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: 2018
• 资助国家: 加拿大
• 起止时间: 2018-01-01 至 2019-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=656808
• 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在核内体中的非规范作用******异三聚体G蛋白(a, β,？亚单位)通常参与由G蛋白偶联受体（gpcr）在质膜（PM）启动的信号转导。然而，积累的证据强调了胞内细胞器上异三聚体G蛋白的存在，从而表明它们在GPCR信号传导和运输中具有潜在作用。因此，我的研究计划是为了更好地理解g蛋白的这些新的非传统方面。******最近的研究证实，气体存在于核内体上，在核内体受体信号传导和运输中具有非传统的作用。现在公认的是典型的GPCR-Gas信号从核内体和PM发生。我们最近发现了气体在gpcr的内体分选到溶酶体降解中的新作用。目前的研究计划分为两个目标，是这些先前发现的逻辑延伸。******假设：与内吞作用的原始概念不一致，我们认为这一过程不会完全终止受体的信号传导，而是将其从一个途径转移到另一个途径。我们提出，一旦GPCR到达内体，它以非规范的方式结合并激活Gas亚基，从而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。******AIM1。为了确定Gas是否以及如何识别核内体上的GPCR ***GPCR以其结合和激活Ga蛋白的能力而闻名。我们注意到受Gas耗尽影响的gpcr（如dOR， CXCR4）在质膜的信号通路中不与Gas偶联。将使用包括共免疫沉淀、GST-pull-down和BRET在内的各种技术来确定这些GPCR是否可以直接或间接（通过ß-arrest或ubiquitin，溶酶体的两种GPCR分选信号）与内体上的Gas相互作用。******AIM2。研究gai偶联gpcr是否触发内体上Gas的激活。***为了验证GPCR内体交通启动气体信号传导的假设，我们将使用交通同步协议来检查GPCR交通“脉冲”到达内体是否会引起该细胞器上的气体信号传导反应。细胞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的量化来检查。******意义：目前的理论表明，gpcr的信号传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，即这些受体的内体信号控制它们的内体运输到溶酶体。*****