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

虽然Gs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在核内体上。该研究项目于5年前获得资助，长期目标是表征Gs在细胞内区室中的非常规作用，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信号从核内体和PM发生。此外，我们小组还指出了Gs在gpcr的内体分选和溶酶体的单一跨膜受体中的新作用。在接下来的5年里，我们计划概述Gs的内体易位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。

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.