Luciferase Reporter Assay System for Deciphering GPCR Pathways.
Luciferase Reporter Assay System for Deciphering GPCR Pathways.
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DOI：
10.2174/1875397301004010084
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发表时间：
2010-12-21
期刊：
影响因子：
--
通讯作者：
Fan F
中科院分区：
文献类型：
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作者：
Cheng Z;Garvin D;Paguio A;Stecha P;Wood K;Fan F
G蛋白偶联受体（GPCR）代表了当前近一半治疗药物的靶标类别，并且仍然是药物发现工作的重点。受体信号传导的复杂性不断发展。现在已知许多 GPCR 与多种 G 蛋白偶联，从而调节下游各自的信号通路。破译这种受体偶联将有助于我们了解 GPCR 功能并最终开发候选药物。在此，我们报告了使用改进的不稳定荧光素酶和各种响应元件（CRE、NFAT-RE、SRE 和 SRF-RE）开发的四种同质生物发光报告基因检测。这些测定允许分别使用相同的报告测定格式测量主要 GPCR 途径，包括 cAMP 产生、细胞内 Ca2+ 动员、ERK/MAPK 活性和小 G 蛋白 RhoA 活性。我们表明，通过使用这四种报告基因检测，我们可以破译 HEK293 细胞中外源性 m3 毒蕈碱受体和内源性 β2-肾上腺素能受体的 G 蛋白激活谱。此外，我们证明这些测定可以很容易地用于激动剂和拮抗剂的效力排名以及高通量筛选。
The G protein coupled receptors (GPCR) represent the target class for nearly half of the current therapeutic drugs and remain to be the focus of drug discovery efforts. The complexity of receptor signaling continues to evolve. It is now known that many GPCRs are coupled to multiple G-proteins, which lead to regulation of respective signaling pathways downstream. Deciphering this receptor coupling will aid our understanding of the GPCR function and ultimately developing drug candidates. Here, we report the development of four homogenous bioluminescent reporter assays using improved destabilized luciferases and various response elements: CRE, NFAT-RE, SRE, and SRF-RE. These assays allowed measurement of major GPCR pathways including cAMP production, intracellular Ca2+ mobilizations, ERK/MAPK activ-ity, and small G protein RhoA activity, respectively using the same reporter assay format. We showed that we can decipher G protein activation profiles for exogenous m3 muscarinic receptor and endogenous β2-adrenergic receptors in HEK293 cells by using these four reporter assays. Furthermore, we demonstrated that these assays can be readily used for potency rankings of agonists and antagonists, and for high throughput screening.