Skip to main content
PMC home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1990 Jun 1;110(6):2185–2193. doi: 10.1083/jcb.110.6.2185

Isolation of a novel integrin receptor mediating Arg-Gly-Asp-directed cell adhesion to fibronectin and type I collagen from human neuroblastoma cells. Association of a novel beta 1-related subunit with alpha v

PMCID: PMC2116123  PMID: 1693626

Abstract

We report the isolation from two human neuroblastoma cell lines of an Arg-Gly-Asp-dependent integrin complex capable of binding to vitronectin, fibronectin, and type I collagen. The two neuroblastoma cell lines, SK-N-SH and IMR-32, exhibit specific attachment to fibronectin and type I collagen. SK-N-SH cells exhibit a much stronger attachment to vitronectin than the IMR-32 cells, which attach poorly to this substrate. Affinity chromatography of octylglucoside extracts of 125I surface-labeled cells on GRGDSPK-Sepharose columns resulted in the specific binding and elution with GRGDSP of three radiolabeled polypeptides with relative molecular masses of 135, 115, and 90 kD when analyzed by SDS-PAGE under nonreducing conditions. In the SK-N-SH cells the 135- and 90-kD polypeptides were more abundant whereas in the IMR- 32 cells the 135- and 115-kD polypeptides were more highly expressed. Liposomes prepared from fractions containing all three polypeptides bound to vitronectin, fibronectin, and type I collagen, whereas liposomes prepared from the 135- and 115-kD polypeptides bound only to fibronectin and type I collagen. Polyclonal antibodies against the alpha/beta complexes of both the vitronectin receptor and the fibronectin receptor immunoprecipitated all three polypeptides. A monoclonal antibody against beta 1 immunoprecipitated only the 135- and the 115-kD polypeptides, whereas a monoclonal antibody against beta 3 subunit immunoprecipitated the 135- and 90-kD polypeptides. Although, the 115-kD polypeptide could be recognized by an anti-beta 1 antibody, a comparison of peptide maps generated by V8 protease digestion of the 115-kD polypeptide and beta 1 subunit immunoprecipitated from GRGDSPK- Sepharose flow-through material indicated that these two polypeptides are distinct. Depletion of the 90-kD polypeptide with an anti-beta 3 monoclonal antibody did not effect the ability of the 115- and 135-kD polypeptides to bind to GRGDSPK-Sepharose. These data indicate that the SK-N-SH and IMR-32 neuroblastoma cells express a novel "beta 1-like" integrin subunit that can associate with alpha v and can bind to RGD. We propose to name this beta 1-like subunit beta n. The data reported here thus demonstrate that in these two cell lines alpha v associates with two beta subunits, beta n and beta 3, forming two heterodimers. The alpha v beta n complex mediates binding to fibronectin and type I collagen, whereas the alpha v beta 3 complex mediates binding to vitronectin.

Full Text

The Full Text of this article is available as a PDF (1.6 MB).

