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. 1992 Apr 2;117(2):449–459. doi: 10.1083/jcb.117.2.449

A synthetic peptide derived from the carboxy terminus of the laminin A chain represents a binding site for the alpha 3 beta 1 integrin

PMCID: PMC2289418  PMID: 1560034

Abstract

The purpose of this study was to identify the binding site(s) within laminin for the alpha 3 beta 1 integrin receptor. It has been previously shown, using proteolytic fragments and anti-laminin antibodies, that the region in laminin for alpha 3 beta 1 integrin binding is localized to the carboxy-terminal region at the end of the long arm (Gehlsen, K. R., E. Engvall, K. Dickerson, W. S. Argraves, and E. Ruoslahti. 1989. J. Biol. Chem. 264:19034-19038; Tomaselli, K. J., D. E. Hall, L. T. Reichardt, L. A. Flier, K. R. Gehlsen, D. C. Turner, and S. Carbonetto. 1990. Neuron. 5:651-662). Using synthetic peptides, we have identified an amino acid sequence within the carboxy-terminal region of the laminin A chain that is recognized by the alpha 3 beta 1 integrin. The amino acid sequence represented by the synthetic peptide GD-6 (KQNCLSSRASFRGCVRNLRLSR residues numbered 3011 to 3032) of the globular domain of the murine A chain supports cell attachment and inhibits cell adhesion to laminin-coated surfaces. By affinity chromatography, peptide GD-6-Sepharose specifically bound solubilized alpha 3 beta 1 from extracts of surface-iodinated cells in a cation- dependent manner, while it did not bind other integrins. In addition, exogenous peptide GD-6 specifically eluted bound alpha 3 beta 1 from laminin-Sepharose columns but did not elute the alpha 3 beta 1 integrin from a fibronectin-Sepharose column. Using integrin subunit-specific monoclonal antibodies, only those antibodies against the alpha 3 and beta 1 subunits inhibited cell adhesion to peptide GD-6-coated surfaces. Finally, a polyclonal antibody made against peptide GD-6 reacted specifically with both murine and human laminin and significantly inhibited cell adhesion to laminin-coated surfaces but not those coated with other matrix proteins. These results identify the laminin A chain amino acid sequence of peptide GD-6 as representing a binding site in laminin for the alpha 3 beta 1 integrin.

