用于检测GPCR激活的可接近和可推广的体外发光测定。

IF 4.6 Q1 CHEMISTRY, ANALYTICAL
Ruby M. Miller, Jennifer Sescil, Marina C. Sarcinella, Ryan C. Bailey and Wenjing Wang*, 
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引用次数: 0

摘要

G蛋白偶联受体(GPCR)是最丰富的受体家族,具有重要的生理作用。在这里,我们描述了一种可推广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和构象特异性粘合剂之间的相互作用,在体外重建分裂的纳米萤光素酶(NanoLuc)。该工具,体外GPCR分裂NanoLuc配体触发报告器(IGNiTR),具有广泛的应用。我们已经证明了IGNiTR与三种Gs偶联的GPCR、两种Gi偶联的GPCr和三类构象特异性粘合剂的用途:纳米体、miniG蛋白和G蛋白肽模拟物。作为一种体外方法,IGNiTR能够使用合成的G蛋白肽模拟物,并为表征GPCR和配体提供易于扩展和便携式的试剂。我们测试了IGNiTR的三种不同应用:(1)使用多巴胺受体D1 IGNiTR进行概念验证GPCR配体筛选;(2) 检测用于护理点检测的阿片类药物;和(3)表征基于纳米盘的重构过程中的GPCR功能。由于IGNiTR的独特优势及其基于细胞裂解物的格式的便利性,该工具将在GPCR配体检测、筛选和GPCR表征中得到广泛应用。
本文章由计算机程序翻译,如有差异,请以英文原文为准。

Accessible and Generalizable in Vitro Luminescence Assay for Detecting GPCR Activation

Accessible and Generalizable in Vitro Luminescence Assay for Detecting GPCR Activation

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.

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来源期刊
ACS Measurement Science Au
ACS Measurement Science Au 化学计量学-
CiteScore
5.20
自引率
0.00%
发文量
0
期刊介绍: ACS Measurement Science Au is an open access journal that publishes experimental computational or theoretical research in all areas of chemical measurement science. Short letters comprehensive articles reviews and perspectives are welcome on topics that report on any phase of analytical operations including sampling measurement and data analysis. This includes:Chemical Reactions and SelectivityChemometrics and Data ProcessingElectrochemistryElemental and Molecular CharacterizationImagingInstrumentationMass SpectrometryMicroscale and Nanoscale systemsOmics (Genomics Proteomics Metabonomics Metabolomics and Bioinformatics)Sensors and Sensing (Biosensors Chemical Sensors Gas Sensors Intracellular Sensors Single-Molecule Sensors Cell Chips Arrays Microfluidic Devices)SeparationsSpectroscopySurface analysisPapers dealing with established methods need to offer a significantly improved original application of the method.
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