Divalent Cations Differentially Regulate Integrin alpha IIb Cytoplasmic Tail Binding to beta 3 and to Calcium- and Integrin-binding Protein*

Laurent VallarDagger , Chantal MelchiorDagger , Sébastien PlançonDagger , Hervé Drobecq§, Guy Lippens§, Véronique Regnault, and Nelly KiefferDagger parallel

From the Dagger  Laboratoire Franco-Luxembourgeois de Recherche Biomédicale (CNRS and CRP-Santé), Centre Universitaire, L-1511 Luxembourg, Grand Duchy of Luxembourg, the § Institut de Biologie de Lille, Laboratoire de RMN, F-59000 Lille, France, and the  Laboratoire d'Hématologie, UMR CNRS 7563, Faculté de Médecine, F-54500 Vandoeuvre-lès-Nancy, France

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We have used recombinant or synthetic alpha IIb and beta 3 integrin cytoplasmic peptides to study their in vitro complexation and ligand binding capacity by surface plasmon resonance. alpha ·beta heterodimerization occurred in a 1:1 stoichiometry with a weak KD in the micromolar range. Divalent cations were not required for this association but stabilized the alpha ·beta complex by decreasing the dissociation rate. alpha ·beta complexation was impaired by the R995A substitution or the KVGFFKR deletion in alpha IIb but not by the beta 3 S752P mutation. Recombinant calcium- and integrin-binding protein (CIB), an alpha IIb-specific ligand, bound to the alpha IIb cytoplasmic peptide in a Ca2+- or Mn2+-independent, one-to-one reaction with a KD value of 12 µM. In contrast, in vitro liquid phase binding of CIB to intact alpha IIbbeta 3 occurred preferentially with Mn2+-activated alpha IIbbeta 3 conformers, as demonstrated by enhanced coimmunoprecipitation of CIB with PAC-1-captured Mn2+-activated alpha IIbbeta 3, suggesting that Mn2+ activation of intact alpha IIbbeta 3 induces the exposure of a CIB-binding site, spontaneously exposed by the free alpha IIb peptide. Since CIB did not stimulate PAC-1 binding to inactive alpha IIbbeta 3 nor prevented activated alpha IIbbeta 3 occupancy by PAC-1, we conclude that CIB does not regulate alpha IIbbeta 3 inside-out signaling, but rather is involved in an alpha IIbbeta 3 post-receptor occupancy event.

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Integrins are alpha beta heterodimeric cell-surface receptors that promote not only adhesion to components present within the extracellular matrix or on the surface of opposite cells but also transfer information into and out of a cell (1). The adhesive functions of integrins can be regulated by intracellular processes referred to as "inside-out signaling." Conversely, ligand binding to the extracellular domain of integrins initiates a cascade of intracellular events termed "outside-in signaling" that generate a large spectrum of cellular responses, such as cell migration, proliferation, differentiation, and gene expression (2). Integrin cytoplasmic tails appear to be key elements in these bidirectional signaling pathways, despite their short size as compared with other signaling receptors and the absence of any demonstrable catalytic activity (3, 4). Integrin alpha  and beta  cytoplasmic domains are thought to mediate signaling events through modifications of their own structural and spatial organization and/or through interactions with specific cytoplasmic components. Various proteins have been identified that bind, at least in vitro, to the cytoplasmic tail of alpha  and beta  subunits and are likely to play a role in regulating integrin signaling functions. These include cytoskeletal components such as talin and alpha -actinin, as well as several signaling or regulatory proteins such as integrin-linked kinase p59ILK, focal adhesion kinase pp125FAK, Grb2, beta 3-endonexin, cytohesin-1, integrin cytoplasmic domain-associated protein ICAP-1, calreticulin and calcium- and integrin-binding protein CIB1 (reviewed in Refs. 5 and 6).

Recently used methods for studying protein-protein interactions, such as the two-hybrid system, have allowed the identification of integrin-specific intracellular ligands (7-12). These methods are based on the use of a unique linear amino acid sequence as a bait and consequently do not take into account the secondary and tertiary structural features of the interacting molecules. However, numerous studies tend to demonstrate that alpha  and beta  cytoplasmic domains adopt a defined conformation and that the preservation of these structural constraints is crucial to maintain the functional properties of integrin receptors (13-20).

One of the best studied integrins is the platelet fibrinogen receptor, integrin alpha IIbbeta 3, that undergoes conformational changes necessary for receptor function. In order to elucidate further the structural relationship of the cytoplasmic tails of alpha IIb and beta 3, we have used surface plasmon resonance biosensor technology to monitor real time assembly of the integrin alpha IIb and beta 3 cytoplasmic tails and to investigate their ligand binding capacity.

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Antibodies and Synthetic Peptides-- The anti-beta 3 monoclonal antibody (mAb) 4D10G3, the anti-beta 3 cytoplasmic domain mAb C3a.19.5, and the anti-alpha IIb mAb S1.3 were kindly provided by Dr. D. R. Phillips (Cor Therapeutics, South San Francisco, CA), the anti-beta 3 mAb D3GP3 by Dr. L. K. Jennings (University of Tennessee, Memphis, TE), and the anti-alpha IIbbeta 3 complex-specific mAb 10E5 by Dr. B. S. Coller (Mount Sinai School of Medicine, New York, NY). The anti-alpha IIbbeta 3 mAb PAC-1 was from Becton-Dickinson (San Jose, CA), and the anti-human alpha v mAb VNR 139 was from Life Technologies, Inc. (Merelbeke, Belgium). Polyclonal anti-alpha IIbbeta 3 antibodies were raised in rabbits against purified human platelet alpha IIbbeta 3. Synthetic peptides corresponding to either the wild type alpha IIb cytoplasmic domain (alpha IIb Lys989-Gln1008), the alpha IIb cytoplasmic sequence with a R995A substitution (alpha IIb R995A), or the alpha IIb sequence deleted of the 989KVGFFKR995 motif (alpha IIb Asn996-Gln1008) were all purchased from Neosystem (Strasbourg, France).

Platelets and Cell Lines-- Outdated platelet concentrates were kindly provided by Dr. J.-C. C. Faber (Luxembourg Red Cross Blood Transfusion Center). The stable transfected CHO cell line A06, expressing high levels of human alpha vbeta 3 integrin (21), was grown in Iscove's modified Dulbecco's medium, and HEL-5J20 cells in RPMI medium (Life Technologies) (22). Culture medium was supplemented with glutamine, penicillin, and streptomycin, and 10% heat-inactivated fetal calf serum. The adherent CHO cells were routinely passaged with EDTA buffer, pH 7.4 (1 mM EDTA, 126 mM NaCl, 5 mM KCl, 50 mM Hepes).

