Identification of Motifs in the Fasciclin Domains of the Transforming Growth Factor-β-induced Matrix Protein βig-h3 That Interact with the αvβ5 Integrin
2002; Elsevier BV; Volume: 277; Issue: 48 Linguagem: Inglês
10.1074/jbc.m207055200
ISSN1083-351X
AutoresJung‐Eun Kim, Ha-Won Jeong, Ju-Ock Nam, Byung-Heon Lee, Je‐Yong Choi, Rang‐Woon Park, Jae Yong Park, In‐San Kim,
Tópico(s)Signaling Pathways in Disease
Resumoβig-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; β2, P4H9; αvβ3, LM609; and αvβ5, P1F6. Confluent cells were detached from the plates by treatment with 0.25% trypsin/0.05% EDTA. After being washed twice in PBS, the cells were suspended in PBS and incubated for 1 h at 4 °C with the appropriate antibodies. Antibodies specific for α1 (FB12), α3 (ASC-1), α4 (P1H4), α5β1 (HA5), α6 (CLB701), αv (P3G8), αvβ3 (LM609), αvβ5 (P1F6), and β1 (12G10) were purchased from Chemicon. Cells were then incubated for 1 h at 4 °C with 10 μg/ml of the secondary antibody, goat-anti-mouse IgG conjugated with fluorescein isothiocyanate (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), and analyzed at 488 nm on the flow cytometer FACScalibur system (BD Biosciences) equipped with a 5-watt argon laser. In our previous report (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 demonstrated that the second and the fourth fas-1 domains but not the first and/or the third domains mediate corneal epithelial cell adhesion. In contrast, in this study, we found that MRC-5 fibroblasts adhere equally well to each of the four fas-1 domains (Fig.1 A). This suggests that each fas-1 domain has a motif for MRC-5 cell adhesion. Furthermore, the MRC-5-bininding motifs in the second and the fourth fas-1 domains may not be the NKDIL and EPDIM regions, respectively, that were found earlier to mediate corneal epithelial cell adhesion. Confirming this, we showed that the NKDIL and EPDIM peptides do not inhibit MRC-5 cell adhesion to any of the fas-1 domains or to the βig-h3-WT protein (Fig. 1 A). To identify the βig-h3 receptor on MRC-5 fibroblast cells, function-blocking monoclonal antibodies specific for various integrin subunits were examined for their ability to block the adhesion of MRC-5 cells to the βig-h3-WT protein or the fas-1 domains (Fig. 1, B and C). MRC-5 cell adhesion to the βig-h3-WT protein and all of the fas-1 domains was specifically inhibited by the antibody to the αvβ5 subunit but not by any of the other antibodies (Fig. 1, B and C). Thus, βig-h3 has a motif in each fas-1 domain that interacts with the αvβ5 integrin. To confirm that MRC-5 cells express the αvβ5 integrin on their surface, we performed FACS analysis using monoclonal antibodies to several integrins (Fig. 1 D). Several integrins, including the αvβ5 integrin, were observed on the MRC-5 cell surface, but the αvβ3 integrin was not detected. To identify the αvβ5 integrin-interacting motif(s) in βig-h3, we made five N-terminal- and C-terminal-deleted recombinant proteins of the fourth fas-1 domain, which consists of amino acids 498–637. 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 Scholar) (Fig.2 A). ΔH1 and ΔH2 represent the fourth fas-1 domain lacking one of two conserved fas-1 regions known as H1 (amino acids 498–547) and H2 (amino acids 621–637). ΔH1H2 lacks both H1 and H2. 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) 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 Scholar) are ΔH2 and ΔH1H2 that lack in addition the C-terminal amino acids containing the α3β1 integrin-interacting motif EPDIM. We tested the ability of each protein to promote MRC-5 cell adhesion (Fig. 2 A). All, including the smallest mutant protein, 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 (Fig 2 A). Thus, H1 and H2 are not involved and the cell adhesion motif is present within amino acids 548–614. To further identify the cell-adhesion motif within this fragment, we performed a computer search using Prodom 2001.2 searching for homologies not only among the four βig-h3 domains but also among the fas-1 domains from other proteins. Interestingly, many fas-1 domains, including the four fas-1 domains