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Originally published In Press as doi:10.1074/jbc.M402159200 on April 9, 2004

J. Biol. Chem., Vol. 279, Issue 26, 27286-27293, June 25, 2004
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ICln, a Novel Integrin {alpha}IIb{beta}3-Associated Protein, Functionally Regulates Platelet Activation*

Deirdre Larkin{ddagger}, Derek Murphy§, Dermot F. Reilly{ddagger}, Martha Cahill{ddagger}, Ellen Sattler{ddagger}, Pat Harriott¶, Dolores J. Cahill§, and Niamh Moran{ddagger}||

From the {ddagger}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

Received for publication, February 26, 2004 , and in revised form, April 8, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
A critical role for the conserved {alpha}-integrin cytoplasmic motif, KVGFFKR, is recognized in the regulation of activation of the platelet integrin {alpha}IIb{beta}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 peptide, biotin-KVGFFKR, to probe a high density protein expression array (37,200 recombinant human proteins) for high affinity interactions. A number of potential integrin-binding proteins were identified. One such protein, a chloride channel regulatory protein, ICln, was characterized further because its affinity for the integrin peptide was highest as was its expression in platelets. We verified the presence of ICln in human platelets by PCR, Western blots, immunohistochemistry, and its co-association with {alpha}IIb{beta}3 by surface plasmon resonance. The affinity of this interaction was 82.2 ± 24.4 nM in a cell free assay. ICln co-immunoprecipitates with {alpha}IIb{beta}3 in platelet lysates demonstrating that this interaction is physiologically relevant. Furthermore, immobilized KVGFFKR peptides, but not control KAAAAAR peptides, specifically extract ICln from platelet lysates. Acyclovir (100 µM to 5 mM), a pharmacological inhibitor of the ICln chloride channel, specifically inhibits integrin activation (PAC-1 expression) and platelet aggregation 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 µM) also inhibits platelet function. Thus, we have identified, verified, and characterized a novel functional interaction between the platelet integrin and ICln, in the platelet membrane.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Integrins are heterodimeric cell adhesion molecules composed of {alpha}- and {beta}-subunits that mediate cell-cell and cell-matrix adhesion and coordinate bi-directional signaling events across a cell membrane. The platelet-specific integrin {alpha}IIb{beta}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 conformation. However, an understanding of integrin activation mechanisms has only recently been elaborated.

