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.
We 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-EP(1) 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 EP(1) 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 EP(1) 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.
Durocher
Y
Y
Bioprocess Sector, Biotechnology Research Institute, 6100 Royalmount Avenue, Montreal, Quebec, H4P 2R2, Canada. yves.durocher@nrc.ca
Perret
S
S
Thibaudeau
E
E
Gaumond
M H
MH
Kamen
A
A
Stocco
R
R
Abramovitz
M
M
D001483:Base Sequence; D018929:Cell Culture Techniques; D002460:Cell Line; D004247:DNA; D019309:Epstein-Barr Virus Nuclear Antigens; D019204:GTP-Binding Proteins / Q000378:metabolism*; D017930:Genes, Reporter; D006801:Humans; D008969:Molecular Sequence Data; D010957:Plasmids; D011956:Receptors, Cell Surface / Q000235:genetics / Q000378:metabolism*
D019309:Epstein-Barr Virus Nuclear Antigens; D011956:Receptors, Cell Surface; D004247:DNA; D019204:GTP-Binding Proteins