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

虽然Gas亚基经典地已知将细胞外信号从位于质膜（PM）上的G蛋白偶联受体（GPCR）传递到下游效应物，但最近也已证明其定位于内体上。这项研究计划的长期目标是5年前资助的，也是我实验室的长期兴趣，是表征气体在细胞内室的非常规作用。在过去的几年中，大量的数据和工具强调了Gas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信号传导发生在内体以及PM。此外，我们小组还指出了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的内体易位机制及其对内体微结构域的分子组织和调控。还将开发新的工具来回答这些问题。AIM 1.为了研究Gas在内体上的募集，虽然Gas在内体上的定位现在被广泛接受，但对Gas进出内体的运输知之甚少。在这一目标下，将使用标记到可光活化的绿色至红色荧光蛋白的气体的活细胞显微镜来监测特定气体池（在PM或内体处光活化）的细胞内动态。CRISPR/Cas9基因编辑也将用于标记内源性Gas，以更好地进行功能分析。将在刺激在其信号传导途径中与Gas偶联或不偶联的各种受体后检查内体上的气体募集。将使用特异性抑制剂/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）试验检查活性形式气体的分布。目标2.为了表征内体微结构域上的气体分布和功能，将内体划分为不同的功能亚结构域，例如信号传导、再循环和分选到用于溶酶体降解的管腔内囊泡中。在这一目标下，活细胞和超分辨率显微镜（STED）显微镜将用于表征各种受体激活和内吞作用后Gas在内体亚结构域中的分布，动力学和分子组织。 重要性：在过去的十年中，我们已经了解了很多关于气体组织在质膜，但仍然有很多要了解的性质，其内体招聘，组织，调节和生理功能。我们的工作将导致对这些基本问题的新见解，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.