INTEGRIN ALPHA-4 CYSTEINES 278 AND 717 MODULATE VLA-4 LIGAND BINDING AND ALSO CONTRIBUTE TO ALPHA(4/180) FORMATION

摘要

Here we describe experiments in which we mutated four oPthe six integrin alpha(4) subunit cysteine residues that are not present in most other integrin alpha subunits that lack an I domain. In four different types of ligand binding assay we found that optimal integrin alpha(4) beta(1) (VLA-4) binding to vascular cell adhesion molecule 1 (VCAM-1) and/or to CS1 peptide required the presence of both alpha(4) Cys(278) and Cys(717). In addition, optimal ligand binding required divalent cations and reduced cysteines, as evidenced by EDTA and N-ethylmaleimide inhibition results. In a control experiment, an alpha(4) mutation that completely eliminated the alpha(4) 80/70 proteolytic cleavage site had no effect on ligand binding. Notably, although Cys(278) and Cys(717) mutations markedly altered ligand binding, they had no adverse effect on cell adhesion. Thus, compared with cell adhesion, ligand binding is a distinct and apparently more stringent test of VLA-4 integrin-ligand interactions. In addition, we have established that the formation of the previously described alpha(4/180) [Parker, Pujades, Brenner and Hemler (1993) J. Biol. Chem. 268, 7028-7035] also requires Cys(278) and Cys(717), divalent cations and reduced cysteines. Thus alpha(4/180) appears to be more functionally relevant than alpha(4/150). [References: 49]