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Selected References

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  1. Beatty P. G., Ledbetter J. A., Martin P. J., Price T. H., Hansen J. A. Definition of a common leukocyte cell-surface antigen (Lp95-150) associated with diverse cell-mediated immune functions. J Immunol. 1983 Dec;131(6):2913–2918. [PubMed] [Google Scholar]
  2. Begovac P. C., Hall D. E., Shur B. D. Laminin fragment E8 mediates PC12 cell neurite outgrowth by binding to cell surface beta 1,4 galactosyltransferase. J Cell Biol. 1991 May;113(3):637–644. doi: 10.1083/jcb.113.3.637. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Carter W. G., Ryan M. C., Gahr P. J. Epiligrin, a new cell adhesion ligand for integrin alpha 3 beta 1 in epithelial basement membranes. Cell. 1991 May 17;65(4):599–610. doi: 10.1016/0092-8674(91)90092-d. [DOI] [PubMed] [Google Scholar]
  4. Carter W. G., Wayner E. A., Bouchard T. S., Kaur P. The role of integrins alpha 2 beta 1 and alpha 3 beta 1 in cell-cell and cell-substrate adhesion of human epidermal cells. J Cell Biol. 1990 Apr;110(4):1387–1404. doi: 10.1083/jcb.110.4.1387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Charonis A. S., Skubitz A. P., Koliakos G. G., Reger L. A., Dege J., Vogel A. M., Wohlhueter R., Furcht L. T. A novel synthetic peptide from the B1 chain of laminin with heparin-binding and cell adhesion-promoting activities. J Cell Biol. 1988 Sep;107(3):1253–1260. doi: 10.1083/jcb.107.3.1253. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. 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]
  7. Chung A. E., Jaffe R., Freeman I. L., Vergnes J. P., Braginski J. E., Carlin B. Properties of a basement membrane-related glycoprotein synthesized in culture by a mouse embryonal carcinoma-derived cell line. Cell. 1979 Feb;16(2):277–287. doi: 10.1016/0092-8674(79)90005-9. [DOI] [PubMed] [Google Scholar]
  8. Clément B., Segui-Real B., Savagner P., Kleinman H. K., Yamada Y. Hepatocyte attachment to laminin is mediated through multiple receptors. J Cell Biol. 1990 Jan;110(1):185–192. doi: 10.1083/jcb.110.1.185. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Deutzmann R., Huber J., Schmetz K. A., Oberbäumer I., Hartl L. Structural study of long arm fragments of laminin. Evidence for repetitive C-terminal sequences in the A-chain, not present in the B-chains. Eur J Biochem. 1988 Oct 15;177(1):35–45. doi: 10.1111/j.1432-1033.1988.tb14342.x. [DOI] [PubMed] [Google Scholar]
  10. Dillner L., Dickerson K., Manthorpe M., Ruoslahti E., Engvall E. The neurite-promoting domain of human laminin promotes attachment and induces characteristic morphology in non-neuronal cells. Exp Cell Res. 1988 Jul;177(1):186–198. doi: 10.1016/0014-4827(88)90036-5. [DOI] [PubMed] [Google Scholar]
  11. Ehrig K., Leivo I., Argraves W. S., Ruoslahti E., Engvall E. Merosin, a tissue-specific basement membrane protein, is a laminin-like protein. Proc Natl Acad Sci U S A. 1990 May;87(9):3264–3268. doi: 10.1073/pnas.87.9.3264. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. 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]
  13. Elices M. J., Urry L. A., Hemler M. E. Receptor functions for the integrin VLA-3: fibronectin, collagen, and laminin binding are differentially influenced by Arg-Gly-Asp peptide and by divalent cations. J Cell Biol. 1991 Jan;112(1):169–181. doi: 10.1083/jcb.112.1.169. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Engvall E., Davis G. E., Dickerson K., Ruoslahti E., Varon S., Manthorpe M. Mapping of domains in human laminin using monoclonal antibodies: localization of the neurite-promoting site. J Cell Biol. 1986 Dec;103(6 Pt 1):2457–2465. doi: 10.1083/jcb.103.6.2457. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Gehlsen K. R., Dickerson K., Argraves W. S., Engvall E., Ruoslahti E. Subunit structure of a laminin-binding integrin and localization of its binding site on laminin. J Biol Chem. 1989 Nov 15;264(32):19034–19038. [PubMed] [Google Scholar]
  16. 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]
