Targeted Elimination of G Proteins and Arrestins Defines Their Specific Contributions to Both Intensity and Duration of G Protein-coupled Receptor Signaling.
Targeted Elimination of G Proteins and Arrestins Defines Their Specific Contributions to Both Intensity and Duration of G Protein-coupled Receptor Signaling.
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DOI：
10.1074/jbc.m116.754887
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发表时间：
2016-12-30
期刊：
影响因子：
--
通讯作者：
Milligan G
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文献类型：
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作者：
Alvarez-Curto E;Inoue A;Jenkins L;Raihan SZ;Prihandoko R;Tobin AB;Milligan G
G蛋白偶联受体（GPCR）可以通过偶联到一系列异源三聚体G蛋白和抑制蛋白接头蛋白来启动细胞内信号级联反应。由于缺乏选择性干扰受体功能的工具，理解这些偶联选择中的每一种对GPCR信号传导的贡献受到阻碍。在这里，我们采用CRISPR/Cas9基因组编辑来消除HEK 293细胞中选定的G蛋白（Gαq和Gα11）或arrestin 2和arrestin 3，同时消除受体磷酸化位点，以确定G蛋白、arrestin和受体磷酸化对游离脂肪酸受体4（FFA 4）信号传导结果的相对贡献。在Gαq/Gα11-null细胞中缺乏FFA 4介导的细胞内Ca 2+升高以及在arrestin 2/3-null细胞中缺乏激动剂介导的受体内化证实了先前报道的该受体的典型信号传导特征，从而验证了基因组编辑的HEK 293细胞。FFA 4介导的ERK 1/2激活完全依赖于Gq/11，但有趣的是，对于缺乏调节磷酸化位点的FFA 4受体，ERK 1/2激活显著增强。这不是由于简单缺乏Gq/11信号转导的脱敏作用，因为Gq/11依赖性钙反应被受体磷酸化和arrestin依赖性机制脱敏，而ERK 1/2反应的显著增强仅在缺乏磷酸化位点的受体中观察到，而在arrestin 2/3-null细胞中未观察到。总之，我们验证了缺乏Gq/11或arrestin 2/3的CRISPR/Cas9工程化HEK 293细胞作为GPCR信号转导研究的系统，并利用这些细胞揭示了以前未被认识到的信号转导途径的相互作用，其中受体磷酸化可以通过可能独立于arrestins的机制影响ERK 1/2信号转导。
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.