Molecular determinants of Arg-Gly-Asp ligand specificity for beta(3) integrins

The Arg-Tyr-Asp (RYD) and Arg-Gly-Asp (RGD) sequences within the third complementarity-determining region of the heavy chain (H3) of murine recombinant Feb molecules OPG2 and AP7, respectively, are responsible for their specific binding to the platelet integrin alpha(IIb)beta(3). In this study, we evaluated the influence of divalent cation composition and single amino acid substitutions at key positions within H3 on the selectivity of these Fab molecules for integrin alpha(IIb)beta(3) versus the vitronectin receptor alpha(v) beta(3). The parent Fab molecule OPG2 (H3 sequence, HPFYRYDGGN) binds selectively to alpha(IIb)beta(3) and not at all to any other RGD-cognitive integrin, particularly alpha(IIb)beta(3), under any divalent cation conditions. The binding of the AP7 Fab molecule (HPFYRGDGGN) to alpha(IIb)beta(3) is not affected by the relative composition of calcium, magnesium or manganese. However, AP7 binding to alpha(v) beta(3), either expressed by M21 cells or as the purified integrin, is supported by manganese and inhibited by calcium. If the flanking asparagine 108 residue within the AP7 H3 loop is replaced by alanine (HPFYRGDGGA), the resulting Fab molecule AP7.4 binds selectively to alpha(v) beta(3) in a cation-dependent manner, but does not bind at all to alpha(IIb)beta(3) under any conditions. AP7.4 binding to alpha(IIb)beta(3) is supported by manganese, completely inhibited by calcium, and largely unaffected by magnesium. This behavior mimics that of the adhesive protein, osteopontin, another ligand that binds preferentially to alpha(v) beta(3). Despite these differences in specificity for alpha(IIb)beta(3) and alpha(v) beta(3), AP7 and AP7.4 remain selective for the beta(3) integrins and do not bind to cell lines that express the RGD-cognitive integrins alpha(v) beta(5) or alpha(5) beta(1). These results confirm that subtle changes in the amino acid composition immediately flanking the RGD or RYD moths can have a profound effect on beta(3) integrin specificity, most likely because they influence the juxtaposition of the arginine and aspartate side chains within the extended RGD loop sequence.