The Integrin α9β1 Binds to a Novel Recognition Sequence (SVVYGLR) in the Thrombin-cleaved Amino-terminal Fragment of Osteopontin
1999; Elsevier BV; Volume: 274; Issue: 51 Linguagem: Inglês
10.1074/jbc.274.51.36328
ISSN1083-351X
AutoresYasuyuki Yokosaki, Nariaki Matsuura∥, Tomohiro Sasaki, Isao Murakami, Holm Schneider, Shigeki Higashiyama, Yoshiki Saitoh, Michio Yamakido, Yasuyuki Taooka, Dean Sheppard,
Tópico(s)Protease and Inhibitor Mechanisms
ResumoThe integrin α9β1 mediates cell adhesion to tenascin-C and VCAM-1 by binding to sequences distinct from the common integrin-recognition sequence, arginine-glycine-aspartic acid (RGD). A thrombin-cleaved NH2-terminal fragment of osteopontin containing the RGD sequence has recently been shown to also be a ligand for α9β1. In this report, we used site-directed mutagenesis and synthetic peptides to identify the α9β1 recognition sequence in osteopontin. α9-transfected SW480, Chinese hamster ovary, and L-cells adhered to a recombinant NH2-terminal osteopontin fragment in which the RGD site was mutated to RAA (nOPN-RAA). Adhesion was completely inhibited by anti-α9 monoclonal antibody Y9A2, indicating the presence of a non-RGD α9β1recognition sequence within this fragment. Alanine substitution mutagenesis of 13 additional conserved negatively charged amino acid residues in this fragment had no effect on α9β1-mediated adhesion, but adhesion was dramatically inhibited by either alanine substitution or deletion of tyrosine 165. A synthetic peptide, SVVYGLR, corresponding to the sequence surrounding Tyr165, blocked α9β1-mediated adhesion to nOPN-RAA and exposed a ligand-binding-dependent epitope on the integrin β1 subunit on α9-transfected, but not on mock-transfected cells. These results demonstrate that the linear sequence SVVYGLR directly binds to α9β1 and is responsible for α9β1-mediated cell adhesion to the NH2-terminal fragment of osteopontin. The integrin α9β1 mediates cell adhesion to tenascin-C and VCAM-1 by binding to sequences distinct from the common integrin-recognition sequence, arginine-glycine-aspartic acid (RGD). A thrombin-cleaved NH2-terminal fragment of osteopontin containing the RGD sequence has recently been shown to also be a ligand for α9β1. In this report, we used site-directed mutagenesis and synthetic peptides to identify the α9β1 recognition sequence in osteopontin. α9-transfected SW480, Chinese hamster ovary, and L-cells adhered to a recombinant NH2-terminal osteopontin fragment in which the RGD site was mutated to RAA (nOPN-RAA). Adhesion was completely inhibited by anti-α9 monoclonal antibody Y9A2, indicating the presence of a non-RGD α9β1recognition sequence within this fragment. Alanine substitution mutagenesis of 13 additional conserved negatively charged amino acid residues in this fragment had no effect on α9β1-mediated adhesion, but adhesion was dramatically inhibited by either alanine substitution or deletion of tyrosine 165. A synthetic peptide, SVVYGLR, corresponding to the sequence surrounding Tyr165, blocked α9β1-mediated adhesion to nOPN-RAA and exposed a ligand-binding-dependent epitope on the integrin β1 subunit on α9-transfected, but not on mock-transfected cells. These results demonstrate that the linear sequence SVVYGLR directly binds to α9β1 and is responsible for α9β1-mediated cell adhesion to the NH2-terminal fragment of osteopontin. Chinese hamster ovary NH2-terminal osteopontin Dulbecco's modified Eagle's medium polymerase chain reaction Integrins are cell surface heterodimeric receptors that mediate cell-cell and cell-extracellular matrix adhesion (1Sheppard D. Bioessays. 1996; 18: 655-660Crossref PubMed Scopus (121) Google Scholar, 2Hynes R.O. Cell. 1992; 69: 11-25Abstract Full Text PDF PubMed Scopus (8966) Google Scholar). Upon ligation by a wide variety of ligands, integrins can initiate signaling cascades that regulate cell growth, cell death, migration, polarization, and tissue remodeling (3Clark E.A. Brugge J.S. Science. 1995; 268: 233-239Crossref PubMed Scopus (2809) Google Scholar). Integrins recognize a surprisingly large number of functionally diverse proteins as ligands, and the list of known integrin ligands continues to grow. New integrin ligands have been identified, and drugs targeting integrins have been developed as a consequence of the description of short linear amino acid sequences that directly bind to integrins. For example, the integrins α5β1, α8β1, αvβ1, αvβ3, αIIbβ3, αvβ5, αvβ6, and αvβ8 bind to sequences containing the tri-peptide sequence Arg-Gly-Asp (RGD). Several new and biologically important integrin ligands have been identified based on the presence of this sequence (4Munger J.S. Huang X. Kawakatsu H. Griffiths M.J. Dalton S.L. Wu J. Pittet J.F. Kaminski N. Garat C. Matthay M.A. Rifkin D.B. Sheppard D. Cell. 1999; 96: 319-328Abstract Full Text Full Text PDF PubMed Scopus (1620) Google Scholar, 5Ruoslahti E. Annu. Rev. Cell Dev. Biol. 1996; 12: 697-715Crossref PubMed Scopus (2512) Google Scholar). Drugs modeled on the structure of the RGD sequence are being used or tested to inhibit integrin function for treatment of thrombosis, inflammation, atherosclerosis, osteoporosis, and cancer (5Ruoslahti E. Annu. Rev. Cell Dev. Biol. 1996; 12: 697-715Crossref PubMed Scopus (2512) Google Scholar). The RGD sequence has also been exploited to target cell surface integrins to enhance gene delivery (6Hart S. Harbottle R.P. Cooper R. Miller A. Williamson R Coutelle C. Gene Ther. 1995; 2: 552-554PubMed Google Scholar). We have previously identified the recognition sequence for the integrin α9β1 in tenascin-C and found that this sequence did not include RGD, but was homologous to the α4β1 recognition sequence in the inducible endothelial adhesion molecule VCAM-1 (7Yokosaki