Tyr178 of β3 is critical for αIIb maturation and macromolecular ligand binding to αIIbβ3

Abstract

To explore the structural basis of ligand binding to αIIbβ3, we conducted a site-directed mutagenesis of Y178, which is located in the ligand-specificity region (C177–C184) of the β3 subunit. Two mutant β3 constructs, Y178A and Y178I, were transfected into CHO cells and co-expressed with human αIIb subunit on the cell surface. Our results showed that the Y178A mutation affected processing and cell surface exposure of recombinant αIIbβ3 receptor, abrogated the binding of PAC-1, a ligand-mimetic antibody, to αIIbβ3 pre-treated with the known activator DTT. The Y178A mutation also resulted in reduced adhesion of αIIbβ3 on immobilized fibrinogen. In contrast, the interaction of αIIbβ3 with the small molecular ligand RGDS was unaffected by Y178A mutation, as evidenced by the elevated LIBS-1 epitope expression following RGDS addition. Interestingly however, Y178I mutation did not affect the receptor synthesis and function at all. As for post-receptor occupancy, neither Y178A nor Y178I prevented αIIbβ3 translocation to focal adhesion contacts. These results suggest that Y178 is involved in αIIb maturation and αIIbβ3 complex expression. This residue is also critical for αIIbβ3 interaction with its macromolecular ligand or ligand-mimetic mAb, possibly due to its involvement in other ligand-binding sites distinct from the RGD-binding pocket. We also propose that a residue with appropriate side-chain size and hydrophobicity at position 178 is spatially required for formation of the correct tertiary structure of the site.