Skip to main content
PMC home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1988 Jun 1;106(6):2171–2182. doi: 10.1083/jcb.106.6.2171

Cell surface distribution of fibronectin and vitronectin receptors depends on substrate composition and extracellular matrix accumulation

PMCID: PMC2115138  PMID: 2454933

Abstract

We used antibodies against the alpha subunits of the human fibronectin receptor (FNR) and vitronectin receptor (VNR) to localize simultaneously FNR and VNR at major substrate adhesion sites of fibroblasts and melanoma cells with double-label immunofluorescence microscopy. In early (2-6-h) serum-containing cultures, both FNR and VNR coaccumulated in focal contacts detected by interference reflection microscopy. Under higher resolution immunoscanning electron microscopy, FNR and VNR were also observed to be distributed randomly on the dorsal cell surface. As fibronectin-containing extracellular matrix fibers accumulated beneath the cells at 24 h, FNR became concentrated at contacts with these fibers and was no longer detected at focal contacts. VNR was not observed at matrix contacts but remained strikingly localized in focal contacts of the 24-h cells. Since focal contacts represent the sites of strongest cell-to-substrate adhesion, these results suggest that FNR and VNR together play critical roles in the maintenance of stable contacts between the cell and its substrate. In addition, the accumulation of FNR at extracellular matrix contacts implies that this receptor might also function in the process of cellular migration along fibronectin-containing matrix cables. To define the factors governing accumulation of FNR and VNR at focal contacts, fibroblasts in serum-free media were plated on substrates coated with purified ligands. Fibronectin-coated surfaces fostered accumulation of FNR but not VNR at focal contacts. On vitronectin- coated surfaces, or substrata derivatized with a tridecapeptide containing the cell attachment sequence Arg-Gly-Asp, both FNR and VNR became concentrated at focal contacts. These observations suggest that the availability of ligand is critical to the accumulation of FNR and VNR at focal contacts, and that FNR might also recognize substrate- bound vitronectin.

Full Text

The Full Text of this article is available as a PDF (5.6 MB).

Selected References

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

  1. Akiyama S. K., Hasegawa E., Hasegawa T., Yamada K. M. The interaction of fibronectin fragments with fibroblastic cells. J Biol Chem. 1985 Oct 25;260(24):13256–13260. [PubMed] [Google Scholar]
  2. Akiyama S. K., Yamada S. S., Yamada K. M. Characterization of a 140-kD avian cell surface antigen as a fibronectin-binding molecule. J Cell Biol. 1986 Feb;102(2):442–448. doi: 10.1083/jcb.102.2.442. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Argraves W. S., Pytela R., Suzuki S., Millán J. L., Pierschbacher M. D., Ruoslahti E. cDNA sequences from the alpha subunit of the fibronectin receptor predict a transmembrane domain and a short cytoplasmic peptide. J Biol Chem. 1986 Oct 5;261(28):12922–12924. [PubMed] [Google Scholar]
  4. Bronner-Fraser M. Alterations in neural crest migration by a monoclonal antibody that affects cell adhesion. J Cell Biol. 1985 Aug;101(2):610–617. doi: 10.1083/jcb.101.2.610. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Buck C. A., Shea E., Duggan K., Horwitz A. F. Integrin (the CSAT antigen): functionality requires oligomeric integrity. J Cell Biol. 1986 Dec;103(6 Pt 1):2421–2428. doi: 10.1083/jcb.103.6.2421. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Burridge K., Connell L. A new protein of adhesion plaques and ruffling membranes. J Cell Biol. 1983 Aug;97(2):359–367. doi: 10.1083/jcb.97.2.359. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Chen W. T., Hasegawa E., Hasegawa T., Weinstock C., Yamada K. M. Development of cell surface linkage complexes in cultured fibroblasts. J Cell Biol. 1985 Apr;100(4):1103–1114. doi: 10.1083/jcb.100.4.1103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Chen W. T., Singer S. J. Immunoelectron microscopic studies of the sites of cell-substratum and cell-cell contacts in cultured fibroblasts. J Cell Biol. 1982 Oct;95(1):205–222. doi: 10.1083/jcb.95.1.205. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Chen W. T., Wang J., Hasegawa T., Yamada S. S., Yamada K. M. Regulation of fibronectin receptor distribution by transformation, exogenous fibronectin, and synthetic peptides. J Cell Biol. 1986 Nov;103(5):1649–1661. doi: 10.1083/jcb.103.5.1649. [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. Human endothelial cells synthesize and express an Arg-Gly-Asp-directed adhesion receptor involved in attachment to fibrinogen and von Willebrand factor. Proc Natl Acad Sci U S A. 1987 Sep;84(18):6471–6475. doi: 10.1073/pnas.84.18.6471. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Cheresh D. A., Pytela R., Pierschbacher M. D., Klier F. G., Ruoslahti E., Reisfeld R. A. An Arg-Gly-Asp-directed receptor on the surface of human melanoma cells exists in an divalent cation-dependent functional complex with the disialoganglioside GD2. J Cell Biol. 1987 Sep;105(3):1163–1173. doi: 10.1083/jcb.105.3.1163. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Dahlbäck K., Löfberg H., Dahlbäck B. Localization of vitronectin (S-protein of complement) in normal human skin. Acta Derm Venereol. 1986;66(6):461–467. [PubMed] [Google Scholar]
  14. Damsky C. H., Knudsen K. A., Bradley D., Buck C. A., Horwitz A. F. Distribution of the cell substratum attachment (CSAT) antigen on myogenic and fibroblastic cells in culture. J Cell Biol. 1985 May;100(5):1528–1539. doi: 10.1083/jcb.100.5.1528. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Duband J. L., Rocher S., Chen W. T., Yamada K. M., Thiery J. P. Cell adhesion and migration in the early vertebrate embryo: location and possible role of the putative fibronectin receptor complex. J Cell Biol. 1986 Jan;102(1):160–178. doi: 10.1083/jcb.102.1.160. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Engvall E., Ruoslahti E. Binding of soluble form of fibroblast surface protein, fibronectin, to collagen. Int J Cancer. 1977 Jul 15;20(1):1–5. doi: 10.1002/ijc.2910200102. [DOI] [PubMed] [Google Scholar]
  17. Fitzgerald L. A., Steiner B., Rall S. C., Jr, Lo S. S., Phillips D. R. Protein sequence of endothelial glycoprotein IIIa derived from a cDNA clone. Identity with platelet glycoprotein IIIa and similarity to "integrin". J Biol Chem. 1987 Mar 25;262(9):3936–3939. [PubMed] [Google Scholar]
  18. Geiger B. A 130K protein from chicken gizzard: its localization at the termini of microfilament bundles in cultured chicken cells. Cell. 1979 Sep;18(1):193–205. doi: 10.1016/0092-8674(79)90368-4. [DOI] [PubMed] [Google Scholar]
  19. Giancotti F. G., Comoglio P. M., Tarone G. A 135,000 molecular weight plasma membrane glycoprotein involved in fibronectin-mediated cell adhesion. Immunofluorescence localization in normal and RSV-transformed fibroblasts. Exp Cell Res. 1986 Mar;163(1):47–62. doi: 10.1016/0014-4827(86)90557-4. [DOI] [PubMed] [Google Scholar]
  20. Ginsberg M. H., Loftus J., Ryckwaert J. J., Pierschbacher M., Pytela R., Ruoslahti E., Plow E. F. Immunochemical and amino-terminal sequence comparison of two cytoadhesins indicates they contain similar or identical beta subunits and distinct alpha subunits. J Biol Chem. 1987 Apr 25;262(12):5437–5440. [PubMed] [Google Scholar]
  21. Grinnell F. Focal adhesion sites and the removal of substratum-bound fibronectin. J Cell Biol. 1986 Dec;103(6 Pt 2):2697–2706. doi: 10.1083/jcb.103.6.2697. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Hall D. E., Neugebauer K. M., Reichardt L. F. Embryonic neural retinal cell response to extracellular matrix proteins: developmental changes and effects of the cell substratum attachment antibody (CSAT). J Cell Biol. 1987 Mar;104(3):623–634. doi: 10.1083/jcb.104.3.623. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Hayman E. G., Pierschbacher M. D., Ohgren Y., Ruoslahti E. Serum spreading factor (vitronectin) is present at the cell surface and in tissues. Proc Natl Acad Sci U S A. 1983 Jul;80(13):4003–4007. doi: 10.1073/pnas.80.13.4003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Hayman E. G., Pierschbacher M. D., Suzuki S., Ruoslahti E. Vitronectin--a major cell attachment-promoting protein in fetal bovine serum. Exp Cell Res. 1985 Oct;160(2):245–258. doi: 10.1016/0014-4827(85)90173-9. [DOI] [PubMed] [Google Scholar]
