Identification of the vinculin-binding site in the cytoskeletal protein α-actinin

Using low-speed sedimentation equilibrium we have established that vinculin binds to alpha-actinin with a Kd of 1.3 x 10(-5) M. Electron microscopy of negatively stained preparations of vinculin revealed spherical particles (diameter 11.2 nm; S.D. 1.7 nm, n = 21), whereas alpha-actinin appeared as a rod-shaped particle (length 33 nm; S.D. 3.3 nm, n = 23). Mixtures of the two proteins contained both ‘lollipop’- and ‘dumbell’-shaped particles which we interpret as either one or two spherical vinculin molecules associated with the ends of the alpha-actinin rod. We have further defined the vinculin-binding site in alpha-actinin using 125I-vinculin and a gel-blot assay in which proteolytic fragments of alpha-actinin and fragments of alpha-actinin expressed in Escherichia coli were resolved by SDS/PAGE and blotted to nitrocellulose. 125I-vinculin bound to polypeptides derived from the spectrin-like repeat region of alpha-actinin, but did not bind to the actin-binding domain. Binding was inhibited by a 100-fold molar excess of unlabelled vinculin. Using a series of glutathione S-transferase fusion proteins we have mapped the vinculin-binding site to a region toward the C-terminal end of the molecule (alpha-actinin residues 713-749). 125I-vinculin also bound to fusion proteins containing this sequence which had been immobilized on glutathione-agarose beads. The vinculin-binding site is localized in a highly conserved region of the molecule close to the first of two EF-hand calcium-binding motifs.

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

Journal of Cell Science ◽

10.1242/jcs.103.3.719 ◽

1992 ◽

Vol 103 (3) ◽

pp. 719-731 ◽

Cited By ~ 4

Author(s):

A.P. Gilmore ◽

P. Jackson ◽

G.T. Waites ◽

D.R. Critchley

Keyword(s):

Fusion Protein ◽

Fusion Proteins ◽

Binding Activity ◽

Cytoskeletal Proteins ◽

Cytoskeletal Protein ◽

Glutathione S Transferase ◽

Agarose Beads ◽

Cell Matrix ◽

Sds Page ◽

Alternatively Spliced

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.

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Talin contains three actin-binding sites each of which is adjacent to a vinculin-binding site

Journal of Cell Science ◽

10.1242/jcs.109.11.2715 ◽

1996 ◽

Vol 109 (11) ◽

pp. 2715-2726 ◽

Cited By ~ 19

Author(s):

L. Hemmings ◽

D.J. Rees ◽

V. Ohanian ◽

S.J. Bolton ◽

A.P. Gilmore ◽

...

Keyword(s):

Fusion Protein ◽

Binding Site ◽

Binding Sites ◽

Fusion Proteins ◽

Focal Adhesions ◽

Actin Binding ◽

Mouse Protein ◽

Protein Alignments ◽

Terminal Site ◽

Actin Binding Site

We have determined the sequence of chicken talin (2,541 amino acids, M(r) 271,881) which is very similar (89% identity) to that of the mouse protein. Alignments with the Caenorhabditis elegans and Dictyostelium discoideum talin sequences show that the N- and C-terminal regions of the protein are conserved whereas the central part of the molecule is more divergent. By expressing overlapping talin polypeptides as fusion proteins, we have identified at least three regions of the protein which can bind F-actin: residues 102–497, 951–1,327 and 2,269-2,541. The N-terminal binding site contains a region with homology to the ERM family of actin-binding proteins, and the C-terminal site is homologous to the yeast actin-binding protein Sla2p. Each of the actin-binding sites is close to, but distinct from a binding site for vinculin, a protein which also binds actin. The Pro1176 to Thr substitution found in talin from Wistar-Furth rats does not destroy the capacity of this region of the protein to bind actin or vinculin. Microinjection studies showed that a fusion protein containing the N-terminal actin-binding site localised weakly to stress fibres, whereas one containing the C-terminal site initially localised predominantly to focal adhesions. The former was readily solubilised, and the latter was resistant to Triton extraction. The N-terminal talin polypeptide eventually disrupted actin stress fibres whereas the C-terminal polypeptide was without effect. However, a larger C-terminal fusion protein also containing a vinculin-binding site did disrupt stress fibres and focal adhesions. The results suggest that, although both the N- and C-terminal regions of talin bind actin, the properties of these two regions of the protein are distinct.

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