Departments of Psychiatry & Molecular Pharmacology and Therapeutics, Vagelos College of Physicians & Surgeons, Columbia University, New York, New York, USA; Division of Molecular Therapeutics, New York State Psychiatric Institute, New York, New York, USA; NNF Center for Basic Metabolic Research, Section for Metabolic Receptology, Faculty of Health and Medical Sciences, University of Copenhagen, Denmark.
Multispan Inc, Hayward, California, USA.
J Biol Chem. 2021 Jan-Jun;296:100503. doi: 10.1016/j.jbc.2021.100503. Epub 2021 Mar 5.
G protein-coupled receptors (GPCRs) signal through activation of G proteins and subsequent modulation of downstream effectors. More recently, signaling mediated by β-arrestin has also been implicated in important physiological functions. This has led to great interest in the identification of biased ligands that favor either G protein or β-arrestin-signaling pathways. However, nearly all screening techniques for measuring β-arrestin recruitment have required C-terminal receptor modifications that can in principle alter protein interactions and thus signaling. Here, we have developed a novel luminescence-based assay to measure β-arrestin recruitment to the membrane or early endosomes by unmodified receptors. Our strategy uses NanoLuc, an engineered luciferase from Oplophorus gracilirostris (deep-sea shrimp) that is smaller and brighter than other well-established luciferases. Recently, several publications have explored functional NanoLuc split sites for use in complementation assays. We have identified a unique split site within NanoLuc and fused the corresponding N-terminal fragment to either a plasma membrane or early endosome tether and the C-terminal fragment to β-arrestins, which form the basis for the MeNArC and EeNArC assays, respectively. Upon receptor activation, β-arrestin is recruited to the membrane and subsequently internalized in an agonist concentration-dependent manner. This recruitment promotes complementation of the two NanoLuc fragments, thereby reconstituting functional NanoLuc, allowing for quantification of β-arrestin recruitment with a single luminescence signal. Our assay avoids potential artifacts related to C-terminal receptor modification and has promise as a new generic assay for measuring β-arrestin recruitment to diverse GPCR types in heterologous or native cells.
G 蛋白偶联受体 (GPCRs) 通过 G 蛋白的激活和下游效应器的后续调节来传递信号。最近，β-arrestin 介导的信号转导也被认为与重要的生理功能有关。这导致人们对鉴定偏向配体产生了极大的兴趣，这些配体有利于 G 蛋白或β-arrestin 信号通路。然而，几乎所有用于测量β-arrestin 募集的筛选技术都需要对受体的 C 端进行修饰，这在原则上可以改变蛋白质相互作用，从而改变信号转导。在这里，我们开发了一种新的基于发光的测定法，用于测量未修饰受体与膜或早期内体的β-arrestin 募集。我们的策略使用了来自 Oplophorus gracilirostris（深海虾）的工程化荧光素酶 NanoLuc，它比其他已建立的荧光素酶更小、更亮。最近，有几篇出版物探讨了功能性 NanoLuc 分裂位点在互补测定中的应用。我们在 NanoLuc 内部确定了一个独特的分裂位点，并将相应的 N 端片段融合到质膜或早期内体连接物上，将 C 端片段融合到β-arrestin 上，这分别是 MeNArC 和 EeNArC 测定的基础。受体激活后，β-arrestin 被募集到膜上，并随后以激动剂浓度依赖的方式内化。这种募集促进了两个 NanoLuc 片段的互补，从而重新构建了功能性 NanoLuc，允许通过单个发光信号定量β-arrestin 的募集。我们的测定法避免了与受体 C 端修饰相关的潜在假象，有望成为一种新的通用测定法，用于测量异源或天然细胞中不同 GPCR 类型的β-arrestin 募集。
