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Identification of a talin binding site in the cytoskeletal protein vinculin

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posted on 2016-03-04, 10:25 authored by P. Jones, P. Jackson, G. J. Price, B. Patel, V. Ohanion, A. L. Lear, D. R. Critchley
Binding of the cytoskeletal protein vinculin to talin is one of a number of interactions involved in linking F-actin to cell-matrix junctions. To identify the talin binding domain in vinculin, we expressed the NH2-terminal region of the molecule encoded by two closely similar, but distinct vinculin cDNAs, using an in vitro transcription translation system. The 5' Eco RI-Bam HI fragment of a partial 2.89-kb vinculin cDNA encodes a 45-kD polypeptide containing the first 398 amino acids of the molecule. The equivalent restriction enzyme fragment of a second vinculin cDNA (cVin5) lacks nucleotides 746-867, and encodes a 41-kD polypeptide missing amino acids 167-207. The radiolabeled 45-kD vinculin polypeptide bound to microtiter wells coated with talin, but not BSA, and binding was inhibited by unlabeled vinculin. In contrast, the 41-kD vinculin polypeptide was devoid of talin binding activity. The role of residues 167-207 in talin binding was further analyzed by making a series of deletions spanning this region, each deletion of seven amino acids contiguous with the next. Loss of residues 167-173, 174-180, 181-187, 188-194, or 195-201 resulted in a marked reduction in talin binding activity, although loss of residues 202-208 had much less effect. When the 45-kD vinculin polypeptide was expressed in Cos cells, it localized to cell matrix junctions, whereas the 41-kD polypeptide, lacking residues 167-207, was unable to do so. Interestingly, some deletion mutants with reduced ability to bind talin in vitro, were still able to localize to cell matrix junctions.

Funding

This work was supported by project grants to D. R. Critchley from the Cancer Research Campaign and the Medical Research Council (UK).

History

Citation

The Journal of Cell Biology, 1989 vol. 109 no. 6 2917-2927

Author affiliation

/Organisation/COLLEGE OF MEDICINE, BIOLOGICAL SCIENCES AND PSYCHOLOGY/MBSP Non-Medical Departments/Molecular & Cell Biology

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  • VoR (Version of Record)

Published in

The Journal of Cell Biology

Publisher

Rockefeller University Press

issn

0021-9525

eissn

1540-8140

Available date

2016-03-04

Publisher version

http://jcb.rupress.org/content/109/6/2917

Language

en

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