Development of a novel DnaE intein-based assay for quantitative analysis of G-protein-coupled receptor internalization.
Development of a novel DnaE intein-based assay for quantitative analysis of G-protein-coupled receptor internalization.
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DOI：
10.1016/j.ab.2011.06.005
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发表时间：
2011-10
影响因子：
2.9
通讯作者：
Yaping Zhang;Wen Yang;Linjie Chen;Ying Shi;Guo Li;N. Zhou
中科院分区：
文献类型：
--
作者：
Yaping Zhang;Wen Yang;Linjie Chen;Ying Shi;Guo Li;N. Zhou
G蛋白偶联受体（GPCR）内化提供了一种G蛋白亚型独立的方法，用于测定激动剂刺激的受体活化。我们已经开发了一种新的测定方法，该方法允许基于活化的GPCR和β-arrestin 2之间的相互作用以及基于Nostoc punctiforme DnaE内含肽介导的海肾荧光素酶片段的重建来定量分析GPCR内化。使用用激动剂和拮抗剂处理的四种功能不同的GPCR验证该测定系统。已知激动剂和拮抗剂的EC 50值与先前报道的结果非常一致，表明该测定系统足够灵敏，可以定量GPCR内化。因此，这种快速和定量的检测方法，可以普遍使用的GPCR高通量筛选药物发现过程中的功能性细胞为基础的检测。
G-protein-coupled receptor (GPCR) internalization provides a G-protein-subtype-independent method for assaying agonist-stimulated activation of receptors. We have developed a novel assay that allows quantitative analysis of GPCR internalization based on the interaction between activated GPCRs and β-arrestin2 and on Nostoc punctiforme DnaE intein-mediated reconstitution of Renilla luciferase fragments. This assay system was validated using four functionally divergent GPCRs treated with agonists and antagonists. The EC50values obtained for the known agonists and antagonists are in close agreement with the results of previous reports, indicating that this assay system is sensitive enough to permit quantification of GPCR internalization. This rapid and quantitative assay, therefore, could be used universally as a functional cell-based assay for GPCR high-throughput screening during drug discovery.