Translocation of arrestin induced by human A3 adenosine receptor ligands in an engineered cell line: Comparison with G protein-dependent pathways
Introduction
Arrestins are a family of four cytoplasmic scaffolding proteins primarily responsible for dual roles in inactivating the signaling ability of a G protein-coupled receptor (GPCR) following the binding of an agonist and in modulating intracellular signaling [1], [2]. There are four isoforms of arrestins. Arrestin1 and arrestin4 are only expressed in the visual transduction system. Arrestin2 (β-arrestin1) and arrestin3 (β-arrestin2) are expressed ubiquitously in the body. Arrestins translocate and bind to an activated GPCR that has been phosphorylated by a G protein-coupled receptor kinase (GRK). It has been suggested that arrestins bind to phosphorylated sites on an intracellular loop of the GPCR to prevent its association with the Gα subunit [3]. Consequently, the G protein-dependent signaling is terminated. Additionally, arrestins act as scaffold proteins linking the GPCR to internalization proteins, such as clathrin. The GPCRs are either recycled to the cell surface or degraded in the lyzosomes after moving to the clathrin pits for endocytosis. Recent evidence has shown that arrestins can also function to activate signaling cascades independently of G protein activation [1]. The ubiquitination of β-arrestin has been shown to be dispensable for the cytosol-to-membrane transition, but essential for the formation of a tight complex with the GPCR [2]. The ability of arrestin to modulate ERK activation, an important signal in cellular survival and proliferation, has been linked to this ubiquitination.
The A3 adenosine receptor (AR) is an important target for a number of inflammatory, neoplastic, and neurodegenerative conditions [4], [5], [6], [7]. Both agonists and antagonists [8] are of potential clinical application. The selective A3AR agonists IB-MECA and Cl-IB-MECA are currently in clinical trials for the autoimmune inflammatory disease rheumatoid arthritis and cancer, respectively [5], [6]. We have previously studied G protein-mediated signaling after agonist binding to the A3AR. It was found that some adenosine derivatives, previously assumed to be full AR agonists, are partial agonists or antagonists at the A3 subtype [9]. It is important to know how these agonists and antagonists behave in A3AR-mediated pathways that are independent of G proteins, such as arrestin. The A3AR has been reported to be a rapidly desensitizing receptor [10], [11], and receptor regulation appears to be important in the clinical actions of A3AR agonists [12]. GRKs have been suggested to be involved in the desensitization and internalization process. However, it has been reported that the arrestin-translocation was not observed following the activation of the rat A3AR endogenously expressed in the RBL-2H3 cells [13]. Previously, it was not clear if the human A3AR couples to arrestins.
In order to test whether the human A3AR can induce arrestin translocation and how the known A3AR agonists and antagonists behave, a PathHunter™ cell line engineered for human β-arrestin translocation in response to A3AR activation was utilized [14]. The focus of this paper is both to demonstrate the utility in drug screening of a cell line engineered for detecting arrestin translocation and to identify ligands that show biased agonism. The response was initially measured in a 96-well, and later a 384-well plate format with detection through a luminescent reaction based on enzyme fragment complementation [15]. A bioactive chemical library [16] was selected for this purpose.
This study takes into consideration that a given agonist may have differential functional effects on multiple signaling pathways downstream of the A3AR. Since multiple pathways may be involved in the clinically relevant action of A3 agonists, it would be important to identify biased agonists that favor one pathway over another for use as pharmacological tools and potential leads for therapeutic agents.
Section snippets
Materials
NECA (adenosine-5′-N-ethyluronamide), CCPA (2-chloro-N6-cyclopentyladenosine), Cl-IB-MECA (2-chloro-N6-(3-iodobenzyl)adenosine-5′-N-methyluronamide), adenosine and inosine were from Sigma (MO, USA). LUF6000 (N-(3,4-dichloro-phenyl)-2-cyclohexyl-1H-imidazo[4,5-c]quinolin-4-amine) was synthesized as described at Leiden/Amsterdam Center for Drug Research (Leiden, The Netherlands) [17]. Compound 20 [18] was synthesized by Liesbet Cosyn and Prof. Serge Van Calenbergh at Ghent University, Ghent,
Results
The assay method for arrestin translocation consists of an engineered PathHunter cell system. The chemiluminescent signal is dependent on the proximity of two fragments of beta-galactosidase, one of which (termed the enzyme acceptor) is fused to the human β-arrestin2 protein and the other (termed the ProLink tag) is fused to the C-terminus of the GPCR. When arrestin translocates to its natural binding site on the GPCR, the two inactive fragments form an active complex that converts an
Discussion
The present study clearly demonstrates for the first time that the human A3AR mediates an arrestin translocation. Despite the artificial nature of the engineered cell line used, this study suggests that different agonists of a given GPCR can selectively trigger certain effector pathways. The system allowed us to study agonist efficacy of a variety of nucleoside derivatives that have been previously shown to be A3AR agonists or antagonists. Comparison of different A3AR-mediated signaling
Acknowledgements
This work was supported by the NIDDK Intramural Research Program, National Institutes of Health (Bethesda, MD, USA). We thank Justin Mika, Dr. Vashti Lacaille, and Dr. Keith R. Olson of DiscoveRx Corp. (Fremont, CA) for helpful discussions. We thank Prof. Lak Shin Jeong (EWHA Womens Univ., Seoul, Korea), Liesbet Cosyn and Prof. Serge Van Calenbergh (Ghent Univ., Ghent, Belgium), Prof. Ad P. IJzerman (Leiden Univ., Leiden, The Netherlands), Prof. Christa Müller (Univ. of Bonn, Bonn, Germany),
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