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UNDERSTANDING THE ROLE OF CONFORMATIONAL DYNAMICS IN PHOSPHOLIPASE Cβ FUNCTIONIgi Vilza (12450744) 25 April 2022 (has links)
<p>Phospholipase C β (PLCβ) is an enzyme that plays a key role in cardiovascular function by hydrolyzing phosphatidylinositides (PIs) at the plasma membrane in response to the activation of G protein-coupled receptors at the membrane. PLCβ normally has low basal activity, and its activation is driven by direct binding of the heterotrimeric G protein subunits Gβγ and Gαq. Recent work from our lab and others has shown that the PH domain and first two EF-hands (EF1/2) are conformationally dynamic in solution. This opens up a potential avenue in looking at the dyamics of PLCβ as structures only depict the lipase in a more compact globular shape. We want to address how these known conformational changes PLCβ interacts with the membrane and protein-protein interactions necessary for fuction. In this study, we are using an intramolecular disulfide crosslink to stabilize defined conformational states of PLCβ. These conformational variants will be assessed for their basal activity and their ability to interact with liposomes. In addition, we are laying the initial groundwork to use these variants in single molecule tracking experiments on supported lipid bilayers. These experiments will provide significant insights into how the conformational state of PLCβ contributes to membrane binding and Gβγ stimulation at the membrane interface.</p>
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The dissection of the molecular mechanism underlying the facilitative action of prostaglandin E receptor EP1 on dopamine D1 receptor-induced cAMP production / ドパミンD1受容体によるcAMP産生におけるプロスタグランジンE受容体EP1の促進的作用を担う分子機構の解明Aliza Toby Ehrlich 24 September 2013 (has links)
京都大学 / 0048 / 新制・課程博士 / 博士(生命科学) / 甲第17931号 / 生博第294号 / 新制||生||38(附属図書館) / 30751 / 京都大学大学院生命科学研究科高次生命科学専攻 / (主査)教授 垣塚 彰, 教授 渡邉 大, 教授 松崎 文雄 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM
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Structural studies of Gαq signaling and regulationShankaranarayanan, Aruna 07 November 2012 (has links)
Gαq signaling is implicated in a number of physiological processes that include platelet activation, cardiovascular development and smooth muscle function. Historically, Gαq is known to function by activating its effector, phospholipase Cβ. Desensitization of Gαq signaling is mediated by G-protein coupled receptor kinases (GRK) such as GRK2 that phosphorylates the activated receptor and also sequesters activated Gαq and Gβγ subunits. Our crystal structure of Gαq-GRK2-Gβγ complex shows that Gαq forms effector-like interactions with the regulator of G-protein signaling (RGS) homology domain of GRK2 involving the classic effector-binding site of Gα subunits, raising the question if GRK2 can itself be a Gáq effector and initiate its own signaling cascade. In the structure, Gα and Gβγ subunits are completely dissociated from one another and the orientation of activated Gαq with respect to the predicted cell membrane is drastically different from its position in the inactive Gαβγ heterotrimer. Recent studies have identified a novel Gαq effector, p63RhoGEF that activates RhoA. Our crystal structure of the Gαq-p63RhoGEF-RhoA complex reveals that Gαq interacts with both the Dbl homology (DH) and pleckstrin homology (PH) domains of p63RhoGEF with its C-terminal helix and its effector-binding site, respectively. The structure predicts that Gαq relieves auto-inhibition of the catalytic DH domain by the PH domain. We show that Gαq activates p63RhoGEF-related family members, Trio and Kalirin, revealing several conduits by which RhoA is activated in response to Gq-coupled receptors. The Gαq effector-site interaction with p63RhoGEF/GRK2 does not overlap with the Gαq-binding site of RGS2/RGS4 that function as GTPase activating proteins (GAPs). This suggests that activated G proteins, effectors, RGS proteins, and activated receptors can form high-order complexes at the cell membrane. We confirmed the formation of RGS-Gαq-effector complexes and our results suggest that signaling pathways initiated by GRK2 and p63RhoGEF are regulated by RGS proteins via both allosteric and GAP mechanisms. Our structural studies of Gαq signaling provide insight into protein-protein interactions that induce profound physiological changes. Understanding such protein interfaces is a key step towards structure-based drug design that can be targeted to treat diseases concerned with impaired Gαq signaling. / text
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