Identification of amino acid residues that form part of the ligand-binding pocket of integrin α5β1

Arg-Arg-Glu-Thr-Ala-Trp-Ala (RRETAWA) is a novel ligand peptide for integrin alpha(5)beta(1), which blocks alpha(5)beta(1)-mediated cell adhesion to fibronectin (Koivunen, E., Wang, B., and Ruoslahti, E. (1994) J. Cell Biol. 124, 373-380), Here we ha ve localized the binding site for RRETAWA on alpha(5)beta(1) using inhibitory monoclonal antibodies (mAbs) and site-directed mutagenesis. A cyclic peptide containing this sequence (*CRRETAWAC*) had little effect on the binding of most anti-alpha(5) and anti-beta(1) mAbs to alpha(5)beta(1) but completely blocked binding of the anti-alpha(5) mAb 16 in a directly competitive manner. Hence, the binding site of RRETAWA appears to closely overlap with the epitope of mAb 16, *CRRETAWAC* also acted as a direct competitive inhibitor of the binding of Arg-Gly-Asp (RGD)-containing fibronectin fragments to alpha(5)beta(1), suggesting that the binding site for RRETAWA is also closely overlapping with that for RGD. However, differences between the binding sites of RRETAWA and RGD were apparent in that (i) RGD peptides allosterically inhibited the binding of mAb 16 to alpha(5)beta(1), and (ii) several mAbs that perturbed binding of alpha(5)beta(1) to RGD had little effect on binding of alpha(5)beta(1) to RRETAWA. A double mutation in alpha(5) (S156G/W157S) blocked the interaction of both RRETAWA and mAb 16 with alpha(5)beta(1) but had no effect on fibronectin binding or on the binding of other anti-alpha(5) mAbs. Ser(156)-Trp(157) is located near the apex of a putative loop region on the upper surface of a predicted beta-propeller structure formed by the NH2-terminal repeats of alpha(5). Our findings suggest that this sequence forms part of the ligand-binding pocket of alpha(5)beta(1). Furthermore, as Ser(156)-Trp(157) is unique to the alpha(5) subunit, it may be responsible for the specific recognition of RRETAWA by alpha(5)beta(1).