Asp-698 and Asp-811 of the integrin alpha4-subunit are critical for the formation of a functional heterodimer.

The amino acid motif LDV is the principal binding site for alpha4 integrins in fibronectin, and homologous motifs are recognized in vascular cell adhesion molecule-1 and MAdCAM-1. Three conserved LDV motifs (LDV-1 to 3) occur in the ectodomain of the human and mouse alpha4-subunit, the functions of which are unknown. We demonstrate here that alpha4-transfected fibroblasts with mutation in LDV-1 (D489N) behaved like alpha4-wild type but that LDV-2 (D698N) and LDV-3 (D811N) mutants were impaired in binding and spreading on alpha4-specific substrates. On the RGD-containing fibronectin fragment FN-120 there was an inverse behavior; now the alpha4-wild type and the LDV-1 mutant could not adhere whereas the two other mutants could. The beta1 chain was critical for the differential integrin response. Biochemical analysis demonstrated that the LDV-2 and -3 mutations reduced the strength of the alpha4beta1 association, favored the formation of alpha5beta1, and prevented the expression of alpha4beta7 on the cell surface. Our results indicate that LDV-2 and LDV-3 are critical for the formation of a functional heterodimer. The presence of similar amino acid motifs in ligands and the alpha4-subunit suggest that metal coordination plays an important role in integrin-ligand binding as well as for heterodimer formation.