The Cryo-electron Microscopy Structure Of The Human Glucagon-like Peptide-2 Receptor

G protein-coupled receptors?GPCRs?are a family of membrane proteins with seven transmembrane domains.GPCRs can recognize a variety of external stimuli and then couple to downstream effector proteins thereby activating intracellular signaling pathways.GPCR-driven signaling pathways are involved in almost all physiological and pathological processes,therefore GPCRs are currently the most widely studied drug targets.Glucagon-like peptide-2 receptor?GLP-2R?belongs to class B1 GPCR subfamily and its endogenous ligand is glucagon-like peptide-2?GLP-2?.As an intestinal nutrition factor,the main physiological function of GLP-2 is to promote the growth of intestinal mucosa and increase nutrient digestion and absorption.GLP-2analog,Teduglutide,is mainly used in the treatment of short bowel syndrome.Recent studies in rodents have shown that the function of GLP-2 may not be limited to the intestine.It is also related to glucose metabolism and energy balance,pointing to a new regulatory mechanism for glucose control and insulin sensitivity.Moreover,GLP-2 can play a role in the central nervous system such as hypotension and antidepression.Class B1 GPCRs have poor receptor stability and high crystallization difficulty due to their huge extracellular domains.It is very hard to obtain complex structures by X-ray.In recent years,with the continuous development of cryo-electron microscopy,more than half of the class B1 GPCR structures in complex with G_s protein have been solved without crystallization.Structural determination of GLP-2R will provide valuable insights into new functions of GLP-2 in addition to that in the intestine as well as important information for the development of small molecule GLP-2R modulators.In this thesis,the bac-to-bac system was used to obtain the target protein by co-expressing GLP-2R and G proteins in insect cells.By optimizing the fusion tag,C-terminal truncation,detergent,and other purification conditions,a small amount of GLP-2-GLP-2R-Gs complexes were obtained.However,these proteins had poor stability and were easy to aggregate,making it impossible for subsequent cryo-electron microscopy data collection.The NanoBiT strategy was then used to modify the receptor and G proteins.Through the strong interaction between LgBiT and HiBiT,the receptor and G proteins were further stabilized.Uniform and intact complex proteins were eventually obtained by using this method.The cryo-electron microscopy was then employed to collect imaging data of the GLP-2-GLP-2R-G_s complex and a three-dimensional cryo-electron microscopy structure was obtained with a resolution of 3.0?.The GLP-2-GLP-2R-Gs structure laid a solid foundation for revealing the interaction mode between GLP-2 and GLP-2R,showing the molecular mechanism of how G_s protein was recruited by the receptor and provided a theoretical basis for structure-based drug discovery.This thesis also conducted functional studies of biased agonists of the5-hydroxytryptamine 2C receptor(5-HT_2CR).Biased agonists can specifically select downstream signaling pathways to trigger a specific activity while shutting down other pathways that may cause side-effects.Shui Wenjing's group at ShanghaiTech University discovered a biased G_q signaling pathway agonist of 5-HT_2CR in herbal extracts through previous research.In this thesis,bioluminescence resonance energy transfer experiments were carried out and found that this agonist selectively inhibited the recruitment of?-arrestin2 to 5-HT_2CR,thus verifying its bias nature towards the G_q signaling pathway.