Targeted elimination of G proteins and arrestins defines their specific contributions to both intensity and duration of G protein-coupled receptor signalling

Alvarez-Curto, E. , Inoue, A., Jenkins, L. , Raihan, S. Z., Prihandoko, R., Tobin, A. B. and Milligan, G. (2016) Targeted elimination of G proteins and arrestins defines their specific contributions to both intensity and duration of G protein-coupled receptor signalling. Journal of Biological Chemistry, 291(53), pp. 27147-27159. (doi:10.1074/jbc.M116.754887) (PMID:27852822) (PMCID:PMC5207144)

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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.

Item Type:Articles
Status:Published
Refereed:Yes
Glasgow Author(s) Enlighten ID:Milligan, Professor Graeme and Prihandoko, Dr Rudi and Jenkins, Mrs Laura and Alvarez-Curto, Dr Elisa and Tobin, Andrew
Authors: Alvarez-Curto, E., Inoue, A., Jenkins, L., Raihan, S. Z., Prihandoko, R., Tobin, A. B., and Milligan, G.
College/School:College of Medical Veterinary and Life Sciences > Institute of Molecular Cell and Systems Biology
Journal Name:Journal of Biological Chemistry
Publisher:American Society for Biochemistry and Molecular Biology, Inc.
ISSN:0021-9258
ISSN (Online):1083-351X
Published Online:16 November 2016

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Project CodeAward NoProject NamePrincipal InvestigatorFunder's NameFunder RefLead Dept
618921GPR120: a G protein-coupled receptor with the potential to regulate insulin secretion and inflammationGraeme MilliganBiotechnology and Biological Sciences Research Council (BBSRC)BB/K019864/1RI MOLECULAR CELL & SYSTEMS BIOLOGY
744711GPR120: a G protein-coupled receptor with the potential to regulate insulin secretion and inflammationAndrew TobinBiotechnology and Biological Sciences Research Council (BBSRC)BB/K019856/1RI MOLECULAR CELL & SYSTEMS BIOLOGY