Deng Huayun, Sun Haiyan, Fang Ye
Biochemical Technologies, Science and Technology Division, Corning Incorporated, Corning, NY 14831, United States.
J Pharmacol Toxicol Methods. 2013 Nov-Dec;68(3):323-33. doi: 10.1016/j.vascn.2013.07.005. Epub 2013 Aug 9.
Efficacy describes the property of a ligand that enables the receptor to change its behavior towards the host cell, while biased agonism defines the ability of a ligand to differentially activate some of the vectorial pathways over others mediated through the receptor. However, little is known about the molecular basis defining the efficacy of ligands at G protein-coupled receptors. Here we characterize the biased agonism and cell phenotypic efficacy of seven agonists at the endogenous muscarinic M3 receptors in six different cell lines including HT-29, PC-3, HeLa, SF268, CCRF-CEM and HCT-15 cells.
Quantitative real-time PCR and multiple label-free whole cell dynamic mass redistribution (DMR) assays were used to determine the functional muscarinic receptors in each cell line. DMR pathway deconvolution assay was used to determine the pathway biased activity of the muscarinic agonists. Operational agonism model was used to quantify the pathway bias, while macro-kinetic data reported in literature was used to analyze the biochemical mechanism of action of these agonists.
Quantitative real-time PCR and ligand pharmacology studies showed that all the native cell lines endogenously express functional M3 receptors. Furthermore, different agonists triggered distinct DMR signals in a specific cell line as well as in different cell lines. DMR pathway deconvolution using known G protein modulators revealed that the M3 receptor in all the six cell lines signals through multiple G protein-mediated pathways, and certain agonists display biased agonism in a cell line-dependent manner. The whole cell efficacy and potency of these agonists were found to be sensitive to the assay time as well as the cell background. Correlation analysis suggested that the whole cell efficacy of agonists is correlated well with their macro-dissociation rate constants.
This study implicates that the endogenous M3 receptors are coupled to multiple pathways, and the muscarinic agonists can display distinct biased agonism and whole cell phenotypic efficacy.
效能描述了配体使受体改变其对宿主细胞行为的特性，而偏向性激动作用则定义了配体在通过受体介导的其他途径中差异性激活某些矢量途径的能力。然而，关于决定配体在G蛋白偶联受体上效能的分子基础知之甚少。在此，我们表征了七种激动剂在六种不同细胞系（包括HT-29、PC-3、HeLa、SF268、CCRF-CEM和HCT-15细胞）中内源性毒蕈碱M3受体上的偏向性激动作用和细胞表型效能。
采用定量实时PCR和多标记无标记全细胞动态质量重分布（DMR）测定法来确定每个细胞系中的功能性毒蕈碱受体。DMR途径反卷积测定法用于确定毒蕈碱激动剂的途径偏向性活性。使用操作激动作用模型来量化途径偏向性，同时利用文献中报道的宏观动力学数据来分析这些激动剂的生化作用机制。
定量实时PCR和配体药理学研究表明，所有天然细胞系均内源性表达功能性M3受体。此外，不同的激动剂在特定细胞系以及不同细胞系中引发了不同的DMR信号。使用已知G蛋白调节剂进行的DMR途径反卷积显示，所有六个细胞系中的M3受体均通过多种G蛋白介导的途径发出信号，并且某些激动剂以细胞系依赖性方式表现出偏向性激动作用。发现这些激动剂的全细胞效能和效价对测定时间以及细胞背景敏感。相关性分析表明，激动剂的全细胞效能与其宏观解离速率常数密切相关。
本研究表明内源性M3受体与多种途径偶联，并且毒蕈碱激动剂可表现出明显的偏向性激动作用和全细胞表型效能。
