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

异源三聚体G蛋白Gas在内体上的非典型作用 ** 异源三聚体G蛋白（a，b，？亚基）典型地参与由G蛋白偶联受体（GPCR）在质膜（PM）处启动的信号转导。然而，越来越多的证据强调了细胞内细胞器上存在异源三聚体G蛋白，从而表明它们在GPCR信号传导和运输中具有潜在作用。因此，我的研究计划旨在更好地了解G蛋白的这些新的非传统方面。最近的研究已经证实Gas存在于内体上，并且在内体受体信号传导和运输中具有非常规作用。现在公认的是，典型的GPCR-气体信号传导发生在内体以及PM。我们最近发现Gas在GPCR内体分选到溶酶体降解中的新作用。本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，我们认为这个过程不会完全终止受体的信号传导，而是可以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，它就以非经典的方式结合并激活Gas亚基，其调节或招募这些膜上的分选组分以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。AIM 1.为了确定Gas是否以及如何识别核内体上的GPCR，已知GPCR具有结合和激活Ga蛋白的能力。我们注意到受Gas耗尽影响的GPCR（如dOR、CXCR 4）在质膜的信号传导途径中不与Gas偶联。包括免疫共沉淀、GST-下拉和BRET在内的各种技术将用于确定这些GPCR是否可以与内体上的Gas直接或间接（通过β-抑制蛋白或泛素，溶酶体的两种GPCR分选信号）相互作用。目标2.研究Gai偶联的GPCR是否触发Gas在内体上的活化。* 为了检验GPCR内体运输启动的Gas信号传导的假设，我们将使用运输同步协议来检查GPCR运输“脉冲”到达内体是否引起该细胞器上的Gas信号传导响应。细胞内膜上活性气体的存在将通过使用特异性活性气体生物传感器的共聚焦显微镜成像以及通过cAMP的定量来检查。**意义：目前的教条表明，GPCR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，其中这些受体的内体信号传导控制它们的内体运输到溶酶体。

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.*****