Definition of an Unexpected Ligand Recognition Motif for αvβ6 Integrin

Abstract

Integrin interactions with extracellular matrix proteins are mediated by brief oligopeptide recognition sequences, and synthetic peptides containing such sequences can inhibit integrin binding to the matrix. The RGD peptide motif is recognized by many integrins including αvβ6, a specific receptor for fibronectin thought to support epithelial cell proliferation during wound healing and carcinoma progression. We report here the discovery of an unexpected non-RGD recognition motif for integrin αvβ6. We compared the recognition profiles of recombinant αvβ6 and αvβ3 integrins by using phage display screening employing 7-mer and 12-mer peptide libraries. As predicted, phages binding strongly to αvβ3 contained ubiquitous RGD sequences. However, on αvβ6, in addition to RGD- containing phages, one-quarter of the population from the 12-mer library contained the distinctive consensus motif DLXXL. A synthetic DLXXL peptide, RTDLDSLRTYTL, selected from the phage sequences (clone-1) was a selective inhibitor of RGD-dependent ligand binding to αvβ6 in isolated receptor assays (IC50 = 20 nm), and in cell adhesion assays (IC50 = 50 μm). DLXXL peptides were highly specific inhibitors of αvβ6-fibronectin interaction as synthetic scrambled or reversed DLXXL peptides were inactive. NH2- and COOH-terminal modifications of the flanking amino acids suggested that the preceding two and a single trailing amino acid were also involved in interaction with αvβ6. The DLXXL sequence is present in several matrix components and in the β chain of many integrins. Although there is as yet no precise biological role known for DLXXL, it is clearly a specific inhibitory sequence for integrin αvβ6 which has been unrecognized previously. Integrin interactions with extracellular matrix proteins are mediated by brief oligopeptide recognition sequences, and synthetic peptides containing such sequences can inhibit integrin binding to the matrix. The RGD peptide motif is recognized by many integrins including αvβ6, a specific receptor for fibronectin thought to support epithelial cell proliferation during wound healing and carcinoma progression. We report here the discovery of an unexpected non-RGD recognition motif for integrin αvβ6. We compared the recognition profiles of recombinant αvβ6 and αvβ3 integrins by using phage display screening employing 7-mer and 12-mer peptide libraries. As predicted, phages binding strongly to αvβ3 contained ubiquitous RGD sequences. However, on αvβ6, in addition to RGD- containing phages, one-quarter of the population from the 12-mer library contained the distinctive consensus motif DLXXL. A synthetic DLXXL peptide, RTDLDSLRTYTL, selected from the phage sequences (clone-1) was a selective inhibitor of RGD-dependent ligand binding to αvβ6 in isolated receptor assays (IC50 = 20 nm), and in cell adhesion assays (IC50 = 50 μm). DLXXL peptides were highly specific inhibitors of αvβ6-fibronectin interaction as synthetic scrambled or reversed DLXXL peptides were inactive. NH2- and COOH-terminal modifications of the flanking amino acids suggested that the preceding two and a single trailing amino acid were also involved in interaction with αvβ6. The DLXXL sequence is present in several matrix components and in the β chain of many integrins. Although there is as yet no precise biological role known for DLXXL, it is clearly a specific inhibitory sequence for integrin αvβ6 which has been unrecognized previously. enzyme-linked immunosorbent assay high performance liquid chromatography bovine serum albumin. Integrins are a family of heterodimeric class I transmembrane receptors involved in numerous cell-matrix and cell-cell adhesion phenomena (1Tuckwell D.S. Weston S.A. Humphries M.J. Symp. Soc. Exp. Biol. 1993; 47: 107-136PubMed Google Scholar). They can be grouped roughly into three classes: the β1 series, which are ubiquitous receptors for extracellular matrix (2Ruoslahti E. Noble N.A. Kagami S. Border W.A. Kidney Int. Suppl. 