Title: Investigation of the mechanisms underlying endosomal Gq signalling
Funder: Biotechnology and Biological Sciences Research Council

Abstract: G protein-coupled receptors (GPCRs) are the largest receptor family and regulate the entire physiology. Hormones bind to GPCRs resulting in G protein activation and signalling mediating responses. Canonically, G protein activation occurs at plasma membrane (PM) and is ended by GPCR internalisation into endosomes. Recent evidence show that it also occurs non-canonically from endosomes and that endosomal signalling leads to responses that are distinct from PM signalling. As drug discovery efforts have focused on compounds binding to GPCRs at PM, a vast repertoire of therapeutic targets have been unexplored. The first GPCRs reported to activate G proteins from endomembranes were GPCRs activating Gs. Recently, endosomal Gq activation by other GPCRs was reported to be involved in Ca2+ homeostasis, pain perception, and inflammation. However, the effector and messengers engaged by Gq are different from those engaged by Gs. The molecular events translating Gq activation from endosomes to cell responses are obscure. Upon Gq activation, the activity of phospholipase C beta (PLCb) is increased, which converts PIP2, a lipid at PM, into diacylglycerol and IP3, which are messengers binding to proteins mediating responses. PLCb is abundant at PM but little is known about its potential presence in endosomes. Are there isoforms trafficking to endosomes with the GPCR and others strictly at PM? Answering this question could underlie a new mechanism of functional selectivity between cell types expressing a same GPCR. Moreover, a major paradox surrounding endosomal Gq signalling is the low PIP2 levels in endosomes. How does endosomal Gq activation is translated to a response in quasi absence of PIP2 in endosomes? Is there a local system replenishing PIP2 levels when needed? Such system could constitute a key target to switch off a specific cell response elicited by a GPCR signalling through Gq in endosomes in a therapeutic context. Our project aims to answer these fundamental questions.

Lay abstract: 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._x000D__x000D_ _x000D__x000D_ 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._x000D__x000D_ _x000D__x000D_ 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._x000D__x000D_ _x000D__x000D_ 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._x000D__x000D_ _x000D__x000D_ 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.

Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: GENERIC HEALTH RELEVANCE