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. 1987 Sep 1;105(3):1163–1173. doi: 10.1083/jcb.105.3.1163

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

PMCID: PMC2114807  PMID: 2443507

Abstract

The disialogangliosides GD2 and GD3 play a major role in the ability of human melanoma cells to attach to Arg-Gly-Asp-containing substrates such as fibronectin and vitronectin, since pretreatment of these cells with monoclonal antibodies to the oligosaccharide of GD2 and GD3 can inhibit their attachment and spreading on such adhesive proteins. This report demonstrates that human melanoma cells (M21) synthesize and express a glycoprotein receptor that shares antigenic epitopes with the vitronectin receptor on human fibroblasts and is capable of specifically recognizing the Gly-Arg-Gly-Asp-Ser-Pro sequence. In the presence of calcium, GD2, the major ganglioside of M21 cells, colocalized with this receptor on the surface of human melanoma cells and their focal adhesion plaques as demonstrated by double-label transmission immunoelectron microscopy and indirect immunofluorescence. Biochemical evidence is presented indicating that the vitronectin receptor on M21 human melanoma cells contains associated calcium and GD2. This ganglioside copurified with the glycoprotein receptor for vitronectin on affinity columns containing either an Arg-Gly-Asp- containing peptide, concanavalin A, or lentil lectin. This major Arg- Gly-Asp-directed receptor on M21 cells could be metabolically labeled with 45Ca2+. Chelation of this ion with EDTA caused the dissociation of GD2 from the receptor and rendered the remaining glycoprotein incapable of binding to an Arg-Gly-Asp-containing peptide. Reconstitution experiments demonstrated a requirement for calcium, and not magnesium, for receptor binding to Arg-Gly-Asp and indicated that addition of ganglioside can enhance this interaction.

