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。这种构成活性已经在几种gpcr的天然细胞或组织中建立，并且可以代表高达50%的最大激动剂依赖活性，表明其高度的生理相关性。然而，在对GPCR病理生理影响的理解中，组成活性经常被忽视，并且主要被证明与G蛋白依赖的信号传导有关。gpcr是否也可以组成性地激活非规范（不依赖于G蛋白）信号通路仍有待确定。为了解决这些问题，该项目将使用三种gs偶联5-羟色胺（5-HT）受体，包括5-HT4、5-HT6和5-HT7受体，这些受体显示出高水平的组成活性，作为GPCR模型，并追求以下两个互补目标：1)在典型和非典型（例如，mTOR和Rho-GTPase激活，淀粉样前体蛋白切割）信号通路中解读其构成活性的机制，特别关注受体相互作用组，磷酸化和区隔的作用。2)表征这些受体不依赖激动剂激活的病理生理结果。我们将特别探讨其在生理和病理条件下对神经发育的影响，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规范和非规范信号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伙伴的受体关联来调节，作为正或负变构调节剂；（ii）确定构成型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双方合作团队在血清素受体药理学、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方面的互补技术技能（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）。双方合作团队已经合作多年，破译信号转导参与的血清素受体和机制控制其功能状态在各种生物模型。这种合作非常富有成效（自2002年以来发表了6篇联合论文），并产生了初步结果，作为当前提案概念的证明，有力地保证了该项目的成功。

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.