Investigating the compartment-specific functions of G protein-coupled receptor kinase 2 in the central circadian clock of mice

研究小鼠中央生物钟中 G 蛋白偶联受体激酶 2 的区室特异性功能

• 批准号: RGPIN-2016-05563
• 负责人: Cheng, HaiYing
• 金额: $ 5.1万
• 依托单位: University of Toronto
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2022
• 资助国家: 加拿大
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=745998
• 关键词: Investigating; compartment; specific; functions; protein

Most living organisms possess an intrinsic timekeeping mechanism, known as the circadian clock, that temporally organizes behavioural and physiological processes to near-24h cycles. The circadian clock oscillates in a self-sustaining fashion, but is able to reset its phase in response to light or other external time cues. This ability to synchronize (or entrain) behaviour and physiology to environmental light-dark cycles is vital for the principal function of the circadian clock: to serve as an internal predictor of external time, and in doing so to facilitate adaptation to environmental changes. In mammals, the central circadian pacemaker resides in the suprachiasmatic nucleus (SCN) of the hypothalamus, which serves as the gateway through which light acts on the rest of the circadian timing system. Inter- as well as intra-cellular communication are essential for the proper functioning of the SCN clock. The overarching goal of my research program is to identify and understand the molecular mechanisms that govern SCN clock entrainment and rhythmicity. Using pan-SCN grk2 knockout mice, we recently uncovered a role of the G protein-coupled receptor (GPCR) kinase, GRK2, in fine-tuning the entrainment, amplitude and period properties of the SCN. Our data suggest that GRK2 may be interacting at various levels with different GPCRs and core clock elements to regulate the SCN clock. However, the exact molecular mechanisms remain to be clearly elucidated. Moreover, given that the SCN is anatomically divided into light-responsive (core) and robustly rhythmic (shell) regions, it is unclear how region-specific GRK2 activity influences entrainment and oscillatory properties of the SCN, or what the mechanisms are that underlie compartment-specific effects of GRK2. Hence, this proposal aims to dissect the precise roles of GRK2 in the SCN by selectively ablating GRK2 in the subsets of SCN neurons that comprise the two functionally distinct compartments, and by using a multi-pronged approach that incorporates behavioural, pharmacological, biochemical, histological, cell culture-based and bioluminescent imaging techniques to tackle mechanistic questions. We will examine the functional interactions between GRK2 and key neuropeptide-GPCR systems, as well as interactions between GRK2 and core clock proteins, in the SCN shell and core regions. In doing so, we will provide fresh mechanistic insights into how GRK2 regulates the ability of the SCN to respond to environmental light cues and maintain rhythmicity with defined yet tunable characteristics. Overall, the proposed research, which offers exceptional opportunities for training graduate and undergraduate students, will greatly expand our current knowledge of the cellular mechanisms that are essential for proper clock function, and will lead to important insights of broad relevance to diverse biological systems that are regulated by GRK2.

大多数生物体都拥有一种内在的计时机制，称为生物钟，可以将行为和生理过程暂时组织到近 24 小时的周期。生物钟以自我维持的方式振荡，但能够根据光或其他外部时间提示重置其相位。这种将行为和生理学与环境明暗周期同步（或引导）的能力对于生物钟的主要功能至关重要：作为外部时间的内部预测器，并以此促进对环境变化的适应。在哺乳动物中，中央昼夜节律起搏器位于下丘脑的视交叉上核（SCN）中，它是光作用于昼夜节律计时系统其余部分的门户。细胞间和细胞内通信对于 SCN 时钟的正常运行至关重要。我的研究项目的首要目标是识别和理解控制 SCN 时钟夹带和节律性的分子机制。使用泛 SCN grk2 敲除小鼠，我们最近发现了 G 蛋白偶联受体 (GPCR) 激酶 GRK2 在微调 SCN 的夹带、幅度和周期特性中的作用。我们的数据表明GRK2可能在不同水平上与不同的GPCR和核心时钟元件相互作用以调节SCN时钟。然而，确切的分子机制仍有待明确阐明。此外，鉴于 SCN 在解剖学上分为光响应（核心）和强节奏（外壳）区域，目前尚不清楚区域特异性 GRK2 活性如何影响 SCN 的夹带和振荡特性，也不清楚 GRK2 区室特异性作用的机制是什么。因此，本提案旨在通过选择性消融包含两个功能不同区室的 SCN 神经元子集中的 GRK2，并使用结合行为、药理学、生化、组织学、基于细胞培养和生物发光成像技术的多管齐下的方法来解决机制问题，从而剖析 GRK2 在 SCN 中的精确作用。我们将研究 GRK2 与关键神经肽-GPCR 系统之间的功能相互作用，以及 SCN 外壳和核心区域中 GRK2 与核心时钟蛋白之间的相互作用。在此过程中，我们将为 GRK2 如何调节 SCN 响应环境光信号的能力提供新的机制见解，并通过定义但可调节的特性保持节律性。总体而言，拟议的研究为培训研究生和本科生提供了绝佳的机会，将极大地扩展我们目前对正常时钟功能所必需的细胞机制的了解，并将带来与 GRK2 调节的不同生物系统广泛相关的重要见解。