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Divalent Cations Differentially Regulate Integrin αIIb Cytoplasmic Tail Binding to β3 and to Calcium- and Integrin-binding Protein

1999; Elsevier BV; Volume: 274; Issue: 24 Linguagem: Inglês

10.1074/jbc.274.24.17257

1083-351X

Laurent Vallar, Chantal Melchior, Sébastien Plançon, Hervé Drobecq, Guy Lippens, Véronique Regnault, Nelly Kieffer,

Biochemical and Structural Characterization

We have used recombinant or synthetic αIIb and β3 integrin cytoplasmic peptides to study their in vitro complexation and ligand binding capacity by surface plasmon resonance. α·β heterodimerization occurred in a 1:1 stoichiometry with a weakK D in the micromolar range. Divalent cations were not required for this association but stabilized the α·β complex by decreasing the dissociation rate. α·β complexation was impaired by the R995A substitution or the KVGFFKR deletion in αIIb but not by the β3 S752P mutation. Recombinant calcium- and integrin-binding protein (CIB), an αIIb-specific ligand, bound to the αIIbcytoplasmic peptide in a Ca2+- or Mn2+-independent, one-to-one reaction with aK D value of 12 μm. In contrast,in vitro liquid phase binding of CIB to intact αIIbβ3 occurred preferentially with Mn2+-activated αIIbβ3conformers, as demonstrated by enhanced coimmunoprecipitation of CIB with PAC-1-captured Mn2+-activated αIIbβ3, suggesting that Mn2+activation of intact αIIbβ3 induces the exposure of a CIB-binding site, spontaneously exposed by the free αIIb peptide. Since CIB did not stimulate PAC-1 binding to inactive αIIbβ3 nor prevented activated αIIbβ3 occupancy by PAC-1, we conclude that CIB does not regulate αIIbβ3 inside-out signaling, but rather is involved in an αIIbβ3 post-receptor occupancy event. We have used recombinant or synthetic αIIb and β3 integrin cytoplasmic peptides to study their in vitro complexation and ligand binding capacity by surface plasmon resonance. α·β heterodimerization occurred in a 1:1 stoichiometry with a weakK D in the micromolar range. Divalent cations were not required for this association but stabilized the α·β complex by decreasing the dissociation rate. α·β complexation was impaired by the R995A substitution or the KVGFFKR deletion in αIIb but not by the β3 S752P mutation. Recombinant calcium- and integrin-binding protein (CIB), an αIIb-specific ligand, bound to the αIIbcytoplasmic peptide in a Ca2+- or Mn2+-independent, one-to-one reaction with aK D value of 12 μm. In contrast,in vitro liquid phase binding of CIB to intact αIIbβ3 occurred preferentially with Mn2+-activated αIIbβ3conformers, as demonstrated by enhanced coimmunoprecipitation of CIB with PAC-1-captured Mn2+-activated αIIbβ3, suggesting that Mn2+activation of intact αIIbβ3 induces the exposure of a CIB-binding site, spontaneously exposed by the free αIIb peptide. Since CIB did not stimulate PAC-1 binding to inactive αIIbβ3 nor prevented activated αIIbβ3 occupancy by PAC-1, we conclude that CIB does not regulate αIIbβ3 inside-out signaling, but rather is involved in an αIIbβ3 post-receptor occupancy event. Integrins are αβ 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 (1Hynes O.R. Cell. 1992; 69: 11-25Abstract Full Text PDF PubMed Scopus (9002) Google Scholar). 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 (2Clark E.A. Brugge J.S. Science. 1995; 268: 233-239Crossref PubMed Scopus (2812) Google Scholar). 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 (3Sastry S.K. Horwitz A.F. Curr. Opin. Cell Biol. 1993; 5: 819-831Crossref PubMed Scopus (407) Google Scholar, 4Schwartz M.A. Schaller M.D. Ginsberg M.H. Annu. Rev. Cell Dev. Biol. 1995; 11: 549-599Crossref PubMed Scopus (1467) Google Scholar). Integrin α and β 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 α and β subunits and are likely to play a role in regulating integrin signaling functions. These include cytoskeletal components such as talin and α-actinin, as well as several signaling or regulatory proteins such as integrin-linked kinase p59ILK, focal adhesion kinase pp125FAK, Grb2, β3-endonexin, cytohesin-1, integrin cytoplasmic domain-associated protein ICAP-1, calreticulin and calcium- and integrin-binding protein CIB 1The 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 1The 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 (reviewed in Refs. 5Dedhar S. Hannigan G.E. Curr. Opin. Cell Biol. 1996; 8: 657-669Crossref PubMed Scopus (344) Google Scholar and 6Shattil S.J. Kashiwagi H. Pampori N. Blood. 