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首頁> 美國衛(wèi)生研究院文獻(xiàn)>Frontiers in Endocrinology >Homogeneous Time-Resolved Fluorescence-Based Assay to Monitor Extracellular Signal-Regulated Kinase Signaling in a High-Throughput Format
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Homogeneous Time-Resolved Fluorescence-Based Assay to Monitor Extracellular Signal-Regulated Kinase Signaling in a High-Throughput Format

機(jī)譯：基于均相時間分辨熒光的方法以高通量格式監(jiān)測細(xì)胞外信號調(diào)節(jié)的激酶信號

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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
機(jī)譯：細(xì)胞外信號調(diào)節(jié)激酶（ERKs）是調(diào)節(jié)多種生物學(xué)反應(yīng)的多個重要細(xì)胞信號通路的關(guān)鍵組成部分。該信號轉(zhuǎn)導(dǎo)的特征是磷酸化級聯(lián)反應(yīng)導(dǎo)致ERK1 / 2活化，并被各種細(xì)胞表面受體（包括G蛋白偶聯(lián)受體（GPCR）和酪氨酸激酶（RTK））促進(jìn)。我們報告了一種新的基于細(xì)胞的Phospho-ERK1 / 2測定法（稱為Phospho-ERK）的開發(fā)，這是一種使用均相時間分辨熒光技術(shù)的基于三明治鄰近度的測定法。我們已經(jīng)驗(yàn)證了被表皮生長因子激活的內(nèi)源性表達(dá)ERK1 / 2作為典型RTK的測定方法，以及屬于不同類別并偶聯(lián)至不同異三聚體G蛋白的各種GPCR。使用內(nèi)源，瞬時或穩(wěn)定表達(dá)不同受體的各種細(xì)胞系，成功地在384孔板中將測定法微型化。在劑量反應(yīng)以及動力學(xué)分析中對激動劑，拮抗劑和抑制劑進(jìn)行了驗(yàn)證，并復(fù)制了不同受體的信號傳導(dǎo)和藥理特性。此外，Z'因子值為0.7的確定表明Phospho-ERK分析可能用于高通量篩選可能調(diào)節(jié)ERK1 / 2信號傳導(dǎo)的化合物。最后，我們的研究對目前有關(guān)ERK1 / 2信號轉(zhuǎn)導(dǎo)的研究產(chǎn)生了濃厚的興趣，涉及偏配基，G蛋白依賴性/非依賴性ERK1 / 2激活以及GPCR和RTK之間的功能性反激活等新興概念。 ERK1 / 2途徑在細(xì)胞信號轉(zhuǎn)導(dǎo)中的作用

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