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 f or 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 us e of the secreted form of the human placental alkaline phosphatase (SEAP) a s the reporter enzyme transcriptionally regulated by 5-cyclic AMP (cAMP) re sponse elements (CREs) (Chen et al., Anal. Biochem. 226, 349-354 (1995)), m akes this CRE-SEAP assay potentially attractive for high-throughput screeni ng (HTS). A 293E clonal cell line, stably transfected with the CRE-SEAP pla smid, was initially characterized with compounds known to activate intracel lular signal transduction pathways similar to those activated by GPCRs. For skolin and cAMP analogues were potent at inducing SEAP expression but calci um ionophores (A23187 and ionomycin) were without effect. The forskolin res ponse was also potentiated by the protein kinase C activator phorbol myrist ate acetate as well as the phosphodiesterase inhibitor isobutylmethylxanthi ne. Previously established cell lines expressing the G(alpha s)-coupled DP or the G(alpha q)-coupled-EP1 prostanoid receptors were stably transfected with the reporter gene construct and clones were selected based on their ab ility to secrete SEAP upon agonist challenge. Pharmacological characterizat ion of the DP and EP1 receptors displayed a similar rank order of potency f or several known prostanoids and related compounds to that previously repor ted 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 assa y for GPCRs that couple to both G(alpha s) and G(alpha q) and is amenable t o HTS of both agonists and antagonists. (C) 2000 Academic Press.