J Biol Chem, Vol. 273, Issue 12, 6786-6795, March 20, 1998

Asp-698 and Asp-811 of the Integrin ₄-Subunit Are Critical for the Formation of a Functional Heterodimer

Yvonka Zeller, Jens Lohr, Marei Sammar, Eugene C. Butcher, and Peter Altevogt

From the Tumor Immunology Programme, 0710, German Cancer Research Center, D-69120 Heidelberg, Federal Republic of Germany, the  Laboratory of Immunology and Vascular Biology, Department of Pathology and Digestive Disease Center, Stanford University, Stanford, California 94305, and the Center of Molecular Biology in Medicine, Veterans Administration Medical Center, Palo Alto, California 94304

The amino acid motif LDV is the principal binding site for ₄ 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 ₄-subunit, the functions of which are unknown. We demonstrate here that ₄-transfected fibroblasts with mutation in LDV-1 (D489N) behaved like ₄-wild type but that LDV-2 (D698N) and LDV-3 (D811N) mutants were impaired in binding and spreading on ₄-specific substrates. On the RGD-containing fibronectin fragment FN-120 there was an inverse behavior; now the ₄-wild type and the LDV-1 mutant could not adhere whereas the two other mutants could. The ₁ chain was critical for the differential integrin response. Biochemical analysis demonstrated that the LDV-2 and -3 mutations reduced the strength of the ₄₁ association, favored the formation of ₅₁, and prevented the expression of ₄₇ 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 ₄-subunit suggest that metal coordination plays an important role in integrin-ligand binding as well as for heterodimer formation.