Activation of {alpha}IIb{beta}3 in response to cellular stimulation involves a conformational change in the large extracellular components of the integrin, possibly coordinated by the endogenous thiol-isomerase activity of the {beta}-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, membrane-adjacent KXGFFKR motif in {alpha}-integrins results in constitutive integrin activation (2, 3). Furthermore, we and others have shown that addition of exogenous cell-permeable peptides corresponding to this sequence, KVGFFKR or KLGFFKR, directly activates integrins {alpha}IIb{beta}3 or {alpha}2{beta}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 {beta}-cytoplasmic tail can modulate integrin activation state (6, 7). It has been demonstrated that the membrane-adjacent cytoplasmic regions of the integrin {alpha}- and {beta}-subunits form a salt bridge that facilitates their interaction in resting integrins (8). Cellular activation 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). However, the nature of the cytosolic stimulus that initiates integrin activation following cellular activation is not understood. Because 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 {alpha}-actinin (11), cytosolic proteins such as CIB (12), or {beta}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 potassium currents, leading to hyperpolarization of monocytes and enhanced Ca2+ influx (30). Integrin co-localization with ion channels has been observed in LOX tumor cells where {beta}1-integrins associate laterally with potassium channels during adhesion contributing to the regulation of integrin function in these cells (31). Co-localization of {beta}1-integrins with the {alpha}-subunits of Na,K-ATPase, the epithelial sodium channel, and the voltage-activated calcium channel has also been shown (23). However, indirect evidence for integrin modulation of specific chloride ion currents comes from studies that demonstrate that conformational change of {beta}2-integrins in polymorphonuclear leukocytes is inhibited by hypertonic saline (32) and is dependent on altered chloride fluxes in these cells (33). In addition, CLCA (chloride channel, calcium activatable) protein has been identified as ligands for {beta}4-integrins facilitating tumor metastasis. This interaction involves transcellular contacts of extracellular 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 indicates that ICln lacks the transmembrane helices necessary for channel-pore formation by most vertebrates. When expressed in Xenopus oocytes, it mediates a nucleotide-sensitive outwardly rectifying chloride channel closely resembling the biophysical 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 associated with the cell membrane following a hypotonic challenge. Thus, physiological cell swelling stimulates cytosol to membrane transposition of ICln (21). However, controversy arises over this hypothesis (38). Furthermore, reconstitution of recombinant ICln into lipid bilayers yields a cationic permeability (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 cytoplasmic binding partners by screening a large human recombinant expression library for specific interactions with a tagged synthetic 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 {alpha}IIb{beta}3. Pharmacological modulators of ICln function specifically to modify integrin-mediated platelet responses, verifying a physiological role for ICln in platelet activation events. Because ICln is thought to have many functional roles, we propose that one such role is in the regulation of the specific platelet integrin {alpha}IIb{beta}3 through the KVGFFKR motif. In addition, a novel synthetic peptide, identified as a potential integrin-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
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
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 (accession no. AF074376 [GenBank] ) in E. coli strain SCS-1, which permits IPTG2-inducible expression of His6-tagged fusion proteins directly on a membrane (43, 44). The arrays consist of 2 PVDF membranes (22.2 x 22.2 cm, Millipore) containing a total of 37,200 clones arrayed in duplicate in a 5 x 5 pattern. The membranes are incubated overnight on 2YT agar (supplemented with 100 µg/ml ampicillin, 15 µg/ml kanamycin, and 2% glucose), and recombinant protein expression is induced on 2YT agar containing 1 mM IPTG for 3–4 h. Next, the membranes are transferred onto pre-soaked blotting paper (Whatman 3MM) with denaturing solution (0.5 M NaOH, 1.5 M NaCl) for 10 min to lyse the bacterial cells, followed by 5-min incubation in neutralizing solution (1 M Tris-HCl, pH 7.5, 1.5 M NaCl) and 15 min in 2x 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 mM Tris-HCl, pH 7.4, 0.5 M NaCl, 0.05% Tween 20, 0.5% Triton X-100). The protein array filters were incubated with 100 µM biotin-labeled KVGFFKR or KAAAAAR peptide in TBST (20 mM Tris-HCl, pH 7.4, 0.5 M NaCl, 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 Typhoon 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 mM IPTG. Bacteria were lysed by a freeze-thaw cycle and incubated in 1 ml of lysis buffer (50 mM Tris, 300 mM NaCl, pH 8.0, 10 mM imidazole, 1 mM phenylmethylsulfonyl fluoride, 0.6 mg/ml lysozyme 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 µl/ml lysate). The purification of the proteins was verified by SDS-PAGE.

Dot Blot Verification of Protein-Peptide Interactions—The 19 proteins 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 {alpha}-biotin secondary antibody (1:25,000). CIB (gift of Dr. Nelly Kieffer, Luxembourg) was used as a positive control, to ascertain the efficiency of the peptide interactions. Antibody detection was analyzed with SuperSignal according to the manufacturer's instructions (Pierce).

RNA Isolation and Purification from Platelets—1 ml of Tri reagent was added to 108 platelets from a highly purified platelet preparation (45) and allowed to lyse on ice for 15 min to 1 h. 1-Bromo-3-chloropropane (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 x g at 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 x g for 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 x g for 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 assessed 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 µg) was denatured at 65 °C for 10 min and incubated at 37 °C overnight in the presence of 1x reverse transcription buffer, 1 µl of Moloney murine leukemia virus reverse transcriptase, 25 µM deoxycholate triphosphates (dNTPs), and 1 µl of random hexamers in a final volume of 8 µl. The reaction was stopped by heating at 95 °C for 5 min. A final assay volume of 50 µl containing 1.5 mM MgCl2, 250 µM dNTPs, 10 pmol/µl of each sense ICln (5'-GAAGACAGTGATGATGATGTTGAACC-3') and antisense primer ICln (5'-TTCCACATCATATTCTTCTCCATCGTA-3'), 2 units of TaqDNA polymerase (Promega), and 4 µl 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 µg/ml ethidium bromide and visualized on an ultraviolet transilluminator.

