Abstract
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 eight newly discovered positive and negative mediators (NUP93, PRIM1, RUVBL1, PKMYT1, TP53, SF3A2, HRAS, RBM12), 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.
Highlights
The G protein-coupled receptors (GPCRs)/cyclic AMP (cAMP) cascade is a key signaling axis coordinating the ability of cells to respond to fluctuations in their environment
The pathway is initiated by ligand binding to the transmembrane receptor, which leads to the stimulation of adenylyl cyclase enzymes (ACs) by the dissociated G protein subunit, Gαs, and to subsequent cAMP production. cAMP binds to intracellular effectors, including its main effector, protein kinase A (PKA), and triggers substrate phosphorylation, and gene expression reprogramming through phosphorylation and activation of the nuclear cAMP-response binding protein (CREB)
The CRISPR interference (CRISPRi) technology relies on transcriptional repression and has been applied successfully in pooled screens to assess the functional impacts of silencing specific genes [5]
Summary
The GPCR/cAMP cascade is a key signaling axis coordinating the ability of cells to respond to fluctuations in their environment. Recent technological advances using CRISPR/Cas9-based methods have revolutionized the field of functional genomics and enabled large-scale unbiased screens to probe the regulation of signaling cascades While these approaches have been successfully implemented to characterize a number of important mammalian pathways [2], to our best knowledge there have been no efforts to date to systematically identify genes mediating GPCR/cAMP signaling. In large part, this can be attributed to the lack of robust selectable phenotypic readouts that such pooled screening methods necessitate. The high-throughput screening paradigm described here provides a novel platform for unbiased dissection of regulators of receptor function, and pinpoints target genes for future manipulation of the pathway and its signaling consequences
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