Integrin alpha5beta1 is a key receptor for the
extracellular matrix protein fibronectin. Antagonists of human
integrin alpha5beta1 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 alpha5beta1 is poorly understood. In the present study, we demonstrate that zebrafish alpha5beta1
integrins do not interact with human
fibronectin or the human alpha5beta1 antagonists
JSM6427 and
cyclic peptide CRRETAWAC. Zebrafish alpha5beta1
integrins do bind zebrafish fibronectin-1, and mutagenesis of residues on the upper surface and side of the zebrafish alpha5 subunit beta-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 alpha5 subunit with the corresponding regions of human alpha5 we show that blades 1-4 of the beta-propeller are required for human
fibronectin recognition, suggesting that
fibronectin binding involves a broad interface on the side and upper face of the beta-propeller domain. We find that the loop connecting blades 2 and 3 of the beta-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 alpha5beta1 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 alpha5beta1 functions in vivo.