Expression and Function of Calcium Binding Domain Chimeras of the Integrins αIIb and α5
2000; Elsevier BV; Volume: 275; Issue: 9 Linguagem: Inglês
10.1074/jbc.275.9.6680
ISSN1083-351X
AutoresSusan Gidwitz, Suzanne Lyman, Gilbert White,
Tópico(s)Platelet Disorders and Treatments
ResumoTo further identify amino acid domains involved in the ligand binding specificity of αIIbβ3, chimeras of the conserved calcium binding domains of αIIb and the α subunit of the fibronectin receptor α5β1 were constructed. Chimeras that replaced all four calcium binding domains, replaced all but the second calcium binding domain of αIIb with those of α5, or deleted all four calcium binding domains were synthesized but not expressed on the cell surface. Additional chimeras exchanged subsets or all of the variant amino acids in the second calcium binding domain, a region implicated in ligand binding. Cell surface expression of each second calcium binding domain mutant complexed with β3 was observed. Each second calcium binding domain mutant was able to 1) bind to immobilized fibrinogen, 2) form fibrinogen-dependent aggregates after treatment with dithiothreitol, and 3) bind the activation-dependent antibody PAC1 after LIBS 6 treatment. Soluble fibrinogen binding studies suggested that there were only small changes in either the K d orB max of any mutant. We conclude that chimeras of αIIb containing the second calcium binding domain sequences of α5 are capable of complexing with β3, that the complexes are expressed on the cell surface, and that mutant complexes are capable of binding both immobilized and soluble fibrinogen, suggesting that the second calcium binding domain does not determine ligand binding specificity. To further identify amino acid domains involved in the ligand binding specificity of αIIbβ3, chimeras of the conserved calcium binding domains of αIIb and the α subunit of the fibronectin receptor α5β1 were constructed. Chimeras that replaced all four calcium binding domains, replaced all but the second calcium binding domain of αIIb with those of α5, or deleted all four calcium binding domains were synthesized but not expressed on the cell surface. Additional chimeras exchanged subsets or all of the variant amino acids in the second calcium binding domain, a region implicated in ligand binding. Cell surface expression of each second calcium binding domain mutant complexed with β3 was observed. Each second calcium binding domain mutant was able to 1) bind to immobilized fibrinogen, 2) form fibrinogen-dependent aggregates after treatment with dithiothreitol, and 3) bind the activation-dependent antibody PAC1 after LIBS 6 treatment. Soluble fibrinogen binding studies suggested that there were only small changes in either the K d orB max of any mutant. We conclude that chimeras of αIIb containing the second calcium binding domain sequences of α5 are capable of complexing with β3, that the complexes are expressed on the cell surface, and that mutant complexes are capable of binding both immobilized and soluble fibrinogen, suggesting that the second calcium binding domain does not determine ligand binding specificity. fetal bovine serum Chinese hamster ovary K1, DMEM, Dulbecco's modified Eagle medium, high glucose formula phosphate-buffered saline monoclonal antibody fluorescein isothiocyanate bovine serum albumin dithiothreitol Integrins function as adhesion receptors that mediate cell-extracellular matrix, cell-cell, and cell-soluble ligand interactions (1.Hynes R.O. Cell. 1992; 69: 11-25Abstract Full Text PDF PubMed Scopus (9013) Google Scholar). The platelet integrin αIIbβ3 (membrane glycoprotein IIb-IIIa) functions to mediate platelet-platelet and platelet-subendothelial matrix interactions through the binding of its ligands: fibrinogen, von Willebrand factor, thrombospondin, fibronectin, and vitronectin (2.Phillips D.R. Charo I.F. Parise L.V. Fitzgerald L.A. Blood. 