Identification of Motifs in the Fasciclin Domains of the Transforming Growth Factor-β-induced Matrix Protein βig-h3 That Interact with the αvβ5 Integrin

Abstract

βig-h3 is a TGF-β-induced matrix protein known to mediate the adhesion of several cell types. In this study, we found that all four of the fas-1 domains in βig-h3 mediate MRC-5 fibroblast adhesion and that this was specifically inhibited by a function-blocking monoclonal antibody specific for the αvβ5 integrin. Using deletion mutants of the fourth fas-1 domain revealed the MRC-5 cell adhesion motif (denoted the YH motif) is located in amino acids 548–614. Experiments with substitution mutants showed that tyrosine 571, histidine 572, and their flanking leucine and isoleucine amino acids, which are all highly conserved in many fas-1 domains, are essential for mediating MRC-5 cell adhesion. A synthetic 18-amino acid peptide encompassing these conserved amino acids could effectively block MRC-5 cell adhesion to βig-h3. Using HEK293 cells stably transfected with the β5 integrin cDNA, we confirmed that the αvβ5 integrin is a functional receptor for the YH motif. In conclusion, we have identified a new αvβ5 integrin-interacting motif that is highly conserved in the fas-1 domains of many proteins. This suggests that fas-1 domain-containing proteins may perform their biological functions by interacting with integrins. βig-h3 is a TGF-β-induced matrix protein known to mediate the adhesion of several cell types. In this study, we found that all four of the fas-1 domains in βig-h3 mediate MRC-5 fibroblast adhesion and that this was specifically inhibited by a function-blocking monoclonal antibody specific for the αvβ5 integrin. Using deletion mutants of the fourth fas-1 domain revealed the MRC-5 cell adhesion motif (denoted the YH motif) is located in amino acids 548–614. Experiments with substitution mutants showed that tyrosine 571, histidine 572, and their flanking leucine and isoleucine amino acids, which are all highly conserved in many fas-1 domains, are essential for mediating MRC-5 cell adhesion. A synthetic 18-amino acid peptide encompassing these conserved amino acids could effectively block MRC-5 cell adhesion to βig-h3. Using HEK293 cells stably transfected with the β5 integrin cDNA, we confirmed that the αvβ5 integrin is a functional receptor for the YH motif. In conclusion, we have identified a new αvβ5 integrin-interacting motif that is highly conserved in the fas-1 domains of many proteins. This suggests that fas-1 domain-containing proteins may perform their biological functions by interacting with integrins. βig-h3 is an extracellular matrix protein whose expression in several cell types, including fibroblasts, is strongly induced by TGF-β. 1The abbreviations used for: TGF-β, transforming growth factor β; fas-1, fasciclin 1 homologous domain; HEK293, human embryonic kidney cell; BSA, bovine serum albumin; PBS, phosphate-buffered saline; WT, wild type; FACS, fluorescence-activated cell sorting. The gene encoding βig-h3 was first identified by Skonier et al. (1Skonier J. Neubauer M. Madisen L. Bennett K. Plowman G.D. Purchio A.F. DNA Cell Biol. 1992; 11: 511-522Crossref PubMed Scopus (505) Google Scholar), who isolated it by screening a cDNA library made from a human lung adenocarcinoma cell line (A549) that had been treated with TGF-β. The βig-h3 protein comprises 683 amino acids containing four homologous internal repeat domains. These domains are homologous to similar motifs in the Drosophila protein fasciclin-I and thus are denoted fas-1 domains. The fas-1 domain has highly conserved sequences found in secretory and membrane proteins of several species, including mammals, insects, sea urchins, plants, yeast, and bacteria (2Kawamoto T. Noshiro M. Shen M. Nakamasu K. Hashimoto K. Kawashima-Ohya Y. Gotoh O. Kato Y. Biochim. Biophys. Acta. 1998; 1395: 288-292Crossref PubMed Scopus (105) Google Scholar). Mutations in βig-h3 have been shown to be responsible for 5q31-linked human autosomal dominant corneal dystrophies. It has a fibrillar structure and interacts with several extracellular matrix proteins such as fibronectin and collagen (3Kim J.