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

异源三聚体 G 蛋白 Galphas (Gas) 对内涵体的非典型作用

• 批准号: RGPIN-2015-06138
• 负责人: Lavoie, Christine
• 金额: $ 2.77万
• 依托单位: Université de Sherbrooke
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2019
• 资助国家: 加拿大
• 起止时间: 2019-01-01 至 2020-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=687176
• 关键词: Non; canonical; role; heterotrimeric; protein

Non canonical role of the heterotrimeric G protein Gas on endosomes******Heterotrimeric G proteins (a,ß,? subunits) are classically involved in signal transduction initiated at the plasma membrane (PM) by G protein-coupled receptors (GPCRs). However, accumulated evidence has highlighted the presence of heterotrimeric G proteins on intracellular organelles thereby suggesting that they have a potential role in GPCR signalling and trafficking. My research program is thus geared toward a better understanding of these new non-traditional aspects of the G-proteins.******Recent studies have confirmed that Gas is present on endosomes and has non-conventional roles in endosomal receptor signalling and trafficking. It is now accepted that canonical GPCR-Gas signalling occurs from endosomes as well as the PM. We have recently discovered a novel role of Gas on the endosomal sorting of GPCRs to lysosome for degradation. The present research program is divided into two aims that are a logical extension of these previous findings.******Hypothesis: At odds with the original concept of endocytosis, we believe that this process does not completely terminate the signalling of a receptor, but can instead divert it from one pathway to another. We propose that once the GPCR reaches the endosomes, it binds and activates Gas subunit in a non-canonical way, which regulates or recruits sorting components on these membranes to modulate receptor targeting to lysosomes. Our overall objective for the next 5 years is to outline the molecular composition and the mode of operation of this Gas-based traffic-detection-and-response.******AIM1. To determine whether and how Gas recognizes GPCR on endosomes***GPCRs are known for their capacity to bind and activate Ga proteins. We noticed that the GPCRs affected by Gas depletion (such as dOR, CXCR4) are not coupled to Gas in their signalling pathway at the plasma membrane. Various techniques including co-immunoprecipitation, GST-pull-down and BRET will be used to determine whether these GPCRs can interact directly or indirectly (via ß-arrestin or ubiquitin, two GPCR sorting signal for lysosome) with Gas on endosomes.******AIM2. To investigate whether Gai-coupled GPCRs trigger the activation of Gas on endosomes. ***To test the hypothesis of GPCR endosomal traffic-initiated Gas signalling, we will use traffic-synchronization protocols to examine whether the arrival of a GPCR traffic `pulse' at the endosomes elicits a Gas signalling response on this organelle. The presence of active Gas on intracellular membranes will be examined by confocal microscopy imaging using specific active Gas biosensor as well as by the quantification of cAMP.******Significance: The current dogma suggests that the signalling of GPCRs is tightly controlled by the endocytic pathways. Our work will highlight a novel concept in which the endosomal signalling of these receptors control their endosomal trafficking to lysosome.*****

异三聚体G蛋白Gas对内体G蛋白(a，？)的非正则作用亚基)是由G蛋白偶联受体(GPCRs)在质膜(PM)启动的信号转导过程。然而，越来越多的证据表明，细胞内细胞器上存在异三聚体G蛋白，从而表明它们在GPCR信号转导和运输中具有潜在的作用。因此，我的研究计划是为了更好地了解G-蛋白的这些新的非传统方面。*最近的研究证实，GAS存在于内体上，在内体受体信号传递和运输中具有非常规作用。现在公认的是，典型的GPCR-Gas信号既来自内体，也来自PM我们最近发现了一种新的作用，即GAS在GPCRs内体分选到溶酶体进行降解。目前的研究计划分为两个目标，这两个目标是这些先前发现的合乎逻辑的延伸。*假设：与最初的内吞作用概念不同，我们认为这个过程并不完全终止受体的信号传递，而是可以将其从一条途径转移到另一条途径。我们认为，一旦GPCR到达内小体，它就以一种非规范的方式结合并激活Gas亚基，调节或招募这些膜上的分选成分来调节受体靶向溶酶体。我们未来5年的总体目标是概述这种基于气体的交通检测和响应的分子组成和操作模式。为了确定GAS是否以及如何识别内体上的GPCRs*GPCRs以其结合和激活Ga蛋白的能力而闻名。我们注意到，受气体耗竭影响的GPCRs(如DOR、CXCR4)在质膜上的信号通路中不与气体偶联。不同的技术，包括免疫共沉淀，GST-下拉和Bret将被用来确定这些GPCRs是否可以直接或间接地(通过？arrestin或ubiquitin，两个溶酶体的GPCR分选信号)与内体上的GAS相互作用。目的：研究GAI偶联的GPCRs是否触发GAS对内含体的激活。*为了验证GPCR内体流量启动的Gas信号的假说，我们将使用流量同步协议来检查GPCR流量“脉冲”到达内体是否会在这个细胞器上引起Gas信号反应。细胞内膜上活性气体的存在将通过使用特定活性气体生物传感器的共聚焦显微镜成像以及cAMP的定量来检测。*意义：目前的教条表明，GPCRs的信号转导受到内吞途径的严格控制。我们的工作将突出一个新的概念，在这个概念中，这些受体的内体信号控制它们向溶酶体的内体运输。