Construction of pGEX-4T-2 Expression Plasmids-- The cDNA encoding the wild type or S752P mutant human beta 3 integrin cytoplasmic tail (Lys716-Thr762) was generated by the polymerase chain reaction (PCR) using full-length pBJ1-beta 3 plasmids as templates (21). The upstream (sense) primer was a 28-mer with a BamHI site (Gdown-arrow GATCC) corresponding to the beta 3 nucleotide sequence 2245-2266, 5'-GGATCCAAACTCCTCATCACCATCCACG-3'. The downstream (antisense) primer was a 30-mer corresponding to the beta 3 nucleotide sequence 2365-2388 and comprising an SmaI restriction site (CCCdown-arrow GGG) followed by a stop codon 5'-CCCGGGTTAAGTGCCCCGGTACGTGATATT-3'. PCR amplification was performed using the Takara PCR kit (Shiga, Japan). The full-length cDNA encoding human CIB was obtained by reverse transcriptase-PCR (RT-PCR) of HEL-5J20 cell mRNA. Briefly, total RNA was isolated from 5 × 106 cells according to the method of Chomczynski and Sacchi (23), and RT-PCR was performed using the RNA-PCR kit from Promega (Madison, WI). The sense primer was a 36-mer corresponding to the published CIB nucleotide sequence 1-30 (12) with an additional BamHI restriction site (Gdown-arrow GATCC), 5'-GGATCCATGGGGGGCTCGGGCAGTCGCCTGTCCAAG-3'. The downstream antisense primer was a 32-mer corresponding to the CIB nucleotide sequence 551-576 followed by a stop codon and an EcoRI restriction site (Gdown-arrow GATTC), 5'-GGATTCTCACAGGACAATCTTAAAGGAGCTGG-3'. All the primers used to generate cDNA fragments were obtained from Life Technologies, Inc. PCR and RT-PCR products were purified using the PCR Preps DNA Purification System from Promega. They were digested with the corresponding restriction enzymes and inserted into the glutathione S-transferase (GST) vector pGEX-4T-2 (Amersham Pharmacia Biotech, Uppsala, Sweden) containing a thrombin cleavage site. The expected nucleotide sequence was confirmed for each construct by direct sequencing using the T7 sequencing kit from Amersham Pharmacia Biotech.

Expression and Purification of Recombinant Fusion Proteins-- Native GST and in-frame GST fusion proteins were expressed in Escherichia coli JM105 (Amersham Pharmacia Biotech) according to the manufacturer's instructions. Bacterial pellets were suspended in PBS (140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.4) containing 10 mM EDTA and 50 µM aminoethylbenzenesulfonyl fluoride and were incubated for 30 min at room temperature in the presence of 200 µg/ml lysozyme. The bacterial suspension was submitted to short bursts of sonication and further treated with 1% Triton X-100 for 30 min at 4 °C. The insoluble material was pelleted by a 20-min centrifugation at 10,400 × g at 4 °C. Supernatants were filtered on a 0.8-µm membrane and submitted to affinity chromatography on a glutathione-Sepharose 4B (Amersham Pharmacia Biotech) column (100 × 20-mm inner diameter) previously equilibrated with PBS. Bound fusion proteins were eluted with 10 mM reduced glutathione in 50 mM Tris-HCl, pH 8.0. GST-beta 3 and GST-beta 3(S752P) fusion proteins were further purified by immunoaffinity chromatography using the anti-beta 3 cytoplasmic tail mAb C3a.19.5 immobilized on CNBr-activated Sepharose CL4B (Amersham Pharmacia Biotech) in order to eliminate free GST.

Proteolytic Cleavage of GST Fusion Proteins and Purification of Recombinant Products-- Cleavage of recombinant CIB or wild type beta 3 cytoplasmic tail peptide from GST was achieved by incubating 2 NIH units of thrombin protease (Amersham Pharmacia Biotech) per mg of purified material under gentle stirring for 5 h at room temperature. beta 3 peptide was purified by preparative reverse-phase high performance liquid chromatography (HPLC) using a C4 column (100 × 20-mm inner diameter) with a 0-40% linear gradient of acetonitrile in 0.05% trifluoroacetic acid. The amino acid sequence of the recombinant peptide was checked by microsequencing on an Applied Biosystems Procise sequencer. The purified peptide was stored dessicated at 4 °C. For CIB purification, the thrombin hydrolysate was extensively dialyzed against PBS and then passed through a glutathione-Sepharose column in order to remove GST. The flow-through fraction containing CIB was kept frozen at -20 °C until use.

Preparation of alpha IIbbeta 3- or alpha vbeta 3-enriched Glycoprotein Concentrates-- For alpha IIbbeta 3-enriched glycoprotein concentrates, outdated platelets were washed and then lysed in 150 mM NaCl, 1 mM CaCl2, 1 mM MgCl2, 20 mM Tris-HCl (TBS), pH 7.4, containing 1% Triton X-100, 10 µM leupeptin, 500 µM PMSF, 2 mM N-ethylmaleimide, 0.02% NaN3, according to the procedure described by Fitzgerald et al. (24). The lysate was applied to a concanavalin A (ConA)-Sepharose 4B (Amersham Pharmacia Biotech) column (100 × 20-mm inner diameter) equilibrated with TBS, pH 7.0, 0.1% Triton X-100 (TBS/ConA), and bound glycoproteins were eluted with 100 mM alpha -methyl-D-mannose in running buffer. For alpha vbeta 3-enriched glycoprotein concentrates, alpha vbeta 3-expressing CHO cells (cell clone A06) were detached with EDTA buffer, pH 7.4, for 10 min at 37 °C and then washed in cold PBS. Cells (5-9 × 106) were lysed for 30 min in 500 µl of ice-cold lysis buffer, pH 7.5 (150 mM NaCl, 50 µM aminoethylbenzenesulfonyl fluoride, 1% Triton X-100, 10 mM Tris-HCl), and the lysate was precleared by centrifugation at 10,000 × g for 10 min at 4 °C. The supernatant was incubated for 2 h at 4 °C with 1 volume of 50% slurry ConA-Sepharose 4B suspension. After extensive washes with cold TBS/ConA, bound glycoproteins were eluted as described above and kept on ice until use.

alpha IIbbeta 3 Binding to Immobilized Human Fibrinogen-- alpha IIbbeta 3 binding to fibrinogen was determined according to Kouns et al. (25) with some modifications. 96-well microtiter plates (Costar, Cambridge, MA) were coated overnight at 4 °C with 100 µl/well purified human fibrinogen (Sigma, Bornem, Belgium) at 5 µg/ml in TBS. The plates were then saturated with TBS containing 3.5% bovine serum albumin and 0.05% NaN3 (125 µl/well) overnight at 4 °C. The ConA-enriched platelet glycoprotein fraction was serially diluted in TBS/ELISA alone (TBS containing 1% bovine serum albumin, 0.035% Triton X-100) or in TBS/ELISA containing either 2 µg/ml D3GP3 mAb or 10 mM MnCl2. 100-µl aliquots were added to the wells and incubated 4 h at room temperature. After three washes with TBS, 1 µg/ml polyclonal rabbit anti-alpha IIbbeta 3 antibodies in TBS/ELISA were added (100 µl/well) for 2 h at room temperature. The wells were washed three times with TBS, followed by 90 min incubation at room temperature with 100 µl/well donkey anti-rabbit Ig antibodies conjugated to horseradish peroxidase (Amersham Pharmacia Biotech). After three washes with TBS, 100 µl of 0.1 mg/ml 3,3',5,5'-tetramethylbenzidine, 0.01% H2O2 in 140 mM sodium acetate-citrate buffer, pH 6.0, were dispensed into each well. The enzymatic reaction was stopped by addition of 25 µl of 2 M H2SO4, and the absorbance was measured at 450 nm.

