AMP and adenosine are both ligands for adenosine 2B receptor signaling

https://doi.org/10.1016/j.bmcl.2017.11.019Get rights and content

Highlights

  • After renal ischemia both AMP and adenosine levels are increased.

  • Computational modelling shows AMP binds to A2BR.

  • Biological assays in CHO-hA2B cells show that AMP binds to A2BR.

  • Binding of AMP leads to Gq activation.

Abstract

Adenosine is considered the canonical ligand for the adenosine 2B receptor (A2BR). A2BR is upregulated following kidney ischemia augmenting post ischemic blood flow and limiting tubular injury. In this context the beneficial effect of A2BR signaling has been attributed to an increase in the pericellular concentration of adenosine. However, following renal ischemia both kidney adenosine monophosphate (AMP) and adenosine levels are substantially increased. Using computational modeling and calcium mobilization assays, we investigated whether AMP could also be a ligand for A2BR.

The computational modeling suggested that AMP interacts with more favorable energy to A2BR compared with adenosine. Furthermore, AMPαS, a non-hydrolyzable form of AMP, increased calcium uptake by Chinese hamster ovary (CHO) cells expressing the human A2BR, indicating preferential signaling via the Gq pathway. Therefore, a putative AMP-A2BR interaction is supported by the computational modeling data and the biological results suggest this interaction involves preferential Gq activation. These data provide further insights into the role of purinergic signaling in the pathophysiology of renal IRI.

Introduction

Adenosine monophosphate (AMP) is generated by the sequential hydrolysis of extracellular adenosine triphosphate (ATP) and adenosine diphosphate (ADP) by the ectonucleotidase CD39 (NTPDase1, ectonucleoside triphosphate diphosphohydrolase-1). AMP is hydrolyzed by CD73 (ecto – 5′ – nucleotidase) to adenosine, which in turn is rapidly metabolized and cleared from the circulation. CD73 and the adenosine receptors (A1R, A2AR, A2BR, A3R) are widely expressed, implicating adenosine signaling in a diverse range of physiological and pathophysiological processes.

The signaling properties of extracellular AMP remain controversial. Over 30 years ago it was first reported that phosphorylated adenosine derivatives were low-affinity adenosine receptor agonists.1 In 2004 it was proposed that the G-protein-coupled-receptor (GPCR) orphan receptor GPR80/99 was a receptor for extracellular AMP.2 However, this was quickly discounted because of the endogenous expression of adenosine receptors on HEK293 cells used in the experiments.3 In 2011 the authors of the update on the classification of adenosine receptors noted “there [was] no good evidence that adenine nucleotides can act on adenosine receptors without being degraded to nucleosides first”.4 However, in 2013 AMP was shown to play a role in thermoregulation mediated by the A1R.5 This was supported by in vitro studies using HEK293 cells6 in which AMP activated the human A1R with equivalent potency to adenosine.

The canonical ligand for all four receptors is adenosine, the potency of which varies according to receptor density and the type of response measured. The receptors are localized on the cell surface and belong to the GPCR family.4 Structural information of the GPCR family as a whole has expanded rapidly.7 Within the adenosine family of receptors the A2AR has been most thoroughly studied and the crystalline structure determined,8 which has enhanced the understanding of both orthosteric and allosteric binding sites. In addition a number of studies have demonstrated that adenosine receptors can exist as homodimers and heterodimers, which may impact GPCR function (reviewed in 7).

The A2BR has been implicated in the pathophysiology of renal ischemic-reperfusion injury (IRI) by increasing post-ischemic blood flow9 and limiting TNF-α release from neutrophils.10 The A2BR is a Gs-coupled protein receptor and its activation increases intracellular adenylyl cyclase activity and cAMP levels. A2BR is also coupled to Gq proteins, which upon A2BR engagement activate phosphatidylinositol-phospholipase C, triggering a series of steps resulting in the opening of calcium channels. In many cells Gs coupling appears preferred.4 The A2BR is widely expressed throughout the body. However, because of the relatively low potency of adenosine for this receptor it may not be fully activated under physiological conditions but rather activated in pathological states, such as hypoxia, when the pericellular adenosine concentration is significantly elevated. Indeed the A2BR is upregulated on the renal vasculature following ischemia,10 mediated by the transcription factor HIF-1α,11 and this heightened expression is maintained for at least 4 weeks.12 The coordinated increase in the expression of A2BR and local concentration of adenosine in response to kidney ischemia implicates purinergic signaling in the pathophysiology of renal IRI. Indeed augmenting adenosine signaling, particularly via the A2BR, prior to acute kidney ischemia disease mitigates injury (reviewed in 13).

Although the effects of adenosine receptor signaling in acute renal IRI have been attributed to the generation of high concentrations of adenosine, we have recently reported an increase in kidney levels of both AMP and adenosine, with a concomitant fall in ATP and ADP.14 We have also previously demonstrated that both serum AMP and adenosine concentrations were increased following the intravenous injection of pro-inflammatory collagen.15 This observation together with the evidence of increased A2BR expression in acute renal IRI, led to the hypothesis that AMP, in addition to adenosine, may be a ligand for the A2BR. This potential interaction is investigated here using computational modeling and calcium mobilization assays.

Section snippets

Homology modeling and docking

A homology model of A2BR was created based on the model of A2AR in complex with adenosine17 and the first published A2AR crystal structure16 using Modeller.24 The stereochemical quality of the model was checked using ProCheck V 3.5.18 This model was then used to dock adenosine, AMP and AMPαS using the Dock-Geom module within Sybyl-X 2.1.1 (Certara L.P.). A protomol was created based upon the position of adenosine as seen in the A2AR crystal structure. The bloat was increased to 2 Å (default 0)

Results and discussion

To explore the hypothesis that AMP was a ligand for A2BR, we analyzed the docking potential of adenosine, AMP and AMPαS using computational modeling. Adenosine was chosen as the canonical ligand; AMP as the putative ligand and AMPαS as it is a non-hydrolysable analogue of AMP and was used for in vitro studies.

A homology model of A2BR was created using the combined A2AR crystal structures as a template.16, 17 The quality of the model was assessed, with 98.9% of the amino acids residing in the

Acknowledgements

K.M.D. current address: School of Medicine, Faculty of Health, Deakin University, Geelong, Australia. Funding from the Victorian Government Operational Infrastructure Support Scheme to St Vincent’s Institute is acknowledged. M.W.P. is a National Health and Medical Research Council of Australia Research Fellow and J.K.H. is a 5point Foundation Christine Martin Post-Doctoral Fellow.

Conflict of interest

The authors declare that they have no conflicts of interest with the contents of this article.

Authors’ contributions

JKH designed and performed experiments in Fig. 1, Fig. 2. BS, ES and VR designed and performed the experiments in Fig. 3. MWP contributed to analysis and editing of manuscript. PJC contributed to analysis and editing of manuscript. KMD contributed to the analysis and wrote the manuscript.

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