Franken, S. | bioRxiv Science

bioRxiv2023

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

BackgroundAdenosine 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 {beta}-arrestin 2 recruitment. We have applied this assay to a set of different compounds that interact with this receptor. MethodsBased on NanoBit(R) 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 {beta}-arrestin 2. Stimulation of A1AR 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 GloSensorTM assay. ResultsThe 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 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 {beta}-arrestin 2 by stimulation with a valerian extract. ConclusionThe presented assay is a useful tool for the quantitative study of the A1AR mediated {beta}-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.

pharmacology and toxicology↗

bioRxiv2018

Live-cell single-molecule analysis of β2-adrenergic receptor diffusion dynamics and confinement

Signal transduction mechanisms and successive regulatory processes alter the lateral mobility of {beta}2-adrenergic receptors ({beta}2AR). In this work we combined modern single particle tracking methods in order to analyze the diffusion dynamics of SNAP-tagged {beta}2AR in HEK wild-type cells and HEK {beta}-arrestin knockout cells before and after agonist stimulation. For analysis of trajectories we first used mean squared displacement (MSD) analysis. Secondly, we applied an advanced variational Bayesian treatment of hidden Markov models (vbSPT) in combination with the recently introduced packing coefficient (Pc), which together provided a detailed model of three discrete diffusive states, state transitioning and spatial confinement. Interesting to note, state switching between S3 (fast-diffusing) and S1 (slow-diffusing) occurred sequentially over an intermediate state S2. After ligand stimulation more SNAP-tagged {beta}2AR in HEK wild-type cells switched occupancy into the slow-diffusing state, whereas less receptors were found in the fast diffusive state. Unexpectedly, all three states showed a fraction of confined receptor mobility that increased under stimulation, but confinement sizes were unaffected. Receptor diffusion characteristics were comparable in HEK {beta}-arrestin knockout cells under basal conditions and only minor but non-significant changes occurred upon stimulation, as expected from the depletion of {beta}-arrestin, an important regulatory protein. The data presented here on the occurrence of different diffusion states, their transitioning and variable spatial confinements clearly indicate that lateral mobility of {beta}2AR is much more complex than previously thought.

cell biology↗