MRC Laboratory of Molecular Biology, Cambridge, UK.
Methods Mol Biol. 2022;2507:313-325. doi: 10.1007/978-1-0716-2368-8_16.
The thyroid-stimulating hormone receptor (TSHR) is a Class A G protein-coupled receptor (GPCR) that mediates signalling through the hypothalamic-pituitary-thyroid axis. Inappropriate activation of TSHR by autoantibodies or mutations, results in human disease such as Grave's disease and Hashimito's thyroiditis. Therefore, there is a need to develop novel therapeutics targeting the TSHR. Understanding the structure and mechanism of activation of this receptor would help elucidate the pathogenesis of disease and aid drug development. Here, we describe a method for the expression of the human TSHR in a mammalian cell line generated through a lentiviral expression system. The receptor is then purified by affinity chromatography in the ligand-free state and is suitable for structure determination by single-particle electron cryo-microscopy (cryo-EM).
促甲状腺激素受体 (TSHR) 是一种 A 类 G 蛋白偶联受体 (GPCR)，通过下丘脑-垂体-甲状腺轴介导信号转导。自身抗体或突变导致 TSHR 异常激活可导致 Graves 病和桥本甲状腺炎等人类疾病。因此，需要开发针对 TSHR 的新型治疗方法。了解该受体的结构和激活机制将有助于阐明疾病的发病机制并辅助药物开发。在这里，我们描述了一种通过慢病毒表达系统在哺乳动物细胞系中表达人 TSHR 的方法。然后通过亲和层析在无配体状态下纯化该受体，并且适合通过单颗粒电子低温冷冻显微镜 (cryo-EM) 进行结构测定。
