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A Role of Dystroglycan in Schwannoma Cell Adhesion to Laminin

1997; Elsevier BV; Volume: 272; Issue: 21 Linguagem: Inglês

10.1074/jbc.272.21.13904

1083-351X

Kiichiro Matsumura, Atsuro Chiba, Hiroki Yamada, Hiroko Fukuta‐Ohi, Sachiko Fujita, Tamao Endo, Akira Kobata, Louise V.B. Anderson, Ichiro Kanazawa, Kevin P. Campbell, Teruo Shimizu,

Silk-based biomaterials and applications

Dystroglycan is encoded by a single gene and cleaved into two proteins α- and β-dystroglycan by posttranslational processing. Recently, α-dystroglycan was demonstrated to be an extracellular laminin-binding protein anchored to the cell membrane by a transmembrane protein β-dystroglycan in striated muscle and Schwann cells. However, the biological functions of the dystroglycan-laminin interaction remain obscure, and in particular, it is still unclear if dystroglycan plays a role in cell adhesion. In the present study, we characterized the role of dystroglycan in the adhesion of schwannoma cells to laminin-1. Immunochemical analysis demonstrated that the dystroglycan complex, comprised of α- and β-dystroglycan, was a major laminin-binding protein complex in the surface membrane of rat schwannoma cell line RT4. It also demonstrated the presence of α-dystroglycan, but not β-dystroglycan, in the culture medium, suggesting secretion of α-dystroglycan by RT4 cells. RT4 cells cultured on dishes coated with laminin-1 became spindle in shape and adhered to the bottom surface tightly. Monoclonal antibody IIH6 against α-dystroglycan was shown previously to inhibit the binding of laminin-1 to α-dystroglycan. In the presence of IIH6, but not several other control antibodies in the culture medium, RT4 cells remained round in shape and did not adhere to the bottom surface. The adhesion of RT4 cells to dishes coated with fibronectin was not affected by IIH6. The known inhibitors of the interaction of α-dystroglycan with laminin-1, including EDTA, sulfatide, fucoidan, dextran sulfate, heparin, and sialic acid, also perturbed the adhesion of RT4 cells to laminin-1, whereas the reagents which do not inhibit the interaction, including dextran, chondroitin sulfate, dermatan sulfate, and GlcNAc, did not. Altogether, these results support a role for dystroglycan as a major cell adhesion molecule in the surface membrane of RT4 cells. Dystroglycan is encoded by a single gene and cleaved into two proteins α- and β-dystroglycan by posttranslational processing. Recently, α-dystroglycan was demonstrated to be an extracellular laminin-binding protein anchored to the cell membrane by a transmembrane protein β-dystroglycan in striated muscle and Schwann cells. However, the biological functions of the dystroglycan-laminin interaction remain obscure, and in particular, it is still unclear if dystroglycan plays a role in cell adhesion. In the present study, we characterized the role of dystroglycan in the adhesion of schwannoma cells to laminin-1. Immunochemical analysis demonstrated that the dystroglycan complex, comprised of α- and β-dystroglycan, was a major laminin-binding protein complex in the surface membrane of rat schwannoma cell line RT4. It also demonstrated the presence of α-dystroglycan, but not β-dystroglycan, in the culture medium, suggesting secretion of α-dystroglycan by RT4 cells. RT4 cells cultured on dishes coated with laminin-1 became spindle in shape and adhered to the bottom surface tightly. Monoclonal antibody IIH6 against α-dystroglycan was shown previously to inhibit the binding of laminin-1 to α-dystroglycan. In the presence of IIH6, but not several other control antibodies in the culture medium, RT4 cells remained round in shape and did not adhere to the bottom surface. The adhesion of RT4 cells to dishes coated with fibronectin was not affected by IIH6. The known inhibitors of the interaction of α-dystroglycan with laminin-1, including EDTA, sulfatide, fucoidan, dextran sulfate, heparin, and sialic acid, also perturbed the adhesion of RT4 cells to laminin-1, whereas the reagents which do not inhibit the interaction, including dextran, chondroitin sulfate, dermatan sulfate, and GlcNAc, did not. Altogether, these results support a role for dystroglycan as a major cell adhesion molecule in the surface membrane of RT4 cells. There is now mounting evidence that the intracellular signal transduction pathways activated by the adhesion of cells to other cells or the extracellular matrix (ECM) 1The abbreviations used are: ECM, extracellular matrix; BSA, bovine serum albumin; cLSM, confocal laser scanning microscope; WGA, wheat germ agglutinin; PBS, phosphate-buffered saline. 1The abbreviations used are: ECM, extracellular matrix; BSA, bovine serum albumin; cLSM, confocal laser scanning microscope; WGA, wheat germ agglutinin; PBS, phosphate-buffered saline. play crucial roles in cellular differentiation, migration, and proliferation. The prototypical cell adhesion molecules are the cell surface receptors for the ECM glycoproteins. Dystroglycan, originally identified as a member of the sarcolemmal glycoproteins complexed with dystrophin, is encoded by a single gene and cleaved into two proteins α- and β-dystroglycan by posttranslational processing (1Ibraghimov-Beskrovnaya O. Ervasti J.M. Leveille C.J. Slaughter C.A. Sernett S.W. Campbell K.P. Nature. 