Development of a Generic Dual-Reporter Gene Assay for Screening G-Protein-Coupled Receptors

¹ Research School of Biosciences, University of Kent, Canterbury, Kent, UK; Present address: DI&A Functional Genomics, Aventis Pharma Deutschland GmbH, Frankfurt am Main, Germany
² Akubio Ltd., Cambridge, UK
³ Research School of Biosciences, University of Kent, Canterbury, Kent, UK

^* To whom correspondence should be addressed. E-mail: L.H.Naylor{at}kent.ac.uk.

	Abstract

Multiple assay formats have been developed for the pharmacological characterization of G-protein-coupled receptors (GPCRs) and for screening orphan receptors. However, the increased pace of target identification and the rapid expansion of compound libraries present the need to develop novel assay formats capable of screening multiple GPCRs simultaneously. To address this need, the authors have developed a generic dual-reporter gene assay that can detect ligand activity at 2 GPCRs within the same assay. Two stable HEK293 cell lines were generated expressing either a firefly (Photinus) luciferase gene under the control of multiple cAMP-response elements (CREs) or a Renilla luciferase gene under the control of multiple 12-O-tetradecanoyl-phorbol-13-acetate (TPA)--responsive elements (TREs). Coseeded reporter cells were used to assess ligand-binding activity at both G_s- and G_q-coupled receptors. By selectively coexpressing receptors with a chimeric G-protein, agonist activity was assessed at G_i/o-coupled receptors in combination with either G_s- or G_q-coupled receptors. The dual-reporter gene assay was shown to be capable of simultaneously performing duplexed screens for a variety of agonist and/or antagonist combinations. The data generated from the duplexed reporter assays were pharmacologically relevant, and Z' factor analysis indicated the suitability of both agonist and antagonist screens for use in high-throughput screening.

Key Words: drug discovery, G-protein-coupled receptor, intracellular signaling, reporter gene, high-throughput screening, multiplexed

First published on June 24, 2005, doi:10.1177/1087057105275033

Journal of Biomolecular Screening 2005;10:437.

A more recent version of this article appeared on August 1, 2005


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