1988; 71: 831-843Crossref PubMed Google Scholar, 3.Plow E.F. Ginsberg M.H. Prog. Hemostasis Thromb. 1989; 9: 117-156PubMed Google Scholar). Although progress has been made in elucidating ligand structure and potential ligand binding sites in integrins, questions regarding areas that determine both ligand binding and ligand binding specificity in αIIbβ3 remain. While integrins bind diverse ligands, three themes are apparent in ligand binding. First, many ligands have an acidic amino acid, usually aspartic acid, in the recognition sequence (4.Ginsberg M.H. Xiaoping D. O'Toole T.E. Loftus J.C. Plow E.F. Thromb. Haemostasis. 1993; 70: 87-93Crossref PubMed Scopus (107) Google Scholar, 5.Loftus J.C. Smith J.W. Ginsberg M.H. J. Biol. Chem. 1994; 269: 25235-25238Abstract Full Text PDF PubMed Google Scholar). Certain integrins, including αIIbβ3 and α5β1, bind ligands that contain the tripeptide sequence arginine-glycine-aspartic acid (RGD) (6.Pierschbacher M.D. Ruoslahti E. Nature. 1984; 309: 30-33Crossref PubMed Scopus (2882) Google Scholar), while the recognition motif of others is more varied (7.Humphries M.J. J. Cell Sci. 1990; 97: 585-592Crossref PubMed Google Scholar). Second, the ligand recognition sequence is usually a short peptide presented on an extended loop containing a β-turn (5.Loftus J.C. Smith J.W. Ginsberg M.H. J. Biol. Chem. 1994; 269: 25235-25238Abstract Full Text PDF PubMed Google Scholar). For example, the crystal and NMR structures of the ligand recognition sequence of fibronectin have been determined (8.Dickinson C.D. Veerapandian B. Dai X.P. Hamlin R.C. Xuong N.H. Ruoslahti E. Ely K.R. J. Mol. Biol. 1994; 236: 1079-1092Crossref PubMed Scopus (183) Google Scholar, 9.Main A.L. Harvey T.S. Baron M. Boyd J. Campbell I.D. Cell. 1992; 71: 671-678Abstract Full Text PDF PubMed Scopus (422) Google Scholar), revealing the RGD sequence to be present on an extended loop with a β-turn. Although not as well defined, NMR studies have shown that the fibrinogen C-terminal γ-chain peptide assumes a helical conformation in solution with a β-turn centered at residues 408 and 409 (10.Mayo K.H. Burke C. Lindon J.N. Kloczewiak M. Biochemistry. 1990; 29: 3277-3286Crossref PubMed Scopus (21) Google Scholar, 11.Blumenstein M. Matsueda G.R. Timmons S. Hawiger J. Biochemistry. 1992; 31: 10692-10698Crossref PubMed Scopus (50) Google Scholar). Third, ligand binding specificity is varied. For example, while α5β1 binds primarily to fibronectin, αIIbβ3 is a more promiscuous receptor and binds to multiple ligands. Much work has been done to determine areas of αIIbβ3 involved in ligand binding. Previous studies have implicated multiple ligand interaction areas on both subunits, leading to models in which the ligand binding pocket is proposed to be formed by amino acids contributed by both subunits (12.Rocco M. Spotorno B. Hantgan R.R. Protein Sci. 1993; 2: 2154-2166Crossref PubMed Scopus (20) Google Scholar,13.Calvete J.J. Thromb. Haemostasis. 1994; 72: 1-15Crossref PubMed Scopus (177) Google Scholar). The αIIbβ3 complex must undergo a conformational change before it is capable of high affinity binding of fibrinogen, but the resting complex is still capable of binding small peptide ligands. Site-directed mutagenesis (14.Bajt M.L. Loftus J.C. J. Biol. Chem. 1994; 269: 20913-20919Abstract Full Text PDF PubMed Google Scholar), peptide and antibody inhibition (15.Charo I.F. Nannizzi L. Phillips D.R. Hsu M.A. Scarborough R.M. J. Biol. Chem. 1991; 266: 1415-1421Abstract Full Text PDF PubMed Google Scholar, 16.Cook J.J. Trybulek M. Lasz E.C. Khan S. Niewiarowski S. Biochim. Biophys. Acta. 1992; 1119: 312-321Crossref PubMed Scopus (37) Google Scholar, 17.Calvete J.J. Arias J. Alvarez M.V. Lopez M.M. Henschen A. Gonzalez-Rodriguez J. Biochem. J. 1991; 274: 457-463Crossref PubMed Scopus (49) Google Scholar, 18.Andrieux A. Rabiet M.J. Chapel A. Concord E. Marguerie G. J. Biol. Chem. 1991; 266: 14202-14207Abstract Full Text PDF PubMed Google Scholar), chemical cross-linking studies (19.D'Souza S.E. Ginsberg M.H. Burke T.A. Lam S.C. Plow E.F. Science. 