Defining extracellular integrin alpha-chain sites that affect cell adhesion and adhesion strengthening without altering soluble ligand binding

It was previously shown that mutations of integrin alpha 4 chain sites, within putative EF-hand-type divalent cation-binding domains, each caused a marked reduction in alpha 4 beta 1-dependent cell adhesion. Some reports have suggested that alpha-chain ''EF-hand'' sites may interact directly with ligands. However, we show here that mutations of three different alpha 4 ''EF-hand'' sites each had no effect on binding of soluble monovalent or bivalent vascular cell adhesion molecule 1 whether measured indirectly or directly. Furthermore, these mutations had minimal effect on alpha 4 beta 1-dependent cell tethering to vascular cell adhesion molecule 1 under shear. However, EF-hand mutants did show severe impairments in cellular resistance to detachment under shear flow. Thus, mutation of integrin alpha 4 ''EF-hand-like'' sites may impair 1) static cell adhesion and 2) adhesion strengthening under shear flow by a mechanism that does not involve alterations of initial ligand binding.