Molecular Endocrinology and Pharmacology, Harry Perkins Institute of Medical Research and Centre for Medical Research, The University of Western Australia , Nedlands, WA , Australia.
Cisbio Bioassays , Codolet , France.
Front Endocrinol (Lausanne). 2014 Jun 23;5:94. doi: 10.3389/fendo.2014.00094. eCollection 2014.
The extracellular signal-regulated kinases (ERKs) are key components of multiple important cell signaling pathways regulating diverse biological responses. This signaling is characterized by phosphorylation cascades leading to ERK1/2 activation and promoted by various cell surface receptors including G protein-coupled receptors (GPCRs) and receptor tyrosine kinases (RTKs). We report the development of a new cell-based Phospho-ERK1/2 assay (designated Phospho-ERK), which is a sandwich proximity-based assay using the homogeneous time-resolved fluorescence technology. We have validated the assay on endogenously expressed ERK1/2 activated by the epidermal growth factor as a prototypical RTK, as well as various GPCRs belonging to different classes and coupling to different heterotrimeric G proteins. The assay was successfully miniaturized in 384-well plates using various cell lines endogenously, transiently, or stably expressing the different receptors. The validation was performed for agonists, antagonists, and inhibitors in dose-response as well as kinetic analysis, and the signaling and pharmacological properties of the different receptors were reproduced. Furthermore, the determination of a Z'-factor value of 0.7 indicates the potential of the Phospho-ERK assay for high-throughput screening of compounds that may modulate ERK1/2 signaling. Finally, our study is of great interest in the current context of investigating ERK1/2 signaling with respect to the emerging concepts of biased ligands, G protein-dependent/independent ERK1/2 activation, and functional transactivation between GPCRs and RTKs, illustrating the importance of considering the ERK1/2 pathway in cell signaling.
细胞外信号调节激酶（ERK）是调节多种生物反应的多个重要细胞信号通路的关键组成部分。这种信号转导的特征是磷酸化级联反应，导致 ERK1/2 激活，并由各种细胞表面受体促进，包括 G 蛋白偶联受体（GPCR）和受体酪氨酸激酶（RTK）。我们报告了一种新的基于细胞的磷酸化 ERK1/2 检测（命名为 Phospho-ERK）的开发，这是一种使用均相时间分辨荧光技术的夹心接近检测。我们已经通过表皮生长因子激活的内源性 ERK1/2 验证了该检测，表皮生长因子是一种典型的 RTK，以及属于不同类别和与不同异三聚体 G 蛋白偶联的各种 GPCR。该检测已成功地在 384 孔板中使用各种内源性、瞬时或稳定表达不同受体的细胞系进行了微型化。在剂量反应和动力学分析中，对激动剂、拮抗剂和抑制剂进行了验证，并再现了不同受体的信号和药理学特性。此外，Z'因子值为 0.7 表明 Phospho-ERK 检测具有用于筛选可能调节 ERK1/2 信号的化合物的高通量筛选的潜力。最后，我们的研究对于当前研究 ERK1/2 信号转导具有重要意义，特别是对于新兴的偏向配体、G 蛋白依赖性/非依赖性 ERK1/2 激活以及 GPCR 和 RTK 之间的功能转激活的概念，说明了考虑 ERK1/2 途径在细胞信号转导中的重要性。
