Department of Pharmacology and Cancer Biology, Duke University, Durham, North Carolina, United States of America.
Institute for Neurodegenerative Diseases, Department of Biochemistry and Biophysics, University of California, San Francisco, California, United States of America.
PLoS Genet. 2020 Oct 14;16(10):e1009103. doi: 10.1371/journal.pgen.1009103. eCollection 2020 Oct.
G protein-coupled receptors (GPCRs) allow cells to respond to chemical and sensory stimuli through generation of second messengers, such as cyclic AMP (cAMP), which in turn mediate a myriad of processes, including cell survival, proliferation, and differentiation. In order to gain deeper insights into the complex biology and physiology of these key cellular pathways, it is critical to be able to globally map the molecular factors that shape cascade function. Yet, to this date, efforts to systematically identify regulators of GPCR/cAMP signaling have been lacking. Here, we combined genome-wide screening based on CRISPR interference with a novel sortable transcriptional reporter that provides robust readout for cAMP signaling, and carried out a functional screen for regulators of the pathway. Due to the sortable nature of the platform, we were able to assay regulators with strong and moderate phenotypes by analyzing sgRNA distribution among three fractions with distinct reporter expression. We identified 45 regulators with strong and 50 regulators with moderate phenotypes not previously known to be involved in cAMP signaling. In follow-up experiments, we validated the functional effects of seven newly discovered mediators (NUP93, PRIM1, RUVBL1, PKMYT1, TP53, SF3A2, and HRAS), and showed that they control distinct steps of the pathway. Thus, our study provides proof of principle that the screening platform can be applied successfully to identify bona fide regulators of GPCR/second messenger cascades in an unbiased and high-throughput manner, and illuminates the remarkable functional diversity among GPCR regulators.
G 蛋白偶联受体（GPCRs）通过生成第二信使（如环腺苷酸[cAMP]）使细胞能够对化学和感官刺激做出反应，而 cAMP 又介导了包括细胞存活、增殖和分化在内的多种过程。为了更深入地了解这些关键细胞途径的复杂生物学和生理学，能够全局绘制塑造级联功能的分子因素至关重要。然而，迄今为止，系统性鉴定 GPCR/cAMP 信号转导调节剂的努力一直缺乏。在这里，我们将基于 CRISPR 干扰的全基因组筛选与一种新型可分选转录报告基因相结合，该报告基因可提供 cAMP 信号的强大读数，并对该途径的调节剂进行功能筛选。由于该平台具有可分选的性质，我们能够通过分析三个具有不同报告基因表达的分数中的 sgRNA 分布来检测具有强表型和中表型的调节剂。我们鉴定出 45 个强表型调节剂和 50 个以前不参与 cAMP 信号转导的中表型调节剂。在后续实验中，我们验证了七个新发现的介质（NUP93、PRIM1、RUVBL1、PKMYT1、TP53、SF3A2 和 HRAS）的功能效应，并表明它们控制了该途径的不同步骤。因此，我们的研究证明了筛选平台可以成功地应用于以无偏和高通量的方式识别 GPCR/第二信使级联的真正调节剂，并阐明了 GPCR 调节剂之间显著的功能多样性。
