Abstract
G protein–coupled receptors (GPCRs) and their downstream signaling cascades contribute to most physiological processes and a variety of human diseases. Isolating the effects of GPCR activation in an in vivo experimental setting is challenging as exogenous ligands have off-target effects and endogenous ligands constantly modulate the activity of native receptors. Highly specific designer drug–designer receptor complexes are a valuable tool for elucidating the effects of activating particular receptors and signaling pathways within selected cell types in vivo. In this study, we describe a generic protocol for the directed molecular evolution of designer receptors exclusively activated by designer drugs (DREADDs). First, the yeast system is validated with the template receptor. Second, a mutant library is generated by error-prone PCR. Third, the library is screened by drug-dependent yeast growth assays. Mutants exhibiting the desired properties are selected for further rounds of mutagenesis or for characterization in mammalian systems. In total, these steps should take 6–8 weeks of experimentation and should result in the evolution of a receptor to be activated by the chosen ligand. This protocol should help improve the experimental targeting of select cell populations.
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Acknowledgements
We thank Blaine N. Armbruster for his work developing this technique. This study was supported by a NARSAD Distinguished Investigator Award and by the following grants from the National Institutes of Health: GM07040 and GM008719 from the National Institute of General Medical Sciences (S.C.R.); and MH087074 (S.C.R.), MH082441 and MH061887 (B.L.R.) from the National Institute of Mental Health.
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S.D., S.C.R. and B.L.R. prepared the paper.
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Dong, S., Rogan, S. & Roth, B. Directed molecular evolution of DREADDs: a generic approach to creating next-generation RASSLs. Nat Protoc 5, 561–573 (2010). https://doi.org/10.1038/nprot.2009.239
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DOI: https://doi.org/10.1038/nprot.2009.239
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