Investigation of the mechanisms underlying endosomal Gq signalling

G蛋白偶联受体(GPCRs)是最大的受体家族，可将多种信息传递给细胞反应，调节几乎所有的生理功能。就像打开一扇门的钥匙一样，荷尔蒙等分子与它们的gpr结合在一起，导致G蛋白激活，激活一个信号级联，介导重要的细胞反应。这种信号被受体内化到称为内小体的膜小泡中终止，在内小体中，它被降解或循环到质膜。长期以来，人们一直认为G蛋白的激活只发生在质膜上。然而，越来越多的证据表明，它也可以发生在内体的GPCR内化。值得注意的是，来自内体的G蛋白信号导致了与质膜信号不同的细胞效应，这一概念被称为空间偏向。最早报道的G蛋白信号是通过GPCRs激活Gs家族中的G蛋白。最近，人们进行了大量的努力，以了解这些受体介导的空间偏向如何塑造细胞生物学和人类生理学。对这些问题的研究有助于更好地了解胰岛素分泌、甲状腺基因转录、心脏收缩、排卵、维生素D合成和骨形成等广泛的生理功能。最近，也有报道称，GQ家族中一些激活G蛋白的受体是通过内体激活的。这些研究表明，这种局部的GQ激活是许多额外的生理功能(钙稳态、痛觉、神经传递和炎症)所必需的。这一发现引起了人们的极大热情，因为这些功能是治疗血清钙调节失调、慢性疼痛、肠易激疾病和偏头痛的有趣的治疗靶点。然而，Gq激活所涉及的一系列信号蛋白与Gs信号所涉及的一系列信号蛋白不同，并出现了许多基本的机制问题。将GQ激活从内体转化为细胞反应的一系列分子事件尚不清楚。当GPCR激活GQ蛋白时，这种蛋白激活一种名为磷脂酶Cβ(PLCB)的酶，该酶将质膜上丰富的磷脂酶PIP2转化为二酰甘油和IP3。二酰甘油和IP3是与蛋白质结合的信使分子，指示细胞进行特定的行动。虽然PLCB在质膜中含量丰富，但对其在内体中的潜在存在知之甚少。有没有PLCB亚型与gpr一起内化？内体中是否已经存在异构体，其他异构体是否仅存在于质膜中？这些都是至关重要的问题，因为每种细胞类型都有自己的PLCB亚型表达谱，所以PLCB亚型的不同位置可以解释为什么GPCR在给定的细胞类型中产生反应，而在另一种细胞类型中产生不同的反应。另一个需要研究的谜团是，与质膜相比，内体中可获得的PIP2水平较低。如果局部存在低水平的PIP2，细胞如何将GQ激活转化为内体中的一种作用？在内体中是否存在补充PIP2水平的局部系统？我们建议回答这些重要的问题，因为这个系统可以构成一个靶点，在临床环境中关闭特定的有害细胞反应。由于到目前为止，药物发现工作主要集中在鉴定与质膜上的GPCRs结合的分子，由内体发出的G蛋白信号控制的细胞功能尚未成为靶点。以这些新的位置为目标开启了更好地了解生物过程及其临床意义的许多机会。然而，在将这些细胞功能用于治疗目的之前，我们需要更好地了解来自内吞体内GQ蛋白激活的信息是如何转化为细胞行为的。

G protein-coupled receptors (GPCRs) are the largest receptor family and transduce a wide array of inputs into cellular responses regulating almost all physiological functions. Like a key into a lock opening a door, molecules such as hormones bind to their GPCR, which leads to activation of G proteins activating a signalling cascade mediating important cellular responses. This signalling is terminated by receptor internalisation into membrane vesicles called endosomes, where it is targeted for degradation or recycled to plasma membrane. It was since a long time believed that G protein activation only occurs at plasma membrane. However, accumulating evidence suggest that it can also occur upon GPCR internalisation from endosomes. Remarkably, G protein signalling from endosomes leads to cellular effects that are distinct from signalling at plasma membrane, a concept termed as spatial bias. The first reports of G protein signalling from endosomes were for GPCRs activating G proteins from the Gs family. A lot of efforts have been recently deployed to understand how spatial bias mediated by these receptors shapes cellular biology and human physiology. Investigation of these questions led to a better understanding of a wide range of physiological functions, such as insulin secretion, gene transcription in thyroids, heart contraction, ovulation, vitamin D synthesis, and bone formation.Recently, some receptors activating G proteins from the Gq family were also reported to be activated from endosomes. These studies demonstrate that this local Gq activation is required for many additional physiological functions (calcium homeostasis, pain perception, neurotransmission and inflammation). This discovery generated a lot of enthusiasm as these functions are interesting therapeutic targets to treat dysregulation of calcium in serum, chronic pain, irritable bowel disease, and migraine. However, the series of signalling proteins engaged by Gq activation are different from those involved in Gs signalling and a lot of fundamental mechanistic questions arise. The series of molecular events translating Gq activation from endosomes to cellular responses remain obscure.When a GPCR activates Gq protein, this protein activates an enzyme called phospholipase C beta (PLCb), which transforms a lipid abundant at the plasma membrane called PIP2 into diacylglycerol and IP3. Diacylglycerol and IP3 are messengers molecules binding to proteins to instruct the cell to do a specific action. While PLCb is abundant at plasma membrane, little is known about its potential presence in endosomes. Are there PLCb isoforms internalising together with the GPCR? Are there isoforms already present in endosomes and others strictly at plasma membrane? These are crucial questions because each cell type has his own PLCb isoform expression profile, so different location of PLCb isoforms could explains why a GPCR produces a response in a given cell type but a different one in another one. Another mystery to investigate is the low levels of PIP2 available in endosomes compared to plasma membrane. How does the cell translate Gq activation into an action in endosomes if there is low local levels of PIP2? Is there a local system in endosomes replenishing PIP2 levels? We propose to answer these important questions because this system could constitute a target to switch off specific unwanted cellular responses in a clinical context.As drug discovery efforts have focused so far on identification of molecules binding to GPCRs at plasma membrane, cellular functions controlled by G protein signalling from endosomes have not been targeted yet. Targeting these new sites unlocks many opportunities to better understand biological processes and their clinical significance. However, before to target these cellular functions for therapeutic purposes, we need to better understand how the message coming from Gq protein activation in endosomes is translated into a cellular action.