1992; 355: 696-702Google Scholar, 2Ervasti J.M. Campbell K.P. Cell. 1991; 66: 1121-1131Google Scholar). α-Dystroglycan is an extracellular glycoprotein anchored to the cell membrane by a transmembrane glycoprotein β-dystroglycan, and the complex comprised of α- and β-dystroglycan is called the dystroglycan complex (2Ervasti J.M. Campbell K.P. Cell. 1991; 66: 1121-1131Google Scholar, 3Ervasti J.M. Campbell K.P. J. Cell Biol. 1993; 122: 809-823Google Scholar, 4Deyst K.A. Bowe M.A. Leszyk J.D. Fallon J.R. J. Biol. Chem. 1995; 270: 25956-25959Google Scholar). In striated muscle, α-dystroglycan binds the ECM components laminin-1 and -2 in a Ca2+-dependent manner (3Ervasti J.M. Campbell K.P. J. Cell Biol. 1993; 122: 809-823Google Scholar, 5Sunada Y. Bernier S.M. Kozak C.A. Yamada Y. Campbell K.P. J. Biol. Chem. 1994; 269: 13729-13732Google Scholar, 6Pall E.A. Bolton K.M. Ervasti J.M. J. Biol. Chem. 1996; 271: 3817-3821Google Scholar). On the cytoplasmic side of the sarcolemma, β-dystroglycan is anchored to the cytoskeletal proteins dystrophin or its homologues (7Suzuki A. Yoshida M. Hayashi K. Mizuno Y. Hagiwara Y. Ozawa E. Eur. J. Biochem. 1994; 220: 283-292Google Scholar, 8Jung D. Yang B. Meyer J. Chamberlain J.S. Campbell K.P. J. Biol. Chem. 1995; 270: 27305-27310Google Scholar). Besides this structural role, β-dystroglycan is also proposed to play a role in signal transduction, based on the finding that its cytoplasmic domain contains a phosphotyrosine consensus sequence and several proline-rich regions that could associate with SH2 and SH3 domains of signaling proteins (1Ibraghimov-Beskrovnaya O. Ervasti J.M. Leveille C.J. Slaughter C.A. Sernett S.W. Campbell K.P. Nature. 1992; 355: 696-702Google Scholar). Dystrophin deficiency causes a drastic reduction of the dystroglycan complex in the sarcolemma and, thus, the loss of the linkage between the subsarcolemmal cytoskeleton and the ECM, eventually leading to muscle cell death in Duchenne muscular dystrophy and its animal model mdx mice (for reviews, see Refs. 9Campbell K.P. Cell. 1995; 80: 675-679Google Scholar and 10Ozawa E. Yoshida M. Suzuki A. Mizuno Y. Hagiwara Y. Noguchi S. Hum. Mol. Genet. 1995; 4: 1711-1716Google Scholar).The dystroglycan complex is also expressed in non-muscle tissues (1Ibraghimov-Beskrovnaya O. Ervasti J.M. Leveille C.J. Slaughter C.A. Sernett S.W. Campbell K.P. Nature. 1992; 355: 696-702Google Scholar, 3Ervasti J.M. Campbell K.P. J. Cell Biol. 1993; 122: 809-823Google Scholar,11Gee S.H. Blacher R.W. Douville P.J. Provost P.R. Yurchenco P.D. Carbonetto S. J. Biol. Chem. 1993; 268: 14972-14980Google Scholar, 12Smalheiser N.R. Kim E. J. Biol. Chem. 1995; 270: 15425-15433Google Scholar, 13Yamada H. Shimizu T. Tanaka T. Campbell K.P. Matsumura K. FEBS Lett. 1994; 352: 49-53Google Scholar). In the peripheral nervous system, it is expressed in the Schwann cell membrane, and the Schwann cell α-dystroglycan binds not only laminin-1 but also laminin-2, a major component of the endoneurium, in a Ca2+-dependent manner (13Yamada H. Shimizu T. Tanaka T. Campbell K.P. Matsumura K. FEBS Lett. 1994; 352: 49-53Google Scholar, 14Yamada H. Chiba A. Endo T. Kobata A. Anderson L.V.B. Hori H. Fukuta-Ohi H. Kanazawa I. Campbell K.P. Shimizu T. Matsumura K. J. Neurochem. 1996; 66: 1518-1524Google Scholar, 15Yamada H. Denzer A.J. Hori H. Tanaka T. Anderson L.V.B. Fujita S. Fukuta-Ohi H. Shimizu T. Ruegg M.A. Matsumura K. J. Biol. Chem. 1996; 271: 23418-23423Google Scholar). Recently, laminin-2 was shown to be deficient in congenital muscular dystrophy and its animal model dy mice, which are characterized by peripheral dysmyelination as well as muscular dystrophy (16Arahata K. Hayashi Y.K. Koga R. Goto K. Lee J.H. Miyagoe Y. Ishii H. Tsukahara T. Takeda S. Woo M. Nonaka I. Matsuzaki T. Sugita H. Proc. Jpn. Acad. Ser. B Phys. Biol. Sci. 1993; 69: 259-264Google Scholar, 17Sunada Y. Bernier S.M. Utani A. Yamada Y. Campbell K.P. Hum. Mol. Genet. 1995; 4: 1055-1061Google Scholar, 18Xu H. Christmas P. Wu X.R. Wewer U.M. Engvall E. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5572-5576Google Scholar, 19Xu H. Wu X.-R. Wewer U.M. Engvall E. Nat. Genet. 1994; 8: 297-302Google Scholar, 20Tomé F.M.S. Evangelista T. Leclerc A. Sunada Y. Manole E. Estournet B. Barois A. Campbell K.P. Fardeau M. C. R. Acad. Sci. Ser. III Sci. Vie. 1994; 317: 351-357Google Scholar, 21Shorer Z. Philpot J. Muntoni F. Sewry C. Dubowitz V. J. Child Neurol. 1995; 10: 472-475Google Scholar, 22Helbling-Leclerc A. Zhang X. Topaloglu H. Cruaud C. Tesson F. Weissenbach J. Tomé F.M.S. Schwartz K. Fardeau M. Tryggvason K. Guicheney P. Nat. Genet. 1995; 11: 216-218Google Scholar). These findings have suggested roles for the dystroglycan-laminin interaction in not only the maintenance of sarcolemmal architecture but also peripheral myelinogenesis.Despite these recent developments, the biological functions of the dystroglycan complex remain obscure, and in particular, it has not yet been established if the dystroglycan complex is indeed involved in the process of cell adhesion. In the present study, we have identified the dystroglycan complex as a major laminin-binding protein complex in the surface membrane of rat schwannoma cell line RT4 and characterized its role in RT4 cell adhesion to laminin-1.DISCUSSIONα-Dystroglycan, which is an extracellular peripheral membrane glycoprotein anchored to the cell membrane by a transmembrane glycoprotein β-dystroglycan, binds laminin and agrin in striated muscle, neuromuscular junction, and Schwann cells (1Ibraghimov-Beskrovnaya O. Ervasti J.M. Leveille C.J. Slaughter C.A. Sernett S.W. Campbell K.P. Nature. 1992; 355: 696-702Google Scholar, 2Ervasti J.M. Campbell K.P. Cell. 1991; 66: 1121-1131Google Scholar, 3Ervasti J.M. Campbell K.P. J. Cell Biol. 1993; 122: 809-823Google Scholar, 4Deyst K.A. Bowe M.A. Leszyk J.D. Fallon J.R. J. Biol. Chem. 1995; 270: 25956-25959Google Scholar, 5Sunada Y. Bernier S.M. Kozak C.A. Yamada Y. Campbell K.P. J. Biol. Chem. 1994; 269: 13729-13732Google Scholar, 6Pall E.A. Bolton K.M. Ervasti J.M. J. Biol. Chem. 1996; 271: 3817-3821Google Scholar, 13Yamada H. Shimizu T. Tanaka T. Campbell K.P. Matsumura K. FEBS Lett. 1994; 352: 49-53Google Scholar, 14Yamada H. Chiba A. Endo T. Kobata A. Anderson L.V.B. Hori H. Fukuta-Ohi H. Kanazawa I. Campbell K.P. Shimizu T. Matsumura K. J. Neurochem. 