1994; 44: S17-S22PubMed Google Scholar); the β2 series, which are activatable on leukocytes and are triggered during the inflammatory response (3Gahmberg C.G. Tolvanen M. Kotovuori P. Eur. J. Biochem. 1997; 245: 215-232Crossref PubMed Scopus (192) Google Scholar); and the αv series, which bind and mediate the cell response to provisional extracellular matrices found during wound repair and other pathological processes (4Marshall J.F. Hart I.R. Semin. Cancer Biol. 1996; 7: 129-138Crossref PubMed Scopus (94) Google Scholar). The integrins α5β1 (5Pytela R. Pierschbacher M.D. Ruoslahti E. Cell. 1985; 40: 191-198Abstract Full Text PDF PubMed Scopus (688) Google Scholar), αIIbβ3 (6Pytela R. Pierschbacher M.D. Ginsberg M.H. Plow E.F. Ruoslahti E. Science. 1986; 231: 1559-1562Crossref PubMed Scopus (664) Google Scholar), α8β1 (7Schnapp L.M. Hatch N. Ramos D.M. Klimanskaya I.V. Sheppard D. Pytela R. J. Biol. Chem. 1995; 270: 23196-23202Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar), αvβ1 (8Vogel B.E. Tarone G. Giancotti F.G. Gailit J. Ruoslahti E. J. Biol. Chem. 1990; 265: 5934-5937Abstract Full Text PDF PubMed Google Scholar), αvβ3 (9Charo I.F. Nannizzi L. Smith J.W. Cheresh D.A. J. Cell Biol. 1990; 111: 2795-2800Crossref PubMed Scopus (231) Google Scholar), and αvβ6 (10Busk M. Pytela R. Sheppard D. J. Biol. Chem. 1992; 267: 5790-5796Abstract Full Text PDF PubMed Google Scholar) all bind the Arg-Gly-Asp- (RGD) peptide sequence in fibronectin, where it is presented in a constrained loop (11Leahy D.J. Aukhil I. Erickson H.P. Cell. 1996; 84: 155-164Abstract Full Text Full Text PDF PubMed Scopus (587) Google Scholar). Soluble RGD-containing peptides can inhibit the interaction of each of these integrins with fibronectin. However, to analyze the function of individual fibronectin receptors in a particular cellular environment it is useful to have more specific inhibitors, and a variety of inhibitory antibodies, modified peptides, and non-peptidic substances has been developed. However, as yet no inhibitor has been discovered specific for αvβ6. αvβ6 is a rare integrin, induced during repair processes in epithelia (10Busk M. Pytela R. Sheppard D. J. Biol. Chem. 1992; 267: 5790-5796Abstract Full Text PDF PubMed Google Scholar, 12Breuss J.M. Gillett N. Lu L. Sheppard D. Pytela R. J. Histochem. Cytochem. 1993; 41: 1521-1527Crossref PubMed Scopus (201) Google Scholar). Its only known specificities are for fibronectin (10Busk M. Pytela R. Sheppard D. J. Biol. Chem. 1992; 267: 5790-5796Abstract Full Text PDF PubMed Google Scholar), where it can be the dominant receptor mediating cell adhesion (13Weinacker A. Chen A. Agrez M. Cone R.I. Nishimura S. Wayner E. Pytela R. Sheppard D. J. Biol. Chem. 1994; 269: 6940-6948Abstract Full Text PDF PubMed Google Scholar), and for tenascin (14Prieto A.L. Edelman G.M. Crossin K.L. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10154-10158Crossref PubMed Scopus (216) Google Scholar). αvβ6 is believed to be involved in supporting the proliferation of epithelia during repair processes (15Breuss J.M. Gallo J. DeLisser H.M. Klimanskaya I.V. Folkesson H.G. Pittet J.F. Nishimura S.L. Aldape K. Landers D.V. Carpenter W. et al.J. Cell Sci. 1995; 108: 2241-2251Crossref PubMed Google Scholar), and it can promote the proliferation of carcinoma cells (16Agrez M. Chen A. Cone R.I. Pytela R. Sheppard D. J. Cell Biol. 1994; 127: 547-556Crossref PubMed Scopus (149) Google Scholar). Phage display technology has proved useful for identifying novel specific peptide sequences that act as ligand mimetics (17Scott J.K. Smith G.P. Science. 1990; 249: 386-390Crossref PubMed Scopus (1900) Google Scholar, 18Devlin J.J. Panganiban L.C. Devlin P.E. Science. 1990; 249: 404-406Crossref PubMed Scopus (824) Google Scholar). Accordingly, both constrained cyclic and linear peptide libraries have been used to discover novel peptides that interact with integrins (19O'Neil K.T. Hoess R.H. Jackson S.A. Ramachandran N.S. Mousa S.A. DeGrado W.F. Proteins. 1992; 14: 509-515Crossref PubMed Scopus (275) Google Scholar, 20Koivunen E. Gay D.A. Ruoslahti E. J. Biol. Chem. 1993; 268: 20205-20210Abstract Full Text PDF PubMed Google Scholar, 21Koivunen E. Wang B. Ruoslahti E. J. Cell Biol. 1994; 124: 373-380Crossref PubMed Scopus (297) Google Scholar, 22Koivunen E. Wang B. Ruoslahti E. Bio/Technology. 1995; 13: 265-270Crossref PubMed Scopus (446) Google Scholar, 23Healy J.M. Murayama O. Maeda T. Yoshino K. Sekiguchi K. Kikuchi M. Biochemistry. 1995; 34: 3948-3955Crossref PubMed Scopus (123) Google Scholar). But, with few exceptions, these peptides contain RGD sequences, whereas the non-RGD sequences found have only bound weakly. Here we have used recombinant αvβ6 expressed as a transmembrane truncated soluble receptor to screen phage libraries displaying peptides of 7 or 12 amino acids. This revealed a strong and previously unpredicted recognition motif for αvβ6 integrin which we describe here. Similar motifs are displayed in several extracellular matrix molecules. The ligands fibronectin (24Ruoslahti E. Hayman E.G. Pierschbacher M. Engvall E. Methods Enzymol. 1982; 82: 803-831Crossref PubMed Scopus (470) Google Scholar), vitronectin (25Yatohgo T. Izumi M. Kashiwagi H. Hayashi M. Cell Struct. Funct. 1988; 13: 281-292Crossref PubMed Scopus (439) Google Scholar), and fibrinogen (26Kazal L.A. Ansel S. Miller O.P. Tocantins L.M. Proc. Soc. Exp. Biol. Med. 1963; 113: 989-994Crossref PubMed Scopus (210) Google Scholar) were purified from human blood and biotinylated as described previously (27Mitjans F. Sander D. Adan J. Sutter A. Martinez J.M. Jaggle C.S. Moyano J.M. Kreysch H.G. Piulats J. Goodman S.L. J. Cell Sci. 1995; 108: 2825-2838PubMed Google Scholar, 28Mehta R.J. Diefenbach B. Brown A. Cullen E. Jonczyk A. Güssow D. Luckenbach G.A. Goodman S.L. Biochem. J. 1998; 330: 861-869Crossref PubMed Scopus (69) Google Scholar). Human integrins were extracted from native sources (αvβ5, αIIbβ3) or expressed as recombinant soluble receptors using the αIIbβ3 was from human (6Pytela R. Pierschbacher M.D. Ginsberg M.H. Plow E.F. Ruoslahti E. Science. 1986; 231: 1559-1562Crossref PubMed Scopus (664) Google Scholar) as previously (27Mitjans F. Sander D. Adan J. Sutter A. Martinez J.M. Jaggle C.S. Moyano J.M. Kreysch H.G. Piulats J. Goodman S.L. J. Cell Sci. 1995; 108: 2825-2838PubMed Google Scholar) by chromatography on The was with the the containing αIIbβ3 was and was purified from human J.W. D.J. Cheresh D.A. J. Biol. Chem. 1990; 265: Full Text PDF PubMed Google Scholar). was in of A in A by and The was extracted by in of in and The was a (13Weinacker A. Chen A. Agrez M. Cone R.I. Nishimura S. Wayner E. Pytela R. Sheppard D. J. Biol. Chem. 1994; 269: 6940-6948Abstract Full Text PDF PubMed Google Scholar). with in and bound was with to The was with and using The purified receptor was αvβ3 was purified in a soluble transmembrane truncated from a as previously R.J. Diefenbach B. Brown A. Cullen E. Jonczyk A. Güssow D. Luckenbach G.A. Goodman S.L. Biochem. J. 1998; 330: 861-869Crossref PubMed Scopus (69) Google Scholar) with modifications using chromatography (27Mitjans F. Sander D. Adan J. Sutter A. Martinez J.M. Jaggle C.S. Moyano J.M. Kreysch H.G. Piulats J. Goodman S.L. J. Cell Sci. 1995; 108: 2825-2838PubMed Google Scholar). the extracellular of αv and human integrin were into the the recombinant containing both were used to and the soluble receptor was from the of by the and were and and The soluble human receptor ligand binding specificities from the native receptor isolated from (27Mitjans F. Sander D. Adan J. Sutter A. Martinez J.M. Jaggle C.S. Moyano J.M. Kreysch H.G. Piulats J. Goodman S.L. J. Cell Sci. 1995; 108: 2825-2838PubMed Google Scholar). αvβ6 was purified in a soluble transmembrane truncated (13Weinacker A. Chen A. Agrez M. Cone R.I. Nishimura S. Wayner E. Pytela R. Sheppard D. J. Biol. Chem. 1994; 269: 6940-6948Abstract Full Text PDF PubMed Google Scholar) from a as previously for αvβ3 R.J. Diefenbach B. Brown A. Cullen E. Jonczyk A. Güssow D. Luckenbach G.A. Goodman S.L. Biochem. J. 1998; 330: 861-869Crossref PubMed Scopus (69) Google Scholar) using chromatography (27Mitjans F. Sander D. Adan J. Sutter A. Martinez J.M. Jaggle C.S. Moyano J.M. Kreysch H.G. Piulats J. Goodman S.L. J. Cell Sci. 1995; 108: 2825-2838PubMed Google Scholar). The was the of D. Sheppard of The and the be R. J. A. E. J. D. R. S. L. and D. in the of two and was used to recombinant transmembrane truncated αvβ6. The of truncated αv was as described R.J. Diefenbach B. Brown A. Cullen E. Jonczyk A. Güssow D. Luckenbach G.A. Goodman S.L. Biochem. J. 1998; 330: 861-869Crossref PubMed Scopus (69) Google Scholar). truncated αv was from R.J. Diefenbach B. Brown A. Cullen E. Jonczyk A. Güssow D. Luckenbach G.A. Goodman S.L. Biochem. J. 1998; 330: 861-869Crossref PubMed Scopus (69) Google Scholar) using and and into the of of the by truncated was from (13Weinacker A. Chen A. Agrez M. Cone R.I. Nishimura S. Wayner E. Pytela R. Sheppard D. J. Biol. Chem. 1994; 269: 6940-6948Abstract Full Text PDF PubMed Google Scholar) using and and into the of of the by The containing truncated αv and truncated were used to recombinant as described R.J. Diefenbach B. Brown A. Cullen E. Jonczyk A. Güssow D. Luckenbach G.A. Goodman S.L. Biochem. J. 1998; 330: 861-869Crossref PubMed Scopus (69) Google Scholar). The recombinant were used to and the soluble receptor was from the of by the and processes were and in the of were and and The recombinant soluble human receptor is and ligand (13Weinacker A. Chen A. Agrez M. Cone R.I. Nishimura S. Wayner E. Pytela R. Sheppard D. J. Biol. Chem. 1994; 269: 6940-6948Abstract Full Text PDF PubMed Google Scholar). The integrin and were as by using and β and by were and as described R. R. Diefenbach B. Goodman S.L. Jonczyk A. H. J. Chem. Soc. 1996; Scopus Google Scholar) using an with chain on Wang using a peptide was by reversed and by and The peptide is as and and all inhibit cell adhesion mediated by integrins and have been described in (27Mitjans F. Sander D. Adan J. Sutter A. Martinez J.M. Jaggle C.S. Moyano J.M. Kreysch H.G. Piulats J. Goodman S.L. J. Cell Sci. 1995; 108: 2825-2838PubMed Google Scholar). phage display libraries displaying linear peptides of 7 or 12 amino acids from were used to was as described in the with the Integrins were to in 50 and for for with in of the phage display libraries were in containing and phages were to bind for phages were by with containing and bound phages were with and in were from the by and used for the of the of selected phage were and was using the with the Integrins were as described for the from single and purified by were in containing 20 and and to bind to integrins for bound phages were with an and The of the integrins to the was using Integrins were by on and the were with as described of specific for the αv chain of all αv integrins (27Mitjans F. Sander D. Adan J. Sutter A. Martinez J.M. Jaggle C.S. Moyano J.M. Kreysch H.G. Piulats J. Goodman S.L. J. Cell Sci. 1995; 108: 2825-2838PubMed