1988; 242: 91-93Crossref PubMed Scopus (292) Google Scholar), and analysis of thrombasthenic mutations (20.Loftus J.C. O'Toole T.E. Plow E.F. Glass A. Frelinger III, A.L. Ginsberg M.H. Science. 1990; 249: 915-918Crossref PubMed Scopus (327) Google Scholar, 21.Lanza F. Stierle A. Fournier D. Morales M. Andre G. Nurden A.T. Cazenave J.-P. J. Clin. Invest. 1992; 89: 1995-2004Crossref PubMed Google Scholar, 22.Bajt M.L. Ginsberg M.H. Frelinger III, A.L. Berndt M.C. Loftus J.C. J. Biol. Chem. 1992; 267: 3789-3794Abstract Full Text PDF PubMed Google Scholar, 23.Newman P.J. Weyerbusch-Bottum S. Visentin G.P. Gidwitz S. White G.C., II. Thromb. Haemostasis. 1993; 69 (abstr.): 1017Google Scholar) have implicated two areas of β3, amino acids 109–133 and 212–222, in ligand binding. Recently, a third area, amino acids 274–368, has been reported to bind fibrinogen (24.Alemany M. Concord E. Garin J. Vincon M. Giles A. Marguerie G. Gulino D. Blood. 1996; 87: 592-601Crossref PubMed Google Scholar). Multiple areas of αIIb appear to be important in ligand binding. A recombinant truncated fragment, amino acids 171–464, has been shown to bind fibrinogen in a calcium-dependent manner (25.Gulino D. Boudignon C. Zhang L.Y. Concord E. Rabiet M.J. Marguerie G. J. Biol. Chem. 1992; 267: 1001-1007Abstract Full Text PDF PubMed Google Scholar), while ligand binding specificity has been localized to the first 334 amino acids of αIIb using αIIbαv chimeras (26.Loftus J.C. Halloran C.E. Ginsberg M.H. Feigen L.P. Zablocki J.A. Smith J.W. J. Biol. Chem. 1996; 271: 2033-2039Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). Site-directed mutagenesis of residues in a predicted β-turn in the third N-terminal repeat of αIIb, α4, and α5(amino acids 184–193 of αIIb) has shown the region to be critical for ligand binding (27.Kamata T. Irie A. Tokuhira M. Takada Y. J. Biol. Chem. 1996; 271: 18610-18615Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 28.Irie A. Kamata T. Puzon-McLaughlin W. Takada Y. EMBO J. 1995; 14: 5550-5556Crossref PubMed Scopus (91) Google Scholar). A synthetic peptide corresponding to αIIb-(656–667) has been demonstrated to bind to soluble fibrinogen, and both the peptide and antibodies to the peptide inhibit platelet aggregation (29.Calvete J.J. Rivas G. Schafer W. McLane M.A. Niewiarowski S. FEBS Lett. 1993; 335: 132-135Crossref PubMed Scopus (12) Google Scholar). Only one area of the αIIbβ3 has been shown to bind the fibrinogen γ-chain HHLGGAKQAGDV (H12) peptide in a calcium-dependent manner. Chemical cross-linking of the H12 peptide followed by proteolysis identified the second calcium binding domain of αIIb, amino acids 296–314, as a critical region for binding the fibrinogen carboxyl-terminal γ-chain peptide (30.D'Souza S.E. Ginsberg M.H. Burke T.A. Plow E.F. J. Biol. Chem. 1990; 265: 3440-3446Abstract Full Text PDF PubMed Google Scholar). High affinity binding of the H12 peptide requires calcium and αIIbβ3 to be in the activated conformation (31.Andrieux A. Hudry-Clergeon G. Ryckewaert J.J. Chapel A. Ginsberg M.H. Plow E.F. Marguerie G. J. Biol. Chem. 1989; 264: 9258-9265Abstract Full Text PDF PubMed Google Scholar). Peptides corresponding to amino acids 296–306 or 300–312 blocked platelet aggregation and bound directly to fibrinogen. Antibodies against αIIb-(296–306) blocked binding of fibrinogen to either the peptide or αIIbβ3(32.D'Souza S.E. Ginsberg M.H. Matsueda G.R. Plow E.F. Nature. 