title: Targeted Elimination of G Proteins and Arrestins Defines Their Specific Contributions to Both Intensity and Duration of G Protein-coupled Receptor Signaling.
abstract: 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 (Gαqand 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/11but intriguingly was substantially enhanced for FFA4 receptors lacking sites of regulated phosphorylation. This was not due to a simple lack of desensitization of Gq/11signaling 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/11or 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.
authors.affiliation: From the Centre for Translational Pharmacology, Institute of Molecular, Cell and Systems Biology, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow G12 8QQ, Scotland, United Kingdom, Elisa.Alvarez-Curto@glasgow.ac.uk.
authors.forename: Elisa
authors.initials: E
authors.lastname: Alvarez-Curto
authors.affiliation: the Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Miyagi 980-8578, Japan, and.|the Japan Science and Technology Agency (JST), Precursory Research for Embryonic Science and Technology (PRESTO), Kawaguchi, Saitama 332-0012, Japan.
authors.forename: Asuka
authors.initials: A
authors.lastname: Inoue
authors.affiliation: From the Centre for Translational Pharmacology, Institute of Molecular, Cell and Systems Biology, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow G12 8QQ, Scotland, United Kingdom.
authors.forename: Laura
authors.initials: L
authors.lastname: Jenkins
authors.affiliation: From the Centre for Translational Pharmacology, Institute of Molecular, Cell and Systems Biology, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow G12 8QQ, Scotland, United Kingdom.
authors.forename: Sheikh Zahir
authors.initials: SZ
authors.lastname: Raihan
authors.affiliation: From the Centre for Translational Pharmacology, Institute of Molecular, Cell and Systems Biology, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow G12 8QQ, Scotland, United Kingdom.
authors.forename: Rudi
authors.initials: R
authors.lastname: Prihandoko
authors.affiliation: From the Centre for Translational Pharmacology, Institute of Molecular, Cell and Systems Biology, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow G12 8QQ, Scotland, United Kingdom.
authors.forename: Andrew B
authors.initials: AB
authors.lastname: Tobin
authors.affiliation: From the Centre for Translational Pharmacology, Institute of Molecular, Cell and Systems Biology, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow G12 8QQ, Scotland, United Kingdom, Graeme.Milligan@glasgow.ac.uk.
authors.forename: Graeme
authors.identifier: 0000-0002-6946-3519
authors.initials: G
authors.lastname: Milligan
mesh_terms: D019390:Arrestins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D064113:CRISPR-Cas Systems / Q000235:genetics*; D002118:Calcium / Q000378:metabolism; D043802:GTP-Binding Protein alpha Subunits, Gq-G11 / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism*; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism*; D010766:Phosphorylation; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism*; D015398:Signal Transduction
chemical_list: D019390:Arrestins; D043562:Receptors, G-Protein-Coupled; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D002118:Calcium
keywords: CRISPR/Cas; G protein; G protein-coupled receptor (GPCR); arrestin; calcium intracellular release; extracellular-signal-regulated kinase (ERK); fatty acid