of βig-h3, contain two amino acid residues, a tyrosine and a histidine, in the domain center (Fig.2 B). In the fourth domain of βig-h3, the tyrosine and the histidine are amino acids 571 and 572, respectively. We assessed whether the tyrosine and histidine are needed for MRC-5 cell adhesion, first by using the two deleted proteins βig-h3 D-IV-569 and βig-h3 D-IV-575. These proteins encode amino acids 569–632 and 575–632, respectively, and thus βig-h3 D-IV-575 lacks the tyrosine and histidine residues. As shown in Fig. 2 C, the mutant proteins were both significantly less able to mediate MRC-5 cell adhesion compared 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 Scholar) proteins tested earlier (Fig. 2 A), suggesting the potential importance of residues 548–569. However, the ability of D-IV-575 to promote MRC-5 cell adhesion was less than that of βig-h3 D-IV-569. Thus, tyrosine 571 and histidine 572 may play an essential role in the cell-adhesion activity of βig-h3. To confirm this, we generated substitution mutant βig-h3 proteins whose tyrosine 571 and/or histidine 571 residues were replaced with alanine (Fig. 2 C). Unexpectedly, the single point mutations did not significantly affect cell-adhesion activity. However, the double point mutation significantly inhibited the cell adhesion activity of βig-h3, although not completely. Thus, tyrosine 571 and histidine 572 are clearly required in βig-h3-mediated MRC-5 cell adhesion. However, flanking amino acids may also be needed. To confirm that tyrosine 571 and histidine 572 are necessary for MRC-5 cell adhesion, a synthetic peptide corresponding to amino acids 569–574 (YH6) was generated and used to compete cell adhesion to βig-h3. However, this peptide could not inhibit MRC-5 cell adhesion to βig-h3. We then used the larger YH10 peptide corresponding to amino acids 567–576 but it also could not inhibit cell adhesion. However, when we tested YH18, which corresponds to amino acids 563–580, MRC-5 cell adhesion to βig-h3 was almost completely abolished. The control peptide YH18-con, which contains the amino acids of YH18 but in a scrambled sequence, did not affect cell adhesion to βig-h3 (Fig.3 A). We obtained similar results when each of the four fas-1 domains was used as the substrate (Fig. 3 B). The competition of cell adhesion by the YH18 peptide was dose-dependent (Fig. 3 C). To confirm that the YH18-like sequences of the other three fas-1 domains of βig-h3 are also effective in competing MRC-5 cell adhesion to βig-h3, we synthesized three peptides representing these sequences. As shown in Fig. 3 D, these peptides also inhibited MRC-5 cell adhesion to βig-h3 in a dose-dependent manner. Whereas peptides from D-I, -II, and -III regions showed similar inhibitory activity, the peptide from the D-IV showed slightly higher activity compared with the others. Although all of these peptides were effective in the micromolar range (D-IV peptide inhibited 50% cell adhesion at 200 μm), they displayed far less activity compared with the recombinant domain IV protein which inhibited cell adhesion by 50% between 5 μm and 10 μm(Fig. 3 E). This suggests that, although a region of 18 amino acids comprises the basic element required for minimal activity, longer stretches are required for full activity. Taken together, these results suggest that the YH18-like sequences encompassing the conserved tyrosine and histidine residues are essential for MRC-5 cell adhesion, confirming that not only the tyrosine and histidine but also flanking amino acids are minimally required for βig-h3-mediated MRC-5 cell adhesion. Because the leucine and isoleucine residues that flank the tyrosine and histidine are also well conserved among the fas-1 domains from different proteins (Fig.4 A), we examined their role in βig-h3-mediated MRC-5 cell adhesion. We constructed a number of substitution mutants of βig-h3 whose tyrosine, histidine, leucine, and isoleucine residues were substituted in various combinations by serine (leucine and isoleucine) or alanine (tyrosine and histidine) (Fig. 4 B). These mutants were then used in cell adhesion