Identification of a talin binding site in the cytoskeletal protein vinculin.
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.
Jones
P
P
Department of Biochemistry, University of Leicester, United Kingdom.
Jackson
P
P
Price
G J
GJ
Patel
B
B
Ohanion
V
V
Lear
A L
AL
Critchley
D R
DR
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D002645:Chickens; D003598:Cytoskeletal Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D005822:Genetic Vectors; D008565:Membrane Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009154:Mutation; D015183:Restriction Mapping; D016608:Talin; D014162:Transfection; D016596:Vinculin
D003598:Cytoskeletal Proteins; D008565:Membrane Proteins; D016608:Talin; D016596:Vinculin