The high-affinity interaction of integrin alpha 5 beta 1 with the central cell-binding domain of fibronectin requires both the Arg-Gly-Asp (RGD) sequence (in the tenth type III repeat) and a second site Pro-His-Ser-Arg-Asn (PHSRN) in the adjacent ninth type III repeat, which synergizes with RGD. Arg-Arg-Glu-Thr-Ala-Trp-Ala (RRETAWA) is a novel peptidic ligand for alpha 5 beta 1, identified by phage display, which blocks alpha 5 beta 1-mediated cell adhesion to fibronectin. A key question is the location of the binding sites for these ligand sequences within the integrin. In this study we have identified residues that form part of the epitopes of three inhibitory anti-alpha 5 monoclonal antibodies (mAbs): 16, P1D6 and SNAKA52. These mAbs have distinct functional properties. mAb 16 blocks the recognition of RGD and RRETAWA, whereas P1D6 blocks binding to the synergy sequence. The binding of SNAKA52 is inhibited by anti-beta 1 mAbs, indicating that its epitope is close to the interface between the alpha and beta subunits. Residues in human alpha 5 were replaced with the corresponding residues in mouse alpha 5 by site-directed mutagenesis; wild-type or mutant human alpha 5 was expressed on the surface of alpha 5-deficient Chinese hamster ovary cells. mAb binding was assessed by flow cytometry and by adhesion to the central cell-binding domain of fibronectin or RRETAWA by cell attachment assay. All three epitopes were located to different putative loops in the N-terminal domain of alpha 5. As expected, disruption of these epitopes had no effect on ligand recognition by alpha 5 beta 1. The locations of these epitopes are consistent with the beta-propeller model for integrin alpha-subunit structure and allow us to propose a topological image of the integrin-ligand complex.
