TALIN CONTAINS 3 ACTIN-BINDING SITES EACH OF WHICH IS ADJACENT TO A VINCULIN-BINDING SITE

We have determined the sequence of chicken talin (2,541 amino acids, M (r) 271,881) which is very similar (89% identity) to that of the mouse protein, Alignments with the Caenorhabditis elegans and Dictyostelium discoideum talin sequences show that the N- and C-terminal regions of the protein are conserved whereas the central part of the molecule is more divergent, By expressing overlapping talin polypeptides as fusio n proteins, we have identified at least three regions of the protein w hich can bind F-actin: residues 102-497, 951-1,327 and 2,269-2,541. Th e N-terminal binding site contains a region with homology to the ERM f amily of actin-binding proteins, and the C-terminal site is homologous to the yeast actin-binding protein Sla2p, Each of the actin-binding s ites is close to, but distinct from a binding site for vinculin, a pro tein which also binds actin, The Pro1176 to Thr substitution found in talin from Wistar-Furth rats does not destroy the capacity of this reg ion of the protein to hind actin or vinculin, Microinjection studies s howed that a fusion protein containing the N-terminal actin-binding si te localised weakly to stress fibres, whereas one containing the C-ter minal site initially localised predominantly to focal adhesions, The f ormer was readily solubilised, and the latter was resistant to Triton extraction, The N-terminal talin polypeptide eventually disrupted acti n stress fibres whereas the C-terminal polypeptide was without effect, However, a larger C-terminal fusion protein also containing a vinculi n-binding site did disrupt stress fibres and focal adhesions, The resu lts suggest that, although both the N- and C-terminal regions of talin bind actin, the properties of these two regions of the protein are di stinct.