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Changing Ligand Specificities of αvβ1 and αvβ3 Integrins by Swapping a Short Diverse Sequence of the β Subunit

1997; Elsevier BV; Volume: 272; Issue: 32 Linguagem: Inglês

10.1074/jbc.272.32.19794

1083-351X

Junichi Takagi, Tetsuji Kamata, Jere E. Meredith, Wilma Puzon-McLaughlin, Yoshikazu Takada,

Biochemical and Structural Characterization

Integrins mediate signal transduction through interaction with multiple cellular or extracellular matrix ligands. Integrin αvβ3 recognizes fibrinogen, von Willebrand factor, and vitronectin, while αvβ1 does not. We studied the mechanisms for defining ligand specificity of these integrins by swapping the highly diverse sequences in the I domain-like structure of the β1 and β3 subunits. When the sequence CTSEQNC (residues 187–193) of β1 is replaced with the corresponding CYDMKTTC sequence of β3, the ligand specificity of αvβ1 is altered. The mutant (αvβ1–3-1), like αvβ3, recognizes fibrinogen, von Willebrand factor, and vitronectin (a gain-of-function effect). The αvβ1–3-1 mutant is recruited to focal contacts on fibrinogen and vitronectin, suggesting that the mutant transduces intracellular signals on adhesion. The reciprocal β3–1-3 mutation blocks binding of αvβ3 to these multiple ligands and to LM609, a function-blocking anti-αvβ3 antibody. These results suggest that the highly divergent sequence is a key determinant of integrin ligand specificity. Also, the data support a recent hypothetical model of the I domain of β, in which the sequence is located in the ligand binding site. Integrins mediate signal transduction through interaction with multiple cellular or extracellular matrix ligands. Integrin αvβ3 recognizes fibrinogen, von Willebrand factor, and vitronectin, while αvβ1 does not. We studied the mechanisms for defining ligand specificity of these integrins by swapping the highly diverse sequences in the I domain-like structure of the β1 and β3 subunits. When the sequence CTSEQNC (residues 187–193) of β1 is replaced with the corresponding CYDMKTTC sequence of β3, the ligand specificity of αvβ1 is altered. The mutant (αvβ1–3-1), like αvβ3, recognizes fibrinogen, von Willebrand factor, and vitronectin (a gain-of-function effect). The αvβ1–3-1 mutant is recruited to focal contacts on fibrinogen and vitronectin, suggesting that the mutant transduces intracellular signals on adhesion. The reciprocal β3–1-3 mutation blocks binding of αvβ3 to these multiple ligands and to LM609, a function-blocking anti-αvβ3 antibody. These results suggest that the highly divergent sequence is a key determinant of integrin ligand specificity. Also, the data support a recent hypothetical model of the I domain of β, in which the sequence is located in the ligand binding site. Integrins are a family of α/β heterodimers of cell adhesion receptors that mediate cell-extracellular matrix and cell-cell interactions (1Springer T.A. Cell. 1994; 76: 301-314Abstract Full Text PDF PubMed Scopus (6355) Google Scholar, 2Ruoslahti E. J. Clin. Invest. 1991; 87: 1-7Crossref PubMed Scopus (1477) Google Scholar, 3Hynes R.O. Cell. 1992; 69: 11-25Abstract Full Text PDF PubMed Scopus (8941) Google Scholar, 4Hemler M.E. Annu. Rev. Immunol. 1990; 8: 365-400Crossref PubMed Google Scholar, 5Yamada K.M. J. Biol. Chem. 1991; 266: 12809-12812Abstract Full Text PDF PubMed Google Scholar). Integrin-ligand interactions are critically involved in the pathogenesis of many diseases in human and animal models. Although integrin-ligand interaction is a therapeutic target, we poorly understand at the molecular level how integrins recognize multiple ligands. Evidence suggests that the I or A domain, a set of inserted sequences consisting of about 200 amino acid residues, of several integrin α subunits (αM, αL, α1, α2) is important in ligand binding and receptor activation (reviewed in Ref. 6Puzon-McLaughlin W. Takada Y. J. Biol. Chem. 1996; 271: 20438-20443Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar and references therein). The presence of an I domain-like structure within the β subunit has been suggested based on the similarity in hydropathy profiles between the I domain and part of the β subunit (7Lee J.