The cytoskeletal protein talin contains at least two distinct vinculin binding domains

We have mapped the vinculin-binding sites in the cytoskeletal protein talin as well as those sequences which target the talin molecule to focal contacts. Using a series of overlapping talin-fusion proteins expressed in E. coli and (125)I-vinculin in both gel-overlay and microtitre well binding assay...

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Bibliographic Details
Published in:The Journal of cell biology 1993-07, Vol.122 (2), p.337-347
Main Authors: Gilmore, A.P, Wood, C, Ohanian, V, Jackson, P, Patel, B, Rees, D.J.G, Hynes, R.O, Critchley, D.R
Format: Article
Language:English
Subjects:
3T3 Cells
Animals
Binding Sites
Biological and medical sciences
Cell Line
Cell lines
Cell structures and functions
Cells
Cellular biology
Chickens
Complementary DNA
COS cells
Cytoskeleton, cytoplasm. Intracellular movements
ESTRUCTURA CELULAR
Focal adhesions
Fundamental and applied biological sciences. Psychology
Integrins
Mice
Molecular and cellular biology
Molecules
NIH 3T3 cells
POLLO
POULET
Poultry
PROTEINAS
PROTEINE
Proteins
Recombinant Fusion Proteins - metabolism
STRUCTURE CELLULAIRE
Talin - chemistry
Talin - genetics
Talin - metabolism
Transfection
Vinculin - metabolism
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Summary:We have mapped the vinculin-binding sites in the cytoskeletal protein talin as well as those sequences which target the talin molecule to focal contacts. Using a series of overlapping talin-fusion proteins expressed in E. coli and (125)I-vinculin in both gel-overlay and microtitre well binding assays, we present evidence for three separable binding sites for vinculin. All three are in the tail segment of talin (residues 434-2541) and are recognized by the same fragment of vinculin (residues 1-258). Two sites are adjacent to each other and span residues 498-950, and the third site is more than 700 residues distant in the primary sequence. Scatchard analysis of (125)I-vinculin binding to talin also indicates three sites, each with a similar affinity (Kd = 2-6 x 10-(7) M). We also detect a substoichiometric interaction of higher affinity (Kd = 3 x 10(-8) M) which remains unexplained. By expressing regions of the chicken talin molecule in heterologous cells, we have shown that the sequences required to target talin to focal contacts overlap those which bind vinculin
ISSN:0021-9525
1540-8140
DOI:10.1083/jcb.122.2.337