An intramolecular association between the head and tail domains of vinculin modulates talin binding.
Three independent lines of evidence demonstrate an interaction between the head and tail domains of vinculin. First, both the purified 30-kDa tail fragment and a fusion protein containing tail domain, vinculin residues 884-1066, protect purified 95-kDa head domain from cleavage by protease V8 in a specific, dose-dependent manner. Second, direct binding studies between the 95-kDa head and the V884-1066 fusion protein and carboxyl-terminal truncations of the fusion protein indicate a tight interaction (Kd approximately 50 nM) that requires vinculin residues 1013-1043. Finally, cross-linking of vinculin demonstrates cross-linkable head-tail complexes in the intact protein, which are almost exclusively intramolecular. Talin and the tail domain compete for binding to the vinculin head domain. Therefore, we propose that the loss of the intramolecular interaction of head and tail accounts for the increased affinity for talin of the head domain compared with intact vinculin. The observation that a head-tail interaction in the molecule affects the ability of vinculin to interact with another adherens junction component suggests that modulation of head-tail binding is a way to control recruitment of vinculin into an adherens junction.
Johnson
R P
RP
Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
Craig
S W
SW
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002645:Chickens; D003432:Cross-Linking Reagents; D005022:Ethyldimethylaminopropyl Carbodiimide; D005982:Glutathione Transferase / Q000378:metabolism; D006868:Hydrolysis; D008969:Molecular Sequence Data; D009154:Mutation; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D012697:Serine Endopeptidases; D016608:Talin / Q000378:metabolism*; D016596:Vinculin / Q000737:chemistry / Q000378:metabolism*
D003432:Cross-Linking Reagents; D011993:Recombinant Fusion Proteins; D016608:Talin; D016596:Vinculin; D005982:Glutathione Transferase; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase; D005022:Ethyldimethylaminopropyl Carbodiimide