Sub-cellular propagation of GPCR signaling; functional imaging in space and time

GPCR 信号传导的亚细胞传播；

• 批准号: RGPIN-2018-05595
• 负责人: Bouvier, Michel
• 金额: $ 3.06万
• 依托单位: Université de Montréal
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2018
• 资助国家: 加拿大
• 起止时间: 2018-01-01 至 2019-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=646068
• 关键词: Sub; cellular; propagation; GPCR; signaling

G protein-coupled receptors (GPCR) form the largest family of proteins involved in the transfer of information across biological membranes. They are involved in the control of cellular homeostasis and in orchestrating a large number of physiological responses. In their classical representation, GPCR detect signals at the cell surface and transmit the information across the plasma membrane through conformational rearrangements leading to the engagement and activation of heterotrimeric G proteins attached to the inner face of the plasma membrane. The G proteins then interact and regulate the activity of effectors such as enzymes and channels. Following activation, phosphorylation of the receptors promotes the recruitment of the accessory protein βarrestin that leads to signal arrest and initiates receptor endocytosis. This traditional view is however rapidly evolving and ligand-promoted sub-cellular targeting of signaling complexes is emerging as an important determinant of GPCR signaling activities. We and others showed that βarrestin promotes G protein-independent signaling and may play a role in sustained endosomal G protein signaling. Nuclear translocation of GPCR and βarrestin has also been reported and linked to gene transcription regulation. Yet, most of the evidence for organelle-specific GPCR signaling are indirect and, in some cases, controversial. Directly monitoring organelle-specific signal propagation and its dynamic regulation remains a daunting challenge. We therefore propose a research program aimed at addressing this challenge by directly studying the dynamics of GPCR signal propagation in specific subcellular compartments. For this purpose, we will: 1) Develop new BRET-based biosensors detecting organelle-specific GPCR signalling activity, 2) Use high-resolution and time-resolved microscopy imaging to visualize the compartmentalized signalling activity. Using biosensors that directly monitor the real-time activation of the receptors' downstream effectors, we propose to monitor the organelle-specific signaling activity by targeting BRET probes engineered to restrict their localization to individual compartments. In addition to generate new fundamental knowledge about the dynamics and role of compartmentalized signaling of one of the most important class of signal transducers, the program will lead to the development of biophysical and imaging tools that will be useful for the study of other organelle-specific biological processes.

G蛋白偶联受体是参与跨生物膜信息传递的最大蛋白质家族。它们参与细胞内稳态的控制和协调大量的生理反应。在经典的表达方式中，GPCR检测细胞表面的信号，并通过构象重排将信息传递到质膜上，导致附着在质膜内表面的异源三聚体G蛋白的结合和激活。然后，G蛋白相互作用，调节酶和通道等效应器的活动。激活后，受体的磷酸化促进辅助蛋白βarrestin的募集，导致信号停滞并启动受体内吞作用。然而，这种传统的观点正在迅速演变，配体促进的信号复合体的亚细胞靶向正在成为GPCR信号活性的一个重要决定因素。我们和其他人表明，βarrestin促进G蛋白非依赖性信号转导，并可能在持续的内体G蛋白信号转导中发挥作用。GPCRs和βarrestin的核易位也被报道，并与基因转录调控有关。然而，大多数细胞器特异性GPCR信号的证据都是间接的，在某些情况下，还存在争议。直接监测细胞器特异的信号传播及其动态调节仍然是一个艰巨的挑战。因此，我们提出了一个旨在通过直接研究GPCR信号在特定亚细胞间传播的动力学来应对这一挑战的研究计划。为此，我们将：1)开发新的基于BRET的生物传感器来检测细胞器特异性的GPCR信号活性；2)使用高分辨率和时间分辨的显微成像来可视化划分的信号活动。使用直接监控受体下游效应器实时激活的生物传感器，我们建议通过靶向Bret探针来监控细胞器特异的信号活性，这些探针被设计成将它们的定位限制在单个隔室。除了产生关于最重要的信号传导器之一的分隔化信号的动力学和作用的新的基础知识外，该计划还将导致生物物理和成像工具的开发，这些工具将有助于研究其他细胞器特定的生物过程。