Structural And Functional Studies Of Adhesion G Protein-Coupled Receptors

G protein-coupled receptors(GPCRs)are featured with numerous members and various functions,and are involved in the regulation of almost all the physiological processes.The abnormal expression and dysfunction of GPCRs result in a broad spectrum of diseases,making them attractive therapeutic targets for disease treatment.As the second largest family in the GPCR superfamily,the B2 family of adhesion GPCRs(a GPCRs)are widely associated with diverse physiological activities and diseases.They are unique in many structural and functional characteristics,with a huge N-terminal extracellular domain(ECD)and a classical seven-transmembrane domain(TMD)which play a dual role in cell adhesion and signal transduction.The N-terminal ECD of the a GPCR contains varied adhesion domains that interact with extracellular adhesion molecules to mediate cell–cell communications,while the TMD undergoes conformational changes to recruit downstream effector proteins upon activation to initiate signal transduction.Located between these domains is the GPCRautoproteolysis inducing domain(GAIN domain),where autoproteolysis occurs in most of a GPCRs and generates two protein fragments,NTF(N-terminal fragment)and CTF(C terminal fragment).Most of the a GPCRs are still orphan receptors until now with their endogenous ligands,physiological functions and downstream signaling pathways unknown,greatly impeding the understanding of this GPCR family and hampering their potential as drug targets.Therefore,it is of significance to elucidate the molecular mechanism of a GPCR activation and the dual role regulation in cell adhesion and signal transduction from structural and functional perspectives.Several representative a GPCRs were selected as research objects in this study considering their features in autoproteolysis,ligand recognition and downstream signaling pathways.The main contents include studying the activation mechanisms of the non-autoproteolytic ADGRA2 and autoproteolytic ADGRE5,ADGRL3,ADGRF1 and ADGRD1,and investigating the receptor–protein ligand interaction patterns of the ADGRE5–CD55 and ADGRL3–FLRT3 complexes.These studies would uncover the activation modes of the a GPCRs and the modulation mechanism of the protein ligands on the conformation and function of the receptors,which would illustrate the diversity of a GPCR family in a more comprehensive way and provide valuable information for a GPCR research.In the study of the activation mechanism of the a GPCRs,we systematically optimized the genetic modification,expression and purification conditions on multiple receptors,and successfully obtained several high-quality protein samples,which were used for structural studies of the inactive state of the receptors with X-ray crystallography or single particle cryo-electron microscopy.Subsequently,we prepared the protein samples of some a GPCR–G protein complexes in order to determine the active a GPCR structures.With these efforts,we finally solved the structures of active ADGRF1 and ADGRD1 in complex with G proteins and investigated the new discoveries with various functional assays.Combined with functional studies,the structures,for the first time,revealed the unique tethered-stalk peptide agonism of the a GPCRs and the interaction mode between the stalk peptide and receptor TMD.We also disclosed the possible effects of autoproteolysis on a GPCR activation.In addition,we compared the conformational changes of some key motifs and the G protein binding interfaces in the active a GPCR structures,which highlighted diversity of the structural and functional characteristics of the a GPCRs.Furthermore,the ADGRF1 structures also revealed a specific interaction between this receptor and a lipid molecule lysophosphatidylcholine(LPC),which was identified by mass spectrometry.This lipid regulates the receptor activation and its binding pocket may serve as the potential binding site for synaptamide,which was previously reported as a small molecular agonist for ADGRF1.These results not only expand our knowledge about the activation mechanism of the a GPCRs,but also provide a basis for further structural and functional research of the a GPCR family.In the structural studies of the a GPCR–protein ligand complexes,we performed genetic modification,expression and purification optimization to improve the protein quality of the protein ligands CD55 and FLRT3.We then screened different ADGRE5 isoforms and purification protocols for the ADGRE5–CD55 complex.Moreover,we carried out a series of optimization screening for the ADGLR3–FLRT3 complex and obtained high-quality complex sample for cryo-electron microscopy experiments.These works characterized and verified different a GPCR-protein ligand complexes,and made an exploration towards the structure determination and mechanism understanding of the a GPCR–protein ligand complex.