Investigation of the mechanisms underlying endosomal Gq signalling

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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.