Surface Plasmon Resonance—Real-time binding and kinetic analyses were performed at the University of Reading on a BIAcore 3000 biosensor system (Amersham Biosciences Biosensor AB) using surface plasmon resonance measurements. Purified {alpha}IIb{beta}3 (1 µM, Calbiochem) was immobilized on carboxymethylated sensor chips (type CM5) in 10 mM sodium 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 µM to 10 µM) in 150 mM NaCl, 100 mM HEPES (pH 7.4), 0.05% Surfactant p20, 1.8 mM CaCl2, and 0.15 mg/ml bovine serum albumin under a continuous flow of 5 µl/min at 25 °C. 30 µl of ICln-containing solutions were flowed over the surface of the chip for 2 min. Binding of ICln to {alpha}IIb{beta}3-immobilized flow cells was corrected for binding to control flow cells. Flow cells were regenerated with running buffer, following removal of ICln from {alpha}IIb{beta}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 Biosystems automated peptide synthesizer (model 432A, Norwalk, CT) using a standard solid-phase Fmoc (n-(9-fluorenyl)methoxycarbonyl) procedure, 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-permeable 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 µM U46619 [GenBank] , 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 mM Tris, pH 7.4, 50 mM NaCl, 2 mM CaCl2, 2 mM MgCl2, 1 mM Na3VO4, 1% Brij-35, protease inhibitor mixture (Calbiochem, 500 µM 4-(2-aminoethyl)benzenesulfonylfluoride hydrochloride, 500 µM EDTA, 1 µM E-64, 1 µM leupeptin, 1 µg/ml aprotinin)). Aliquots of platelet lysates were incubated with a monoclonal antibody to ICln (1 µg, Clone 32, BD Transduction laboratories) or a control IgG overnight, then immunoprecipitated with blocked protein-G-Sepharose beads for 3 h, washed extensively in ice-cold buffers. Proteins binding to the beads were removed by boiling in 1x (2% SDS, 50 µM dithiothreitol, 125 mM Tris, pH 6.8, 20% glycerol, 0.0004% bromphenol blue) sample buffer, and separated on 7.5% SDS-PAGE before being transferred to PVDF membranes and Western-blotted with SZ22, an antibody detecting {alpha}IIb. Peptide pull-down assays were performed in an identical manner using a biotinylated KVGFFKR peptide (350 µM), a control biotinylated KAAAAAR peptide (350 µM), and 40 µl 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 x 103/µl were pre-treated with acyclovir (up to 5 mM) or Me2SO vehicle control (up to 0.5%) for 1 min before activation with 1 unit/ml thrombin, or 10 µM U46619 [GenBank] 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 µl, Molecular Probes) and fixed in 0.1% formaldehyde. A methanol vehicle control was used for the AKFEEE and ELFNDG peptides. Platelet-associated fluorescence was estimated on a FACSCalibur flow cytometer.

Immunohistochemistry—Gel-filtered platelets (50 x 103/µl) were adhered to fibrinogen (20 µg/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 Laboratories) and the SZ22 (Immunotech) antibodies were directly labeled prior to use. The platelets were probed simultaneously for ICln and {alpha}IIb{beta}3 or stained for polymerized actin using Alexa 488-phalloidin (Molecular Probes) in the platelet time course. Slides were viewed using a Zeiss LSM501 confocal microscope.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
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 transmembrane domain of the platelet {alpha}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 corresponding 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 redundant 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 interactions. 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 proteins. The veracity of the technique was displayed by the specificity 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 biotin 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 hypothetical protein "MGC" (clone 6), demonstrated highly specific binding (Fig. 1B). Calcium-integrin-binding protein (CIB) identified from a yeast-two-hybrid screen with the {alpha}-cytoplasmic tail is included as a positive control at equimolar concentrations and binds KVGFFKR weakly in comparison to both ICln and MGC.