-E. Park R.-W. Choi J.-Y. Bae Y.-C. Kim K.-S. Joo C.-K. Kim I.-S. Invest. Ophthalmol. Vis. Sci. 2002; 43: 656-661PubMed Google Scholar). In addition, βig-h3 has been reported to be involved in cell growth and differentiation, wound healing, and cell adhesion (4Skonier J. Benenett K. Rothwell V. Kosowski S. Plowman G. Wallace P. Edelhoff S. Disteche C. Neubauer M. Marquardt H. Rodgers J. Purchio A.F. DNA Cell Biol. 1994; 13: 571-584Crossref PubMed Scopus (260) Google Scholar, 5Dieudonne S.C. Kerr K.M. Xu T. Sommer B. DeRubeis A.R. Kuznetsov S.A. Kim I.-S. Robey P.G. Young M.F. J. Cell. Biochem. 1999; 76: 231-243Crossref PubMed Scopus (61) Google Scholar, 6Kim J.-E. Kim E.-H. Han E.-H. Park R.-W. Park I.-H. Jun S.-H. Kim J.-C. Young M.F. Kim I.-S. J. Cell. Biochem. 2000; 77: 169-178Crossref PubMed Scopus (110) Google Scholar, 7Rawe I.M. Zhan Q. Burrows R. Bennett K. Cintron C. Invest. Ophthalmol. Vis. Sci. 1997; 38: 893-900PubMed Google Scholar, 8LeBaron R.G. Bezverkov K.I. Zimber M.P. Pavele R. Skonier J. Purchio A.F. J. Invest. Dermatol. 1995; 104: 844-849Abstract Full Text PDF PubMed Scopus (198) Google Scholar, 9Ohno S. Noshiro M. Makihira S. Kawamoto T. Shen M. Yan W. Kawashim-Ohya Y. Fujimoto K. Tanne K. Kato Y. Biochim. Biophys. Acta. 1999; 1451: 196-205Crossref PubMed Scopus (101) Google Scholar). βig-h3 mediates the adhesion of many different cell types, including corneal epithelial cells, chondrocytes, and fibroblasts (8LeBaron R.G. Bezverkov K.I. Zimber M.P. Pavele R. Skonier J. Purchio A.F. J. Invest. Dermatol. 1995; 104: 844-849Abstract Full Text PDF PubMed Scopus (198) Google Scholar, 9Ohno S. Noshiro M. Makihira S. Kawamoto T. Shen M. Yan W. Kawashim-Ohya Y. Fujimoto K. Tanne K. Kato Y. Biochim. Biophys. Acta. 1999; 1451: 196-205Crossref PubMed Scopus (101) Google Scholar, 10Kim J.-E. Kim S.-J. Lee B.-H. Park R.-W. Kim K.-S. Kim I.-S. J. Biol. Chem. 2000; 275: 30907-30915Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar). We reported previously that βig-h3 mediates corneal epithelial cell adhesion by binding to α3β1 integrin. Two motifs interacting with the α3β1 integrin were located within the second and the fourth fas-1 domains of βig-h3. Interestingly, however, we found that these two motifs are not involved in βig-h3-mediated fibroblastic cell adhesion. Furthermore, all four fas-1 domains of βig-h3 mediate fibroblastic cell adhesion, whereas corneal epithelial cell adhesion is supported by just the second and the fourth fas-1 domains. This suggests that βig-h3 has additional motifs that can mediate the adhesion of other cell types. Confirming this, we show in the present study that βig-h3 has a motif that promotes fibroblastic cell adhesion by interacting with the αvβ5 integrin on the fibroblast cell surface. This motif is well conserved in many fas-1 domains found in various proteins from different species. Together with our previous findings, these observations suggest that βig-h3 mediates cell functions through multiple cell-adhesion motifs that interact with different integrins on various cell types. This also suggests that other fas-I-containing proteins could regulate cell functions by interacting with integrins. Bacterial expression vectors for the wild-type βig-h3 and each fas-1 domain have been described previously (10Kim J.