Namkung Yoon, Radresa Olivier, Armando Sylvain, Devost Dominic, Beautrait Alexandre, Le Gouill Christian, Laporte Stephane A
Department of Medicine, McGill University Health Center Research Institute, McGill University, Montréal, Canada.
Department of Pharmacology and Therapeutics, McGill University, Montréal, Canada.
Methods. 2016 Jan 1;92:5-10. doi: 10.1016/j.ymeth.2015.04.010. Epub 2015 Apr 15.
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.
人们越来越认识到，通过特定配体可以实现G蛋白偶联受体（GPCR）的功能选择性（即偏向性信号传导），尤其是在G蛋白依赖性信号传导和β-抑制蛋白依赖性信号传导之间，并且这种定向信号传导是提高药物疗效和治疗效果的一个有前景的途径。因此，对于任何给定的GPCR，重要的是确定从药理学角度表征和分类药物偏向信号传导倾向的方法。在这里，我们描述了一种实验方案和逐步方法，用于评估在HEK 293细胞中表达的典型血管紧张素II（AngII）1型受体（AT1R）的Gαq和β-抑制蛋白依赖性反应之间的功能选择性。该方案描述了用于Gαq或β-抑制蛋白与AT1R的生物发光共振能量转移（BRET）传感器的表达，以及使用药理学激动作用的操作模型来量化配体偏向性。这些方法同样适用于其他GPCR及其下游信号效应器。
