Constitutive activity of Gs-coupled serotonin receptors: from underlying mechanisms to pathophysiological outcomes

G蛋白偶联受体（GPCR）药理学的一个重要概念演变是证明它们可以在没有激动剂的情况下偶联并激活G蛋白。已经在天然细胞或组织中建立了几种GPCR的这种组成性活性，并且可以代表高达50%的最大激动剂依赖性活性，表明其具有高度的生理相关性。然而，组成型活动往往被忽视的GPCR病理生理影响的理解，并已被证明主要是对G蛋白依赖性信号。GPCR是否也可以组成性激活非典型为了解决这些问题，该项目将使用三种GS偶联5-HT受体，包括5-HT 4、5-HT 6和5-HT 7受体，它们显示出高水平的组成型活性作为GPCR模型，并追求以下两个互补目标：1）在规范和非规范两种情况下破译其组成性活性的机制（例如，mTOR和Rho-GT3激活、淀粉样前体蛋白裂解）信号传导途径，特别关注受体相互作用组、磷酸化和区室化的作用。2）表征这些受体的激动剂非依赖性激活的病理生理学结果。我们将特别探讨其在生理和病理条件下对神经元发育的影响，特别关注神经退行性疾病。因此，ContAct_5-HT提案的最终目标是（i）证明GPCR经典和非经典信号传导不仅通过与细胞外配体的结合来调节，而且还通过与相互作用伴侣的受体缔合来调节，作为正或负变构调节剂，和（ii）建立组成型GPCR活性在健康和疾病中的重要性，并阐明其与药物发现的相关性。这个跨学科项目依赖于两个合作团队在5-羟色胺受体药理学，信号传导和功能方面的长期经验，以及他们在蛋白质组学和小鼠行为（合作伙伴#2）和最先进的成像技术（包括共聚焦和双光子FRET和TIRF-FRET成像（合作伙伴#1））方面的互补技术技能。这两个合作团队多年来一直在合作，以破译5-羟色胺受体参与的信号转导以及在各种生物模型中控制其功能状态的机制。这一合作卓有成效（自2002年以来共发表了6篇联合出版物），并产生了初步成果，证明了当前提案的概念，有力地保证了这一项目的成功。

An important conceptual evolution in G protein-coupled receptor (GPCR) pharmacology is the demonstration that they can couple to and activate G proteins in absence of agonists. This constitutive activity has been established in native cells or tissues for several GPCRs and can represent up to 50% of maximal agonist-dependent activity, suggesting its high physiological relevance. However, constitutive activity is often neglected in the understanding of GPCR pathophysiological influence and has mostly been demonstrated toward G protein-dependent signaling. Whether GPCRs can also constitutively activate non-canonical (G protein-independent) signaling pathways remains to be established.To address these issues, the project will use three Gs-coupled serotonin (5-HT) receptors, including 5-HT4, 5-HT6 and 5-HT7 receptors, that display a high level of constitutive activity as GPCR models, and pursue the two following complementary objectives: 1) Deciphering the mechanisms underlying their constitutive activity at both canonical and non-canonical (e.g., mTOR and Rho-GTPase activation, amyloid precursor protein cleavage) signaling pathways, with a special focus on the role of receptor interactome, phosphorylation and compartmentation.2) Characterizing the pathophysiological outcomes of agonist-independent activation of these receptors. We will specifically explore its impact on neuronal development under physiological and pathological conditions with a particular focus on neurodegenerative disorders.The ultimate goal of the ContAct_5-HT proposal thus is (i) to demonstrate that GPCR canonical and non-canonical signaling is not only modulated by the binding to an extracellular ligand, but also by the receptor association with interacting partners, acting as positive or negative allosteric modulators, and (ii) to establish the importance of constitutive GPCR activity in health and disease and elucidate its relevance for drug discovery. This transdisciplinary project relies on the longstanding experience of both partner teams in serotonin receptor pharmacology, signaling and functions, and on their complementary technical skills in proteomics and mouse behavior (Partner #2) and state-of-the-art imaging technologies, including confocal and 2-photon FRET and TIRF-FRET imaging (Partner #1). Both partner teams have been collaborating for many years to decipher signal transduction engaged by serotonin receptors and mechanisms controlling their functional status in various biological models. This collaboration has been very fruitful (6 joint publications since 2002) and generated preliminary results that serve as proof of concept of the current proposal, which strongly guarantees the success of this project.