-E. Kim S.-J. Lee B.-H. Park R.-W. Kim K.-S. Kim I.-S. J. Biol. Chem. 2000; 275: 30907-30915Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar). βig-h3 d-IV cDNA that encodes amino acids 498–637 (the fourth fas-1 domain) was used to clone several deletion mutant constructs, as follows. Several cDNAs encoding fragments of βig-h3, namely, amino acids 548–637, 498–620, 498–614, 548–620, 548–614, 569–637, and 575–637, were generated by PCR using βigh3 D-IV cDNA as template. These fragments were cloned into theEcoRV/XhoI sites of the pET-29b(+) vector (Novagen) and were denoted as ΔH1, ΔH2, ΔH2 (6Kim J.-E. Kim E.-H. Han E.-H. Park R.-W. Park I.-H. Jun S.-H. Kim J.-C. Young M.F. Kim I.-S. J. Cell. Biochem. 2000; 77: 169-178Crossref PubMed Scopus (110) Google Scholar), ΔH1H2, ΔH1H2 (6Kim J.-E. Kim E.-H. Han E.-H. Park R.-W. Park I.-H. Jun S.-H. Kim J.-C. Young M.F. Kim I.-S. J. Cell. Biochem. 2000; 77: 169-178Crossref PubMed Scopus (110) Google Scholar), 569, and 575, respectively. Substitution mutants in which conserved tyrosine, histidine, leucine and isoleucine residues in βig-h3 D-IV were substituted by alanine or serine, were generated by two-step PCR as described previously (11Kim I.-S. Sinha S. deCrombrugghe B. Maity S.N. Mol. Cell. Biol. 1996; 16: 4003-4013Crossref PubMed Scopus (127) Google Scholar). The mutations were confirmed by DNA sequencing. Recombinant βig-h3 proteins were induced and purified as described previously (6Kim J.-E. Kim E.-H. Han E.-H. Park R.-W. Park I.-H. Jun S.-H. Kim J.-C. Young M.F. Kim I.-S. J. Cell. Biochem. 2000; 77: 169-178Crossref PubMed Scopus (110) Google Scholar). MRC-5 (human lung fibroblast) cells were cultured at 37 °C in 5% CO2 in RPMI 1640 medium (Invitrogen) supplemented with 10% fetal bovine serum. Human embryonic kidney (HEK293) cells stably transfected with an empty vector (pcDNA3) or a human integrin β5 expression vector were donated by Dr. Jeffrey Smith (Burnham Institute, San Diego, CA). These stable cell lines were cultured in Dulbecco's modified Eagle medium supplemented with 10% fetal bovine serum and antibiotics. The cell adhesion assay was performed as described previously (11Kim I.-S. Sinha S. deCrombrugghe B. Maity S.N. Mol. Cell. Biol. 1996; 16: 4003-4013Crossref PubMed Scopus (127) Google Scholar). Briefly, flat-bottomed 96-well enzyme-linked immunosorbent assay (ELISA) plates (Costar Corning Inc., New York) were incubated overnight at 4 °C with 10 μg/ml recombinant βig-h3 proteins or other extracellular matrix proteins and then blocked for 1 h at room temperature with phosphate-buffered saline (PBS) containing 2% bovine serum albumin (BSA). Cells were suspended in medium at a density of 3 × 105 cells/ml, and 0.1 ml of the cell suspension was added to each well of the coated plates. After incubation for 30 min at 37 °C, unattached cells were removed by rinsing once with PBS. Attached cells were then incubated for 1 h at 37 °C in 50 mm citrate buffer, pH 5.0, containing 3.75 mm p-nitrophenyl-N-acetyl-β-d-glycosaminide and 0.25% Triton X-100. Enzyme activity was blocked by adding 50 mm glycine buffer, pH 10.4, containing 5 mmEDTA, and the absorbance was measured at 405 nm in a Bio-Rad model 550 microplate reader. Synthetic peptides purchased from AnyGen Co. Ltd. (Kwangju, Korea) were tested for their ability to inhibit cells from adhering to the protein substrates coating the wells. The cell-adhesion assay was performed in the presence or absence of the indicated concentrations of each peptide. To identify the receptor for βig-h3, monoclonal antibodies to different types of integrins (Chemicon, International Inc. Temecula, CA) at