Studies On The Structure-function Relationship Of GPCR-the Glycosylation Of ?₂ Adrenergic Receptor, Dynamic Of The Conserved Disulfide Bond And Interaction Mechanism With Arrestin

G protein coupled receptors?GPCRs?constitute the largest class of cell surface receptors.GPCRs can be activated by a variety of ligands,including biogenetic amines,peptides,neurotransmitters,ions and fatty acids.They participate in regulating human physiology process,including cardiovascular system,immune system and nervous system.GPCRs have been important targets for diagnosis and treatment of many diseases.About 50% of the drugs,many of which are the top 100 drugs in the world,currently on the market have been developed using GPCRs as the targets.Therefore,the studies on the structure and function of GPCRs will provide important foundation for diagnosis and treatment of diseases.In the present thesis,we investigate the structure-function relationship of GPCRs in the following three aspects: 1)the role of glycosylation in ?₂ adrenergic receptor??₂AR?function;2)the study on the dynamics of the conserved disulfide bond in GPCRs;3)the study on the molecular mechanism of ?₁ adrenergic receptor??₁AR?and ?-arrestin1 interaction in the presence of the biased ligand.N-glycosylation is a common post-translational modification of GPCRs.However,it remains unknown how N-glycosylation regulates GPCRs signaling.?₂AR has three N-glycosylation sites: Asn6,Asn15 at the N-terminus and Asn187 at the second extracellular loop?ECL2?.We investigated the role of glycosylation in ?₂AR function by combining site directed mutagenesis,enzyme digestion,cAMP assay,covalent cross-linking,co-immunoprecipitation and Tag-lite assay.We found that deletion of the N-glycosylation did not affect receptor expression and ligand binding.Deletion of the N-glycosylation at the N-terminus rather than Asn187 showed decreased effects on isoproterenol-promoted G protein dependent signaling,?-arrestin2 recruitment and receptor internalization.Based on these results,we further explored the mechanism of N-glycosylation regulating receptor function.Both N6 Q and N15 Q showed decreased receptor dimerization,while N187 Q did not influence receptor dimerization.As decreased ?₂AR homodimer accompanied with reduced efficiency for receptor function,we proposed that the N-glycosylation of ?₂AR regulated receptor function by influencing receptor dimerization.To verify this hypothesis,we further paid attention to the residues at the dimerization interface,Lys60 and Glu338.The double mutant K60A/E338 A showed decreased ?₂AR dimerization and its effects on receptor function was similar to N6 Q and N15 Q,which further supported the important role of dimerization in receptor function.This work provides new insights into the relationship among glycosylation,dimerization and function of GPCRs.The disulfide bond that links TM3 and ECL2 is conserved in about 92% of GPCRs superfamily.In class A GPCRs,disruption the conserved disulfide bridge can affect receptor structure and function.The disulfide bond is observed in agonist-bound P2Y12 R structure.However,in the structure of the antagonist-bound P2Y12 R,the conserved disulfide bridge between TM3 and ECL2 is not observed and instead appears to be labile and dynamic.Based on these phenomenons we doubt the prevailing view that the disulfide bond is a basic structural feature of GPCRs.At present,the crystal structures have shown the information about the conserved disulfide bridge in active and inactive GPCRs.Before ligand binding,GPCRs exist in the basic or apo state;however,the characteristics of the conserved two disulfide bond in this state are rarely studied.Because of the high dynamics of the apo state,only the crystal structure of ?₁AR in apo state has been resolved.The apo ?₁AR exhibits the disulfide bond,but in other GPCRs whether the disulfide bond exists in the apo states remains ambiguous.So we combined molecular dynamics and MTSEA-biotin specific labeling to investigate the conserved disulfide bond in apo P2Y12 R,?₁AR and ?₂AR.Our results showed that the disulfide bond in apo P2Y12 R and ?₂AR was labile;however,the conserved disulfide bond in apo ?₁AR was stable.Moreover,our results showed that the disulfide bond in P2Y12 R and ?₂AR was dynamic in the presence of agonist.This result might show that the conserved disulfide bond is not essential for receptor activation.Our findings updated the understanding of the conserved disulfide bond in GPCRs and provided a new aspect for the studies on structure-function relationship of GPCRs.?-arrestins can regulate the desensitization,internalization and intracellular trafficking of a majority of GPCRs.Recent studies reveal that the biased ligands can selectively activate ?-arrestin dependent signaling but not G protein dependent signaling.In clinical studies,biased ligands exhibit reduced side effects.Thus studies on the molecular mechanism of GPCR-arrestin interaction are critical for understanding the mechanism of ligands biase.We applied disulfide bond crosslinking to study the interaction interface between ?₁AR and ?-arrestin1.Our results exhibited that when exposed to biased ligand carvedilol,?-arrestin1 Finger loop could form crosslinking with receptor helix8;the Middle loop could crosslink with receptor TM4 and ICL1/2;the C loop could crosslink with receptor TM5 and ICL2.Based on the crosslinking results,we constructed ?₁AR-?-arrestin1 complex model in the presence of biased ligand.The model exhibited biased ligand specific receptor-arrestin interaction and provided structural basis to understand ?-arrestin dependent signal transduction.