analysis of integrin cytoplasmic tails
strongly supports association of membrane-adjacent
␣
-helices
from
␣
- and

-cytoplasmic tails. However, substantial differ-
ences exist in the proposed structures (24, 35). In particular the
models predict binding of

3
to opposite sides of the
␣
IIb
N-
terminal helix. This may reflect the trapping of the peptides in
structures corresponding to differing activation states of the
integrin. The NMR models show marked sensitivity to ionic
interactions and could only be determined in low ionic strength
buffers, indicative of sensitivity in this region to activation
conditions (35). Coordinate activation of an outwardly rectify-
ing chloride channel could produce local alterations in intra-
platelet ionic environment facilitating the change in integrin
tail conformation and mutual recognition sites. We would
therefore suggest that the KVGFFKR motif acts as a recogni-
tion site on
␣
IIb
that can alternatively bind to

3
or ICln
depending on the ionic environment, reflecting alternate inte-
grin activation states. Further investigations will need to be
carried out to determine the complete significance of this
interaction.
The AKFEEE sequence in the

3
cytoplasmic tail has also
been implicated in other integrin activation events. Sampath et
al. (58) shows an inducible interaction of
␣
-actinin with the
AKFEEE homologous region in

2
integrins, but
␣
-actinin
bindsconstitutively tothe

1
-homologousregion. Thusactivation-
dependent conformational changes can critically alter cytoplas-
mic contact in this vital

-integrin area.
In summary, our data identify an interaction between the
integrin
␣
-motif and a ubiquitous chloride channel ICln
through unique use of high density protein expression arrays
and suggest that this interaction can modulate platelet func-
tion. We have demonstrated that the ICln protein is ex-
pressed in platelets. Its inhibition by acyclovir manifests as
specific inhibition of integrin activation and platelet aggre-
gation. We identified a putative recognition domain, AK-
FEEE, on ICln that may mediate interaction with the KVG-
FFKR motif. A cell-permeable peptide corresponding to this
sequence specifically and completely inhibits platelet aggre-
gation and integrin activation without affecting integrin-
independent aspects of platelet function, such as secretion.
We propose that this motif on the

-integrin and ICln may act
as an alternative binding partner for the
␣
-integrin thereby
regulating integrin activation.
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ICln Associates Platelet Integrin
␣
IIb

3
27293
by guest on April 19, 2017http://www.jbc.org/Downloaded from
Harriott, Dolores J. Cahill and Niamh Moran
Deirdre Larkin, Derek Murphy, Dermot F. Reilly, Martha Cahill, Ellen Sattler, Pat
Activation
-Associated Protein, Functionally Regulates Platelet
3
β
IIb
αICln, a Novel Integrin
doi: 10.1074/jbc.M402159200 originally published online April 9, 2004
2004, 279:27286-27293.J. Biol. Chem.
10.1074/jbc.M402159200Access the most updated version of this article at doi:
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