a concentration of 5 μg/ml were preincubated at 37 °C for 30 min with MRC-5 in 0.1 ml of incubation solution (3 × 105 cells/ml). The cells were then transferred onto plates precoated with recombinant βig-h3 proteins and incubated further for 30 min at 37 °C. The attached cells were then quantified as described above. Function-blocking monoclonal antibodies to the following to integrin subunits were used: α1, FB12; α2, P1E6; α3, P1B5; α4, P1H4; α5, P1D6; α6, GoH3; αv, P3G8; β1, 6S6; and cells were from the plates by with 0.25% After in PBS, the cells were suspended in and incubated for 1 h at 4 °C with the specific for αvβ5 and were purchased from Cells were then incubated for 1 h at 4 °C with 10 μg/ml of the with Inc., and at nm on the with a In our previous (10Kim J.-E. Kim S.-J. Lee B.-H. Park R.-W. Kim K.-S. Kim I.-S. J. Biol. Chem. 2000; 275: 30907-30915Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar), we that the second and the fourth fas-1 domains not the first the domains mediate corneal epithelial cell adhesion. In in this study, we found that MRC-5 fibroblasts well to each of the four fas-1 domains This suggests that each fas-1 domain has a motif for MRC-5 cell adhesion. Furthermore, the motifs in the second and the fourth fas-1 domains may not be the and that were found to mediate corneal epithelial cell adhesion. Confirming this, we showed that the and peptides not inhibit MRC-5 cell adhesion to of the fas-1 domains or to the protein 1 To identify the βig-h3 receptor on MRC-5 fibroblast cells, function-blocking monoclonal antibodies specific for various integrin subunits were for their ability to block the adhesion of MRC-5 cells to the protein or the fas-1 domains and MRC-5 cell adhesion to the protein and all of the fas-1 domains was specifically inhibited by the antibody to the αvβ5 not by of the other antibodies and βig-h3 has a motif in each fas-1 domain that interacts with the αvβ5 integrin. To that MRC-5 cells the αvβ5 integrin on their we performed using monoclonal antibodies to several integrins 1 Several including the αvβ5 were on the MRC-5 cell the integrin was not To identify the αvβ5 integrin-interacting in βig-h3, we made and recombinant proteins of the fourth fas-1 which of amino acids The proteins were denoted ΔH1, ΔH2, ΔH2 (6Kim J.-E. Kim E.-H. Han E.-H. Park R.-W. Park I.-H. Jun S.-H. Kim J.-C. Young M.F. Kim I.-S. J. Cell. Biochem. 2000; 77: 169-178Crossref PubMed Scopus (110) Google Scholar), ΔH1H2, and ΔH1H2 (6Kim J.-E. Kim E.-H. Han E.-H. Park R.-W. Park I.-H. Jun S.-H. Kim J.-C. Young M.F. Kim I.-S. J. Cell. Biochem. 2000; 77: 169-178Crossref PubMed Scopus (110) Google and ΔH2 the fourth fas-1 domain of two conserved fas-1 known as acids and acids ΔH1H2 and ΔH2 (6Kim J.-E. Kim E.-H. Han E.-H. Park R.-W. Park I.-H. Jun S.-H. Kim J.-C. Young M.F. Kim I.-S. J. Cell. Biochem. 2000; 77: 169-178Crossref PubMed Scopus (110) Google and ΔH1H2 (6Kim J.-E. Kim E.-H. Han E.-H. Park R.-W. Park I.-H. Jun S.-H. Kim J.-C. Young M.F. Kim I.-S. J. Cell. Biochem. 2000; 77: 169-178Crossref PubMed Scopus (110) Google are ΔH2 and ΔH1H2 that in the amino acids containing the α3β1 integrin-interacting motif We tested the ability of each protein to MRC-5 cell adhesion including the mutant ΔH1H2 (6Kim J.-E. Kim E.-H. Han E.-H. Park R.-W. Park I.-H. Jun S.-H. Kim J.-C. Young M.F. Kim I.-S. J. Cell. Biochem. 