Measuring ligand efficacy at the mu-opioid receptor using a conformational biosensor.
Measuring ligand efficacy at the mu-opioid receptor using a conformational biosensor.
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DOI：
10.7554/elife.32499
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发表时间：
2018-06-22
期刊：
影响因子：
7.7
通讯作者：
Traynor JR
中科院分区：
文献类型：
--
作者：
Livingston KE;Mahoney JP;Manglik A;Sunahara RK;Traynor JR
作用于 G 蛋白偶联受体 (GPCR) 的正位配体的内在功效反映了它们稳定活性受体状态 (R*) 的能力，并且是其生理效应的主要决定因素。在这里，我们提出了一种直接的方法来量化配体的功效，通过使用干涉测量法测量 R* 特异性生物传感器与纯化受体的结合。例如，我们使用 mu-阿片受体 (μ-OR)、A 类 GPCR 原型及其活性状态传感器 nanobody-39 (Nb39)。我们证明配体将 Nb39 招募到 µ-OR 的能力各不相同，并将美沙酮、洛哌丁胺和 PZM21 描述为支持 µ-OR 独特 R* 构象的配体。我们进一步表明，μ-OR 的正变构调节剂除了增强正构激动剂的促进作用外，还促进 R* 的形成。最后，我们证明该技术可用于异源三聚体 G 蛋白。该方法是无细胞的、不依赖于信号转导的，并且通常适用于 GPCR。
The intrinsic efficacy of orthosteric ligands acting at G-protein-coupled receptors (GPCRs) reflects their ability to stabilize active receptor states (R*) and is a major determinant of their physiological effects. Here, we present a direct way to quantify the efficacy of ligands by measuring the binding of a R*-specific biosensor to purified receptor employing interferometry. As an example, we use the mu-opioid receptor (µ-OR), a prototypic class A GPCR, and its active state sensor, nanobody-39 (Nb39). We demonstrate that ligands vary in their ability to recruit Nb39 to µ-OR and describe methadone, loperamide, and PZM21 as ligands that support unique R* conformation(s) of µ-OR. We further show that positive allosteric modulators of µ-OR promote formation of R* in addition to enhancing promotion by orthosteric agonists. Finally, we demonstrate that the technique can be utilized with heterotrimeric G protein. The method is cell-free, signal transduction-independent and is generally applicable to GPCRs.