title: Characterization of two F-actin-binding and oligomerization sites in the cell-contact protein vinculin.
abstract: Vinculin, a structural protein of animal cells, is critically involved in the assembly of microfilament/plasma membrane junctions at cell contacts. To understand its role in organizing the distal portions of microfilaments into specific, morphologically distinct structures at these sites in more detail, we characterized its interaction with filamentous actin and with itself by means of in vitro assays. Using recombinant proteins comprising different parts of the vinculin tail fused to the maltose-binding protein of Escherichia coli, we show in sedimentation assays that this part of vinculin harbors two discrete sites that can bind to actin independently. They reside within amino acid residues 893-985 and 1016-1066 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 and bundling are alternatives determined by the molar ratio of fusion protein to F-actin. Both actin-binding sequences are capable of oligomer formation, as shown in chemical-cross-linking and dot-overlay assays. These data allow us to propose a possible role for vinculin in organizing the distal ends of microfilaments at the plasma membrane into the point-like structure characteristic for cell-matrix contacts.
authors.affiliation: Cell Biology, Zoological Institute, Technical University of Braunschweig, Germany.
authors.forename: S
authors.initials: S
authors.lastname: Hüttelmaier
authors.forename: P
authors.initials: P
authors.lastname: Bubeck
authors.forename: M
authors.initials: M
authors.lastname: Rüdiger
authors.forename: B M
authors.initials: BM
authors.lastname: Jockusch
mesh_terms: D000199:Actins / Q000378:metabolism*; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D001704:Biopolymers; D017931:DNA Primers; D018076:DNA, Complementary; D018482:Muscle, Skeletal / Q000378:metabolism; D011817:Rabbits; D016596:Vinculin / Q000235:genetics / Q000378:metabolism*
chemical_list: D000199:Actins; D001704:Biopolymers; D017931:DNA Primers; D018076:DNA, Complementary; D016596:Vinculin