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是证明它们可以在没有激动剂的情况下与G蛋白偶联并激活G蛋白。这种结构性活性已经在天然细胞或组织中建立了几个GPCRs，并且可以代表最大激动剂依赖活性的50%，这表明它具有很高的生理相关性。然而，在理解GPCR的病理生理影响时，结构活性往往被忽视，并且大多被证明是针对G蛋白依赖的信号转导。为了解决这些问题，该项目将使用三个Gs偶联的5-羟色胺(5-HT)受体，包括5-HT4，5-HT6和5-HT7受体，它们作为GPCR模型显示出高水平的结构性活性，并追求以下两个互补的目标：1)破译它们在典型和非典型信号通路(例如，mTOR和RHO-GTP酶激活，淀粉样前体蛋白切割)的构成活性的机制，特别关注受体相互作用的作用，磷酸化和区隔2)表征这些受体非激动剂激活的病理生理结果。我们将特别探讨它在生理和病理条件下对神经元发育的影响，尤其是神经退行性疾病。因此，Contact_5-HT方案的最终目标是(I)证明GPCR规范和非规范信号不仅受与细胞外配体的结合调节，还受与相互作用的伙伴的受体联系的调节，充当正负变构调节器，以及(Ii)确定结构性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。这个跨学科的项目依赖于两个合作伙伴团队在5-羟色胺受体药理学、信号和功能方面的长期经验，以及他们在蛋白质组学和老鼠行为方面的互补技术技能(合作伙伴2)，以及最先进的成像技术，包括共聚焦和双光子FRET和TIRF-FRET成像(合作伙伴1)。这两个合作团队多年来一直在合作破译由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年以来出版了6份联合出版物)，并产生了初步成果，作为当前提案的概念证明，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.