CHARACTERIZATION OF AN F-ACTIN-BINDING DOMAIN IN THE CYTOSKELETAL PROTEIN VINCULIN

Vinculin, a major structural component of vertebrate cell-cell and cel l-matrix adherens junctions, has been found to interact with several o ther junctional components. In this report, we have identified and cha racterized a binding site for filamentous actin. These results include d studies with gizzard vinculin, its proteolytic head and tail fragmen ts, and recombinant proteins containing various gizzard vinculin seque nces fused to the maltose binding protein (MBP) of Escherichia coli. I n cosedimentation assays, only the vinculin tail sequence mediated a d irect interaction with actin filaments. The binding was saturable, wit h a dissociation constant value in the micromolar range. Experiments w ith deletion clones localized the actin-binding domain to a region con fined by residues 893-1016 in the 170-residue-long carboxyterminal seg ment, while the proline-rich hinge connecting the globular head to the rodlike tail was not required for this interaction. In fixed and perm eabilized cells (cell models), as well as after microinjection, protei ns containing the actin-binding domain specifically decorated stress f ibers and the cortical network of fibroblasts and epithelial cells, as well as of brush border type microvilli. These results corroborated t he sedimentation experiments. Our data support and extend previous wor k showing that vinculin binds directly to actin filaments. They are co nsistent with a model suggesting that in adhesive cells, the NH2-termi nal head piece of vinculin directs this molecule to the focal contact sites, while its tail segment causes bundling of the actin filament en ds into the characteristic spear tip-shaped structures.