Skip to main content
NCBI home page
Biochemical Journal logoLink to Biochemical Journal
. 2000 Sep 1;350(Pt 2):485–493.

Monoclonal antibodies identify residues 199-216 of the integrin alpha2 vWFA domain as a functionally important region within alpha2beta1.

D S Tuckwell 1, L Smith 1, M Korda 1, J A Askari 1, S Santoso 1, M J Barnes 1, R W Farndale 1, M J Humphries 1
PMCID: PMC1221276  PMID: 10947963

Abstract

Integrin alpha2beta1 is the major receptor for collagens in the human body, and the collagen-binding site on the alpha2 subunit von Willebrand factor A-type domain (vWFA domain) is now well defined. However, the biologically important conformational changes that are associated with collagen binding, and the means by which the vWFA domain is integrated into the whole integrin are not completely understood. We have raised monoclonal antibodies against recombinant alpha2 vWFA domain for use as probes of function. Three antibodies, JA202, JA215 and JA218, inhibited binding to collagen, collagen I C-propeptide and E-cadherin, demonstrating that their function is important for structurally diverse alpha2beta1 ligands. Cross-blocking studies grouped the epitopes into two clusters: (I) JA202, the inhibitory antibody, Gi9, and a non-inhibitory antibody, JA208; (II) JA215 and JA218. Both clusters were sensitive to events at the collagen binding site, as binding of Gi9, JA202, JA215 and JA218 were inhibited by collagen peptide, JA208 binding was enhanced by collagen peptide, and binding of JA202 was decreased after mutagenesis of the cation-binding residue Thr(221) to alanine. Binding of cluster I antibodies was inhibited by the anti-functional anti-beta1 antibody Mab13, and binding of Gi9 and JA218 to alpha2beta1 was inhibited by substituting Mn(2+) for Mg(2+), demonstrating that these antibodies were sensitive to changes initiated outside the vWFA domain. Mapping of epitopes showed that JA202 and Gi9 bound between residues 212-216, while JA208 bound between residues 199-216. We have therefore identified two epitope clusters with novel properties; i.e. they are intimately associated with the collagen-binding site, responsive to conformational changes at the collagen-binding site and sensitive to events initiated outside the vWFA domain.

Full Text

The Full Text of this article is available as a PDF (243.8 KB).

