Quantifying biased signaling in GPCRs using BRET-based biosensors.
Quantifying biased signaling in GPCRs using BRET-based biosensors.
复制标题
使用基于 BRET 的生物传感器量化 GPCR 中的偏差信号。
DOI：
10.1016/j.ymeth.2015.04.010
复制
发表时间：
2016
期刊：
影响因子：
4.8
通讯作者：
S. Laporte
中科院分区：
文献类型：
--
作者：
Y. Namkung;O. Radresa;Sylvain Armando;D. Devost;Alexandre Beautrait;C. Le Gouill;S. Laporte
人们越来越认识到G蛋白偶联受体（GPCR）功能选择性（即偏导信号），特别是G蛋白和β-抑制蛋白依赖的信号之间的偏导信号，可以通过特定配体实现，这种定向信号代表了改善药物疗效和治疗的有希望的途径。因此，对于任何给定的gpcr来说，重要的是确定药物的药理学特征和分类方法，因为它们倾向于偏倚信号。在这里，我们描述了一个实验方案和一步一步的方法，利用HEK 293细胞中表达的血管紧张素II (AngII) 1型受体（AT1R）来评估Gαq和β-抑制素依赖反应之间的功能选择性。该方案描述了生物发光共振能量转移（BRET）传感器在AT1R中表达Gαq或β-阻滞蛋白，并使用药理学激动作用的操作模型来量化配体偏差。这些方法同样适用于其他gpcr及其下游信号效应物。
There has been a growing appreciation that G protein-coupled receptor (GPCR) functional selectivity (viz. biased signaling), in particular between G protein- and β-arrestin-dependent signaling, can be achieved with specific ligands, and that such directed signaling represents a promising avenue for improving drug efficacy and therapy. Thus, for any given GPCRs it is important to define means to pharmacologically characterize and classify drugs for their propensity to bias signaling. Here we describe an experimental protocol and step-by-step approach to assess functional selectivity between Gαq and β-arrestin-dependent responses using the prototypical angiotensin II (AngII) type 1 receptor (AT1R) expressed in HEK 293 cells. The protocol describes the expression of Bioluminescence Resonance Energy Transfer (BRET) sensors for either Gαq or β-arrestin with AT1R, and the use of the operational model of pharmacological agonism to quantify ligand bias. Such methods are equally applicable to other GPCRs and their downstream signaling effectors.