  25. 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]
  26. Horwitz A., Duggan K., Greggs R., Decker C., Buck C. The cell substrate attachment (CSAT) antigen has properties of a receptor for laminin and fibronectin. J Cell Biol. 1985 Dec;101(6):2134–2144. doi: 10.1083/jcb.101.6.2134. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. 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]
  28. Hynes R. O., Yamada K. M. Fibronectins: multifunctional modular glycoproteins. J Cell Biol. 1982 Nov;95(2 Pt 1):369–377. doi: 10.1083/jcb.95.2.369. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Izzard C. S., Lochner L. R. Formation of cell-to-substrate contacts during fibroblast motility: an interference-reflexion study. J Cell Sci. 1980 Apr;42:81–116. doi: 10.1242/jcs.42.1.81. [DOI] [PubMed] [Google Scholar]
  30. Johnson D. A., Elder J. H. Antibody directed to determinants of a Moloney virus derived MCF GP70 recognizes a thymic differentiation antigen. J Exp Med. 1983 Nov 1;158(5):1751–1756. doi: 10.1084/jem.158.5.1751. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Kishimoto T. K., O'Connor K., Lee A., Roberts T. M., Springer T. A. Cloning of the beta subunit of the leukocyte adhesion proteins: homology to an extracellular matrix receptor defines a novel supergene family. Cell. 1987 Feb 27;48(4):681–690. doi: 10.1016/0092-8674(87)90246-7. [DOI] [PubMed] [Google Scholar]
  32. Manigley C., Roth J. Applications of immunocolloids in light microscopy. IV. Use of photochemical silver staining in a simple and efficient double-staining technique. J Histochem Cytochem. 1985 Dec;33(12):1247–1251. doi: 10.1177/33.12.2415576. [DOI] [PubMed] [Google Scholar]
  33. Pierschbacher M. D., Hayman E. G., Ruoslahti E. The cell attachment determinant in fibronectin. J Cell Biochem. 1985;28(2):115–126. doi: 10.1002/jcb.240280205. [DOI] [PubMed] [Google Scholar]
  34. 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]
  35. 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]
  36. Pytela R., Pierschbacher M. D., Ginsberg M. H., Plow E. F., Ruoslahti E. Platelet membrane glycoprotein IIb/IIIa: member of a family of Arg-Gly-Asp--specific adhesion receptors. Science. 1986 Mar 28;231(4745):1559–1562. doi: 10.1126/science.2420006. [DOI] [PubMed] [Google Scholar]
  37. Pytela R., Pierschbacher M. D., Ruoslahti E. A 125/115-kDa cell surface receptor specific for vitronectin interacts with the arginine-glycine-aspartic acid adhesion sequence derived from fibronectin. Proc Natl Acad Sci U S A. 1985 Sep;82(17):5766–5770. doi: 10.1073/pnas.82.17.5766. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. 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]
  39. Ruoslahti E., Pierschbacher M. D. New perspectives in cell adhesion: RGD and integrins. Science. 1987 Oct 23;238(4826):491–497. doi: 10.1126/science.2821619. [DOI] [PubMed] [Google Scholar]
  40. Singer I. I. Association of fibronectin and vinculin with focal contacts and stress fibers in stationary hamster fibroblasts. J Cell Biol. 1982 Feb;92(2):398–408. doi: 10.1083/jcb.92.2.398. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Singer I. I., Kawka D. W., Scott S., Mumford R. A., Lark M. W. The fibronectin cell attachment sequence Arg-Gly-Asp-Ser promotes focal contact formation during early fibroblast attachment and spreading. J Cell Biol. 1987 Mar;104(3):573–584. doi: 10.1083/jcb.104.3.573. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Singer I. I., Kazazis D. M., Kawka D. W., Rupp E. A., Bayne E. K. Extracellular matrix-cytoskeletal interactions in rheumatoid arthritis. I. Immunoelectron microscopic analysis of the fibronexus at the adhesive surface of normal porcine type B synoviocytes in vitro. Arthritis Rheum. 1985 Oct;28(10):1105–1116. doi: 10.1002/art.1780281005. [DOI] [PubMed] [Google Scholar]
  43. Singer I. I., Paradiso P. R. A transmembrane relationship between fibronectin and vinculin (130 kd protein): serum modulation in normal and transformed hamster fibroblasts. Cell. 1981 May;24(2):481–492. doi: 10.1016/0092-8674(81)90339-1. [DOI] [PubMed] [Google Scholar]
  44. Singer I. I., Scott S., Kawka D. W., Hassell J. R. Extracellular matrix fibers containing fibronectin and basement membrane heparan sulfate proteoglycan coalign with focal contacts and microfilament bundles in stationary fibroblasts. Exp Cell Res. 1987 Dec;173(2):558–571. doi: 10.1016/0014-4827(87)90295-3. [DOI] [PubMed] [Google Scholar]
  45. 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]
  46. Springer T. A., Dustin M. L., Kishimoto T. K., Marlin S. D. The lymphocyte function-associated LFA-1, CD2, and LFA-3 molecules: cell adhesion receptors of the immune system. Annu Rev Immunol. 1987;5:223–252. doi: 10.1146/annurev.iy.05.040187.001255. [DOI] [PubMed] [Google Scholar]
  47. Suzuki S., Argraves W. S., Pytela R., Arai H., Krusius T., Pierschbacher M. D., Ruoslahti E. cDNA and amino acid sequences of the cell adhesion protein receptor recognizing vitronectin reveal a transmembrane domain and homologies with other adhesion protein receptors. Proc Natl Acad Sci U S A. 1986 Nov;83(22):8614–8618. doi: 10.1073/pnas.83.22.8614. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Suzuki S., Oldberg A., Hayman E. G., Pierschbacher M. D., Ruoslahti E. Complete amino acid sequence of human vitronectin deduced from cDNA. Similarity of cell attachment sites in vitronectin and fibronectin. EMBO J. 1985 Oct;4(10):2519–2524. doi: 10.1002/j.1460-2075.1985.tb03965.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. 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]
  50. Takada Y., Strominger J. L., Hemler M. E. The very late antigen family of heterodimers is part of a superfamily of molecules involved in adhesion and embryogenesis. Proc Natl Acad Sci U S A. 1987 May;84(10):3239–3243. doi: 10.1073/pnas.84.10.3239. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. 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]

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

RESOURCES