Skip to main content
PMC home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1991 Apr 2;113(2):451–461. doi: 10.1083/jcb.113.2.451

Adhesive properties of the beta 3 integrins: comparison of GP IIb-IIIa and the vitronectin receptor individually expressed in human melanoma cells

PMCID: PMC2288943  PMID: 1707057

Abstract

Glycoprotein IIb-IIIa (alpha IIb beta 3) and the vitronectin receptor (alpha v beta 3), two integrins that share the common beta 3 subunit, have been reported to function as promiscuous receptors for the RGD- containing adhesive proteins fibrinogen, vitronectin, fibronectin, von Willebrand factor, and thrombospondin. The present study was designed to establish a cell system for the expression of either GP IIb-IIIa or the vitronectin receptor in an otherwise identical cellular environment and to compare the adhesive properties of these two integrins with those of native GP IIb-IIIa and the vitronectin receptor constitutively expressed in HEL cells or platelets. M21 human melanoma cells lack GP IIb-IIIa and use the vitronectin receptor to attach to vitronectin, fibrinogen, fibronectin, and von Willebrand factor. To study the functional properties of GP IIb-IIIa in these cells, we transfected GP IIb into M21-L cells, a variant of M21 cells (Cheresh, D.A., and R.C. Spiro. 1987. J. Biol. Chem. 262:17703-17711), which lack the expression of functional alpha v and are therefore unable to attach to vitronectin, fibrinogen, and von Willebrand factor. Transfectants expressing GP IIb were isolated by immunomagnetic beads and surface expression of the GP IIb-IIIa complex was documented by FACS analysis and immunoprecipitation experiments performed with 125I-labeled M21- L/GP IIb cells. Comparative functional studies demonstrated that GP IIb- IIIa expressed in M21-L/GPIIb cells as well as native GP IIb-IIIa constitutively expressed in HEL-5J20 cells (an HEL variant lacking alpha v beta 3) mediated cell attachment to immobilized fibrinogen, but not to vitronectin or von Willebrand factor, whereas the vitronectin receptor expressed in M21 cells and HEL-AD1 cells (an HEL variant expressing alpha v beta 3) mediated cell attachment to fibrinogen, vitronectin, and von Willebrand factor. Similarly, PGl2-treated resting platelets attached to immobilized fibrinogen but not to vitronectin or von Willebrand factor, and this attachment could be inhibited by mAb A2A9 (directed against a functional site on the GP IIb-IIIa complex). However, in contrast to platelets, which adhered to vitronectin and von Willebrand factor after stimulation by thrombin or PMA, activation of the protein kinase C pathway in M21-L/GP IIb or HEL cells did not induce cell adhesion to vitronectin or von Willebrand factor.(ABSTRACT TRUNCATED AT 400 WORDS)

Full Text

The Full Text of this article is available as a PDF (2.4 MB).