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  		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 x 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 membranes 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.

 
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 {alpha}-integrin subunits; {alpha}IIb, found exclusively in platelets, and {alpha}L and {alpha}X, found in neutrophils. Because the expression library on the protein array is derived from human fetal brain, the observed interactions, though specific, might not be physiologically 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 stringently prepared by repeated differential centrifugation and analyzed for white cell contamination by estimating T-cell receptor 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{alpha}. Only platelet preparations that lacked T-cell receptor and CD45 and expressed strong positive bands for GPIb{alpha} 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 {alpha}IIb{beta}3 interaction with ICln in platelets.



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  		FIG. 2.
Identification of the chloride channel ICln in human platelets. A, ICln was amplified by RT-PCR from human platelets (PLT) as a 168-bp product 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), conditions were probed in parallel. Both cell types have a single reactive band observed at 42 kDa. C, distribution of ICln (green) relative to {alpha}IIb{beta}3 (red) in human platelets. Measurement bars represent 2 µm. Gel-filtered platelets adhered to fibrinogen-coated glass slides (20 µg/ml) were examined by confocal microscopy on a Zeiss LSM510 microscope using a 63x oil immersion lens. High zoom images (upper panel) showing a single platelet or low zoom images (lower panel) showing multiple platelets are presented. Data represents a single dataset representative of five similar independent observations.

 
Western blotting and immunofluorescent microscopy confirmed the presence of ICln in platelets using commercial monoclonal antibodies (Clone 32) (Fig. 2, B and C) and polyclonal 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 lysates 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 {alpha}IIb{beta}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 ascertain if there is co-localization with {alpha}IIb{beta}3 due to the low abundance of ICln. To confirm the association of ICln with {alpha}IIb{beta}3 in platelets, therefore, we needed to use more sensitive techniques. Two types of co-precipitation studies were carried out. First, immunoprecipitations using a monoclonal antibody to ICln (Clone 32) demonstrated that {alpha}IIb{beta}3 co-precipitated with ICln from platelet lysate (Fig. 3A). Second, to confirm the specificity of interaction with the peptide motif, a biotin-peptide pull-down assay was performed using immobilized KVG-FFKR or control KAAAAAR peptides to extract platelet proteins. Western blots of these precipitates for ICln showed a selective interaction of ICln with the KVGFFKR peptide verifying 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 peptide motif and confirms that the interaction between ICln and {alpha}IIb{beta}3 is physiological and occurs in platelets.



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  		FIG. 3.
Association of platelet integrin {alpha}IIb{beta}3 and the chloride channel ICln. A, a specific association between the chloride channel and {alpha}IIb{beta}3 is demonstrated by co-immunoprecipitation of ICln from a platelet lysate. Anti-Icln-immunoprecipitates were Western-blotted with an antibody specific to {alpha}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 precipitate {alpha}IIb. The proteins are separated on 7.5% SDS-PAGE and Western blotted with SZ22 to detect {alpha}IIb. Data represents a single dataset representative 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 represents a single dataset representative of two similar independent observations. C, the specific interaction between {alpha}IIb{beta}3 and ICln was examined by surface plasmon resonance (BIAcore 3000). The recombinant ICln protein binds in a dose-dependent manner (0.625 µM to 10 µM as indicated) to purified {alpha}IIb{beta}3 immobilized on a sensor CM5 chip. Data carboxymethylated represent a single dataset representative of three similar independent observations.

