Characterisation of the cytoskeletal protein talin

Talin is a 270 kDa cytoskeletal protein implicated in integrin-mediated cellular interactions with the extracellular matrix. In the current study, the three vinculin-binding sites in talin have been further defined, using a yeast two-hybrid approach, and were found to be contained within short amphipathic -helices encompassing residues 607-636, 852-876 and 1945-1969. Each of these helices was found to compete for the same binding site within the vinculin head, and to bind with similar rate constants. The talin-binding site in vinculin was further defined to residues 1-167. Fluorescence assays have been used to demonstrate that a talin polypeptide encompassing residues 2270-2541 binds and bundles F-actin, and that native talin does not nucleate polymerisation of actin.;In order to identify novel binding partners for talin, a polypeptide encompassing talin residues 1856-2301 was used to screen a cDNA expression library using a yeast two-hybrid approach. The library screen identified a single novel talin-binding protein that has been named TIP1 (talin-interacting protein-1). TIP1 binds specifically to talin in both the two-hybrid system and in vitro binding assays, and searches of genomic and EST databases have uncovered homologues in human, mouse and rat. The full-length human cDNA has been mapped and is predicted to encode a 300 kDa protein that does not bear similarity to known proteins. The size of the TIP1 protein has been confirmed by production of a polyclonal antibody followed by Western blotting of MEF protein extracts.