Selected References

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

  1. Bennett J. S., Hoxie J. A., Leitman S. F., Vilaire G., Cines D. B. Inhibition of fibrinogen binding to stimulated human platelets by a monoclonal antibody. Proc Natl Acad Sci U S A. 1983 May;80(9):2417–2421. doi: 10.1073/pnas.80.9.2417. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bodary S. C., Napier M. A., McLean J. W. Expression of recombinant platelet glycoprotein IIbIIIa results in a functional fibrinogen-binding complex. J Biol Chem. 1989 Nov 15;264(32):18859–18862. [PubMed] [Google Scholar]
  3. Bray P. F., Rosa J. P., Lingappa V. R., Kan Y. W., McEver R. P., Shuman M. A. Biogenesis of the platelet receptor for fibrinogen: evidence for separate precursors for glycoproteins IIb and IIIa. Proc Natl Acad Sci U S A. 1986 Mar;83(5):1480–1484. doi: 10.1073/pnas.83.5.1480. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Cheresh D. A., Berliner S. A., Vicente V., Ruggeri Z. M. Recognition of distinct adhesive sites on fibrinogen by related integrins on platelets and endothelial cells. Cell. 1989 Sep 8;58(5):945–953. doi: 10.1016/0092-8674(89)90946-x. [DOI] [PubMed] [Google Scholar]
  5. 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]
  6. Coller B. S. Interaction of normal, thrombasthenic, and Bernard-Soulier platelets with immobilized fibrinogen: defective platelet-fibrinogen interaction in thrombasthenia. Blood. 1980 Feb;55(2):169–178. [PubMed] [Google Scholar]
  7. Coller B. S., Seligsohn U., Little P. A. Type I Glanzmann thrombasthenia patients from the Iraqi-Jewish and Arab populations in Israel can be differentiated by platelet glycoprotein IIIa immunoblot analysis. Blood. 1987 Jun;69(6):1696–1703. [PubMed] [Google Scholar]
  8. Conforti G., Zanetti A., Pasquali-Ronchetti I., Quaglino D., Jr, Neyroz P., Dejana E. Modulation of vitronectin receptor binding by membrane lipid composition. J Biol Chem. 1990 Mar 5;265(7):4011–4019. [PubMed] [Google Scholar]
  9. D'Souza S. E., Ginsberg M. H., Burke T. A., Lam S. C., Plow E. F. Localization of an Arg-Gly-Asp recognition site within an integrin adhesion receptor. Science. 1988 Oct 7;242(4875):91–93. doi: 10.1126/science.3262922. [DOI] [PubMed] [Google Scholar]
  10. D'Souza S. E., Ginsberg M. H., Burke T. A., Plow E. F. The ligand binding site of the platelet integrin receptor GPIIb-IIIa is proximal to the second calcium binding domain of its alpha subunit. J Biol Chem. 1990 Feb 25;265(6):3440–3446. [PubMed] [Google Scholar]
  11. Doolittle R. F., Watt K. W., Cottrell B. A., Strong D. D., Riley M. The amino acid sequence of the alpha-chain of human fibrinogen. Nature. 1979 Aug 9;280(5722):464–468. doi: 10.1038/280464a0. [DOI] [PubMed] [Google Scholar]
  12. Duperray A., Berthier R., Chagnon E., Ryckewaert J. J., Ginsberg M., Plow E., Marguerie G. Biosynthesis and processing of platelet GPIIb-IIIa in human megakaryocytes. J Cell Biol. 1987 Jun;104(6):1665–1673. doi: 10.1083/jcb.104.6.1665. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Duperray A., Troesch A., Berthier R., Chagnon E., Frachet P., Uzan G., Marguerie G. Biosynthesis and assembly of platelet GPIIb-IIIa in human megakaryocytes: evidence that assembly between pro-GPIIb and GPIIIa is a prerequisite for expression of the complex on the cell surface. Blood. 1989 Oct;74(5):1603–1611. [PubMed] [Google Scholar]
  14. Elices M. J., Hemler M. E. The human integrin VLA-2 is a collagen receptor on some cells and a collagen/laminin receptor on others. Proc Natl Acad Sci U S A. 1989 Dec;86(24):9906–9910. doi: 10.1073/pnas.86.24.9906. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Engvall E., Ruoslahti E., Miller E. J. Affinity of fibronectin to collagens of different genetic types and to fibrinogen. J Exp Med. 1978 Jun 1;147(6):1584–1595. doi: 10.1084/jem.147.6.1584. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Felgner P. L., Gadek T. R., Holm M., Roman R., Chan H. W., Wenz M., Northrop J. P., Ringold G. M., Danielsen M. Lipofection: a highly efficient, lipid-mediated DNA-transfection procedure. Proc Natl Acad Sci U S A. 1987 Nov;84(21):7413–7417. doi: 10.1073/pnas.84.21.7413. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Fitzgerald L. A., Phillips D. R. Calcium regulation of the platelet membrane glycoprotein IIb-IIIa complex. J Biol Chem. 1985 Sep 15;260(20):11366–11374. [PubMed] [Google Scholar]
