Department of Psychiatry, Vagelos College of Physicians and Surgeons, Columbia University, New York, NY, USA.
Department of Molecular Pharmacology and Therapeutics, Vagelos College of Physicians and Surgeons, Columbia University, New York, NY, USA.
Nat Methods. 2021 Apr;18(4):397-405. doi: 10.1038/s41592-021-01081-y. Epub 2021 Mar 8.
Class C G protein-coupled receptors (GPCRs) are known to form stable homodimers or heterodimers critical for function, but the oligomeric status of class A and B receptors, which constitute >90% of all GPCRs, remains hotly debated. Single-molecule fluorescence resonance energy transfer (smFRET) is a powerful approach with the potential to reveal valuable insights into GPCR organization but has rarely been used in living cells to study protein systems. Here, we report generally applicable methods for using smFRET to detect and track transmembrane proteins diffusing within the plasma membrane of mammalian cells. We leverage this in-cell smFRET approach to show agonist-induced structural dynamics within individual metabotropic glutamate receptor dimers. We apply these methods to representative class A, B and C receptors, finding evidence for receptor monomers, density-dependent dimers and constitutive dimers, respectively.
C 类 G 蛋白偶联受体 (GPCR) 已知可形成稳定的同源二聚体或异源二聚体，这对其功能至关重要，但构成所有 GPCR 超过 90%的 A 类和 B 类受体的寡聚状态仍存在激烈争论。单分子荧光共振能量转移 (smFRET) 是一种强大的方法，具有揭示 GPCR 组织有价值见解的潜力，但在活细胞中很少用于研究蛋白质系统。在这里，我们报告了一般适用于使用 smFRET 检测和跟踪在哺乳动物细胞膜内扩散的跨膜蛋白的方法。我们利用这种细胞内 smFRET 方法来显示激动剂诱导的单个代谢型谷氨酸受体二聚体的结构动力学。我们将这些方法应用于代表性的 A、B 和 C 类受体，分别发现了受体单体、密度依赖的二聚体和组成性二聚体的证据。
