THE CYTOSKELETAL PROTEIN TALIN CONTAINS AT LEAST 2 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 fo cal contacts. Using a series of overlapping talin-fusion proteins expr essed in E. coli and I-125-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 (residu es 434-2541) and are recognized by the same fragment of vinculin (resi dues 1-258). Two sites are adjacent to each other and span residues 49 8-950, and the third site is more than 700 residues distant in the pri mary sequence. Scatchard analysis of I-125-vinculin binding to talin a lso indicates three sites, each with a similar affinity (Kd = 2-6 x 10 (-7) M). We also detect a substoichiometric interaction of higher affi nity (Kd = 3 x 10(-8) M) which remains unexplained. By expressing regi ons 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.