Research article

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

Cristina PUJADES, Joaquin TEIXIDÓ, Gianfranco BAZZONI, Martin E. HEMLER


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 α4/180 [Parker, Pujades, Brenner and Hemler (1993) J. Biol. Chem. 268, 7028–7035] also requires Cys278 and Cys717, divalent cations and reduced cysteines. Thus α4/180 appears to be more functionally relevant than α4/150.