Non-canonical role of the heterotrimeric G protein Galphas (Gas) on endosomes

虽然气体亚基通常被认为是将位于质膜（PM）上的g蛋白偶联受体（gpcr）的细胞外信号传递给下游效应器，但最近也证明它定位于核内体上。该研究项目于5年前获得资助，长期目标是表征气体在细胞内的非传统作用，也是我实验室的长期兴趣所在。在过去的几年中，大量的数据和工具强调了气体在受体信号传导和运输中的内体功能。现在公认的是典型的GPCR-Gas信号从核内体和PM发生。此外，本小组还指出了Gas在gpcr的内体分选和溶酶体的单跨膜受体中的新作用。在接下来的5年里，我们计划概述Gas内体易位的机制及其在内体微域的分子组织和调控。为了回答这些问题，还将开发新的工具。AIM1。虽然气体在核内体上的定位已被广泛接受，但对气体进出核内体的运输知之甚少。在此目的下，Gas的活细胞显微镜标记为光激活的绿到红荧光蛋白，将用于监测特定Gas池的细胞内动态（在PM或核内体处光激活）。CRISPR/Cas9基因编辑也将用于标记内源性Gas，以便更好地进行功能分析。在信号通路中与气体偶联或不偶联的各种受体受到刺激后，内体上的气体募集将被检测。将使用特定抑制剂/siRNA和共定位各种细胞内标记物来确定气体运输途径。使用特定的生物传感器，将通过共聚焦显微镜成像和生物发光共振能量转移（BRET）检测气体活性形式的分布。AIM2。为了表征气体在核内体微域上的分布和功能，核内体被划分为不同的功能亚域，如信号传导、再循环和分选到腔内囊泡中进行溶酶体降解。在这一目标下，活细胞和超分辨率显微镜（STED）显微镜将被用来表征各种受体激活和内吞作用后，气体在内体亚域的分布、动力学和分子组织。意义：在过去的十年中，我们对质膜上的气体组织有了很多了解，但关于其内体募集、组织、调节和生理功能的性质仍有待了解。我们的工作将为这些基本问题带来新的见解，这些问题将影响我们对受体信号传导的理解。

While Gas subunit is classically known to relay extracellular signals from G-protein coupled receptors (GPCRs) located on plasma membrane (PM) to downstream effectors, it has also been recently demonstrated to be localized on endosomes. The long-term objective of this research program, funded 5 years ago, and long-time interest of my lab, is to characterize the non-conventional role of Gas on this intracellular compartment. In the last few years, a plethora of data and tools highlighted the endosomal functions of Gas in receptor signaling and trafficking. It is now accepted that canonical GPCR-Gas signalling occurs from endosome as well as the PM. Furthermore, our group has pointed out a novel role of Gas on the endosomal sorting of GPCRs and single transmembrane receptor to lysosome. Over the next 5 years, we plan to outline the mechanism of endosomal translocation of Gas and its molecular organization and regulation on endosomal microdomains. New tools will also be developed in order to answer these questions. AIM1. To investigate Gas recruitment on endosomes Although Gas localization on endosome is now well accepted, much less is understood about the trafficking of Gas to and from endosomes. Under this aim, live-cell microscopy of Gas tagged to a photoactivatable green-to red fluorescent proteins will be used to monitor the intracellular dynamic of a specific pool of Gas (photactivated at the PM or endosome). CRISPR/Cas9 gene editing will also be used to tag the endogenous Gas for better functional analysis. Gas recruitment on endosome will be examined following stimulation of various receptors that are coupled or not to Gas in their signaling pathway. Gas trafficking pathway will be determines using specific inhibitors/siRNA and colocalization various intracellular markers. Using a specific biosensor, the distribution of the active form of Gas will be examined by confocal microscopy imaging and Biolumiescence Resonance Energy Transfer (BRET) assays. AIM2. To characterize Gas distribution and function on endosome microdomains Endosomes are compartmentalized into distinct functional subdomains such as signaling, recycling and sorting into intraluminal vesicles for lysosomal degradation. Under this aim, live-cell and super-resolution microscopy (STED) microsopy will be used to characterize the distribution, dynamics and molecular organization of Gas in endosomal subdomains following activation and endocytosis of various receptors. Significance: In the past decade, we have learned much about Gas organization at the plasma membrane, but much still remains to be learned both with regards to the nature of its endosomal recruitment, organization, regulation, and the physiological function. Our work will lead to novel insights into these fundamental questions that will impact our understanding of receptor signaling.