title: EWI-2 modulates lymphocyte integrin alpha4beta1 functions.
abstract: The most prominent cell-surface integrin alpha4beta1 partner, a 70-kDa protein, was isolated from MOLT-4 T leukemia cells, using anti-alpha4beta1 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. Wild-type EWI-2 overexpression had no effect on MOLT-4 cell tethering and adhesion strengthening on the alpha4beta1 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 alpha4beta1 and CD81. The endogenous wild-type EWI-2-CD81-alpha4beta1 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 x 10(6) Da to 4 x 10(6) 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, CD81-alpha4beta1, and alpha4beta1-alpha4beta1 associations, and increased the apparent size of CD81-alpha4beta1 complexes. We suggest that EWI-2-dependent reorganization of alpha4beta1-CD81 complexes on the cell surface is responsible for EWI-2 effects on integrin-dependent morphology and motility functions.
authors.affiliation: Dana-Farber Cancer Institute, Brigham and Women's Hospital, Department of Pathology, Harvard Medical School, Boston, MA 02115, USA.
authors.forename: Tatiana V
authors.initials: TV
authors.lastname: Kolesnikova
authors.forename: Christopher S
authors.initials: CS
authors.lastname: Stipp
authors.forename: Ravi M
authors.initials: RM
authors.lastname: Rao
authors.forename: William S
authors.initials: WS
authors.lastname: Lane
authors.forename: Francis W
authors.initials: FW
authors.lastname: Luscinskas
authors.forename: Martin E
authors.initials: ME
authors.lastname: Hemler
mesh_terms: D015703:Antigens, CD / Q000378:metabolism; D001483:Base Sequence; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000276:immunology / Q000378:metabolism; D002465:Cell Movement; D004274:DNA, Recombinant / Q000235:genetics; D006801:Humans; D007136:Immunoglobulins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D039041:Integrin alpha4beta1 / Q000378:metabolism*; D015458:Leukemia, T-Cell / Q000276:immunology / Q000378:metabolism / Q000473:pathology; D008024:Ligands; D046911:Macromolecular Substances; D008565:Membrane Proteins / Q000378:metabolism; D009154:Mutation; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013601:T-Lymphocytes / Q000276:immunology* / Q000473:pathology; D060225:Tetraspanin 28
chemical_list: D015703:Antigens, CD; C554798:CD81 protein, human; D004274:DNA, Recombinant; C440028:IGSF8 protein, human; D007136:Immunoglobulins; D039041:Integrin alpha4beta1; D008024:Ligands; D046911:Macromolecular Substances; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D060225:Tetraspanin 28