Forskolin-free cAMP assay for Gi-coupled receptors
Forskolin-free cAMP assay for Gi-coupled receptors
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DOI：
10.1016/j.bcp.2015.09.010
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发表时间：
2015-12-01
影响因子：
5.8
通讯作者：
Hanson, Julien
中科院分区：
文献类型：
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作者：
Gilissen, Julie;Geubelle, Pierre;Hanson, Julien
G蛋白偶联受体（GPCR）代表了用于治疗人类疾病的最成功的受体家族。许多配体的特征很差，报告的配体很少，或者完全是孤儿。因此，越来越需要筛选兼容和敏感的测定。细胞内环AMP（cAMP）水平的测量是用于测量GPCR活化的经验证的策略。然而，G（i）偶联受体的激动剂配体难以追踪，因为必须使用诸如毛喉素（FSK）的诱导剂，并且是变化和误差的来源。作为概念证明，我们选择了琥珀酸受体1（SUCNR 1或GPR 91），这可能是一个有吸引力的药物靶点。它从来没有被验证，因为很少配体已被描述。以下分析SUCNR 1信号通路，我们表明，GloSensor系统允许真实的时间，FSK无激动剂效应的检测。这种不含FSK的激动剂信号在其他G（i）偶联受体（例如CXCR 4）上得到了证实。在对SUCNR 1的测试筛选中，我们比较了用FSK与无FSK方案获得的结果，并且能够用两种方法鉴定激动剂，但在测量基础水平时具有较少的假阳性。我们验证了一种用于检测G（i）偶联受体激动剂的无cAMP诱导剂的方法，该方法与高通量筛选相容。该方法将有助于G（i）-受体激动剂的研究和筛选。活性配体的偶联受体。(C)2015 Elsevier Inc. All rights reserved.
G protein-coupled receptors (GPCRs) represent the most successful receptor family for treating human diseases. Many are poorly characterized with few ligands reported or remain completely orphans. Therefore, there is a growing need for screening-compatible and sensitive assays. Measurement of intracellular cyclic AMP (cAMP) levels is a validated strategy for measuring GPCRs activation. However, agonist ligands for G(i)-coupled receptors are difficult to track because inducers such as forskolin (FSK) must be used and are sources of variations and errors.We developed a method based on the GloSensor system, a kinetic assay that consists in a luciferase fused with cAMP binding domain. As a proof of concept, we selected the succinate receptor 1 (SUCNR1 or GPR91) which could be an attractive drug target. It has never been validated as such because very few ligands have been described.Following analyses of SUCNR1 signaling pathways, we show that the GloSensor system allows real time, FSK-free detection of an agonist effect. This FSK-free agonist signal was confirmed on other G(i)-coupled receptors such as CXCR4. In a test screening on SUCNR1, we compared the results obtained with a FSK vs FSK-free protocol and were able to identify agonists with both methods but with fewer false positives when measuring the basal levels.In this report, we validate a cAMP-inducer free method for the detection of G(i)-coupled receptors agonists compatible with high-throughput screening.This method will facilitate the study and screening of G(i)-coupled receptors for active ligands. (C) 2015 Elsevier Inc. All rights reserved.