  18. Fitzgerald L. A., Poncz M., Steiner B., Rall S. C., Jr, Bennett J. S., Phillips D. R. Comparison of cDNA-derived protein sequences of the human fibronectin and vitronectin receptor alpha-subunits and platelet glycoprotein IIb. Biochemistry. 1987 Dec 15;26(25):8158–8165. doi: 10.1021/bi00399a021. [DOI] [PubMed] [Google Scholar]
  19. George J. N., Caen J. P., Nurden A. T. Glanzmann's thrombasthenia: the spectrum of clinical disease. Blood. 1990 Apr 1;75(7):1383–1395. [PubMed] [Google Scholar]
  20. Giltay J. C., Leeksma O. C., Breederveld C., van Mourik J. A. Normal synthesis and expression of endothelial IIb/IIIa in Glanzmann's thrombasthenia. Blood. 1987 Mar;69(3):809–812. [PubMed] [Google Scholar]
  21. Gluzman Y. SV40-transformed simian cells support the replication of early SV40 mutants. Cell. 1981 Jan;23(1):175–182. doi: 10.1016/0092-8674(81)90282-8. [DOI] [PubMed] [Google Scholar]
  22. Haverstick D. M., Cowan J. F., Yamada K. M., Santoro S. A. Inhibition of platelet adhesion to fibronectin, fibrinogen, and von Willebrand factor substrates by a synthetic tetrapeptide derived from the cell-binding domain of fibronectin. Blood. 1985 Oct;66(4):946–952. [PubMed] [Google Scholar]
  23. Hawiger J., Timmons S., Kloczewiak M., Strong D. D., Doolittle R. F. gamma and alpha chains of human fibrinogen possess sites reactive with human platelet receptors. Proc Natl Acad Sci U S A. 1982 Mar;79(6):2068–2071. doi: 10.1073/pnas.79.6.2068. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Hemler M. E., Crouse C., Takada Y., Sonnenberg A. Multiple very late antigen (VLA) heterodimers on platelets. Evidence for distinct VLA-2, VLA-5 (fibronectin receptor), and VLA-6 structures. J Biol Chem. 1988 Jun 5;263(16):7660–7665. [PubMed] [Google Scholar]
  25. 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]
  26. KAZAL L. A., AMSEL S., MILLER O. P., TOCANTINS L. M. THE PREPARATION AND SOME PROPERTIES OF FIBRINOGEN PRECIPITATED FROM HUMAN PLASMA BY GLYCINE. Proc Soc Exp Biol Med. 1963 Aug-Sep;113:989–994. doi: 10.3181/00379727-113-28553. [DOI] [PubMed] [Google Scholar]
  27. Kieffer N., Debili N., Wicki A., Titeux M., Henri A., Mishal Z., Breton-Gorius J., Vainchenker W., Clemetson K. J. Expression of platelet glycoprotein Ib alpha in HEL cells. J Biol Chem. 1986 Dec 5;261(34):15854–15862. [PubMed] [Google Scholar]
  28. Kirchhofer D., Languino L. R., Ruoslahti E., Pierschbacher M. D. Alpha 2 beta 1 integrins from different cell types show different binding specificities. J Biol Chem. 1990 Jan 15;265(2):615–618. [PubMed] [Google Scholar]
  29. Kloczewiak M., Timmons S., Lukas T. J., Hawiger J. Platelet receptor recognition site on human fibrinogen. Synthesis and structure-function relationship of peptides corresponding to the carboxy-terminal segment of the gamma chain. Biochemistry. 1984 Apr 10;23(8):1767–1774. doi: 10.1021/bi00303a028. [DOI] [PubMed] [Google Scholar]
  30. Kunicki T. J., Nugent D. J., Staats S. J., Orchekowski R. P., Wayner E. A., Carter W. G. The human fibroblast class II extracellular matrix receptor mediates platelet adhesion to collagen and is identical to the platelet glycoprotein Ia-IIa complex. J Biol Chem. 1988 Apr 5;263(10):4516–4519. [PubMed] [Google Scholar]
  31. Kunkel T. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci U S A. 1985 Jan;82(2):488–492. doi: 10.1073/pnas.82.2.488. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Lam S. C., Plow E. F., D'Souza S. E., Cheresh D. A., Frelinger A. L., 3rd, Ginsberg M. H. Isolation and characterization of a platelet membrane protein related to the vitronectin receptor. J Biol Chem. 1989 Mar 5;264(7):3742–3749. [PubMed] [Google Scholar]
  33. Lam S. C., Plow E. F., Smith M. A., Andrieux A., Ryckwaert J. J., Marguerie G., Ginsberg M. H. Evidence that arginyl-glycyl-aspartate peptides and fibrinogen gamma chain peptides share a common binding site on platelets. J Biol Chem. 1987 Jan 25;262(3):947–950. [PubMed] [Google Scholar]
  34. Lindon J. N., McManama G., Kushner L., Merrill E. W., Salzman E. W. Does the conformation of adsorbed fibrinogen dictate platelet interactions with artificial surfaces? Blood. 1986 Aug;68(2):355–362. [PubMed] [Google Scholar]
  35. Marguerie G. A., Plow E. F., Edgington T. S. Human platelets possess an inducible and saturable receptor specific for fibrinogen. J Biol Chem. 1979 Jun 25;254(12):5357–5363. [PubMed] [Google Scholar]