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Selected References

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  1. Abramson M. B., Yu R. K., Zaby V. Ionic properties of beef brain gangliosides. Biochim Biophys Acta. 1972 Oct 5;280(2):365–372. doi: 10.1016/0005-2760(72)90105-1. [DOI] [PubMed] [Google Scholar]
  2. Behr J. P., Lehn J. M. The binding of divalent cations by purified gangliosides. FEBS Lett. 1973 May 1;31(3):297–300. doi: 10.1016/0014-5793(73)80126-7. [DOI] [PubMed] [Google Scholar]
  3. Bremer E. G., Hakomori S., Bowen-Pope D. F., Raines E., Ross R. Ganglioside-mediated modulation of cell growth, growth factor binding, and receptor phosphorylation. J Biol Chem. 1984 Jun 10;259(11):6818–6825. [PubMed] [Google Scholar]
  4. Bremer E. G., Schlessinger J., Hakomori S. Ganglioside-mediated modulation of cell growth. Specific effects of GM3 on tyrosine phosphorylation of the epidermal growth factor receptor. J Biol Chem. 1986 Feb 15;261(5):2434–2440. [PubMed] [Google Scholar]
  5. Brown P. J., Juliano R. L. Selective inhibition of fibronectin-mediated cell adhesion by monoclonal antibodies to a cell-surface glycoprotein. Science. 1985 Jun 21;228(4706):1448–1451. doi: 10.1126/science.4012302. [DOI] [PubMed] [Google Scholar]
  6. Bumol T. F., Reisfeld R. A. Unique glycoprotein-proteoglycan complex defined by monoclonal antibody on human melanoma cells. Proc Natl Acad Sci U S A. 1982 Feb;79(4):1245–1249. doi: 10.1073/pnas.79.4.1245. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. 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]
  8. Cheresh D. A., Harper J. R., Schulz G., Reisfeld R. A. Localization of the gangliosides GD2 and GD3 in adhesion plaques and on the surface of human melanoma cells. Proc Natl Acad Sci U S A. 1984 Sep;81(18):5767–5771. doi: 10.1073/pnas.81.18.5767. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Cheresh D. A., Klier F. G. Disialoganglioside GD2 distributes preferentially into substrate-associated microprocesses on human melanoma cells during their attachment to fibronectin. J Cell Biol. 1986 May;102(5):1887–1897. doi: 10.1083/jcb.102.5.1887. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Cheresh D. A., Pierschbacher M. D., Herzig M. A., Mujoo K. Disialogangliosides GD2 and GD3 are involved in the attachment of human melanoma and neuroblastoma cells to extracellular matrix proteins. J Cell Biol. 1986 Mar;102(3):688–696. doi: 10.1083/jcb.102.3.688. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Cheresh D. A., Reisfeld R. A., Varki A. P. O-acetylation of disialoganglioside GD3 by human melanoma cells creates a unique antigenic determinant. Science. 1984 Aug 24;225(4664):844–846. doi: 10.1126/science.6206564. [DOI] [PubMed] [Google Scholar]
  12. Cheresh D. A. Structural and functional properties of ganglioside antigens on human tumors of neuroectodermal origin. Surv Synth Pathol Res. 1985;4(2):97–109. doi: 10.1159/000156968. [DOI] [PubMed] [Google Scholar]
  13. Cheresh D. A., Varki A. P., Varki N. M., Stallcup W. B., Levine J., Reisfeld R. A. A monoclonal antibody recognizes an O-acylated sialic acid in a human melanoma-associated ganglioside. J Biol Chem. 1984 Jun 25;259(12):7453–7459. [PubMed] [Google Scholar]
  14. Feizi T. Demonstration by monoclonal antibodies that carbohydrate structures of glycoproteins and glycolipids are onco-developmental antigens. Nature. 1985 Mar 7;314(6006):53–57. doi: 10.1038/314053a0. [DOI] [PubMed] [Google Scholar]
  15. Gabius H. J., Springer W. R., Barondes S. H. Receptor for the cell binding site of discoidin I. Cell. 1985 Sep;42(2):449–456. doi: 10.1016/0092-8674(85)90102-3. [DOI] [PubMed] [Google Scholar]
  16. Gardner J. M., Hynes R. O. Interaction of fibronectin with its receptor on platelets. Cell. 1985 Sep;42(2):439–448. doi: 10.1016/0092-8674(85)90101-1. [DOI] [PubMed] [Google Scholar]
  17. Garner J. A., Culp L. A. Aggregation competence of proteoglycans from the substratum adhesion sites of murine fibroblasts. Biochemistry. 1981 Dec 22;20(26):7350–7359. doi: 10.1021/bi00529a005. [DOI] [PubMed] [Google Scholar]
  18. Giancotti F. G., Tarone G., Knudsen K., Damsky C., Comoglio P. M. Cleavage of a 135 kD cell surface glycoprotein correlates with loss of fibroblast adhesion to fibronectin. Exp Cell Res. 1985 Jan;156(1):182–190. doi: 10.1016/0014-4827(85)90272-1. [DOI] [PubMed] [Google Scholar]
  19. Ginsberg M., Pierschbacher M. D., Ruoslahti E., Marguerie G., Plow E. Inhibition of fibronectin binding to platelets by proteolytic fragments and synthetic peptides which support fibroblast adhesion. J Biol Chem. 1985 Apr 10;260(7):3931–3936. [PubMed] [Google Scholar]
  20. Hakomori S., Kannagi R. Glycosphingolipids as tumor-associated and differentiation markers. J Natl Cancer Inst. 1983 Aug;71(2):231–251. [PubMed] [Google Scholar]
  21. Hakomori S. Tumor-associated carbohydrate antigens. Annu Rev Immunol. 1984;2:103–126. doi: 10.1146/annurev.iy.02.040184.000535. [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. Heath T. D., Martin F. J., Macher B. A. Association of ganglioside-protein conjugates into cell and Sendai virus. Requirement for the HN subunit in viral fusion. Exp Cell Res. 1983 Nov;149(1):163–175. doi: 10.1016/0014-4827(83)90389-0. [DOI] [PubMed] [Google Scholar]