Y. Matsuura N. Higashiyama S. Murakami I. Obara M. Yamakido M. Shigeto N. Chen J. Sheppard D. J. Biol. Chem. 1998; 273: 11423-11428Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). This finding led to our identification of α9β1 as a receptor for VCAM-1 (8Taooka Y. Chen J. Yednock T. Sheppard D. J. Cell Biol. 1999; 145: 413-420Crossref PubMed Scopus (235) Google Scholar).Osteopontin is a phosphorylated acidic glycoprotein with diverse functions (9Denhardt D.T. Guo X. FASEB J. 1993; 7: 1475-1482Crossref PubMed Scopus (1001) Google Scholar) including cell adhesion, chemoattraction (10Singh R.P. Patarca R. Schwartz J. Singh P. Canter H. J. Exp. Med. 1990; 171: 1931-1942Crossref PubMed Scopus (211) Google Scholar), and immunomodulation (11Weber G.F. Canter H. Cytokine Growth Fact. Rev. 1996; 7: 241-248Crossref PubMed Scopus (113) Google Scholar). Osteopontin is present at high concentrations in diseases associated with tissue remodeling, including granuloma formation (12O'Regan A.W. Chupp G.L. Lowry J.A. Goetschkes M. Mulligan N. Berman J.S. J. Immunol. 1999; 162: 1024-1031PubMed Google Scholar) and coronary re-stenosis (13O'Brien E.R. Garvin M.R. Stewart D.K. Hinohara T. Simpson J.B. Schwartz S.M. Giachelli C.M. Arteriosc. Thromb. 1994; 14: 1648-1656Crossref PubMed Google Scholar, 14Panda D. Kundu G.C. Lee B.I. Peri A. Fohl D. Chackalaparmpil I. Mukherjee B.B. Li X.D. Mukherjee D.C. Seides S. Rosenberg J. Stark K. Mukherjee A.B. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9308-9313Crossref PubMed Scopus (153) Google Scholar), suggesting that this molecule might contribute to the process of remodeling. Osteopontin contains a predicted thrombin cleavage site (15Senger D.R. Perruzzi C.A. Gracey C.F. Papadopoulos A. Tenen D.G. Cancer Res. 1988; 48: 5770-5774PubMed Google Scholar) and appears to be cleaved at this site in vivo (16Senger D.R. Perruzzi C.A. Papadopoulous A. Anticancer Res. 1989; 9: 1291-1299PubMed Google Scholar, 17Senger D.R. Perruzzi C.A. Papadopoulous S.A. Van de Water L. Mol. Biol. Cell. 1994; 5: 565-574Crossref PubMed Scopus (180) Google Scholar). Several integrins have been identified as osteopontin receptors. Of these, α5β1 (18Nasu K. Ishida T. Setoguchi M. Higuchi Y. Akizuki S. Yamamoto S. Biochem. J. 1995; 307: 257-265Crossref PubMed Scopus (49) Google Scholar), α8β1(19Denda S. Reichardt L.F. Muller U. Mol. Biol. Cell. 1998; 9: 1425-1435Crossref PubMed Scopus (162) Google Scholar), αvβ1 (20Liaw L. Skinner M.P. Raines E.W. Ross R. Cheresh D.A. Schwartz S.M. Giachelli C.M. J. Clin. Invest. 1995; 95: 713-724Crossref PubMed Google Scholar, 21Hu D.D. Lin E.C. Kovach N.L. Hoyer J.R. Smith J.W. J. Biol. Chem. 1995; 270: 26232-26238Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar), αvβ3 (22Miyauchi A. Alvarez J. Greenfield E.M. Teti A. Grano M. Colucci S. Zanbinin-Zallone A. Ross F.P. Teitelbaum S.L. Cheresh D. J. Biol. Chem. 1991; 266: 20347-20369Google Scholar), and αvβ5 (20Liaw L. Skinner M.P. Raines E.W. Ross R. Cheresh D.A. Schwartz S.M. Giachelli C.M. J. Clin. Invest. 1995; 95: 713-724Crossref PubMed Google Scholar, 21Hu D.D. Lin E.C. Kovach N.L. Hoyer J.R. Smith J.W. J. Biol. Chem. 1995; 270: 26232-26238Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar) recognize an RGD sequence that is present within the NH2-terminal fragment of cleaved osteopontin. However, two integrins that generally bind to non-RGD sequences, α4β1 (23Bayless K.J. Meininger G.A. Scholtz J.M. Davis G.E. J. Cell Sci. 1998; 111: 1165-1174Crossref PubMed Google Scholar) and α9β1 (24Smith L.L. Cheung H.K. Ling L.E. Chen J. Sheppard D. Pytela R. Giachelli C.M. J. Biol. Chem. 1996; 271: 28485-28491Abstract Full Text Full Text PDF PubMed Scopus (231) Google Scholar), have also been reported to be osteopontin receptors. In contrast to the other integrin osteopontin receptors, α9β1 recognizes only the NH2-terminal fragment produced by thrombin cleavage, but does not appear to bind to full-length osteopontin, at least in vitro (24Smith L.L. Cheung H.K. Ling L.E. Chen J. Sheppard D. Pytela R. Giachelli C.M. J. Biol. Chem. 1996; 271: 28485-28491Abstract Full Text Full Text PDF PubMed Scopus (231) Google Scholar). In the present study we have used substitution and deletion mutagenesis and synthetic peptides to identify the sequence within this fragment that serves as the binding site for α9β1. The sequence identified (SVVYGLR) is a novel integrin-binding site that could serve as a basis for identifying additional α9β1 ligands or for developing specific inhibitors of α9β1function.DISCUSSIONPrevious reports identified osteopontin as a ligand for the integrin α9β1 and localized the binding site to a thrombin-cleaved NH2-terminal osteopontin fragment (24Smith L.L. Cheung H.K. Ling L.E. Chen J. Sheppard D. Pytela R. Giachelli C.M. J. Biol. Chem. 1996; 271: 28485-28491Abstract Full Text Full Text PDF PubMed Scopus (231) Google Scholar). In the present study, we have mapped the ligand-binding region within this osteopontin fragment in more detail by site-directed mutagenesis and synthetic peptides. The dramatic reduction of adhesion of α9-transfected cells to the mutant fragment in which tyrosine 165 in the C-terminal region of the fragment was replaced with alanine identified this tyrosine residue as critical for ligand binding. Two different deletion mutations in the C-terminal region also abolished α9β1-mediated adhesion. The effects of the peptide corresponding to the region surrounding Tyr165, SVVYGLR, inhibition of adhesion of α9-transfected cells to the NH2-terminal osteopontin fragment, and induction of a ligand binding-dependent epitope on the integrin β1subunit, provide further evidence that this region includes the ligand binding site.One previous report suggested that the