  36. Martin P., Papayannopoulou T. HEL cells: a new human erythroleukemia cell line with spontaneous and induced globin expression. Science. 1982 Jun 11;216(4551):1233–1235. doi: 10.1126/science.6177045. [DOI] [PubMed] [Google Scholar]
  37. McManama G., Lindon J. N., Kloczewiak M., Smith M. A., Ware J. A., Hawiger J., Merrill E. W., Salzman E. W. Platelet aggregation by fibrinogen polymers crosslinked across the E domain. Blood. 1986 Aug;68(2):363–371. [PubMed] [Google Scholar]
  38. Niewiarowski S., Regoeczi E., Stewart G. J., Senyl A. F., Mustard J. F. Platelet interaction with polymerizing fibrin. J Clin Invest. 1972 Mar;51(3):685–699. doi: 10.1172/JCI106857. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Nurden A. T., Didry D., Kieffer N., McEver R. P. Residual amounts of glycoproteins IIb and IIIa may be present in the platelets of most patients with Glanzmann's thrombasthenia. Blood. 1985 Apr;65(4):1021–1024. [PubMed] [Google Scholar]
  40. O'Toole T. E., Loftus J. C., Plow E. F., Glass A. A., Harper J. R., Ginsberg M. H. Efficient surface expression of platelet GPIIb-IIIa requires both subunits. Blood. 1989 Jul;74(1):14–18. [PubMed] [Google Scholar]
  41. Phillips D. R., Charo I. F., Parise L. V., Fitzgerald L. A. The platelet membrane glycoprotein IIb-IIIa complex. Blood. 1988 Apr;71(4):831–843. [PubMed] [Google Scholar]
  42. Piotrowicz R. S., Orchekowski R. P., Nugent D. J., Yamada K. Y., Kunicki T. J. Glycoprotein Ic-IIa functions as an activation-independent fibronectin receptor on human platelets. J Cell Biol. 1988 Apr;106(4):1359–1364. doi: 10.1083/jcb.106.4.1359. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Poncz M., Eisman R., Heidenreich R., Silver S. M., Vilaire G., Surrey S., Schwartz E., Bennett J. S. Structure of the platelet membrane glycoprotein IIb. Homology to the alpha subunits of the vitronectin and fibronectin membrane receptors. J Biol Chem. 1987 Jun 25;262(18):8476–8482. [PubMed] [Google Scholar]
  44. Rosa J. P., McEver R. P. Processing and assembly of the integrin, glycoprotein IIb-IIIa, in HEL cells. J Biol Chem. 1989 Jul 25;264(21):12596–12603. [PubMed] [Google Scholar]
  45. 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]
  46. Santoro S. A., Cowan J. F. Thrombin enhanced adhesion of platelets to von Willebrand factor substrates. Thromb Res. 1986 Jul 1;43(1):57–72. doi: 10.1016/0049-3848(86)90044-7. [DOI] [PubMed] [Google Scholar]
  47. Smith J. W., Cheresh D. A. The Arg-Gly-Asp binding domain of the vitronectin receptor. Photoaffinity cross-linking implicates amino acid residues 61-203 of the beta subunit. J Biol Chem. 1988 Dec 15;263(35):18726–18731. [PubMed] [Google Scholar]
  48. Solowska J., Guan J. L., Marcantonio E. E., Trevithick J. E., Buck C. A., Hynes R. O. Expression of normal and mutant avian integrin subunits in rodent cells. J Cell Biol. 1989 Aug;109(2):853–861. doi: 10.1083/jcb.109.2.853. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Steiner B., Cousot D., Trzeciak A., Gillessen D., Hadváry P. Ca2+-dependent binding of a synthetic Arg-Gly-Asp (RGD) peptide to a single site on the purified platelet glycoprotein IIb-IIIa complex. J Biol Chem. 1989 Aug 5;264(22):13102–13108. [PubMed] [Google Scholar]
  50. Takebe Y., Seiki M., Fujisawa J., Hoy P., Yokota K., Arai K., Yoshida M., Arai N. SR alpha promoter: an efficient and versatile mammalian cDNA expression system composed of the simian virus 40 early promoter and the R-U5 segment of human T-cell leukemia virus type 1 long terminal repeat. Mol Cell Biol. 1988 Jan;8(1):466–472. doi: 10.1128/mcb.8.1.466. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Thiagarajan P., Shapiro S. S., Sweterlitsch L., McCord S. A human erythroleukemia cell line synthesizes a functionally active glycoprotein IIb-IIIa complex capable of binding fibrinogen. Biochim Biophys Acta. 1987 Apr 16;924(1):127–134. doi: 10.1016/0304-4165(87)90079-1. [DOI] [PubMed] [Google Scholar]
  52. Werb Z., Tremble P. M., Behrendtsen O., Crowley E., Damsky C. H. Signal transduction through the fibronectin receptor induces collagenase and stromelysin gene expression. J Cell Biol. 1989 Aug;109(2):877–889. doi: 10.1083/jcb.109.2.877. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Yatohgo T., Izumi M., Kashiwagi H., Hayashi M. Novel purification of vitronectin from human plasma by heparin affinity chromatography. Cell Struct Funct. 1988 Aug;13(4):281–292. doi: 10.1247/csf.13.281. [DOI] [PubMed] [Google Scholar]

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

RESOURCES