  17. Goodman S. L., Aumailley M., von der Mark H. Multiple cell surface receptors for the short arms of laminin: alpha 1 beta 1 integrin and RGD-dependent proteins mediate cell attachment only to domains III in murine tumor laminin. J Cell Biol. 1991 May;113(4):931–941. doi: 10.1083/jcb.113.4.931. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Goodman S. L., Deutzmann R., von der Mark K. Two distinct cell-binding domains in laminin can independently promote nonneuronal cell adhesion and spreading. J Cell Biol. 1987 Jul;105(1):589–598. doi: 10.1083/jcb.105.1.589. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Graf J., Iwamoto Y., Sasaki M., Martin G. R., Kleinman H. K., Robey F. A., Yamada Y. Identification of an amino acid sequence in laminin mediating cell attachment, chemotaxis, and receptor binding. Cell. 1987 Mar 27;48(6):989–996. doi: 10.1016/0092-8674(87)90707-0. [DOI] [PubMed] [Google Scholar]
  20. 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]
  21. Hemler M. E. VLA proteins in the integrin family: structures, functions, and their role on leukocytes. Annu Rev Immunol. 1990;8:365–400. doi: 10.1146/annurev.iy.08.040190.002053. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. Ignatius M. J., Large T. H., Houde M., Tawil J. W., Barton A., Esch F., Carbonetto S., Reichardt L. F. Molecular cloning of the rat integrin alpha 1-subunit: a receptor for laminin and collagen. J Cell Biol. 1990 Aug;111(2):709–720. doi: 10.1083/jcb.111.2.709. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Kouzi-Koliakos K., Koliakos G. G., Tsilibary E. C., Furcht L. T., Charonis A. S. Mapping of three major heparin-binding sites on laminin and identification of a novel heparin-binding site on the B1 chain. J Biol Chem. 1989 Oct 25;264(30):17971–17978. [PubMed] [Google Scholar]
  25. Kramer R. H., Cheng Y. F., Clyman R. Human microvascular endothelial cells use beta 1 and beta 3 integrin receptor complexes to attach to laminin. J Cell Biol. 1990 Sep;111(3):1233–1243. doi: 10.1083/jcb.111.3.1233. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Kramer R. H., Vu M. P., Cheng Y. F., Ramos D. M., Timpl R., Waleh N. Laminin-binding integrin alpha 7 beta 1: functional characterization and expression in normal and malignant melanocytes. Cell Regul. 1991 Oct;2(10):805–817. doi: 10.1091/mbc.2.10.805. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Kyte J., Doolittle R. F. A simple method for displaying the hydropathic character of a protein. J Mol Biol. 1982 May 5;157(1):105–132. doi: 10.1016/0022-2836(82)90515-0. [DOI] [PubMed] [Google Scholar]
  28. Languino L. R., Gehlsen K. R., Wayner E., Carter W. G., Engvall E., Ruoslahti E. Endothelial cells use alpha 2 beta 1 integrin as a laminin receptor. J Cell Biol. 1989 Nov;109(5):2455–2462. doi: 10.1083/jcb.109.5.2455. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Malinoff H. L., Wicha M. S. Isolation of a cell surface receptor protein for laminin from murine fibrosarcoma cells. J Cell Biol. 1983 May;96(5):1475–1479. doi: 10.1083/jcb.96.5.1475. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Mould A. P., Komoriya A., Yamada K. M., Humphries M. J. The CS5 peptide is a second site in the IIICS region of fibronectin recognized by the integrin alpha 4 beta 1. Inhibition of alpha 4 beta 1 function by RGD peptide homologues. J Biol Chem. 1991 Feb 25;266(6):3579–3585. [PubMed] [Google Scholar]
  31. Olsen D., Nagayoshi T., Fazio M., Peltonen J., Jaakkola S., Sanborn D., Sasaki T., Kuivaniemi H., Chu M. L., Deutzmann R. Human laminin: cloning and sequence analysis of cDNAs encoding A, B1 and B2 chains, and expression of the corresponding genes in human skin and cultured cells. Lab Invest. 1989 Jun;60(6):772–782. [PubMed] [Google Scholar]
  32. Phillips D. R., Charo I. F., Scarborough R. M. GPIIb-IIIa: the responsive integrin. Cell. 1991 May 3;65(3):359–362. doi: 10.1016/0092-8674(91)90451-4. [DOI] [PubMed] [Google Scholar]
  33. Ruoslahti E. Integrins. J Clin Invest. 1991 Jan;87(1):1–5. doi: 10.1172/JCI114957. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Sanes J. R., Engvall E., Butkowski R., Hunter D. D. Molecular heterogeneity of basal laminae: isoforms of laminin and collagen IV at the neuromuscular junction and elsewhere. J Cell Biol. 1990 Oct;111(4):1685–1699. doi: 10.1083/jcb.111.4.1685. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Sasaki M., Kleinman H. K., Huber H., Deutzmann R., Yamada Y. Laminin, a multidomain protein. The A chain has a unique globular domain and homology with the basement membrane proteoglycan and the laminin B chains. J Biol Chem. 1988 Nov 15;263(32):16536–16544. [PubMed] [Google Scholar]