Selected References

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

  1. Bazzoni G., Shih D. T., Buck C. A., Hemler M. E. Monoclonal antibody 9EG7 defines a novel beta 1 integrin epitope induced by soluble ligand and manganese, but inhibited by calcium. J Biol Chem. 1995 Oct 27;270(43):25570–25577. doi: 10.1074/jbc.270.43.25570. [DOI] [PubMed] [Google Scholar]
  2. Bergelson J. M., Shepley M. P., Chan B. M., Hemler M. E., Finberg R. W. Identification of the integrin VLA-2 as a receptor for echovirus 1. Science. 1992 Mar 27;255(5052):1718–1720. doi: 10.1126/science.1553561. [DOI] [PubMed] [Google Scholar]
  3. Binnerts M. E., van Kooyk Y., Edwards C. P., Champe M., Presta L., Bodary S. C., Figdor C. G., Berman P. W. Antibodies that selectively inhibit leukocyte function-associated antigen 1 binding to intercellular adhesion molecule-3 recognize a unique epitope within the CD11a I domain. J Biol Chem. 1996 Apr 26;271(17):9962–9968. doi: 10.1074/jbc.271.17.9962. [DOI] [PubMed] [Google Scholar]
  4. Brookman J. L., Stott A. J., Cheeseman P. J., Burns N. R., Adams S. E., Kingsman A. J., Gull K. An immunological analysis of Ty1 virus-like particle structure. Virology. 1995 Feb 20;207(1):59–67. doi: 10.1006/viro.1995.1051. [DOI] [PubMed] [Google Scholar]
  5. Calderwood D. A., Tuckwell D. S., Eble J., Kühn K., Humphries M. J. The integrin alpha1 A-domain is a ligand binding site for collagens and laminin. J Biol Chem. 1997 May 9;272(19):12311–12317. doi: 10.1074/jbc.272.19.12311. [DOI] [PubMed] [Google Scholar]
  6. Colombatti A., Bonaldo P., Doliana R. Type A modules: interacting domains found in several non-fibrillar collagens and in other extracellular matrix proteins. Matrix. 1993 Jul;13(4):297–306. doi: 10.1016/s0934-8832(11)80025-9. [DOI] [PubMed] [Google Scholar]
  7. Davies D., Tuckwell D. S., Calderwood D. A., Weston S. A., Takigawa M., Humphries M. J. Molecular characterisation of integrin-procollagen C-propeptide interactions. Eur J Biochem. 1997 Jun 1;246(2):274–282. doi: 10.1111/j.1432-1033.1997.t01-1-00274.x. [DOI] [PubMed] [Google Scholar]
  8. Depraetere H., Wille C., Gansemans Y., Stanssens P., Lauwereys M., Baruch D., De Reys S., Deckmyn H. The integrin alpha 2 beta 1 (GPIa/IIa)-I-domain inhibits platelet-collagen interaction. Thromb Haemost. 1997 May;77(5):981–985. [PubMed] [Google Scholar]
  9. Dickeson S. K., Mathis N. L., Rahman M., Bergelson J. M., Santoro S. A. Determinants of ligand binding specificity of the alpha(1)beta(1) and alpha(2)beta(1) integrins. J Biol Chem. 1999 Nov 5;274(45):32182–32191. doi: 10.1074/jbc.274.45.32182. [DOI] [PubMed] [Google Scholar]
  10. Dickeson S. K., Walsh J. J., Santoro S. A. Contributions of the I and EF hand domains to the divalent cation-dependent collagen binding activity of the alpha2beta1 integrin. J Biol Chem. 1997 Mar 21;272(12):7661–7668. doi: 10.1074/jbc.272.12.7661. [DOI] [PubMed] [Google Scholar]
  11. Edwards C. P., Champe M., Gonzalez T., Wessinger M. E., Spencer S. A., Presta L. G., Berman P. W., Bodary S. C. Identification of amino acids in the CD11a I-domain important for binding of the leukocyte function-associated antigen-1 (LFA-1) to intercellular adhesion molecule-1 (ICAM-1) J Biol Chem. 1995 May 26;270(21):12635–12640. doi: 10.1074/jbc.270.21.12635. [DOI] [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. Emsley J., King S. L., Bergelson J. M., Liddington R. C. Crystal structure of the I domain from integrin alpha2beta1. J Biol Chem. 1997 Nov 7;272(45):28512–28517. doi: 10.1074/jbc.272.45.28512. [DOI] [PubMed] [Google Scholar]
  14. Emsley J., Knight C. G., Farndale R. W., Barnes M. J., Liddington R. C. Structural basis of collagen recognition by integrin alpha2beta1. Cell. 2000 Mar 31;101(1):47–56. doi: 10.1016/S0092-8674(00)80622-4. [DOI] [PubMed] [Google Scholar]
  15. Fleischmajer R., Perlish J. S., MacDonald E. D., 2nd, Schechter A., Murdoch A. D., Iozzo R. V., Yamada Y. There is binding of collagen IV to beta 1 integrin during early skin basement membrane assembly. Ann N Y Acad Sci. 1998 Oct 23;857:212–227. doi: 10.1111/j.1749-6632.1998.tb10118.x. [DOI] [PubMed] [Google Scholar]
  16. Hangan D., Uniyal S., Morris V. L., MacDonald I. C., von Ballestrem C., Chau T., Schmidt E. E., Chambers A. F., Groom A. C., Chan B. M. Integrin VLA-2 (alpha2beta1) function in postextravasation movement of human rhabdomyosarcoma RD cells in the liver. Cancer Res. 1996 Jul 1;56(13):3142–3149. [PubMed] [Google Scholar]
  17. Jenkins G., Redwood K. L., Meadows L., Green M. R. Effect of gel re-organization and tensional forces on alpha2beta1 integrin levels in dermal fibroblasts. Eur J Biochem. 1999 Jul;263(1):93–103. doi: 10.1046/j.1432-1327.1999.00468.x. [DOI] [PubMed] [Google Scholar]
  18. Kamata T., Liddington R. C., Takada Y. Interaction between collagen and the alpha(2) I-domain of integrin alpha(2)beta(1). Critical role of conserved residues in the metal ion-dependent adhesion site (MIDAS) region. J Biol Chem. 1999 Nov 5;274(45):32108–32111. doi: 10.1074/jbc.274.45.32108. [DOI] [PubMed] [Google Scholar]
  19. Kamata T., Puzon W., Takada Y. Identification of putative ligand binding sites within I domain of integrin alpha 2 beta 1 (VLA-2, CD49b/CD29) J Biol Chem. 1994 Apr 1;269(13):9659–9663. [PubMed] [Google Scholar]
  20. Kamata T., Takada Y. Direct binding of collagen to the I domain of integrin alpha 2 beta 1 (VLA-2, CD49b/CD29) in a divalent cation-independent manner. J Biol Chem. 1994 Oct 21;269(42):26006–26010. [PubMed] [Google Scholar]
  21. King S. L., Cunningham J. A., Finberg R. W., Bergelson J. M. Echovirus 1 interaction with the isolated VLA-2 I domain. J Virol. 1995 May;69(5):3237–3239. doi: 10.1128/jvi.69.5.3237-3239.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Kirchhofer D., Grzesiak J., Pierschbacher M. D. Calcium as a potential physiological regulator of integrin-mediated cell adhesion. J Biol Chem. 1991 Mar 5;266(7):4471–4477. [PubMed] [Google Scholar]
  23. Knight C. G., Morton L. F., Onley D. J., Peachey A. R., Messent A. J., Smethurst P. A., Tuckwell D. S., Farndale R. W., Barnes M. J. Identification in collagen type I of an integrin alpha2 beta1-binding site containing an essential GER sequence. J Biol Chem. 1998 Dec 11;273(50):33287–33294. doi: 10.1074/jbc.273.50.33287. [DOI] [PubMed] [Google Scholar]
  24. Knight C. G., Morton L. F., Peachey A. R., Tuckwell D. S., Farndale R. W., Barnes M. J. The collagen-binding A-domains of integrins alpha(1)beta(1) and alpha(2)beta(1) recognize the same specific amino acid sequence, GFOGER, in native (triple-helical) collagens. J Biol Chem. 2000 Jan 7;275(1):35–40. doi: 10.1074/jbc.275.1.35. [DOI] [PubMed] [Google Scholar]
  25. 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]
  26. Langholz O., Röckel D., Mauch C., Kozlowska E., Bank I., Krieg T., Eckes B. Collagen and collagenase gene expression in three-dimensional collagen lattices are differentially regulated by alpha 1 beta 1 and alpha 2 beta 1 integrins. J Cell Biol. 1995 Dec;131(6 Pt 2):1903–1915. doi: 10.1083/jcb.131.6.1903. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Lee J. O., Rieu P., Arnaout M. A., Liddington R. Crystal structure of the A domain from the alpha subunit of integrin CR3 (CD11b/CD18). Cell. 1995 Feb 24;80(4):631–638. doi: 10.1016/0092-8674(95)90517-0. [DOI] [PubMed] [Google Scholar]
  28. Li R., Rieu P., Griffith D. L., Scott D., Arnaout M. A. Two functional states of the CD11b A-domain: correlations with key features of two Mn2+-complexed crystal structures. J Cell Biol. 1998 Dec 14;143(6):1523–1534. doi: 10.1083/jcb.143.6.1523. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Messent A. J., Tuckwell D. S., Knäuper V., Humphries M. J., Murphy G., Gavrilovic J. Effects of collagenase-cleavage of type I collagen on alpha2beta1 integrin-mediated cell adhesion. J Cell Sci. 1998 Apr;111(Pt 8):1127–1135. doi: 10.1242/jcs.111.8.1127. [DOI] [PubMed] [Google Scholar]
  30. Michishita M., Videm V., Arnaout M. A. A novel divalent cation-binding site in the A domain of the beta 2 integrin CR3 (CD11b/CD18) is essential for ligand binding. Cell. 1993 Mar 26;72(6):857–867. doi: 10.1016/0092-8674(93)90575-b. [DOI] [PubMed] [Google Scholar]
  31. Mould A. P., Akiyama S. K., Humphries M. J. Regulation of integrin alpha 5 beta 1-fibronectin interactions by divalent cations. Evidence for distinct classes of binding sites for Mn2+, Mg2+, and Ca2+. J Biol Chem. 1995 Nov 3;270(44):26270–26277. doi: 10.1074/jbc.270.44.26270. [DOI] [PubMed] [Google Scholar]
  32. Mould A. P., Akiyama S. K., Humphries M. J. The inhibitory anti-beta1 integrin monoclonal antibody 13 recognizes an epitope that is attenuated by ligand occupancy. Evidence for allosteric inhibition of integrin function. J Biol Chem. 1996 Aug 23;271(34):20365–20374. doi: 10.1074/jbc.271.34.20365. [DOI] [PubMed] [Google Scholar]
  33. Mould A. P., Askari J. A., Akiyama S. K., Yamada K. M., Humphries M. J. An assessment of the efficacy of anti-integrin alpha subunit monoclonal antibody production using affinity purified beta 1-integrin dimers as immunogen. Biochem Soc Trans. 1991 Nov;19(4):361S–361S. doi: 10.1042/bst019361s. [DOI] [PubMed] [Google Scholar]
  34. Mould A. P., Garratt A. N., Askari J. A., Akiyama S. K., Humphries M. J. Identification of a novel anti-integrin monoclonal antibody that recognises a ligand-induced binding site epitope on the beta 1 subunit. FEBS Lett. 1995 Apr 17;363(1-2):118–122. doi: 10.1016/0014-5793(95)00301-o. [DOI] [PubMed] [Google Scholar]
  35. Mould A. P. Getting integrins into shape: recent insights into how integrin activity is regulated by conformational changes. J Cell Sci. 1996 Nov;109(Pt 11):2613–2618. doi: 10.1242/jcs.109.11.2613. [DOI] [PubMed] [Google Scholar]
  36. Nieuwenhuis H. K., Akkerman J. W., Houdijk W. P., Sixma J. J. Human blood platelets showing no response to collagen fail to express surface glycoprotein Ia. Nature. 1985 Dec 5;318(6045):470–472. doi: 10.1038/318470a0. [DOI] [PubMed] [Google Scholar]
  37. Oxvig C., Lu C., Springer T. A. Conformational changes in tertiary structure near the ligand binding site of an integrin I domain. Proc Natl Acad Sci U S A. 1999 Mar 2;96(5):2215–2220. doi: 10.1073/pnas.96.5.2215. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Riikonen T., Westermarck J., Koivisto L., Broberg A., Kähäri V. M., Heino J. Integrin alpha 2 beta 1 is a positive regulator of collagenase (MMP-1) and collagen alpha 1(I) gene expression. J Biol Chem. 1995 Jun 2;270(22):13548–13552. doi: 10.1074/jbc.270.22.13548. [DOI] [PubMed] [Google Scholar]
  39. Santoro S. A. Identification of a 160,000 dalton platelet membrane protein that mediates the initial divalent cation-dependent adhesion of platelets to collagen. Cell. 1986 Sep 12;46(6):913–920. doi: 10.1016/0092-8674(86)90073-5. [DOI] [PubMed] [Google Scholar]
  40. Santoso S., Kalb R., Walka M., Kiefel V., Mueller-Eckhardt C., Newman P. J. The human platelet alloantigens Br(a) and Brb are associated with a single amino acid polymorphism on glycoprotein Ia (integrin subunit alpha 2). J Clin Invest. 1993 Nov;92(5):2427–2432. doi: 10.1172/JCI116849. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Schiro J. A., Chan B. M., Roswit W. T., Kassner P. D., Pentland A. P., Hemler M. E., Eisen A. Z., Kupper T. S. Integrin alpha 2 beta 1 (VLA-2) mediates reorganization and contraction of collagen matrices by human cells. Cell. 1991 Oct 18;67(2):403–410. doi: 10.1016/0092-8674(91)90191-z. [DOI] [PubMed] [Google Scholar]
  42. Smith C., Estavillo D., Emsley J., Bankston L. A., Liddington R. C., Cruz M. A. Mapping the collagen-binding site in the I domain of the glycoprotein Ia/IIa (integrin alpha(2)beta(1)). J Biol Chem. 2000 Feb 11;275(6):4205–4209. doi: 10.1074/jbc.275.6.4205. [DOI] [PubMed] [Google Scholar]
  43. Takada Y., Puzon W. Identification of a regulatory region of integrin beta 1 subunit using activating and inhibiting antibodies. J Biol Chem. 1993 Aug 15;268(23):17597–17601. [PubMed] [Google Scholar]
  44. Tuckwell D. S., Ayad S., Grant M. E., Takigawa M., Humphries M. J. Conformation dependence of integrin-type II collagen binding. Inability of collagen peptides to support alpha 2 beta 1 binding, and mediation of adhesion to denatured collagen by a novel alpha 5 beta 1-fibronectin bridge. J Cell Sci. 1994 Apr;107(Pt 4):993–1005. doi: 10.1242/jcs.107.4.993. [DOI] [PubMed] [Google Scholar]
  45. Tuckwell D. S., Reid K. B., Barnes M. J., Humphries M. J. The A-domain of integrin alpha 2 binds specifically to a range of collagens but is not a general receptor for the collagenous motif. Eur J Biochem. 1996 Nov 1;241(3):732–739. doi: 10.1111/j.1432-1033.1996.00732.x. [DOI] [PubMed] [Google Scholar]
  46. Tuckwell D., Calderwood D. A., Green L. J., Humphries M. J. Integrin alpha 2 I-domain is a binding site for collagens. J Cell Sci. 1995 Apr;108(Pt 4):1629–1637. doi: 10.1242/jcs.108.4.1629. [DOI] [PubMed] [Google Scholar]
  47. Tuckwell D. Evolution of von Willebrand factor A (VWA) domains. Biochem Soc Trans. 1999 Dec;27(6):835–840. doi: 10.1042/bst0270835. [DOI] [PubMed] [Google Scholar]
  48. Vandenberg P., Kern A., Ries A., Luckenbill-Edds L., Mann K., Kühn K. 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. J Cell Biol. 1991 Jun;113(6):1475–1483. doi: 10.1083/jcb.113.6.1475. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Weston S. A., Hulmes D. J., Mould A. P., Watson R. B., Humphries M. J. Identification of integrin alpha 2 beta 1 as cell surface receptor for the carboxyl-terminal propeptide of type I procollagen. J Biol Chem. 1994 Aug 19;269(33):20982–20986. [PubMed] [Google Scholar]
  50. Zhang L., Plow E. F. A discrete site modulates activation of I domains. Application to integrin alphaMbeta2. J Biol Chem. 1996 Nov 22;271(47):29953–29957. doi: 10.1074/jbc.271.47.29953. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES