Expression Of The OGPCRs-Gi1α Fusion Proteins And The Exploring Of GPR80 Function

G protein-coupled receptors (GPCRs), which mediate cell communication and signal transduction, play important physiological roles and pathological significances. They are the most important drug targets and regarded as the major subjects of pharmacology. It is estimated that the total numbers of human GPCRs are around 900, in which 100 members are called orphan GPCRs (oGPCRs) due to their unidentified ligands and functions.Given the importance of GPCR, how to de-orphanize oGPCRs and to further explore their roles has become the key research content of oGPCR. This study consists of two parts:1) Fusions of three oGPCR genes with the a subunit of G protein and then expressions by insect sf9 cells; 2) Preliminary exploring of the newly de-orphanized GPR80 receptor. The main results are summarized as the following.In the first part of this study, the whole open reading frames of orphan receptor GPR45 (GenBank Accession number:NM₀07227), GPR85 (GenBank Accession number:NM₀18970), GPR174 (GenBank Accession number: NM₀32553) and Gilαwere cloned separately, and the corresponding fusion genes were amplified by overlap extension PCR. Then, the fusion gene was cloned into the donor plasmid pFASTBacl, from which the recombinant pBacmid-oGPCR-Gila was successfully constructed with Bac-to-Bac baculovirus expression system indicated by specific transposition. The insect sf9 cells were transfected with pBacmid-oGPCRs-Gilα, and the supernatant containing recombinant virus were harvested.With the supernatant, insect sf9 cells were infected under an optimized condition (moi=5, infection time=72h) and the fusion proteins were prepared and detected by Western blotting.In the second part of our study, the possible role of GPR80 on the renal tubular epithelial-mesenchymal transdifferentiation (Epithelial-mesenchymal transdifferentiati-on, EMT) was investigated. HK-2 cells, a human proximal tubular epithelial cell line, were used to induce EMT by TGFβ-1. The optional conditions are 5ng/ml and 72 h for TGFβ-1 induction. By using the model, the effects of a-ketoglutarate sodium on EMT were examined. It was showed that a-ketoglutarate sodium at 100 or 500μM exerted a significant inhibition on EMT changes, indicated by the increase of E-cadherin mRNA expression (P<0.01), an epithelial marker, and the decrease of Vimentin expression (P<0.01), a mesenchymal marker. Meanwhile, the expression of GPR80 mRNA was also detected to be upregulated. These findings provided clues to further explore the physiological and pathological roles of GPR80.Conclusions:Gene fusions of three oGPCRs with Gilαand their protein expressions were both achieved sucessfully. The fusion proteins can be utilized in the high-throughput ligand screening for oGPCR. It was first time proven that GPR80 and its ligand might be involved in the occurrence and development of EMT, suggesting the receptor could serve as a potential target for the related deseases.