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

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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.