Skip to main content
PMC home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1990 Aug 1;111(2):699–708. doi: 10.1083/jcb.111.2.699

Inhibition of binding of fibronectin to matrix assembly sites by anti- integrin (alpha 5 beta 1) antibodies

PMCID: PMC2116209  PMID: 2380248

Abstract

Fibroblasts have cell surface sites that mediate assembly of plasma and cellular fibronectin into the extracellular matrix. Cell adhesion to fibronectin can be mediated by the interaction of an integrin (alpha 5 beta 1) with the Arg-Gly-Asp-Ser (RGDS)-containing cell adhesion region of fibronectin. We have attempted to elucidate the role of the alpha 5 beta 1 fibronectin receptor in assembly of fibronectin in matrices. Rat monoclonal antibody mAb 13, which recognizes the integrin beta 1 subunit, completely blocked binding and matrix assembly of 125I- fibronectin as well as binding of the 125I-70-kD amino-terminal fragment of fibronectin (70 kD) to fibroblast cell layers. Fab fragments of the anti-beta 1 antibody were also inhibitory. Antibody mAb 16, which recognizes the integrin alpha 5 subunit, partially blocked binding of 125I-fibronectin and 125I-70-kD. When cell layers were coincubated with fluoresceinated fibronectin and either anti-beta 1 or anti-alpha 5, anti-beta 1 was a more effective inhibitor than anti- alpha 5 of binding of labeled fibronectin to the cell layer. Inhibition of 125I-fibronectin binding by anti-beta 1 IgG occurred within 20 min. Inhibition of 125I-fibronectin binding by anti-beta 1 Fab fragments or IgG could not be overcome with increasing concentrations of fibronectin, suggesting that anti-beta 1 and exogenous fibronectin may not compete for the same binding site. No beta 1-containing integrin bound to immobilized 70 kD. These data indicate that the beta 1 subunit plays an important role in binding and assembly of exogenous fibronectin, perhaps by participation in the organization, regeneration, or cycling of the assembly site rather than by a direct interaction with fibronectin.

Full Text

The Full Text of this article is available as a PDF (1.9 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Akiyama S. K., Yamada K. M. The interaction of plasma fibronectin with fibroblastic cells in suspension. J Biol Chem. 1985 Apr 10;260(7):4492–4500. [PubMed] [Google Scholar]
  2. Akiyama S. K., Yamada S. S., Chen W. T., Yamada K. M. Analysis of fibronectin receptor function with monoclonal antibodies: roles in cell adhesion, migration, matrix assembly, and cytoskeletal organization. J Cell Biol. 1989 Aug;109(2):863–875. doi: 10.1083/jcb.109.2.863. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Allen-Hoffmann B. L., Crankshaw C. L., Mosher D. F. Transforming growth factor beta increases cell surface binding and assembly of exogenous (plasma) fibronectin by normal human fibroblasts. Mol Cell Biol. 1988 Oct;8(10):4234–4242. doi: 10.1128/mcb.8.10.4234. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Allen-Hoffmann B. L., Mosher D. F. Matrix assembly sites for exogenous fibronectin are decreased on human fibroblasts after treatment with agents which increase intracellular cAMP. J Biol Chem. 1987 Oct 15;262(29):14361–14365. [PubMed] [Google Scholar]
  5. Allio A. E., McKeown-Longo P. J. Extracellular matrix assembly of cell-derived and plasma-derived fibronectins by substrate-attached fibroblasts. J Cell Physiol. 1988 Jun;135(3):459–466. doi: 10.1002/jcp.1041350313. [DOI] [PubMed] [Google Scholar]
  6. Argraves W. S., Dickerson K., Burgess W. H., Ruoslahti E. Fibulin, a novel protein that interacts with the fibronectin receptor beta subunit cytoplasmic domain. Cell. 1989 Aug 25;58(4):623–629. doi: 10.1016/0092-8674(89)90097-4. [DOI] [PubMed] [Google Scholar]
  7. Barry E. L., Mosher D. F. Factor XIII cross-linking of fibronectin at cellular matrix assembly sites. J Biol Chem. 1988 Jul 25;263(21):10464–10469. [PubMed] [Google Scholar]