In Vitro Liquid Phase alpha IIbbeta 3-CIB Binding Assays-- ConA-purified platelet glycoproteins (250 µg) or alpha vbeta 3-enriched CHO-A06 cell glycoproteins (1 mg) were incubated with 50 µg of purified GST-CIB or GST alone for 2 h at 4 °C under gentle stirring. Experiments were carried out in ice-cold 0.1% Triton X-100, 150 mM NaCl, 50 mM Tris-HCl, pH 7.4, containing either 2 mM CaCl2, 2.5 mM EGTA, or 1 mM CaCl2, 1 mM MgCl2 ± 10 mM MnCl2. In Mn2+-related assays, platelet glycoproteins were first preincubated for 30 min at room temperature in 10 mM MnCl2 or in Mn2+-free buffer before incubation with GST fusion proteins. For alpha IIbbeta 3 capture, the mixtures were incubated with 5 µg of the 10E5 mAb for 2 h at 4 °C or, alternatively, with 4 µg of the mAb PAC-1 for 2 h at room temperature followed by 8.5 µg of rabbit anti-mouse µ-chain-specific antibodies (Jackson Immunoresearch, West Grove, PA) for an additional 2 h at 4 °C. Protein A-Sepharose CL 4B beads (80 µl of a 50% slurry suspension) were added and incubated for 2 h at 4 °C. For GST fusion protein capture, the mixtures were incubated for 2 h at 4 °C with 80 µl of 50% slurry glutathione-Sepharose 4B suspension. Control experiments were performed using non-substituted Sepharose CL4B. The adsorbents were washed three times with 500 µl of the respective ice-cold incubation buffer, and the captured proteins were recovered by boiling the beads in 30-50 µl of 5% beta -mercaptoethanol, 2% SDS, 10% glycerol, 25 µg/ml bromphenol blue in 15.625 mM Tris-HCl, pH 6.8. Each sample was analyzed by SDS-polyacrylamide gel electrophoresis (PAGE) followed by immunoblotting as described below.

Protein Assay, Electrophoresis, and Western Blot Analysis-- Protein concentration was determined using the Bio-Rad Protein assay reagent. SDS-PAGE was performed using the mini-Protean II electrophoresis system (Bio-Rad), and Tris-Tricine SDS-PAGE was carried out according to the method described by Schagger and von Jagow (26). Electrophoresed samples were transferred onto Hybond-C nitrocellulose membrane (Amersham Pharmacia Biotech) using a semi-dry transblot apparatus (Amersham Pharmacia Biotech). The membranes were blocked overnight in blotting buffer (5% dry milk, 0.1% Tween 20, 150 mM NaCl, 20 mM Tris-HCl, pH 7.4), and incubated for 2 h with the anti-beta 3 4D10G3 mAb mixed with either the anti-alpha IIb S1.3 mAb or the anti-alpha v VNR 139 mAb diluted in blotting buffer. After three washes in blotting buffer, membranes were incubated for 1 h with diluted sheep anti-mouse Ig conjugated to horseradish peroxidase (Amersham Pharmacia Biotech). Membranes were again washed three times in blotting buffer and then in 137 mM NaCl, 20 mM Tris-HCl, pH 7.4 (TBS/WB), and developed using the chemiluminescence ECL kit (Pierce) according to the manufacturer's instructions. The membranes were then stripped by successive washes in TBS/WB containing 100 mM beta -mercaptoethanol, 2% SDS, 52.5 mM Tris-HCl, pH 6.7, for 30 min at 50 °C, and again in TBS/WB. After an overnight incubation in blotting buffer, the membranes were reprobed for 2 h with polyclonal goat anti-GST antibodies (Amersham Pharmacia Biotech), and antibody binding was detected as described above with horseradish peroxidase-conjugated rabbit anti-goat IgG (Jackson Immunoresearch).

Surface Plasmon Resonance Binding Studies-- Real time biomolecular interaction analysis was performed using the BialiteTM or Biacore XTM instruments (Biacore, Uppsala, Sweden). Purified proteins were covalently attached to carboxymethyl dextran (CM5) chips (Biacore) previously activated with a mixture of N-hydroxysuccinimide and N-ethyl-N'-dimethylaminopropyl carbodiimide according to the manufacturer's instructions. Experiments were performed at 25 °C using as running buffers either Biacore HBS (150 mM NaCl, 3.4 mM EDTA, 0.005% Tween 20, 10 mM Hepes, pH 7.4) or TBS/Bia (150 mM NaCl, 0.005% Tween 20, 50 mM Tris-HCl, pH 7.4) ± 2 mM CaCl2. The sensorchips were regenerated with a short pulse of either 200 mM glycine HCl, pH 2.2 (anti-GST antibody-coated chip), or 10 mM HCl (GST fusion protein-derivatized chip). The amount of analyte bound to the immobilized ligand was monitored by measuring the variation of the surface plasmon resonance angle as a function of time. Results were expressed in resonance units (RU), an arbitrary unit specific for the Biacore instrument (1000 RU correspond to approximately 1 ng of bound protein/mm2 and are recorded for a change of 0.1° in resonance angle) (27). The transformation of crude data, the preparation of overlay plots, and the determination of kinetic parameters of the binding reactions were performed using the Biaevaluation 3.0 software. The association rate constant (kon) and the dissociation rate constant (koff) were determined separately from individual association and dissociation phases, respectively, assuming a one-to-one interaction. The affinity constant KD was calculated as koff/kon. Experimental values from the first 20 s at the beginning of each phase were not considered in the fitting to avoid distortions due to injection and mixing.

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Generation of Recombinant Wild Type or Mutant beta 3 Integrin Cytoplasmic Peptides and of CIB-- In order to perform in vitro studies of alpha IIb and beta 3 cytoplasmic tail association, we generated recombinant GST fusion proteins containing a thrombin cleavage site and corresponding to the entire wild type or S752P mutant cytoplasmic domain of the beta 3 integrin (residues 716-762) and to the alpha IIb-binding protein CIB (residues 1-191). The fusion proteins were isolated from bacterial cell lysates by glutathione affinity chromatography, and the GST-beta 3 proteins were further immunopurified using the anti-beta 3 monoclonal antibody (mAb) C3a.19.5. Thrombin-released wild type beta 3 cytoplasmic tail peptide and CIB were purified free of GST using reverse-phase HPLC and glutathione affinity chromatography, respectively. The accurate amino acid sequence of the beta 3 peptides was confirmed by microsequencing. SDS-PAGE analysis of isolated proteins revealed a purity greater than 98%, as evaluated by densitometric scanning of the gel (Fig. 1). The apparent molecular masses of both GST-beta 3 and GST-beta 3 (S752P) (34 kDa), GST-CIB (48 kDa), CIB (25 kDa), and GST (29 kDa) were in good agreement with the predicted mass deduced from their amino acid composition. When analyzed with higher resolutive SDS-PAGE and on a C18 HPLC column, the 5.6-kDa beta 3 peptide appeared homogeneous with only slight impurities (Fig. 2).


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Fig. 1.   SDS-PAGE analysis of purified GST fusion proteins. Proteins were electrophoresed under reducing conditions in a 12.5% Tris glycine SDS gel, and stained with Coomassie Brilliant Blue: lane 1, immunoaffinity purified GST-beta 3, and lane 2, GST-beta 3(S752P); lane 3, thrombin digest of GST-beta 3; lane 4, purified recombinant beta 3 cytoplasmic domain peptide; lane 5, native GST; lane 6, affinity isolated GST-CIB before, and lane 7, after thrombin proteolysis; lane 8, purified CIB after removal of GST on a glutathione-Sepharose affinity column; lane 9, molecular mass standards.