1991; 350: 66-68Crossref PubMed Scopus (150) Google Scholar, 33.Taylor D.B. Gartner T.K. J. Biol. Chem. 1992; 267: 11729-11733Abstract Full Text PDF PubMed Google Scholar). Amino acids 296–312 in αIIb define one of four calcium binding domains of αIIb. The integrin calcium binding domains are homologous with the “EF-hand” helix-loop-helix motif found in calmodulin and other proteins (34.Tuckwell D.S. Brass A. Humphries M.J. Biochem. J. 1992; 285: 325-331Crossref PubMed Scopus (94) Google Scholar, 35.Tuckwell D.S. Humphries M.J. Brass A. Cell Adhes. Commun. 1994; 2: 385-402Crossref PubMed Scopus (35) Google Scholar). However, integrin cation binding domains are missing the flanking helices and one of six divalent cation coordination sites. These coordination sites are presented as a linear array of amino acids having precisely spaced oxygenated side chains at positions 1, 3, 5, 9, and (in true EF-hands) 12. Position 7 contributes a coordination site through its main chain carbonyl. The divalent cation binding domains in integrins are missing the oxygenated, nearly invariant, glutamic acid residue at position 12, having a small hydrophobic residue instead. It has been postulated that the acidic residue in the ligand contributes the final coordination site (4.Ginsberg M.H. Xiaoping D. O'Toole T.E. Loftus J.C. Plow E.F. Thromb. Haemostasis. 1993; 70: 87-93Crossref PubMed Scopus (107) Google Scholar) and that the metal ion, the ligand, and the receptor form a transient ternary complex before the metal ion dissociates (36.D'Souza S.E. Haas T.A. Piotrowicz R.S. Byers-Ward V. McGrath D.E. Soule H.R. Cierniewski C. Plow E.F. Smith J.W. Cell. 1994; 79: 659-667Abstract Full Text PDF PubMed Scopus (204) Google Scholar). The second calcium binding domain of αIIb and α5 occur in a region of high homology, having 80% sequence identity over 35 amino acids. In this paper, we examine the expression, complex formation, and function of a series of chimeric αIIbα5β3 molecules in which we substituted clusters of the divergent α5 amino acids into the second calcium binding domain of αIIb. The mutations were made as chimeras in order to preserve both calcium and RGD binding, while probing ligand binding specificity. The results indicate that these mutants are expressed on the cell surface, complex with β3 but not β1, and function in a manner indistinguishable from wild type αIIbβ3, indicating that the second calcium binding domain of αIIbβ3 does not determine ligand binding specificity. EA.hy 926, an endothelial-adenocarcinoma hybrid cell, λ-gt11 cDNA library was provided by C.-J. Edgell (University of North Carolina, Chapel Hill, NC) (37.Edgell C.J. McDonald C.C. Graham J.B. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 3734-3737Crossref PubMed Scopus (1360) Google Scholar). A partial-length αIIb was the gift of M. Poncz (University of Pennsylvania, Philadelphia, PA). A λ-gt11 liver cDNA library was provided by L. Brass (University of Pennsylvania). Cloned α5 cDNA was provided by R. Juliano (University of North Carolina). Bluescript KS was from Stratagene (San Diego, CA). The plasmids pcDNAI/AMP and pRc/CMV were from Invitrogen (San Diego, CA). Oligonucleotides were obtained from the University of North Carolina Department of Pathology Oligonucleotide Synthesis Facility. The T7-GEN In Vitro Mutagenesis Kit was from U. S. Biochemical Corp. (Cleveland, OH). Horseradish peroxidase-conjugated streptavidin, LipofectAMINE, and tissue culture media and supplements, except for fetal bovine serum (FBS),1 were from Life Technologies, Inc. FBS was from either HyClone (Logan, UT) or Irvine Scientific (Santa Clara, CA). Pefabloc SC was obtained