-O. Rieu P. Arnaout M.A. Liddington R. Cell. 1995; 80: 631-638Abstract Full Text PDF PubMed Scopus (792) Google Scholar). Interestingly, this region of β has been reported to be critical for ligand binding and its regulation (reviewed in Ref. 8Loftus J.C. Smith J.W. Ginsberg M.H. J. Biol. Chem. 1994; 269: 25235-25238Abstract Full Text PDF PubMed Google Scholar) (Fig. 1). The Asp-119 (β3) (9Loftus J.C. O'Toole T.E. Plow E.F. Glass A. Frelinger A.L. Ginsberg M.H. Science. 1990; 249: 915-918Crossref PubMed Scopus (324) Google Scholar) and Asp-130 (β1) (10Takada Y. Ylanne J. Mandelman D. Puzon W. Ginsberg M. J. Cell Biol. 1992; 119: 913-921Crossref PubMed Scopus (98) Google Scholar, 11Kamata T. Puzon W. Takada Y. Biochem. J. 1995; 305: 945-951Crossref PubMed Scopus (123) Google Scholar) and the corresponding residues in β2 and β6 are critical for ligand binding (12Bajt M. Loftus J. J. Biol. Chem. 1994; 269: 20913-20919Abstract Full Text PDF PubMed Google Scholar, 13Huang X. Chen A. Agrez M. Sheppard D. Am. J. Resp. Cell Mol. Biol. 1995; 13: 245-251Crossref PubMed Scopus (14) Google Scholar). A synthetic peptide of β3 (MDLSYSMKDDLWSI, residues 118–131) has been shown to produce a ternary complex with cations and ligand (14D'Souza S.E. Haas T.A. Piotrowicz R.S. Byers-Ward V. McGrath 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). Also, the sequence DDLW (residues 126–129 of β3) was shown to be critical for interaction with the RGD sequence using a phage display system (15Pasqualini R. Koivunen E. Ruoslahti E. J. Cell Biol. 1995; 130: 1189-1196Crossref PubMed Scopus (201) Google Scholar). A synthetic peptide of β3, DAPEGGFDAIMQATV (residues 217–231 of β3), has been shown to bind to immobilized fibrinogen (Fg), 1The abbreviations used are: Fg, fibrinogen; CHO, Chinese hamster ovary; Fn, fibronectin; Vn, vitronectin; vWf, von Willebrand's factor; mAb, monoclonal antibody; WT, wild type; PBS, phosphate-buffered saline; PIPES, 1,4-piperazinediethanesulfonic acid. 1The abbreviations used are: Fg, fibrinogen; CHO, Chinese hamster ovary; Fn, fibronectin; Vn, vitronectin; vWf, von Willebrand's factor; mAb, monoclonal antibody; WT, wild type; PBS, phosphate-buffered saline; PIPES, 1,4-piperazinediethanesulfonic acid. von Willebrand's factor (vWf), and fibronectin (Fn) (16Cook J. Trybulec M. Lasz E. Khan S. Niewiarowski S. Biochim. Biophys. A. 1992; 1119: 312-321Crossref PubMed Scopus (37) Google Scholar, 17Lasz E. McLane M. Trybulec M. Kowalska M. Khan S. Budzynski A. Niewiarowski S. Biochem. Biophys. Res. Commun. 1993; 190: 118-124Crossref PubMed Scopus (21) Google Scholar). A synthetic peptide of β3, SVSRNRDAPEG (residues 211–221 of β3), has been reported to block binding of Fg to αIIbβ3 (18Charo 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, 19Steiner B. Trzeciak A. Pfenninger G. Kouns W. J. Biol. Chem. 1993; 268: 6870-6873Abstract Full Text PDF PubMed Google Scholar). We identified a small region of β1 (residues 207–218, a regulatory epitope) that is recognized by both activating and inhibiting anti-β1 antibodies (20Takada Y. Puzon W. J. Biol. Chem. 1993; 268: 17597-17601Abstract Full Text PDF PubMed Google Scholar). These antibodies probably induce high or low affinity states, respectively, by changing the conformation of the β1 subunit through binding to the non-ligand binding site (20Takada Y. Puzon W. J. Biol. Chem. 1993; 268: 17597-17601Abstract Full Text PDF PubMed Google Scholar). We and researchers at other laboratories have recently identified residues critical for ligand binding in the putative I domain-like structure of β1 (6Puzon-McLaughlin W. Takada Y. J. Biol. Chem. 1996; 271: 20438-20443Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar), β2 (21Goodman T. Bajt M. J. Biol. Chem. 1996; 271: 23729-23736Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar), and β3 (22Tozer E. Liddington R. Sutcliffe M. Smeeton A. Loftus J. J. Biol. Chem. 1996; 271: 21978-21984Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). In β1, eight critical oxygenated residues are located in several separate predicted loop structures, which probably constitute multiple ligand/cation binding sites within the I domain-like structure of the β subunit. These critical oxygenic residues are conserved among integrin β subunits, indicating that these residues are ubiquitously involved in ligand binding regardless of ligand and integrin species. We observed that a large predicted loop region (residues 176–199 of β1) is diverse among the β subunits (Fig. 1). Furthermore, a recent structural model (23Tuckwell D. Humphries M. FEBS Lett. 1997; 400: 297-303Crossref PubMed Scopus (99) Google Scholar) and our preliminary model (not shown) of the I domain-like structure of β suggest that the sequence is also on the same side of the domain as residues critical for ligand binding. We hypothesized that the predicted loop (especially the disulfide-linked short sequences, e.g. residues 187–193 of β1) is involved in ligand specificity of integrins. αvβ3 has been shown to recognize a wide variety of ligands, including Fn, Fg, vWf, and vitronectin (Vn); αvβ1 is specific to Fn. We designed experiments, using αvβ1 and αvβ3 integrins, to determine whether a diverse sequence in the predicted loop (e.g.residues 176–199 in β1, residues 166–190 in β3) is involved in ligand specificity of integrins. mAb 4B4 (to human β1) (24Morimoto C. Letvin N.L. Boyd A.W. Hagan M. Brown H. Kormacki M. Schlossman S.F. J. Immunol. 1985; 134: 3762-3769PubMed Google Scholar) was kindly provided by C. Morimoto (Dana-Farber Cancer Institute, Boston, MA); 8A2 (to human β1) (25Kovach N.L. Carlos T.M. Yee E. Harlan J.M. J. Cell Biol. 1992; 116: 499-509Crossref PubMed Scopus (179) Google Scholar) by N. Kovach and J. Harlan (University of Washington, Seattle, WA); A1A5 (to human β1) (26Hemler M.E. Sanchez-Madrid F. Flotte T.J. Krensky A.M. Burakoff S.J. Bhan A.K. Springer T.A. Strominger J.L. J. Immunol. 1984; 132: 3011-3018PubMed Google Scholar) by M. Hemler (Dana-Farber Cancer Institute, Boston, MA); LM142 (to human αv), LM609 (to αvβ3) (27Cheresh D.A. Smith J.W. Cooper H.M. Quaranta V. Cell. 1989; 57: 59-69Abstract Full Text PDF PubMed Scopus (254) Google Scholar), and P3G2 (to αvβ5) (28Wayner E.A. Orlando R.A. Cheresh D.A. J. Cell Biol. 1991; 113: 919-929Crossref PubMed Scopus (296) Google Scholar) by D. Cheresh (Scripps); 15 (to human β3) (29Frelinger III, A. Cohen I. Plow E. Smith M. Roberts J. Lam S. Ginsberg M. J. Biol. Chem. 1990; 265: 6346-6352Abstract Full Text PDF PubMed Google Scholar) by M. H. Ginsberg (Scripps); and 15/7 (to human β1) (30Yednock T.A. Cannon C. Vandevert C. Goldbach E.G. Shaw G. Ellis D.K. Liaw C. Fritz L.C. Tanner L.I. J. Biol. Chem. 1995; 270: 28740-28750Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar) by T. Yednock (Athena Neurosciences, San Francisco, CA). P5D2 (to human β1) and polyclonal anti-αv cytoplasmic peptide antibody were purchased from Chemicon (Temecula, CA). A Fn 110-kDa fragment was prepared from bovine plasma Fn (Life Technologies, Inc.) as described (31Pierschbacher M.D. Ruoslahti E. Sundelin J. Lind P. Peterson P.A. J. Biol. Chem. 1982; 257: 9593-9597Abstract Full Text PDF PubMed Google Scholar). Bovine Fg was purchased from Daiichi Chemical (Tokyo, Japan). Purified vWf was provided by Z. Ruggeri (Scripps). Bovine Vn was purified according to Yatohgo et al. (32Yatohgo T. Izumi M. Kashiwagi H. Hayashi M. Cell Struct. Funct. 1988; 13: 282-292Crossref Scopus (437) Google Scholar). Fg, Vn, and Fn 110-kDa fragment were coupled to CNBr-activated Sepharose 4B (Pharmacia) according to the manufacturer's instructions. The ligand concentration was 4.0, 1.2, and 2.3 mg/ml gel for Fg-, Vn-, and Fn 110-kDa fragment-Sepharose, respectively. The GRGDS peptide (6 mg/ml gel, Peptide Institute, Osaka, Japan) was coupled at the 6-carbon spacing arm of CH-Sepharose (Sigma) according to the manufacturer's instructions. Human αv and β3 cDNAs were provided by J. Loftus (Scripps). Ten μg of wild-type human αv cDNA in pBJ-1 vector (33Takebe Y. Seiki M. Fujisawa J.