Integrin α⁴ cysteines 278 and 717 modulate VLA-4 ligand binding and also contribute to α^4/180 formation

Here we describe experiments in which we mutated four of the six integrin α⁴ subunit cysteine residues that are not present in most other integrin α subunits that lack an I domain. In four different types of ligand binding assay we found that optimal integrin α⁴β₁ (VLA-4) binding to vascular cell adhesion molecule 1 (VCAM-1) and/or to CS1 peptide required the presence of both α⁴ Cys²⁷⁸ and Cys⁷¹⁷. 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 α⁴ mutation that completely eliminated the α⁴ 80/70 proteolytic cleavage site had no effect on ligand binding. Notably, although Cys²⁷⁸ and Cys⁷¹⁷ 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 α^4/180 [Parker, Pujades, Brenner and Hemler (1993) J. Biol. Chem. 268, 7028–7035] also requires Cys²⁷⁸ and Cys⁷¹⁷, divalent cations and reduced cysteines. Thus α^4/180 appears to be more functionally relevant than α^4/150.