HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY EWI-2 modulates lymphocyte integrin 4 1 functions

The most prominent cell-surface integrin 4 1 partner, a 70-kDa protein, was isolated from MOLT-4 T leukemia cells, using anti– 4 1 integrin antibody-coated beads. By mass spectrometry, this protein was identified as EWI-2, a previously described cell-surface partner for tetraspanin proteins CD9 and CD81. Wildtype EWI-2 overexpression had no effect on MOLT-4 cell tethering and adhesion strengthening on the 4 1 ligand, vascular cell adhesion molecule-1 (VCAM-1), in shear flow assays. However, EWI-2 markedly impaired spreading and ruffling on VCAM-1. In contrast, a mutant EWI-2 molecule, with a different cytoplasmic tail, neither impaired cell spreading nor associated with 4 1 and CD81. The endogenous wild-type EWI-2–CD81– 4 1 complex was fully soluble, and highly specific as seen by the absence of other MOLT-4 cell-surface proteins. Also, it was relatively small in size (0.5 106 Da to 4 106 Da), as estimated by size exclusion chromatography. Overexpression of EWI-2 in MOLT-4 cells caused reorganization of cell-surface CD81, increased the extent of CD81-CD81, CD814 1, and 4 14 1 associations, and increased the apparent size of CD814 1 complexes. We suggest that EWI-2–dependent reorganization of 4 1-CD81 complexes on the cell surface is responsible for EWI-2 effects on integrin-dependent morphology and motility functions. (Blood. 2004;103: 3013-3019)