A region of the integrin VLA alpha 4 subunit involved in homotypic cell aggregation and in fibronectin but not vascular cell adhesion molecule-1 binding.

The VLA-4 (alpha 4 beta 1) integrin is involved in the adhesion of cells to fibronectin and vascular cell adhesion molecule-1 (VCAM-1). In order to study alpha 4 structure-function relationships, we have expressed mutated alpha 4 subunit by transfection into VLA-4-negative K562 cells. Substitutions at alpha 4 residues Arg89-Asp90, which show the highest surface probability indexes inside the N-terminal alpha 4/80 fragment, resulted in a reduction in the reactivity of all anti-alpha 4 epitope A monoclonal antibodies (mAbs) tested, compared with the reactivity with anti-alpha 4 epitopes B1, B2, and C mAb, both by transfectant flow cytometry, and by immunoprecipitation and SDS-polyacrylamide gel electrophoresis analysis of transfectant surface-iodinated proteins. In contrast, substitutions at nearby residues, Gln101, Pro102, and Ile105 did not affect the reactivity of any anti-alpha 4 mAb representing the known alpha 4 epitopes. Homotypic cell aggregation triggered by anti-alpha 4 epitope A mAb was prevented in the transfectants expressing mutated alpha 4 Arg89-Asp90Asp residues, while cell aggregation was fully achieved with either anti-alpha 4 epitope B2 or anti-beta 1 mAb. Mutations at alpha 4 residues Gln101, Pro102, and Ile108 did not affect the homotypic cell aggregation of the transfectants expressing these mutations. In addition, the adhesion of mutant Arg89-Asp90 alpha 4 transfectants to the connecting segment-1-containing fibronectin-40 (FN-40) fragment of fibronectin was diminished compared to wild type alpha 4 transfectants, as well as to other mutant alpha 4 transfectants. This adhesion to FN-40 was restored when the activating anti-beta 1 TS2/16 mAb was present in the adhesion assays. In contrast, adhesion to VCAM-1 was not affected by mutations at Arg89-Asp90, nor at Gln101, Pro102, and Ile108 alpha 4 residues. Altogether, these results indicate that alpha 4 residues Arg89 and Asp90 are included in a region involved in homotypic cell aggregation, as well as in adhesion to FN-40, but not to VCAM-1.