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. 1991 Jun 2;113(6):1475–1483. doi: 10.1083/jcb.113.6.1475

Characterization of a type IV collagen major cell binding site with affinity to the alpha 1 beta 1 and the alpha 2 beta 1 integrins

PMCID: PMC2289033  PMID: 1646206

Abstract

The aim of this investigation was to identify the domains of type IV collagen participating in cell binding and the cell surface receptor involved. A major cell binding site was found in the trimeric cyanogen bromide-derived fragment CB3, located 100 nm away from the NH2 terminus of the molecule, in which the triple-helical conformation is stabilized by interchain disulfide bridges. Cell attachment assays with type IV collagen and CB3 revealed comparable cell binding activities. Antibodies against CB3 inhibited attachment on fragment CB3 completely and on type IV collagen to 80%. The ability to bind cells was strictly conformation dependent. Four trypsin derived fragments of CB3 allowed a closer investigation of the binding site. The smallest, fully active triple-helical fragment was (150)3-amino acid residues long. It contained segments of 27 and 37 residues, respectively, at the NH2 and COOH terminus, which proved to be essential for cell binding. By affinity chromatography on Sepharose-immobilized CB3, two receptor molecules of the integrin family, alpha 1 beta 1 and alpha 2 beta 1, were isolated. Their subunits were identified by sequencing the NH2 termini or by immunoblotting. The availability of fragment CB3 will allow for a more in-depth study of the molecular interaction of a short, well defined triple-helical ligand with collagen receptors alpha 1 beta 1 and alpha 2 beta 1.

