On the G protein-coupling selectivity of the native A2B adenosine receptor
Graphical abstract
Introduction
The A2B adenosine receptor (A2BAR) is involved in many important physiological and pathophysiological functions, such as the hematopoietic stem cell emergence [1], inflammation [2], [3], [4], cell proliferation [5], [6], [7]; reactive oxygen species (ROS) production [8], erectile function [9], bone homeostasis [10], cardiac protection [11] and diabetes mellitus [12]. However, the signaling mechanisms related to those functions are not well understood. In particular, the mechanisms leading to ERK1/2 phosphorylation, one of those canonical signaling processes, have not been well explored and currently published data are inconsistent. For example, it has been suggested A2BAR-mediated ERK1/2 activity in HEK293 cells is via the Gq/11 protein [13]. However, Schulte and Fredholm [14] suggested that A2BAR-mediated ERK1/2 activity in CHO cells expressing the recombinant A2BAR is mediated via Gs rather than Gq/11. Epperson et al. [15] also found a lack of A2BAR-coupling to Gq protein in cardiac fibroblasts. PKC is a typical modulator of the A2BAR- and other GPCR-mediated ERK1/2 activity [16]. However, Fang and Olah [17] showed that EPAC1 but not PKC is involved in A2BAR-mediated ERK1/2 phosphorylation in human vascular endothelial cells (HUVEC). In rat skeletal muscle, A2BAR activation was found not to modulate ERK1/2, although it stimulated cAMP accumulation [18]. In trophoblast cells, it was found that A2BAR downregulates ERK1/2 activity [19]. ERK1/2 activity stimulated by adenosine-5′-N-ethyluronamide (NECA) in A2BAR-expressing CHO is via PI3K, but not PKA [14]. However, Sun et al. [20] showed that A2BAR activation stimulates ERK1/2 activity via PKA in sickle erythrocytes. Thus, it seems signal mechanisms diverge in various studies and in different cell types under different conditions. Indeed, A2BAR has been shown to cause both proliferation and antiproliferation in different cell types [5], [21], [22], [23], [24], [25], [26], [27]. A2BAR has been reported to involve both pro- and anti-inflammatory actions [2], [28], [29], [30], which might be related the different G protein-coupling in different types of immune cells. Thus, it is important to understand the types of G proteins or signaling pathways involved in A2BAR-mediated functions.
Recent studies show that various A2BAR agonists have different maximum effect (Emax) and signaling bias in different signaling pathways [31], [32]. Nonnucleoside agonist BAY60-6583 stimulates ERK1/2 activity in HEK293 cells but not in T24 bladder cancer cells, both endogenously expressing the A2BAR, although nucleosides NECA and adenosine-5′-N-cyclopropyluronamide (CPCA) stimulated ERK1/2 activity in both cell types [31]. It was demonstrated that BAY60-6583 behaves as an antagonist and induces insulin secretion in a mouse pancreatic cell line, MIN-6 [31]. Some controversies concerning the A2BAR might also be due to different agonists used in those earlier studies.
In the present study, we explore the coupling of the native human A2BAR to Gi, Gq/11 and Gs in three different cell types, 1321 N1 astrocytoma, HEK293, and T24 bladder cancer cells. We used NECA and BAY60-6583, selective blockers for Gi, Gs and Gq/11, as well as the clustered regularly interspaced short palindromic repeat-associated protein-9 nuclease (CRISPR/Cas9)-based Gq/11- and Gs-null HEK293 cells [33], [34]. As ERK1/2 activity and calcium mobilization are involved in many A2B-mediated functions and are the convergence of multiple G protein-signaling pathways directed under the A2BAR, we explored the contributions of various G proteins and downstream signaling molecules to ERK1/2 activity and calcium mobilization. These pathways were compared in three different cell types endogenously expressing almost solely the A2BAR with the other three AR subtypes being at very low levels. We found that the A2BAR may couple to different downstream signaling events via different G proteins in the same cell type, and the A2BAR may couple to the same downstream pathway via different G proteins in different cell types. Gi and Gs proteins activated by the A2BAR may play opposing or synergistic roles in different cell types and in different signaling pathways. Any one of the three G proteins, Gi, Gq/11, and Gs, may be solely responsible for a specific signaling event mediated via the A2BAR depending on cell type. The findings could be related to controversial roles of the A2BAR-mediated effects reported in literature, e.g. proliferation and antiproliferation, or proinflammatory vs. anti-inflammatory. These findings could be applicable to the study of signaling pathways of other GPCRs, and should be of considerable relevance to future drug discovery and development.
Section snippets
Materials
GO6983, CE3F4, ESI-09, HJC0350, H89, PSB603, NECA (adenosine-5′-N-ethyluronamide) and MRS2365 ([[(1R,2R,3S,4R,5S)-4-[6-amino-2-(methylthio)-9H-purin-9-yl]-2,3-dihydroxybicyclo[3.1.0]hex-1-yl]methyl] diphosphoric acid monoester trisodium salt) were from Tocris (Ellisville, MO). CGS21680, Carbachol, CTX and PTX were from Sigma (St. Louis, MO). UBO-QIC was purchased from University of Bonn (Bonn, Germany). BAY60-6583 (LUF6210, termed hereafter ‘BAY’) was synthesized at Leiden/Amsterdam Center for
Gene expression of four AR subtypes in three cell types
In order to study the coupling of the endogenous human A2BAR to different G proteins, we selected three cell types, HEK293, T24 and 1321 N1, which endogenously express the A2BAR highly in comparison to the other three AR subtypes. The reason for our use of AR agonist NECA is that it is still the only established full agonist for the A2BAR that is commercially available, although nonselective. The nonnucleoside agonist BAY60-6583 is a partial agonist, albeit selective. Previously, we have found
Discussion
By using three cell types, HEK293 (including wild-type, Gs- and Gq/11-null HEK293), 1321 N1 astrocytoma and T24 bladder cancer cells, it was demonstrated in the present study that the endogenous A2BAR may couple to the same downstream signaling pathway via different G proteins in different cell types, and it may couple to different signaling pathways via different G proteins in the same type of cells (Table 1). Furthermore, Gs and Gi may have both opposing effects and synergistic effects in
Acknowledgements
Supported the Intramural Research Program of the National Institute of Diabetes and Digestive and Kidney Diseases (ZIADK031117), National Institutes of Health, Bethesda, MD, USA. AI was funded by the PRIME from Japan Agency for Medical Research and Development (AMED) and Japan Society for the Promotion of Science KAKENHI Grant Number 17 K08264. The authors thank Jurgen Wess (NIDDK, NIH) for advice, Ad IJzerman (Leiden, The Netherlands) for providing BAY60-6583 (LUF6210), E. Kiselev (NIDDK, NIH)
Conflict of interest
The authors have no conflict of interest to declare.
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