Structural Studies On G Protein-coupled Receptor Signal Transduction

The G protein-coupled receptor(GPCR)family is the largest group of cell membrane receptors responsible for sensing and transmitting various extracellular signals.Upon activation by extracellular signals,GPCRs induce G protein dissociation or β-arrestin recruitment on the cytoplasmic side of the cell membrane,leading to a series of biochemical events such as the accumulation of intracellular second messengers,ion channel opening/closing,kinase cascade activation and receptor internalization,thereby allowing the cell to respond to extracellular signals.The crucial role of the GPCR family in cell signal transduction has made it a target for numerous known and ongoing drug developments.A key question in GPCR biology is how various receptors recognize their respective ligands,activate,and transmit signals downstream.In recent years,the peptide receptor subfamily of the A family has become one of the most studied groups of GPCRs targeted by drugs.However,the signaling mechanisms of most peptide receptors is still not well understood.One of the research subjects in this study,the galanin receptors(GALRs),is a subfamily containing GALR1-3 that specifically recognizes the neuropeptide galanin(GAL).GAL and GALRs are associated with physiological processes such as feeding,metabolism,pain,sleep behavior as well as pathological processes such as depression and schizophrenia.Although researchers discovered this signaling transduction system decades ago,the principles of ligand recognition and binding,the mechanisms of highly specific coupling with certain types of G proteins by GALR subtypes remain unclear.In this research,through the single-particle cryo-electron microscopy analysis method(SPA),the respective structures of the GALR1 and GALR2 signaling complexes in the activated state were solved.It was observed that GAL folds into a typical helix-turn-helix structure and lays on the top of receptor plane.Through structural analysis and extensive point mutation studies,the current research demonstrated that there are significant differences in the recognition of GAL among GALR subtypes and further proved that GALRs maintain strict G protein subtype specificity through the sequence differences of their ICL2(Intracellular loop 2)and ICL3.In addition,through analysis of GALR1’s histidine-rich ligand binding pocket,the current research proposed that Zn2+ serves as a GALR1-specific negative allosteric modulator(NAM)involved in the regulation of receptor activation.Another key question in GPCR biology is desensitization of the signal transduction process.The desensitization mechanism prevents the receptor from being overactivated under constant exposure to ligands,which can cause damage to the cell.The classical desensitization systems are the GRK/β-arrestin system and the RGS system.Recently,it was reported that when GPCRs are activated,the free Gβγ subunit is recognized by a member of the potassium channel tetramerization domain-containing protein family(KCTD),KCTD5,which then recruits the E3 ubiquitin ligase Cullin-3 to ubiquitinate and degrade Gβγ.This is the first reported desensitization mechanism against Gβγ by ubiquitination and degradation.Compared to other desensitization systems,the biochemical and structural details of specific recognition and degradation of Gβγ by the Cullin-3/KCTD5 E3 ligase system are not yet clear.Therefore,in the second part of this research,the novel desensitization mechanism was explored using the SPA method.Firstly,it was determined that KCTD5 and Gβγ bind in a maximal stoichiometry of 5:5.The complex structure was subsequently resolved at high resolution,providing a detailed explanation of the structural basis of KCTD5 recruiting Gβγ.To overcome the instability issue of the KCTD5/Cullin-3 complex,the structure of the KCTD5 homolog,KCTD7,in complex with Cullin-3 was resolved.Based on the conserved interaction mode,the first Cullin-3/KCTD5/Gβγ E3 ubiquitin ligase/substrate complex model was successfully constructed.In summary,this study used the SPA method to investigate two major questions in GPCR biology.Through a combination of biochemical and molecular biology methods,we described the basis and details of GALRs activation and specific coupling to G proteins,providing detailed references for potential agonist and antagonist design and a deeper understanding of the G protein selectivity of various GPCRs.Further,this study described the structural basis of KCTD5 as an adaptor protein that recruits Cullin-3 and activates Gβγ,and for the first time,systematically reported the complex structure of Cullin3/KCTD family E3 ligase in complex with substrates.Additionally,solvation of the structure of KCTD7,the pathogenic gene for Progressive myoclonic epilepsy,helps us expand our understanding of the structural diversity of the KCTD family.These findings provide a detailed reference for further understanding and studying the physiological and pathological mechanisms of the KCTD family.