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|Title:||Molecular studies on the cytoskeletal proteins vinculin and talin.|
|Authors:||Gilmore, Andrew Peter.|
|Presented at:||University of Leicester|
|Abstract:||Vinculin and talin are components of adherens type cell/ECM junctions (adhesion plaques) where they are thought to form part of the linkage between the cytoplasmic domain of the beta-subunit of integrins and the actin cytoskeleton. Vinculin has been shown to interact with the adhesion plaque proteins talin, paxillin and beta-actinin, and talin has been shown to interact with vinculin, integrins beta1 and beta3, and actin. This study has examined the domain structure of vinculin and talin with regard to the interaction between them. Saturation binding analysis using purified chicken vinculin and talin demonstrated that the interaction was biphasic, with two dissociation constants of 3x10.;-8Mand 5.5x10.;-7M. The lower affinity interaction indicated that multiple bindingsites existed, with three moles of vinculin binding per mole of talin. The high affinity interaction was substoichiometric, and this remains to be explained. To localise the talin binding domain in vinculin, contiguous regions of the molecule were expressed as fusion proteins in E. coli and tested for binding iodinated talin in solid phase assays. Binding was localised to the N-terminal 258 amino acids of vinculin, and no further truncation of this region left detectable talin binding activity. This region was found to bind talin as effectively as intact vinculin, and no evidence was obtained to suggest that short linear sequences within this region could account for the interaction. Two independently isolated vinculin cDNA clones, one of which lacked 123bp of coding sequence, had suggested that alternative splicing of the vinculin mRNA within the region encoding the talin binding domain could be a mechanism for regulating talin binding. Though this region was found to be contained on a single exon within the chicken vinculin gene, no evidence was found to authenticate the existence of such a spliced message using a reverse transcriptase/PCR protocol to map vinculin transcripts. Vinculin binding sites were mapped in talin by expressing a series of overlapping talin fusion proteins and investigating their ability to bind vinculin in solid phase assays. This identified three non-overlapping regions of talin within its 190kDa domain which bound vinculin with dissociation constants around 10-7M. Two of these were within residues 498-950, whilst the most C-terminal mapped to between residues 1554-2268. Further experiments on the most C-terminal of these sites suggested that a 40 amino acid sequence was able to bind vinculin.|
|Rights:||Copyright © the author. All rights reserved.|
|Appears in Collections:||Theses, Dept. of Biochemistry|
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