  8. Barry E. L., Mosher D. F. Factor XIIIa-mediated cross-linking of fibronectin in fibroblast cell layers. Cross-linking of cellular and plasma fibronectin and of amino-terminal fibronectin fragments. J Biol Chem. 1989 Mar 5;264(7):4179–4185. [PubMed] [Google Scholar]
  9. Bretscher M. S. Endocytosis and recycling of the fibronectin receptor in CHO cells. EMBO J. 1989 May;8(5):1341–1348. doi: 10.1002/j.1460-2075.1989.tb03514.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Cheresh D. A., Harper J. R. Arg-Gly-Asp recognition by a cell adhesion receptor requires its 130-kDa alpha subunit. J Biol Chem. 1987 Feb 5;262(4):1434–1437. [PubMed] [Google Scholar]
  11. Cheresh D. A., Smith J. W., Cooper H. M., Quaranta V. A novel vitronectin receptor integrin (alpha v beta x) is responsible for distinct adhesive properties of carcinoma cells. Cell. 1989 Apr 7;57(1):59–69. doi: 10.1016/0092-8674(89)90172-4. [DOI] [PubMed] [Google Scholar]
  12. Cheresh D. A., Spiro R. C. Biosynthetic and functional properties of an Arg-Gly-Asp-directed receptor involved in human melanoma cell attachment to vitronectin, fibrinogen, and von Willebrand factor. J Biol Chem. 1987 Dec 25;262(36):17703–17711. [PubMed] [Google Scholar]
  13. Choi M. G., Hynes R. O. Biosynthesis and processing of fibronectin in NIL.8 hamster cells. J Biol Chem. 1979 Dec 10;254(23):12050–12055. [PubMed] [Google Scholar]
  14. Cuatrecasas P. Protein purification by affinity chromatography. Derivatizations of agarose and polyacrylamide beads. J Biol Chem. 1970 Jun;245(12):3059–3065. [PubMed] [Google Scholar]
  15. Ehrismann R., Roth D. E., Eppenberger H. M., Turner D. C. Arrangement of attachment-promoting, self-association, and heparin-binding sites in horse serum fibronectin. J Biol Chem. 1982 Jul 10;257(13):7381–7387. [PubMed] [Google Scholar]
  16. Hemler M. E., Huang C., Schwarz L. The VLA protein family. Characterization of five distinct cell surface heterodimers each with a common 130,000 molecular weight beta subunit. J Biol Chem. 1987 Mar 5;262(7):3300–3309. [PubMed] [Google Scholar]
  17. Horwitz A., Duggan K., Buck C., Beckerle M. C., Burridge K. Interaction of plasma membrane fibronectin receptor with talin--a transmembrane linkage. Nature. 1986 Apr 10;320(6062):531–533. doi: 10.1038/320531a0. [DOI] [PubMed] [Google Scholar]
  18. Hynes R. O., Destree A. Extensive disulfide bonding at the mammalian cell surface. Proc Natl Acad Sci U S A. 1977 Jul;74(7):2855–2859. doi: 10.1073/pnas.74.7.2855. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Hynes R. O. Integrins: a family of cell surface receptors. Cell. 1987 Feb 27;48(4):549–554. doi: 10.1016/0092-8674(87)90233-9. [DOI] [PubMed] [Google Scholar]
  20. Hynes R. O., Marcantonio E. E., Stepp M. A., Urry L. A., Yee G. H. Integrin heterodimer and receptor complexity in avian and mammalian cells. J Cell Biol. 1989 Jul;109(1):409–420. doi: 10.1083/jcb.109.1.409. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kantor R. R., Mattes M. J., Lloyd K. O., Old L. J., Albino A. P. Biochemical analysis of two cell surface glycoprotein complexes, very common antigen 1 and very common antigen 2. Relationship to very late activation T cell antigens. J Biol Chem. 1987 Nov 5;262(31):15158–15165. [PubMed] [Google Scholar]
  22. Keski-Oja J., Mosher D. F., Vaheri A. Dimeric character of fibronectin, a major cell surface-associated glycoprotein. Biochem Biophys Res Commun. 1977 Jan 24;74(2):699–706. doi: 10.1016/0006-291x(77)90359-x. [DOI] [PubMed] [Google Scholar]
  23. Kessler S. W. Use of protein A-bearing staphylococci for the immunoprecipitation and isolation of antigens from cells. Methods Enzymol. 1981;73(Pt B):442–459. doi: 10.1016/0076-6879(81)73084-2. [DOI] [PubMed] [Google Scholar]
  24. Kramer R. H., Marks N. Identification of integrin collagen receptors on human melanoma cells. J Biol Chem. 1989 Mar 15;264(8):4684–4688. [PubMed] [Google Scholar]
  25. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  26. Lebien T. W., Boué D. R., Bradley J. G., Kersey J. H. Antibody affinity may influence antigenic modulation of the common acute lymphoblastic leukemia antigen in vitro. J Immunol. 1982 Nov;129(5):2287–2292. [PubMed] [Google Scholar]
