Investigation of adenosine A1 receptor-mediated β-arrestin 2 recruitment using a split-luciferase assay

Background: Adenosine A1 receptor (A(1)AR) 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 A(1)AR 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 A(1)AR compared to the activation of G proteins. In this work, we characterized a live cell assay for A(1)AR-mediated beta-arrestin 2 recruitment. We have applied this assay to a set of different compounds that interact with this receptor. Methods: Based on NanoBit((R)) technology, a protein complementation assay was developed in which the A(1)AR is coupled to the large part of the nanoluciferase (LgBiT), whereas its small part (SmBiT) is fused to the N-terminus of beta-arrestin 2. Stimulation of A(1)AR results in the recruitment of beta-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 T 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 beta-arrestin 2, whereas it shows full agonism in the case of the inhibitory effect of A(1)AR 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 A(1)AR-mediated recruitment of beta-arrestin 2 by stimulation with a valerian extract. Conclusion: The presented assay is a useful tool for the quantitative study of A(1)AR-mediated beta-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.

Automated summary

In this study, a live cell assay for A(1)AR-mediated beta-arrestin 2 recruitment was characterized. The assay showed that Capadenoson exhibited partial agonism in beta-arrestin 2 recruitment, but full agonism in the inhibitory effect on cAMP production. Furthermore, it was demonstrated for the first time that a valerian extract can stimulate A(1)AR-mediated beta-arrestin 2 recruitment.