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-2019-05556
• 负责人: Bouvier, Michel
• 金额: $ 4.37万
• 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: 2020
• 资助国家: 加拿大
• 起止时间: 2020-01-01 至 2021-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=714584
• 关键词: Sub; cellular; propagation; GPCR; signaling

Proteostasis and protein trafficking are central processes controlling the homeostasis of cells and their ability to respond to external stimuli. One of the protein families for which these processes play a central role is the G protein-coupled receptors (GPCR). These receptors form the largest family of proteins involved in the transfer of information across biological membranes. In their classical representation, GPCR detect signals at the cell surface and transmit the information across the plasma membrane, leading to the 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 receptor promotes the recruitment of the accessory protein arrestin, leading to receptor endocytosis and signal arrest. This traditional view is however rapidly evolving and ligand-promoted trafficking or direct targeting of signaling complexes to lipid raft, caveolae, endosomes, nucleus, Golgi, mitochondria and endoplasmic reticulum, have been linked to non-canonical signaling as well as regulatory functions in these organelles. Yet, most of the evidence for organelle-specific GPCR activity are indirect and, in some cases, controversial. Directly monitoring organelle-specific protein trafficking and signal propagation remains a daunting challenge. The long-term objective of our NSERC-supported research program is to develop a comprehensive biophysical platform allowing the real-time monitoring of organelle-specific trafficking and activity in living cells. GPCR will be used as prototypical models to develop this platform. In the present application, we propose to: 1) develop a collection of new bioluminescence resonance energy transfer (BRET)-based biosensors detecting organelle-specific GPCR trafficking and, 2) use high-resolution and time-resolved microscopy to visualize the compartmentalized signalling activity. To reach these specific objectives, we will design and validate a collection of BRET probes engineered to restrict their localization to individual compartments. The studies will generate new fundamental knowledge about the dynamics and role of compartmentalized signaling. Because of their portability, the biophysical and imaging tools developed will be used in longer-term projects to asses a diversity of other organelle-specific biological processes.

蛋白质稳态和蛋白质转运是控制细胞内稳态及其对外部刺激反应能力的核心过程。这些过程起核心作用的蛋白质家族之一是G蛋白偶联受体（GPCR）。这些受体构成了参与跨生物膜信息传递的最大的蛋白质家族。在它们的经典表达中，GPCR检测细胞表面的信号并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蛋白的激活。然后G蛋白相互作用并调节效应器（如酶和通道）的活性。激活后，受体的磷酸化促进辅助蛋白阻滞蛋白的募集，导致受体内吞作用和信号阻滞。然而，这一传统观点正在迅速发展，配体促进的转运或信号复合物直接靶向脂质筏、小泡、核内体、高尔基体、线粒体和内质网，与这些细胞器中的非规范信号传导和调节功能有关。然而，大多数关于细胞器特异性GPCR活性的证据是间接的，在某些情况下是有争议的。直接监测细胞器特异性蛋白质运输和信号传播仍然是一个艰巨的挑战。我们nserc支持的研究项目的长期目标是开发一个全面的生物物理平台，允许实时监测活细胞中细胞器特异性的运输和活动。GPCR将作为开发该平台的原型模型。在目前的应用中，我们建议：1)开发一系列新的基于生物发光共振能量转移（BRET）的生物传感器来检测细胞器特异性GPCR运输；2)使用高分辨率和时间分辨率显微镜来可视化区分开的信号活动。为了达到这些具体目标，我们将设计和验证一系列BRET探针，以限制其定位到单个隔室。这些研究将产生关于区隔信号的动力学和作用的新的基础知识。由于其可移植性，开发的生物物理和成像工具将用于长期项目，以评估其他细胞器特异性生物过程的多样性。