Ligand binding to integrin αvβ3requires tyrosine 178 in the αv subunit

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Abstract
Integrin αvβ3 has been implicated in angiogenesis and other biological processes. However, the ligand-binding sites in αv, a non–I-domain α subunit, remain to be identified. Recently in αIIb, the other partner of the β3 subunit, several discontinuous residues important for ligand binding were identified in the predicted loops between repeats 2 and 3 (W3 4-1 loop) and within repeat 3 (W3 2-3 loop). Based on these findings, alanine-scanning mutagenesis in 293 cells was used to investigate the role of these loops (cysteine [C]142-C155 and glycine [G]172-G181) of αv in ligand binding. Wild-type αvβ3 was able to bind soluble fibrinogen following integrin activation either by 0.5 mM manganese dichloride (MnCl2) or a mutation of β3 threonine (T)562 to asparagine. However, mutation of tyrosine (Y)178 to alanine in the predicted G172-G181 loop of αv abolished fibrinogen binding, and alanine (A) substitutions at adjacent residues phenylalanine (F)177 and tryptophan (W)179 had a similar effect. Cells expressing Y178Aαvalso failed to bind to immobilized fibrinogen. Moreover, the Y178A mutation abolished the binding of WOW-1 Fab, a monovalent ligand-mimetic anti-αvβ3 antibody, and the expression of β3 ligand–induced binding sites (LIBS) induced by arginine-glycine-aspartic acid-tryptophan (RGDW). In sharp contrast to the data obtained with αIIb, none of the mutations in the predicted W3 4-1 loop in αv impaired ligand binding. These results implicate αv Y178 in ligand binding to αvβ3, and they suggest that there are key structural differences in the adhesive ligand-binding sites of αvβ3 and αIIbβ3.