FLIM-FRET-Based Structural Characterization of a Class-A GPCR Dimer in the Cell Membrane
FLIM-FRET-Based Structural Characterization of a Class-A GPCR Dimer in the Cell Membrane
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基于 FLIM-FRET 的细胞膜中 A 类 GPCR 二聚体的结构表征
DOI：
10.1016/j.jmb.2020.06.009
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发表时间：
2020
影响因子：
5.6
通讯作者：
Zhang Wei-Ping
中科院分区：
文献类型：
--
作者：
Yang Ju;Gong Zhou;Lu Yun-Bi;Xu Chan-Juan;Wei Tao-Feng;Yang Meng-Shi;Zhan Tian-Wei;Yang Yu-Hong;Lin Li;Liu Jianfeng;Tang Chun;Zhang Wei-Ping
已知a类G蛋白偶联受体（gpcr）在膜中同质二聚化。然而，缺乏表征天然环境中GPCR二聚体结构的方法。因此，a类GPCR二聚化的分子基础和功能相关性尚不清楚。在这里，我们提出了细胞膜中GPR17的二聚体结构模型。该二聚体主要涉及界面处的跨膜螺旋5 (TM5)，而TM5中的F229是一个关键残基。无论受体的表达水平如何，F229A突变都会使GPR17成为单体。GPR17的单体突变体显示ERK1/2激活受损，并且在激动剂治疗后不能正常内化。相反，F229C突变体作为二聚体交联，其行为与野生型相似。重要的是，利用荧光寿命成像显微镜获得的稀疏原聚体间FRET距离限制，对GPR17二聚体结构进行了建模。同样的方法可以应用于描述细胞中其他重要膜蛋白的相互作用。
Class-A G protein-coupled receptors (GPCRs) are known to homo-dimerize in the membrane. Yet, methods to characterize the structure of GPCR dimer in the native environment are lacking. Accordingly, the molecular basis and functional relevance of the class-A GPCR dimerization remain unclear. Here, we present the dimeric structural model of GPR17 in the cell membrane. The dimer mainly involves transmembrane helix 5 (TM5) at the interface, with F229 in TM5, a critical residue. An F229A mutation makes GPR17 monomeric regardless of the expression level of the receptor. Monomeric mutants of GPR17 display impaired ERK1/2 activation and cannot be properly internalized upon agonist treatment. Conversely, the F229C mutant is cross-linked as a dimer and behaves like wild-type. Importantly, the GPR17 dimer structure has been modeled using sparse inter-protomer FRET distance restraints obtained from fluorescence lifetime imaging microscopy. The same approach can be applied to characterizing the interactions of other important membrane proteins in the cell.