CHARACTERIZATION OF 2 F-ACTIN-BINDING AND OLIGOMERIZATION SITES IN THE CELL-CONTACT PROTEIN VINCULIN

Vinculin, a structural protein of animal cells, is critically involved in the assembly of microfilament/plasma membrane junctions at cell co ntacts, To understand its role in organizing the distal portions of mi crofilaments into specific, morphologically distinct structures at the se sites in more detail, we characterized its interaction with filamen tous actin and with itself by means of in vitro assays. Using recombin ant proteins comprising different parts of the vinculin tail fused to the maltose-binding protein of Escherichia coli. we show in sedimentat ion assays that this part of vinculin harbors two discrete sites that can hind to actin independently. They reside within amino acid residue s 893-985 and 1010-1066 of the 1066-residue polypeptide chain, However , both sites are necessary to cross-link or bundle actin filaments. as demonstrated by low shear viscometry. Crosslinking anti bundling are alternatives determined by the molar ratio of fusion protein to F-acti n, Both actin-binding sequences are capable of oligomer formation, as shown in chemical-cross-linking and dot-overlay assays. These data all ow us to propose a possible role for vinculin in organizing the distal ends of microfilament, at the plasma membrane into the point-like str ucture characteristic for cell-matrix contacts.