| G protein coupled receptor(GPCR)is the largest family of transmembrane receptor proteins in the human genome.The human genome encodes more than 800 GPCRs,which are widely distributed in a variety of tissues and organs.GPCR can convert extracellular stimuli such as light,neurotransmitters,hormones and immune inflammatory factors into intracellular signals by recruiting downstream G protein and Arrestin,and participate in the regulation of most physiological processes in the human body.In addition,GPCR plays a key role in the occurrence and development of a variety of diseases,so it is an important clinical drug target.At present,more than one-third of FDA-approved drugs target GPCR.Bile acids are a kind of amphiphilic metabolites produced by liver cells and secreted into the intestine by bile ducts.They have an important emulsifying effect on the fat ingested by human body in the process of digestion.One of the main ways for bile acids to play a role in the body is to bind to their membrane receptor GPBAR and play an important regulatory role in a variety of physiological processes related to metabolism and inflammation.However,it is still unclear how GPBAR recognizes different bile acids and mediates downstream signals,and there are very few drugs targeting the bile acid receptor GPBAR in clinic.In this study,we analyzed the high-resolution structure of the complex formed by bile acid receptor GPBAR and downstream Gs protein trimer under the action of small molecule compound P395 and bile acid analogue INT-777 by biochemical,cellular studies and cryo-EM,observed in the ligand binding pocket,all polar groups of the bile acid analogue INT-777 face to one side,and stabilized by hydrogen bonding with Y240,S270 of the receptor and other polar interactions,all hydrophobic groups are stabilized by the hydrophobic pocket of the receptor toward the other side,which reveals the recognition mechanism of bile acid receptor GPBAR for amphiphilic ligands.We used cAMP accumulation assay to analyze the signal differences caused by different structures of endogenous bile acids and their analogue,clarified the the recognition mechanism of bile acid receptor GPBAR to amphiphilic ligands.clarified the fingerprints of GPBAR identifying various endogenous bile acids.We used arrestin recruitment assay,cAMP accumulation assay and bias calculation formula to reveal the structural basis of GPBAR mediated downstream preference signal pathway.At the same time,we also found a new mechanism of GPCR activation by comparing the activation process of GPBAR with that of other classical GPCRs and the function of the third intracellular loop of GPCR that has not been solved before.This study will have a broadimpact on understanding the key role of GPCR in metabolic process and the activation of GPCR.Because GPBAR plays an important role in metabolism and immune regulation,the high-resolution structure of GPBAR and agonists and our research on the recognition and activation mechanism of GPBAR ligands will also promote the drug design of targeting GPBAR for the treatment of these diseases.Pseudoallergic receptor MRGPRX2 also plays an important role in the regulation of immune inflammation,it is also an important itch receptor.MRGPRX2 is a member of human Mas-related GPCR family.It is one of the important receptors that activate mast cells and cause mast cell degranulation which is mainly expressed in mast cells.Endogenous and exogenous secretagogues,such as compound 48/80,PAMP-12,substance P,cortistatin 14,act on MRGPRX2 on mast cells and activate the receptor.The receptor binds to the downstream G protein,causing changes in intracellular second messengers such as calcium and cAMP,leading to degranulation of mast cells,causing immune inflammation and activating itch neurons in the brain through the afferent nervous system,producing itching sensation.MRGPRX2 can not only mediate the allergic reaction caused by non-histaminergic peptides,but also serves as an important factor in the occurrence of red man syndrome,specific dermatitis and other diseases.In addition,clinically,many FDA approved drugs can cause allergy,pruritus and other drug side effects by activating MRGPRX2.Even if these drugs have good curative effect,patients cannot take them for a long time or in large doses,which seriously restricts the exertion of their efficacy.Therefore,intensive study of the mechanism of ligand recognition and signal transduction of MRGPRX2 plays a vital role in solving the above problems.In this study:we first characterized in detail the downstream signal pathway caused by a variety of small molecule compounds and polypeptide ligands through MRGPRX2.Then,with the help of biochemical,cellular and single particle cryoelectron microscopy techniques,we found that MRGPRX2 is different from traditional GPCR in that its ligand binding pocket is very shallow,and the N-terminal and extracellular ring of the receptor oscillate greatly,this phenomenon explained why itch receptors can recognize a variety of drugs as their ligands in clinic.According to the structural model and the detection of multiple drug-mediated signal pathways,we revealed the general mechanism of MRGPRX2 recognizing polycation compounds.We also found that MRGPRX2 can recognize polycation compound ligands through two specific acidic amino acid pockets,one consists of D184 and E164,and the other consists of D254,and this kind of small molecule ligands have one common feature:two positively charged atoms are separated by 8-10 carbon atoms,and the space distance is about 13 ?,clinical compound drugs with these two properties can often cause itching through MRGPRX2.We found a consensus motif for MRGPRX2 to recognize short peptide ligands,φp9(X0-1)R/Kp10(X2)φp13(X2-3)φP16(X3)R/Kp20,a short peptide with this motif,can usually activate MRGPRX2 and mediate downstream signal transduction.These important findings provide a theoretical basis for the clinical development of drugs to avoid side effects such as allergy,pruritus and inflammation.In addition,we also found that MRGPRX2 has a unique activation mode from signal recognition to transmission by forming kink,and the reasons for the difference of itch perception between different SNPs in the population is that SNPs in the receptor weaken the ability to sense ligands or bind downstream G proteins.These studies systematically revealed the molecular mechanism of pseudoallergic reaction perception,which provided a theoretical basis and structural basis for the development of drugs targeting these receptors.In conclusion,in this study,we deeply analyzed the ligand recognition and activation mechanisms of two important GPCRs that play an important role in the process of metabolism and immune inflammation,which provided theoretical basis and structural support for the drug development targeting bile acid receptor GPBAR and pseudoallergic receptor MRGPRX2.At the same time,these new findings also expanded the previous understanding of GPCR ligand recognition and activation mechanism. |
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