A universal bioluminescence resonance energy transfer sensor design enables high-sensitivity screening of GPCR activation dynamics
A universal bioluminescence resonance energy transfer sensor design enables high-sensitivity screening of GPCR activation dynamics
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DOI：
10.1038/s42003-018-0072-0
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发表时间：
2018-01-01
影响因子：
5.9
通讯作者：
Maiellaro, Isabella
中科院分区：
文献类型：
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作者：
Schihada, Hannes;Vandenabeele, Sylvie;Maiellaro, Isabella
G 蛋白偶联受体 (GPCR) 代表最重要的药物靶点类别之一。新 GCPR 疗法的发现将极大地受益于通用高通量检测的开发，以直接监测其激活或失活。在这里，我们筛选了插入第三个细胞内环和 α(2 Lambda)-肾上腺素受体 C 末端的各种标记，并使用荧光 (FRET) 和生物发光共振能量转移 (BRET) 来监测配体结合和激活动力学。然后，我们开发了一种通用分子内 BRET 受体传感器设计，用于实时量化完整细胞中 GPCR 配体的功效和效力。我们通过克隆 β(2)-肾上腺素能和 PTH1-受体 BRET 传感器来证明传感器设计的可转移性，并监测其功效和效力。对于所有生物传感器，Z 因子远高于 0.5，表明这种设计适用于微量滴定板测定。该技术将有助于鉴定新型 GPCR 配体。
G-protein-coupled receptors (GPCRs) represent one of the most important classes of drug targets. The discovery of new GCPR therapeutics would greatly benefit from the development of a generalizable high-throughput assay to directly monitor their activation or de-activation. Here we screened a variety of labels inserted into the third intracellular loop and the C-terminus of the alpha(2 Lambda)-adrenergic receptor and used fluorescence (FRET) and bioluminescence resonance energy transfer (BRET) to monitor ligand-binding and activation dynamics. We then developed a universal intramolecular BRET receptor sensor design to quantify efficacy and potency of GPCR ligands in intact cells and real time. We demonstrate the transferability of the sensor design by cloning beta(2)-adrenergic and PTH1-receptor BRET sensors and monitored their efficacy and potency. For all biosensors, the Z factors were well above 0.5 showing the suitability of such design for microtiter plate assays. This technology will aid the identification of novel types of GPCR ligands.