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Fig. 2.   Analysis of purified wild type beta 3 cytoplasmic domain peptide. The purity of the recombinant beta 3 peptide was checked by reverse-phase HPLC using an analytical C18 column (50 × 4.6-mm inner diameter) with a 0-40% linear gradient of acetonitrile in 0.05% trifluoroacetic acid and by 15% Tris-Tricine SDS-PAGE. The position of the 5.6-kDa beta 3 cytoplasmic peptide is indicated by an arrow. AU, absorbance arbitrary units (214 nm).

In Vitro Complex Formation of alpha IIb and beta 3 Cytoplasmic Domains as Monitored by SPR Analysis-- In vitro complex formation of wild type alpha IIb and beta 3 integrin cytoplasmic tails was investigated by surface plasmon resonance (SPR) in order to monitor real time biomolecular interactions. We first examined whether the synthetic alpha IIb peptide was able to bind to the beta 3 cytoplasmic tail fused to GST. Purified anti-GST polyclonal antibodies were immobilized on a sensor chip through amine coupling and were allowed to stably capture native GST or the GST-beta 3 fusion protein before injection of the alpha IIb peptide. As shown in Fig. 3A, a characteristic binding signal was monitored when the peptide was brought into contact with captured GST-beta 3 but not with either an uncoated surface, immobilized antibodies alone, or GST-antibody complexes. In these latter control experiments, the rapid change in the resonance signal was due to a dilution buffer-induced, nonspecific change in the bulk refractive index. The maximum response monitored at the end of the peptide injection phase was about 60-70 RU for 1100 RU of initially captured GST-beta 3 protein. The corresponding molar ratio was estimated at ~0.8 mol/mol and was consistent with a 1:1 interaction. Further studies showed that alpha IIb binding was dose-dependent and could be almost completely inhibited by soluble GST-beta 3 protein but not by GST alone (Fig. 3, B and C). Taken together, these data demonstrate that alpha IIb specifically interacts with the beta 3 cytoplasmic tail and that this interaction can be monitored using SPR technology.


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Fig. 3.   SPR analysis of the interaction between alpha IIb and beta 3 cytoplasmic tails. The experimental sensorgrams were recorded on a Bialite apparatus using TBS/Bia as running buffer and a flow rate of 5 µl/min (except for B which is 50 µl/min). Data are given as absolute responses. A, response curves recorded during and after injection of 41.7 µM alpha IIb peptide on an uncoated surface (curve 1) or on a chip with immobilized polyclonal anti-GST antibodies before (curve 4) and after capture of ~1100 RU of GST (curve 3) or GST-beta 3 (curve 2). GST/antibody interaction was very stable over time with a decrease in signal of only 1-2 RU/min. B, overlaid dose-response binding curves obtained with various concentrations of alpha IIb peptide injected over a GST-beta 3-coated chip surface (~12,500 RU). C, interaction of covalently immobilized GST-beta 3 (~12,500 RU) with 41.7 µM of alpha IIb peptide preincubated with 1.5 mg/ml free purified GST (curve 2) or GST-beta 3 (curve 1). The binding curve obtained in the absence of the competitor is shown as a reference (curve 3).

The alpha IIbbeta 3 Cytoplasmic Domain Complex Is Stabilized by Divalent Cations-- In order to determine the role of divalent cations in integrin alpha IIbbeta 3 cytoplasmic domain association, we investigated the binding of alpha IIb to immobilized GST-beta 3 in the presence of 2 mM CaCl2 or MgCl2. The influence of Ca2+ on the interaction was apparent from the slopes of the sensorgrams in Fig. 4. Interestingly, the rates of association of alpha IIb with GST-beta 3 were similar, independent of the presence or absence of Ca2+. In contrast, the dissociation of alpha IIb was more rapid in the absence of Ca2+, as demonstrated by a greater slope in the curve. Similar results were obtained with a low cation concentration (50 µM) or with a Mg2+-containing buffer (data not shown). To characterize further the dynamic parameters of the interaction, we determined binding isotherms in the presence or absence of Ca2+ by injecting alpha IIb peptide solutions ranging from 4.2 to 83.3 µM over a GST-beta 3 fusion protein-coated chip (Fig. 3B). From these curves, the association and dissociation rates and the apparent KD of the binding were determined as indicated under "Experimental Procedures." As shown in Table I, the on rates with or without Ca2+ were very similar. In contrast, the peptide dissociation was slower when Ca2+ ions were present in the flow as compared with Ca2+-free buffer. This resulted in an increased affinity, although the KD value was still in the range of weak interactions. Taken together, these data suggest that divalent ions have a different effect on the association and dissociation of alpha IIb and beta 3 cytoplasmic tails.


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Fig. 4.   Effect of Ca2+ on the interaction of soluble alpha IIb peptide with immobilized GST-beta 3 fusion protein. Sensorgrams were collected during and after the injection of 41.7 µM alpha IIb peptide at a flow rate of 5 µl/min using a Bialite apparatus and Ca2+-free TBS/Bia as running buffer. Peptide dilutions were prepared either in running buffer or in TBS/Bia complemented with 2 mM CaCl2. In Ca2+-related experiments, the Ca2+-containing buffer was injected during the dissociation phase to replace the running buffer (arrow). Data are expressed as absolute responses. The sensor chip used was the same as in Fig. 3, B and C.

                              
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Table I
Kinetics of alpha IIb peptide interaction with GST-beta 3 fusion protein as a function of [Ca2+]
Experiments were performed on a Bialite instrument using a flow rate of 50 µl/min and TBS/Bia buffer ± 2 mM CaCl2 as running and dilution buffer. The association (kon) and dissociation (koff) rate constants were generated using the Biaevaluation analysis software, from data recorded for a range of synthetic alpha IIb peptide concentrations (4.2-83.3 µM) injected over a GST-beta 3 fusion protein-coated surface (~12,500 RU). The affinity constants (KD) were calculated as koff/kon from curve-fitting analysis as described under "Experimental Procedures." The kinetic data presented are the mean values ± S.D. of at least two separate experiments.

