A high-throughput CRISPR interference screen for dissecting functional regulators of GPCR/cAMP signaling

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 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.

DNA primase subunit 1 deteriorated progression of hepatocellular carcinoma by activating AKT/mTOR signaling and UBE2C-mediated P53 ubiquitination

“…A recent work using CRISPR-based-genome-wide screening determined PRIM1 as a robust regulator of cAMP-PKA signaling. Further validation also confirmed that deletion of PRIM1 significantly blunted the activation of cAMP signaling via multi-stimuli [ 22 ]. As an important second messenger, cAMP is competent in multiple signaling transduction and pathway linking.…”

Genomic analyses identify key molecules and significant signaling pathways in AZIN1 regulated prostate cancer cells

“…G proteincoupled receptors (GPCRs) promote cells to respond to several stimuli through generation of second messengers, which in turn regulate a variety of processes such as cell survival, proliferation, migration, and differentiation [18][19][20][21][22][23][24][25][26][27][28] . As a key GTPase, HRAS was found to be involved in controlling the GPCR/cAMP signaling pathways, thereby mediating the cancer cells 29 . Mingming Wang et al found the microRNA-4873-3p inhibits the progression of prostate cancer by targeting CCND1 30 .…”

Endosome positioning coordinates spatially selective GPCR signaling

“…Next, we set off to establish a complementary optical readout of bulk β2-AR/cAMP-dependent transcription. We recently published a fluorescent transcriptional reporter for the cAMP response element-binding protein (CREB) 31 . This reporter contains two cAMP response elements (CREs) recognized by CREB that drive the expression of the zsGreen fluorescent protein and a destabilizing domain (DD).…”

“…A previously described lentiviral plasmid encoding a transcriptional reporter for CREB activity 31 was used as the backbone for the CREB transcriptional reporter in this study. To express this construct in the nucleus, a nuclear localization sequence (NLS) was amplified from dCas9-BFP-KRAB (a gift from Martin Kampmann, UCSF) and inserted into the BamHI-digested reporter backbone using Gibson cloning.…”