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

Background Adenosine A1 receptor (A₁AR) plays a prominent role in neurological and cardiac diseases as well as during 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, 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 for signal transduction and regulation of A₁AR compared to the activation of G proteins. In this work, we characterized a live cell assay for the 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 is a full agonist 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 were able to 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 the A₁AR mediated β-arrestin 2 recruitment. It allows data collection for stimulatory, inhibitory as well as modulatory substances and is also suitable for more complex substance mixtures such as valerian extract.