Department of Pharmacology, Vanderbilt University, Nashville, Tennessee.
Departments of Psychiatry and Molecular Pharmacology and Therapeutics, Columbia University Vagelos College of Physicians and Surgeons, New York, New York.
Curr Protoc. 2023 Oct;3(10):e890. doi: 10.1002/cpz1.890.
G protein-coupled receptors (GPCRs) represent ∼30% of current drug targets. Ligand binding to these receptors activates G proteins and arrestins, which function in different signaling pathways. Given that functionally selective or biased ligands preferentially activate one of these two groups of pathways, they may be superior medications for certain disease states. The identification of such ligands requires robust drug screening assays for both G protein and arrestin activity. This unit describes protocols for assays that monitor reversible arrestin recruitment to GPCRs in living cells using either bioluminescence resonance energy transfer (BRET) or nanoluciferase complementation (NanoLuc). Two types of assays can be used: one configuration directly measures arrestin recruitment to a GPCR fused to a protein tag at its intracellular C-terminus, whereas the other configuration detects arrestin translocation to the plasma membrane in response to activation of an unmodified GPCR. Together, these assays are powerful tools for studying dynamic interactions between GPCRs and arrestins. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Receptor-arrestin BRET assay to measure ligand-induced recruitment of arrestin to receptors Basic Protocol 2: Receptor-arrestin NANOBIT assay to measure ligand-induced recruitment of arrestin to receptors Alternative Protocol 1: BRET assay to measure ligand-induced recruitment of arrestin to the plasma membrane Alternative Protocol 2: NANOBIT assay to measure ligand-induced recruitment of arrestin to the plasma membrane Support Protocol 1: Optimization of polyethylenimine (PEI) concentration for transfection.
G 蛋白偶联受体（GPCRs）约占当前药物靶点的 30%。配体与这些受体结合会激活 G 蛋白和 arrestin，它们在不同的信号通路中发挥作用。鉴于功能选择性或偏向性配体优先激活这两组途径中的一种，它们可能是某些疾病状态的更好药物。这些配体的鉴定需要针对 G 蛋白和 arrestin 活性的强大药物筛选测定法。本单元描述了使用生物发光共振能量转移（BRET）或纳米荧光素酶互补（NanoLuc）在活细胞中监测可逆 arrestin 募集到 GPCR 的测定法的协议。可以使用两种类型的测定法：一种构型直接测量与在其细胞内 C 末端融合有蛋白标签的 GPCR 募集的 arrestin，而另一种构型检测在未修饰的 GPCR 激活时 arrestin 向质膜的易位。这些测定法共同成为研究 GPCR 和 arrestin 之间动态相互作用的有力工具。© 2023 威立公司。基本方案 1：受体- arrestin BRET 测定法，用于测量配体诱导的 arrestin 募集到受体基本方案 2：受体- arrestin NANOBIT 测定法，用于测量配体诱导的 arrestin 募集到受体替代方案 1：BRET 测定法，用于测量配体诱导的 arrestin 募集到质膜替代方案 2：NANOBIT 测定法，用于测量配体诱导的 arrestin 募集到质膜支持方案 1：转染用聚乙烯亚胺（PEI）浓度的优化。
