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Title:The membrane lipid composition regulates talin mediated integrin activation
Author(s):Ye, Xin
Director of Research:Sligar, Stephen
Doctoral Committee Chair(s):Sligar, Stephen
Doctoral Committee Member(s):Morrissey, James; Tajkhorshid, Emad; Fratti, Rutilio
Department / Program:Biochemistry
Degree Granting Institution:University of Illinois at Urbana-Champaign
Abstract:Integrins are a family of transmembrane receptors that mediate cell-cell and cell-extra cellular matrix (ECM) interaction and signaling. Inside-out activation of integrin receptors often requires the binding of the cytoplasmic domain of the subunits by talin. This interaction leads to separation of the integrin and transmembrane domain and significant conformational changes in the extracellular domains, resulting in a dramatic increase in integrin's affinity for ligands. Membrane bilayers containing anionic lipids are indispensable for proper talin-integrin interaction, yet the detail picture of the interplay between protein and membrane has remained elusive. This thesis describe a series of fluorescence based assay for measuring talin-membrane interactions with bilayers of controlled composition using Nanodiscs technology. Results show that recruitment of talin head domain (THD) to the membrane surface is governed by electrostatics in the absence of other adapter proteins. In addition, distance measurements reveal that anionic lipids stimulate a conformational change in the talin head domain allowing interaction of the F3 domain with the phospholipid bilayer. The magnitude of this conformational change is regulated by the identity of the phospholipid headgroup, with phosphatidylinositides promoting the largest change. This emphasizes the importance of PIP2 in converting talin to a conformation optimized for interactions with integrin cytoplasmic tails. Moreover, a phenylalanine-rich region in F2 serves as a hidden hydrophobic anchor was initially indicated by computational simulation and later observed experimentally. It inserts into membrane after initial electrostatic contact providing a stabilizing force for membrane bound talin. Insertion of these phenylalanine may be critical for triggering the F3 conformational change described above. In the cytosol, talin adopts an auto-inhibited conformation, that C-terminal rod binds the N-terminal head domain, preventing talin interactions with the membrane surface and the integrin cytoplasmic domain. A Fluorescence Resonance Energy Transfer (FRET) based binding assay reveals that R9 and R12R13 segments of the talin rod domain inhibit the binding of the talin head to anionic lipid bilayers. In contrast, the binding of talin to bilayers containing PIP2 is insensitive to the presence of the inhibitor domains thereby directly implicating PIP2 as an effective activator of talin. The interaction of PIP2 with talin head for activation was located at F2F3 domain. The Nanodisc technology also offer an ideal platform for assembling unclustered integrin transmembrane and cytoplasmic domain in lipid bilayer. Measurements of the THD binding to integrin inserted Nanodiscs reveal that integrin provides most binding free energy of talin-membrane recruitment. The conformational change of THD F3, which is critical for optimal talin-integrin interactions, remains sensitive to lipid headgroup identity in the presence of integrin tail and PIP2 promotes the largest change. Further investigation also demonstrates that, with integrin inserted Nanodiscs, talin self-inhibitory rod domains impede THD binding to PS membrane in different degrees. PIP2 membrane renders THD insensitive to inhibition by talin R9 and R12R13. Results also implicates that talin R9 sterically blocks THD binding integrin on F3 domain and R12R13 may hinder the membrane interaction on F2 domain. Thus, this thesis work shows that PIP2 plays a central role in the regulation of the auto-inhibited form of talin and stimulates recruitment of talin to the membrane and integrin, which is essential for integrin inside-out activation.
Issue Date:2016-07-25
Rights Information:Copyright 2016 Xin Ye
Date Available in IDEALS:2017-03-01
Date Deposited:2016-12

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