In Vitro alpha /beta Heterodimerization Is Impaired by the R995A Substitution or the KVGFFKR Deletion within the alpha IIb Cytoplasmic Tail but Not by the S752P Substitution in the beta 3 Cytoplasmic Domain-- Several mutations or deletions within the alpha IIb and beta 3 cytoplasmic tails have been shown to disturb alpha IIbbeta 3-mediated signaling, such as the alpha IIb (R995A) substitution, the alpha IIb membrane-proximal GFFKR truncation, or the beta 3 (S752P) point mutation (15, 28, 29). To investigate the influence of these mutations on the in vitro alpha ·beta complexation, a chip coated with GST-beta 3 was used. The binding curves obtained following the injection of equimolar solutions of alpha IIb (R995A) or alpha IIb (Asn996-Gln1008) peptides were strongly reduced as compared with the curve monitored with wild type alpha IIb (Fig. 5A). Residual binding calculated from these curves 20 s after the end of the sample injection, using wild type alpha IIb peptide binding as a 100% reference, were only ~15% for the substituted mutant and ~20% for the deleted mutant. In contrast, sensorgrams obtained when wild type alpha IIb peptide was injected over GST-beta 3 or GST-beta 3 (S752P) were superimposable (Fig. 5B). The presence of 2 mM Ca2+ in the running buffer only slightly improved alpha IIb mutant binding to GST-beta 3 (22% residual binding for both). Calcium stabilized the interaction of the wild type alpha IIb peptide with GST-beta 3 (S752P) in the same way as with GST-beta 3, and kinetic parameters determined for the alpha IIb/GST-beta 3 (S752P) interaction in the absence or presence of 2 mM CaCl2 were essentially the same as those obtained with wild type beta 3 (data not shown). These results demonstrate that the alpha IIb membrane-proximal KVGFFKR sequence and the alpha IIb residue Arg995 are crucial for the formation of alpha ·beta complexes, as well as their subsequent stabilization by divalent cations. In contrast, the S752P mutation in the beta 3 cytoplasmic tail does not affect alpha ·beta dimerization and stabilization, suggesting that the beta 3 C-terminal part is not involved in these processes.


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Fig. 5.   SPR analysis of the binding properties of alpha IIb and beta 3 cytoplasmic tail mutants. Sensorgrams were monitored on a Biacore X instrument using Ca2+-free TBS as running and dilution buffer and a flow rate of 20 µl/min. Data are expressed as relative responses after subtraction of the background signal recorded on a reference surface made up of ethanolamine-substituted dextran matrix. A, 100 µM peptide solutions of either wild type alpha IIb (curve 1), alpha IIb (R995A) (curve 2), or alpha IIb (Asn996-Gln1008) (curve 3) were passed over a chip with immobilized GST-beta 3 (~7300 RU). B, 50 µM wild type alpha IIb peptide solution was injected over a chip with ~2000 RU of antibody-captured GST-beta 3 (curve 1) or GST-beta 3 (S752P) (curve 2).

Calcium-independent Interaction of CIB with the alpha IIb Cytoplasmic Tail-- We have used purified recombinant CIB as a control reporter protein to monitor alpha IIb cytoplasmic tail ligand binding functions. As shown in Fig. 6A, a binding signal was recorded when the alpha IIb peptide was passed over sensorchips with antibody-captured GST-CIB but not with anti-GST antibodies alone, GST, or ethanolamine-substituted dextran, indicating that the interaction occurred specifically between alpha IIb and CIB. The molar ratio calculated from these curves was ~0.7 mol/mol and was consistent with a 1:1 stoichiometry. Binding experiments performed in the presence or absence of 2 mM CaCl2 in the running buffer showed that CaCl2 did not significantly modify either the association or the dissociation phase (Fig. 6B), demonstrating that the alpha IIb interaction with CIB was Ca2+-independent, despite the fact that CIB is a Ca2+-binding protein (12). The on and off rates, and the apparent KD, calculated as described previously from overlaid sensorgrams, are summarized in Table II. As predicted from the sensorgrams in Fig. 6B, all the dynamic parameters were essentially the same, independent of the presence or absence of Ca2+, and revealed a weak affinity of 12 µM.


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Fig. 6.   Characterization by SPR analysis of the interaction between alpha IIb cytoplasmic domain and CIB. The experiments were performed on a Biacore X instrument using TBS/Bia as running and dilution buffer and a flow rate of 20 µl/min. Data are given as relative responses. A, alpha IIb peptide (41.7 µM) was passed over a chip with immobilized polyclonal anti-GST antibodies before (curve 2) or after capture of ~1300 RU of GST (curve 3) or GST-CIB (curve 1). B, sensorgrams recorded during the interaction of 41.7 µM of alpha IIb peptide with GST-CIB (~1200 RU) previously captured by immobilized anti-GST antibodies. The experiments were performed with TBS/Bia either alone or complemented with 2 mM CaCl2.

                              
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Table II
Kinetics of the interaction of alpha IIb peptide with GST-CIB fusion protein as a function of [Ca2+]
Sensorgrams were collected for a range of alpha IIb peptide concentrations (4.2-83.3 µM) interacting with antibody-captured GST-CIB fusion protein (1200-1400 RU). Measurements were carried out on a Biacore X instrument at a flow rate of 50 µl/min using TBS/Bia ± 2 mM CaCl2 as running and dilution buffer. On and off rates (with KD = koff/kon) were evaluated using Biaevaluation software as described under "Experimental Procedures." Kinetic data presented are the mean values ± S.D. of three separate experiments.

The alpha IIb Cytoplasmic Peptide Can Bind Simultaneously to the beta 3 Cytoplasmic Tail and to CIB-- We further examined whether the binding of the alpha IIb peptide to antibody-captured GST-CIB was influenced by the beta 3 cytoplasmic tail. Samples of each of the two isolated peptides were injected sequentially or simultaneously after an in vitro incubation in the presence or absence of CaCl2, in order to allow alpha ·beta complex formation. Experiments were performed in a Ca2+-free running buffer. As expected from previous results, the alpha IIb peptide always bound to captured GST-CIB when injected alone (Fig. 7A). In contrast, the purified beta 3 peptide did not. Injection of the alpha IIb and beta 3 peptides preincubated in a Ca2+-free buffer led to a response almost similar to that monitored with the alpha IIb peptide alone, whereas the alpha /beta mixture preincubated in the presence of Ca2+ supported an enhanced resonance signal (Fig. 7B). Comparable results were obtained when the experiments were carried out with running buffer containing 2 mM CaCl2 (data not shown). Considering that the SPR response is directly related to the mass of the analyte adsorbed on the chip surface, we conclude from these results that binding of preformed alpha IIb·beta 3 complexes was responsible for the difference observed with the reference signal recorded with the alpha IIb peptide alone. These data indicate that alpha IIb and beta 3 cytoplasmic tails can form a multimolecular complex together with CIB, suggesting that the regions in the alpha IIb amino acid sequence implicated in the interaction with beta 3 and CIB are distinct. Since the alpha IIb membrane-proximal region has been shown to contain the key contact sites for beta 3 binding, CIB interaction is likely to involve the alpha IIb C terminus.


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Fig. 7.   Interaction of the alpha IIb·beta 3 cytoplasmic tail complex with GST-CIB fusion protein. Experiments were carried out on a Biacore X apparatus at a flow rate of 20 µl/min. TBS/Bia without CaCl2 was used as running and dilution buffer. Data are expressed as relative responses. Both alpha IIb and beta 3 peptides were injected at a concentration of 40 µM. Sensorgrams were obtained using an antibody-captured GST-CIB chip surface (~1700-1800 RU) after sequential injection (A) of each of the peptides (curve 1, alpha IIb followed by beta 3; curve 2, beta 3 followed by alpha IIb) or after a single injection (B) of the two peptides previously incubated together for at least 15 min at room temperature with (curve 3) or without 2 mM CaCl2 (curve 4). Sample injections are indicated by arrows.

