Integrin α2β1 is the major receptor for collagens in the human body, and the collagen-binding site on the α2 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 α2 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 α2β1 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 Thr221 to alanine. Binding of cluster I antibodies was inhibited by the anti-functional anti-β1 antibody Mab13, and binding of Gi9 and JA218 to α2β1 was inhibited by substituting Mn2+ for Mg2+, 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.
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September 2000
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Research Article|
August 23 2000
Monoclonal antibodies identify residues 199–216 of the integrin α2 vWFA domain as a functionally important region within α2β1
Danny S. TUCKWELL;
Danny S. TUCKWELL
1
*Wellcome Trust Centre for Cell-Matrix Research, School of Biological Sciences, University of Manchester, Oxford Road, Manchester M13 9PT, U.K.
1To whom correspondence should be addressed (e-mail danny.tuckwell@man.ac.uk).
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Lyndsay SMITH;
Lyndsay SMITH
*Wellcome Trust Centre for Cell-Matrix Research, School of Biological Sciences, University of Manchester, Oxford Road, Manchester M13 9PT, U.K.
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Michelle KORDA;
Michelle KORDA
*Wellcome Trust Centre for Cell-Matrix Research, School of Biological Sciences, University of Manchester, Oxford Road, Manchester M13 9PT, U.K.
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Janet A. ASKARI;
Janet A. ASKARI
*Wellcome Trust Centre for Cell-Matrix Research, School of Biological Sciences, University of Manchester, Oxford Road, Manchester M13 9PT, U.K.
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Sentot SANTOSO;
Sentot SANTOSO
†Institute for Clinical Immunology and Transfusion Medicine, Justus-Liebig University, Giessen, Germany
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Michael J. BARNES;
Michael J. BARNES
‡Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 1QW, U.K.
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Richard W. FARNDALE;
Richard W. FARNDALE
‡Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 1QW, U.K.
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Martin J. HUMPHRIES
Martin J. HUMPHRIES
*Wellcome Trust Centre for Cell-Matrix Research, School of Biological Sciences, University of Manchester, Oxford Road, Manchester M13 9PT, U.K.
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Publisher: Portland Press Ltd
Received:
March 10 2000
Revision Received:
May 25 2000
Accepted:
June 23 2000
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London © 2000
2000
Biochem J (2000) 350 (2): 485–493.
Article history
Received:
March 10 2000
Revision Received:
May 25 2000
Accepted:
June 23 2000
Citation
Danny S. TUCKWELL, Lyndsay SMITH, Michelle KORDA, Janet A. ASKARI, Sentot SANTOSO, Michael J. BARNES, Richard W. FARNDALE, Martin J. HUMPHRIES; Monoclonal antibodies identify residues 199–216 of the integrin α2 vWFA domain as a functionally important region within α2β1. Biochem J 1 September 2000; 350 (2): 485–493. doi: https://doi.org/10.1042/bj3500485
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