-I. Hoy P. Yokota K. Arai K.-I. Yoshida M. Arai N. Mol. Cell. Biol. 1988; 8: 466-472Crossref PubMed Google Scholar, 34Lin A.Y. Devaux B. Green A. Sagerstrom C. Elliott J.F. Davis M. Science. 1990; 249: 677-679Crossref PubMed Scopus (191) Google Scholar) was transfected into parental CHO-K1 cells (8 × 106 cells) together with 1 μg of pFneo plasmid containing a neomycin-resistant gene by electroporation as described (35Irie A. Kamata T. Puzon-McLaughlin W. Takada Y. EMBO J. 1995; 14: 5542-5549Crossref PubMed Scopus (91) Google Scholar). After they were selected for G418 resistance, cells expressing αv were cloned by cell sorting in FACStar cell sorter (Becton-Dickinson) with mAb LM142 (the cloned cells are designated αv-CHO cells). Human β1 or β3 (WT/mutant) cDNA in pBJ-1 vector was transfected into αv-CHO cells together with 1 μg of pCD-hygro plasmid with a hygromycin-resistant gene or into parent CHO cells together with 1 μg of pFneo; cells were then selected with hygromycin (500 μg/ml; Calbiochem) or G418 essentially as described above. Cells expressing human β1 or β3 were cloned by sorting with mAb A1A5 or 15 as described above. The flow cytometric analysis was carried out using FACScan (Becton-Dickinson). Wells of 96-well Immulon-2 microtiter plates (Dynatech Laboratories, Chantilly, VA) were coated with 100 μl of PBS (10 mm phosphate buffer, 0.15 m NaCl, pH 7.4) containing Fg, vWf, Fn, and Vn at a concentration of 10 μg/ml overnight at 4 °C. The remaining protein binding sites were blocked by incubating with 1% bovine serum albumin (Calbiochem) for 1 h at room temperature. Cells (105 cells/well) in 100 μl of Dulbecco's modified Eagle's medium containing 0.5 mg/ml bovine serum albumin were added to the wells and incubated at 37 °C for 1 h. After gently rinsing the wells three times with PBS to remove unbound cells, bound cells were quantified using endogenous phosphatase activity (36Prater C.A. Plotkin J. Jaye D. Frazier W.A. J. Cell Biol. 1991; 112: 1031-1040Crossref PubMed Scopus (188) Google Scholar). Cells were harvested with 3.5 mm EDTA in PBS and washed with PBS. Cells (about 5 × 106) were then surface-labeled with 125I by using IODO-GEN (Pierce) (37Braciale T.J. Henkel T.J. Lukacher A. Braciale V.L. J. Immunol. 1986; 137: 995-1002PubMed Google Scholar), washed three times with PBS, and solubilized in 1 ml of 100 mm octyl glucoside in 10 mm Tris-HCl, 0.15 m NaCl, pH 7.4 (TBS), containing 2.5 mm MnCl2, 1 mmphenylmethylsulfonyl fluoride (Sigma) at 4 °C for 15 min. The insoluble materials were removed by centrifugation at 15,000 ×g for 10 min. The supernatant was then incubated with a small amount of underivatized Sepharose 4B at 4 °C for 15 min to remove nonspecific binding material. The supernatant was incubated at 4 °C for 1 h with 200–500 μl of packed Fg-, Vn-, Fn 110-kDa fragment-, or GRGDS-Sepharose that had been equilibrated with TBS containing 2.5 mm MnCl2, 1 mmphenylmethylsulfonyl fluoride, 25 mm octyl glucoside (washing buffer). The unbound materials were washed with a 20 × column volume of washing buffer, and the bound materials were eluted with 20 mm EDTA instead of 1 mmMnCl2 in washing buffer; and then 0.5-ml fractions were collected. Twenty-μl aliquots from each fraction was analyzed by SDS-polyacrylamide gel electrophoresis using 7% polyacrylamide gel followed by autoradiography. Glass coverslips (Fisher) were treated with 10% KOH in methanol for 1 h at room temperature, washed three times with distilled H2O, and stored in ethanol. Etched coverslips were then coated with 50 μg/ml Fg, 50 μg/ml Fn, or 22 μg/ml Vn in PBS overnight at 4 °C and then blocked with 10 mg/ml heat-denatured bovine serum albumin (Calbiochem) in PBS for 10 min at room temperature. For plating experiments, cells were washed and then detached with 2.5 mm EDTA/PBS. Detached cells were isolated, washed, resuspended in Dulbecco's modified Eagle's medium, and then replated on coated coverslips. Cells were allowed to attach and spread for 2 h. Prior to fixation, cells were chilled on ice for 5 min, washed with cold PBS, and then extracted with cold PIPES buffer (0.1 m PIPES, pH 6.8, 1 mmMgCl2, and 1 mm EGTA) containing 1% glycerol and 0.5% Nonidet P-40 for 1–2 min. Extracted cells were washed with cold PIPES buffer and then fixed with 3.7% methanol-free formaldehyde (Polysciences) in PIPES buffer for 20 min at room temperature. Following fixation, cells were washed with PBS and then blocked with 10% normal goat serum (Life Technologies, Inc.)