Conformational dynamics between transmembrane domains and allosteric modulation of a metabotropic glutamate receptor
Conformational dynamics between transmembrane domains and allosteric modulation of a metabotropic glutamate receptor
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DOI：
10.7554/elife.45116
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发表时间：
2019-06-07
期刊：
影响因子：
7.7
通讯作者：
Levitz, Joshua
中科院分区：
文献类型：
--
作者：
Gutzeit, Vanessa A.;Thibado, Jordana;Levitz, Joshua
代谢型谷氨酸受体（mGluR）是C类突触G蛋白偶联受体（GPCR），其含有大的细胞外配体结合结构域（LBD）并形成组成型二聚体。尽管存在一个详细的图片间LBD构象动力学和结构快照的隔离域和全长受体，它仍然不清楚如何mGluR激活进行跨膜结构域（TMD）的水平，以及如何TMD靶向变构药物发挥其作用。在这里，我们使用时间分辨的功能和构象分析来剖析变构药物激活和调节mGluR2的机制。单分子亚基计数和间TMD荧光共振能量转移测量在活细胞中揭示LBD无关的TMD二聚体之间的构象重排在受体调节。使用这些检测沿着功能读数，我们发现了阳性和阴性变构药物作用的大小、方向和时间的异质性。我们的实验一起导致TMD激活的三态模型，这为理解亚基间重排如何驱动C类GPCR激活提供了一个框架。
Metabotropic glutamate receptors (mGluRs) are class C, synaptic G-protein-coupled receptors (GPCRs) that contain large extracellular ligand binding domains (LBDs) and form constitutive dimers. Despite the existence of a detailed picture of inter-LBD conformational dynamics and structural snapshots of both isolated domains and full-length receptors, it remains unclear how mGluR activation proceeds at the level of the transmembrane domains (TMDs) and how TMD-targeting allosteric drugs exert their effects. Here, we use time-resolved functional and conformational assays to dissect the mechanisms by which allosteric drugs activate and modulate mGluR2. Single-molecule subunit counting and inter-TMD fluorescence resonance energy transfer measurements in living cells reveal LBD-independent conformational rearrangements between TMD dimers during receptor modulation. Using these assays along with functional readouts, we uncover heterogeneity in the magnitude, direction, and the timing of the action of both positive and negative allosteric drugs. Together our experiments lead to a three-state model of TMD activation, which provides a framework for understanding how inter-subunit rearrangements drive class C GPCR activation.