A reporter gene assay for high-throughput screening of G-protein-coupled receptors stably or transiently expressed in HEK293 EBNA cells grown in suspension culture

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DOIResolve DOI: http://doi.org/10.1006/abio.2000.4698
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TypeArticle
Journal titleAnalytical Biochemistry
ISSN0003-2697
Volume284
Issue2
Pages316326; # of pages: 11
SubjectProstanoid receptors; cyclic AMP response element; secreted alkaline phosphatase; gene transcription; antagonists; base sequence; cell culture techniques; cell line; DNA; Epstein-Barr virus nuclear antigens; genes, reporter; GTP-binding proteins; humans; molecular sequence data; plasmids; receptors; cell surface
AbstractWe describe in detail a robust, sensitive, and versatile functional assay for G-protein-coupled receptors (GPCRs) expressed in human embryonic kidney (HEK) 293-EBNA (Epstein-Barr virus nuclear antigen) (designated 293E) cells. The ability to grow these cells in suspension, in conjunction with the use of the secreted form of the human placental alkaline phosphatase (SEAP) as the reporter enzyme transcriptionally regulated by 5-cyclic AMP (cAMP) response elements (CREs) (Chen et al., Anal. Biochem. 226, 349-354 (1995)), makes this CRE-SEAP assay potentially attractive for high-throughput screening (HTS). A 293E clonal cell line, stably transfected with the CRE-SEAP plasmid, was initially characterized with compounds known to activate intracellular signal transduction pathways similar to those activated by GPCRs. Forskolin and cAMP analogues were potent at inducing SEAP expression but calcium ionophores (A23187 and ionomycin) were without effect. The forskolin response was also potentiated by the protein kinase C activator phorbol myristate acetate as well as the phosphodiesterase inhibitor isobutylmethylxanthine. Previously established cell lines expressing the G(alphas)-coupled DP or the G(alphaq)-coupled-EP₁ prostanoid receptors were stably transfected with the reporter gene construct and clones were selected based on their ability to secrete SEAP upon agonist challenge. Pharmacological characterization of the DP and EP₁ receptors displayed a similar rank order of potency for several known prostanoids and related compounds to that previously reported using classical binding assays or other functional assays. The CRE-SEAP assay was also used to characterize the EP₁ receptor antagonists SC-51322, SC-51089, and AH6809. In summary, we have established a reporter gene assay for GPCRs that couple to both G(alphas) and G(alphaq) and is amenable to HTS of both agonists and antagonists.
Publication date
LanguageEnglish
AffiliationNational Research Council Canada; NRC Biotechnology Research Institute
Peer reviewedNo
Identifier10269630
NRC number37684
NPARC number12328481
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Record identifier9ff98f61-f02d-47b4-9532-dba8a746f12c
Record created2009-09-10
Record modified2016-06-01
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