/PBS for 20 min at 37 °C. Human integrins were detected using either the anti-human β1 antibody P5D2 or the anti-human β3 antibody 15. Cells were immunostained for 1 h at 37 °C, washed, and then stained with a fluorescein isothiocyanate-conjugated sheep anti-mouse IgG secondary antibody (Molecular Probes) for 30 min at 37 °C; cells were also labeled with rhodamine phalloidin (Molecular Probes) to detect actin stress fibers. Stained cells were mounted in Fluoromount-G (Fisher) and photographed using a Nikon Diaphot inverted microscope. Site-directed mutagenesis of the β1 and β3 cDNA in a pBJ-1 vector was carried out using unique restriction site elimination (38Deng W.P. Nickoloff J.A. Anal. Biochem. 1992; 200: 81-88Crossref PubMed Scopus (1078) Google Scholar). The presence of mutations was confirmed by DNA sequencing. Immunoprecipitation was carried out as described previously (20Takada Y. Puzon W. J. Biol. Chem. 1993; 268: 17597-17601Abstract Full Text PDF PubMed Google Scholar). To determine whether a diverse sequence in the predicted loop (residues 176–199 in β1 and residues 166–190 in β3) is involved in ligand specificity of integrins, we replaced the CTSEQNC sequence of β1 with the corresponding CYDMKTTC sequence of β3 by site-directed mutagenesis. The CYDMKTTC sequence of β3 has been reported to be disulfide-linked (39Calvete J.J. Henschen A. Gonzalez-Rodriguez J. Biochem. J. 1991; 274: 63-71Crossref PubMed Scopus (158) Google Scholar). The resulting mutant β1–3-1, wild-type β1, or wild-type β3 cDNA constructs were transfected into either parental CHO cells or CHO cells expressing wild-type human αv (αv-CHO). Parent CHO cells have been reported to express endogenous hamster αv (40Ferrer M. Fernandez-Pinel M. Gonzalez-Manchon C. Gonzalez J. Ayuso M. Parrilla R. Thromb. Haemostasis. 1996; 76: 292-301Crossref PubMed Scopus (25) Google Scholar) but not β3 (41Ylanne J. Chen Y. O'Toole T. Loftus J.C. Takada Y. Ginsberg M.H. J. Cell Biol. 1993; 122: 223-233Crossref PubMed Scopus (194) Google Scholar). Consistent with these findings, we found that CHO cells express αvβ5 using mAb P3G2 (data not shown). The cloned cells expressing WT or mutant β1 in association with exogenous human αv are designated αvβ1-, αvβ1–3-1-, αvβ3-CHO cells, and those with only endogenous hamster αv are designated as β1-, β1–3-1-, β3-CHO cells. αvβ3 recognizes multiple ligands, including Fn, Fg, vWf, and Vn; αvβ1 is specific to Fn on CHO cells. Therefore, we tested the ligand specificity of the β1–3-1 mutant. As shown in Fig.2 A, we found that cells expressing αvβ3 or αvβ1–3-1, but not αvβ1, adhered to both Fg and vWf. Adhesion of the αvβ1–3-1- but not αvβ3-CHO cells was blocked by the inhibitory anti-human β1 mAb 4B4 (Fig.2 B), indicating that adhesion of the αvβ1–3-1-expressing cells to Fg and vWf is mediated by human β1 sequences. Similar results were obtained with the β1-, β3-, and β1–3-1-CHO cells (Fig. 2, A and B). These results suggest that the region spanning residues 187–193 of β1 or 177–184 of β3 is involved in the regulation of ligand specificity. The specificity of the interaction between αvβ1–3-1 and ligands was further analyzed by affinity chromatography. Lysates from surface 125I-labeled αvβ1–3-1-CHO cells (as well as control αvβ1- and αvβ3-CHO cells) were incubated with immobilized Fg or Fn 110-kDa fragments, and bound materials were eluted with EDTA. As shown in Fig.3 A, bands corresponding to αv and β1 in size were eluted from Fg-Sepharose using a lysate of αvβ1–3-1-CHO cells, while bands corresponding to human αv and β3 were eluted from Fg-Sepharose