Department of Pharmacology, University of North Carolina School of Medicine, Chapel Hill, North Carolina, USA.
Nat Protoc. 2010 Mar;5(3):561-73. doi: 10.1038/nprot.2009.239. Epub 2010 Feb 25.
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.
G 蛋白偶联受体（GPCRs）及其下游信号级联反应参与了大多数生理过程和多种人类疾病。在体内实验环境中分离 GPCR 激活的影响具有挑战性，因为外源性配体具有脱靶效应，而内源性配体则不断调节天然受体的活性。高度特异性的设计药物-设计受体复合物是阐明在特定细胞类型中激活特定受体和信号通路的影响的有价值的工具。在这项研究中，我们描述了一种通用的方案，用于定向进化专门由设计药物（DREADD）激活的设计受体。首先，通过易错 PCR 生成突变文库。第三，通过依赖药物的酵母生长测定筛选文库。表现出所需特性的突变体被选择用于进一步的诱变或在哺乳动物系统中进行表征。总的来说，这些步骤应该需要 6-8 周的实验，并且应该导致受体被所选配体激活。本方案应有助于提高选择细胞群体的实验靶向性。
