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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Ngo, T., Coleman, J.L.J., Smith, N.J. (2015). Using Constitutive Activity to Define Appropriate High-Throughput Screening Assays for Orphan G Protein-Coupled Receptors. In: Prazeres, D.M.F., Martins, S.A.M. (eds) G Protein-Coupled Receptor Screening Assays. Methods in Molecular Biology, vol 1272. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-2336-6_7
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DOI: https://doi.org/10.1007/978-1-4939-2336-6_7
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Online ISBN: 978-1-4939-2336-6
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