Targeted Elimination of G Proteins and Arrestins Defines Their Specific Contributions to Both Intensity and Duration of G Protein-coupled Receptor Signaling.pdf (1.82 MB)
Targeted Elimination of G Proteins and Arrestins Defines Their Specific Contributions to Both Intensity and Duration of G Protein-coupled Receptor Signaling.
journal contribution
posted on 2019-09-18, 14:02 authored by E Alvarez-Curto, A Inoue, L Jenkins, SZ Raihan, R Prihandoko, AB Tobin, G MilliganG 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.
Funding
This work was supported by Biotechnology and Biological Sciences Research Council Grants BB/K019864/1 (to G. M.) and BB/K019856/1 (to A. B. T.) and PRESTO, Japan Science and Technology Agency (to A. I.). The authors declare that they have no conflicts of interest with the contents of this article.
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Citation
Journal of Biological Chemistry, 2016, 291 (53), pp. 27147-27159Author affiliation
/Organisation/COLLEGE OF LIFE SCIENCES/Biological Sciences/Molecular & Cell BiologyVersion
- VoR (Version of Record)
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Journal of Biological ChemistryPublisher
American Society for Biochemistry and Molecular Biologyeissn
1083-351XCopyright date
2016Available date
2019-09-18Publisher DOI
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http://www.jbc.org/content/291/53/27147Language
enAdministrator link
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CRISPR/CasG proteinG protein-coupled receptor (GPCR)arrestincalcium intracellular releaseextracellular-signal-regulated kinase (ERK)fatty acidArrestinsCRISPR-Cas SystemsCalciumGTP-Binding Protein alpha Subunits, Gq-G11HEK293 CellsHumansMitogen-Activated Protein Kinase 1Mitogen-Activated Protein Kinase 3PhosphorylationReceptors, G-Protein-CoupledSignal Transduction
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