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Selected References

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

  1. Albelda S. M., Buck C. A. Integrins and other cell adhesion molecules. FASEB J. 1990 Aug;4(11):2868–2880. [PubMed] [Google Scholar]
  2. Allmann H., Fietzek P. P., Glanville R. W., Kühn K. The covalent structure of calf skin type III collagen. VI. The amino acid sequence of the carboxyterminal cyanogen bromide peptide alpha 1(III)CB9B (position 928--1028). Hoppe Seylers Z Physiol Chem. 1979 Jul;360(7):861–868. [PubMed] [Google Scholar]
  3. Aumailley M., Mann K., von der Mark H., Timpl R. Cell attachment properties of collagen type VI and Arg-Gly-Asp dependent binding to its alpha 2(VI) and alpha 3(VI) chains. Exp Cell Res. 1989 Apr;181(2):463–474. doi: 10.1016/0014-4827(89)90103-1. [DOI] [PubMed] [Google Scholar]
  4. Aumailley M., Nurcombe V., Edgar D., Paulsson M., Timpl R. The cellular interactions of laminin fragments. Cell adhesion correlates with two fragment-specific high affinity binding sites. J Biol Chem. 1987 Aug 25;262(24):11532–11538. [PubMed] [Google Scholar]
  5. Aumailley M., Timpl R. Attachment of cells to basement membrane collagen type IV. J Cell Biol. 1986 Oct;103(4):1569–1575. doi: 10.1083/jcb.103.4.1569. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Borg T. K., Rubin K., Lundgren E., Borg K., Obrink B. Recognition of extracellular matrix components by neonatal and adult cardiac myocytes. Dev Biol. 1984 Jul;104(1):86–96. doi: 10.1016/0012-1606(84)90038-1. [DOI] [PubMed] [Google Scholar]
  7. Brazel D., Pollner R., Oberbäumer I., Kühn K. Human basement membrane collagen (type IV). The amino acid sequence of the alpha 2(IV) chain and its comparison with the alpha 1(IV) chain reveals deletions in the alpha 1(IV) chain. Eur J Biochem. 1988 Feb 15;172(1):35–42. doi: 10.1111/j.1432-1033.1988.tb13852.x. [DOI] [PubMed] [Google Scholar]
  8. Bächinger H. P., Bruckner P., Timpl R., Prockop D. J., Engel J. Folding mechanism of the triple helix in type-III collagen and type-III pN-collagen. Role of disulfide bridges and peptide bond isomerization. Eur J Biochem. 1980 May;106(2):619–632. doi: 10.1111/j.1432-1033.1980.tb04610.x. [DOI] [PubMed] [Google Scholar]
  9. Chelberg M. K., McCarthy J. B., Skubitz A. P., Furcht L. T., Tsilibary E. C. Characterization of a synthetic peptide from type IV collagen that promotes melanoma cell adhesion, spreading, and motility. J Cell Biol. 1990 Jul;111(1):261–270. doi: 10.1083/jcb.111.1.261. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Clyman R. I., Turner D. C., Kramer R. H. An alpha 1/beta 1-like integrin receptor on rat aortic smooth muscle cells mediates adhesion to laminin and collagen types I and IV. Arteriosclerosis. 1990 May-Jun;10(3):402–409. doi: 10.1161/01.atv.10.3.402. [DOI] [PubMed] [Google Scholar]
  11. Collier I. E., Wilhelm S. M., Eisen A. Z., Marmer B. L., Grant G. A., Seltzer J. L., Kronberger A., He C. S., Bauer E. A., Goldberg G. I. H-ras oncogene-transformed human bronchial epithelial cells (TBE-1) secrete a single metalloprotease capable of degrading basement membrane collagen. J Biol Chem. 1988 May 15;263(14):6579–6587. [PubMed] [Google Scholar]
  12. Dennis J., Waller C., Timpl R., Schirrmacher V. Surface sialic acid reduces attachment of metastatic tumour cells to collagen type IV and fibronectin. Nature. 1982 Nov 18;300(5889):274–276. doi: 10.1038/300274a0. [DOI] [PubMed] [Google Scholar]
  13. Dieringer H., Hollister D. W., Glanville R. W., Sakai L. Y., Kühn K. Structural studies of human basement-membrane collagen with the use of a monoclonal antibody. Biochem J. 1985 Apr 1;227(1):217–222. doi: 10.1042/bj2270217. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Dölz R., Engel J., Kühn K. Folding of collagen IV. Eur J Biochem. 1988 Dec 15;178(2):357–366. doi: 10.1111/j.1432-1033.1988.tb14458.x. [DOI] [PubMed] [Google Scholar]
  15. Engvall E., Bell M. L., Carlsson R. N., Miller E. J., Ruoslahti E. Nonhelical, fibronectin-binding basement-membrane collagen from endodermal cell culture. Cell. 1982 Jun;29(2):475–482. doi: 10.1016/0092-8674(82)90164-7. [DOI] [PubMed] [Google Scholar]
  16. Fauvel F., Legrand Y. J., Bentz H., Fietzek P. P., Kuhn K., Caen J. P. Platelet-collagen interaction: adhesion of human blood platelets to purified (CB4) peptide from type III collagen. Thromb Res. 1978 May;12(5):841–850. doi: 10.1016/0049-3848(78)90278-5. [DOI] [PubMed] [Google Scholar]
  17. Fessler L. I., Duncan K. G., Fessler J. H., Salo T., Tryggvason K. Characterization of the procollagen IV cleavage products produced by a specific tumor collagenase. J Biol Chem. 1984 Aug 10;259(15):9783–9789. [PubMed] [Google Scholar]
  18. Forsberg E., Paulsson M., Timpl R., Johansson S. Characterization of a laminin receptor on rat hepatocytes. J Biol Chem. 1990 Apr 15;265(11):6376–6381. [PubMed] [Google Scholar]
  19. 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]
  20. Gillies R. J., Didier N., Denton M. Determination of cell number in monolayer cultures. Anal Biochem. 1986 Nov 15;159(1):109–113. doi: 10.1016/0003-2697(86)90314-3. [DOI] [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. Hostikka S. L., Tryggvason K. The complete primary structure of the alpha 2 chain of human type IV collagen and comparison with the alpha 1(IV) chain. J Biol Chem. 1988 Dec 25;263(36):19488–19493. [PubMed] [Google Scholar]
  23. 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]
  24. 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]
  25. 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]
  26. Kleinman H. K., McGarvey M. L., Liotta L. A., Robey P. G., Tryggvason K., Martin G. R. Isolation and characterization of type IV procollagen, laminin, and heparan sulfate proteoglycan from the EHS sarcoma. Biochemistry. 1982 Nov 23;21(24):6188–6193. doi: 10.1021/bi00267a025. [DOI] [PubMed] [Google Scholar]
