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.
Article|
December 01 1989
Identification of a talin binding site in the cytoskeletal protein vinculin.
P Jones,
P Jones
Department of Biochemistry, University of Leicester, United Kingdom.
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P Jackson,
P Jackson
Department of Biochemistry, University of Leicester, United Kingdom.
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G J Price,
G J Price
Department of Biochemistry, University of Leicester, United Kingdom.
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B Patel,
B Patel
Department of Biochemistry, University of Leicester, United Kingdom.
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V Ohanion,
V Ohanion
Department of Biochemistry, University of Leicester, United Kingdom.
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A L Lear,
A L Lear
Department of Biochemistry, University of Leicester, United Kingdom.
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D R Critchley
D R Critchley
Department of Biochemistry, University of Leicester, United Kingdom.
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P Jones
Department of Biochemistry, University of Leicester, United Kingdom.
P Jackson
Department of Biochemistry, University of Leicester, United Kingdom.
G J Price
Department of Biochemistry, University of Leicester, United Kingdom.
B Patel
Department of Biochemistry, University of Leicester, United Kingdom.
V Ohanion
Department of Biochemistry, University of Leicester, United Kingdom.
A L Lear
Department of Biochemistry, University of Leicester, United Kingdom.
D R Critchley
Department of Biochemistry, University of Leicester, United Kingdom.
Online ISSN: 1540-8140
Print ISSN: 0021-9525
J Cell Biol (1989) 109 (6): 2917–2927.
Citation
P Jones, P Jackson, G J Price, B Patel, V Ohanion, A L Lear, D R Critchley; Identification of a talin binding site in the cytoskeletal protein vinculin.. J Cell Biol 1 December 1989; 109 (6): 2917–2927. doi: https://doi.org/10.1083/jcb.109.6.2917
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