ASPARTATE-698 WITHIN A NOVEL CATION-BINDING MOTIF IN ALPHA(4) INTEGRIN IS REQUIRED FOR CELL-ADHESION

The interactions of alpha(4) beta(1) integrin with vascular cell adhesion molecule (VCAM) and fibronectin play important roles in many physiological and pathological processes, To understand the mechanism of alpha(1) beta(1) integrin-mediated cell adhesion, we made mutant alpha(4) constructs, Three aspartic acid (Asp) residues in alpha(4), Asp 489, Asp-698, and Asp-811, were replaced with glutamic acids (Glu), The wild-type and mutant alpha(4) constructs were transfected into K562 cells, and stable transfectants with similar levels of alpha(4) surface expression were established, The Asp --> Glu substitutions did not affect alpha(4) beta(1) association or heterodimer formation as demonstrated by immunoprecipitation analysis. However, the glutamate substitutions at Asp-489 and Asp-698 severely impaired cell adhesion to VCAM and fibronectin, whereas the substitution at Asp-811 had no detectable effect on cell adhesion. In contrast to these results, isolated alpha(4) beta(1), containing the D489E or D698E substitution, was able to bind to VCAM, suggesting that these two residues are not critical for ligand recognition. In searching for a mechanism to explain inhibition of adhesion by Asp-489 and Asp 698 mutations, we found that the sequences flanking Asp 698 resemble the DxxxxxD-S-Sx divalent cation/ligand binding motif in beta integrins and the I-domains of alpha integrins. This suggests that Asp-698 in the alpha(4) integrin, which does not possess an I-domain, may also be involved in cation binding and may be part of a sequence functionally similar to that found in the I-domains of other alpha integrins.