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Conformational Regulation of Membrane Localization and Activation of Talin

Dwivedi, Pallavi
http://rave.ohiolink.edu/etdc/view?acc_num=csu1405078702

Abstract Details

2014, Master of Science in Biology, Cleveland State University, College of Sciences and Health Professions.
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, tissue redmodeling. 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 conformationaly 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 the talin-R9 (talin-3E mutant) to make it more negatively charged. Our cosedimentation experiment demonstrates that the talin mutant has significantly reduced ability to associate with membrane as compared to the WT talin, thus supporting the hypothesis I. To test the hypothesis II, we made a triple mutant (D1676R, E1770K, M319A) to weaken the talin-F3/R9 autoinhibitory interaction (talin-activation mutant). The co-sedimentation experiment demonstrates that the talin-activation mutant had substantially increased capacity to bind to membrane as compared to WT talin, indicating that the relief of the autoinhibition promoted the “pulling” of talin-H to membrane and “pushing” away of talin-R from membrane. Consistently, the talin-activation mutant had increased integrin-binding, leading to significantly enhanced talin-mediated integrin activation as compared to WT talin. Consistent with previous studies, we found the presence of three actin binding sites on talin. Interestingly, full length talin has little binding to actin, suggesting that the full length talin adopts a conformation that not only prevents integrin binding but also the actin binding. Disruption of the autoinhibitory interface that masks the integrin binding increased the talin binding to integrin but not to actin, suggesting that the two binding events are regulated by different conformational activation mechanisms.
Jun Qin, PhD (Advisor)
Sadashiva Karnik, PhD (Committee Member)
Barsanjit Mazumder, PhD (Committee Member)
Biochemistry; Biology

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Citations

  • Dwivedi, P. (2014). Conformational Regulation of Membrane Localization and Activation of Talin [Master's thesis, Cleveland State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=csu1405078702

    APA Style (7th edition)

  • Dwivedi, Pallavi. Conformational Regulation of Membrane Localization and Activation of Talin. 2014. Cleveland State University, Master's thesis. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=csu1405078702.

    MLA Style (8th edition)

  • Dwivedi, Pallavi. "Conformational Regulation of Membrane Localization and Activation of Talin." Master's thesis, Cleveland State University, 2014. http://rave.ohiolink.edu/etdc/view?acc_num=csu1405078702

    Chicago Manual of Style (17th edition)

csu1405078702
326
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This open access ETD is published by Cleveland State University and OhioLINK.