IDENTIFICATION OF AMINO-ACID-RESIDUES THAT FORM PART OF THE LIGAND-BINDING POCKET OF INTEGRIN ALPHA-5-BETA-1

Arg-Arg-Glu-Thr-Ala-Trp-Ala (RRETAWA) is a novel ligand peptide for in tegrin alpha(5)beta(1), which blocks alpha(5)beta(1)-mediated cell adh esion 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 thi s 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 bloc ked binding of the anti-alpha(5) mAb 16 in a directly competitive mann er. 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 RRE TAWA is also closely overlapping with that for RGD. However, differenc es between the binding sites of RRETAWA and RGD were apparent in that (i) RGD peptides allosterically inhibited the binding of mAb 16 to alp ha(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 RRET AWA. A double mutation in alpha(5) (S156G/W157S) blocked the interacti on of both RRETAWA and mAb 16 with alpha(5)beta(1) but had no effect o n fibronectin binding or on the binding of other anti-alpha(5) mAbs. S er(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 t he NH2-terminal repeats of alpha(5). Our findings suggest that this se quence forms part of the ligand-binding pocket of alpha(5)beta(1). Fur thermore, as Ser(156)-Trp(157) is unique to the alpha(5) subunit, it m ay be responsible for the specific recognition of RRETAWA by alpha(5)b eta(1).