G protein-coupled receptor signaling analysis using homogenous time-resolved Förster resonance energy transfer (HTRF®) technology.
G protein-coupled receptor signaling analysis using homogenous time-resolved Förster resonance energy transfer (HTRF®) technology.
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DOI：
10.3390/ijms15022554
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发表时间：
2014-02-13
影响因子：
5.6
通讯作者：
Bräuner-Osborne H
中科院分区：
文献类型：
--
作者：
Nørskov-Lauritsen L;Thomsen AR;Bräuner-Osborne H
研究 G 蛋白偶联受体 (GPCR) 的多维信号传导以寻找新的更好的治疗方法需要采用高通量筛选 (HTS) 形式的灵活、可靠和灵敏的检测方法。如今，高温超导中使用的检测技术一半以上基于荧光，因为其灵敏度高且信号丰富，但猝灭、光学干涉和光散射是严重的缺点。 20 世纪 90 年代，开发了基于时间分辨均质格式福斯特共振能量转移 (FRET) 的 HTRF®（Cisbio Bioassays，Codolet，法国）技术。这项改进的技术消除了传统的缺点。这里描述的基于 HTRF® 技术的优化方案用于研究钙敏感受体 CaSR 的激活和信号传导途径，CaSR 是一种负责维持钙稳态的 GPCR。激动剂对 CaSR 的刺激激活了多种途径，这些途径可通过测量第二信使 d-肌醇 1-磷酸 (IP1) 和环腺苷 3',5'-单磷酸 (cAMP) 的积累以及通过测量细胞外信号调节激酶 1 和 2 (ERK1/2) 的磷酸化来检测。在这里，我们展示了与以前的检测相比具有众多优势的优化 HTRF® 平台如何提供研究 GPCR 信号传导的实质性和稳健的模式。此外还讨论了如何优化和小型化这些测定，以满足 HTS 要求和筛选化合物库。
Studying multidimensional signaling of G protein-coupled receptors (GPCRs) in search of new and better treatments requires flexible, reliable and sensitive assays in high throughput screening (HTS) formats. Today, more than half of the detection techniques used in HTS are based on fluorescence, because of the high sensitivity and rich signal, but quenching, optical interferences and light scattering are serious drawbacks. In the 1990s the HTRF® (Cisbio Bioassays, Codolet, France) technology based on Förster resonance energy transfer (FRET) in a time-resolved homogeneous format was developed. This improved technology diminished the traditional drawbacks. The optimized protocol described here based on HTRF® technology was used to study the activation and signaling pathways of the calcium-sensing receptor, CaSR, a GPCR responsible for maintaining calcium homeostasis. Stimulation of the CaSR by agonists activated several pathways, which were detected by measuring accumulation of the second messengers d-myo-inositol 1-phosphate (IP1) and cyclic adenosine 3′,5′-monophosphate (cAMP), and by measuring the phosphorylation of extracellular signal-regulated kinase 1 and 2 (ERK1/2). Here we show how an optimized HTRF® platform with numerous advantages compared to previous assays provides a substantial and robust mode of investigating GPCR signaling. It is furthermore discussed how these assays can be optimized and miniaturized to meet HTS requirements and for screening compound libraries.
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影响因子：
1.8
作者：
Bergsdorf, Christian;Kropp-Goerkis, Carmen;Bader, Benjamin
通讯作者：
Bader, Benjamin
影响因子：
3.3
作者：
ALPHA, B;BALLARDINI, R;SABBATINI, N
通讯作者：
SABBATINI, N
影响因子：
--
作者：
FORSTER, T
通讯作者：
FORSTER, T
DOI：
10.1124/jpet.110.173948
发表时间：
2011-02-01
影响因子：
3.5
作者：
Kenakin, Terry
通讯作者：
Kenakin, Terry
影响因子：
4.8
作者：
BROWN, EM;El-Hajj Fuleihan, G;KIFOR, O
通讯作者：
KIFOR, O