The Membrane-proximal KVGFFKR Domain and the Arg995 Residue of alpha IIb Cytoplasmic Tail Are Also Required for Optimal alpha IIb Binding to CIB-- To delineate further the contact site of CIB within the alpha IIb cytoplasmic tail, we investigated the binding of alpha IIb (R995A) and alpha IIb (Asn996-Gln1008) peptides to GST-CIB. Interestingly, the sensorgrams obtained with both mutants were markedly reduced, as compared with the binding curve monitored with the wild type alpha IIb peptide (Fig. 8), although residual binding of ~30% persisted for the two mutants independent of the presence or absence of calcium. These results indicate that CIB binding to alpha IIb does not rely exclusively on the C-terminal part of alpha IIb and that the KVGFFKR sequence or the Arg995 residue within alpha IIb are required for optimal binding.


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Fig. 8.   Interaction of the alpha IIb mutant peptides with GST-CIB fusion protein. Experiments were carried out on a Biacore X instrument using TBS/Bia as running and dilution buffer and a flow rate of 20 µl/min. Data are given as relative responses. The sensorgrams were recorded using 50 µM peptide solutions of either wild type alpha IIb (curve 1), alpha IIb(R995A) (curve 2), or alpha IIb (Asn996-Gln1008) (curve 3) injected over a chip with antibody-captured GST-CIB (~1600 RU).

Calcium-independent Binding of CIB to Intact Platelet alpha IIbbeta 3-- To confirm further the calcium-independent interaction of CIB with the alpha IIb cytoplasmic domain, we performed in vitro liquid phase binding assays using intact alpha IIbbeta 3. For this purpose, an alpha IIbbeta 3-rich glycoprotein fraction was prepared from platelets by ConA affinity chromatography. Similarly, an alpha vbeta 3-enriched glycoprotein concentrate was prepared from CHO cells expressing human alpha vbeta 3 for control experiments. SDS-PAGE and Western blot analysis demonstrated that these samples contained appreciable levels of beta 3 integrins (Fig. 9A, lanes 2 and 3). Platelet or CHO-A06 cell glycoproteins were incubated with GST-CIB or GST alone in the presence of 2 mM CaCl2 or 2.5 mM EGTA. GST fusion proteins were recovered using glutathione-Sepharose beads, and bound proteins were analyzed by SDS-PAGE and Western blotting using anti-alpha IIb, anti-alpha v, anti-beta 3, and anti-GST antibodies. As shown in Fig. 9B (panel 1), GST-CIB was able to retain alpha IIbbeta 3 independent of the presence or absence of Ca2+, whereas GST alone was not. In contrast, no alpha vbeta 3 integrin was detected in either GST-CIB- or GST-containing samples incubated with the alpha vbeta 3-enriched fraction (Fig. 9B, panel 2). In competitive inhibition experiments, an excess of free alpha IIb cytoplasmic peptide abrogated alpha IIbbeta 3/GST-CIB coprecipitation (Fig. 9B, panel 3). Taken together, these results demonstrate that CIB binds specifically to intact platelet alpha IIbbeta 3 in a Ca2+-independent manner.


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Fig. 9.   In vitro binding of GST-CIB to platelet alpha IIbbeta 3. A, ConA-purified glycoproteins from either nontransfected CHO cell lysate (100 µg, lane 1), human alpha vbeta 3-expressing CHO-A06 cell lysate (100 µg, lane 2), or alpha IIbbeta 3-rich platelet extract (25 µg, lane 3) were resolved by 7.8% reducing SDS-PAGE (upper panel) and subjected to Western blot analysis using specific anti-beta 3 (4D10G3), anti-alpha IIb (S1.3), or anti-alpha v (VNR 139) mAbs (lower panel). Considering the chemiluminescence signal obtained using an equivalent dilution of anti-beta 3 mAb and the same film exposure time, the level of alpha IIbbeta 3 in the platelet glycoprotein extract was estimated to be ~4 times higher than that of alpha vbeta 3 in the CHO-A06 glycoprotein fraction. The slight difference in the electrophoretic mobility of alpha IIb and alpha v is not apparent in this mini-gel. B, ConA-purified glycoproteins from platelet lysate (250 µg, panel 1) or alpha vbeta 3-expressing CHO-A06 cell lysate (1 mg, panel 2), both containing approximately equal amounts of beta 3 integrins, were incubated with 50 µg of purified GST or GST-CIB in the presence of 2 mM CaCl2 (+) or 2.5 mM EGTA (-). Binding inhibition experiments were performed by adding 25 nmol of alpha IIb cytoplasmic peptide to the platelet glycoproteins/GST-CIB or GST mixture (panel 3). Proteins were captured onto glutathione-Sepharose beads and were analyzed by 7.8% reducing SDS-PAGE and Western blot. The membrane was first probed with anti-beta 3 (4D10G3) mAbs combined with either (panels 1 and 3) anti-alpha IIb (S1.3) or (panel 2) anti-alpha v (VNR 139) mAbs (upper panel), stripped and reprobed with anti-GST antibodies (lower panel). The weak band observed in some GST immunoblot analysis corresponds to an unidentified protein distinct from GST-CIB, as demonstrated by slightly different electrophoretic mobilities.

CIB Binds Preferentially to Mn2+-activated alpha IIbbeta 3-- Since both inactive and active alpha IIbbeta 3 conformers are isolated from platelet lysate by ConA affinity chromatography (30), we determined the activation state of the alpha IIbbeta 3 heterodimers present in our ConA-purified platelet extract by examining the binding capacity of alpha IIbbeta 3 to immobilized fibrinogen. Fig. 10A shows that specific alpha IIbbeta 3 binding was significantly weaker with crude sample than with fractions incubated either with the activating anti-beta 3 mAb D3GP3 or with 10 mM Mn2+, demonstrating that the ConA-purified platelet extract contained essentially inactive alpha IIbbeta 3 and minor amounts of activated alpha IIbbeta 3.


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Fig. 10.   In vitro binding of ConA-purified alpha IIbbeta 3 to immobilized fibrinogen and GST-CIB as a function of MnCl2. A, various concentrations of crude ConA-purified platelet glycoproteins were incubated for 4 h at room temperature with 2 µg/ml D3GP3 mAb (black-triangle), 10 mM MnCl2 (black-square), or buffer alone () in 96-well microtiter plates coated with 5 µg/ml human fibrinogen. After washing, bound alpha IIbbeta 3 was detected by ELISA using rabbit anti-alpha IIbbeta 3 antibodies as stated under "Experimental Procedures." The figure is representative of two independent experiments, each data point corresponding to the mean of duplicates. B, ConA-purified platelet glycoproteins (250 µg) were incubated with 50 µg of purified GST-CIB or GST in the presence (+) or absence (-) of 10 mM MnCl2. Binding inhibition experiment was performed using 25 nmol of alpha IIb cytoplasmic peptide. Proteins were recovered using glutathione-Sepharose beads and were analyzed by 7.8% reducing SDS-PAGE and by Western blot. The membrane was first probed with anti-beta 3 (4D10G3) and anti-alpha IIb (S1.3) mAbs (upper panel), stripped and reprobed with anti-GST antibodies (lower panel). Two nonspecific bands of high electrophoretic mobility were observed with the GST-CIB-containing samples.