with a lysate of αvβ3-CHO cells. Immunoprecipitation of the eluate from αvβ1–3-1 cells using anti-β1 mAb A1A5 (Fig. 3 C, lane 5) and anti-αv mAb LM142 (Fig. 3 D, lane 5) confirmed that these two bands are human αv and β1 (β1–3-1). In contrast, very low levels of αv and β1 were detected in the Fg-Sepharose eluate with lysate of αvβ1-CHO cells. These results suggest that αvβ1–3-1 exhibits a much higher affinity for Fg than αvβ1. Similar results were obtained with Vn-Sepharose (data not shown), suggesting that αvβ1–3-1 shows a much higher affinity to Vn as well. In experiments done in parallel, we have detected bands corresponding to αvβ1, αvβ1–3-1, and αvβ3 in the eluate from Fn 110-kDa fragment-Sepharose with lysates from αvβ1-, αvβ1–3-1, and αvβ3-CHO cells, respectively (Fig.3 B). Immunoprecipitation confirmed that the major β subunits in the eluates are β1, β1–3-1, and β3, respectively (Fig. 3 C). These results suggest that the αvβ1–3-1 mutant, like αvβ3, binds to Fg, Vn, and Fn 110-kDa fragments in a solubilized form. Next we determined if the altered ligand specificity of the αvβ1–3-1 chimera affected intracellular signaling. Cells were plated on Fn, Vn, or Fg, and localization of the human integrin was determined by immunostaining with anti-human β1 (αvβ1 and αvβ1–3-1) or anti-human β3 (αvβ3). While all three receptors localized to focal adhesions in cells plated on Fn (Fig. 4, A,C, and F), only β1–3-1 and β3 localized to focal adhesions in cells on Vn; αvβ1-CHO cells did attach and spread on Vn due to endogenous αvβ5. However, αvβ1 exhibited a diffuse staining pattern. This result is consistent with the binding data and indicates that the αvβ1–3-1 chimera is able to generate intracellular signals. In addition, we found that the αvβ1–3-1 chimera, like αvβ3, induced cell spreading and focal adhesion formation in cells plated on Fg; αvβ1 cells did not adhere to Fg. Similar results were obtained with the β1-, β1–3-1-, and β3-CHO cells that express lower levels of the transfected integrins (data not shown). These results indicate that the αvβ1–3-1 chimera is a functional receptor and has the same signaling properties as αvβ3. To determine whether the reciprocal swapping mutation has any effect on the ligand specificity of αvβ3, we replaced the CYDMKTTC sequence of β3 (residues 177–184) with the corresponding CTSEQNC sequence of β1 (the β3–1-3 mutation). The resulting mutant β3–1-3 and WT β3 cDNA constructs were transfected into αv-CHO cells, and cells stably expressing αvβ3–1-3 or αvβ3 were cloned by sorting (αvβ3–1-3-CHO and αvβ3-CHO cells, respectively). The levels of αv and β3 expression were comparable in clonal WT αvβ3-CHO and αvβ3–1-3-CHO cells used. αvβ3–1-3-CHO cells showed significantly lower adhesion activity than αvβ3-CHO cells to both Fg and vWf; αvβ3–1-3-CHO cells required higher ligand concentrations for adhesion than WT αvβ3-CHO cells (Fig.5, A and B). In addition, solubilized αvβ3–1-3 did not bind to either Fg or Vn immobilized to Sepharose, although solubilized WT αvβ3 did (Fig.5 C). These results suggest that the β3–1-3 mutation significantly reduces binding of αvβ3 to Fg, Vn, and vWf. Although we observed that αvβ3–1-3 mutant binds to Fn 110-kDa fragments and to the GRGDS peptide on affinity chromatography (data not shown), we could not determine whether the β3–1-3 mutation changes the binding affinity of αvβ3 to Fn 110-kDa fragment or the GRGDSP peptide (because of the presence of other fibronectin receptors, endogenous αvβ1 and α5β1). The immunoprecipitation of whole lysate of αvβ3–1-3-CHO cells using anti-αv and anti-β3 mAbs showed that anti-αv and anti-β3 co-precipitated β3–1-3 and αv subunits, respectively, suggesting that the β3–1-3 mutation does not affect the α-β association. However, the αvβ3–1-3 mutant did not react with LM609, a function-blocking anti-αvβ3 mAb, upon immunoprecipitation (Fig.6) and flow cytometric analysis (data not shown), suggesting that the β3–1-3 mutation destroyed the LM609 epitope and that the CYDMKTTC sequence of β3 is closely located to ligand binding sites