REGULATION OF G PROTEIN MEDIATED PHOSPHOLIPID SIGNALING

G 蛋白介导的磷脂信号传导的调节

• 批准号: 2732629
• 负责人: MARK M. RASENICK
• 金额: $ 17.82万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-09-01 至 2002-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2732629
• 关键词: Baculoviridae; G protein; Sf9 cell line; biological signal transduction; confocal scanning microscopy; electron microscopy; enzyme activity; guanosine triphosphate; immunocytochemistry; intermolecular interaction; microtubule associated protein; phosphatidylinositols; phospholipase C; transfection; tubulin; video microscopy

Studies of the cellular and molecular aspects of Alzheimer's disease have focused upon cytoskeletal proteins, particularly the microtubule associated protein, Tan, which makes up neurofibrillary tangles. Neurotransmitter dysfunction has also been implicated in the Alzheimer's disease brain and such dysfunction is likely to result in or originate from some aspect of G protein-mediated signal transduction. This project represents an attempt to correlate the changes in G-protein mediated signaling with polymerization of Tau in the hope that clear linkage might be made between the cytoskeletal changes and the neurochemical changes of Alzheimer's disease, especially in relation to neurotransmitter responsiveness. This laboratory has been engaged, for the past several years, in efforts to demonstrate that cytoskeletal proteins, especially tubulin, interact directly with specific G proteins to regulate neurotransmitter- responsive enzymes such as adenylyl cyclase and phospholipase C. Tubulin regulation of phospholipase is biphasic. At low concentrations, tubulin activates Gq (the G protein which activates phospholipase Cbeta1) by forming a complex and transferring GTP directly. At high concentrations, tubulin inhibits the enzymes activation both by disrupting the coupling between muscarinic acetylcholine receptors and Gq as well as by binding the substrate, PIP2, directly. Thus, increased tubulin dimer concentration, resulting from increased calcium, could "feedback inhibit" phospholipase C. In the Alzheimer's brain, this regulatory process may be disrupted by Tau and Tau polymers, since it appears that PIP2 may be crucial for the assembly of Tau as well. This project will attempt to test the interaction between Gq and tubulin and how Tau or PIP2 might modify that process. This will be done by studying the binding of the species (fluorescence and microscopy) and the activation by GTP transfer (fluorescence, photoaffinity and binding studies). Experiments will be done with purified protein components as well as with SF9 cells infected with baculovirus containing cDNA for (in various combinations) ml receptors, Gq and phospholipase Cbeta1. Effects of Tau and tubulin on the enzyme activity will be determined in the hope that a logical pathway for this richly articulated regulation can be described.

研究阿尔茨海默氏病细胞和分子方面的研究 专注于细胞骨架蛋白，尤其是微管 相关蛋白质棕褐色，构成神经纤维缠结。 神经递质功能障碍也与 阿尔茨海默氏病大脑和这种功能障碍可能导致 或源自G蛋白介导的信号的某些方面 转导。该项目代表着将 G蛋白介导的信号传导随着Tau聚合的变化而变化 希望可以在细胞骨架之间建立明确的联系 阿尔茨海默氏病的变化和神经化学变化， 特别是与神经递质的反应性有关。这 在过去的几年中，实验室一直在参与 证明细胞骨架蛋白，尤其是微管蛋白相互作用 直接使用特定的G蛋白来调节神经递质 - 反应性酶，例如腺苷酸环化酶和磷脂酶C. 磷脂酶的微管蛋白调节是双相。在低浓度下， 微管蛋白激活GQ（激活磷脂酶的G蛋白 CBETA1）直接形成复合物和转移GTP。在高 浓度，微管蛋白抑制酶的激活 破坏毒蕈碱乙酰胆碱受体和 GQ以及通过直接结合底物PIP2。因此，增加了 微管蛋白二聚体浓度是由钙增加引起的，可以 “反馈抑制”磷脂酶C.在阿尔茨海默氏症的大脑中， tau和tau聚合物可能会破坏调节过程，因为它 看来PIP2对于TAU的组装也可能至关重要。这 项目将尝试测试GQ和微管蛋白和小管之间的相互作用 tau或pip2如何修改该过程。这将由 研究物种的结合（荧光和显微镜）和 GTP转移的激活（荧光，光接触和结合 研究）。实验将使用纯化的蛋白质成分作为 以及与含有baculovirus的SF9细胞一起 （在各种组合中）ML受体，GQ和磷脂酶CBETA1。 将确定tau和微管蛋白对酶活性的影响 希望这是这一鲜明表达的逻辑途径 可以描述调节。