Establishment And Application Of A Detection Model For G Protein-coupled Receptor Signaling Pathways And Drug Screening For Orphan GPCRs

GPCRs regulate the downstream signaling molecules mainly through activating G proteins and then involve in certain physiological and pathological reactions.β-arrestin as an important adaptor protein in the cells, play important roles in signal transduction, such as GPCRs desensitization, internalization, cell proliferation and gene transcription. So rapid quantitative methods for elucidating GPCRs andβ-arrestin1/2 interactions have become necessary. In this research, we describe a technique for monitoring AGTR1(β2AR, GCGR) and β-arrestin1/2 interactions in living cells based on complementation of split luciferase fragments from click beetle.We transfected plasmids which encoding AGTR1/β2AR/GCGR-ELucC or ELucN-β-arrestin1/2 into HEK293 cells. Their expression in HEK293 cells were detected by immunofluorescence staining and western blot. Calcium mobilization assay was conducted to examine the activity of AGTR1(β2AR, GCGR). Then co-transfected AGTR1/β2AR/GCGR-ELucC and ELucN-β-arrestin1/2 into HEK293 cells. After the optimization of cell transfection conditions as well as the time of compounds stimulated, we detected the luciferase activity under the ligands stimulation. And also we use the model to test the interactions between AGTR1/β2AR and β-arrestin1/2 when stimulated by biased-agonist. In our research, we found the significant increase of interactions between AGTR1 /β2AR and β-arrestin1/2 when stimulated by agonist, and the results of biased-agonit is consistent with we all known.Above all the results demonstrate that we had successfully established a detection model for AGTR1 /β2AR and β-arrestin1/2 interactions. We can further research on the GPCRs signal transduction and their biased-agonist.In addition, we using the split Emerald luciferase-fragment complementation assay established a detection model for β2AR and G protein interactions. Then we studied the interactions of β2AR and Gα16. In our research, we found there isn’t significant changes of β2AR-Gα16 or β2AR-Gαs interactions when β2AR is activated.In the second part, we established a high throughput screening model for GPR85 and GPR98 by cAMP assay. We used about 10,000 compounds to carry out large-scale random screening. Then we found 8 compounds need to further research.