To investigate further whether Mn2+-induced activation of platelet alpha IIbbeta 3 was able to influence alpha IIbbeta 3/CIB interaction, we performed in vitro binding studies in the presence or absence of MnCl2. Interestingly, the binding of alpha IIbbeta 3 to GST-CIB was more pronounced in the presence of 10 mM MnCl2 than in Mn2+-free buffer (Fig. 10B). Also, alpha IIbbeta 3 heterodimers were undetectable in competitive assays using an excess of alpha IIb cytoplasmic peptide or in control experiments performed with purified GST alone, demonstrating that the specificity of the interaction was not modified by Mn2+. To confirm further these results, we studied the binding of CIB to platelet alpha IIbbeta 3 as a function of Mn2+ by immunoprecipitation experiments. Crude or Mn2+-treated platelet glycoproteins were first incubated with purified GST-CIB and then with the anti-alpha IIbbeta 3 complex-specific mAb 10E5 to retain total alpha IIbbeta 3, or with the anti-beta 3 fibrinogen-mimetic mAb PAC-1 to capture activated alpha IIbbeta 3. The immunoprecipitates were analyzed by SDS-PAGE and Western blotting using anti-alpha IIb, anti-beta 3, and anti-GST antibodies. As shown in Fig. 11, alpha IIbbeta 3 binding to PAC-1 was weak in the absence of Mn2+ (B, lane 1) and was significantly enhanced in 10 mM MnCl2 buffer (B, lane 2), demonstrating that the activated alpha IIbbeta 3 conformers were increased in the Mn2+-treated platelet glycoprotein concentrate. In Mn2+-free buffer, few GST-CIB coimmunoprecipitated with 10E5-captured alpha IIbbeta 3 (A, lane 3), whereas GST-CIB was not detectable with PAC-1-bound alpha IIbbeta 3, probably on account of the small recovery in alpha IIbbeta 3 obtained with this sample, containing a weak proportion of active heterodimers (B, lane 3). Conversely, Mn2+ treatment of the platelet glycoproteins led to a marked increase in GST-CIB coprecipitation in experiments performed with either 10E5 or PAC-1 mAb (A and B, lane 4). This effect was particularly apparent from the level of GST-CIB coprecipitated in 10E5 assays, which was significantly greater in the presence of 10 mM Mn2+ than in Mn2+-free buffer, although the amount of captured alpha IIbbeta 3 remained unchanged (A, lanes 3 and 4). As expected from our previous assays, addition of alpha IIb cytoplasmic peptides to the incubation mixture almost completely inhibited GST-CIB binding to platelet alpha IIbbeta 3 (A and B, lane 5), and no GST was coprecipitated with alpha IIbbeta 3 (A and B, lane 6). Taken together, these results demonstrate that CIB binds preferentially to the Mn2+-treated, activated form of alpha IIbbeta 3 integrin. These data further suggest that CIB is unlikely to have a regulatory effect on alpha IIbbeta 3 ligand binding function, since its interaction with alpha IIbbeta 3 does not stimulate PAC-1 binding to inactive alpha IIbbeta 3 nor inhibit Mn2+-activated alpha IIbbeta 3 occupancy by PAC-1.


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Fig. 11.   In vitro GST-CIB binding to platelet alpha IIbbeta 3 as a function of MnCl2. ConA-purified platelet glycoproteins (250 µg) were incubated with GST-CIB (50 µg), and alpha IIbbeta 3 complexes were immunoprecipitated using either the anti-alpha IIbbeta 3 mAb 10E5 (A) or the fibrinogen-mimetic mAb PAC-1 (B) and analyzed by SDS-PAGE and Western blot as described in the legend to Fig. 10. Samples were as follows: platelet glycoproteins in Mn2+-free buffer (lane 1) or in 10 mM MnCl2 buffer (lane 2); platelet glycoproteins incubated with GST-CIB in Mn2+-free buffer (lane 3), 10 mM MnCl2 buffer (lane 4), 10 mM MnCl2 buffer containing 25 nmol alpha IIb cytoplasmic peptide (lane 5); platelet glycoproteins incubated with purified GST in 10 mM MnCl2 buffer (lane 6).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

SPR technology has been successfully used by several authors to analyze the interaction of structural domains of receptor cytoplasmic tails with their intracellular targets, such as the interaction of cytoplasmic tail sorting sequences with the adaptor proteins AP-1, AP-2, and AP-3 (31-33) or of growth factor receptors with the Src homology 2 (SH2) or the phosphotyrosine-binding (PTB) domains of tyrosine kinases (34, 35). SPR has also been used to characterize the interaction of various integrin receptors with extracellular matrix proteins (36-38), ligand-mimetic antibodies (39, 40), or with integrin counter-receptors (41). In this report, we have used SPR technology to study in vitro complexation of the alpha - and beta -cytoplasmic tails of integrin alpha IIbbeta 3, as data from mutagenesis, spectroscopic, and computer modeling studies indicate this heterodimerization (14-16, 19). SPR provided a direct and more informative approach to investigate alpha ·beta complexation, since continuous real time monitoring of the interaction allowed a detailed characterization of specificity, affinity, and kinetics. Our experimental data are consistent with a 1:1 interaction that proceeds transiently according to affinity constants (KD) in the micromolar range. These affinities are low when compared with those obtained by SPR for receptor cytoplasmic tails (32, 33, 35) or signaling molecules (42-45) which usually range from 10-7 to 10-9 M. As our kinetic data are close to the limits specified for the BiacoreTM instruments (46), they should be considered as an indication of an order of magnitude rather than definite numerical data. Nevertheless, the affinities found here are in agreement with transient and potentially easily modulatory processes, such as those expected intuitively in signal transduction pathways, and predict that the interactions actually proceed in vivo if the binding partners are locally concentrated. This is easily conceivable for integrin alpha  and beta  cytoplasmic tails which are maintained in close proximity at the cytoplasmic face of the plasma membrane through tight interactions of the integrin extracellular domains.

In contrast to previous studies, in which alpha  or beta  cytoplasmic tail peptides were linked to an helical coiled-coil motif mimicking the transmembrane domain (14, 20), the alpha ·beta intersubunit binding shown here occurs in the absence of any additional structural motifs. Furthermore, no homodimerization of beta 3 peptides was observed in our study, although this process has been recently described in cells transfected with a beta 3 cytoplasmic tail connected to an alpha -helical domain (47), suggesting that transmembrane domains are necessary for integrin oligomerization but not for alpha ·beta cytoplasmic tail complexation. Interestingly, our data demonstrate that the S752P substitution in beta 3 did not affect alpha ·beta heterodimerization, indicating that the C-terminal part of the beta 3 cytoplasmic tail is not involved in this process. As the beta 3(S752P) mutation has previously been shown to disrupt bidirectional signaling in platelets and transfected CHO cells (28, 29), our results suggest that this defect cannot be attributed to an alteration in alpha ·beta cytoplasmic tail interaction but rather to a disruption in a specific beta 3 interaction with intracellular regulatory molecules. Actually, the beta 3 (S752P) mutation has been shown to markedly reduce beta 3-endonexin-specific binding to beta 3 and to impair its modulating effect on the alpha IIbbeta 3 affinity state (8, 48). Our data further indicate that the alpha IIb membrane-proximal region is critical for alpha ·beta heterodimer assembly, as both alpha IIb (R995A) and alpha IIb (Asn996-Gln1008) peptides failed to interact efficiently with the beta 3 cytoplasmic tail. Our data apparently contradict previous biophysical data which demonstrated that the interactive sites involved in alpha ·beta dimerization are located within the beta 3 Ile721-Asp740 and alpha IIb Pro999-Gln1008 sequences (16). However, it should be noted that their data were primarily derived from terbium luminescence spectroscopy experiments which did not allow investigation of an interaction between the N-terminal regions of the alpha IIb and beta 3 cytoplasmic tails, since neither alpha IIb Leu985-Pro998 nor beta 3 were able to bind Tb3+ ions necessary for the generation of a specific fluorescence energy transfer signal. In contrast, our data are in good agreement with previous mutagenesis studies (15) and also indicate that the alpha IIb KVGFFKR motif is necessary but not sufficient to support alpha ·beta heterodimerization. Indeed, alterations within this sequence did not totally abolish the alpha IIb·beta 3 interaction, suggesting that binding sites distinct from the alpha IIb membrane-proximal domain are involved in beta 3 engagement. These additional contact sites are probably located within the alpha IIb C-terminal acidic tail.

