title: Using constitutive activity to define appropriate high-throughput screening assays for orphan g protein-coupled receptors.
abstract: Orphan G protein-coupled receptors represent an underexploited resource for drug discovery but pose a considerable challenge for assay development because their cognate G protein signaling pathways are often unknown. In this methodological chapter, we describe the use of constitutive activity, that is, the inherent ability of receptors to couple to their cognate G proteins in the absence of ligand, to inform the development of high-throughput screening assays for a particular orphan receptor. We specifically focus on a two-step process, whereby constitutive G protein coupling is first determined using yeast Gpa1/human G protein chimeras linked to growth and β-galactosidase generation. Coupling selectivity is then confirmed in mammalian cells expressing endogenous G proteins and driving accumulation of transcription factor-fused luciferase reporters specific to each of the classes of G protein. Based on these findings, high-throughput screening campaigns can be performed on the already miniaturized mammalian reporter system.
authors.affiliation: Molecular Cardiology Division, Victor Chang Cardiac Research Institute, Lowy Packer Building, 405 Liverpool St, Darlinghurst, Sydney, NSW, 2010, Australia.
authors.forename: Tony
authors.initials: T
authors.lastname: Ngo
authors.forename: James L J
authors.initials: JL
authors.lastname: Coleman
authors.forename: Nicola J
authors.initials: NJ
authors.lastname: Smith
mesh_terms: D000818:Animals; D002453:Cell Cycle / Q000187:drug effects / Q000235:genetics; D005576:Colforsin / Q000494:pharmacology; D050756:Cyclin-Dependent Kinase Inhibitor Proteins / Q000235:genetics / Q000378:metabolism; D043802:GTP-Binding Protein alpha Subunits, Gq-G11 / Q000235:genetics / Q000378:metabolism; D015966:Gene Expression Regulation, Fungal; D017930:Genes, Reporter; D057809:HEK293 Cells; D057166:High-Throughput Screening Assays; D006801:Humans; D015759:Ionomycin / Q000494:pharmacology; D008024:Ligands; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism; D043562:Receptors, G-Protein-Coupled / Q000819:agonists / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014162:Transfection; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
chemical_list: D050756:Cyclin-Dependent Kinase Inhibitor Proteins; C081353:FAR1 protein, S cerevisiae; D008024:Ligands; D043562:Receptors, G-Protein-Coupled; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D005576:Colforsin; D015759:Ionomycin; D008156:Luciferases; D001616:beta-Galactosidase; C084293:GPA1 protein, S cerevisiae; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D013755:Tetradecanoylphorbol Acetate