G Protein Regulation of Golgi Structure and Function

摘要 异三聚体G蛋白(αβγ)具有连接G蛋白偶联受体的功能 (GPCRs)对各种细胞内的反应，从而在传递广泛的 各种细胞外信号转化为调节无数的生理过程。在教科书中，G 蛋白质在与细胞质膜的细胞质表面相联系时发挥其功能。 与质膜受限的G蛋白信号的经典观点不同，它正变得越来越多 认识到G蛋白定位是动态的和受调节的，因此它们可以可逆地从 质膜到细胞内的位置，以及G蛋白在 细胞内的位置。该方案中的研究侧重于理解Gβγ的非正则功能 亚基，特别是Gβγ在高尔基体信号调节中的作用。我们之前的工作揭示了一个 高尔基体定位的G-βγ在高尔基体细胞质表面信号通路调控中的重要作用 控制发往质膜的精选蛋白质货物高尔基出口的膜。这个 目前这项提案中的研究将检验Gβγ调节信号通路的假设 通过调节生理和生理状态下高尔基体的碎裂来控制高尔基体的完整性 病理生理条件。可逆高尔基碎裂是一种细胞现象，在 正常状态，如在有丝分裂期间，以及在疾病状态下发生，如感染、癌症和 神经退行性疾病。本申请将重点定义Gβγ在监管 有丝分裂高尔基体碎裂检查点，并通过询问Gβγ在微管调节中的新角色- 依赖型高尔基体碎裂。此外，该应用程序还将定义直接 在高尔基推广Gβγ的信号。这些目标将通过各种试验性的 方法包括培养细胞、免疫荧光显微镜、活体荧光显微镜 细胞、生物传感器、药理抑制剂、突变分析和生化分析。

Abstract Heterotrimeric G proteins (αβγ) are well known for their function in linking G protein-coupled receptors (GPCRs) to a variety of intracellular responses, and thereby playing essential roles in transmitting a wide variety of extracellular signals into regulation of countless physiological processes. In the textbook view, G proteins carry out their function while associated with the cytoplasmic surface of a cell’s plasma membrane. In contrast to the classical view of plasma membrane-limited G protein signaling, it is becoming increasingly recognized that G protein localization is dynamic and regulated, such that they can reversibly traffic from the plasma membrane to intracellular locations, and that G proteins can have important cellular functions at intracellular sites. The research in this proposal focuses on understanding non-canonical functions of Gβγ subunits, and specifically roles for Gβγ in regulating signaling at the Golgi. Our previous work revealed an important role for Golgi-localized Gβγ in regulating a signaling pathway on the cytoplasmic surface of Golgi membranes that controls the Golgi exit of select protein cargo destined for the plasma membrane. The research in this current proposal will examine the hypothesis that Gβγ regulates signaling pathways that control Golgi integrity by regulating the fragmentation of the Golgi under physiological and pathophysiological conditions. Reversible Golgi fragmentation is a cellular phenomenon that occurs under normal conditions, such as during mitosis, and that occurs in disease states, such as infection, cancer and neurodegenerative disease. This application will focus on defining a novel role for Gβγ in regulating the mitotic Golgi fragmentation checkpoint and by interrogating a novel role for Gβγ in regulating microtubule- dependent Golgi fragmentation. In addition, this application will define upstream mechanisms that directly promote signaling by Gβγ at the Golgi. These objectives will be pursued by a variety of experimental approaches, including cultured cells, immunofluorescence microscopy, fluorescence microscopy of live cells, biosensors, pharmacological inhibitors, mutational analysis, and biochemical assays.