We have also provided direct evidence that Ca2+ and Mg2+ are not required for alpha IIb·beta 3 complexation but rather stabilize the heterodimeric structure by reducing the dissociation rate. Since alpha ·beta association proceeded with the same extent, independent of the presence of cations, the cation coordination sites within alpha IIb and beta 3 cytoplasmic tails are likely to be distinct from those involved in intersubunit binding, namely the membrane-proximal region of each integrin subunit. Interestingly, our study indicates that the alpha IIb membrane-proximal region is involved in divalent cation-induced alpha ·beta complex stabilization, since an alteration in the KVGFFKR sequence impaired the stability of the heterodimers. In support of these results, a functional cation binding domain has previously been mapped to the negatively charged acidic C-terminal region of alpha IIb (residues 999-1008) and was found to bind divalent cations in coordination with sites located in the alpha IIb 985-998 sequence (16, 19). Based on the structural model of alpha IIbbeta 3, Haas and Plow (19) speculated that a cation coordination site rearrangement could occur upon alpha IIb·beta 3 cytoplasmic tail complexation. The faster complex dissociation observed here in the absence of cations actually suggests that additional intra- and/or intersubunit cation coordination site(s) probably appear(s) during the complexation resulting in a more stable structure.

Because CIB is the only intracellular protein identified so far that specifically interacts with the alpha IIb integrin subunit cytoplasmic tail, we used recombinant CIB as a reporter protein to monitor the ligand binding capacity of alpha IIb either as a monomer or as a heterodimer in association with beta 3. Our data clearly demonstrate that CIB interacts with the alpha IIb peptide in a one-to-one, weak affinity reaction (KD = 12 µM) and also binds to the preformed alpha IIb·beta 3 cytoplasmic complex, suggesting that the contact sites within the alpha IIb amino acid sequence involved in the interaction with CIB are distinct from those engaged in beta 3 binding. As CIB has been shown to interact only with alpha IIb and not with alpha v, alpha 2, or alpha 5 in the yeast two-hybrid system, Naik et al. (12) concluded that CIB was unlikely to bind to the highly conserved GFFKR motif common to all alpha  subunits but rather to the highly acidic C-terminal part of the alpha IIb cytoplasmic tail. Our data, however, provide evidence that the KVGFFKR sequence is necessary for optimal CIB·alpha IIb interaction, since both alpha IIb (R995A) and alpha IIb (Asn996-Gln1008) peptides failed to interact efficiently with CIB. In the three-dimensional model of alpha IIbbeta 3, the negatively charged C terminus of the alpha IIb cytoplasmic tail is predicted to fold back onto itself and to interact with the positively charged N terminus through several side chain and backbone contacts (19). Thus, it is conceivable that the deletion of the KVGFFKR sequence or the R995A substitution disrupts some of the intrasubunit contacts that stabilize the alpha IIb conformation, leading to an alteration in the capacity of the cytoplasmic tail peptide to bind to CIB. Interestingly, although CIB has two conserved EF-hand motifs within its protein structure, and has been found to bind Ca2+ in vitro (12), our data demonstrate a Ca2+-independent binding of CIB to the alpha IIb cytoplasmic peptide. This finding indicates that the potential Ca2+-dependent regulatory function of CIB, based on its sequence homology with calmodulin and calcineurin B (12), is not involved in the association with alpha IIb but rather in the activity or in the binding to an as yet unidentified CIB-associated protein. Most strikingly, when binding studies were performed with intact alpha IIbbeta 3, CIB bound preferentially to Mn2+-activated alpha IIbbeta 3, suggesting that the accessibility of the CIB-binding site within alpha IIb is increased in active alpha IIbbeta 3 conformers. Since Mn2+ did not affect the binding characteristics of the GST-CIB·alpha IIb cytoplasmic peptide complex in SPR studies (data not shown), our results suggest that Mn2+ activation of intact alpha IIbbeta 3 induces a conformational change that is transmitted from the alpha IIbbeta 3 extracellular domains to the cytoplasmic tails. Such long range transmembrane structural alterations have been anticipated from studies showing that specific epitopes are exposed or disappear within the cytoplasmic tails of alpha IIb and beta 3 subunits following alpha IIbbeta 3 activation or ligand occupancy (49, 50). Alternatively, an increase in alpha IIbbeta 3 avidity for CIB cannot be excluded, since the fibrinogen-mimetic mAb PAC-1 used to capture active alpha IIbbeta 3 is a multimeric IgM antibody and is thus likely to trigger oligomerization of alpha IIbbeta 3 complexes that mimic integrin clustering (51). Finally, our data provide evidence that CIB is unlikely to have a regulatory effect on alpha IIbbeta 3 ligand binding function, since its interaction with alpha IIb does not trigger ligand binding to inactive alpha IIbbeta 3 nor inhibit activated alpha IIbbeta 3 occupancy by a ligand, suggesting that CIB is most likely involved in alpha IIbbeta 3 post-receptor occupancy events.

    ACKNOWLEDGEMENTS

We thank Dr. J.-C. Mani for critical reading of the manuscript and Drs. B. S. Coller, L. K. Jennings, and D. R. Phillips for their generous gifts of monoclonal antibodies. We are also grateful to Dr. J.-C. Faber for providing outdated platelet concentrates.

    FOOTNOTES

* This work was supported by grants from Centre de Recherche Public-Santé (CRP-Santé, Luxembourg), CNRS (France), Fondation Luxembourgeoise Contre le Cancer (Luxembourg), and EC Biomed-Project Grant BMH4-CT98-3517.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.

parallel To whom correspondence should be addressed: Laboratoire Franco-Luxembourgeois de Recherche Biomédicale, (CNRS and CRP-Santé), Centre Universitaire, 162A Ave. de la Faïencerie, L-1511 Luxembourg, Grand Duchy of Luxembourg. Tel.: 352-466644-440; Fax: 352-466644-442; E-mail: kieffer{at}cu.lu.

    ABBREVIATIONS

The abbreviations used are: CIB, calcium- and integrin-binding protein; CHO, Chinese hamster ovary; ConA, concanavalin A; GST, glutathione S-transferase; HEL, human erythroleukemia; HPLC, high performance liquid chromatography; mAb, monoclonal antibody; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; RT-PCR, reverse transcriptase-polymerase chain reaction; RU, resonance unit; SPR, surface plasmon resonance; TBS, Tris-buffered saline; ELISA, enzyme-linked immunosorbent assay; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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