assays. D-IV-L represents the fourth fas-1 domain whose leucine 565, isoleucine 568, and leucine 569 were replaced with serine. D-IV-R represents the fourth fas-1 domain whose isoleucine 573, isoleucine 577, and leucine 578 were replaced with serine. D-IV-LAA and D-IV-AAR represents D-IV-L and D-IV-R whose tyrosine 571 and histidine 572 were replaced with alanine. D-IV-LYHR represents the fourth fas-1 domain whose conserved amino acids mentioned above were replaced with serine or alanine. Although D-IV-L, D-IV-R, D-IV-LAA, D-IV-AAR, and D-IV-LYHR show less cell-adhesion activity compared with the fourth fas-1 domain, they still promote some cell adhesion (Fig. 4 B). In contrast, the D-IV-LAAR mutant, where all conserved amino acids had been substituted, was very poor in mediating MRC-5 cell adhesion. Thus, multiple amino acids are required to form a conformation that mediates MRC-5 cell adhesion to βig-h3. To confirm that βig-h3 mediates cell adhesion through the αvβ5 integrin, we used stably transfected HEK293 cells (β5/293) that express human β5 integrin. HEK293 cells do not normally express the β5 integrin although they do express many β1 integrins, including α1β1, α3β1, α5β1, α6β1, and αvβ1 (12Bossy B. Reichardt L.F. Biochemistry. 1990; 29: 10191-10198Crossref PubMed Scopus (45) Google Scholar). We confirmed that β5/293 cells express the αvβ5 integrin by FACS analysis (data not shown). β5/293 cells strongly adhered to surfaces coated with βig-h3-WT or vitronectin, unlike pc/293 cells, which are HEK293 cells stably transfected with an empty vector (Fig. 5 A). β5/293 cell adhesion to βig-h3 was specifically inhibited by an antibody recognizing the αvβ5 integrin (Fig. 5 B) and by the YH18 peptide (Fig. 5 C). These results confirm that βig-h3 mediates cell adhesion by interacting with the αvβ5 integrin. We previously identified two α3β1 integrin-interacting motifs that are required 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). However, these motifs are not involved in the βig-h3-mediated adhesion of mesenchymal cells, including fibroblasts, vascular smooth muscle cells, and osteoblasts. This observation led us to seek the motifs in βig-h3 that are involved in the βig-h3-mediated adhesion of fibroblasts. Here we report a new βig-h3 motif that interacts with the αvβ5 integrin and is necessary for the βig-h3-mediated adhesion of fibroblasts. Unlike corneal epithelial cells, MRC-5 fibroblasts adhere to all four fas-1 domains of βig-h3. Furthermore, this adhesion was not blocked by the presence of the α3β1 integrin-interacting peptides EPDIM and NKDIL. Thus, MRC-5 cells must adhere to βig-h3 through an integrin other than the α3β1 integrin. We used several function-blocking integrin-specific antibodies and found that only anti-αvβ5 integrin antibody could eliminate MRC-5 cell adhesion to βig-h3 and the fas-1 domains. We confirmed that MRC-5 cells express the αvβ5 integrin on their cell surface. Ohno et al. (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) previously reported that βig-h3 mediates MRC-5 fibroblast cell adhesion through the α1β1 integrin. However, we consistently found that α1 and β1 integrin-function blocking antibodies did not significantly block MRC-5 cell adhesion to βig-h3. We confirmed that α1 and β1 integrin function-blocking antibodies used effectively inhibited the adhesion of several cell lines to specific ligands including collagen and fibronectin (data not shown). Thus, it is very unlikely that this discrepancy is due to using different antibodies. Unfortunately, they did not test whether αvβ5 function-blocking antibody could inhibit MRC-5 cell adhesion to βig-h3. Thus, we are as yet unable to explain the discrepancy clearly. Interestingly, even though 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 Scholar) express both the α3β1 and αvβ5 integrins, the two cell lines use only one but not the same integrin type for adhesion to βig-h3 (this report and Ref. 