1996; 66: 1518-1524Google Scholar, 15Yamada H. Denzer A.J. Hori H. Tanaka T. Anderson L.V.B. Fujita S. Fukuta-Ohi H. Shimizu T. Ruegg M.A. Matsumura K. J. Biol. Chem. 1996; 271: 23418-23423Google Scholar, 27Gee S.H. Montanaro F. Lindenbaum M.H. Carbonetto S. Cell. 1994; 77: 675-686Google Scholar,28Campanelli J.T. Roberds S.L. Campbell K.P. Scheller R.H. Cell. 1994; 77: 663-674Google Scholar, 31Sugiyama J. Bowen D.C. Hall Z.W. Neuron. 1994; 13: 103-115Google Scholar, 32Bowe M.A. Deyst K.A. Leszyk J.D. Fallon J.R. Neuron. 1994; 12: 1173-1180Google Scholar, 33Fallon J.R. Hall Z.W. Trends Neurosci. 1994; 17: 469-473Google Scholar, 34Carbonetto S. Lindenbaum M. Curr. Opin. Neurobiol. 1995; 5: 596-605Google Scholar). On the other hand, the cytoplasmic domain of β-dystroglycan contains a phosphotyrosine consensus sequence and several proline-rich regions that could associate with SH2 and SH3 domains of signaling proteins (1Ibraghimov-Beskrovnaya O. Ervasti J.M. Leveille C.J. Slaughter C.A. Sernett S.W. Campbell K.P. Nature. 1992; 355: 696-702Google Scholar, 35Yang B. Jung D. Motto D. Meyer J. Koretzky G. Campbell K.P. J. Biol. Chem. 1995; 270: 11711-11714Google Scholar). Indeed, Grb2, an adaptor protein in the signal transduction pathways, was recently demonstrated to bind to the cytoplasmic proline-rich regions of β-dystroglycan via the two SH3 domains (35Yang B. Jung D. Motto D. Meyer J. Koretzky G. Campbell K.P. J. Biol. Chem. 1995; 270: 11711-11714Google Scholar). These findings have suggested a possible role for the dystroglycan complex, comprised of α- and β-dystroglycan, as a signaling receptor involved in the maintenance of sarcolemmal architecture, peripheral synaptogenesis, and myelinogenesis. In addition, the dystroglycan complex has also been implicated in kidney epithelial development, although its ECM ligand in kidney has not yet been identified (29Durbeej M. Larsson E. Ibraghimov-Beskrovnaya O. Roberds S.L. Campbell K.P. Ekblom P. J. Cell Biol. 1995; 130: 79-91Google Scholar). Despite these recent developments, the precise roles of the interaction of the dystroglycan complex with ECM ligands in these specialized biological processes remain obscure, and in particular, it has not yet been confirmed if the dystroglycan complex plays a role in cell adhesion.Under these circumstances, identification of cell lines that express the dystroglycan complex in the surface membrane would provide us useful tools for testing the proposed functions of the dystroglycan complex in vivo. In the present study, we have demonstrated, by immunochemical analyses, that the dystroglycan complex, comprised of α- and β-dystroglycan, is a major laminin-binding protein complex in the surface membrane of rat schwannoma cell line RT4. Similar to Schwann cells (14Yamada H. Chiba A. Endo T. Kobata A. Anderson L.V.B. Hori H. Fukuta-Ohi H. Kanazawa I. Campbell K.P. Shimizu T. Matsumura K. J. Neurochem. 1996; 66: 1518-1524Google Scholar), utrophin, which has the binding capacity for the cytoplasmic domain of β-dystroglycan (7Suzuki A. Yoshida M. Hayashi K. Mizuno Y. Hagiwara Y. Ozawa E. Eur. J. Biochem. 1994; 220: 283-292Google Scholar, 8Jung D. Yang B. Meyer J. Chamberlain J.S. Campbell K.P. J. Biol. Chem. 1995; 270: 27305-27310Google Scholar, 36Matsumura K. Ervasti J.M. Ohlendieck K. Kahl S.D. Campbell K.P. Nature. 1992; 360: 588-591Google Scholar), was localized diffusely in the cytoplasm of RT4 cells and not associated with the dystroglycan complex. Thus, the putative membrane-associated cytoskeletal protein anchoring the dystroglycan complex to the underlying submembranous cytoskeleton remains to be elucidated.In the present study, we have also tested the role of α-dystroglycan in RT4 cell adhesion to laminin-1. When RT4 cells were cultured on laminin-1, they became spindle in shape immediately and adhered to the bottom surface tightly. However, when RT4 cells were cultured on laminin-1 in the presence of the known inhibitors of the interaction of α-dystroglycan with laminin-1, including EDTA, sulfatide, fucoidan, dextran sulfate, heparin, and sialic acid, they remained round in shape and did not adhere to the bottom surface. Because these reagents may also perturb the interaction of laminin-1 with the cell surface adhesion molecules other than α-dystroglycan, such as the members of the integrin family for instance, we have looked at the effects of monoclonal antibody IIH6 against α-dystroglycan, which inhibits the interaction of α-dystroglycan with laminin-1, and found that this antibody drastically reduces the adhesion of RT4 cells to laminin-1. Furthermore, IIH6 did not perturb the adhesion of RT4 cells to fibronectin. Together with the previous demonstration of high affinity binding of laminin-1 to α-dystroglycan (1Ibraghimov-Beskrovnaya O. Ervasti J.M. Leveille C.J. Slaughter C.A. Sernett S.W. Campbell K.P. Nature. 1992; 355: 696-702Google Scholar, 3Ervasti J.M. Campbell K.P. J. Cell Biol. 1993; 122: 809-823Google Scholar), these results indicate a role for α-dystroglycan as a major cell adhesion molecule in the surface membrane of RT4 cells and suggest that the dystroglycan complex may play an important role in cell adhesion in vivo. Finally, we have demonstrated the results which suggest the secretion of α-dystroglycan by RT4 cells. In the future, it would be interesting to see if α-dystroglycan is secreted in vivoand if the secreted α-dystroglycan has inhibitory, and potentially regulatory, effects on the interaction of the cell surface α-dystroglycan with the ECM ligands.The mechanism by which the dystroglycan complex may mediate such diverse and specific biological processes as sarcolemmal stabilization, epithelial morphogenesis, synaptogenesis, and myelinogenesis remains unclear. For instance, it has been disputed if the dystroglycan complex is actively involved in the acetylcholine receptor clustering in the neuromuscular junction as a signaling receptor of agrin (31Sugiyama J. Bowen D.C. Hall Z.W. Neuron. 1994; 13: 103-115Google Scholar, 33Fallon J.R. Hall Z.W. Trends Neurosci. 