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 c aused a marked reduction in alpha 4 beta 1-dependent cell adhesion. So me reports have suggested that alpha-chain ''EF-hand'' sites may inter act directly with ligands. However, we show here that mutations of thr ee different alpha 4 ''EF-hand'' sites each had no effect on binding o f soluble monovalent or bivalent vascular cell adhesion molecule 1 whe ther 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 sh ow severe impairments in cellular resistance to detachment under shear flow. Thus, mutation of integrin alpha 4 ''EF-hand-like'' sites may i mpair 1) static cell adhesion and 2) adhesion strengthening under shea r flow by a mechanism that does not involve alterations of initial lig and binding.