RGD sequence itself played an important role in α9β1-mediated adhesion to the NH2-terminal fragment of osteopontin (30Smith L.L. Giachelli C.M. Exp. Cell Res. 1998; 242: 351-360Crossref PubMed Scopus (91) Google Scholar). In that report, a melanoma cell line that did not express any αvintegrins (Mo) and had previously been shown to attach to the NH2-terminal fragment, demonstrated markedly less adhesion to a fragment in which the RGD site was changed to RAA. In that study, deletion of the carboxyl-terminal region of the fragment, including both the RGD sequence and the adjacent SVVYGLR completely abolished adhesion of these cells, whereas the RAA mutation partially inhibited adhesion. These results are consistent with a role for the SVVYGLR peptide in α9β1-mediated adhesion. In the current study, we also found some inhibition of adhesion of α9-expressing cells by the RAA mutation, but this effect could have been entirely due to the effects of this mutation on other RGD-binding integrins expressed on the cells we used. Our results do not allow us to completely exclude any contribution of the RGD site to α9β1-mediated adhesion, but any such contribution, if present, appears to be minimal. We cannot fully explain the difference in the magnitude of inhibition by the RAA mutation in our study and the previous report. One possible explanation would be differential effects of the RAA mutation on the conformation of the SVVYGLR binding site under the conditions used to purify the recombinant fragments in the two studies. Another possibility is that the cell line used in the previous report expresses higher levels of other RGD-binding integrins or lower levels of α9β1 than the three transfected cell lines used in the current study.We have previously reported that α9β1 binds to two other ligands, tenascin-C (7Yokosaki Y. Matsuura N. Higashiyama S. Murakami I. Obara M. Yamakido M. Shigeto N. Chen J. Sheppard D. J. Biol. Chem. 1998; 273: 11423-11428Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, 25Yokosaki Y. Palmer E.L. Prieto A.L. Crossin K.L. Bourdon M.A. Pytela R. Sheppard D. J. Biol. Chem. 1994; 269: 26691-26696Abstract Full Text PDF PubMed Google Scholar) and VCAM-1 (8Taooka Y. Chen J. Yednock T. Sheppard D. J. Cell Biol. 1999; 145: 413-420Crossref PubMed Scopus (235) Google Scholar), at sites that do not include an RGD sequence. In those studies it was possible to definitively exclude any contribution of RGD sites. Since VCAM-1 does not contain an RGD, the attachment of α9-transfected CHO cells and SW480 cells to recombinant VCAM-1 (8Taooka Y. Chen J. Yednock T. Sheppard D. J. Cell Biol. 1999; 145: 413-420Crossref PubMed Scopus (235) Google Scholar) is definitely RGD-independent. α9β1 mediates adhesion to the third fibronectin type III repeat in tenascin-C, a repeat that does contain an RGD site that serves as a ligand binding site for the integrins αvβ3 (31Prieto A.L. Edelman G.M. Crossin K.L. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10154-10158Crossref PubMed Scopus (215) Google Scholar), αvβ6 (31Prieto A.L. Edelman G.M. Crossin K.L. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10154-10158Crossref PubMed Scopus (215) Google Scholar, 32Yokosaki Y. Monis H. Chen J. Sheppard D. J. Biol. Chem. 1996; 271: 24144-24150Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar), and α8β1 (33Schnapp L.M. Hatch N. Ramos D. Kliminskaya I.V. Sheppard D. Pytela R. J. Biol. Chem. 1995; 270: 23196-23202Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar). However, the α9-transfected SW480 cells we used to study interaction with tenascin-C do not express any of these integrins. These cells adhered equally well to a wild type tenascin-C fragment as to a fragment in which the RGD sequence had been changed to RAA, and an RGD-containing peptide had no effect on adhesion.Most previously described integrin recognition motifs, including the EIDGIEL-recognition sequence we described in tenascin-C, include a central negatively charged amino acid residue. We were therefore surprised that mutation of each of the conserved negatively charged amino acids in the NH2-terminal fragment of osteopontin had little effect on α9β1-mediated adhesion to this fragment, and that the binding site we identified did not contain any negatively charged amino acids. Rather, the critical sequence includes a central tyrosine residue. Thus far, the only integrin fragments for which there are solved crystal structures are the inserted (or I) domains present in a subset of integrin α subunits. In the case of these integrins, the negatively charged aspartic acid residue present in many integrin ligands has been suggested as one of the coordination sites for a metal ion predicted to be required at the ligand binding site (34Lee J.O. Rieu P. Arnaout M.A. Liddington R. Cell. 1995; 80: 631-638Abstract Full Text PDF PubMed Scopus (798) Google Scholar). However, the α9 subunit does not contain an I domain (35Palmer E.L. Ruegg C. Ferrando R. Pytela R. Sheppard D. J. Cell Biol. 1993; 123: 1289-1297Crossref PubMed Scopus (215) Google Scholar), and the structural basis of interactions of non-I domain-containing integrins with their ligands remains to be determined. The identification of an integrin ligand binding site without any acidic residues suggests that alternative mechanisms must exist for integrin-ligand interactions. However, it is possible that the free carboxyl terminus could be an alternative source of a negative charge. In any case, the recognition sequence we have described could also allow identification of additional α9β1 ligands and improved design of specific inhibitors.Based on the results of in vitro cell adhesion assays, the adhesive sequence in the NH2-terminal osteopontin fragment appears to