Targeted Elimination of G proteins and Arrestins Defines their Specific Contributions to both Intensity and Duration of G protein-Coupled Receptor Signalling

Abstract G protein-coupled receptors (GPCRs) can initiate intracellular signalling 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 signalling 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 signalling 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 signalling 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 signalling because the Gq/11-dependent calcium response was desensitized by both receptor phosphorylation and arrestin-dependent mechanisms whilst 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 signalling research and employ these cells to reveal a previously unappreciated interplay of signalling pathways where receptor phosphorylation can impact on ERK1/2 signalling through a mechanism that is likely independent of arrestins.