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

虽然Gs亚基是经典已知的从位于质膜（PM）上的G蛋白偶联受体（GPCR）向下游效应物传递细胞外信号，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，5年前资助，和我的实验室的长期兴趣，是表征的非传统作用的Gs在这个细胞内室。在过去的几年中，大量的数据和工具强调了Gs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-Gs信号传导发生在内体以及PM。此外，我们小组还指出了Gs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年中，我们计划概述Gs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.研究Gs在核内体上的募集 虽然Gs在核内体上的定位现在被广泛接受，但对Gs进出核内体的运输知之甚少。根据这一目标，活细胞显微镜的Gs标记到一个光活化的绿色到红色的荧光蛋白将被用来监测细胞内动态的特定池的Gs（光激活的PM或内体）。CRISPR/Cas9基因编辑也将用于标记内源性Gs，以更好地进行功能分析。将在刺激在其信号传导途径中与Gs偶联或不偶联的各种受体后检查Gs在内体上的募集。Gs运输途径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。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）测定来检查Gs活性形式的分布。 目标2.表征Gs在核内体微区上的分布和功能 内体被划分为不同的功能亚结构域，例如信号传导、再循环和分选到管腔内囊泡中用于溶酶体降解。根据这一目标，活细胞和超分辨率显微镜（STED）显微镜将被用来表征的分布，动态和分子组织的Gs在内体亚域激活和内吞的各种受体。 重要性：在过去的十年中，我们已经了解了很多关于Gs组织在质膜，但仍有很多要了解的性质，其内涵体招聘，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 Gs 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 Gs on this intracellular compartment. In the last few years, a plethora of data and tools highlighted the endosomal functions of Gs in receptor signaling and trafficking. It is now accepted that canonical GPCR-Gs signalling occurs from endosome as well as the PM. Furthermore, our group has pointed out a novel role of Gs 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 Gs and its molecular organization and regulation on endosomal microdomains. New tools will also be developed in order to answer these questions. AIM1. To investigate Gs recruitment on endosomes Although Gs localization on endosome is now well accepted, much less is understood about the trafficking of Gs to and from endosomes. Under this aim, live-cell microscopy of Gs tagged to a photoactivatable green-to red fluorescent proteins will be used to monitor the intracellular dynamic of a specific pool of Gs (photactivated at the PM or endosome). CRISPR/Cas9 gene editing will also be used to tag the endogenous Gs for better functional analysis. Gs recruitment on endosome will be examined following stimulation of various receptors that are coupled or not to Gs in their signaling pathway. Gs 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 Gs will be examined by confocal microscopy imaging and Biolumiescence Resonance Energy Transfer (BRET) assays. AIM2. To characterize Gs 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 Gs in endosomal subdomains following activation and endocytosis of various receptors. Significance: In the past decade, we have learned much about Gs 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.