be cryptic, and is not recognized by α9β1 in the full-length form of osteopontin (24Smith L.L. Cheung H.K. Ling L.E. Chen J. Sheppard D. Pytela R. Giachelli C.M. J. Biol. Chem. 1996; 271: 28485-28491Abstract Full Text Full Text PDF PubMed Scopus (231) Google Scholar). Osteopontin cleavage by thrombin thus appears to be critical for induction of accessibility to this site. Since the crystal structure of the osteopontin protein is not available at this time, we cannot predict the nature of any conformational change that results from thrombin cleavage. However, the localization of the recognition site to a linear peptide sequence immediately adjacent to the cleavage site fits well with the idea that this sequence is made accessible by thrombin cleavage.The biological significance of the thrombin-cleaved fragment that is present in vivo has not been previously determined (16Senger D.R. Perruzzi C.A. Papadopoulous A. Anticancer Res. 1989; 9: 1291-1299PubMed Google Scholar, 17Senger D.R. Perruzzi C.A. Papadopoulous S.A. Van de Water L. Mol. Biol. Cell. 1994; 5: 565-574Crossref PubMed Scopus (180) Google Scholar). The results of this study together with the previous report provide insights into the potential physiological role of thrombin cleavage. We have previously shown that α9β1 is expressed on neutrophils (8Taooka Y. Chen J. Yednock T. Sheppard D. J. Cell Biol. 1999; 145: 413-420Crossref PubMed Scopus (235) Google Scholar), smooth muscle cells, and epithelial cells (35Palmer E.L. Ruegg C. Ferrando R. Pytela R. Sheppard D. J. Cell Biol. 1993; 123: 1289-1297Crossref PubMed Scopus (215) Google Scholar), and that ligation of α9β1 can contribute to cell migration (8Taooka Y. Chen J. Yednock T. Sheppard D. J. Cell Biol. 1999; 145: 413-420Crossref PubMed Scopus (235) Google Scholar) and proliferation (32Yokosaki Y. Monis H. Chen J. Sheppard D. J. Biol. Chem. 1996; 271: 24144-24150Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). Upon thrombin cleavage of osteopontin at sites of inflammation or remodeling, the generation of an α9β1 ligand could thus enhance the cell migration and proliferation that are required for tissue remodeling to occur. In addition, osteopontin has another thrombin cleavage site at Arg160-Gly161 (16Senger D.R. Perruzzi C.A. Papadopoulous A. Anticancer Res. 1989; 9: 1291-1299PubMed Google Scholar). The sequence of the fragment produced by cleavage at both sites would be GDSVVYGLR, a fragment that would be expected to inhibit α9β1-mediated effects on cell behavior, and thereby potentially contribute to the termination of the events described above. Integrins are cell surface heterodimeric receptors that mediate cell-cell and cell-extracellular matrix adhesion (1Sheppard D. Bioessays. 1996; 18: 655-660Crossref PubMed Scopus (121) Google Scholar, 2Hynes R.O. Cell. 1992; 69: 11-25Abstract Full Text PDF PubMed Scopus (8966) Google Scholar). Upon ligation by a wide variety of ligands, integrins can initiate signaling cascades that regulate cell growth, cell death, migration, polarization, and tissue remodeling (3Clark E.A. Brugge J.S. Science. 1995; 268: 233-239Crossref PubMed Scopus (2809) Google Scholar). Integrins recognize a surprisingly large number of functionally diverse proteins as ligands, and the list of known integrin ligands continues to grow. New integrin ligands have been identified, and drugs targeting integrins have been developed as a consequence of the description of short linear amino acid sequences that directly bind to integrins. For example, the integrins α5β1, α8β1, αvβ1, αvβ3, αIIbβ3, αvβ5, αvβ6, and αvβ8 bind to sequences containing the tri-peptide sequence Arg-Gly-Asp (RGD). Several new and biologically important integrin ligands have been identified based on the presence of this sequence (4Munger J.S. Huang X. Kawakatsu H. Griffiths M.J. Dalton S.L. Wu J. Pittet J.F. Kaminski N. Garat C. Matthay M.A. Rifkin D.B. Sheppard D. Cell. 1999; 96: 319-328Abstract Full Text Full Text PDF PubMed Scopus (1620) Google Scholar, 5Ruoslahti E. Annu. Rev. Cell Dev. Biol. 1996; 12: 697-715Crossref PubMed Scopus (2512) Google Scholar). Drugs modeled on the structure of the RGD sequence are being used or tested to inhibit integrin function for treatment of thrombosis, inflammation, atherosclerosis, osteoporosis, and cancer (5Ruoslahti E. Annu. Rev. Cell Dev. Biol. 1996; 12: 697-715Crossref PubMed Scopus (2512) Google Scholar). The RGD sequence has also been exploited to target cell surface integrins to enhance gene delivery (6Hart S. Harbottle R.P. Cooper R. Miller A. Williamson R Coutelle C. Gene Ther. 1995; 2: 552-554PubMed Google Scholar). We have previously identified the recognition sequence for the integrin α9β1 in tenascin-C and found that this sequence did not include RGD, but was homologous to the α4β1 recognition sequence in the inducible endothelial adhesion molecule VCAM-1 (7Yokosaki Y. Matsuura N. Higashiyama S. Murakami I. Obara M. Yamakido M. Shigeto N. Chen J. Sheppard D. J. Biol. Chem. 1998; 273: 11423-11428Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). This finding led to our identification of α9β1 as a receptor for VCAM-1 (8Taooka Y. Chen J. Yednock T. Sheppard D. J. Cell Biol. 1999; 145: 413-420Crossref PubMed Scopus (235) Google Scholar). Osteopontin is a phosphorylated acidic glycoprotein with diverse functions (9Denhardt D.T. Guo X. FASEB J. 1993; 7: 1475-1482Crossref PubMed Scopus (1001) Google Scholar) including cell adhesion, chemoattraction (10Singh R.P. Patarca R. Schwartz J. Singh P. Canter H. J. Exp. Med. 1990; 171: 1931-1942Crossref PubMed Scopus (211) Google Scholar), and immunomodulation (11Weber G.F. Canter H. Cytokine Growth Fact. Rev. 1996; 7: 241-248Crossref PubMed Scopus (113) Google Scholar). Osteopontin is present at high concentrations in diseases associated with tissue remodeling, including granuloma formation (12O'Regan A.W. Chupp G.L. Lowry J.A. Goetschkes M. Mulligan N. Berman J.S. J. Immunol. 