title: A fluorescent reporter assay for the detection of ligands acting through Gi protein-coupled receptors.
abstract: 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 Gi protein-coupled receptors. Two Gi-GPCRs, mu-opioid receptor (mu-OPR) and 5-hydroxytryptamine receptor la (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 Gq/i5 protein (which re-directs a negative Gi-type signal to a positive Gq-type response), (2) a given Gi-GPCR, and (3) a beta-lactamase (beta1a) reporter gene responsive to Gi-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.
authors.affiliation: Aurora Biosciences Corporation, San Diego, CA 92121, USA.
authors.forename: H
authors.initials: H
authors.lastname: Xing
authors.forename: H C
authors.initials: HC
authors.lastname: Tran
authors.forename: T E
authors.initials: TE
authors.lastname: Knapp
authors.forename: P A
authors.initials: PA
authors.lastname: Negulescu
authors.forename: B A
authors.initials: BA
authors.lastname: Pollok
mesh_terms: D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D006224:Cricetinae; D017931:DNA Primers / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics; D005456:Fluorescent Dyes; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000235:genetics / Q000378:metabolism*; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D017930:Genes, Reporter; D020962:Heterotrimeric GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism; D008024:Ligands; D050778:NFATC Transcription Factors; D009687:Nuclear Proteins; D011956:Receptors, Cell Surface / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D014157:Transcription Factors / Q000235:genetics; D014162:Transfection; D001618:beta-Lactamases / Q000235:genetics
chemical_list: D017931:DNA Primers; D004268:DNA-Binding Proteins; D005456:Fluorescent Dyes; D008024:Ligands; D050778:NFATC Transcription Factors; D009687:Nuclear Proteins; D011956:Receptors, Cell Surface; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D001618:beta-Lactamases; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D020962:Heterotrimeric GTP-Binding Proteins