Post-translational Modifications of  5  1 Integrin by Glycosaminoglycan Chains THE  5  1 INTEGRIN IS A FACULTATIVE PROTEOGLYCAN* (Received for publication, October 2, 1996, and in revised form, December 23, 1996) Silvio S. Veiga‡§¶, Maria Carolina Q. B. Elias‡, Waldemiro Gremski‡§, Marimelia A. Porcionatto, Roseli da Silva, Helena B. Nader, and Ricardo R. Brentani‡ From the ‡Ludwig Institute for Cancer Research, R. Prof. Antonio Prudente, 109, 4 A, 01509-010, Sa ˜ o Paulo, SP, Brazil, the §Department of Cell Biology, Federal University of Parana, Curitiba, Centro Polite ´cnico, Seter de Cie ˆncias Biolo ´gicas, Jardim des Americas, 81531-990, Curitiba, PR, and the Department of Biochemistry, Federal University of Sa ˜ o Paulo, Universidade Federal de Sa ˜ o Paulo, Escola Paulista de Medicina, rea 3 de maio, 100 4 andar, 04044-020, Sa ˜ o Paulo, SP, Brazil Cell-fibronectin interactions, mediated through sev- eral different receptors, have been implicated in a wide variety of cellular properties. Among the cell surface receptors for fibronectin, integrins are the best charac- terized, particularly the prototype  5  1 integrin. Using [ 125 I]iodine cell surface labeling or metabolic radiolabel- ing with sodium [ 35 S]sulfate, we identified  5  1 integrin as the only sulfated integrin among  1 integrin het- erodimers expressed by the human melanoma cell line Mel-85. This facultative sulfation was confirmed not only by immunoprecipitation reactions using specific monoclonal antibodies but also by fibronectin affinity chromatography, two-dimensional electrophoresis, and chemical reduction. The covalent nature of  5  1 integrin sulfation was evidenced by its resistance to treatments with high ionic, chaotrophic, and denaturing agents such as 4 M NaCl, 4 M MgCl 2 ,8 M urea, and 6 M guanidine HCl. Based on deglycosylation procedures as chemical -elimination, proteinase K digestion, and susceptibility to glycosaminoglycan lyases (chondroitinase ABC and heparitinases I and II), it was demonstrated that the  5  1 heterodimer and  5 and  1 integrin subunits were proteoglycans. The importance of  5  1 sulfation was strengthened by the finding that this molecule is also sulfated in MG-63 (human osteosarcoma) and HCT-8 (hu- man colon adenocarcinoma) cells. Proteoglycans are complex molecules formed by a core pro- tein to which one or more glycosaminoglycan (GAG) 1 chains are linked. This basic definition, although true, hides the molecu- lar complexity shown by these molecules. They encompass an exceptionally large range of structures involving different core proteins, different classes of GAGs, and different numbers and lengths of individual GAG chains. Other post-translation mod- ifications such as N- and O-glycosylation increase the complex- ity of these molecules (for review see Refs. 1 and 2). The biological functions of proteoglycans are numerous. They have been involved in several biological effects (1, 3–5), such as extracellular matrix (ECM) assembly (6) and cell surface-ECM receptors for growth factors and hormones (2, 5, 7) or have had a role in biological processes such as cell-cell recognition (8) and control of cell growth (9). The fact that several ECM proteins, such as fibronectin (10), laminin (11), thrombospondin (12), vitronectin (13), type IV collagen (14), and tenascin (15), have GAG binding sites adds credence to the postulated multiple roles of proteoglycans. Supporting the idea of proteoglycans as ECM receptors, syndecan type I binds fibronectin, throm- bospondin, collagens (5), and tenascin (16); the heparan sulfate proteoglycan of Schwann cells binds laminin (17); a cell surface chondroitin sulfate proteoglycan is apparently involved in cell adhesion to laminin (18); and a cell surface phosphatidyl inos- itol-anchored heparan sulfate proteoglycan mediates mela- noma cell adhesion to fibronectin (19). Strong corroboration for these proteoglycan-ECM interactions comes from the presence of a heparan sulfate proteoglycan that co-localizes with  1 integrins as a widespread component of focal adhesion (20). Among the several ECM molecules that bind proteoglycans, the role of fibronectin should be emphasized not only because of its GAG binding domains but also because of the adhesive properties conferred to this molecule by these domains together with the RGD cell-binding fragment (21–23). Cells devoid of proteoglycans or bearing proteoglycans with altered GAG chains have a reduced capacity of adhesion to fibronectin and have a defective focal adhesion plaque formation in response to this molecule (24, 25). The best studied receptors for fibronectin that bear adhesive- ness and focal adhesion plaque formation are integrins that are / heterodimers widely expressed by almost all animal cells (26, 27). Integrins represent good examples of how post-trans- lational modifications can alter the structure of a molecule, thus modulating its biological activity. Integrin glycosylations represent a kind of regulation by which a wide variety of these receptors have their specificity and affinity modulated in sev- eral cell lines (28 –30). However, the versatility of cells to mod- ulate the binding properties of integrins is not restricted to glycosylation. Integrin functions can be modulated by acylation of membrane lipid (31), by divalent metal binding (32), and, for the cytoplasmic domain, by tyrosine phosphorylation, which is the best understood example of this type of biological modifi- cation, especially in leukocytes and platelets (27, 33). In the present study, we characterize  5  1 integrin as a part-time proteoglycan containing both heparan and chon- * This work was supported by grants from Conselho Nacional de Pesquisas and Fundac ¸a ˜ o de Amparo a ` Pesquisa do estado de Sa ˜ o Paulo. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ¶ To whom correspondence should be addressed: Ludwig Inst. for Cancer Research, R. Prof. Antonio Prudente, 109, 4 A, cep: 01509-010, Sa ˜ o Paulo, SP, Brazil. 1 The abbreviations used are: GAG, glycosaminoglycan chains; RGD, peptide Arg-Gly-Asp; PAGE, polyacrylamide gel electrophoresis; ECM, extracellular matrix. THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 272, No. 19, Issue of May 9, pp. 12529 –12535, 1997 © 1997 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. This paper is available on line at http://www-jbc.stanford.edu/jbc/ 12529 by guest on April 19, 2016 http://www.jbc.org/ Downloaded from