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 choice of the integrin used may depend on the activation state of the integrins in each cell line. There are a variety of intracellular cues that cause cells to modulate the affinity and avidity of their integrins for their extracellular matrix ligands. For example, R-Ras and H-Ras are known to regulate cell adhesion by modulating the affinity of multiple integrins for the extracellular matrix (13Zhang Z. Vuori K. Wang H. Reed J.C. Ruoslahti E. Cell. 1996; 85: 61-69Abstract Full Text Full Text PDF PubMed Scopus (379) Google Scholar, 14Hughes P.E. Renshaw M.W. Pfaff M. Forsyth J. Keivens V.M. Schwartz M.A. Ginsberg M.H. Cell. 1997; 88: 521-530Abstract Full Text Full Text PDF PubMed Scopus (434) Google Scholar). Furthermore, phosphorylation of the cytoplasmic domain of integrins alters the conformation of the integrins and thereby changes their affinity for their ligands (15Datta A. Huber F. Boettiger D. J. Biol. Chem. 2002; 277: 3943-3949Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). We are currently engaged in investigating why MRC-5 and corneal epithelial cells use different integrin types to interact with βig-h3. The finding that the anti-αvβ5 integrin antibody blocks 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 investigated this issue by constructing a series of deletion mutants of the fourth fas-1 domain and assessing their ability to promote MRC-5 cell adhesion. Two regions denoted H1 and H2 are highly conserved in each of the fas-1 domains, but deletion of either or both of these regions did not affect cell adhesion. Because all four fas-1 domains mediate cell adhesion through the same integrin type, it is likely that they all carry the same conserved consensus sequence that facilitates this interaction. We performed sequence analysis of the fas-1 domains of βig-h3 and other fas-1-containing proteins and found a tyrosine, a histidine, and many leucine/isoleucine residues flanking the tyrosine and histidine, which are highly conserved. 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, indicating they are part of the αvβ5 integrin-binding motif in βig-h3. Unlike the α3β1 integrin-interacting motifs NKDIL and EPDIM in βig-h3, the cell-adhesion activity of this new motif does not depend on a few crucial amino acid residues because single point mutations of tyrosine and histidine did not significantly affect the cell adhesion activity of βig-h3. Even substitution mutations of several of the conserved tyrosine, histidine, leucine, and isoleucine residues could not completely abolish the cell adhesion activity. Only the massive mutation of all these conserved amino acids almost completely abrogated the cell adhesion activity of βig-h3. This is supported by competition studies using synthetic peptides that showed that six- and ten-amino acid peptides containing the tyrosine and histidine residues could not compete cell adhesion, unlike the much larger 18-amino acid peptide YH18. Furthermore, although a peptide of 18 amino acids had activity, it was much less active compared with the recombinant domain IV protein, suggesting that the region of 18 amino acids only comprises the minimal element required for the cell adhesion activity of the fas-1 domain. Amino acids flanking this minimally conserved region could possibly contribute to the conformation of the αvβ5 integrin-binding site, thereby enhancing its activity. It is, however, very hard to predict which amino acids are critical because we could not find any conserved amino acids other than tyrosine, histidine, and leucine/isoleucine among several fas-1 domains although the computer analysis showed a few weakly conserved alipathic or hydrophobic amino acids were present around the conserved tyrosine and histidine residues. Thus, although the 18-amino acid region comprising the conserved histidine, tyrosine, and leucine/isoleucine residues contains the basic requirements for cell adhesion, a more extensive region is required for full potentiation of this activity. The αvβ5 integrin is thought to bind to a number of extracellular matrix proteins, including vitronectin and osteopontin (16Smith J.W. Vestal D.J. Irwin S.V. Burke T.A. Cheresh D.A. J. Biol. Chem. 1990; 265: 11008-11013Abstract Full