1994; 17: 469-473Google Scholar, 34Carbonetto S. Lindenbaum M. Curr. Opin. Neurobiol. 1995; 5: 596-605Google Scholar,37Gesemann M. Denzer A.J. Ruegg M.A. J. Cell Biol. 1995; 128: 625-636Google Scholar, 38Hopf C. Hoch W. J. Biol. Chem. 1996; 271: 5231-5236Google Scholar, 39Gesemann M. Cavalli V. Denzer A.J. Brancaccio A. Schumacher B. Ruegg M.A. Neuron. 1996; 16: 755-767Google Scholar, 40Meier T. Gesemann M. Cavalli V. Ruegg M.A. Wallace B.G. EMBO J. 1996; 15: 2625-2631Google Scholar, 41Campanelli J.T. Gayer G.G. Scheller R.H. Development. 1996; 122: 1663-1672Google Scholar, 42Glass D.J. Bowen D.C. Stitt T.N. Radziejewski C. Bruno J. Ryan T.E. Gies D.R. Shah S. Mattsson K. Burden S.J. DiStefano P.S. Valenzuela D.M. DeChiara T.M. Yancopoulos G.D. Cell. 1996; 85: 513-523Google Scholar, 43O'Toole J.J. Deyst K.A. Bowe M.A. Nastuk M.A. McKechnie B.A. Fallon J.R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7369-7374Google Scholar). Among others, our results seem consistent with at least two possibilities. First, the dystroglycan complex may function as a helper protein in these processes; the initial and high affinity binding of the ECM ligands to the dystroglycan complex may enable the more specific and functional cell surface receptors, such as the members of the integrin family or the putative myotube-associated specificity component (MASC), which was recently proposed to work in concert with the receptor tyrosine kinase MuSK in the neuromuscular junction formation, to interact with these ligands (31Sugiyama J. Bowen D.C. Hall Z.W. Neuron. 1994; 13: 103-115Google Scholar, 33Fallon J.R. Hall Z.W. Trends Neurosci. 1994; 17: 469-473Google Scholar, 34Carbonetto S. Lindenbaum M. Curr. Opin. Neurobiol. 1995; 5: 596-605Google Scholar, 37Gesemann M. Denzer A.J. Ruegg M.A. J. Cell Biol. 1995; 128: 625-636Google Scholar, 38Hopf C. Hoch W. J. Biol. Chem. 1996; 271: 5231-5236Google Scholar, 42Glass D.J. Bowen D.C. Stitt T.N. Radziejewski C. Bruno J. Ryan T.E. Gies D.R. Shah S. Mattsson K. Burden S.J. DiStefano P.S. Valenzuela D.M. DeChiara T.M. Yancopoulos G.D. Cell. 1996; 85: 513-523Google Scholar). Second, the dystroglycan complex may function as a structural protein in the maturational stages of these processes. In this scenario, it would be intriguing to postulate that the binding of the ECM ligands to the dystroglycan complex may trigger the reorganization of the submembranous dystrophin/utrophin-cytoskeleton and lead to the stabilization of the cell membrane (44Cody R.L. Wicha M.S. Exp. Cell Res. 1986; 165: 107-116Google Scholar). The fact that the binding sites for dystrophin/utrophin and Grb2 overlap in the C terminus of β-dystroglycan raises a possibility that this process may be mediated by Grb2 and other signaling/adaptor proteins (8Jung D. Yang B. Meyer J. Chamberlain J.S. Campbell K.P. J. Biol. Chem. 1995; 270: 27305-27310Google Scholar, 35Yang B. Jung D. Motto D. Meyer J. Koretzky G. Campbell K.P. J. Biol. Chem. 1995; 270: 11711-11714Google Scholar). There is now mounting evidence that the intracellular signal transduction pathways activated by the adhesion of cells to other cells or the extracellular matrix (ECM) 1The abbreviations used are: ECM, extracellular matrix; BSA, bovine serum albumin; cLSM, confocal laser scanning microscope; WGA, wheat germ agglutinin; PBS, phosphate-buffered saline. 1The abbreviations used are: ECM, extracellular matrix; BSA, bovine serum albumin; cLSM, confocal laser scanning microscope; WGA, wheat germ agglutinin; PBS, phosphate-buffered saline. play crucial roles in cellular differentiation, migration, and proliferation. The prototypical cell adhesion molecules are the cell surface receptors for the ECM glycoproteins. Dystroglycan, originally identified as a member of the sarcolemmal glycoproteins complexed with dystrophin, is encoded by a single gene and cleaved into two proteins α- and β-dystroglycan by posttranslational processing (1Ibraghimov-Beskrovnaya O. Ervasti J.M. Leveille C.J. Slaughter C.A. Sernett S.W. Campbell K.P. Nature. 1992; 355: 696-702Google Scholar, 2Ervasti J.M. Campbell K.P. Cell. 1991; 66: 1121-1131Google Scholar). α-Dystroglycan is an extracellular glycoprotein anchored to the cell membrane by a transmembrane glycoprotein β-dystroglycan, and the complex comprised of α- and β-dystroglycan is called the dystroglycan complex (2Ervasti J.M. Campbell K.P. Cell. 1991; 66: 1121-1131Google Scholar, 3Ervasti J.M. Campbell K.P. J. Cell Biol. 1993; 122: 809-823Google Scholar, 4Deyst K.A. Bowe M.A. Leszyk J.D. Fallon J.R. J. Biol. Chem. 1995; 270: 25956-25959Google Scholar). In striated muscle, α-dystroglycan binds the ECM components laminin-1 and -2 in a Ca2+-dependent manner (3Ervasti J.M. Campbell K.P. J. Cell Biol. 1993; 122: 809-823Google Scholar, 5Sunada Y. Bernier S.M. Kozak C.A. Yamada Y. Campbell K.P. J. Biol. Chem. 1994; 269: 13729-13732Google Scholar, 6Pall E.A. Bolton K.M. Ervasti J.M. J. Biol. Chem. 1996; 271: 3817-3821Google Scholar). On the cytoplasmic side of the sarcolemma, β-dystroglycan is anchored to the cytoskeletal proteins dystrophin or its homologues (7Suzuki A. Yoshida M. Hayashi K. Mizuno Y. Hagiwara Y. Ozawa E. Eur. J. Biochem. 1994; 220: 283-292Google Scholar, 8Jung D. Yang B. Meyer J. Chamberlain J.S. Campbell K.P. J. Biol. Chem. 1995; 270: 27305-27310Google Scholar). Besides this structural role, β-dystroglycan is also proposed to play a role in signal transduction, based on the finding that its cytoplasmic domain contains a phosphotyrosine consensus sequence and several proline-rich regions that could associate with SH2 and SH3 domains of signaling proteins (1Ibraghimov-Beskrovnaya O. Ervasti J.M. Leveille C.J. Slaughter C.A. Sernett S.W. Campbell K.P. Nature. 