from Roche Molecular Biochemicals. Protein A-Sepharose, GammaBind Plus Sepharose, and gelatin Sepharose were from Amersham Pharmacia Biotech. NHS-LC-biotin was obtained from Pierce. Biotinylated molecular weight markers, gelatin-agarose, and fluorescein isothiocyanate (FITC)-Celite were from Sigma. The ECL chemiluminescence detection system was from Amersham Pharmacia Biotech. Peptides were from the University of North Carolina Protein Chemistry Laboratory. Purified fibrinogen and fibronectin were the gift of L. Parise (University of North Carolina). Human fibrinogen, plasminogen- and von Willebrand factor-depleted, for soluble fibrinogen binding studies, was purchased from Enzyme Research Laboratories, Inc. (South Bend, IN). Chelex 100 was from Bio-Rad. Other reagents were from standard sources. The αIIbβ3complex-specific monoclonal antibody (mAb) AP2 (38.Pidard D. Montgomery R.R. Bennett J.S. Kunicki T.J. J. Biol. Chem. 1983; 258: 12582-12586Abstract Full Text PDF PubMed Google Scholar) was supplied by T. Kunicki (Scripps Research Institute, La Jolla, CA). Anti-β3 mAb AP3 (39.Newman P.J. Allen R.W. Kahn R.A. Kunicki T.J. Blood. 1983; 65: 227-232Crossref Google Scholar) and rabbit polyclonal anti-αIIb SEW 8 were provided by P. Newman (Blood Center of Southeastern Wisconsin, Milwaukee, WI). The mAb Tab (40.McEver R.P. Bennett E.M. Martin M.N. J. Biol. Chem. 1983; 258: 5269-5275Abstract Full Text PDF PubMed Google Scholar), specific for αIIb, was provided by R. McEver (University of Oklahoma Health Sciences Center, Oklahoma City, OK). The mAbs A2A9 (41.Bennett J.S. Hoxie J.A. Leitman S.F. Vilaire G. Cines D.B. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 2417-2421Crossref PubMed Scopus (170) Google Scholar), recognizing the αIIbβ3 complex, and B1B5 (42.Silver S.M. McDonough M.M. Vilaire G. Bennett J.S. Blood. 1987; 69: 1031-1037Crossref PubMed Google Scholar), recognizing an epitope on αIIb, were provided by J. Bennett (University of Pennsylvania, Philadelphia, PA). The αIIbβ3 complex-specific mAb 10E5 (43.Coller B.S. Peerschke I.L. Scudder L.E. Sullivan C.A. J. Clin. Invest. 1983; 72: 325-338Crossref PubMed Scopus (562) Google Scholar) was provided by B. Coller (Albert Einstein University, New York, NY). The anti-hamster β1 mAb 7E2 (44.Brown P.J. Juliano R.L. Exp. Cell Res. 1988; 177: 303-318Crossref PubMed Scopus (44) Google Scholar) was provided by R. Juliano (University of North Carolina). The mAb LIBS 6, specific for β3 (45.Frelinger III, A.L. Du X.P. Plow E.F. Ginsberg M.H. J. Biol. Chem. 1991; 266: 17106-17111Abstract Full Text PDF PubMed Google Scholar), was provided by M. Ginsberg (Scripps Research Institute). PAC1 murine IgM (46.Shattil S.J. Hoxie J.A. Cunningham M. Brass L.F. J. Biol. Chem. 1985; 260: 11107-11114Abstract Full Text PDF PubMed Google Scholar) was from University of Pennsylvania Cell Center (Philadelphia, PA). Monoclonal antibodies against synthetic peptides containing the N- and C-terminal fibrinogen Aα RGD motifs (Aα 87–100 and Aα 566–580, respectively) were provided by Z. Ruggeri (Scripps Research Institute). Monoclonal antibody 4A5 (47.Taubenfield S.M. Song Y. Sheng D. Ball E.L. Matsueda G.R. Thromb. Haemostasis. 1995; 74: 923-927Crossref PubMed Scopus (19) Google Scholar) against the fibrinogen γ-chain H12 sequence was provided by Gary Matsueda (Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, NJ). FITC-conjugated goat anti-mouse IgG F(ab′)2was from Fisher. FITC-conjugated rabbit anti-mouse IgM was fromZymed Laboratories Inc. (South San Francisco, CA). FITC-conjugated chicken anti-human fibrinogen was from Biopool International (Ventura, CA). Normal mouse serum, goat anti-mouse IgG, goat anti-rabbit IgG, horseradish peroxidase-goat anti-mouse IgG, horseradish peroxidase-goat