1998; 91: 2645-2657Crossref PubMed Google Scholar). Recently used methods for studying protein-protein interactions, such as the two-hybrid system, have allowed the identification of integrin-specific intracellular ligands (7Chang D.D. Wong C. Smith H. Liu J. J. Cell Biol. 1997; 138: 1149-1157Crossref PubMed Scopus (149) Google Scholar, 8Shattil S.J. O'Toole T. Eigenthaler M. Thon V. Williams M. Babior B.M. Ginsberg M.H. J. Cell Biol. 1995; 131: 807-816Crossref PubMed Scopus (164) Google Scholar, 9Kolanus W. Nagel W. Schiller B. Zeitlmann L. Godar S. Stockinger H. Seed B. Cell. 1996; 86: 233-242Abstract Full Text Full Text PDF PubMed Scopus (401) Google Scholar, 10Tanaka T. Yamaguchi R. Sabe H. Sekiguchi K. Healy J.M. FEBS Lett. 1996; 399: 53-58Crossref PubMed Scopus (33) Google Scholar, 11Hannigan G.E. Leung-Hagesteijn C. Fitz-Gibbon L. Coppolino M.G. Radeva G. Filmus J. Bell J.C. Dedhar S. Nature. 1996; 379: 91-96Crossref PubMed Scopus (965) Google Scholar, 12Naik U.P. Patel P.M. Parise L.V. J. Biol. Chem. 1997; 272: 4651-4654Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar). 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 α and β cytoplasmic domains adopt a defined conformation and that the preservation of these structural constraints is crucial to maintain the functional properties of integrin receptors (13Filardo E.J. Cheresh D.A. J. Biol. Chem. 1994; 269: 4641-4647Abstract Full Text PDF PubMed Google Scholar, 14Muir T.W. Williams M.J. Ginsberg M.H. Kent S.B.H. Biochemistry. 1994; 33: 7701-7708Crossref PubMed Scopus (82) Google Scholar, 15Hughes P.E. Diaz-Gonzalez F. Leong L. Wu C. McDonald J.A. Shattil S.J. Ginsberg M.H. J. Biol. Chem. 1996; 271: 6571-6574Abstract Full Text Full Text PDF PubMed Scopus (506) Google Scholar, 16Haas T.A. Plow E.F. J. Biol. Chem. 1996; 271: 6017-6026Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 17Wang R.G. Shattil S.J. Ambruso D.R. Newman P.J. J. Clin. Invest. 1997; 100: 2393-2403Crossref PubMed Scopus (102) Google Scholar, 18de Melker A.A. Kramer D. Kuikman I. Sonnenberg A. Biochem. J. 1997; 328: 529-537Crossref PubMed Scopus (28) Google Scholar, 19Haas T.A. Plow E.F. Protein Eng. 1997; 10: 1395-1405Crossref PubMed Scopus (42) Google Scholar, 20Pfaff M. Liu S. Erle D.J. Ginsberg M.H. J. Biol. Chem. 1998; 273: 6104-6109Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar). One of the best studied integrins is the platelet fibrinogen receptor, integrin αIIbβ3, that undergoes conformational changes necessary for receptor function. In order to elucidate further the structural relationship of the cytoplasmic tails of αIIb and β3, we have used surface plasmon resonance biosensor technology to monitor real time assembly of the integrin αIIb and β3 cytoplasmic tails and to investigate their ligand binding capacity. The anti-β3monoclonal antibody (mAb) 4D10G3, the anti-β3 cytoplasmic domain mAb C3a.19.5, and the anti-αIIb mAb S1.3 were kindly provided by Dr. D. R. Phillips (Cor Therapeutics, South San Francisco, CA), the anti-β3 mAb D3GP3 by Dr. L. K. Jennings (University of Tennessee, Memphis, TE), and the anti-αIIbβ3 complex-specific mAb 10E5 by Dr. B. S. Coller (Mount Sinai School of Medicine, New York, NY). The anti-αIIbβ3 mAb PAC-1 was from Becton-Dickinson (San Jose, CA), and the anti-human αvmAb VNR 139 was from Life Technologies, Inc. (Merelbeke, Belgium). Polyclonal anti-αIIbβ3 antibodies were raised in rabbits against purified human platelet αIIbβ3. Synthetic peptides corresponding to either the wild type αIIb cytoplasmic domain (αIIb Lys989–Gln1008), the αIIb cytoplasmic sequence with a R995A substitution (αIIb R995A), or the αIIb sequence deleted of the 989KVGFFKR995 motif (αIIbAsn996–Gln1008) were all purchased from Neosystem (Strasbourg, France). 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 αvβ3integrin (21Kieffer N. Melchior C. Guinet J.M. Michels S. Gouon V. Bron N. Cell Adhes. Commun. 