Structural And Functional Studies Of Orphan Receptor GPR3

The orphan receptor GPR34 belongs to the family of Rhodopsin G protein-coupled receptor（GPCR）,which is evolutionarily well conserved in vertebrates.There are evidences that induction of the highly expressed of GPR34 in microglia by its endogenous ligand lysophosphatidylserine（Lyso PS）leads to an increased neuroinflammatory response.Compared to the normal cells,in a variety of tumors such as colon,gastric,cervical and lymphoma cells,the expression of GPR34 is significantly elevated,while mouse GPR34 gene knock-out experiments showed that GPR34 was closely associated with enhanced survival,invasion and migration of tumor cells.These results suggested that the molecule inhibitors targeting GPR34protein may be effective clinical agents for the treatment of neuroinflammatory pain and various cancers.Unfortunately,there is no report of the antagonists targeting GPR34,and one of the most important reasons for this phenomenon is the lacking of the three-dimensional spatial structure of GPR34.In this thesis,I tried to overcome the problem of structural resolution of GPR34 by using the Bac-to-Bac insect expression system to purify the GPR34 protein in vitro.And resolve the high-resolution protein structure of GPR34 in the inactive state by optimizing the expression vector and screening the descaler.The study was divided into three parts.Firstly,the fusion protein MBP and the apocytochrome b562 fusion protein（BRIL）were inserted at the N-terminal end of GPR34 and its more flexible third intracellular loop（ICL3）respectively,by molecular cloning technique to improve the expression of the target protein in vitro,stabilizing the overall conformational the protein GPR34.Secondly,the detergents combinations were screened to optimize the protein.Finally,I added an antibody fragment BAG2 with highly affinity to BRIL as benchmark labels for molecular images and co-incubated with molecule inhibitors during the in vitro purification process to maintain the overall protein energy at a low threshold,further stabilizing the protein conformation and resulting in protein particles with well stability and particle homogeneity.Eventually,we resolved the three-dimensional spatial structure of the GPR34 in complex with the exogenous inhibitor M244B by cryo-electron microscopy with a resolution of 3.6（?）.Subsequently,the Glosensor^TMassay based on the intracellular cyclic adenosine monophosphate（c AMP）concentration change.Two amino acid residues around the M244B binding pocket which are M136 and I143 were found to affect the inhibitory activity of small molecules on the target protein GPR34 after mutation to alanine without side chains.The results speculate that M136 is associated with the entry of small molecules,while the I143 is closely associated with the binding of small molecules.In conclusion,there is the first resolve of the inactive GPR34 three-dimensional structure.In addition,the structure of the GPR34 with M244B complex would help to further elucidate the signaling mechanism of the GPR34 in the host,which also provides the structural basis for the screening of selective and active antagonist drugs which targeting GPR34at a later stage.