  36. Seftor R. E., Seftor E. A., Gehlsen K. R., Stetler-Stevenson W. G., Brown P. D., Ruoslahti E., Hendrix M. J. Role of the alpha v beta 3 integrin in human melanoma cell invasion. Proc Natl Acad Sci U S A. 1992 Mar 1;89(5):1557–1561. doi: 10.1073/pnas.89.5.1557. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Skubitz A. P., Letourneau P. C., Wayner E., Furcht L. T. Synthetic peptides from the carboxy-terminal globular domain of the A chain of laminin: their ability to promote cell adhesion and neurite outgrowth, and interact with heparin and the beta 1 integrin subunit. J Cell Biol. 1991 Nov;115(4):1137–1148. doi: 10.1083/jcb.115.4.1137. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Skubitz A. P., McCarthy J. B., Zhao Q., Yi X. Y., Furcht L. T. Definition of a sequence, RYVVLPR, within laminin peptide F-9 that mediates metastatic fibrosarcoma cell adhesion and spreading. Cancer Res. 1990 Dec 1;50(23):7612–7622. [PubMed] [Google Scholar]
  39. Sonnenberg A., Gehlsen K. R., Aumailley M., Timpl R. Isolation of alpha 6 beta 1 integrins from platelets and adherent cells by affinity chromatography on mouse laminin fragment E8 and human laminin pepsin fragment. Exp Cell Res. 1991 Dec;197(2):234–244. doi: 10.1016/0014-4827(91)90428-w. [DOI] [PubMed] [Google Scholar]
  40. Sonnenberg A., Linders C. J., Modderman P. W., Damsky C. H., Aumailley M., Timpl R. Integrin recognition of different cell-binding fragments of laminin (P1, E3, E8) and evidence that alpha 6 beta 1 but not alpha 6 beta 4 functions as a major receptor for fragment E8. J Cell Biol. 1990 Jun;110(6):2145–2155. doi: 10.1083/jcb.110.6.2145. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Sonnenberg A., Modderman P. W., Hogervorst F. Laminin receptor on platelets is the integrin VLA-6. Nature. 1988 Dec 1;336(6198):487–489. doi: 10.1038/336487a0. [DOI] [PubMed] [Google Scholar]
  42. Tashiro K., Sephel G. C., Greatorex D., Sasaki M., Shirashi N., Martin G. R., Kleinman H. K., Yamada Y. The RGD containing site of the mouse laminin A chain is active for cell attachment, spreading, migration and neurite outgrowth. J Cell Physiol. 1991 Mar;146(3):451–459. doi: 10.1002/jcp.1041460316. [DOI] [PubMed] [Google Scholar]
  43. Tashiro K., Sephel G. C., Weeks B., Sasaki M., Martin G. R., Kleinman H. K., Yamada Y. A synthetic peptide containing the IKVAV sequence from the A chain of laminin mediates cell attachment, migration, and neurite outgrowth. J Biol Chem. 1989 Sep 25;264(27):16174–16182. [PubMed] [Google Scholar]
  44. Terranova V. P., Rao C. N., Kalebic T., Margulies I. M., Liotta L. A. Laminin receptor on human breast carcinoma cells. Proc Natl Acad Sci U S A. 1983 Jan;80(2):444–448. doi: 10.1073/pnas.80.2.444. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Tomaselli K. J., Hall D. E., Flier L. A., Gehlsen K. R., Turner D. C., Carbonetto S., Reichardt L. F. A neuronal cell line (PC12) expresses two beta 1-class integrins-alpha 1 beta 1 and alpha 3 beta 1-that recognize different neurite outgrowth-promoting domains in laminin. Neuron. 1990 Nov;5(5):651–662. doi: 10.1016/0896-6273(90)90219-6. [DOI] [PubMed] [Google Scholar]
  46. Welch D. R., Bisi J. E., Miller B. E., Conaway D., Seftor E. A., Yohem K. H., Gilmore L. B., Seftor R. E., Nakajima M., Hendrix M. J. Characterization of a highly invasive and spontaneously metastatic human malignant melanoma cell line. Int J Cancer. 1991 Jan 21;47(2):227–237. doi: 10.1002/ijc.2910470211. [DOI] [PubMed] [Google Scholar]
  47. Wewer U. M., Taraboletti G., Sobel M. E., Albrechtsen R., Liotta L. A. Role of laminin receptor in tumor cell migration. Cancer Res. 1987 Nov 1;47(21):5691–5698. [PubMed] [Google Scholar]
  48. Wilke M. S., Skubitz A. P. Human keratinocytes adhere to multiple distinct peptide sequences of laminin. J Invest Dermatol. 1991 Jul;97(1):141–146. doi: 10.1111/1523-1747.ep12479311. [DOI] [PubMed] [Google Scholar]
  49. Wright S. D., Rao P. E., Van Voorhis W. C., Craigmyle L. S., Iida K., Talle M. A., Westberg E. F., Goldstein G., Silverstein S. C. Identification of the C3bi receptor of human monocytes and macrophages by using monoclonal antibodies. Proc Natl Acad Sci U S A. 1983 Sep;80(18):5699–5703. doi: 10.1073/pnas.80.18.5699. [DOI] [PMC free article] [PubMed] [Google Scholar]

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