2000; 77: 169-178Crossref PubMed Scopus (110) Google Scholar), could mediate MRC-5 cell adhesion and are not involved and the cell adhesion motif is present within amino acids 548–614. To further identify the cell-adhesion motif within this we performed a using for not the four βig-h3 domains also the fas-1 domains from other proteins. Interestingly, many fas-1 domains, including the four fas-1 domains of βig-h3, two amino acid a tyrosine and a histidine, in the domain In the fourth domain of βig-h3, the tyrosine and the histidine are amino acids and 572, respectively. We the tyrosine and histidine are for MRC-5 cell adhesion, first by using the two proteins βig-h3 and βig-h3 These proteins amino acids and and thus βig-h3 the tyrosine and histidine shown in the mutant proteins were to mediate MRC-5 cell adhesion with the ΔH1, ΔH2, ΔH2 (6Kim J.-E. Kim E.-H. Han E.-H. Park R.-W. Park I.-H. Jun S.-H. Kim J.-C. Young M.F. Kim I.-S. J. Cell. Biochem. 2000; 77: 169-178Crossref PubMed Scopus (110) Google Scholar), ΔH1H2, and ΔH1H2 (6Kim J.-E. Kim E.-H. Han E.-H. Park R.-W. Park I.-H. Jun S.-H. Kim J.-C. Young M.F. Kim I.-S. J. Cell. Biochem. 2000; 77: 169-178Crossref PubMed Scopus (110) Google proteins tested the of residues the ability of to MRC-5 cell adhesion was that of βig-h3 tyrosine and histidine may an essential in the cell-adhesion activity of βig-h3. To this, we generated substitution mutant βig-h3 proteins whose tyrosine histidine residues were with alanine the mutations not cell-adhesion the inhibited the cell adhesion activity of βig-h3, not tyrosine and histidine are in βig-h3-mediated MRC-5 cell adhesion. flanking amino acids may also be To that tyrosine and histidine are for MRC-5 cell adhesion, a synthetic peptide to amino acids was generated and used to cell adhesion to βig-h3. this peptide could not inhibit MRC-5 cell adhesion to βig-h3. We then used the peptide to amino acids it also could not inhibit cell adhesion. we tested which to amino acids MRC-5 cell adhesion to βig-h3 was The peptide which the amino acids of in a not cell adhesion to βig-h3 We similar each of the four fas-1 domains was used as the 3 The of cell adhesion by the peptide was 3 To that the sequences of the other fas-1 domains of βig-h3 are also in MRC-5 cell adhesion to βig-h3, we peptides these shown in 3 these peptides also inhibited MRC-5 cell adhesion to βig-h3 in a peptides from and showed similar the peptide from the D-IV showed activity with the all of these peptides were in the peptide inhibited cell adhesion at activity with the recombinant domain protein which inhibited cell adhesion by 5 and 10 3 This suggests a of amino acids comprises the for are for these suggest that the sequences encompassing the conserved tyrosine and histidine residues are essential for MRC-5 cell adhesion, that not the tyrosine and histidine also flanking amino acids are for βig-h3-mediated MRC-5 cell adhesion. the leucine and isoleucine residues that the tyrosine and histidine are also well conserved the fas-1 domains from different proteins we their in βig-h3-mediated MRC-5 cell adhesion. We a of substitution mutants of βig-h3 whose tyrosine, histidine, and isoleucine residues were substituted in various by and or alanine and 4 These mutants were then used in cell adhesion the fourth fas-1 domain whose leucine isoleucine and leucine were with the fourth fas-1 domain whose isoleucine isoleucine and leucine were with and and whose tyrosine and histidine were with the fourth fas-1 domain whose conserved amino acids were with or and show cell-adhesion activity with the fourth fas-1 cell adhesion 4 In the all conserved amino acids had been was in mediating MRC-5 cell adhesion. multiple amino acids are to a that mediates MRC-5 cell adhesion to βig-h3. To that βig-h3 mediates cell adhesion through the αvβ5 we used stably transfected HEK293 cells that human β5 integrin. HEK293 cells not the β5 integrin many including and B. PubMed Scopus Google Scholar). We confirmed that cells the αvβ5 integrin by not cells strongly to coated with or cells, which are HEK293 cells stably transfected with an empty vector 5 cell adhesion to βig-h3 was specifically inhibited by an antibody the αvβ5 integrin 5 and by the peptide 5 These that βig-h3 mediates cell adhesion by interacting with the αvβ5 integrin. We previously identified two α3β1 integrin-interacting motifs that are for βig-h3-mediated adhesion of corneal epithelial cells (10Kim J.