anti-rabbit IgG, and alkaline phosphatase-conjugated goat anti-rabbit IgG were from Sigma. Primary antibodies were used as either ascites fluid or as the IgG fraction after purification on either Protein A-Sepharose or GammaBind Plus Sepharose. A full-length αIIb cDNA clone was isolated from a λ-gt11 liver cDNA library using restriction fragments of a partial-length αIIb clone as probes. Partial-length cDNA clones of β3 were isolated from both the liver cDNA library and an EA.hy 926, endothelial-adenocarcinoma hybrid cell, λ-gt11 cDNA library. All cDNA isolates were subcloned into Bluescript KS. Full-length β3 was constructed from two partial clones that overlapped at the internal EcoRI site. The β3clone isolated had a G2069A mutation encoding a C655Y substitution. The mutation was repaired by subcloning a SapI–MluI fragment containing the correct sequence from an EA.hy926 partial clone. All clones were constructed using standard mutagenic and subcloning techniques. A cDNA encoding β3 with a silent mutation destroying the internalEcoRI site, β3ΔE, was prepared using the T7-GEN In Vitro Mutagenesis Kit. Silent mutations in αIIb creating BstBI and NdeI sites at nucleotides 771 and 1411, respectively, were made simultaneously using the dut-, ung- method (48.Kunkel T.A. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 488-492Crossref PubMed Scopus (4900) Google Scholar). This clone, named BN, was used to make IIbα5, a chimeric cDNA where all four calcium binding domains of αIIb were replaced with the calcium binding domains of α5 (amino acids 230–436), and ΔCa, an in-frame deletion of all four calcium binding domains of αIIb (amino acids 234–438) (Fig. 1 A). To construct IIbα5, the calcium binding domains of α5 were amplified by PCR using oligonucleotides that contained BstBI and NdeI sites, respectively, and that reconstructed the αIIb sequence from the restriction site up to the start of the calcium binding domains. ΔCa was made by annealing oligonucleotides CGAACCCAGAGTACTTCGACGGCGCA and TATGCGCCGTCGAAGTACTCTGGGTT and ligating into BN digested withBstBI and NdeI. The mutant D2 was made by subcloning the second calcium binding domain of αIIb into IIbα5 using a subcloning and PCR strategy that precisely transferred αIIb-(286–315) into IIbα5. Second calcium binding domain mutants YAVAA, LD, and LMD were made using the dut-, ung- method after subcloning the cDNA into M13mp18. YAVAA-LD, YAVAA-LMD, and LD-LMD were made by using the appropriate second mutagenic oligonucleotide after isolating the first mutant. YAVAA-LD-LMD was made by using the YAVAA and LMD oligonucleotides simultaneously with the LD cDNA. The sequence of the second calcium binding domain of the mutant constructs is shown in Fig. 1 B. The cDNAs were isolated from double-stranded M13mp18 by digestion withEcoRI and subcloned into Bluescript KS. αIIbwas subcloned into the expression vector pcDNAI/AMP by digestion with EcoRV and XbaI. αIIb was also transferred into pRc/CMV, an expression vector containing theneo gene, by digesting the vector with HindIII, blunting with Klenow, and digesting with XbaI. The vector was then ligated with the EcoRV–XbaI fragment of αIIb. Mutant constructs were transferred to the expression vectors by subcloning the NotI–NruI fragment of each mutant into similarly digested αIIb. β3ΔE and β3ΔEG2069A were subcloned into pcDNAI/Amp and pRc/CMV by digestion with EcoRV andXbaI. The sequence of all clones was confirmed using dideoxy nucleotide sequencing (49.Sanger F. Nicklen S. Coulson A.R. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 5463-5467Crossref PubMed Scopus (52655) Google Scholar). Chinese hamster ovary K1 (CHO) cells were maintained and transfected as described previously (50.Lyman S. Gilmore A. Burridge K. Gidwitz S. White G.C., II J. Biol. Chem. 1997; 272: 22538-22547Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). COS-7 cells were maintained in DMEM supplemented with 10% FBS and 1% penicillin and streptomycin. Wild type or mutant constructs were co-transfected with β3ΔE into COS-7 cells using LipofectAMINE. Cells were plated at 1.3 × 105cells/cm2 in 25-cm2 flasks. One day after plating, cells were washed with serum-free DMEM and incubated with 2.75 ml of serum-free DMEM and 0.5 ml of Opti-MEM I containing a total of 2.5 μg of β3ΔE and αIIb construct, each in pcDNAI/Amp, and 24 μl of LipofectAMINE. After 5 h at 37 °C, 2.5 ml of DMEM containing 20% FBS was added. Twenty-four h later, the medium was replaced with DMEM containing 10% FBS. Cells were assayed 72 h after the start of transfection. Flow cytometry was performed as described previously (50.Lyman S. Gilmore A. Burridge K. Gidwitz S. White G.C., II J. Biol. Chem. 1997; 272: 22538-22547Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar) except that for PAC1 studies, cells were incubated with a 1:100 dilution of LIBS 6 ascites with or without 1 mmGRGDSP peptide for 15 min at 37 °C. Cells were washed and stained with FITC-conjugated rabbit anti-mouse IgM. For studies of subunit dissociation, cells were harvested and washed and then resuspended in PBS containing 2% BSA. Immediately before the start of the experiment, EDTA was added to a final concentration of 5 mm. Cells were incubated at the indicated temperature for the indicated times and then immediately diluted 10-fold with PBS, centrifuged, and resuspended in PBC (PBS containing 2% BSA, 0.1 mm CaCl2, and 0.1 mm MgCl2) containing the αIIbβ3 complex-specific mAb AP2 and processed as above. Surface labeling and immunoprecipitation were performed essentially as described previously (50.Lyman S. Gilmore A. Burridge K. Gidwitz S. White G.C., II J. Biol. Chem. 1997; 272: 22538-22547Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). One mg of mAb AP2 (IgG fraction) was iodinated with 2 mCi of Na125I using 2 mmchloramine T for 5 min at 22 °C, quenched with 2.3 mmNa2S2O5, and separated from free Na125I by gel filtration. Cells were harvested with EDTA and trypsin as described above for flow cytometry and resuspended in Tris-buffered saline plus 0.5% BSA. Typically, 5 × 105 cells were incubated with varying concentrations of [125I]AP2 for 30 min at 22 °C in a total volume of 0.5 ml. Triplicate 50-μl aliquots were layered over 0.4 ml of 20% sucrose, 0.5% BSA in Tris-buffered saline and centrifuged at 12,000 × g for 2 min. The samples were aspirated to dryness, and the radioactivity associated with the cell pellets was measured in a γ-spectrometer. Total radioactivity in the sample was determined by counting triplicate 50-μl aliquots of the reaction mixture. Nonspecific binding was defined as the radioactivity associated with the cell pellets of mock-transfected cells and was subtracted from that of αIIbβ3-expressing cells. The data were fit to equilibrium binding models (51.McPherson G.A. Computer Prog. Biomed. 1983; 17: 107-114Abstract Full Text PDF PubMed Scopus (793) Google Scholar). Cells were harvested as above for flow cytometry and resuspended in PBC. Flat bottomed immunoassay 96-well polystyrene plates were incubated with either 2.5 μg/ml fibronectin or 4 μg/ml fibrinogen in PBS for 16 h at 4 °C. Plates were then incubated with PBS with 2% BSA for 2 h at 37 °C to block nonspecific binding sites on the plates. Typically, 2 × 106cells/ml were incubated with PBC or PBC plus 1 mm GRGDSP for 30 min at 22 °C prior to plating 1 × 105cells/well. Cells were allowed to adhere to the plates for 2 h at 37 °C. Plates were washed three times with PBS to remove nonadherent cells. Adherent cells were stained with 0.5% crystal