Ligand binding to integrin alpha(v)beta(3) requires tyrosine 178 in the alpha(v) subunit

Integrin alpha (v)beta (3) has been implicated in angiogenesis and other bi ological processes. However, the ligand-binding sites in alpha (v) a non-I- domain alpha subunit, remain to be identified. Recently in alpha (IIb) the other partner of the beta (3) subunit, several discontinuous residues impor tant for ligand binding were identified in the predicted loops between repe ats 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 investigat e the role of these loops (cysteine [C]142-C155 and glycine [G]172-G181) of alpha (v) in ligand binding. Wild-type alpha (v)beta (3) was able to bind soluble fibrinogen following integrin activation either by 0.5 mM manganese dichloride (MnCl2) or a mutation of beta (3) threonine (T)562 to asparagin e, However, mutation of tyrosine (Y)178 to alanine in the predicted G172-G1 81 loop of alpha (v) abolished fibrinogen binding, and alanine (A) substitu tions at adjacent residues phenylalanine (F)177 and tryptophan (W)179 had a similar effect. Cells expressing Y178 alpha (v), also failed to bind to im mobilized fibrinogen. Moreover, the Y178A mutation abolished the binding of WOW-1 Fab, a monovalent ligand-mimetic anti-alpha (v)beta (3) antibody, an d the expression of beta (3) ligand-induced binding sites (LIBS) induced by arginine-glycine-aspartic acid-tryptophan (RGDW). In sharp contrast to the data obtained with alpha (IIb) none of the mutations In the predicted W3 4 -1 loop in a, impaired ligand binding. These results implicate alpha (v) Y1 78 in ligand binding to alpha (v)beta (3) and they suggest that there are k ey structural differences in the adhesive ligand-binding sites of alpha (v) beta (3) and alpha (IIb)beta (3) (C) 2001 by The American Society of Hemato logy.