期刊
JOURNAL OF BIOLOGICAL CHEMISTRY
卷 291, 期 53, 页码 27147-27159出版社
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M116.754887
关键词
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资金
- Biotechnology and Biological Sciences Research Council [BB/K019864/1, BB/K019856/1]
- PRESTO, Japan Science and Technology Agency
- Biotechnology and Biological Sciences Research Council [1653478, BB/K019856/2, BB/L02781X/1, BB/K019864/1, BB/K019856/1] Funding Source: researchfish
- BBSRC [BB/K019864/1, BB/K019856/2, BB/L02781X/1, BB/K019856/1] Funding Source: UKRI
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 (G alpha(q) and G alpha(11)) 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 G alpha q/G alpha(11)-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 G(q)/(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 G(q)/(11) signaling because the G(q)/(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 G(q)/(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.
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