-E. Kim S.-J. Lee B.-H. Park R.-W. Kim K.-S. Kim I.-S. J. Biol. Chem. 2000; 275: 30907-30915Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar). these motifs are not involved in the βig-h3-mediated adhesion of cells, including fibroblasts, cells, and This to the motifs in βig-h3 that are involved in the βig-h3-mediated adhesion of we a new βig-h3 motif that interacts with the αvβ5 integrin and is for the βig-h3-mediated adhesion of corneal epithelial cells, MRC-5 fibroblasts to all four fas-1 domains of βig-h3. Furthermore, this adhesion was not blocked by the presence of the α3β1 integrin-interacting peptides and MRC-5 cells to βig-h3 through an integrin other the α3β1 integrin. We used several function-blocking antibodies and found that integrin antibody could MRC-5 cell adhesion to βig-h3 and the fas-1 domains. We confirmed that MRC-5 cells the αvβ5 integrin on their cell surface. et al. S. Noshiro M. Makihira S. Kawamoto T. Shen M. Yan W. Kawashim-Ohya Y. Fujimoto K. Tanne K. Kato Y. Biochim. Biophys. Acta. 1999; 1451: 196-205Crossref PubMed Scopus (101) Google previously reported that βig-h3 mediates MRC-5 fibroblast cell adhesion through the integrin. we found that and antibodies not block MRC-5 cell adhesion to βig-h3. We confirmed that and integrin function-blocking antibodies used effectively inhibited the adhesion of several cell lines to specific including collagen and fibronectin not it is that this is to using different not αvβ5 function-blocking antibody could inhibit MRC-5 cell adhesion to βig-h3. we are as to the Interestingly, MRC-5 cells and corneal epithelial cells (10Kim J.-E. Kim S.-J. Lee B.-H. Park R.-W. Kim K.-S. Kim I.-S. J. Biol. Chem. 2000; 275: 30907-30915Abstract Full Text Full Text PDF PubMed Scopus (241) Google the α3β1 and αvβ5 the two cell lines not the integrin for adhesion to βig-h3 and 10Kim J.-E. Kim S.-J. Lee B.-H. Park R.-W. Kim K.-S. Kim I.-S. J. Biol. Chem. 2000; 275: 30907-30915Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar). The of the integrin used may on the of the integrins in each cell are a of that cells to the and of their integrins for their extracellular matrix and are known to regulate cell adhesion by the of multiple integrins for the extracellular matrix K. H. Cell. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, M. J. Cell. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). Furthermore, of the domain of integrins the of the integrins and their for their J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). We are in MRC-5 and corneal epithelial cells different integrin types to interact with βig-h3. The that the integrin antibody the ability of all the fas-1 domains in βig-h3 to mediate MRC-5 cell adhesion suggests that each domain has a motif that interacts with the αvβ5 integrin. We this by a of deletion mutants of the fourth fas-1 domain and their ability to MRC-5 cell adhesion. Two denoted and are highly conserved in each of the fas-1 domains, deletion of or of these not cell adhesion. all four fas-1 domains mediate cell adhesion through the integrin it is that all the conserved that this We performed of the fas-1 domains of βig-h3 and other proteins and found a tyrosine, a histidine, and many residues flanking the tyrosine and histidine, which are highly Experiments with deletion and substitution mutants revealed that a motif containing these amino acid residues is essential for the ability of βig-h3 to mediate MRC-5 cell adhesion, are of the αvβ5 motif in βig-h3. the α3β1 integrin-interacting motifs and in βig-h3, the cell-adhesion activity of this new motif not on a amino acid residues mutations of tyrosine and histidine not the cell adhesion activity of βig-h3. substitution mutations of several of the conserved tyrosine, histidine, and isoleucine residues could not the cell adhesion the of all these conserved amino acids the cell adhesion activity of βig-h3. This is supported by using synthetic peptides that showed that and acid peptides containing the tyrosine and histidine residues could not cell adhesion, the 18-amino acid peptide Furthermore, a peptide of amino acids had it was with the recombinant domain that the of amino acids comprises the for the cell adhesion activity of the fas-1 acids flanking this conserved could to the of the αvβ5 It however, to which amino acids are we could not conserved amino acids other tyrosine, histidine, and several fas-1 domains the showed a conserved or amino acids were present the conserved tyrosine and histidine the 18-amino acid the conserved histidine, tyrosine, and residues the for cell adhesion, a is for of this The αvβ5 integrin is to to a of extracellular matrix including and J. Biol. Chem. Full Text PDF PubMed Google Scholar, Smith J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar). to these to be peptides containing this motif inhibit αvβ5 integrin the of the αvβ5 motif in from the peptides may the in the αvβ5 integrin MRC-5 cell adhesion to βig-h3 is inhibited by the peptide not Furthermore, MRC-5 cell adhesion to is also inhibited by the peptide not the binding of αvβ5 integrin may the of the other the the integrin also interacts with and in a R. J. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, Smith J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar), it is that the peptide could also to the integrin. We could not this in the study reported MRC-5 cells not the integrin. it is that βig-h3 could interact with the integrin through the YH motif we have found that adhesion to βig-h3 of cell types on the YH motif and the integrin not In this it is that βig-h3 is by cells and cells Bennett T. T. M. H. Purchio Biol. 1996; 16: PubMed Scopus Google Scholar), of which are known to the and αvβ5 integrins for their biological to a of such as M. B. T. J. M. Smith B. M. L. Google and J. M. C. J. 16: Google Scholar). In four proteins are known to fas-1 domains. βig-h3 and K. S. H. M. H. Y. J. 1999; PubMed Scopus Google are secretory proteins with four fas-1 domains, and and are membrane proteins with fas-1 domains O. K. P. S. G. P. C. J. P. S. S. Biochem. J. 2002; PubMed Scopus Google Scholar). the biological functions of these proteins are not it is that all may in and all of the fas-1 domains integrin-interacting YH These four human proteins are the integrin-interacting proteins in that multiple repeat integrin-interacting of the other known to interact with integrins has multiple of motifs that interact with integrin It is of to the biological of this of the human fas-1 proteins. In conclusion, we have identified a new cell-adhesion motif that interacts with the αvβ5 integrin. This motif is present in each of the fas-1 repeat domains of βig-h3 and of tyrosine and histidine by several The that this motif is well conserved in of the fas-1 domains present in many proteins suggests that fas-1 domain-containing proteins may regulate various cell functions by interacting with integrins. We Dr. Jeffrey Smith (Burnham Institute, San for the