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J Biol Chem. 2016 Dec 30;291(53):27147-27159. doi: 10.1074/jbc.M116.754887. Epub 2016 Nov 16.

Targeted Elimination of G Proteins and Arrestins Defines Their Specific Contributions to Both Intensity and Duration of G Protein-coupled Receptor Signaling.

Author information

1
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.
2
the Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Miyagi 980-8578, Japan, and.
3
the Japan Science and Technology Agency (JST), Precursory Research for Embryonic Science and Technology (PRESTO), Kawaguchi, Saitama 332-0012, Japan.
4
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.
5
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.

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 (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.

KEYWORDS:

CRISPR/Cas; G protein; G protein-coupled receptor (GPCR); arrestin; calcium intracellular release; extracellular-signal-regulated kinase (ERK); fatty acid

PMID:
27852822
PMCID:
PMC5207144
DOI:
10.1074/jbc.M116.754887
[Indexed for MEDLINE]
Free PMC Article

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