胰岛素受体与胰岛素样生长因子1受体结构的研究

STRUCTURAL STUDIES OF INSULIN RECEPTOR AND INSULIN-LIKE GROWTH FACTOR 1 RECEPTOR

张溪

11649008

博士

生物医学工程

汪涛

2020-06-01

2020-07-20

哈尔滨工业大学

深圳

胰岛素对糖尿病患者血糖浓度的控制具有至关重要的作用。研究表明，胰岛素通过与胰岛素受体或胰岛素样生长因子1受体结合，进而激活受体相应信号通路来调节细胞的新陈代谢。虽然这两个受体在20世纪70年代已经被发现，但关于它们与胰岛素结合的分子机制至今仍未阐明。同时，由于这两个受体都属于单次跨膜蛋白，蛋白自身又存在很大的结构柔性，所以很难从传统角度对它们开展结构生物学方面的研究。近期，随着单颗粒冷冻电镜技术在膜蛋白质结构研究领域的应用日益成熟，解析受体蛋白相关结构的可能性也在逐步增加。本研究首先构建与筛选出了能够过量表达人源胰岛素受体与胰岛素样生长因子1受体的HEK293T稳定细胞株，进而获得了全长受体蛋白作为单颗粒冷冻电镜技术的研究对象。Western blot实验结果显示两受体蛋白在体外均具有自身磷酸化的生理活性。同时，根据电镜观察结果，对蛋白缓冲液中的去污剂成分进行了优化，最终确定Amphipol A8-35与LMNG满足两受体电镜结构研究的要求。利用单颗粒冷冻电镜技术，无配体状态下的胰岛素受体及胰岛素受体与胰岛素复合物相关结构被解析出来。结果表明：无配体结合状态下的胰岛素受体自身构象较为灵活，电镜结构的分辨率也相对较低，且其与胰岛素形成的复合物在体外也不稳定；在获得的4个胰岛素受体与胰岛素复合物电镜结构中，3个构象与已发表结果基本保持一致。然而，由于本研究得到的电镜结构中FnIII-2与FnIII-3结构域的电子密度更为完整，进而发现胰岛素受体与1个胰岛素分子复合物结构中的两个FnIII-3结构域之间并不相交；同时，根据胰岛素受体与胰岛素结合的动态结构模型，α-CT螺旋对胰岛素受体自身构象影响较大，而结合胰岛素分子的数目则影响FnIII-2与FnIII-3结构域的稳定性。在对胰岛素样生长因子1受体相关结构的研究中，胰岛素样生长因子1受体与胰岛素复合物结构的分辨率达到了4.7埃，而胰岛素样生长因子1受体与胰岛素样生长因子I复合物结构的分辨率为7.7埃。结果显示：胰岛素样生长因子1受体只结合了1个胰岛素或1个胰岛素样生长因子I分子，且两配体分子都能够激活该受体胞内的酪氨酸激酶结构域；通过结构模型对比，两受体结合配体分子数目的差异揭示了二者虽为同源蛋白，且能够形成异源的二聚体，但与胰岛素结合的分子机制有着较大的差异；此外，胰岛素样生长因子1受体与胰岛素分子结合的单体分子Protomer A构象与胰岛素受体相关构象保持一致，而未结合配体分子的单体分子Protomer B的L1结构域则类似“躲”在了两单体的FnIII-3结构域之间，这与胰岛素受体相关构象不同。人源胰岛素受体与胰岛素样生长因子1受体相关结构的研究，揭示了两受体与胰岛素结合的不同分子机制。这将为进一步阐明两受体在胰岛素信号传导过程中的分子机制及针对性药物研发工作提供新思路。

Insulin plays an important role in controlling blood glucose levels for diabetics. Studies show that insulin can be recognized by the insulin receptor and insulin-like growth factor-1 receptor, and further activate their corresponding signaling pathways to regulate cell metabolism. Although both receptors were discovered in the 1970s, the mechanisms that how insulin binds to the receptors are still unclear. As the single-pass transmembrane receptors, the two proteins have structural flexibility, which makes it hard to study their structures from the standpoint of tradition. Nowadays, with the development of the single-particle cryo-electron microscopy (cryo-EM) in the field of membrane protein structural biology, the structures of the two receptors may be resolved in the future. In this project, HEK293T stable cell lines with over-expression of the insulin receptor or insulin-like growth factor 1 receptor were established. And the full-length receptor proteins were purified and prepared for observation under cryo-EM. In addition, the results of the Western blot tests showed that the two receptors had the physiological activity by self-phosphorylation in vitro. According to the results of images, the detergents were adjusted and optimized, then Amphipol A8-35 and detergent LMNG were finally chosen for data collection. Then, the apo insulin receptor and the complexes insulin receptor/insulin were studied and analyzed by single-particle cryo-EM. The results showed that the conformation of the insulin receptor without insulin was flexible, and the resolution was relatively low. However, the complex insulin receptor/insulin was not a stable state. By comparing with the published models of the complexes, three cryo-EM structures of the four preliminary conformations obtained in this project were consistent with the published results. Surprisingly, the FnIII-2 and FnIII-3 domains were more clearer than that of the published ones. According to our results, when the insulin receptor bound only one insulin molecule, the two FnIII-3 domains did not intersect with each other. Meanwhile, α-CT helix had a greater effect on the conformational changes of the insulin receptor, and the number of insulin affected the stability of the FnIII-2 and FnIII-3 domains according to the dynamic binding model.For the cryo-EM structures of insulin-like growth factor-1 receptor with different ligands, the resolution of the complex insulin-like growth factor-1 receptor/insulin was up to 4.7 angstrom, while that of the insulin-like growth factor-1 receptor/insulin-like growth factor-I was 7.7 angstrom. The results proved that only one ligand was found in the complex, whether it's insulin or insulin-like growth factor-I, and just activated intracellular phosphokinase domain. By comparison with structural models, although the two receptors are homologous proteins and can even form heterodimers, the mechanisms of how insulin binds to the two receptors are quite different: the conformation of insulin-bound Protomer A both from the insulin-like growth factor-1 receptor and insulin receptor is similar, while the L1 domain of the insulin-free Protomer B falls into the gap between two FnIII-3 domains, which is very different with the conformations of insulin receptor.The structural analysis of cryo-EM structures of the insulin receptor and insulin-like growth factor-1 receptor reveals the obvious different molecule mechanism between the two receptors in insulin binding, which may provide a new sight for further elucidating the molecular mechanism in the insulin signaling pathway and drug discovery related to the two receptors.

胰岛素受体 胰岛素 胰岛素样生长因子1受体 胰岛素样生长因子I 单颗粒冷冻电镜技术

Insulin receptor Insulin Insulin-like growth factor 1 receptor Insulin-like growth factor I Single-particle cryo-EM

中文

联合培养

南方科技大学

张溪. 胰岛素受体与胰岛素样生长因子1受体结构的研究[D]. 深圳. 哈尔滨工业大学,2020.