  27. Kramer R. H., Marks N. Identification of integrin collagen receptors on human melanoma cells. J Biol Chem. 1989 Mar 15;264(8):4684–4688. [PubMed] [Google Scholar]
  28. Kühn K., Wiedemann H., Timpl R., Risteli J., Dieringer H., Voss T., Glanville R. W. Macromolecular structure of basement membrane collagens. FEBS Lett. 1981 Mar 9;125(1):123–128. doi: 10.1016/0014-5793(81)81012-5. [DOI] [PubMed] [Google Scholar]
  29. Marcantonio E. E., Hynes R. O. Antibodies to the conserved cytoplasmic domain of the integrin beta 1 subunit react with proteins in vertebrates, invertebrates, and fungi. J Cell Biol. 1988 May;106(5):1765–1772. doi: 10.1083/jcb.106.5.1765. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Matsudaira P. Sequence from picomole quantities of proteins electroblotted onto polyvinylidene difluoride membranes. J Biol Chem. 1987 Jul 25;262(21):10035–10038. [PubMed] [Google Scholar]
  31. Murray J. C., Stingl G., Kleinman H. K., Martin G. R., Katz S. I. Epidermal cells adhere preferentially to type IV (basement membrane) collagen. J Cell Biol. 1979 Jan;80(1):197–202. doi: 10.1083/jcb.80.1.197. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Oberbäumer I., Laurent M., Schwarz U., Sakurai Y., Yamada Y., Vogeli G., Voss T., Siebold B., Glanville R. W., Kühn K. Amino acid sequence of the non-collagenous globular domain (NC1) of the alpha 1(IV) chain of basement membrane collagen as derived from complementary DNA. Eur J Biochem. 1985 Mar 1;147(2):217–224. doi: 10.1111/j.1432-1033.1985.tb08739.x. [DOI] [PubMed] [Google Scholar]
  33. Palotie A., Peltonen L., Risteli L., Risteli J. Effect of the structural components of basement membranes on the attachment of teratocarcinoma-derived endodermal cells. Exp Cell Res. 1983 Mar;144(1):31–37. doi: 10.1016/0014-4827(83)90438-x. [DOI] [PubMed] [Google Scholar]
  34. Palotie A., Tryggvason K., Peltonen L., Seppä H. Components of subendothelial aorta basement membrane. Immunohistochemical localization and role in cell attachment. Lab Invest. 1983 Sep;49(3):362–370. [PubMed] [Google Scholar]
  35. 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]
  36. Santoro S. A., Rajpara S. M., Staatz W. D., Woods V. L., Jr Isolation and characterization of a platelet surface collagen binding complex related to VLA-2. Biochem Biophys Res Commun. 1988 May 31;153(1):217–223. doi: 10.1016/s0006-291x(88)81211-7. [DOI] [PubMed] [Google Scholar]
  37. Staatz W. D., Rajpara S. M., Wayner E. A., Carter W. G., Santoro S. A. The membrane glycoprotein Ia-IIa (VLA-2) complex mediates the Mg++-dependent adhesion of platelets to collagen. J Cell Biol. 1989 May;108(5):1917–1924. doi: 10.1083/jcb.108.5.1917. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Staatz W. D., Walsh J. J., Pexton T., Santoro S. A. The alpha 2 beta 1 integrin cell surface collagen receptor binds to the alpha 1 (I)-CB3 peptide of collagen. J Biol Chem. 1990 Mar 25;265(9):4778–4781. [PubMed] [Google Scholar]
  39. Takada Y., Hemler M. E. The primary structure of the VLA-2/collagen receptor alpha 2 subunit (platelet GPIa): homology to other integrins and the presence of a possible collagen-binding domain. J Cell Biol. 1989 Jul;109(1):397–407. doi: 10.1083/jcb.109.1.397. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. 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]
  41. Thorgeirsson U. P., Turpeenniemi-Hujanen T., Williams J. E., Westin E. H., Heilman C. A., Talmadge J. E., Liotta L. A. NIH/3T3 cells transfected with human tumor DNA containing activated ras oncogenes express the metastatic phenotype in nude mice. Mol Cell Biol. 1985 Jan;5(1):259–262. doi: 10.1128/mcb.5.1.259. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Timpl R. Antibodies to collagens and procollagens. Methods Enzymol. 1982;82(Pt A):472–498. doi: 10.1016/0076-6879(82)82079-x. [DOI] [PubMed] [Google Scholar]
  43. Timpl R., Dziadek M. Structure, development, and molecular pathology of basement membranes. Int Rev Exp Pathol. 1986;29:1–112. [PubMed] [Google Scholar]
  44. Timpl R., Wiedemann H., van Delden V., Furthmayr H., Kühn K. A network model for the organization of type IV collagen molecules in basement membranes. Eur J Biochem. 1981 Nov;120(2):203–211. doi: 10.1111/j.1432-1033.1981.tb05690.x. [DOI] [PubMed] [Google Scholar]
  45. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Vlodavsky I., Levi A., Lax I., Fuks Z., Schlessinger J. Induction of cell attachment and morphological differentiation in a pheochromocytoma cell line and embryonal sensory cells by the extracellular matrix. Dev Biol. 1982 Oct;93(2):285–300. doi: 10.1016/0012-1606(82)90118-x. [DOI] [PubMed] [Google Scholar]
  47. Wayner E. A., Carter W. G. Identification of multiple cell adhesion receptors for collagen and fibronectin in human fibrosarcoma cells possessing unique alpha and common beta subunits. J Cell Biol. 1987 Oct;105(4):1873–1884. doi: 10.1083/jcb.105.4.1873. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Weber S., Engel J., Wiedemann H., Glanville R. W., Timpl R. Subunit structure and assembly of the globular domain of basement-membrane collagen type IV. Eur J Biochem. 1984 Mar 1;139(2):401–410. doi: 10.1111/j.1432-1033.1984.tb08019.x. [DOI] [PubMed] [Google Scholar]
  49. Wilke M. S., Furcht L. T. Human keratinocytes adhere to a unique heparin-binding peptide sequence within the triple helical region of type IV collagen. J Invest Dermatol. 1990 Sep;95(3):264–270. doi: 10.1111/1523-1747.ep12484883. [DOI] [PubMed] [Google Scholar]
  50. Yurchenco P. D., Furthmayr H. Self-assembly of basement membrane collagen. Biochemistry. 1984 Apr 10;23(8):1839–1850. doi: 10.1021/bi00303a040. [DOI] [PubMed] [Google Scholar]

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