Date of Award
Biological, Geological and Environmental Sciences
Cell adhesion, Cell adhesion molecules, Integrins -- Structure-activity relationships, Cytology, Biochemistry, Biochemistry, biology
Talin is a cytosolic protein which is known to be one of the key players involved in integrin mediated cell adhesion dependent processes, including blood coagulation, and tissue remodeling. It connects the extracellular matrix with the actin cytoskeleton. Talin comprises of a head domain (talin-H) and a rod domain (talin-R). Talin-H is further subdivided in F0, F1, F2 and F3 domains. Talin-R contains 13 contiguous helical bundle domains (R1-R13) followed by an actin binding dimerization domain (DD). The F3 domain contains a key integrin binding site that regulates integrin activation. In our previous studies, we have shown that cytosolic talin exists in an autoinhibited state where the integrin binding site in F3 domain is self-masked by R9 domain. The autoinhibited talin is randomly distributed in the cytosol but upon activation, talin is rapidly localized to membrane and it binds and activates integrin. The main focus of the present study was to understand the mechanism of plasma membrane localization and activation of talin. Since talin has long been known to also bind to actin, we also investigated the actin binding sites in talin and how they are conformationally regulated. The crystal structure of autoinhibited talin F2F3-R9 complex, previously determined in our lab, revealed a stretch of negatively charged residues on R9 which is located on the same side as the positively charged surface on talin H. This leads to two hypotheses: (I) Electrostatic repulsion between the negatively charged talin-R9 surface and membrane promotes the cytosolic retention of autoinhibited talin (II) upon enrichment of membrane with negatively charged phosphatidylinositol-4,5-bisphosphate (PIP2), PIP2 strongly pulls the positively charged surface on talin-H towards membrane and simultaneously repels the negatively charged surface on R9, thus promoting the membrane localization and activation of autoinhibited talin via a "pull-push" mechanism. To test the hypothesis I, we made a triple mutant (H1711E, T1812E, N1815E) on
Dwivedi, Pallavi, "Conformational Regulation of Membrane Localization and Activation of Talin" (2014). ETD Archive. 612.