1992; 355: 696-702Google Scholar). Dystrophin deficiency causes a drastic reduction of the dystroglycan complex in the sarcolemma and, thus, the loss of the linkage between the subsarcolemmal cytoskeleton and the ECM, eventually leading to muscle cell death in Duchenne muscular dystrophy and its animal model mdx mice (for reviews, see Refs. 9Campbell K.P. Cell. 1995; 80: 675-679Google Scholar and 10Ozawa E. Yoshida M. Suzuki A. Mizuno Y. Hagiwara Y. Noguchi S. Hum. Mol. Genet. 1995; 4: 1711-1716Google Scholar). The dystroglycan complex is also expressed in non-muscle tissues (1Ibraghimov-Beskrovnaya O. Ervasti J.M. Leveille C.J. Slaughter C.A. Sernett S.W. Campbell K.P. Nature. 1992; 355: 696-702Google Scholar, 3Ervasti J.M. Campbell K.P. J. Cell Biol. 1993; 122: 809-823Google Scholar,11Gee S.H. Blacher R.W. Douville P.J. Provost P.R. Yurchenco P.D. Carbonetto S. J. Biol. Chem. 1993; 268: 14972-14980Google Scholar, 12Smalheiser N.R. Kim E. J. Biol. Chem. 1995; 270: 15425-15433Google Scholar, 13Yamada H. Shimizu T. Tanaka T. Campbell K.P. Matsumura K. FEBS Lett. 1994; 352: 49-53Google Scholar). In the peripheral nervous system, it is expressed in the Schwann cell membrane, and the Schwann cell α-dystroglycan binds not only laminin-1 but also laminin-2, a major component of the endoneurium, in a Ca2+-dependent manner (13Yamada H. Shimizu T. Tanaka T. Campbell K.P. Matsumura K. FEBS Lett. 1994; 352: 49-53Google Scholar, 14Yamada H. Chiba A. Endo T. Kobata A. Anderson L.V.B. Hori H. Fukuta-Ohi H. Kanazawa I. Campbell K.P. Shimizu T. Matsumura K. J. Neurochem. 1996; 66: 1518-1524Google Scholar, 15Yamada H. Denzer A.J. Hori H. Tanaka T. Anderson L.V.B. Fujita S. Fukuta-Ohi H. Shimizu T. Ruegg M.A. Matsumura K. J. Biol. Chem. 1996; 271: 23418-23423Google Scholar). Recently, laminin-2 was shown to be deficient in congenital muscular dystrophy and its animal model dy mice, which are characterized by peripheral dysmyelination as well as muscular dystrophy (16Arahata K. Hayashi Y.K. Koga R. Goto K. Lee J.H. Miyagoe Y. Ishii H. Tsukahara T. Takeda S. Woo M. Nonaka I. Matsuzaki T. Sugita H. Proc. Jpn. Acad. Ser. B Phys. Biol. Sci. 1993; 69: 259-264Google Scholar, 17Sunada Y. Bernier S.M. Utani A. Yamada Y. Campbell K.P. Hum. Mol. Genet. 1995; 4: 1055-1061Google Scholar, 18Xu H. Christmas P. Wu X.R. Wewer U.M. Engvall E. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5572-5576Google Scholar, 19Xu H. Wu X.-R. Wewer U.M. Engvall E. Nat. Genet. 1994; 8: 297-302Google Scholar, 20Tomé F.M.S. Evangelista T. Leclerc A. Sunada Y. Manole E. Estournet B. Barois A. Campbell K.P. Fardeau M. C. R. Acad. Sci. Ser. III Sci. Vie. 1994; 317: 351-357Google Scholar, 21Shorer Z. Philpot J. Muntoni F. Sewry C. Dubowitz V. J. Child Neurol. 1995; 10: 472-475Google Scholar, 22Helbling-Leclerc A. Zhang X. Topaloglu H. Cruaud C. Tesson F. Weissenbach J. Tomé F.M.S. Schwartz K. Fardeau M. Tryggvason K. Guicheney P. Nat. Genet. 1995; 11: 216-218Google Scholar). These findings have suggested roles for the dystroglycan-laminin interaction in not only the maintenance of sarcolemmal architecture but also peripheral myelinogenesis. Despite these recent developments, the biological functions of the dystroglycan complex remain obscure, and in particular, it has not yet been established if the dystroglycan complex is indeed involved in the process of cell adhesion. In the present study, we have identified the dystroglycan complex as a major laminin-binding protein complex in the surface membrane of rat schwannoma cell line RT4 and characterized its role in RT4 cell adhesion to laminin-1. DISCUSSIONα-Dystroglycan, which is an extracellular peripheral membrane glycoprotein anchored to the cell membrane by a transmembrane glycoprotein β-dystroglycan, binds laminin and agrin in striated muscle, neuromuscular junction, and Schwann cells (1Ibraghimov-Beskrovnaya O. Ervasti J.M. Leveille C.J. Slaughter C.A. Sernett S.W. Campbell K.P. Nature. 1992; 355: 696-702Google Scholar, 2Ervasti J.M. Campbell K.P. Cell. 1991; 66: 1121-1131Google Scholar, 3Ervasti J.M. Campbell K.P. J. Cell Biol. 1993; 122: 809-823Google Scholar, 4Deyst K.A. Bowe M.A. Leszyk J.D. Fallon J.R. J. Biol. Chem. 1995; 270: 25956-25959Google Scholar, 5Sunada Y. Bernier S.M. Kozak C.A. Yamada Y. Campbell K.P. J. Biol. Chem. 1994; 269: 13729-13732Google Scholar, 6Pall E.A. Bolton K.M. Ervasti J.M. J. Biol. Chem. 1996; 271: 3817-3821Google Scholar, 13Yamada H. Shimizu T. Tanaka T. Campbell K.P. Matsumura K. FEBS Lett. 1994; 352: 49-53Google Scholar, 14Yamada H. Chiba A. Endo T. Kobata A. Anderson L.V.B. Hori H. Fukuta-Ohi H. Kanazawa I. Campbell K.P. Shimizu T. Matsumura K. J. Neurochem. 1996; 66: 1518-1524Google Scholar, 15Yamada H. Denzer A.J. Hori H. Tanaka T. Anderson L.V.B. Fujita S. Fukuta-Ohi H. Shimizu T. Ruegg M.A. Matsumura K. J. Biol. Chem. 1996; 271: 23418-23423Google Scholar, 27Gee S.H. Montanaro F. Lindenbaum M.H. Carbonetto S. Cell. 1994; 77: 675-686Google Scholar,28Campanelli J.T. Roberds S.L. Campbell K.P. Scheller R.H. Cell. 1994; 77: 663-674Google Scholar, 31Sugiyama J. Bowen D.C. Hall Z.W. Neuron. 1994; 13: 103-115Google Scholar, 32Bowe M.A. Deyst K.A. Leszyk J.D. Fallon J.R. Neuron. 1994; 12: 1173-1180Google Scholar, 33Fallon J.R. Hall Z.W. Trends Neurosci. 1994; 17: 469-473Google Scholar, 34Carbonetto S. Lindenbaum M. Curr. Opin. Neurobiol. 1995; 5: 596-605Google Scholar). On the other hand, the cytoplasmic domain of β-dystroglycan contains a phosphotyrosine consensus sequence and several proline-rich regions that could associate with SH2 and SH3 domains of signaling proteins (1Ibraghimov-Beskrovnaya O. Ervasti J.M. Leveille C.J. Slaughter C.A. Sernett S.W. Campbell K.P. Nature. 