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

虽然Gs亚基经典地将细胞外信号从位于质膜(PM)上的G蛋白偶联受体(GPCRs)传递到下游效应器，但最近也被证明定位于内小体。5年前资助的这项研究计划的长期目标，以及我的实验室的长期兴趣，是为了表征Gs在这个细胞内间隔中的非常规作用。在过去的几年里，大量的数据和工具强调了Gs在受体信号转导和运输中的内体功能。现在公认的是，典型的GPCR-Gs信号发生在内体和PM上此外，我们的团队还指出了Gs在GPCRs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内体易位的机制及其对内体微区的分子组织和调控。还将开发新的工具来回答这些问题。 AIM1.研究Gs在内涵体上的募集 虽然Gs在内体上的定位现在已经被广泛接受，但对Gs往返内体的运输却知之甚少。在这一目标下，标记有可光激活的绿色到红色荧光蛋白的Gs的活细胞显微镜将被用来监测特定Gs池(在PM或内体被光激活)的细胞内动态。CRISPR/Cas9基因编辑也将用于标记内源Gs，以便更好地进行功能分析。内体上的GS募集将在刺激信号通路中与Gs偶联或不偶联的各种受体后进行检测。GS转运途径将利用特定的抑制物/siRNA和共定位各种胞内标志物来确定。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)分析来检测Gs的活性形式的分布。 AIM2.内涵体微区Gs分布及功能的研究 内小体被划分为不同的功能亚域，如信号、循环和分选为溶酶体降解的腔内小泡。在这一目标下，将使用活细胞和超分辨显微镜(STED)显微技术来表征Gs在各种受体激活和内吞作用后在内体亚区的分布、动态和分子组织。 意义：在过去的十年里，我们已经了解了许多关于质膜Gs组织的知识，但关于其内体募集的性质、组织、调节和生理功能仍有许多需要了解的地方。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.