Detetion Of Environmental Estrogen And Screening Of Natural Products Base On Fluorescent Labeled GPCR In Recombinant Cell Lines

G protein coupled receptors（GPCRs）are one kind of the most important receptors in cytoplasmic membrane surface,and have been demonstrated that involved in several metabolic and signal transduction pathways.They also have been proved to be the targets of some hormones and drugs for a long time.How to take the advantages of GPCRs to develop high sensitivity,specificity and stability platforms in detection or screening natural products is an urgent challenge.In order to develop new platforms to detect or screening active components in natural samples,four（Green Fluorescent Protein）GFP labeled recombinant cell lines based on High Content Screening（HCS）technology were developed,in which the GFP was fused at the C terminus of receptors to reduce affecting interaction between receptor and ligand.Firstly,a strategy which the ER?is directly labeled with GFP at C termius was used in this study.A shuttle plasmid of pCMV6-GFP-ERαthat include CMV promoter and SV40 ori for eukaryotic expression was constructed,then the plasmid was transfected into U2OS cells and a stable cell lines was established after directional selection.The recombinant cells can react with ligand（Estrogen 2）of ER?and form fluorescent aggregation vesicle in cytoplasm,the fluorescent intensity of visicles can be detected and analyzed with HCS technology.The cell lines showed high specificity toward E2 and TAM with 0.1 nM of EC₅₀ and 1.5 nM of IC₅₀.The lowest of detection limit was determined to be 1x10^-3 nM.This cell line can specifically detect E2 or its analogs in a artificial mixture sample,which implied that this cell line can be used in detection of E2 in environmental samples in future.Secondly,to facilitate the screening of active components from natural products and avoid potential activity interference of the N-terminus modification,a recombinant cell line hGLP-1R-tGFP containing C-terminus tGFP-labeled human glucagon like peptide 1（hGLP-1）receptor has been constructed.Upon the activation of GLP-1 receptors by Exendin 4,fluorescent pits appeared on cell membrane and subsequently internalized to form fluorescent aggregation vesicles inside the cells under fluorescent microscopy examination.The fluorescence imaging of re-distribution from diffusing to aggregate vesicles inside the cells was quantitated and analyzed by HCSassay.The agonistic activity,sensitivity and specificity of the formation of fluorescent aggregate vesicles in hGLP-1R-tGFP cells have been confirmed by the activation of GLP-1R using the GLP-1analogues.The agonistic effects of GLP-1 analogues are blocked by a GLP-1R antagonist of Exendin9-39.The downstream of GLP-1 pathway,the activation of adenylate cyclase and the raising of cellular cAMP levels,remained intact in this tGFP modified C-terminus GLP-1 receptor cells.Combined with High Content Screening image and data automatic acquisition processing,a new screening assay method has been developed.A total of 100 crude extract samples from Chinese herbs have been screened by this method,and the results showed GLP-1R agonist activity in extracts of Astragalus propinquus and Panax notoginseng.Several potential GLP-1R modulator and inhibitors also have been identified in this study.In the end,in order to illuminate the pathway involved in purinergic P2Y1 receptor（P2Y1R）.Two stable cell lines,hP2Y1R-tGFP/U2OS and hP2Y1R-?-AR2-tGFP/U2OSwere constructed with steady expression of fluorescence tGFP tagged P2Y1R orβ-arrestin2.The High Content Screening（HCS）technique was used to investigate P2Y1R mediated signal transduction processes based on the distribution of fluorescent receptors orβ-arrestin2/receptor complex.ADP activated P2Y1R to induce distinguished pattern of fluorescent vesicles formation inside both cell types.Those two cell lines showed different peak fluorescence signal when activated by ADP at 100?M,and the time to form the maximal vesicles were detected as 15 and20 min respectively.The EC₅₀ values of the concentration-response curves of ADP induced formation of fluorescent vesicles were 35.72 nM and 71.8 nM respectively.The ADP effects could be blocked by a P2Y1R specific antagonist of MRS2179.The P2Y1R are coupled to Gq protein to activate PKC pathway which in turn activate phosphatidylcholine specific PLC,and finally the phospholipase D initiatesβ-arrestin2 mediated signal pathway.It took 30 and 45min to complete P2Y1R re-sensitization in these two constructs respectively.Our results demonstrated that P2Y1R not only via heterotrimeric Gq proteins but also throughβ-arrestin2triggered downstream responses.A automatic and visualized detection or screenig platform in detection or screening activities in natural samples has been established,which was based on GFP labeled GPCRs at C terminus combined with HSC technology.This recombinant cell lines can recognize the target ligands with high specificity.This method has presented several overwhelming advantages over traditional detection/screenig methods,such as target diversity,high stability and specificity,processes visualization,automatic operation and easy of modification for applications.It has been thought to be used in detection of environmental estrogen and screening active ingredients from natural products for treating diabetes and cardiovascular diseases.