1992; 355: 696-702Google Scholar, 35Yang B. Jung D. Motto D. Meyer J. Koretzky G. Campbell K.P. J. Biol. Chem. 1995; 270: 11711-11714Google Scholar). Indeed, Grb2, an adaptor protein in the signal transduction pathways, was recently demonstrated to bind to the cytoplasmic proline-rich regions of β-dystroglycan via the two SH3 domains (35Yang B. Jung D. Motto D. Meyer J. Koretzky G. Campbell K.P. J. Biol. Chem. 1995; 270: 11711-11714Google Scholar). These findings have suggested a possible role for the dystroglycan complex, comprised of α- and β-dystroglycan, as a signaling receptor involved in the maintenance of sarcolemmal architecture, peripheral synaptogenesis, and myelinogenesis. In addition, the dystroglycan complex has also been implicated in kidney epithelial development, although its ECM ligand in kidney has not yet been identified (29Durbeej M. Larsson E. Ibraghimov-Beskrovnaya O. Roberds S.L. Campbell K.P. Ekblom P. J. Cell Biol. 1995; 130: 79-91Google Scholar). Despite these recent developments, the precise roles of the interaction of the dystroglycan complex with ECM ligands in these specialized biological processes remain obscure, and in particular, it has not yet been confirmed if the dystroglycan complex plays a role in cell adhesion.Under these circumstances, identification of cell lines that express the dystroglycan complex in the surface membrane would provide us useful tools for testing the proposed functions of the dystroglycan complex in vivo. In the present study, we have demonstrated, by immunochemical analyses, that the dystroglycan complex, comprised of α- and β-dystroglycan, is a major laminin-binding protein complex in the surface membrane of rat schwannoma cell line RT4. Similar to Schwann cells (14Yamada H. Chiba A. Endo T. Kobata A. Anderson L.V.B. Hori H. Fukuta-Ohi H. Kanazawa I. Campbell K.P. Shimizu T. Matsumura K. J. Neurochem. 1996; 66: 1518-1524Google Scholar), utrophin, which has the binding capacity for the cytoplasmic domain of β-dystroglycan (7Suzuki A. Yoshida M. Hayashi K. Mizuno Y. Hagiwara Y. Ozawa E. Eur. J. Biochem. 1994; 220: 283-292Google Scholar, 8Jung D. Yang B. Meyer J. Chamberlain J.S. Campbell K.P. J. Biol. Chem. 1995; 270: 27305-27310Google Scholar, 36Matsumura K. Ervasti J.M. Ohlendieck K. Kahl S.D. Campbell K.P. Nature. 1992; 360: 588-591Google Scholar), was localized diffusely in the cytoplasm of RT4 cells and not associated with the dystroglycan complex. Thus, the putative membrane-associated cytoskeletal protein anchoring the dystroglycan complex to the underlying submembranous cytoskeleton remains to be elucidated.In the present study, we have also tested the role of α-dystroglycan in RT4 cell adhesion to laminin-1. When RT4 cells were cultured on laminin-1, they became spindle in shape immediately and adhered to the bottom surface tightly. However, when RT4 cells were cultured on laminin-1 in the presence of the known inhibitors of the interaction of α-dystroglycan with laminin-1, including EDTA, sulfatide, fucoidan, dextran sulfate, heparin, and sialic acid, they remained round in shape and did not adhere to the bottom surface. Because these reagents may also perturb the interaction of laminin-1 with the cell surface adhesion molecules other than α-dystroglycan, such as the members of the integrin family for instance, we have looked at the effects of monoclonal antibody IIH6 against α-dystroglycan, which inhibits the interaction of α-dystroglycan with laminin-1, and found that this antibody drastically reduces the adhesion of RT4 cells to laminin-1. Furthermore, IIH6 did not perturb the adhesion of RT4 cells to fibronectin. Together with the previous demonstration of high affinity binding of laminin-1 to α-dystroglycan (1Ibraghimov-Beskrovnaya O. Ervasti J.M. Leveille C.J. Slaughter C.A. Sernett S.W. Campbell K.P. Nature. 1992; 355: 696-702Google Scholar, 3Ervasti J.M. Campbell K.P. J. Cell Biol. 1993; 122: 809-823Google Scholar), these results indicate a role for α-dystroglycan as a major cell adhesion molecule in the surface membrane of RT4 cells and suggest that the dystroglycan complex may play an important role in cell adhesion in vivo. Finally, we have demonstrated the results which suggest the secretion of α-dystroglycan by RT4 cells. In the future, it would be interesting to see if α-dystroglycan is secreted in vivoand if the secreted α-dystroglycan has inhibitory, and potentially regulatory, effects on the interaction of the cell surface α-dystroglycan with the ECM ligands.The mechanism by which the dystroglycan complex may mediate such diverse and specific biological processes as sarcolemmal stabilization, epithelial morphogenesis, synaptogenesis, and myelinogenesis remains unclear. For instance, it has been disputed if the dystroglycan complex is actively involved in the acetylcholine receptor clustering in the neuromuscular junction as a signaling receptor of agrin (31Sugiyama J. Bowen D.C. Hall Z.W. Neuron. 1994; 13: 103-115Google Scholar, 33Fallon J.R. Hall Z.W. Trends Neurosci. 1994; 17: 469-473Google Scholar, 34Carbonetto S. Lindenbaum M. Curr. Opin. Neurobiol. 1995; 5: 596-605Google Scholar,37Gesemann M. Denzer A.J. Ruegg M.A. J. Cell Biol. 1995; 128: 625-636Google Scholar, 38Hopf C. Hoch W. J. Biol. Chem. 1996; 271: 5231-5236Google Scholar, 39Gesemann M. Cavalli V. Denzer A.J. Brancaccio A. Schumacher B. Ruegg M.A. Neuron. 1996; 16: 755-767Google Scholar, 40Meier T. Gesemann M. Cavalli V. Ruegg M.A. Wallace B.G. EMBO J. 1996; 15: 2625-2631Google Scholar, 41Campanelli J.T. Gayer G.G. Scheller R.H. Development. 1996; 122: 1663-1672Google Scholar, 42Glass D.J. Bowen D.C. Stitt T.N. Radziejewski C. Bruno J. Ryan T.E. Gies D.R. Shah S. Mattsson K. Burden S.J. DiStefano P.S. Valenzuela D.M. DeChiara T.M. Yancopoulos G.D. Cell. 1996; 85: 513-523Google Scholar, 43O'Toole J.J. Deyst K.A. Bowe M.A. Nastuk M.A. McKechnie B.A. Fallon J.R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7369-7374Google Scholar). Among others, our results seem consistent with at least two possibilities. First, the dystroglycan complex may function as a helper protein in these processes; the initial and high affinity binding of the ECM ligands to the dystroglycan complex may enable the more specific and functional cell surface receptors, such as the members of the integrin family or the putative myotube-associated specificity component (MASC), which was recently proposed to work in concert with the receptor tyrosine kinase MuSK in the neuromuscular junction formation, to interact with these ligands (31Sugiyama J. Bowen D.C. Hall Z.W. Neuron. 