1999; 162: 1024-1031PubMed Google Scholar) and coronary re-stenosis (13O'Brien E.R. Garvin M.R. Stewart D.K. Hinohara T. Simpson J.B. Schwartz S.M. Giachelli C.M. Arteriosc. Thromb. 1994; 14: 1648-1656Crossref PubMed Google Scholar, 14Panda D. Kundu G.C. Lee B.I. Peri A. Fohl D. Chackalaparmpil I. Mukherjee B.B. Li X.D. Mukherjee D.C. Seides S. Rosenberg J. Stark K. Mukherjee A.B. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9308-9313Crossref PubMed Scopus (153) Google Scholar), suggesting that this molecule might contribute to the process of remodeling. Osteopontin contains a predicted thrombin cleavage site (15Senger D.R. Perruzzi C.A. Gracey C.F. Papadopoulos A. Tenen D.G. Cancer Res. 1988; 48: 5770-5774PubMed Google Scholar) and appears to be cleaved at this site in vivo (16Senger D.R. Perruzzi C.A. Papadopoulous A. Anticancer Res. 1989; 9: 1291-1299PubMed Google Scholar, 17Senger D.R. Perruzzi C.A. Papadopoulous S.A. Van de Water L. Mol. Biol. Cell. 1994; 5: 565-574Crossref PubMed Scopus (180) Google Scholar). Several integrins have been identified as osteopontin receptors. Of these, α5β1 (18Nasu K. Ishida T. Setoguchi M. Higuchi Y. Akizuki S. Yamamoto S. Biochem. J. 1995; 307: 257-265Crossref PubMed Scopus (49) Google Scholar), α8β1(19Denda S. Reichardt L.F. Muller U. Mol. Biol. Cell. 1998; 9: 1425-1435Crossref PubMed Scopus (162) Google Scholar), αvβ1 (20Liaw L. Skinner M.P. Raines E.W. Ross R. Cheresh D.A. Schwartz S.M. Giachelli C.M. J. Clin. Invest. 1995; 95: 713-724Crossref PubMed Google Scholar, 21Hu D.D. Lin E.C. Kovach N.L. Hoyer J.R. Smith J.W. J. Biol. Chem. 1995; 270: 26232-26238Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar), αvβ3 (22Miyauchi A. Alvarez J. Greenfield E.M. Teti A. Grano M. Colucci S. Zanbinin-Zallone A. Ross F.P. Teitelbaum S.L. Cheresh D. J. Biol. Chem. 1991; 266: 20347-20369Google Scholar), and αvβ5 (20Liaw L. Skinner M.P. Raines E.W. Ross R. Cheresh D.A. Schwartz S.M. Giachelli C.M. J. Clin. Invest. 1995; 95: 713-724Crossref PubMed Google Scholar, 21Hu D.D. Lin E.C. Kovach N.L. Hoyer J.R. Smith J.W. J. Biol. Chem. 1995; 270: 26232-26238Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar) recognize an RGD sequence that is present within the NH2-terminal fragment of cleaved osteopontin. However, two integrins that generally bind to non-RGD sequences, α4β1 (23Bayless K.J. Meininger G.A. Scholtz J.M. Davis G.E. J. Cell Sci. 1998; 111: 1165-1174Crossref PubMed Google Scholar) and α9β1 (24Smith L.L. Cheung H.K. Ling L.E. Chen J. Sheppard D. Pytela R. Giachelli C.M. J. Biol. Chem. 1996; 271: 28485-28491Abstract Full Text Full Text PDF PubMed Scopus (231) Google Scholar), have also been reported to be osteopontin receptors. In contrast to the other integrin osteopontin receptors, α9β1 recognizes only the NH2-terminal fragment produced by thrombin cleavage, but does not appear to bind to full-length osteopontin, at least in vitro (24Smith L.L. Cheung H.K. Ling L.E. Chen J. Sheppard D. Pytela R. Giachelli C.M. J. Biol. Chem. 1996; 271: 28485-28491Abstract Full Text Full Text PDF PubMed Scopus (231) Google Scholar). In the present study we have used substitution and deletion mutagenesis and synthetic peptides to identify the sequence within this fragment that serves as the binding site for α9β1. The sequence identified (SVVYGLR) is a novel integrin-binding site that could serve as a basis for identifying additional α9β1 ligands or for developing specific inhibitors of α9β1function. DISCUSSIONPrevious reports identified osteopontin as a ligand for the integrin α9β1 and localized the binding site to a thrombin-cleaved NH2-terminal osteopontin fragment (24Smith L.L. Cheung H.K. Ling L.E. Chen J. Sheppard D. Pytela R. Giachelli C.M. J. Biol. Chem. 1996; 271: 28485-28491Abstract Full Text Full Text PDF PubMed Scopus (231) Google Scholar). In the present study, we have mapped the ligand-binding region within this osteopontin fragment in more detail by site-directed mutagenesis and synthetic peptides. The dramatic reduction of adhesion of α9-transfected cells to the mutant fragment in which tyrosine 165 in the C-terminal region of the fragment was replaced with alanine identified this tyrosine residue as critical for ligand binding. Two different deletion mutations in the C-terminal region also abolished α9β1-mediated adhesion. The effects of the peptide corresponding to the region surrounding Tyr165, SVVYGLR, inhibition of adhesion of α9-transfected cells to the NH2-terminal osteopontin fragment, and induction of a ligand binding-dependent epitope on the integrin β1subunit, provide further evidence that this region includes the ligand binding site.One previous report suggested that the RGD sequence itself played an important role in α9β1-mediated adhesion to the NH2-terminal fragment of osteopontin (30Smith L.L. Giachelli C.M. Exp. Cell Res. 1998; 242: 351-360Crossref PubMed Scopus (91) Google Scholar). In that report, a melanoma cell line that did not express any αvintegrins (Mo) and had previously been shown to attach to the NH2-terminal fragment, demonstrated markedly less adhesion to a fragment in which the RGD site was changed to RAA. In that study, deletion of the carboxyl-terminal region of the fragment, including both the RGD sequence and the adjacent SVVYGLR completely abolished adhesion of these cells, whereas the RAA mutation partially inhibited adhesion. These results are consistent with a role for the SVVYGLR peptide in α9β1-mediated adhesion. In the current study, we also found some inhibition of adhesion of α9-expressing cells by