  24. 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]
  25. Jemmerson R., Klier F. G., Fishman W. H. Clustered distribution of human placental alkaline phosphatase on the surface of both placental and cancer cells. Electron microscopic observations using gold-labeled antibodies. J Histochem Cytochem. 1985 Dec;33(12):1227–1234. doi: 10.1177/33.12.4067276. [DOI] [PubMed] [Google Scholar]
  26. Kleinman H. K., Martin G. R., Fishman P. H. Ganglioside inhibition of fibronectin-mediated cell adhesion to collagen. Proc Natl Acad Sci U S A. 1979 Jul;76(7):3367–3371. doi: 10.1073/pnas.76.7.3367. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Klier F. G., Schubert D., Heinemann S. The ultrastructural differentiation of the clonal myogenic cell line L6 in normal and high K+ medium. Dev Biol. 1977 Jun;57(2):440–449. doi: 10.1016/0012-1606(77)90228-7. [DOI] [PubMed] [Google Scholar]
  28. 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]
  29. Mugnai G., Tombaccini D., Ruggieri S. Ganglioside composition of substrate-adhesion sites of normal and virally-transformed Balb/c 3T3 cells. Biochem Biophys Res Commun. 1984 Nov 30;125(1):142–148. doi: 10.1016/s0006-291x(84)80346-0. [DOI] [PubMed] [Google Scholar]
  30. Nicolson G. L. Cell surface molecules and tumor metastasis. Regulation of metastatic phenotypic diversity. Exp Cell Res. 1984 Jan;150(1):3–22. doi: 10.1016/0014-4827(84)90696-7. [DOI] [PubMed] [Google Scholar]
  31. Okada Y., Mugnai G., Bremer E. G., Hakomori S. Glycosphingolipids in detergent-insoluble substrate attachment matrix (DISAM) prepared from substrate attachment material (SAM). Their possible role in regulating cell adhesion. Exp Cell Res. 1984 Dec;155(2):448–456. doi: 10.1016/0014-4827(84)90205-2. [DOI] [PubMed] [Google Scholar]
  32. Parker P. J., Young S., Gullick W. J., Mayes E. L., Bennett P., Waterfield M. D. Monoclonal antibodies against the human epidermal growth factor receptor from A431 cells. Isolation, characterization, and use in the purification of active epidermal growth factor receptor. J Biol Chem. 1984 Aug 10;259(15):9906–9912. [PubMed] [Google Scholar]
  33. Perkins R. M., Kellie S., Patel B., Critchley D. R. Gangliosides as receptors for fibronectin? Comparison of cell spreading on a ganglioside-specific ligand with that on fibronectin. Exp Cell Res. 1982 Oct;141(2):231–243. doi: 10.1016/0014-4827(82)90211-7. [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. Plow E. F., Pierschbacher M. D., Ruoslahti E., Marguerie G. A., Ginsberg M. H. The effect of Arg-Gly-Asp-containing peptides on fibrinogen and von Willebrand factor binding to platelets. Proc Natl Acad Sci U S A. 1985 Dec;82(23):8057–8061. doi: 10.1073/pnas.82.23.8057. [DOI] [PMC free article] [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. Arg-Gly-Asp: a versatile cell recognition signal. Cell. 1986 Feb 28;44(4):517–518. doi: 10.1016/0092-8674(86)90259-x. [DOI] [PubMed] [Google Scholar]
  40. SVENNERHOLM L. CHROMATOGRAPHIC SEPARATION OF HUMAN BRAIN GANGLIOSIDES. J Neurochem. 1963 Sep;10:613–623. doi: 10.1111/j.1471-4159.1963.tb08933.x. [DOI] [PubMed] [Google Scholar]
  41. Schulz G., Cheresh D. A., Varki N. M., Yu A., Staffileno L. K., Reisfeld R. A. Detection of ganglioside GD2 in tumor tissues and sera of neuroblastoma patients. Cancer Res. 1984 Dec;44(12 Pt 1):5914–5920. [PubMed] [Google Scholar]
  42. Sharom F. J., Grant C. W. A model for ganglioside behaviour in cell membranes. Biochim Biophys Acta. 1978 Feb 21;507(2):280–293. doi: 10.1016/0005-2736(78)90423-6. [DOI] [PubMed] [Google Scholar]
  43. Slot J. W., Geuze H. J. Sizing of protein A-colloidal gold probes for immunoelectron microscopy. J Cell Biol. 1981 Aug;90(2):533–536. doi: 10.1083/jcb.90.2.533. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Spiegel S., Schlessinger J., Fishman P. H. Incorporation of fluorescent gangliosides into human fibroblasts: mobility, fate, and interaction with fibronectin. J Cell Biol. 1984 Aug;99(2):699–704. doi: 10.1083/jcb.99.2.699. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Springer W. R., Cooper D. N., Barondes S. H. Discoidin I is implicated in cell-substratum attachment and ordered cell migration of Dictyostelium discoideum and resembles fibronectin. Cell. 1984 Dec;39(3 Pt 2):557–564. doi: 10.1016/0092-8674(84)90462-8. [DOI] [PubMed] [Google Scholar]
  46. 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]
  47. Symington F. W., Bernstein I. D., Hakomori S. Monoclonal antibody specific for lactosylceramide. J Biol Chem. 1984 May 10;259(9):6008–6012. [PubMed] [Google Scholar]
  48. Thompson L. K., Horowitz P. M., Bentley K. L., Thomas D. D., Alderete J. F., Klebe R. J. Localization of the ganglioside-binding site of fibronectin. J Biol Chem. 1986 Apr 15;261(11):5209–5214. [PubMed] [Google Scholar]
  49. Yamada K. M., Kennedy D. W., Grotendorst G. R., Momoi T. Glycolipids: receptors for fibronectin? J Cell Physiol. 1981 Nov;109(2):343–351. doi: 10.1002/jcp.1041090218. [DOI] [PubMed] [Google Scholar]

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