Text PDF PubMed Google Scholar, 17Hu 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 (226) Google Scholar). Binding to these ligands appears to be RGD-dependent because peptides containing this tripeptide motif inhibit αvβ5 integrin binding. Although the sequence of the αvβ5 integrin-binding motif contained in YH18 differs from the RGD motif, both peptides may recognize the same site in the αvβ5 integrin because MRC-5 cell adhesion to βig-h3 is inhibited by the RGD peptide (data not shown). Furthermore, MRC-5 cell adhesion to vitronectin is also inhibited by the YH18 peptide (data not shown). Alternatively, the binding of one αvβ5 integrin site may alter the conformation of the other site, thereby inhibiting the interaction. Because the αvβ3 integrin also interacts with vitronectin and osteopontin in a RGD-dependent manner (18Bayless K.J. Salazar R. Davis G.E. Am. J. Pathol. 2000; 156: 1673-1683Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar, 19Hu D.D. Hoyer J.R. Smith J.W. J. Biol. Chem. 1995; 270: 9917-9925Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar), it is possible that the YH18 peptide could also bind to the αvβ3 integrin. We could not test this notion in the study reported here because MRC-5 cells do not express the αvβ3 integrin. However, it is possible that βig-h3 could interact with the αvβ3 integrin through the YH motif because we have found that adhesion to βig-h3 of some cell types depends both on the YH motif and the αvβ3 integrin (data not shown). In this regard, it is noteworthy that βig-h3 is expressed by endothelial cells and vascular smooth muscle cells (20O'Brien E.R. Bennett K.L. Garvin M.R. Zdric T.W. Hinohara T. Simpson J.B. Kimura T. Nobuyoshi M. Mizgala H. Purchio A. Schwartz S.M. Arterioscler. Thromb. Vasc. Biol. 1996; 16: 576-584Crossref PubMed Scopus (94) Google Scholar), both of which are known to use the αvβ3 and αvβ5 integrins for their biological responses to a variety of conditions such as angiogenesis (21Kumar C.C. Malkowski M. Yin Z. Tanghetti E. Yaremko B. Nechuta T. Varner J. Liu M. Smith E.M. Neustadt B. Presta M. Armstrong L. Cancer Res. 2001; 61: 2232-2238PubMed Google Scholar) and intimal hyperplasia (22Gimeno M.J. Gonzalez J. Rodriguez M. Corrales C. Bellon J.M. Bujan J. Histol. Histopathol. 2001; 16: 821-826PubMed Google Scholar). In humans, four proteins are known to bear fas-1 domains. βig-h3 and periostin (23Horiuchi K. Amizuka N. Takeshita S. Takamatsu H. Katsuura M. Ozawa H. Toyama Y. Bonewald L.F. Kudo A. J. Bone Miner. Res. 1999; 14: 1239-1249Crossref PubMed Scopus (812) Google Scholar) are secretory proteins with four fas-1 domains, and stabilin-1 and stabilin-2 are membrane proteins with seven fas-1 domains (24Politz O. Gratchev A. McCourt P.A. Schledzewski K. Guillot P. Johansson S. Svineng G. Franke P. Kannicht C. Kzhyshkowska J. Longati P. Velten F.W. Johansson S. Goerdt S. Biochem. J. 2002; 362: 155-164Crossref PubMed Scopus (247) Google Scholar). Although the biological functions of these proteins are not comprehensively understood, it is possible that all may function in regulating cell-matrix interactions and cell-cell interactions, because all of the fas-1 domains bear potential integrin-interacting YH motifs. These four human proteins are unique among the integrin-interacting proteins in that they bear multiple repeat integrin-interacting motifs. None of the other molecules known to interact with integrins has multiple repeats of motifs that interact with one integrin type. It is of interest to determine the biological meaning of this unique feature of the human fas-1 domain-bearing 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 consists of tyrosine and histidine flanked by several leucine/isoleucine residues. The fact that this motif is well conserved in most of the fas-1 domains present in many disparate proteins suggests that fas-1 domain-containing proteins may regulate various cell functions by interacting with integrins. We thank Dr. Jeffrey Smith (Burnham Institute, San Diego) for providing the β5/293 cells.
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