Further characterisation of the talin-binding site in the cytoskeletal protein vinculin

The cytoskeletal protein vinculin is a component of adherens-type junctions where it is one of a number of interacting proteins thought to link the cytoplasmic domain of adhesion receptors to F-actin. Vinculin has been shown to bind to at least three other cytoskeletal proteins, talin, paxillin and alpha-actinin. In this study, we further characterise the talin-binding domain in vinculin using a series of chick vinculin polypeptides expressed as glutathione-S-transferase fusion proteins in Escherichia coli. Thus 125I-talin bound to a fusion protein spanning residues 1-398, but not to those spanning residues 399-881 or 881-1066 in an SDS-PAGE gel-blot assay. We have previously characterised two chick vinculin cDNAs (2.89 kb cDNA and cVin5) which are identical in the region of overlap except that cVin5 lacks coding sequence for residues 167-207. Interestingly, a fusion protein spanning residues 1-398, but lacking residues 167-207, was unable to bind talin. However, further analysis showed that residues 167-207 are insufficient to support binding, and deletion of as few as 31 N-terminal residues abolished binding activity. The results of the gel-blot assay were essentially confirmed using purified fusion proteins adsorbed to glutathione-agarose beads. The smallest vinculin fusion protein able to bind talin contained residues 1-258. This fusion protein was as effective as whole vinculin in inhibiting the binding of 125I-vinculin to talin-coated microtitre wells. Interestingly, mutations which altered the charge characteristics of the highly conserved residues 178 and 181 abolished binding, whereas conservative substitutions were without effect. However, such mutations did not abolish the ability of mutant polypeptides spanning residues 1-398 to target to cell-matrix junctions in Cos cells. We have investigated the possible origin of the cDNA clone cVin5 by defining the structure of a 5' portion of the chicken vinculin gene, and by analysing vinculin transcripts in a variety of adult tissues and embryonic fibroblasts using reverse transcriptase and polymerase chain reaction. Although residues 167-207 are encoded on a separate exon, we have been unable to identify a tissue where this exon is alternatively spliced.