1994; 13: 103-115Google Scholar, 33Fallon J.R. Hall Z.W. Trends Neurosci. 1994; 17: 469-473Google Scholar, 34Carbonetto S. Lindenbaum M. Curr. Opin. Neurobiol. 1995; 5: 596-605Google Scholar, 37Gesemann M. Denzer A.J. Ruegg M.A. J. Cell Biol. 1995; 128: 625-636Google Scholar, 38Hopf C. Hoch W. J. Biol. Chem. 1996; 271: 5231-5236Google Scholar, 42Glass D.J. Bowen D.C. Stitt T.N. Radziejewski C. Bruno J. Ryan T.E. Gies D.R. Shah S. Mattsson K. Burden S.J. DiStefano P.S. Valenzuela D.M. DeChiara T.M. Yancopoulos G.D. Cell. 1996; 85: 513-523Google Scholar). Second, the dystroglycan complex may function as a structural protein in the maturational stages of these processes. In this scenario, it would be intriguing to postulate that the binding of the ECM ligands to the dystroglycan complex may trigger the reorganization of the submembranous dystrophin/utrophin-cytoskeleton and lead to the stabilization of the cell membrane (44Cody R.L. Wicha M.S. Exp. Cell Res. 1986; 165: 107-116Google Scholar). The fact that the binding sites for dystrophin/utrophin and Grb2 overlap in the C terminus of β-dystroglycan raises a possibility that this process may be mediated by Grb2 and other signaling/adaptor proteins (8Jung D. Yang B. Meyer J. Chamberlain J.S. Campbell K.P. J. Biol. Chem. 1995; 270: 27305-27310Google Scholar, 35Yang B. Jung D. Motto D. Meyer J. Koretzky G. Campbell K.P. J. Biol. Chem. 1995; 270: 11711-11714Google Scholar). α-Dystroglycan, which is an extracellular peripheral membrane glycoprotein anchored to the cell membrane by a transmembrane glycoprotein β-dystroglycan, binds laminin and agrin in striated muscle, neuromuscular junction, and Schwann cells (1Ibraghimov-Beskrovnaya O. Ervasti J.M. Leveille C.J. Slaughter C.A. Sernett S.W. Campbell K.P. Nature. 1992; 355: 696-702Google Scholar, 2Ervasti J.M. Campbell K.P. Cell. 1991; 66: 1121-1131Google Scholar, 3Ervasti J.M. Campbell K.P. J. Cell Biol. 1993; 122: 809-823Google Scholar, 4Deyst K.A. Bowe M.A. Leszyk J.D. Fallon J.R. J. Biol. Chem. 1995; 270: 25956-25959Google Scholar, 5Sunada Y. Bernier S.M. Kozak C.A. Yamada Y. Campbell K.P. J. Biol. Chem. 1994; 269: 13729-13732Google Scholar, 6Pall E.A. Bolton K.M. Ervasti J.M. J. Biol. Chem. 1996; 271: 3817-3821Google Scholar, 13Yamada H. Shimizu T. Tanaka T. Campbell K.P. Matsumura K. FEBS Lett. 1994; 352: 49-53Google Scholar, 14Yamada H. Chiba A. Endo T. Kobata A. Anderson L.V.B. Hori H. Fukuta-Ohi H. Kanazawa I. Campbell K.P. Shimizu T. Matsumura K. J. Neurochem. 1996; 66: 1518-1524Google Scholar, 15Yamada H. Denzer A.J. Hori H. Tanaka T. Anderson L.V.B. Fujita S. Fukuta-Ohi H. Shimizu T. Ruegg M.A. Matsumura K. J. Biol. Chem. 1996; 271: 23418-23423Google Scholar, 27Gee S.H. Montanaro F. Lindenbaum M.H. Carbonetto S. Cell. 1994; 77: 675-686Google Scholar,28Campanelli J.T. Roberds S.L. Campbell K.P. Scheller R.H. Cell. 1994; 77: 663-674Google Scholar, 31Sugiyama J. Bowen D.C. Hall Z.W. Neuron. 1994; 13: 103-115Google Scholar, 32Bowe M.A. Deyst K.A. Leszyk J.D. Fallon J.R. Neuron. 1994; 12: 1173-1180Google Scholar, 33Fallon J.R. Hall Z.W. Trends Neurosci. 1994; 17: 469-473Google Scholar, 34Carbonetto S. Lindenbaum M. Curr. Opin. Neurobiol. 1995; 5: 596-605Google Scholar). On the other hand, the cytoplasmic domain of β-dystroglycan contains a phosphotyrosine consensus sequence and several proline-rich regions that could associate with SH2 and SH3 domains of signaling proteins (1Ibraghimov-Beskrovnaya O. Ervasti J.M. Leveille C.J. Slaughter C.A. Sernett S.W. Campbell K.P. Nature. 1992; 355: 696-702Google Scholar, 35Yang B. Jung D. Motto D. Meyer J. Koretzky G. Campbell K.P. J. Biol. Chem. 1995; 270: 11711-11714Google Scholar). Indeed, Grb2, an adaptor protein in the signal transduction pathways, was recently demonstrated to bind to the cytoplasmic proline-rich regions of β-dystroglycan via the two SH3 domains (35Yang B. Jung D. Motto D. Meyer J. Koretzky G. Campbell K.P. J. Biol. Chem. 1995; 270: 11711-11714Google Scholar). These findings have suggested a possible role for the dystroglycan complex, comprised of α- and β-dystroglycan, as a signaling receptor involved in the maintenance of sarcolemmal architecture, peripheral synaptogenesis, and myelinogenesis. In addition, the dystroglycan complex has also been implicated in kidney epithelial development, although its ECM ligand in kidney has not yet been identified (29Durbeej M. Larsson E. Ibraghimov-Beskrovnaya O. Roberds S.L. Campbell K.P. Ekblom P. J. Cell Biol. 1995; 130: 79-91Google Scholar). Despite these recent developments, the precise roles of the interaction of the dystroglycan complex with ECM ligands in these specialized biological processes remain obscure, and in particular, it has not yet been confirmed if the dystroglycan complex plays a role in cell adhesion. Under these circumstances, identification of cell lines that express the dystroglycan complex in the surface membrane would provide us useful tools for testing the proposed functions of the dystroglycan complex in vivo. In the present study, we have demonstrated, by immunochemical analyses, that the dystroglycan complex, comprised of α- and β-dystroglycan, is a major laminin-binding protein complex in the surface membrane of rat schwannoma cell line RT4. Similar to Schwann cells (14Yamada H. Chiba A. Endo T. Kobata A. Anderson L.V.B. Hori H. Fukuta-Ohi H. Kanazawa I. Campbell K.P. Shimizu T. Matsumura K. J. Neurochem. 1996; 66: 1518-1524Google Scholar), utrophin, which has the binding capacity for the cytoplasmic domain of β-dystroglycan (7Suzuki A. Yoshida M. Hayashi K. Mizuno Y. Hagiwara Y. Ozawa E. Eur. J. Biochem. 