  27. Matsuyama T., Yamada A., Kay J., Yamada K. M., Akiyama S. K., Schlossman S. F., Morimoto C. Activation of CD4 cells by fibronectin and anti-CD3 antibody. A synergistic effect mediated by the VLA-5 fibronectin receptor complex. J Exp Med. 1989 Oct 1;170(4):1133–1148. doi: 10.1084/jem.170.4.1133. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. McDonald J. A., Quade B. J., Broekelmann T. J., LaChance R., Forsman K., Hasegawa E., Akiyama S. Fibronectin's cell-adhesive domain and an amino-terminal matrix assembly domain participate in its assembly into fibroblast pericellular matrix. J Biol Chem. 1987 Mar 5;262(7):2957–2967. [PubMed] [Google Scholar]
  29. McKeown-Longo P. J., Etzler C. A. Induction of fibronectin matrix assembly in human fibrosarcoma cells by dexamethasone. J Cell Biol. 1987 Mar;104(3):601–610. doi: 10.1083/jcb.104.3.601. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. McKeown-Longo P. J., Mosher D. F. Binding of plasma fibronectin to cell layers of human skin fibroblasts. J Cell Biol. 1983 Aug;97(2):466–472. doi: 10.1083/jcb.97.2.466. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. McKeown-Longo P. J., Mosher D. F. Interaction of the 70,000-mol-wt amino-terminal fragment of fibronectin with the matrix-assembly receptor of fibroblasts. J Cell Biol. 1985 Feb;100(2):364–374. doi: 10.1083/jcb.100.2.364. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. McKeown-Longo P. J., Mosher D. F. Mechanism of formation of disulfide-bonded multimers of plasma fibronectin in cell layers of cultured human fibroblasts. J Biol Chem. 1984 Oct 10;259(19):12210–12215. [PubMed] [Google Scholar]
  33. Mosher D. F. Fibronectin. Prog Hemost Thromb. 1980;5:111–151. [PubMed] [Google Scholar]
  34. Mosher D. F., Johnson R. B. In vitro formation of disulfide-bonded fibronectin multimers. J Biol Chem. 1983 May 25;258(10):6595–6601. [PubMed] [Google Scholar]
  35. Mosher D. F., McKeown-Longo P. J. Assembly of fibronectin-containing extracellular matrix: a glimpse of the machinery. Biopolymers. 1985 Jan;24(1):199–210. doi: 10.1002/bip.360240116. [DOI] [PubMed] [Google Scholar]
  36. Mosher D. F. Physiology of fibronectin. Annu Rev Med. 1984;35:561–575. doi: 10.1146/annurev.me.35.020184.003021. [DOI] [PubMed] [Google Scholar]
  37. Oh E., Pierschbacher M., Ruoslahti E. Deposition of plasma fibronectin in tissues. Proc Natl Acad Sci U S A. 1981 May;78(5):3218–3221. doi: 10.1073/pnas.78.5.3218. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Peters D. M., Mosher D. F. Localization of cell surface sites involved in fibronectin fibrillogenesis. J Cell Biol. 1987 Jan;104(1):121–130. doi: 10.1083/jcb.104.1.121. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Pierschbacher M. D., Hayman E. G., Ruoslahti E. Location of the cell-attachment site in fibronectin with monoclonal antibodies and proteolytic fragments of the molecule. Cell. 1981 Oct;26(2 Pt 2):259–267. doi: 10.1016/0092-8674(81)90308-1. [DOI] [PubMed] [Google Scholar]
  40. Pierschbacher M. D., Ruoslahti E. Cell attachment activity of fibronectin can be duplicated by small synthetic fragments of the molecule. Nature. 1984 May 3;309(5963):30–33. doi: 10.1038/309030a0. [DOI] [PubMed] [Google Scholar]
  41. Plantefaber L. C., Hynes R. O. Changes in integrin receptors on oncogenically transformed cells. Cell. 1989 Jan 27;56(2):281–290. doi: 10.1016/0092-8674(89)90902-1. [DOI] [PubMed] [Google Scholar]
  42. Plow E. F., Ginsberg M. H. Specific and saturable binding of plasma fibronectin to thrombin-stimulated human platelets. J Biol Chem. 1981 Sep 25;256(18):9477–9482. [PubMed] [Google Scholar]
  43. Pytela R., Pierschbacher M. D., Argraves S., Suzuki S., Ruoslahti E. Arginine-glycine-aspartic acid adhesion receptors. Methods Enzymol. 1987;144:475–489. doi: 10.1016/0076-6879(87)44196-7. [DOI] [PubMed] [Google Scholar]
