PDF(Pubmed)
Abstract：
Background: Adenosine A1 receptor (A1AR) plays a prominent role in neurological and cardiac diseases and inflammatory processes. Its endogenous ligand adenosine is known to be one of the key players in the sleep-wake cycle. Like other G protein-coupled receptors (GPCRs), stimulation of A1AR leads to the recruitment of arrestins in addition to the activation of G proteins. So far, little is known about the role of these proteins in signal transduction and regulation of A1AR compared to the activation of G proteins. In this work, we characterized a live cell assay for A1AR-mediated β-arrestin 2 recruitment. We have applied this assay to a set of different compounds that interact with this receptor. Methods: Based on NanoBit® technology, a protein complementation assay was developed in which the A1AR is coupled to the large part of the nanoluciferase (LgBiT), whereas its small part (SmBiT) is fused to the N-terminus of β-arrestin 2. Stimulation of A1AR results in the recruitment of β-arrestin 2 and subsequent complementation of a functional nanoluciferase. For comparison, corresponding data on the effect of receptor stimulation on intracellular cAMP levels were collected for some data sets using the GloSensor™ assay. Results: The assay gives highly reproducible results with a very good signal-to-noise ratio. Capadenoson, in contrast to adenosine, CPA, or NECA, shows only partial agonism in this assay with respect to the recruitment of β-arrestin 2, whereas it shows full agonism in the case of the inhibitory effect of A1AR on cAMP production. By using a GRK2 inhibitor, it becomes clear that the recruitment is at least partially dependent on the phosphorylation of the receptor by this kinase. Interestingly, this was also the first time that we demonstrate the A1AR-mediated recruitment of β-arrestin 2 by stimulation with a valerian extract. Conclusion: The presented assay is a useful tool for the quantitative study of A1AR-mediated β-arrestin 2 recruitment. It allows data collection for stimulatory, inhibitory, and modulatory substances and is also suitable for more complex substance mixtures such as valerian extract.
摘要：
背景：腺苷A1受体（A1AR）在神经和心脏疾病以及炎症过程中起着重要作用。已知其内源性配体腺苷是睡眠-觉醒周期中的关键参与者之一。像其他G蛋白偶联受体（GPCRs），除了激活G蛋白外，A1AR的刺激还导致抑制素的募集。到目前为止,与G蛋白的激活相比，这些蛋白在A1AR的信号转导和调节中的作用知之甚少。在这项工作中,我们表征了A1AR介导的β-抑制蛋白2募集的活细胞测定法。我们已经将该测定应用于一组与该受体相互作用的不同化合物。方法：基于NanoBit®技术，开发了一种蛋白质互补测定，其中A1AR与纳米荧光素酶（LgBiT）的大部分偶联，而其一小部分（SmBiT）融合到β-抑制蛋白2的N端。A1AR的刺激导致β-抑制蛋白2的募集和随后的功能性纳米电容酶的互补。为了比较，使用GloSensor™测定收集一些数据集的受体刺激对细胞内cAMP水平的影响的相应数据。结果:该测定给出具有非常好的信噪比的高度可再现的结果。Capadenoson,与腺苷相比，CPA,或者NECA,关于β-抑制蛋白2的募集，在该测定中仅显示部分激动作用，而在A1AR对cAMP产生抑制作用的情况下，它显示完全激动作用。通过使用GRK2抑制剂，很明显，募集至少部分依赖于该激酶对受体的磷酸化。有趣的是,这也是我们首次证明缬草提取物刺激A1AR介导的β-抑制素2募集.结论：所提出的方法是定量研究A1AR介导的β-抑制素2募集的有用工具。它允许收集刺激数据，抑制性,和调节物质，也适用于更复杂的物质混合物，如缬草提取物。
