Functional mapping of auto-inhibitory sites in talin

Integrin activation by ìnside-out' signaling is a key process in cell migration and adhesion to extracellular matrix. Recent in-vivo and in-vitro studies have provided convincing evidence that the binding of talin, a cytoskeletal protein, to the integrin [beta] cytoplasmic tails is necessary and sufficient for integrin activation. The significance of its role in integrin signaling leads to the expectation that functions of talin are tightly controlled. Indeed, previous data have shown that cellular distribution of talin and its interaction with integrins is highly regulated; talin resides in the cytosol of platelets under resting conditions but, in response to intracellular signals, translocates to the membrane where it interacts with and activate integrins. However, despite intense interests and vigorous efforts, our understanding of molecular basis of talin regulation remains incomplete. In contrast to FL talin, the N-terminal THD binds to and strongly activates integrins without requiring additional signaling. Thus, we hypothesized that a region in the talin rod domain is involved in suppressing the functions of talin. Sequential C-terminal truncations of the talin rod domain and subsequent mutational analysis revealed that an [alpha]-helical bundle termed Domain E negatively regulates membrane recruitment of talin via its inter- domain interaction with the F3 domain of talin. However, increased membrane association of talin caused by disruption of the Domain E-F3 interaction is insufficient for plasma membrane localization of talin, for the talin- integrin interaction, or for integrin activation in cells or in an in-vitro nanodisc system. NMR analysis revealed an inter-domain interaction between another α- helical bundle in the talin rod called VBS1/2a and the F23 domain of talin, and truncation of VBS1/2a led to increased plasma membrane localization of talin. In addition, truncation analysis identified a short fragment within the linker region between THD and the rod domain that interferes with the talin-integrin interaction. In this study, we have mapped three auto-inhibitory sites within talin and have assigned a specific function to each one of them. These findings provide insights as to how talin is regulated and what may happen to talin during the final steps in integrin activatio