Google Scholar), was biotinylated R.J. Diefenbach B. Brown A. Cullen E. Jonczyk A. Güssow D. Luckenbach G.A. Goodman S.L. Biochem. J. 1998; 330: 861-869Crossref PubMed Scopus (69) Google Scholar), in phage and to bind 20 and bound was with and assays were as described R. R. Diefenbach B. Goodman S.L. Jonczyk A. H. J. Chem. Soc. 1996; Scopus Google Scholar). Integrins were as described of peptides in containing were in with biotinylated fibronectin, or fibrinogen a and with bound ligand was by with an by with a The was by the addition of and the The for the interaction was in was biotinylated as described previously for matrix proteins and R.J. Diefenbach B. Brown A. Cullen E. Jonczyk A. Güssow D. Luckenbach G.A. Goodman S.L. Biochem. J. 1998; 330: 861-869Crossref PubMed Scopus (69) Google Scholar). was in and by to for as described of for with in and in biotinylated integrin in was in the of peptides in the and with bound integrin was by with and as described Cell of and and in cell has been described in (27Mitjans F. Sander D. Adan J. Sutter A. Martinez J.M. Jaggle C.S. Moyano J.M. Kreysch H.G. Piulats J. Goodman S.L. J. Cell Sci. 1995; 108: 2825-2838PubMed Google Scholar, S.L. R. K. J. Cell Biol. PubMed Scopus Google Scholar). is a human cell selected for of αv integrins D.A. J. Biol. Chem. Full Text PDF PubMed Google Scholar) which only α5β1 to to fibronectin. is a human carcinoma cell that only αvβ6 integrin to to fibronectin H. E. Exp. Cell 1997; PubMed Scopus Google Scholar). The integrin of the cells was by cell and the of αv integrins on and the of α5β1 and αvβ3 on also were with fibronectin and with peptides or were by cells cells were and cells using an assay cellular S.L. R. K. J. Cell Biol. PubMed Scopus Google Scholar). soluble human integrins αvβ3 and αvβ6 and native and αIIbβ3 were purified by or ligand each the two to the and the β The the for the and transmembrane truncated recombinant integrin was and ligand binding and from the and also as by (13Weinacker A. Chen A. Agrez M. Cone R.I. Nishimura S. Wayner E. Pytela R. Sheppard D. J. Biol. Chem. 1994; 269: 6940-6948Abstract Full Text PDF PubMed Google Scholar, F. Sander D. Adan J. Sutter A. Martinez J.M. Jaggle C.S. Moyano J.M. Kreysch H.G. Piulats J. Goodman S.L. J. Cell Sci. 1995; 108: 2825-2838PubMed Google Scholar, 28Mehta R.J. Diefenbach B. Brown A. Cullen E. Jonczyk A. Güssow D. Luckenbach G.A. Goodman S.L. Biochem. J. 1998; 330: 861-869Crossref PubMed Scopus (69) Google Scholar, R. R. Diefenbach B. Goodman S.L. Jonczyk A. H. J. Chem. Soc. 1996; Scopus Google Scholar). αvβ6 expressed as a transmembrane truncated soluble receptor bound two of phages in phage display from a linear 12-mer library as from the sequences of more containing RGD sequences and containing motif phages contained RGD and also with the motif as whereas displayed the sequence the phages that were bound contained sequences. A of displayed sequences is binding from phage display library RGD DLXXL displayed peptide sequences from isolated by from recombinant αvβ6 and have been selected to the of RGD sequences have been and DLXXL sequences and in a displayed peptide sequences from isolated by from recombinant αvβ6 and have been selected to the of RGD sequences have been and DLXXL sequences and the non-RGD sequences, the amino acid was whereas were with a amino were by a dominant with the sequence peptides were in only of isolated from a 7-mer phage display that sequences COOH-terminal to DLXXL are also involved in integrin binding the sequence COOH-terminal to the peptide in the phage the 7-mer library and sequences were isolated from the 12-mer was found in a DLXXL and it was from the amino acids COOH-terminal of the DLXXL from these in RGD-containing sequences. sequence was found where the motif was as the of DLXXL sequences was a library we also on Here RGD-containing phages were the DLXXL sequence was and was present in of This was from that of the αvβ6 phage display library screen and to that for linear library J.M. Murayama O. Maeda T. Yoshino K. Sekiguchi K. Kikuchi M. Biochemistry. 