  44. Pytela R., Pierschbacher M. D., Ruoslahti E. Identification and isolation of a 140 kd cell surface glycoprotein with properties expected of a fibronectin receptor. Cell. 1985 Jan;40(1):191–198. doi: 10.1016/0092-8674(85)90322-8. [DOI] [PubMed] [Google Scholar]
  45. Quade B. J., McDonald J. A. Fibronectin's amino-terminal matrix assembly site is located within the 29-kDa amino-terminal domain containing five type I repeats. J Biol Chem. 1988 Dec 25;263(36):19602–19609. [PubMed] [Google Scholar]
  46. Roman J., LaChance R. M., Broekelmann T. J., Kennedy C. J., Wayner E. A., Carter W. G., McDonald J. A. The fibronectin receptor is organized by extracellular matrix fibronectin: implications for oncogenic transformation and for cell recognition of fibronectin matrices. J Cell Biol. 1989 Jun;108(6):2529–2543. doi: 10.1083/jcb.108.6.2529. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Ruoslahti E. Fibronectin and its receptors. Annu Rev Biochem. 1988;57:375–413. doi: 10.1146/annurev.bi.57.070188.002111. [DOI] [PubMed] [Google Scholar]
  48. Santoro S. A., Lawing W. J., Jr Competition for related but nonidentical binding sites on the glycoprotein IIb-IIIa complex by peptides derived from platelet adhesive proteins. Cell. 1987 Mar 13;48(5):867–873. doi: 10.1016/0092-8674(87)90083-3. [DOI] [PubMed] [Google Scholar]
  49. Schwarzbauer J. E., Mulligan R. C., Hynes R. O. Efficient and stable expression of recombinant fibronectin polypeptides. Proc Natl Acad Sci U S A. 1987 Feb;84(3):754–758. doi: 10.1073/pnas.84.3.754. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Sekiguchi K., Klos A. M., Hirohashi S., Hakomori S. Human tissue fibronectin: expression of different isotypes in the adult and fetal tissues. Biochem Biophys Res Commun. 1986 Dec 30;141(3):1012–1017. doi: 10.1016/s0006-291x(86)80145-0. [DOI] [PubMed] [Google Scholar]
  51. Singer I. I. The fibronexus: a transmembrane association of fibronectin-containing fibers and bundles of 5 nm microfilaments in hamster and human fibroblasts. Cell. 1979 Mar;16(3):675–685. doi: 10.1016/0092-8674(79)90040-0. [DOI] [PubMed] [Google Scholar]
  52. Takada Y., Huang C., Hemler M. E. Fibronectin receptor structures in the VLA family of heterodimers. Nature. 1987 Apr 9;326(6113):607–609. doi: 10.1038/326607a0. [DOI] [PubMed] [Google Scholar]
  53. Takada Y., Wayner E. A., Carter W. G., Hemler M. E. Extracellular matrix receptors, ECMRII and ECMRI, for collagen and fibronectin correspond to VLA-2 and VLA-3 in the VLA family of heterodimers. J Cell Biochem. 1988 Aug;37(4):385–393. doi: 10.1002/jcb.240370406. [DOI] [PubMed] [Google Scholar]
  54. Tamkun J. W., DeSimone D. W., Fonda D., Patel R. S., Buck C., Horwitz A. F., Hynes R. O. Structure of integrin, a glycoprotein involved in the transmembrane linkage between fibronectin and actin. Cell. 1986 Jul 18;46(2):271–282. doi: 10.1016/0092-8674(86)90744-0. [DOI] [PubMed] [Google Scholar]
  55. Wayner E. A., Carter W. G. Identification of multiple cell adhesion receptors for collagen and fibronectin in human fibrosarcoma cells possessing unique alpha and common beta subunits. J Cell Biol. 1987 Oct;105(4):1873–1884. doi: 10.1083/jcb.105.4.1873. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Wayner E. A., Carter W. G., Piotrowicz R. S., Kunicki T. J. The function of multiple extracellular matrix receptors in mediating cell adhesion to extracellular matrix: preparation of monoclonal antibodies to the fibronectin receptor that specifically inhibit cell adhesion to fibronectin and react with platelet glycoproteins Ic-IIa. J Cell Biol. 1988 Nov;107(5):1881–1891. doi: 10.1083/jcb.107.5.1881. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Wayner E. A., Garcia-Pardo A., Humphries M. J., McDonald J. A., Carter W. G. Identification and characterization of the T lymphocyte adhesion receptor for an alternative cell attachment domain (CS-1) in plasma fibronectin. J Cell Biol. 1989 Sep;109(3):1321–1330. doi: 10.1083/jcb.109.3.1321. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES