A fluorescent reporter assay for the detection of ligands acting through G(1) protein-coupled receptors

摘要

Accompanying the advances in basic biology of G protein-coupled receptors (GPCRs) is the practical need among biopharmaceutical companies for sensitive assays to assess GPCR function, particularly formats that are compatible with high-throughput drug screening. Here we describe a novel cell-based assay format for the high-throughput detection of ligands for G(i) protein-coupled receptors. Two G(i)-GPCRs, mu -opioid receptor (mu -OPR) and 5-hydroxytryptamine receptor 1a (5HT1aR) are employed as model receptor targets. The key feature of this assay system is the isolation of stable, clonal Chinese hamster ovary (CHO) cell lines that carry three separate expression plasmids: (1) a chimeric G(q/i)5 protein (which re-directs a negative G(i)-type signal to a positive Gq-type response), (2) a given G(i)-GPCR, and (3) a beta -lactamase (beta la) reporter gene responsive to G(i)-GPCR signaling. Cell-based assays built using this format show appropriate rank order of potency among a reference set of receptor agonist and antagonist compounds. Such assays are also robust, reliable, and can be used for industrial-scale applications such as high-throughput screening for drug leads. [References: 26]