Investigation of β-arrestin 2 recruitment, downstream signal transduction and lateral mobility of adenosine A₁ receptor after treatment with valerian extract Ze 911

Abstract

Sleep is a complex physiological process with a variety of molecules involved. Even though a lot of research and progress has been made until today, the exact underlying mechanism still eludes us. Many people must deal with sleep disorders that have a great influence on their daily lives. It is known that the endogenous molecule adenosine plays a superior role in the sleep-wake cycle mediating effects at corresponding adenosine A₁ receptor (A₁AR). A₁AR is highly expressed in the central nervous system and regulator of pathological as well as physiological processes like sleep. Stimulation of A₁AR with agonists results in receptor activation and binding of G proteins, particularly G_i or G_o proteins. This causes inhibition of adenylate cyclase and consequently decrease of cyclic adenosine monophosphate (cAMP) concentration. Activation of A₁AR additionally leads to G protein-coupled receptor kinase (GRK)-mediated receptor phosphorylation at the C-terminus and consequently to the recruitment of adaptor proteins like β-arrestins. Four different isoforms of arrestins are described – 1 and 4 are visual arrestins and exclusively expressed in the visual system. The two non-visual isoforms 2 and 3, also known as β-arrestin 1 and 2 play a key role in the regulation of G protein-coupled receptor (GPCRs). Once recruited to A₁AR, binding of β-arrestin 2 can lead to desensitization and/or internalization of the receptor. Besides, β-arrestin 2-mediated downstream signal transduction of A₁AR has been reported. Signal transduction of A₁AR via G protein activation is well studied compared to signalling after recruitment of β-arrestins. However, β-arrestins have gained interest since they are multifunctional proteins that regulate several functions of GPCRs. The preference of activating either G proteins or β-arrestins is called ligand bias – this complex phenomenon can improve safety and efficacy of certain drugs. The mild sleep-inducing agent valerian extract improves sleep quality and is widely used as phytopharmaceutical in several countries. Even though the effect is proven in clinical studies, the exact mechanism of action including potential targets is not fully understood. This present study addresses the influence of valerian extract Ze 911 on A₁AR in detail. One focus was the investigation of β-arrestin 2 recruitment after A₁AR stimulation with valerian extract or synthetic ligands. For that reason, a cell-based β-arrestin 2 recruitment assay was developed. This so-called NanoBiT^® assay worked A₁AR-specific with highly reproducible results. Agonistic effects were measured for endogenous agonist adenosine as well as synthetic agonists like N6-cyclopentyladenosine (CPA) and capadenoson. Moreover, valerian extract Ze 911 showed a robust agonistic effect, which was later attributed to the adenosine detected in the extract. Dicaffeoylquinic acids (DQAs), a substance class present in Ze 911, showed a modulatory effect on A₁AR combined with the endogenous ligand adenosine. Additionally, CPA and Ze 911 treatment decreased cAMP concentration in A₁AR overexpressing HEK 293 GloSensor^TM cells. In a calcium mobilization assay, CPA, Ze 911 and DQAs showed no significant calcium mobilization after single treatment. Interestingly, they modulated the calcium mobilization of the positive control methacholine. Single-particle tracking (SPT) experiments were conducted to investigate lateral mobility of A₁AR after agonist treatment on A₁AR-overexpressing HEK 293 cells. SPT measurements were started 15 minutes after cells were treated with either CPA or Ze 911. No differences in any of the investigated parameters have been detected compared to control cells. Since time played an important part in the response curves of the other assays, a time-dependent approach was conducted next. Five time intervals were investigated from 1 up to 30 minutes after treatment with CPA. Confined fractions and state occupancies of A₁AR were influenced in certain time intervals. Longer incubation times significantly increased those parameters compared to shorter incubation times. Diffusion coefficients and receptor homooligomerization were not altered over time. These findings demonstrate that Ze 911 contains agonistic as well as modulatory substances that influence A₁AR in various ways.