of αvβ3. We established that swapping the CTSEQNC sequence of β1 with the corresponding CYDMKTTC sequence of β3 induces significant changes in ligand specificity of αvβ1. The β1–3-1 mutation markedly increases affinity of αvβ1 to Fg, vWf, and Vn (a gain-of-function effect). Since the αvβ1–3-1 mutant is functional in cultured cells and transduces signals on adhesion to the ligands, the swapping did not induce a detectable adverse effect on the other receptor functions (e.g. α-β association and signal transduction). In reciprocal experiments, swapping a disulfide-linked CYDMKTTC sequence of β3 with the corresponding CTSEQNC sequence of β1 blocks the binding function of αvβ3 to Fg, vWf, and Vn. Taken together, the present study suggests that a small disulfide-linked CYMKTTC sequence of β3 (and the CTSEQNC sequence of β1 as well) defines a novel site of integrin β critical for ligand specificity. Sequence diversity among β subunits and localization within an I domain-like structure of β, close to putative ligand binding sites (see Introduction) is consistent with the proposed function of the sequence. In a preliminary study, we introduced mutations into the corresponding predicted loop of the β2 subunit. We found that these mutations showed profound effects on the ligand binding function of αLβ2 integrin, 2T. Kamata and Y. Takada, unpublished results. indicating that the diverse predicted loops of the β subunits are ubiquitously involved in the regulation of ligand binding functions. Mechanisms by which the disulfide-linked sequences in a predicted loop within the I domain-like structure of the β subunits define ligand specificity of integrins have yet to be studied. In preliminary studies, we did not obtain evidence that the β1–3-1 mutation induces constitutive activation of β1 integrins or induces drastic conformational changes. We determined the reactivity of the β1–3-1 mutant to an activation-dependent anti-β1 mAb 15/7, which recognizes the highly activated form of β1 integrin (30Yednock T.A. Cannon C. Vandevert C. Goldbach E.G. Shaw G. Ellis D.K. Liaw C. Fritz L.C. Tanner L.I. J. Biol. Chem. 1995; 270: 28740-28750Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). The binding profiles of 15/7 to the β1–3-1 mutant and wild-type β1 were identical; binding of 15/7 was dependent on activation in both cases (data not shown). The epitope for 15/7 has been localized within the residues 354–425 of β1 (in the non-ligand binding region outside the I domain-like region) (42Puzon-McLaughlin W. Yednock T. Takada Y. J. Biol. Chem. 1996; 271: 16580-16585Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). Therefore, there is a possibility that the effect of the β1–3-1 mutation on conformation remains local (e.g. within the I domain-like structure of β1) and 15/7 does not detect it. The amino acid residues surrounding the tripeptide RGD of the ligands have been reported to be critical for receptor specificity of snake venom disintegrins (43Lu X. Williams J.A. Deadman J.J. Salmon G.P. Kakkar V.V. Wilkinson J.M. Baruch D. Authi K.S. Rahman S. Biochem. J. 1994; 304: 929-936Crossref PubMed Scopus (48) Google Scholar, 44Scarborough R.M. Rose J.W. Naughton M.A. Phillips D.R. Nannizzi L. Arfsten A. Campbell A.M. Charo I.F. J. Biol. Chem. 1993; 268: 1058-1065Abstract Full Text PDF PubMed Google Scholar, 45Lu X. Rahman S. Kakkar V. Authi K. J. Biol. Chem. 1996; 271: 289-294Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). One possible mechanism is that the predicted loop structures of β3 or β1 interact with the residues surrounding the tripeptide RGD of ligands, if we assume that the predicted loop structure of β is close to the ligand binding site of αvβ3 or αvβ1. Another possibility is that the predicted loops regulate the access of a group of ligands (in the case of αvβ3, Fg, Vn, and vWf) to the ligand binding site. The CTSEQNC sequence of β1 (or the CYDMKTTC sequence of β3) is located within a predicted β-turn in the putative I domain-like structure of the β subunit (6Puzon-McLaughlin W. Takada Y. J. Biol. Chem. 1996; 