Structural Studies Of Somatostatin Receptor SSTR2 And Adhesion Receptors ADGR

G protein-coupled receptors(GPCRs)are the largest human membrane protein family with more than 800 members.These receptors play important roles in cell signal transduction and are involved in a variety of diseases,such as psychiatry disease,cardiovascular disease,metabolic disease and cancer.Up to date,about 34%of FDA approved drugs target GPCRs.Structural and functional studies of GPCRs are of great significance for understanding cell signaling mechanism and drug discovery.GPCRs can recognize a variety of extracellular ligands,which induce conformational changes in the receptor and activate downstream G protein signaling pathways.The GPCRs that bind to peptides are widely distributed in the human body.These recpeors participate in various physiological functions and show great potentials in clinical application.However,the highly flexible conformations of the receptors and complexity of the peptide-receptor recognition pattern hindered drug development targeting the peptide receptors.Structure determination of the peptide GPCRs would provide molecular details of the peptide binding modes and facilitate structure-based drug design.Thus,we conducted structural studies on the somatostatin type II receptor(SSTR2)that recognizes the Somatostatin-14(SST14)and adhesion GPCRs(a GPCRs,ADGRs),which can be activated by their statchel peptides.These studies would deepen our understanding of peptide recognition and signal transduction mechanisms of the GPCR superfamily.SSTR2 belongs to class A GPCR family.In response to the endogenous peptide SST14,this receptor plays multiple roles in hormone secretion,cell cycling,apoptosis and angiogenesis.SSTR2 is the most common SSTR subtype involved in both human neuroendocrine tumors(NETs)and hormone diseases,making it a valuable target for the diagnosis and treatment of tumors and acromegaly.To date,most of the marketed drugs targeting SSTR2 are analogs of SST14,which have longer half life than SST14,but the specificity and safety need to be improved.Therefore,aiming to assist drug discovery,we solved the structure of SST14-SSTR2-G_i complex to elucidate the binding mode of SST14 and the molecular mechanism of signal transduction.By optimizing the receptor construct and screening the expression and purification conditions,we obtained the complex protein sample with high yield and good stability.Using single-particle cryo-electron microscopy,the structure of the SST14-SSTR2-G_icomplex was determined at 3.1(?)overall resolution.Combined with functional studies,the structure provides insights into ligand selectivity,receptor activation and G protein coupling,and offer a molecular model for rational drug design against SSTR2.Adhesion GPCRs,which belong to the class B2 GPCR family,are involved in brain development,cell polarity,cancer cell development and other processes,and have been considered as potential drug targets.The ADGRs have unique features in their extended N-terminal portion,which contains various adhesion domains and a GPCR-autoproteolysis inducing(GAIN)domain.The C-terminalβ-strand in the GAIN domain acts as a tethered agonist(alternatively termed stachel peptide or stalk peptide)to activate the receptor by contacting with the seven transmembrane(7TM)domain,but the underlying molecular mechanism remains unknown.In order to elucidate the binding mode of the statchel peptide and the mechanism of receptor activation,we carried out structural studies on ADGRD1 and ADGRF1.We extensively screened the gene construts of ADGRD1 and ADGRF1 and protein expression systems,and optimized protein purification conditions,which enabled production of stable ADGRD1/F1-G protein complexes,with high protein yield,great purity and good homogeneity.The complex particles showed good homogeneity and dispersion under negative stain electron microscopy.Using the Talos 200 k V cryo-electron microscopy,the cryo-sample preparation conditions were optimized.The 200 k V cryo-EM data of the ADGRF1-G protein complex showed 2D classes of the target complex,and some classes displayed clear secondary structure of the protein.In conclusion,our work was aimed to reveal the peptide binding modes and signal transduction mechanism of the peptide receptors from different GPCR families.We solved the structure of class A GPCR SSTR2 in complex with SST14 and G_i by single-particle cryo-EM.Combined with functional data,this structure uncovers the ligand recognition and signal transduction mechanisms of SSTR2.In the mean while,the structural studies of class B2 adhesion receptors laid a good foundation for furhter structural determination.These studies would facilitate drug development targeting the peptide GPCRs.