Single-molecule analysis reveals that a glucagon-bound extracellular domain of the glucagon receptor is dynamic.
Single-molecule analysis reveals that a glucagon-bound extracellular domain of the glucagon receptor is dynamic.
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DOI：
10.1016/j.jbc.2023.105160
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发表时间：
2023-09
影响因子：
4.8
通讯作者：
Lamichhane, Rajan
中科院分区：
文献类型：
--
作者：
Liu, Ting;Khanal, Susmita;Hertslet, Gillian D.;Lamichhane, Rajan
动态信息对于理解G蛋白偶联受体（GPCRs）的激活机制至关重要。尽管不同构象状态的高分辨率结构的可用性，这些状态在分子水平上的动力学知之甚少。在这里，我们使用全内反射荧光显微镜来研究胰高血糖素受体（GCGR）的细胞外结构域（ECD），GCGR是控制葡萄糖稳态的B类家族GPCR。用单分子荧光共振能量转移法观察了哺乳动物细胞表达和纯化的GCGR分子的ECD动力学。我们观察到，对于apo-GCGR，ECD是动态的，并且主要在闭合构象中花费时间。在胰高血糖素的存在下，ECD是完全开放的，并且也显示出比apo-GCGR更动态的行为，这是以前没有报道过的发现。这些结果表明，无论是apo-GCGR和胰高血糖素结合GCGR显示可逆的打开和关闭的ECD相对于七跨膜（7 TM）结构域。这项工作展示了一种可视化GCGR ECD动力学的分子方法，并为理解GPCR激活背后的构象变化提供了基础，这在新疗法的开发中至关重要。
Dynamic information is vital to understanding the activation mechanism of G protein-coupled receptors (GPCRs). Despite the availability of high-resolution structures of different conformational states, the dynamics of those states at the molecular level are poorly understood. Here, we used total internal reflection fluorescence microscopy to study the extracellular domain (ECD) of the glucagon receptor (GCGR), a class B family GPCR that controls glucose homeostasis. Single-molecule fluorescence resonance energy transfer was used to observe the ECD dynamics of GCGR molecules expressed and purified from mammalian cells. We observed that for apo-GCGR, the ECD is dynamic and spent time predominantly in a closed conformation. In the presence of glucagon, the ECD is wide open and also shows more dynamic behavior than apo-GCGR, a finding that was not previously reported. These results suggest that both apo-GCGR and glucagon-bound GCGRs show reversible opening and closing of the ECD with respect to the seven-transmembrane (7TM) domain. This work demonstrates a molecular approach to visualizing the dynamics of the GCGR ECD and provides a foundation for understanding the conformational changes underlying GPCR activation, which is critical in the development of new therapeutics.
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影响因子：
48
作者：
Asher WB;Geggier P;Holsey MD;Gilmore GT;Pati AK;Meszaros J;Terry DS;Mathiasen S;Kaliszewski MJ;McCauley MD;Govindaraju A;Zhou Z;Harikumar KG;Jaqaman K;Miller LJ;Smith AW;Blanchard SC;Javitch JA
通讯作者：
Javitch JA
影响因子：
3.4
作者：
Aitken, Colin Echeverria;Marshall, R. Andrew;Puglisi, Joseph D.
通讯作者：
Puglisi, Joseph D.
影响因子：
4.8
作者：
Kuszak, Adam J.;Pitchiaya, Sethuramasundaram;Sunahara, Roger K.
通讯作者：
Sunahara, Roger K.
影响因子：
16.8
作者：
通讯作者：
--
影响因子：
8.1
作者：
Neumann UH;Ho JSS;Mojibian M;Covey SD;Charron MJ;Kieffer TJ
通讯作者：
Kieffer TJ