271: 20438-20443Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). A recent model of the β I domain-like structure (23Tuckwell D. Humphries M. FEBS Lett. 1997; 400: 297-303Crossref PubMed Scopus (99) Google Scholar), the folding diagram, appears to be consistent with our previous and present mutagenesis data (Fig.7), and this model is similar to our preliminary model (not shown). All of the residues critical for ligand binding (e.g. Asp-130 and Glu-229 of β1) (6Puzon-McLaughlin W. Takada Y. J. Biol. Chem. 1996; 271: 20438-20443Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 10Takada Y. Ylanne J. Mandelman D. Puzon W. Ginsberg M. J. Cell Biol. 1992; 119: 913-921Crossref PubMed Scopus (98) Google Scholar) are located in the upper face of the model (predicted as the ligand binding site). Also, the regulatory epitope (residues 207–218 of β1) (20Takada Y. Puzon W. J. Biol. Chem. 1993; 268: 17597-17601Abstract Full Text PDF PubMed Google Scholar), which is recognized by both activating and inhibiting anti-β1 mAbs, is located in the non-ligand binding site (in the lower face) of the domain. Interestingly, a diverse sequence in the predicted loop (e.g. residues 176–199 in β1, residues 166–190 in β3), which is involved in ligand specificity of integrins in the present study, is located in the upper face of the domain in this model. The finding that the β3–1-3 mutation blocked binding of the function-blocking anti-αvβ3 antibody LM609 supports the idea that the predicted loop structure is close to the ligand binding site of αvβ3. Taken together, the present and previous mutagenesis data strongly support this model. Recently, Collins Tozer et al.(22Tozer E. Liddington R. Sutcliffe M. Smeeton A. Loftus J. J. Biol. Chem. 1996; 271: 21978-21984Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar) published an interesting atomic model of the putative I domain of β3, which is based on the crystal structure of the αM I domain (7Lee J.-O. Rieu P. Arnaout M.A. Liddington R. Cell. 1995; 80: 631-638Abstract Full Text PDF PubMed Scopus (792) Google Scholar). However, our mutagenesis data do not fit in very well with their model, since 1) the sequence CYDMKTTC of β3, which is critically involved in ligand binding to αvβ3, is not close to the MIDAS site (apparently in a non-ligand binding site) in their model, and 2) although Thr-197 of β3 is located in the MIDAS site of β3 in this model, the corresponding residue of β1 (Thr-206) is very close to the regulatory epitope. This epitope is probably located in a non-ligand binding site of β1 because 1) binding of some mAbs actually activates, instead of inactivating, the β1 integrins, and 2) this epitope has recently been shown to be an allosteric effector site of β1 (46Mould A. Akiyama S. Humphries M. J. Biol. Chem. 1996; 271: 20365-20374Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar), since the binding of an inhibitory anti-β1 mAb 13 to the regulatory epitope is also dramatically attenuated by ligands (Fn fragments or the GRGDS peptide). Further biochemical and structural characterization of this region of the β subunit may be required to substantiate these models. αvβ3 has been shown to be involved in the progression of melanoma and induction of neovascularization by tumor cells. αvβ3 antagonists promote tumor regression by inducing apoptosis of angiogenic blood vessels (47Brooks P. Montgomery A. Rosenfeld M. Reisfeld R. Hu T. Klier G. Cheresh D. Cell. 1994; 79: 1157-1164Abstract Full Text PDF PubMed Scopus (2161) Google Scholar, 48Montgomery A. Reisfeld R. Cheresh D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8856-8860Crossref PubMed Scopus (415) Google Scholar). We identified a critical region for ligand binding and specificity of integrins using a gain of function mutant of the β subunit. The predicted loop sequence of the integrin β3 subunit is a new potential target for designing inhibitors of ligand binding functions of αvβ3. We thank Drs. D. Cheresh, M. H. Ginsberg, M. E. Hemler, R. L. Juliano, J. Loftus, C. Morimoto, and Z. Ruggeri for valuable reagents.