Dual-Color Bioluminescence Analysis for Quantitatively Monitoring G-Protein-Coupled Receptor and β-Arrestin Interactions
Dual-Color Bioluminescence Analysis for Quantitatively Monitoring G-Protein-Coupled Receptor and β-Arrestin Interactions
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DOI：
10.3390/ph4030457
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发表时间：
2011-03-01
期刊：
影响因子：
4.6
通讯作者：
Ozawa, Takeaki
中科院分区：
文献类型：
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作者：
Kafi, A. K. M.;Hattori, Mitsuru;Ozawa, Takeaki
G 蛋白偶联受体 (GPCR) 是哺乳动物信号转导的关键元件，被认为是有效的药物靶标。我们之前在培养细胞中开发了一种 GPCR 检测系统，该系统基于点击甲虫 (Pyrearinus termitluminans) 荧光素酶裂解片段的互补。 GPCR 与其靶标 β-arrestin 相互作用，在其特定配体刺激后产生强烈的生物发光。在本研究中，我们通过使用铁路蠕虫 (Phrixothrix hirtus) 荧光素酶作为内部对照，提高了 GPCR 测定系统的精度。我们生成了含有铁路蠕虫荧光素酶的稳定细胞系，并定量评估了 GPCR-β-arrestin 相互作用的程度。我们展示了四种 GPCR 的浓度依赖性生物发光反应：β2-肾上腺素受体、A 型内皮素受体、α2-肾上腺素受体和人 mu-阿片受体。我们还证明，通过使用铁路蠕虫荧光素酶的生物发光对数据进行标准化，可以显着减少响应的变化。该测定系统代表了一种以高通量方式筛选候选药物的简单而可靠的方法。
G protein-coupled receptors (GPCRs) are crucial elements in mammalian signal transduction, and are considered to represent potent drug targets. We have previously developed a GPCR assay system in cultured cells based on complementation of split fragments of click beetle (Pyrearinus termitilluminans) luciferase. The interaction of GPCRs with its target, beta-arrestin, resulted in strong emission of bioluminescence upon stimulation with its specific ligand. In this study, we improved precision of the GPCR assay system by using railroad worm (Phrixothrix hirtus) luciferase as an internal control. We generated stable cell lines harboring the railroad worm luciferase and quantitatively evaluate the extent of GPCR-beta-arrestin interactions. We showed concentration-dependent bioluminescence responses for four GPCRs: beta 2-adrenoceptor, endothelin receptor type A, alpha 2-adrenoceptor and human mu-opioid receptor. We also demonstrated that the variation of responses was reduced significantly by normalizing the data with bioluminescence from railroad worm luciferase. This assay system represents a simple and reliable approach for screening drug candidates in a high throughput manner.