Methods in molecular biology (Clifton, N.J.), 2015, Vol.1272, p.91
2015
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- Autor(en) / Beteiligte
- Titel
- Using constitutive activity to define appropriate high-throughput screening assays for orphan g protein-coupled receptors
- Ist Teil von
- Methods in molecular biology (Clifton, N.J.), 2015, Vol.1272, p.91
- Ort / Verlag
- United States
- Erscheinungsjahr
- 2015
- Quelle
- MEDLINE
- Beschreibungen/Notizen
- 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.
- Sprache
- Englisch
- Identifikatoren
- eISSN: 1940-6029DOI: 10.1007/978-1-4939-2336-6_7Titel-ID: cdi_pubmed_primary_25563179
- Format
- –
- Schlagworte
- Animals, beta-Galactosidase - genetics, beta-Galactosidase - metabolism, Cell Cycle - drug effects, Cell Cycle - genetics, Colforsin - pharmacology, Cyclin-Dependent Kinase Inhibitor Proteins - genetics, Cyclin-Dependent Kinase Inhibitor Proteins - metabolism, Gene Expression Regulation, Fungal, Genes, Reporter, GTP-Binding Protein alpha Subunits, Gq-G11 - genetics, GTP-Binding Protein alpha Subunits, Gq-G11 - metabolism, HEK293 Cells, High-Throughput Screening Assays, Humans, Ionomycin - pharmacology, Ligands, Luciferases - genetics, Luciferases - metabolism, Plasmids - chemistry, Plasmids - metabolism, Receptors, G-Protein-Coupled - agonists, Receptors, G-Protein-Coupled - antagonists & inhibitors, Receptors, G-Protein-Coupled - genetics, Receptors, G-Protein-Coupled - metabolism, Recombinant Fusion Proteins - genetics, Recombinant Fusion Proteins - metabolism, Saccharomyces cerevisiae - drug effects, Saccharomyces cerevisiae - genetics, Saccharomyces cerevisiae - metabolism, Saccharomyces cerevisiae Proteins - genetics, Saccharomyces cerevisiae Proteins - metabolism, Tetradecanoylphorbol Acetate - pharmacology, Transfection