1996; 4: 25-39Crossref PubMed Google Scholar), was grown in Iscove's modified Dulbecco's medium, and HEL-5J20 cells in RPMI medium (Life Technologies) (22Kieffer N. Debili N. Wicki A. Titeux M. Henri A. Mishal Z. Breton-Gorius J. Vainchenker W. Clemetson K.J. J. Biol. Chem. 1986; 261: 15854-15862Abstract Full Text PDF PubMed Google Scholar). 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 mmHepes). The cDNA encoding the wild type or S752P mutant human β3 integrin cytoplasmic tail (Lys716–Thr762) was generated by the polymerase chain reaction (PCR) using full-length pBJ1-β3 plasmids as templates (21Kieffer N. Melchior C. Guinet J.M. Michels S. Gouon V. Bron N. Cell Adhes. Commun. 1996; 4: 25-39Crossref PubMed Google Scholar). The upstream (sense) primer was a 28-mer with a BamHI site (G↓GATCC) corresponding to the β3 nucleotide sequence 2245–2266, 5′-GGATCCAAACTCCTCATCACCATCCACG-3′. The downstream (antisense) primer was a 30-mer corresponding to the β3 nucleotide sequence 2365–2388 and comprising an SmaI restriction site (CCC↓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 (23Chomczynski P. Sacchi N. Anal. Biochem. 1987; 152: 156-159Crossref Scopus (63148) Google Scholar), 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 (12Naik U.P. Patel P.M. Parise L.V. J. Biol. Chem. 1997; 272: 4651-4654Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar) with an additionalBamHI restriction site (G↓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 (G↓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 glutathioneS-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. 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 μmaminoethylbenzenesulfonyl 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-β3 and GST-β3(S752P) fusion proteins were further purified by immunoaffinity chromatography using the anti-β3cytoplasmic tail mAb C3a.19.5 immobilized on CNBr-activated Sepharose CL4B (Amersham Pharmacia Biotech) in order to eliminate free GST. Cleavage of recombinant CIB or wild type β3 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. β3 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. For αIIbβ3-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. (24Fitzgerald L.A. Leung B. Phillips D.R. Anal. Biochem. 1985; 151: 169-177Crossref PubMed Scopus (101) Google Scholar). 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 α-methyl-d-mannose in running buffer. For αvβ3-enriched glycoprotein concentrates, αvβ3-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 mmNaCl, 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. αIIbβ3 binding to fibrinogen was determined according to Kouns et al. (25Kouns W.C. Kirchhofer D. Hadváry P. Edenhofer A. Weller T. Pfenninger G. Baumgartner H.R. Jennings L.K. Steiner B. Blood. 1992; 80: 2539-2547Crossref PubMed Google Scholar) 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-αIIbβ3 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. ConA-purified platelet glycoproteins (250 μg) or αvβ3-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 mmCaCl2, 2.5 mm EGTA, or 1 mmCaCl2, 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 αIIbβ3 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% β-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 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 (26Schagger H. von Jagow G. Anal. Biochem. 1987; 166: 368-379Crossref PubMed Scopus (10470) Google Scholar). 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-β3 4D10G3 mAb mixed with either the anti-αIIb S1.3 mAb or the anti-αv 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 β-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). 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 andN-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) (27Schuck P. Annu. Rev. Biophys. Biomol. Struct. 1997; 26: 541-566Crossref PubMed Scopus (542) Google Scholar). 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 (k on) and the dissociation rate constant (k off) were determined separately from individual association and dissociation phases, respectively, assuming a one-to-one interaction. The affinity constant K Dwas calculated ask off/k on. 