Elsevier

Analytical Biochemistry

Volume 400, Issue 2, 15 May 2010, Pages 163-172
Analytical Biochemistry

A reporter assay for G-protein-coupled receptors using a B-cell line suitable for stable episomal expression

https://doi.org/10.1016/j.ab.2010.01.036Get rights and content

Abstract

We have established a cAMP response element (CRE)-mediated reporter assay system for G-protein-coupled receptors (GPCRs) using an oriP-based estrogen-inducible expression vector and the B-cell line (GBC53 or GBCC71) that expresses EBNA-1 and is adapted to serum-free culture. GBC53 harbors a GAL4–ER expression unit and a CRE–luciferase gene in the genome, and GBCC71 also harbors expression units for two chimeric Gαs proteins (Gs/q and Gs/i). Introduction of a GPCR expression plasmid into GBC53 or GBCC71 creates polyclonal stable transformants in 2 weeks, and these are easily expanded and used for assays after induction of the GPCR expression. Using GBC53, we detected ligand-dependent signals of Gs-coupled GPCRs such as glucagon-like peptide 1 receptor (GLP1R) and β2 adrenergic receptor (β2AR) with high sensitivity. Interestingly, we also detected constitutive activity of β2AR. Using GBCC71, we detected ligand-dependent signals of Gq- or Gi-coupled GPCRs such as H1 histamine receptor and CXCR1 chemokine receptor in addition to Gs-coupled GPCRs. An agonist, antagonist, or inverse agonist was successfully evaluated in this system. We succeeded in constructing a 384-well high-throughput screening (HTS) system for GLP1R. This system enabled us to easily and rapidly make a large number of efficient GPCR assay systems suitable for HTS as well as ligand hunting of orphan GPCRs.

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.

References (38)

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