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.

阿尔茨海默病的细胞和分子方面的研究 专注于细胞骨架蛋白，特别是微管 相关蛋白 Tan，构成神经原纤维缠结。 神经递质功能障碍也与 阿尔茨海默病的大脑和这种功能障碍很可能导致 或源自 G 蛋白介导的信号的某些方面 转导。该项目试图将 Tau 聚合导致 G 蛋白介导的信号传导发生变化 希望细胞骨架之间能够建立明确的联系 阿尔茨海默病的变化和神经化学变化， 特别是与神经递质反应性有关。这 在过去的几年里，实验室一直致力于 证明细胞骨架蛋白，尤其是微管蛋白，相互作用 直接与特定的G蛋白结合来调节神经递质- 响应酶，如腺苷酸环化酶和磷脂酶 C. 微管蛋白对磷脂酶的调节是双相的。在低浓度下， 微管蛋白激活 Gq（激活磷脂酶的 G 蛋白） Cbeta1)通过形成复合物并直接转移GTP。高时 浓度下，微管蛋白通过以下方式抑制酶的激活 破坏毒蕈碱乙酰胆碱受体和 Gq 以及直接结合底物 PIP2。因此，增加了 由于钙增加而导致的微管蛋白二聚体浓度可以 “反馈抑制”磷脂酶 C。在阿尔茨海默病的大脑中，这种 Tau 和 Tau 聚合物可能会扰乱监管过程，因为 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 转移激活（荧光、光亲和力和结合 研究）。实验将使用纯化的蛋白质成分进行 以及用含有 cDNA 的杆状病毒感染的 SF9 细胞 （各种组合）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 希望能够为这个表达丰富的逻辑路径 调节可以描述。