Selected References

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

  1. Argraves W. S., Suzuki S., Arai H., Thompson K., Pierschbacher M. D., Ruoslahti E. Amino acid sequence of the human fibronectin receptor. J Cell Biol. 1987 Sep;105(3):1183–1190. doi: 10.1083/jcb.105.3.1183. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Calof A. L., Reichardt L. F. Response of purified chick motoneurons to myotube conditioned medium: laminin is essential for the substratum-binding, neurite outgrowth-promoting activity. Neurosci Lett. 1985 Aug 30;59(2):183–189. doi: 10.1016/0304-3940(85)90197-1. [DOI] [PubMed] [Google Scholar]
  3. 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]
  4. Davis G. E., Blaker S. N., Engvall E., Varon S., Manthorpe M., Gage F. H. Human amnion membrane serves as a substratum for growing axons in vitro and in vivo. Science. 1987 May 29;236(4805):1106–1109. doi: 10.1126/science.3576223. [DOI] [PubMed] [Google Scholar]
  5. Davis G. E., Manthorpe M., Engvall E., Varon S. Isolation and characterization of rat schwannoma neurite-promoting factor: evidence that the factor contains laminin. J Neurosci. 1985 Oct;5(10):2662–2671. doi: 10.1523/JNEUROSCI.05-10-02662.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Dedhar S., Argraves W. S., Suzuki S., Ruoslahti E., Pierschbacher M. D. Human osteosarcoma cells resistant to detachment by an Arg-Gly-Asp-containing peptide overproduce the fibronectin receptor. J Cell Biol. 1987 Sep;105(3):1175–1182. doi: 10.1083/jcb.105.3.1175. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Dedhar S., Haqq C., Gray V. Specific overproduction of very late antigen 1 integrin in two human neuroblastoma cell lines selected for resistance to detachment by an Arg-Gly-Asp-containing synthetic peptide. J Biol Chem. 1989 Mar 25;264(9):4832–4836. [PubMed] [Google Scholar]
  8. Dedhar S., Ruoslahti E., Pierschbacher M. D. A cell surface receptor complex for collagen type I recognizes the Arg-Gly-Asp sequence. J Cell Biol. 1987 Mar;104(3):585–593. doi: 10.1083/jcb.104.3.585. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Freed E., Gailit J., van der Geer P., Ruoslahti E., Hunter T. A novel integrin beta subunit is associated with the vitronectin receptor alpha subunit (alpha v) in a human osteosarcoma cell line and is a substrate for protein kinase C. EMBO J. 1989 Oct;8(10):2955–2965. doi: 10.1002/j.1460-2075.1989.tb08445.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Gehlsen K. R., Dillner L., Engvall E., Ruoslahti E. The human laminin receptor is a member of the integrin family of cell adhesion receptors. Science. 1988 Sep 2;241(4870):1228–1229. doi: 10.1126/science.2970671. [DOI] [PubMed] [Google Scholar]
  11. Halfter W., Deiss S. Axonal pathfinding in organ-cultured embryonic avian retinae. Dev Biol. 1986 Apr;114(2):296–310. doi: 10.1016/0012-1606(86)90194-6. [DOI] [PubMed] [Google Scholar]
  12. 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]
  13. Hemler M. E. Adhesive protein receptors on hematopoietic cells. Immunol Today. 1988 Apr;9(4):109–113. doi: 10.1016/0167-5699(88)91280-7. [DOI] [PubMed] [Google Scholar]
  14. Hemler M. E., Crouse C., Sonnenberg A. Association of the VLA alpha 6 subunit with a novel protein. A possible alternative to the common VLA beta 1 subunit on certain cell lines. J Biol Chem. 1989 Apr 15;264(11):6529–6535. [PubMed] [Google Scholar]
  15. 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]
  16. 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]
  17. Kajiji S., Tamura R. N., Quaranta V. A novel integrin (alpha E beta 4) from human epithelial cells suggests a fourth family of integrin adhesion receptors. EMBO J. 1989 Mar;8(3):673–680. doi: 10.1002/j.1460-2075.1989.tb03425.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Krotoski D. M., Domingo C., Bronner-Fraser M. Distribution of a putative cell surface receptor for fibronectin and laminin in the avian embryo. J Cell Biol. 1986 Sep;103(3):1061–1071. doi: 10.1083/jcb.103.3.1061. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Lander A. D., Fujii D. K., Reichardt L. F. Laminin is associated with the "neurite outgrowth-promoting factors" found in conditioned media. Proc Natl Acad Sci U S A. 1985 Apr;82(7):2183–2187. doi: 10.1073/pnas.82.7.2183. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Lander A. D., Fujii D. K., Reichardt L. F. Purification of a factor that promotes neurite outgrowth: isolation of laminin and associated molecules. J Cell Biol. 1985 Sep;101(3):898–913. doi: 10.1083/jcb.101.3.898. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. 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]
  22. 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]
  23. 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]
  24. 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]
  25. 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]
  26. 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]
  27. Rettig W. J., Murty V. V., Mattes M. J., Chaganti R. S., Old L. J. Extracellular matrix-modulated expression of human cell surface glycoproteins A42 and J143. Intrinsic and extrinsic signals determine antigenic phenotype. J Exp Med. 1986 Nov 1;164(5):1581–1599. doi: 10.1084/jem.164.5.1581. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Rogers S. L., Edson K. J., Letourneau P. C., McLoon S. C. Distribution of laminin in the developing peripheral nervous system of the chick. Dev Biol. 1986 Feb;113(2):429–435. doi: 10.1016/0012-1606(86)90177-6. [DOI] [PubMed] [Google Scholar]
  29. 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]
  30. Suzuki S., Argraves W. S., Arai H., Languino L. R., Pierschbacher M. D., Ruoslahti E. Amino acid sequence of the vitronectin receptor alpha subunit and comparative expression of adhesion receptor mRNAs. J Biol Chem. 1987 Oct 15;262(29):14080–14085. [PubMed] [Google Scholar]
  31. Tomaselli K. J., Damsky C. H., Reichardt L. F. Interactions of a neuronal cell line (PC12) with laminin, collagen IV, and fibronectin: identification of integrin-related glycoproteins involved in attachment and process outgrowth. J Cell Biol. 1987 Nov;105(5):2347–2358. doi: 10.1083/jcb.105.5.2347. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Tomaselli K. J., Reichardt L. F., Bixby J. L. Distinct molecular interactions mediate neuronal process outgrowth on non-neuronal cell surfaces and extracellular matrices. J Cell Biol. 1986 Dec;103(6 Pt 2):2659–2672. doi: 10.1083/jcb.103.6.2659. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES