Integrin α5β1 is a key receptor for the extracellular matrix protein fibronectin. Antagonists of human integrin α5β1 have therapeutic potential as anti-angiogenic agents in cancer and diseases of the eye. However, the structure of the integrin is unsolved and the atomic basis of fibronectin and antagonist binding by integrin α5β1 is poorly understood. In the present study, we demonstrate that zebrafish α5β1 integrins do not interact with human fibronectin or the human α5β1 antagonists JSM6427 and cyclic peptide CRRETAWAC. Zebrafish α5β1 integrins do bind zebrafish fibronectin-1, and mutagenesis of residues on the upper surface and side of the zebrafish α5 subunit β-propeller domain shows that these residues are important for the recognition of the Arg-Gly-Asp (RGD) motif and the synergy sequence [Pro-His-Ser-Arg-Asn (PHSRN)] in fibronectin. Using a gain-of-function analysis involving swapping regions of the zebrafish integrin α5 subunit with the corresponding regions of human α5 we show that blades 1–4 of the β-propeller are required for human fibronectin recognition, suggesting that fibronectin binding involves a broad interface on the side and upper face of the β-propeller domain. We find that the loop connecting blades 2 and 3 of the β-propeller, the D3–A3 loop, contains residues critical for antagonist recognition, with a minor role played by residues in neighbouring loops. A new homology model of human integrin α5β1 supports an important function for D3–A3 loop residues Trp157 and Ala158 in the binding of antagonists. These results will aid the development of reagents that block integrin α5β1 functions in vivo.
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December 2009
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Research Article|
November 11 2009
Mapping the ligand-binding pocket of integrin α5β1 using a gain-of-function approach
A. Paul Mould;
A. Paul Mould
*Wellcome Trust Centre for Cell-Matrix Research, Faculty of Life Sciences, Michael Smith Building, University of Manchester, Oxford Road, Manchester M13 9PT, U.K.
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Ewa J. Koper;
Ewa J. Koper
*Wellcome Trust Centre for Cell-Matrix Research, Faculty of Life Sciences, Michael Smith Building, University of Manchester, Oxford Road, Manchester M13 9PT, U.K.
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Adam Byron;
Adam Byron
*Wellcome Trust Centre for Cell-Matrix Research, Faculty of Life Sciences, Michael Smith Building, University of Manchester, Oxford Road, Manchester M13 9PT, U.K.
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Martin J. Humphries
Martin J. Humphries
1
*Wellcome Trust Centre for Cell-Matrix Research, Faculty of Life Sciences, Michael Smith Building, University of Manchester, Oxford Road, Manchester M13 9PT, U.K.
1To whom correspondence should be addressed (email martin.humphries@manchester.ac.uk).
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Biochem J (2009) 424 (2): 179–189.
Article history
Received:
July 01 2009
Revision Received:
September 07 2009
Accepted:
September 14 2009
Accepted Manuscript online:
September 14 2009
Citation
A. Paul Mould, Ewa J. Koper, Adam Byron, Grit Zahn, Martin J. Humphries; Mapping the ligand-binding pocket of integrin α5β1 using a gain-of-function approach. Biochem J 1 December 2009; 424 (2): 179–189. doi: https://doi.org/10.1042/BJ20090992
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