A reporter assay for G-protein-coupled receptors using a B-cell line suitable for stable episomal expression
Section snippets
Reagents
β-Estradiol, glucagon-like peptide 1 (GLP-1) (7-36) amide, isoprenaline, propranolol, histamine, pyrilamine, forskolin, and A23187 were obtained from Sigma–Aldrich (St. Louis, MO). Recombinant human interleukin 8 (IL-8) was obtained from Bachem AG (Bubendorf, Switzerland). Dibutyryl-cAMP (db-cAMP) and phorbol myristate acetate (PMA) were purchased from Wako Pure Chemical Industries (Osaka, Japan). FuGENE 6 Transfection Reagent was purchased from Roche Diagnostics KK (Tokyo, Japan). Blasticidin
Strategy to make a simple and efficient functional GPCR assay system
Our assay system is outlined in Fig. 4. Based on the host–vector system using Namalwa KJM-1 and the oriP-based expression vector, we generated a CRE-mediated reporter assay system. We constructed host cells (GBC53 and GBCC71) suitable for inducible expression and CRE reporter assay from Namalwa KJM-1. GBC53 is applicable to reporter assays of Gs-coupled GPCRs, and GBCC71 is applicable to reporter assays of Gs-, Gq-, and Gi-coupled GPCRs. As estrogen-inducible expression vectors containing oriP,
Discussion
We have established a simple and efficient CRE reporter assay system for GPCRs using an inducible expression plasmid and a human B-cell line suitable for serum-free suspension culture as a host (Fig. 4). As host cells, we constructed GBC53 cells for Gs-coupled GPCRs and constructed GBCC71 cells for Gs-, Gq-, and Gi-coupled GPCRs. Because the host cells express the EBNA-1 gene and the expression plasmid contains oriP, transfection of the plasmid into the host cells yields a large number of
Acknowledgment
We thank Kikuko Funayama, Eri Suzuki, and Megumiko Magara for excellent technical assistance.
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