1994; 220: 283-292Google Scholar, 8Jung D. Yang B. Meyer J. Chamberlain J.S. Campbell K.P. J. Biol. Chem. 1995; 270: 27305-27310Google Scholar, 36Matsumura K. Ervasti J.M. Ohlendieck K. Kahl S.D. Campbell K.P. Nature. 1992; 360: 588-591Google Scholar), was localized diffusely in the cytoplasm of RT4 cells and not associated with the dystroglycan complex. Thus, the putative membrane-associated cytoskeletal protein anchoring the dystroglycan complex to the underlying submembranous cytoskeleton remains to be elucidated. In the present study, we have also tested the role of α-dystroglycan in RT4 cell adhesion to laminin-1. When RT4 cells were cultured on laminin-1, they became spindle in shape immediately and adhered to the bottom surface tightly. However, when RT4 cells were cultured on laminin-1 in the presence of the known inhibitors of the interaction of α-dystroglycan with laminin-1, including EDTA, sulfatide, fucoidan, dextran sulfate, heparin, and sialic acid, they remained round in shape and did not adhere to the bottom surface. Because these reagents may also perturb the interaction of laminin-1 with the cell surface adhesion molecules other than α-dystroglycan, such as the members of the integrin family for instance, we have looked at the effects of monoclonal antibody IIH6 against α-dystroglycan, which inhibits the interaction of α-dystroglycan with laminin-1, and found that this antibody drastically reduces the adhesion of RT4 cells to laminin-1. Furthermore, IIH6 did not perturb the adhesion of RT4 cells to fibronectin. Together with the previous demonstration of high affinity binding of laminin-1 to α-dystroglycan (1Ibraghimov-Beskrovnaya O. Ervasti J.M. Leveille C.J. Slaughter C.A. Sernett S.W. Campbell K.P. Nature. 1992; 355: 696-702Google Scholar, 3Ervasti J.M. Campbell K.P. J. Cell Biol. 1993; 122: 809-823Google Scholar), these results indicate a role for α-dystroglycan as a major cell adhesion molecule in the surface membrane of RT4 cells and suggest that the dystroglycan complex may play an important role in cell adhesion in vivo. Finally, we have demonstrated the results which suggest the secretion of α-dystroglycan by RT4 cells. In the future, it would be interesting to see if α-dystroglycan is secreted in vivoand if the secreted α-dystroglycan has inhibitory, and potentially regulatory, effects on the interaction of the cell surface α-dystroglycan with the ECM ligands. The mechanism by which the dystroglycan complex may mediate such diverse and specific biological processes as sarcolemmal stabilization, epithelial morphogenesis, synaptogenesis, and myelinogenesis remains unclear. For instance, it has been disputed if the dystroglycan complex is actively involved in the acetylcholine receptor clustering in the neuromuscular junction as a signaling receptor of agrin (31Sugiyama J. Bowen D.C. Hall Z.W. Neuron. 1994; 13: 103-115Google Scholar, 33Fallon J.R. Hall Z.W. Trends Neurosci. 1994; 17: 469-473Google Scholar, 34Carbonetto S. Lindenbaum M. Curr. Opin. Neurobiol. 1995; 5: 596-605Google Scholar,37Gesemann M. Denzer A.J. Ruegg M.A. J. Cell Biol. 1995; 128: 625-636Google Scholar, 38Hopf C. Hoch W. J. Biol. Chem. 1996; 271: 5231-5236Google Scholar, 39Gesemann M. Cavalli V. Denzer A.J. Brancaccio A. Schumacher B. Ruegg M.A. Neuron. 1996; 16: 755-767Google Scholar, 40Meier T. Gesemann M. Cavalli V. Ruegg M.A. Wallace B.G. EMBO J. 1996; 15: 2625-2631Google Scholar, 41Campanelli J.T. Gayer G.G. Scheller R.H. Development. 1996; 122: 1663-1672Google Scholar, 42Glass D.J. Bowen D.C. Stitt T.N. Radziejewski C. Bruno J. Ryan T.E. Gies D.R. Shah S. Mattsson K. Burden S.J. DiStefano P.S. Valenzuela D.M. DeChiara T.M. Yancopoulos G.D. Cell. 1996; 85: 513-523Google Scholar, 43O'Toole J.J. Deyst K.A. Bowe M.A. Nastuk M.A. McKechnie B.A. Fallon J.R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7369-7374Google Scholar). Among others, our results seem consistent with at least two possibilities. First, the dystroglycan complex may function as a helper protein in these processes; the initial and high affinity binding of the ECM ligands to the dystroglycan complex may enable the more specific and functional cell surface receptors, such as the members of the integrin family or the putative myotube-associated specificity component (MASC), which was recently proposed to work in concert with the receptor tyrosine kinase MuSK in the neuromuscular junction formation, to interact with these ligands (31Sugiyama J. Bowen D.C. Hall Z.W. Neuron. 1994; 13: 103-115Google Scholar, 33Fallon J.R. Hall Z.W. Trends Neurosci. 1994; 17: 469-473Google Scholar, 34Carbonetto S. Lindenbaum M. Curr. Opin. Neurobiol. 1995; 5: 596-605Google Scholar, 37Gesemann M. Denzer A.J. Ruegg M.A. J. Cell Biol. 1995; 128: 625-636Google Scholar, 38Hopf C. Hoch W. J. Biol. Chem. 1996; 271: 5231-5236Google Scholar, 42Glass D.J. Bowen D.C. Stitt T.N. Radziejewski C. Bruno J. Ryan T.E. Gies D.R. Shah S. Mattsson K. Burden S.J. DiStefano P.S. Valenzuela D.M. DeChiara T.M. Yancopoulos G.D. Cell. 1996; 85: 513-523Google Scholar). Second, the dystroglycan complex may function as a structural protein in the maturational stages of these processes. In this scenario, it would be intriguing to postulate that the binding of the ECM ligands to the dystroglycan complex may trigger the reorganization of the submembranous dystrophin/utrophin-cytoskeleton and lead to the stabilization of the cell membrane (44Cody R.L. Wicha M.S. Exp. Cell Res. 1986; 165: 107-116Google Scholar). The fact that the binding sites for dystrophin/utrophin and Grb2 overlap in the C terminus of β-dystroglycan raises a possibility that this process may be mediated by Grb2 and other signaling/adaptor proteins (8Jung D. Yang B. Meyer J. Chamberlain J.S. Campbell K.P. J. Biol. Chem. 1995; 270: 27305-27310Google Scholar, 35Yang B. Jung D. Motto D. Meyer J. Koretzky G. Campbell K.P. J. Biol. Chem. 1995; 270: 11711-11714Google Scholar).