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

G蛋白偶联受体（GPCR）是参与跨生物膜信息传递的最大的蛋白家族。它们参与控制细胞内稳态和协调大量的生理反应。在它们的经典表述中，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，从而导致附着在质膜内表面的异三聚体G蛋白的结合和激活。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β阻滞蛋白的募集，导致信号阻滞并启动受体内吞作用。然而，这种传统观点正在迅速发展，配体促进的亚细胞靶向信号复合物正在成为GPCR信号活动的重要决定因素。我们和其他人发现β阻滞蛋白促进G蛋白非依赖性信号传导，并可能在持续的内体G蛋白信号传导中发挥作用。GPCR和β阻滞蛋白的核易位也被报道并与基因转录调控有关。然而，大多数关于细胞器特异性GPCR信号的证据是间接的，在某些情况下是有争议的。直接监测细胞器特异性信号传播及其动态调控仍然是一个艰巨的挑战。因此，我们提出了一项研究计划，旨在通过直接研究GPCR信号在特定亚细胞区室中的传播动力学来解决这一挑战。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GPCR信号活性；2)使用高分辨率和时间分辨率的显微镜成像来可视化区隔的信号活性。利用直接监测受体下游效应物实时激活的生物传感器，我们建议通过靶向BRET探针来监测细胞器特异性信号活动，这些探针被设计成限制其定位到单个室。除了产生关于最重要的一类信号换能器之一的区隔信号的动力学和作用的新基础知识外，该计划还将导致生物物理和成像工具的发展，这将有助于研究其他细胞器特异性生物过程。

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.