Label-free cell phenotypic assessment of the biased agonism and efficacy of agonists at the endogenous muscarinic M3 receptors.
Label-free cell phenotypic assessment of the biased agonism and efficacy of agonists at the endogenous muscarinic M3 receptors.
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DOI：
10.1016/j.vascn.2013.07.005
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发表时间：
2013-11
影响因子：
1.9
通讯作者：
Fang Y
中科院分区：
文献类型：
--
作者：
Deng H;Sun H;Fang Y
功效描述了配体的使受体能够改变其对宿主细胞的行为的性质，而偏性激动作用定义了配体与通过受体介导的其他途径相比差异性激活一些载体途径的能力。然而，很少有人知道的分子基础上定义的效力的配体在G蛋白偶联受体。在这里，我们的特点偏激动和细胞表型疗效的内源性毒蕈碱M3受体的激动剂在6个不同的细胞系，包括HT-29，PC-3，HeLa，SF 268，CCRF-CEM和HCT-15细胞。使用定量实时PCR和多个无标记全细胞动态质量再分布（DMR）测定来确定每个细胞系中的功能性毒蕈碱受体。DMR途径去卷积测定用于确定毒蕈碱激动剂的途径偏倚活性。使用操作激动模型来量化通路偏倚，而文献报道的宏观动力学数据用于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，所有的天然细胞系内源性表达功能性M3受体。此外，不同的激动剂在特定的细胞系以及不同的细胞系中触发不同的DMR信号。使用已知G蛋白调节剂的DMR途径去卷积揭示了所有六种细胞系中的M3受体通过多种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显示出偏向性激动作用。发现这些激动剂的全细胞功效和效力对测定时间以及细胞背景敏感。相关性分析表明，激动剂的全细胞效应与其宏观解离速率常数相关性良好。这项研究表明，内源性M3受体是耦合到多个途径，和毒蕈碱激动剂可以显示出不同的偏向激动和全细胞表型功效。
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.
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影响因子：
3.4
作者：
Fang, Ye;Ferrie, Ann M.;Balakrishnan, Jitendra
通讯作者：
Balakrishnan, Jitendra
影响因子：
3.5
作者：
Goral, Vasiliy;Wu, Qi;Fang, Ye
通讯作者：
Fang, Ye
影响因子：
2.9
作者：
Kenakin T
通讯作者：
Kenakin T
影响因子：
3.5
作者：
Fang, Y;Li, GS;Peng, JL
通讯作者：
Peng, JL
DOI：
10.1124/jpet.109.152470
发表时间：
2009-08-01
影响因子：
3.5
作者：
Casarosa, Paola;Bouyssou, Thierry;Pieper, Michael
通讯作者：
Pieper, Michael