violet in PBS containing 20% methanol for 30 min at 22 °C. Excess dye was removed by three washes with water, and cells were solubilized in 1% SDS for 16 h at 22 °C. Adhesion was quantified by measuring the absorbance at 540 nm in a BIO-TEK EL 340 microplate reader (BIO-TEK Instruments, Winooski, VT). Nonspecific binding was defined as the absorbance of cells binding to BSA-coated wells and was subtracted from the absorbance of cells binding to ligand-coated wells. All assays were performed in triplicate. All operations were performed at 22 °C. Cells were harvested as above for flow cytometry and resuspended in HEPES-Tyrode's buffer, pH 7.5, at 2 × 107 cells/ml. Cells were incubated with 10 mm DTT for 20 min, pelleted, and resuspended at the same concentration in HEPES-Tyrode's buffer with or without inhibitors. Cells were incubated for 30 min, and then CaCl2 was added to 0.4 mm. One hundred μl of cells was placed in wells of a 48-well tissue culture plate and incubated with or without 0.25 mg/ml fibrinogen for 30 min at 75 rpm on a gyrotory shaker. Cells were analyzed for aggregation by bright field microscopy. Fibrinogen was passed over a gelatin-agarose column two times to deplete the fibrinogen of fibronectin. Fibrinogen and AP2 were labeled using FITC-Celite essentially as described by Xia et al. (52.Xia Z. Wong T. Liu Q. Kasirer-Friede A. Brown E. Frojmovic M.M. Br. J. Haematol. 1996; 93: 204-214Crossref PubMed Scopus (82) Google Scholar). The fluorescein:protein ratio for fibrinogen ranged from 3 to 5, while the AP2 had a fluorescein:protein ratio of 3. Cells were harvested as above for flow cytometry and resuspended at a final concentration of 3 × 106 cells/ml in DMEM containing 20 mm HEPES, pH 7.5, LIBS 6 ascites at a 1:100 dilution, and either 0.25 mm ligand-blocking peptide RGDW or buffer. Cells were incubated at room temperature for 15 min, and then FITC-fibrinogen was added at the indicated final concentration. Cells were incubated for 30 min at room temperature, washed with PBS, fixed with 1% paraformaldehyde in PBS at 4 °C, and analyzed by flow cytometry. For both fibrinogen and AP2 binding, cytometry was scrupulously gated to analyze only single cells. In an effort to exclude cells lying outside the normal range, only the central 98% of the fluorescent signal (as determined by minimizing the coefficient of variance) was averaged to determine mean fluorescent intensity. Specific binding was defined as the difference in mean fluorescent intensity between cells incubated in the presence and absence of RGDW. Data was fitted to the one-site ligand binding equation y =a 0 *x/(a 1 + x) using the iterative nonlinear curve fitting function of SlideWrite®Plus (Advanced Graphics Software, Inc., Carlsbad, CA). To test the effect of the anti-N-terminal and anti-C-terminal fibrinogen Aα RGD motif and anti-γ-chain antibodies, cells were resuspended in HEPES-Tyrode's buffer without calcium or magnesium that had been treated with Chelex 100. Cells were incubated with 2 mm Ca2+ and LIBS 6 or with 1 mmMn2+ in the presence or absence of 1 mm GRGDSP for 15 min at 22 °C. Fibrinogen and control or anti-fibrinogen peptide IgG were added to final concentrations of 50 nm and 10 μg/ml, respectively. Cells were incubated at 22 °C for 30 min, washed with HEPES-Tyrode's buffer, and then incubated at 22 °C for 30 min with a 1:10 dilution of FITC-chicken anti-human fibrinogen in HEPES-Tyrode's buffer. Cells were washed, fixed, and analyzed by flow cytometry as above. To determine if the various αIIb calcium binding domain mutants were expressed on the cell surface, CHO cells co-transfected with the mutants and wild type β3 were analyzed by flow cytometry using Tab, a murine monoclo
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