2381/45638 E Alvarez-Curto E Alvarez-Curto A Inoue A Inoue L Jenkins L Jenkins SZ Raihan SZ Raihan R Prihandoko R Prihandoko AB Tobin AB Tobin G Milligan G Milligan Targeted Elimination of G Proteins and Arrestins Defines Their Specific Contributions to Both Intensity and Duration of G Protein-coupled Receptor Signaling. University of Leicester 2019 CRISPR/Cas G protein G protein-coupled receptor (GPCR) arrestin calcium intracellular release extracellular-signal-regulated kinase (ERK) fatty acid Arrestins CRISPR-Cas Systems Calcium GTP-Binding Protein alpha Subunits, Gq-G11 HEK293 Cells Humans Mitogen-Activated Protein Kinase 1 Mitogen-Activated Protein Kinase 3 Phosphorylation Receptors, G-Protein-Coupled Signal Transduction 2019-09-18 14:02:01 Journal contribution https://figshare.le.ac.uk/articles/journal_contribution/Targeted_Elimination_of_G_Proteins_and_Arrestins_Defines_Their_Specific_Contributions_to_Both_Intensity_and_Duration_of_G_Protein-coupled_Receptor_Signaling_/10238090 G protein-coupled receptors (GPCRs) can initiate intracellular signaling cascades by coupling to an array of heterotrimeric G proteins and arrestin adaptor proteins. Understanding the contribution of each of these coupling options to GPCR signaling has been hampered by a paucity of tools to selectively perturb receptor function. Here we employ CRISPR/Cas9 genome editing to eliminate selected G proteins (Gαq and Gα11) or arrestin2 and arrestin3 from HEK293 cells together with the elimination of receptor phosphorylation sites to define the relative contribution of G proteins, arrestins, and receptor phosphorylation to the signaling outcomes of the free fatty acid receptor 4 (FFA4). A lack of FFA4-mediated elevation of intracellular Ca2+ in Gαq/Gα11-null cells and agonist-mediated receptor internalization in arrestin2/3-null cells confirmed previously reported canonical signaling features of this receptor, thereby validating the genome-edited HEK293 cells. FFA4-mediated ERK1/2 activation was totally dependent on Gq/11 but intriguingly was substantially enhanced for FFA4 receptors lacking sites of regulated phosphorylation. This was not due to a simple lack of desensitization of Gq/11 signaling because the Gq/11-dependent calcium response was desensitized by both receptor phosphorylation and arrestin-dependent mechanisms, whereas a substantially enhanced ERK1/2 response was only observed for receptors lacking phosphorylation sites and not in arrestin2/3-null cells. In conclusion, we validate CRISPR/Cas9 engineered HEK293 cells lacking Gq/11 or arrestin2/3 as systems for GPCR signaling research and employ these cells to reveal a previously unappreciated interplay of signaling pathways where receptor phosphorylation can impact on ERK1/2 signaling through a mechanism that is likely independent of arrestins.