the RAA mutation, but this effect could have been entirely due to the effects of this mutation on other RGD-binding integrins expressed on the cells we used. Our results do not allow us to completely exclude any contribution of the RGD site to α9β1-mediated adhesion, but any such contribution, if present, appears to be minimal. We cannot fully explain the difference in the magnitude of inhibition by the RAA mutation in our study and the previous report. One possible explanation would be differential effects of the RAA mutation on the conformation of the SVVYGLR binding site under the conditions used to purify the recombinant fragments in the two studies. Another possibility is that the cell line used in the previous report expresses higher levels of other RGD-binding integrins or lower levels of α9β1 than the three transfected cell lines used in the current study.We have previously reported that α9β1 binds to two other ligands, tenascin-C (7Yokosaki Y. Matsuura N. Higashiyama S. Murakami I. Obara M. Yamakido M. Shigeto N. Chen J. Sheppard D. J. Biol. Chem. 1998; 273: 11423-11428Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, 25Yokosaki Y. Palmer E.L. Prieto A.L. Crossin K.L. Bourdon M.A. Pytela R. Sheppard D. J. Biol. Chem. 1994; 269: 26691-26696Abstract Full Text PDF PubMed Google Scholar) and VCAM-1 (8Taooka Y. Chen J. Yednock T. Sheppard D. J. Cell Biol. 1999; 145: 413-420Crossref PubMed Scopus (235) Google Scholar), at sites that do not include an RGD sequence. In those studies it was possible to definitively exclude any contribution of RGD sites. Since VCAM-1 does not contain an RGD, the attachment of α9-transfected CHO cells and SW480 cells to recombinant VCAM-1 (8Taooka Y. Chen J. Yednock T. Sheppard D. J. Cell Biol. 1999; 145: 413-420Crossref PubMed Scopus (235) Google Scholar) is definitely RGD-independent. α9β1 mediates adhesion to the third fibronectin type III repeat in tenascin-C, a repeat that does contain an RGD site that serves as a ligand binding site for the integrins αvβ3 (31Prieto A.L. Edelman G.M. Crossin K.L. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10154-10158Crossref PubMed Scopus (215) Google Scholar), αvβ6 (31Prieto A.L. Edelman G.M. Crossin K.L. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10154-10158Crossref PubMed Scopus (215) Google Scholar, 32Yokosaki Y. Monis H. Chen J. Sheppard D. J. Biol. Chem. 1996; 271: 24144-24150Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar), and α8β1 (33Schnapp L.M. Hatch N. Ramos D. Kliminskaya I.V. Sheppard D. Pytela R. J. Biol. Chem. 1995; 270: 23196-23202Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar). However, the α9-transfected SW480 cells we used to study interaction with tenascin-C do not express any of these integrins. These cells adhered equally well to a wild type tenascin-C fragment as to a fragment in which the RGD sequence had been changed to RAA, and an RGD-containing peptide had no effect on adhesion.Most previously described integrin recognition motifs, including the EIDGIEL-recognition sequence we described in tenascin-C, include a central negatively charged amino acid residue. We were therefore surprised that mutation of each of the conserved negatively charged amino acids in the NH2-terminal fragment of osteopontin had little effect on α9β1-mediated adhesion to this fragment, and that the binding site we identified did not contain any negatively charged amino acids. Rather, the critical sequence includes a central tyrosine residue. Thus far, the only integrin fragments for which there are solved crystal structures are the inserted (or I) domains present in a subset of integrin α subunits. In the case of these integrins, the negatively charged aspartic acid residue present in many integrin ligands has been suggested as one of the coordination sites for a metal ion predicted to be required at the ligand binding site (34Lee J.O. Rieu P. Arnaout M.A. Liddington R. Cell. 1995; 80: 631-638Abstract Full Text PDF PubMed Scopus (798) Google Scholar). However, the α9 subunit does not contain an I domain (35Palmer E.L. Ruegg C. Ferrando R. Pytela R. Sheppard D. J. Cell Biol. 1993; 123: 1289-1297Crossref PubMed Scopus (215) Google Scholar), and the structural basis of interactions of non-I domain-containing integrins with their ligands remains to be determined. The identification of an integrin ligand binding site without any acidic residues suggests that alternative mechanisms must exist for integrin-ligand interactions. However, it is possible that the free carboxyl terminus could be an alternative source of a negative charge. In any case, the recognition sequence we have described could also allow identification of additional α9β1 ligands and improved design of specific inhibitors.Based on the results of in vitro cell adhesion assays, the adhesive sequence in the NH2-terminal osteopontin fragment appears to be cryptic, and is not recognized by α9β1 in the full-length form of osteopontin (24Smith L.L. Cheung H.K. Ling L.E. Chen J. Sheppard D. Pytela R. Giachelli C.M. J. Biol. Chem. 1996; 271: 28485-28491Abstract Full Text Full Text PDF PubMed Scopus (231) Google Scholar). Osteopontin cleavage by thrombin thus appears to be critical for induction of accessibility to this site. Since the crystal structure of the osteopontin protein is not available at this time, we cannot predict the nature of any conformational change that results from thrombin cleavage. However, the localization of the recognition site to a linear peptide sequence immediately adjacent to the cleavage site fits well with the idea that this sequence is made accessible by thrombin cleavage.The biological significance of the thrombin-cleaved fragment that is present in vivo has not been previously determined (16Senger D.R. Perruzzi C.A. Papadopoulous A. Anticancer Res. 1989; 9: 1291-1299PubMed Google Scholar, 17Senger D.R. Perruzzi C.A. Papadopoulous S.A. Van de Water L. Mol. Biol. Cell. 1994; 5: 565-574Crossref PubMed Scopus (180) Google Scholar). The results of this study together with the previous report provide insights into the potential physiological role of thrombin cleavage. We have previously shown that α9β1 is expressed on neutrophils (8Taooka Y. Chen J. Yednock T. Sheppard D. J. Cell Biol. 1999; 145: 413-420Crossref PubMed Scopus (235) Google Scholar), smooth muscle cells, and epithelial cells (35Palmer E.L. Ruegg C. Ferrando R. Pytela R. Sheppard D. J. Cell Biol. 1993; 123: 1289-1297Crossref PubMed Scopus (215) Google Scholar), and that ligation of α9β1 can contribute to cell migration (8Taooka Y. Chen J. Yednock T. Sheppard D. J. Cell Biol. 1999; 145: 413-420Crossref PubMed Scopus (235) Google Scholar) and proliferation (32Yokosaki Y. Monis H. Chen J. Sheppard D. J. Biol. Chem. 1996; 271: 24144-24150Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). Upon thrombin cleavage of osteopontin at sites of inflammation or remodeling, the generation of an α9β1 ligand could thus enhance the cell migration and proliferation that are required for tissue remodeling to occur. In addition, osteopontin has another thrombin cleavage site at Arg160-Gly161 (16Senger D.R. Perruzzi C.A. Papadopoulous A. Anticancer Res. 1989; 9: 1291-1299PubMed Google Scholar). The sequence of the fragment produced by cleavage at both sites would be GDSVVYGLR, a fragment that would be expected to inhibit α9β1-mediated effects on cell behavior, and thereby potentially contribute to the termination of the events described above. Previous reports identified osteopontin as a ligand for the integrin α9β1 and localized the binding site to a thrombin-cleaved NH2-terminal osteopontin fragment (24Smith L.L. Cheung H.K. Ling L.E. Chen J. Sheppard D. Pytela R. Giachelli C.M. J. Biol. Chem. 1996; 271: 28485-28491Abstract Full Text Full Text PDF PubMed Scopus (231) Google Scholar). In the present study, we have mapped the ligand-binding region within this osteopontin fragment in more detail by site-directed mutagenesis and synthetic peptides. The dramatic reduction of adhesion of α9-transfected cells to the mutant fragment in which tyrosine 165 in the C-terminal region of the fragment was replaced with alanine identified this tyrosine residue as critical for ligand binding. Two different deletion mutations in the C-terminal region also abolished α9β1-mediated adhesion. The effects of the peptide corresponding to the region surrounding Tyr165, SVVYGLR, inhibition of adhesion of α9-transfected cells to the NH2-terminal osteopontin fragment, and induction of a ligand binding-dependent epitope on the integrin β1subunit, provide further evidence that this region includes the ligand binding site. One previous report suggested that the RGD sequence itself played an important role in α9β1-mediated adhesion to the NH2-terminal fragment of osteopontin (30Smith L.L. Giachelli C.M. Exp. Cell Res. 1998; 242: 351-360Crossref PubMed Scopus (91) Google Scholar). In that report, a melanoma cell line that did not express any αvintegrins (Mo) and had previously been shown to attach to the NH2-terminal fragment, demonstrated markedly less adhesion to a fragment in which the RGD site was changed to RAA. In that study, deletion of the carboxyl-terminal region of the fragment, including both the RGD sequence and the adjacent SVVYGLR completely abolished adhesion of these cells, whereas the RAA mutation partially inhibited adhesion. These results are consistent with a role for the SVVYGLR peptide in α9β1-mediated adhesion. In the current study, we also found some inhibition of adhesion of α9-expressing cells by the RAA mutation, but this effect could have been entirely due to the effects of this mutation on other RGD-binding integrins expressed on the cells we used. Our results do not allow us to completely exclude any contribution of the RGD site to α9β1-mediated adhesion, but any such contribution, if present, appears to be minimal. We cannot fully explain the difference in the magnitude of inhibition by the RAA mutation in our study and the previous report. One possible explanation would be differential effects of the RAA mutation on the conformation of the SVVYGLR binding site under the conditions used to purify the recombinant fragments in the two studies. Another possibility is that the cell line used in the previous report expresses higher levels of other RGD-binding integrins or lower levels of α9β1 than the three transfected cell lines used in the current study. We have previously reported that α9β1 binds to two other ligands, tenascin-C (7Yokosaki Y. Matsuura N. Higashiyama S. Murakami I. Obara M. Yamakido M. Shigeto N. Chen J. Sheppard D. J. Biol. Chem. 1998; 273: 11423-11428Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, 25Yokosaki Y. Palmer E.L. Prieto A.L. Crossin K.L. Bourdon M.A. Pytela R. Sheppard D. J. Biol. Chem. 1994; 269: 26691-26696Abstract Full Text PDF PubMed Google Scholar) and VCAM-1 (8Taooka Y. Chen J. Yednock T. Sheppard D. J. Cell Biol. 1999; 145: 413-420Crossref PubMed Scopus (235) Google Scholar), at sites that do not include an RGD sequence. In those studies it was possible to definitively exclude any contribution of RGD sites. Since VCAM-1 does not contain an RGD, the attachment of α9-transfected CHO cells and SW480 cells to recombinant VCAM-1 (8Taooka Y. Chen J. Yednock T. Sheppard D. J. Cell Biol. 1999; 145: 413-420Crossref PubMed Scopus (235) Google Scholar) is definitely RGD-independent. α9β1 mediates adhesion to the third fibronectin type III repeat in tenascin-C, a repeat that does contain an RGD site that serves as a ligand binding site for the integrins αvβ3 (31Prieto A.L. Edelman G.M. Crossin K.L. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10154-10158Crossref PubMed Scopus (215) Google Scholar), αvβ6 (31Prieto A.L. Edelman G.M. Crossin K.L. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10154-10158Crossref PubMed Scopus (215) Google Scholar, 32Yokosaki Y. Monis H. Chen J. Sheppard D. J. Biol. Chem. 1996; 271: 24144-24150Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar), and α8β1 (33Schnapp L.M. Hatch N. Ramos D. Kliminskaya I.V. Sheppard D. Pytela R. J. Biol. Chem. 1995; 270: 23196-23202Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar). However, the α9-transfected SW480 cells we used to study interaction with tenascin-C do not express any of these integrins. These cells adhered equally well to a wild type tenascin-C fragment as to a fragment in which the RGD sequence had been changed to RAA, and an RGD-containing peptide had no effect on adhesion. Most previously described integrin recognition motifs, including the EIDGIEL-recognition sequence we described in tenascin-C, include a central negatively charged amino acid residue. We were therefore surprised that mutation of each of the conserved negatively charged amino acids in the NH2-terminal fragment of osteopontin had little effect on α9β1-mediated adhesion to this fragment, and that the binding site we identified did not contain any negatively charged amino acids. Rather, the critical sequence includes a central tyrosine residue. Thus far, the only integrin fragments for which there are solved crystal structures are the inserted (or I) domains present in a subset of integrin α subunits. In the case of these integrins, the negatively charged aspartic acid residue present in many integrin ligands has been suggested as one of the coordination sites for a metal ion predicted to be required at the ligand binding site (34Lee J.O. Rieu P. Arnaout M.A. Liddington R. Cell. 1995; 80: 631-638Abstract Full Text PDF PubMed Scopus (798) Google Scholar). However, the α9 subunit does not contain an I domain (35Palmer E.L. Ruegg C. Ferrando R. Pytela R. Sheppard D. J. Cell Biol. 1993; 123: 1289-1297Crossref PubMed Scopus (215) Google Scholar), and the structural basis of interactions of non-I domain-containing integrins with their ligands remains to be determined. The identification of an integrin ligand binding site without any acidic residues suggests that alternative mechanisms must exist for integrin-ligand interactions. However, it is possible that the free carboxyl terminus could be an alternative source of a negative charge. In any case, the recognition sequence we have described could also allow identification of additional α9β1 ligands and improved design of specific inhibitors. Based on the results of in vitro cell adhesion assays, the adhesive sequence in the NH2-terminal osteopontin fragment appears to be cryptic, and is not recognized by α9β1 in the full-length form of osteopontin (24Smith L.L. Cheung H.K. Ling L.E. Chen J. Sheppard D. Pytela R. Giachelli C.M. J. Biol. Chem. 1996; 271: 28485-28491Abstract Full Text Full Text PDF PubMed Scopus (231) Google Scholar). Osteopontin cleavage by thrombin thus appears to be critical for induction of accessibility to this site. Since the crystal structure of the osteopontin protein is not available at this time, we cannot predict the nature of any conformational change that results from thrombin cleavage. However, the localization of the recognition site to a linear peptide sequence immediately adjacent to the cleavage site fits well with the idea that this sequence is made accessible by thrombin cleavage. The biological significance of the thrombin-cleaved fragment that is present in vivo has not been previously determined (16Senger D.R. Perruzzi C.A. Papadopoulous A. Anticancer Res. 1989; 9: 1291-1299PubMed Google Scholar, 17Senger D.R. Perruzzi C.A. Papadopoulous S.A. Van de Water L. Mol. Biol. Cell. 1994; 5: 565-574Crossref PubMed Scopus (180) Google Scholar). The results of this study together with the previous report provide insights into the potential physiological role of thrombin cleavage. We have previously shown that α9β1 is expressed on neutrophils (8Taooka Y. Chen J. Yednock T. Sheppard D. J. Cell Biol. 1999; 145: 413-420Crossref PubMed Scopus (235) Google Scholar), smooth muscle cells, and epithelial cells (35Palmer E.L. Ruegg C. Ferrando R. Pytela R. Sheppard D. J. Cell Biol. 1993; 123: 1289-1297Crossref PubMed Scopus (215) Google Scholar), and that ligation of α9β1 can contribute to cell migration (8Taooka Y. Chen J. Yednock T. Sheppard D. J. Cell Biol. 1999; 145: 413-420Crossref PubMed Scopus (235) Google Scholar) and proliferation (32Yokosaki Y. Monis H. Chen J. Sheppard D. J. Biol. Chem. 1996; 271: 24144-24150Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). Upon thrombin cleavage of osteopontin at sites of inflammation or remodeling, the generation of an α9β1 ligand could thus enhance the cell migration and proliferation that are required for tissue remodeling to occur. In addition, osteopontin has another thrombin cleavage site at Arg160-Gly161 (16Senger D.R. Perruzzi C.A. Papadopoulous A. Anticancer Res. 1989; 9: 1291-1299PubMed Google Scholar). The sequence of the fragment produced by cleavage at both sites would be GDSVVYGLR, a fragment that would be expected to inhibit α9β1-mediated effects on cell behavior, and thereby potentially contribute to the termination of the events described above. We thank Dr. Ted Yednock (Elan Pharmaceuticals) for antibody 15/7 and Dr. Yasunori Kodama (Director, National Hiroshima Hospital) for generous support of our study.
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