Structural Studies Of Insulin Receptor And Insulin-like Growth Factor 1 Receptor

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 1970 s,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.Therefore,in this study,the structures of insulin receptor and insulin-like growth factor 1 receptor were solved by the single-particle cryo-electron microscopy(cryo-EM),and the main research contents were as follows:In this project,HEK293 T 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,based on single-particle cryo-EM,the conformation of the insulin receptor without insulin was flexible,and two major 3D models were obtained although the resolution was relatively low(15-10 (?)).Meanwhile,the results of Size exclusion chromatography(SEC)and Western blot experiments showed that the complex insulin receptor/insulin was not a stable state: Four different cryo-EM structures were obtained in this project(15-5.9 (?)).Surprisingly,the Fn III-2 and Fn III-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 Fn III-3domains did not intersect with each other.In addition,?-CT helix had a greater effect on the conformational changes of the insulin receptor,and the number of insulin affected the stability of the Fn III-2 and Fn III-3 domains according to the dynamic binding model.For the cryo-EM structures of insulin-like growth factor ? receptor with different ligands,the resolution of the complex insulin-like growth factor ?receptor/insulin was up to 4.2 (?),while that of the insulin-like growth factor 1receptor/insulin-like growth factor 1 was 7.7 (?).From the reconstructed 3D models and Western blot experiments,the results proved that only one ligand was found in the complex,whether it's insulin or insulin-like growth factor 1,and just activated intracellular phosphokinase domain.Furthermore,the conformation of insulinbound Protomer A both from the insulin-like growth factor 1 receptor and insulin receptor was similar,while the L1 domain of the insulin-free Protomer B fell into the gap between two Fn III-3 domains,which was very different with the conformations of insulin receptor.The structural analysis of cryo-EM structures of the insulin receptor and insulin-like growth factor-? 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.