1995; 34: 3948-3955Crossref PubMed Scopus (123) Google Scholar). the that the in phage interactions αvβ3 and αvβ6 were by of integrins during the phage we the of each integrin to the screening the of the screen using an The using the which the αv were in in of and in the of to of the This that of the αv integrins were to the the of interaction of phage we used a to the binding of phages from the αvβ6 screen to purified integrins phages bound only to αvβ6, whereas RGD phages bound both αvβ6 and αvβ3 and to and to This that the isolated in the screen were specific for αvβ6. RGD-containing peptides inhibit the interaction of RGD-containing ligands with integrin DLXXL peptides were to inhibit we the We compared the of peptide and RGD-containing peptides on ligands binding to integrins and αvβ6 The peptide fibronectin binding to αvβ6 with an of 20 ligand binding to or αIIbβ3 (IC50 50 μm). the peptide ligand binding to all integrins with A cyclic peptide inhibitor for αvβ3 (IC50 = αvβ6 and αIIbβ3 (IC50 μm). αvβ3 also to fibronectin. the of DLXXL peptides were a of the ligand used with αvβ6, fibronectin, we also the of peptide on fibronectin binding to αvβ3 The DLXXL peptide also no on fibronectin whereas the peptide (IC50 = the for vitronectin and fibronectin on αvβ3 were of peptides on fibronectin binding to binding to αvβ3 was in the of of or were to of We also the assay was an binding of DLXXL to αvβ6. αvβ6 to fibronectin in the extracellular matrix to the soluble used in the receptor assay here. to the of the αvβ6 integrin and We the of DLXXL peptides on αvβ6 binding to fibronectin the peptide strongly the αvβ6-fibronectin interaction (IC50 = and peptides were also in assay the of DLXXL peptide on αvβ6 was an of integrin to peptides were also in cell carcinoma cell to fibronectin was strongly by peptide and by This was also by the that it was on αv integrins. to fibronectin has been to be mediated by αvβ6. cells to fibronectin, and was by was by the peptide was by or antibodies, that it was mediated by the α5β1 these that the peptide displayed by was an and specific inhibitor of αvβ6 of the RGD We which of were for inhibitory NH2- and COOH-terminal truncated of the peptide were and for to fibronectin binding to αvβ6. The are in of the of DLXXL on inhibitory the was by of the also the that the motif was the acid of fibronectin αvβ6 interaction by synthetic DLXXL fibronectin binding to αvβ6 in the of synthetic linear peptides was using an in a fibronectin binding to αvβ6 in the of synthetic linear peptides was using an Although the specific of DLXXL sequences from a highly display library of we more by the of reversed and scrambled peptides on αvβ6-fibronectin peptides were of the DLXXL peptide The high of the sequence for αvβ6 suggested that it a sequence in a native αvβ6 a of the revealed several extracellular matrix components with consensus sequences including fibrinogen and and the of which has been to bind in an RGD-dependent to αvβ6 (14Prieto A.L. Edelman G.M. Crossin K.L. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10154-10158Crossref PubMed Scopus (216) Google Scholar), of these has been previously as a ligand for αvβ6, the biological of such of human proteins containing the DLXXL β β1 β2 αv in a the sequence is strongly in human integrin β and interact with that the sequence function by the interaction from the of the receptor as a ligand an RGD we a synthetic DLXXL peptide sequence from the for on interaction with the from the peptide was of as an inhibitor of the that the sequences selected from the phage display library were highly We the of the by the flanking sequences from the with of the DLXXL of the a highly as the and the trailing amino acids. the amino acids preceding the DLXXL sequence were also for integrin inhibitory we describe the discovery by phage display screening of an unexpected binding sequence for αvβ6 integrin, which has previously been thought to interact only with RGD sequences in αvβ6 is a rare integrin J.M. Gillett N. Lu L. Sheppard D. Pytela R. J. Histochem. Cytochem. 1993; 41: 1521-1527Crossref PubMed Scopus (201) Google Scholar, J.M. Gallo J. DeLisser H.M. Klimanskaya I.V. Folkesson H.G. Pittet J.F. Nishimura S.L. Aldape K. Landers D.V. Carpenter W. et al.J. Cell Sci. 1995; 108: 2241-2251Crossref PubMed Google Scholar), and from native sources has been αvβ6 has been by recombinant (13Weinacker A. Chen A. Agrez M. Cone R.I. Nishimura S. Wayner E. Pytela R. Sheppard D. J. Biol. Chem. 1994; 269: 6940-6948Abstract Full Text PDF PubMed Google Scholar), and we have technology to of soluble As with αvβ3 R.J. Diefenbach B. Brown A. Cullen E. Jonczyk A. Güssow D. Luckenbach G.A. Goodman S.L. Biochem. J. 1998; 330: 861-869Crossref PubMed Scopus (69) Google Scholar), we found that recombinant αvβ6 the biological and inhibitor profiles of the native receptor in and the of Sheppard and (13Weinacker A. Chen A. Agrez M. Cone R.I. Nishimura S. Wayner E. Pytela R. Sheppard D. J. Biol. Chem. 1994; 269: 6940-6948Abstract Full Text PDF PubMed Google Scholar). the receptor is into the to a highly purified with a of other is an for a phage display is of that RGD-dependent extracellular matrix binding to αvβ6 is strongly by peptides containing DLXXL sequences that no to sequences in fibronectin, a sequence in DLXXL sequences have been found in other phage display on the only described in the an sequence was a inhibitor of αvβ3 J.M. Murayama O. Maeda T. Yoshino K. Sekiguchi K. Kikuchi M. Biochemistry. 1995; 34: 3948-3955Crossref PubMed Scopus (123) Google Scholar). We were to that synthetic DLXXL peptides, strong inhibitors of αvβ6, on the interaction of integrins and αIIbβ3 with Although cell adhesion assays that α5β1 was also The DLXXL sequences, the specific inhibitory peptides for αvβ6 of the of peptides that was a it was by no binding to αvβ6 were found where displayed was by or or was The inhibitory of DLXXL peptides is the of reversed scrambled of highly inhibitory DLXXL peptides have on the αvβ6-fibronectin on the of DLXXL were found in to the biological of the DLXXL DLXXL sequences are found in integrin β in a in ligand binding A. T. J. Biochem. 1997; PubMed Scopus Google Scholar). We that the DLXXL motif was involved in the interaction of integrin and β and that the DLXXL peptide act by with a binding on the by β chain sequences, integrin we a peptide to a DLXXL sequence in the was that the DLXXL peptides were by for chain binding on the αv However, the that the of the DLXXL sequence was flanking amino acids were for inhibitory whereas in the COOH-terminal flanking were present we have no sequences a biological role with αvβ6 As previously for the inhibitory peptide sequence for α5β1 E. Wang B. Ruoslahti E. J. Cell Biol. 1994; 124: 373-380Crossref PubMed Scopus (297) Google bind a from the RGD and act as an inhibitor of in the of present we that sequences are ligand Similar inhibitory binding sequences with no to known ligands are found in the phage display A. J. 1996; Google Scholar, R. Science. 1996; PubMed Scopus Google Scholar). The strong and specific inhibitory binding sequences that we here a from which to αvβ6 biological function We E. for and to the phage display D. Sheppard for and to and R. and I. for