Live cell optical assay for precise characterization of receptors coupling to Gα12.
Live cell optical assay for precise characterization of receptors coupling to Gα12.
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活细胞光学测定精确表征与 Gα12 耦合的受体。
DOI：
10.1111/bcpt.13228
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发表时间：
2020
影响因子：
3.1
通讯作者：
Martemyanov,KirillA
中科院分区：
文献类型：
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作者：
Masuho,Ikuo;Skamangas,NickolasK;Martemyanov,KirillA
异源三聚体G蛋白是G蛋白偶联受体（GPCR）信号传导至细胞内效应物的重要介质。G蛋白亚基有相当大的多样性，它们将GPCR启动的信号引导到特定的结果中。特别是哺乳动物基因组包含16个保守基因，编码具有独特性质的G蛋白α亚基。在Gα 4个亚家族（Gi/o、Gq、Gs和G12/13）中，G12/13家族成员因其在肿瘤发生中的作用而受到广泛关注。然而，我们通过GPCR研究G12/13激活的能力有限，无法区分这两种亚基。在这里，我们提出了一种基于生物发光共振能量转移（BRET）的检测方法，用于特异性监测活细胞中Gα12的活性。在该动力学测定中，使用纳米荧光素酶（Nluc）标记的BRET供体，真实的实时跟踪激动剂诱导的Venus标记的Gβγ亚基从Gα12的释放。用此方法对缓激肽B_2受体（BDKRB_2）进行了鉴定，发现该受体除了与Gαo和Gαq偶联外，还与Gα12偶联，但不与Gαs偶联。我们证明了该检测方法的实用性，以量化G蛋白活化和失活的速率，以及进行剂量反应研究，同时通过单个Gα亚基对信号进行排序。我们通过证明胆囊收缩素A受体（CCKAR）的Gα12偶联，进一步证明了该试验对其他GPCR的实用性。预计Gα12偶联BRET试验的引入将加速GPCR对该未充分研究的G蛋白的作用的表征。
Heterotrimeric G proteins are essential mediators of G protein‐coupled receptors (GPCRs) signalling to intracellular effectors. There is a considerable diversity of G protein subunits that channel signals initiated by GPCRs into specific outcome. In particular, mammalian genomes contain 16 conserved genes encoding G protein α subunits with unique properties. Of four Gα subfamilies (Gi/o, Gq, Gs and G12/13), members of the G12/13 group have received considerable attention for their roles in carcinogenesis. However, our ability to study activation of G12/13 by GPCRs with the power to distinguish between the two subunits is limited. Here, we present an adaptation of the bioluminescence resonance energy transfer (BRET)‐based assay to specifically monitor activity of Gα12 in living cells. In this kinetic assay, agonist‐induced release of Venus‐tagged Gβγ subunits from Gα12 is followed in real time using nano‐luciferase (Nluc)–tagged BRET donor. Using this assay, we characterized bradykinin B2 receptor (BDKRB2) and found that the receptor couples to Gα12 in addition to Gαo, and Gαq, but not to Gαs. We demonstrated the utility of this assay to quantify rates of G protein activation and inactivation as well as performing dose‐response studies while rank ordering signallingviaindividual Gα subunits. We further showed the utility of this assay to other GPCRs by demonstrating Gα12 coupling of cholecystokinin A receptor (CCKAR). Introduction of the Gα12‐coupling BRET assay is expected to accelerate characterization of GPCR actions on this understudied G protein.