Functional characterization of cell lines for high-throughput screening of human neuromedin U receptor subtype 2 specific agonists using a luciferase reporter gene assay.
We developed a functional cell-based high-throughput screening (HTS) assay to identify modulators of the human neuromedin subtype 2 receptor. This assay utilized the signal transduction pathway of hNMU2R, which is positively coupled to adenylyl cyclase and downstream calcium signal pathways. We describe in detail a robust, sensitive, and functional assay for the hNMU2R G-protein-coupled receptor expressed in human embryonic kidney (HEK)-293 cells, whose activity was reflected by a luciferase reporter gene transcriptionally regulated by a 3-repeat serum response element (SRE)-3 repeat multiple response element (MRE)-3 repeat cyclic AMP (cAMP) response element (CRE)-VIP mini promoter. The HEK 293 clonal cell line, stably co-transfected with the 3xSRE/3xMRE/3xCRE/VIP mini promoter-driven luciferase and pCDNA3.1-NMU2R plasmid, was selected by active geneticin sulfate and their ability to express luciferase with a forskolin challenge following hNMU plus forskolin, known to activate intracellular signal transduction. Then the cell density, incubation time, dimethyl sulfoxide (DMSO) concentration used to screen the hNMU receptor subtype 2 specific agonist were optimized, and whether intrinsic luminescent substance of extracts isolated from traditional Chinese herbs disturbs luminescence of luciferase expressed in HEK293 cells was considered. The optimal incubation time was found to be between 8 and 9h, the cell density and DMSO concentrations were optimized from 3x10(4) to 6x10(4), and less than 2%, respectively. Our data show that hNMU2R luci-HEK293 cells and their assay exhibit a low background and ideal model for high-throughput screening. These results demonstrate that this reporter gene assay is useful for pharmacological analysis, and is amenable to HTS for human NMU2R agonists.
Li
Xinping
X
Department of Biosciences, SOOCHOW University, Suzhou, China. lxp67cqu@163.com
Shen
Fuwen
F
Zhang
Yuwu
Y
Zhu
Jiang
J
Huang
Lu
L
Shi
Qunfang
Q
D000818:Animals; D016466:CHO Cells; D002460:Cell Line; D005576:Colforsin / Q000494:pharmacology; D006224:Cricetinae; D003412:Cricetulus; D003627:Data Interpretation, Statistical; D004121:Dimethyl Sulfoxide; D004353:Drug Evaluation, Preclinical; D004365:Drugs, Chinese Herbal / Q000494:pharmacology; D017930:Genes, Reporter / Q000235:genetics*; D006801:Humans; D007202:Indicators and Reagents; D008156:Luciferases / Q000235:genetics*; D049449:Luminescence; D008565:Membrane Proteins / Q000819:agonists*; D010957:Plasmids; D011401:Promoter Regions, Genetic / Q000235:genetics; D017981:Receptors, Neurotransmitter / Q000819:agonists*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D015203:Reproducibility of Results
D004365:Drugs, Chinese Herbal; D007202:Indicators and Reagents; D008565:Membrane Proteins; D017981:Receptors, Neurotransmitter; D011994:Recombinant Proteins; C082444:neuromedin U receptor; D005576:Colforsin; D008156:Luciferases; D004121:Dimethyl Sulfoxide