Chapter title |
Using constitutive activity to define appropriate high-throughput screening assays for orphan g protein-coupled receptors.
|
---|---|
Chapter number | 7 |
Book title |
G Protein-Coupled Receptor Screening Assays
|
Published in |
Methods in molecular biology, December 2014
|
DOI | 10.1007/978-1-4939-2336-6_7 |
Pubmed ID | |
Book ISBNs |
978-1-4939-2335-9, 978-1-4939-2336-6
|
Authors |
Tony Ngo, James L J Coleman, Nicola J Smith, Ngo T, Coleman JL, Smith NJ, Ngo, Tony, Coleman, James L. J., Smith, Nicola J., James L. J. Coleman, Nicola J. Smith |
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. |
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