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.
CITATION STYLE
Ngo, T., Coleman, J. L. J., & Smith, N. J. (2015). Using constitutive activity to defi ne appropriate high-throughput screening assays for orphan G protein-coupled receptors. Methods in Molecular Biology, 1272, 91–106. https://doi.org/10.1007/978-1-4939-2336-6_7
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