 
Finally, surface plasmon resonance analysis of the direct interactions of ICln with purified preparations of {alpha}IIb{beta}3 confirmed a specific and saturable, high affinity interaction between the two proteins in a cell-free system (Fig. 3C). The calculated affinity (Kd) 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 [GenBank] , a thromboxane mimetic (Fig. 4A). Acyclovir causes a dose-dependent inhibition of platelet aggregation by these agonists and, in parallel, inhibits activation of {alpha}IIb{beta}3 as determined by binding of the activation-dependent 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 {alpha}IIb{beta}3 and ICln in the platelet.



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  		FIG. 4.
Effect of acyclovir on thrombin-activated platelets. A, platelet aggregation to the thromboxane mimetic U46619 [GenBank] (10 µM) is determined in the absence (1) or presence of decreasing concentrations of acyclovir, 5 mM (2), 1 mM (3), or 100 µM (4), where platelet aggregation 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 {alpha}IIb{beta}3, in the presence of acyclovir was examined. Resting platelets (light gray histograms) or platelets treated with 1 unit/ml thrombin alone (black line) or in the presence of 5 mM acyclovir 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.

 
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-binding 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 homology 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 {alpha}-integrin motif binds specifically to the corresponding {beta}-tail, we aligned the ICln protein with the {beta}-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 728AKFEEE733 is present in the {beta}-cytoplasmic tail of {alpha}IIb{beta}3, between the known {alpha}-integrin-recognition domain LITIHD and the tyrosine phosphorylation motifs 747 and 759. 84AKFEEE89 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.



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  		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 {beta} cytoplasmic tail (Integrin {beta}3), identified as the red highlight from a T-Coffee alignment. B, the effect of the cell-permeable palmitoylated AKFEEE peptide was examined in platelet aggregation assays in a four-channel Bio-Data aggregometer. At point a AKFEEE is added to channel 2 at 80 µM, to channel 3 at 50 µM, and to channel 4 at 10 µM aggregations. Thrombin alone (0.2 unit/ml) was added to channel 1 at this time point. The AKFEEE (2–3)-treated platelets are then further stimulated 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 thrombin-induced responses. C, a control palmitoylated LEFEEE peptide is added at point a to channels 1 and 2, and a vehicle control of 1% Me2SO 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 µg/ml) was carried out on untreated platelets, vehicle-treated (methanol 2.2%), and control peptide pal-ELFNDG (100 µM)- or AKFEEE (100 µM)-treated platelets. The platelet morphology is altered in the AKFEEE-treated samples only. Measurement bars are representative of 2 µm. Data represent a single dataset representative of three similar independent observations.

