Localization of an integrin binding site to the C terminus of talin

Talin, consisting of a 47-kDa N-terminal head domain (residues 1-433) and a 190-kDa C-terminal rod domain (residues 434-2541), links integrins to the actin cytoskeleton. We previously reported that the binding stoichiometry of integrin alpha (IIb)beta (3):talin is similar to2:1. More recently, an integrin binding site has been localized to the talin head domain. In the present study, we identified another integrin binding site at the C-terminal region of the talin rod domain. In a solid phase binding assay, RGD affinity-purified alpha (IIb)beta (3) bound in a dose-dependent manner to microtiter wells coated with the isolated 190-kDa proteolytic fragment of the talin rod domain. Additionally, alpha (IIb)beta (3) also bound to the talin rod domain captured by 8d4, an anti-talin monoclonal antibody. Polyclonal antibodies raised against a recombinant protein fragment corresponding to the entire talin rod domain (anti-talin-R) inhibited alpha (IIb)beta (3) binding to intact talin by similar to 50% but completely blocked alpha (IIb)beta (3) binding to the talin rod domain. To localize the integrin binding site, we examined alpha (IIb)beta (3) binding to recombinant polypeptide fragments corresponding to partial sequences of the talin rod domain. Whereas alpha (IIb)beta (3) bound effectively to talin(1075-2541.) and talin-(1984-2541), it failed to bind to talin-(434-1076) and talin-(434-1975). Furthermore, the binding of alpha (IIb)beta (3) to talin-(1984-2541) was inhibited by anti-talin-R. These results indicate that an integrin binding site is located within residues 1984-2541 of the talin rod domain. Thus, talin contains two integrin binding sites, one in the homologous FERM (band (f) under bar our-point-one, (e) under bar zrin, (r) under bar adixin, (m) under bar oesin) domain and another near its C terminus. Because talin exists as an anti-parallel homodimer in focal adhesions, the two integrin binding sites in the adjacent talin molecules would be in close proximity with each other.