Accessible and Generalizable in Vitro Luminescence Assay for Detecting GPCR Activation.
Accessible and Generalizable in Vitro Luminescence Assay for Detecting GPCR Activation.
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DOI：
10.1021/acsmeasuresciau.3c00021
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发表时间：
2023-10-18
期刊：
影响因子：
--
通讯作者：
Wang, Wenjing
中科院分区：
文献类型：
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作者：
Miller, Ruby M;Sescil, Jennifer;Sarcinella, Marina C;Bailey, Ryan C;Wang, Wenjing
G蛋白偶联受体(GPCRs)是最丰富的受体家族，发挥着重要的生理作用。在这里，我们描述了一种通用的基于细胞裂解物的方法的设计，该方法利用激动剂激活的GPCR和构象特异结合物之间的相互作用来在体外重建分裂的纳米荧光素酶(NanoLuc)。这一工具，体外GPCR分裂纳米Luc配体触发报告(Ignitr)，具有广泛的应用。我们已经演示了Ignitr与三个Gs偶联的GPCRs、两个GI偶联的GPCRs和三类构象特定结合物的使用：纳米体、mini G蛋白和G蛋白肽仿制。作为一种体外方法，Ignitr能够使用合成的G蛋白多肽模拟物，并提供易于扩展和便携的试剂来表征GPCRs和配体。我们测试了Ignitr的三种不同应用：(1)使用多巴胺受体D1Ignitr进行概念验证GPCR配体筛选；(2)检测阿片类药物用于护理点测试；以及(3)在基于纳米盘的重建过程中表征GPCR功能。由于Ignitr的独特优势及其基于细胞裂解物的形式的方便性，该工具将在GPCR配体检测、筛选和GPCR鉴定中得到广泛应用。
G protein-coupled receptors (GPCRs) serve critical physiological roles as the most abundant family of receptors. Here, we describe the design of a generalizable and cell lysate-based method that leverages the interaction between an agonist-activated GPCR and a conformation-specific binder to reconstitute split nanoluciferase (NanoLuc) in vitro. This tool, In vitro GPCR split NanoLuc ligand Triggered Reporter (IGNiTR), has broad applications. We have demonstrated IGNiTR’s use with three Gs-coupled GPCRs, two Gi-coupled GPCRs and three classes of conformation-specific binders: nanobodies, miniG proteins, and G protein peptidomimetics. As an in vitro method, IGNiTR enables the use of synthetic G protein peptidomimetics and provides easily scalable and portable reagents for characterizing GPCRs and ligands. We tested three diverse applications of IGNiTR: (1) proof-of-concept GPCR ligand screening using dopamine receptor D1 IGNiTR; (2) detection of opioids for point-of-care testing; and (3) characterizing GPCR functionality during Nanodisc-based reconstitution processes. Due to IGNiTR’s unique advantages and the convenience of its cell lysate-based format, this tool will find extensive applications in GPCR ligand detection, screening, and GPCR characterization.