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. In order to performin vitro studies of αIIb and β3cytoplasmic 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 β3 integrin (residues 716–762) and to the αIIb-binding protein CIB (residues 1–191). The fusion proteins were isolated from bacterial cell lysates by glutathione affinity chromatography, and the GST-β3 proteins were further immunopurified using the anti-β3 monoclonal antibody (mAb) C3a.19.5. Thrombin-released wild type β3cytoplasmic 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 β3peptides 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-β3 and GST-β3 (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 β3 peptide appeared homogeneous with only slight impurities (Fig. 2).Figure 2Analysis of purified wild type β3 cytoplasmic domain peptide. The purity of the recombinant β3 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 β3 cytoplasmic peptide is indicated by an arrow. AU, absorbance arbitrary units (214 nm).View Large Image Figure ViewerDownload (PPT) In vitro complex formation of wild type αIIb and β3 integrin cytoplasmic tails was investigated by surface plasmon resonance (SPR) in order to monitor real time biomolecular interactions. We first examined whether the synthetic αIIb peptide was able to bind to the β3 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-β3 fusion protein before injection of the αIIb peptide. As shown in Fig.3 A, a characteristic binding signal was monitored when the peptide was brought into contact with captured GST-β3 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-β3 protein. The corresponding molar ratio was estimated at ∼0.8 mol/mol and was consistent with a 1:1 interaction. Further studies showed that αIIb binding was dose-dependent and could be almost completely inhibited by soluble GST-β3 protein but not by GST alone (Fig. 3,B and C). Taken together, these data demonstrate that αIIb specifically interacts with the β3 cytoplasmic tail and that this interaction can be monitored using SPR technology. In order to determine the role of divalent cations in integrin αIIbβ3cytoplasmic domain association, we investigated the binding of αIIb to immobilized GST-β3 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 αIIb with GST-β3 were similar, independent of the presence or absence of Ca2+. In contrast, the dissociation of αIIb 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 αIIb peptide solutions ranging from 4.2 to 83.3 μm over a GST-β3 fusion protein-coated chip (Fig. 3 B). From these curves, the association and dissociation rates and the apparent K D 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 theK D 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 αIIb and β3 cytoplasmic tails.Table IKinetics of αIIb peptide interaction with GST-β3fusion protein as a function of [Ca2+]Bufferk on ± ςk off ± ςK D ± ς102 × (m−1·s−1)10−2 × (s−1)10−6 × (m)Without CaCl2 (n = 3)4.5 ± 1.02.2 ± 1.050.0 ± 21.02 mm CaCl2(n = 2)5.5 (±1.3)0.41 (±0.05)7.7 (±0.9)Experiments were performed on a Bialite instrument using a flow rate of 50 μl/min and TBS/Bia buffer ± 2 mm CaCl2as running and dilution buffer. The association (k on) and dissociation (k off) rate constants were generated using the Biaevaluation analysis software, from data recorded for a range of synthetic αIIbpeptide concentrations (4.2–83.3 μm) injected over a GST-β3 fusion protein-coated surface (∼12,500 RU). The affinity constants (K D) were calculated ask off/k on 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. Open table in a new tab Experiments were performed on a Bialite instrument using a flow rate of 50 μl/min and TBS/Bia buffer ± 2 mm CaCl2as running and dilution buffer. The association (k on) and dissociation (k off) rate constants were generated using the Biaevaluation analysis software, from data recorded for a range of synthetic αIIbpeptide concentrations (4.2–83.3 μm) injected over a GST-β3 fusion protein-coated surface (∼12,500 RU). The affinity constants (K D) were calculated ask off/k on 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. Several mutations or deletions within the αIIb and β3cytoplasmic tails have been shown to disturb αIIbβ3-mediated signaling, such as the αIIb (R995A) substitution, the αIIbmembrane-proximal GFFKR truncation, or the β3 (S752P) point mutation (15Hug