 
Inhibition of Platelet Function by the Putative Integrin Recognition 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 inhibition of platelet aggregation in response to thrombin (0.1 unit/ml; not shown), thrombin receptor-activating peptide (5 µM; not shown) or the thromboxane mimetic, U46619 [GenBank] (10 µM) was demonstrated (Fig. 5, B and 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 µM AKFEEE compared with controls (Fig. 5D). Thus, the AKFEEE motif, common only to ICln and the {beta}3 integrin, represents an important 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
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
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 expressing recombinant human proteins with a tagged integrin peptide probe, we identified 19 clones that represented potential integrin-binding proteins. Of these, two proteins (three clones) emerged as novel potential integrin-associated proteins. The KVGFFKR sequence from the platelet-specific {alpha}IIb cytoplasmic tail was used to probe these arrays. This sequence is not unique to the platelet integrin. It is also present in the {alpha}L and {alpha}X subunits of the {beta}2-integrin family in polymorphonuclear lymphocytes but not in {alpha}M{beta}2, which has a KLGFFKR sequence. Thus, this experiment has the potential to identify leukocyte proteins that bind to the cytoplasmic tails of the {alpha}L and {alpha}X integrins, in addition to proteins that will bind to the platelet-specific integrin {alpha}IIb. We therefore examined platelet expression 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 involved 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 {alpha}IIb{beta}3 from platelet lysates and interacted strongly and specifically with immobilized KVGFFKR-peptide in a pull-down assay. Surface plasmon resonance confirmed a specific high affinity interaction between {alpha}IIb{beta}3 and ICln in a cell-free system with a measurable affinity of 82 ± 24 nM. Functional verification of this interaction was achieved through the use of a specific inhibitor of the ICln channel protein, acyclovir (47). Acyclovir showed dose-dependent (100 µM to 5 mM) inhibition of platelet aggregation and integrin activation, regardless of the agonists used, but had no effect on integrin-independent indices of platelet activation 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 previously 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 polymorphonuclear lymphocytes that is directly and reversibly associated with {beta}2 integrins and suggest a role for intracellular chloride ions as a second messenger in these cells. Interestingly, polymorphonuclear lymphocytes have been demonstrated to respond to activation with a fall in intracellular chloride concentrations and a corresponding integrin activation (33, 57). Although the authors did not identify which precise chloride channel was responsible for this alteration in chloride homeostasis, they demonstrated that its effect was specific for {alpha}L and {alpha}X integrins (containing KVGFFKR), not the {alpha}M integrin (contains 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 {alpha}-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 {beta}3 cytoplasmic tail. This sequence represents a potential integrin-binding domain. No other common linear sequence was identified between ICln and any other putative integrin-binding 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 {beta}3 within the entire human genome. If this sequence represents the functional site of interaction of ICln with {alpha}IIb{beta}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-dependent 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 {alpha}IIb{beta}3, following 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 {alpha}IIb{beta}3. This was calculated from the knowledge that there are ~55,000 {alpha}IIb{beta}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 lysates (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 significant 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 information is available. NMR analysis of integrin cytoplasmic tails strongly supports association of membrane-adjacent {alpha}-helices from {alpha}- and {beta}-cytoplasmic tails. However, substantial differences exist in the proposed structures (24, 35). In particular the models predict binding of {beta}3 to opposite sides of the {alpha}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 rectifying 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 recognition site on {alpha}IIb that can alternatively bind to {beta}3 or ICln depending on the ionic environment, reflecting alternate integrin activation states. Further investigations will need to be carried out to determine the complete significance of this interaction.

The AKFEEE sequence in the {beta}3 cytoplasmic tail has also been implicated in other integrin activation events. Sampath et al. (58) shows an inducible interaction of {alpha}-actinin with the AKFEEE homologous region in {beta}2 integrins, but {alpha}-actinin bindsconstitutivelytothe{beta}1-homologousregion. Thusactivation-dependent conformational changes can critically alter cytoplasmic contact in this vital {beta}-integrin area.

In summary, our data identify an interaction between the integrin {alpha}-motif and a ubiquitous chloride channel ICln through unique use of high density protein expression arrays and suggest that this interaction can modulate platelet function. We have demonstrated that the ICln protein is expressed in platelets. Its inhibition by acyclovir manifests as specific inhibition of integrin activation and platelet aggregation. We identified a putative recognition domain, AKFEEE, on ICln that may mediate interaction with the KVGFFKR motif. A cell-permeable peptide corresponding to this sequence specifically and completely inhibits platelet aggregation and integrin activation without affecting integrin-independent aspects of platelet function, such as secretion. We propose that this motif on the {beta}-integrin and ICln may act as an alternative binding partner for the {alpha}-integrin thereby regulating integrin activation.


   FOOTNOTES
 
* 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. Back

|| To whom correspondence should be addressed. Tel.: 353-1-402-2153; Fax: 353-1-402-2453; E-mail: nmoran{at}rcsi.ie.

1 BD Transduction Laboratories, pICln data sheet, available at www.bdbioscience.com (2001). Back

2 The abbreviations used are: IPTG, isopropyl-1-thio-{beta}-D-galactopyranoside; MGC, a hypothetical protein; ICln, Homo sapiens nucleotide-sensitive chloride channel (CLNS1a, P54105 [GenBank] ); CIB, calcium integrin-binding protein; CD62P, P-selectin; PVDF, polyvinylidene difluoride; RT, reverse transcription; Back



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