G-protein–coupled receptors (GPCRs) compose the largest family of transmembrane receptors and are targets of approximately one-third of Food and Drug Administration–approved drugs owing to their involvement in almost all physiologic processes. GPCR signaling occurs through the activation of heterotrimeric G-protein complexes and β-arrestins, both of which serve as transducers, resulting in distinct cellular responses. Despite seeming simple at first glance, accumulating evidence indicates that activation of either transducer is not a straightforward process as a stimulation of a single molecule has the potential to activate multiple signaling branches. The complexity of GPCR signaling arises from the aspects of G-protein–coupling selectivity, biased signaling, interpathway crosstalk, and variable molecular modifications generating these diverse signaling patterns. Numerous questions relative to these aspects of signaling remained unanswered until the recent development of CRISPR genome-editing technology. Such genome editing technology presents opportunities to chronically eliminate the expression of G-protein subunits, β-arrestins, G-protein–coupled receptor kinases (GRKs), and many other signaling nodes in the GPCR pathways at one’s convenience. Here, we review the practicality of using CRISPR-derived knockout (KO) cells in the experimental contexts of unraveling the molecular details of GPCR signaling mechanisms. To mention a few, KO cells have revealed the contribution of β-arrestins in ERK activation, Gα protein selectivity, GRK-based regulation of GPCRs, and many more, hence validating its broad applicability in GPCR studies.

中文翻译：

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生成全面的 GPCR 转导器缺陷细胞系以剖析 GPCR 信号传导的复杂性


G 蛋白偶联受体 (GPCR) 构成最大的跨膜受体家族，并且由于其参与几乎所有生理过程，因此成为美国食品和药物管理局批准的大约三分之一药物的靶标。 GPCR 信号传导通过异源三聚体 G 蛋白复合物和β-抑制蛋白的激活而发生，两者都充当传感器，导致不同的细胞反应。尽管乍一看似乎很简单，但越来越多的证据表明，任一传感器的激活都不是一个简单的过程，因为刺激单个分子有可能激活多个信号分支。 GPCR 信号传导的复杂性源于 G 蛋白偶联选择性、偏向信号传导、通路间串扰以及产生这些不同信号传导模式的可变分子修饰等方面。直到最近 CRISPR 基因组编辑技术的发展，与信号传导这些方面相关的许多问题仍然没有得到解答。这种基因组编辑技术提供了长期消除 G 蛋白亚基、 β-抑制蛋白、G 蛋白偶联受体激酶 (GRK) 以及 GPCR 通路中许多其他信号节点的表达的机会。在这里，我们回顾了在揭示 GPCR 信号机制分子细节的实验环境中使用 CRISPR 衍生的敲除 (KO) 细胞的实用性。仅举几例，KO 细胞揭示了β -arrestins 在 ERK 激活、G α蛋白选择性、基于 GRK 的 GPCR 调节等方面的贡献，从而验证了其在 GPCR 研究中的广泛适用性。