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Abstract
The adenosine receptor (AR) subtypes A2A and A2B are rhodopsin-like Gs protein-coupled receptors whose expression is highly regulated under pathological, e.g. hypoxic, ischemic and inflammatory conditions. Both receptors play important roles in inflammatory and neurodegenerative diseases, are blocked by caffeine, and have now become major drug targets in immuno-oncology. By Förster resonance energy transfer (FRET), bioluminescence resonance energy transfer (BRET), bimolecular fluorescence complementation (BiFC) and proximity ligation assays (PLA) we demonstrated A2A-A2BAR heteromeric complex formation. Moreover we observed a dramatically altered pharmacology of the A2AAR when co-expressed with the A2BAR (A2B ≥ A2A) in recombinant as well as in native cells. In the presence of A2BARs, A2A-selective ligands lost high affinity binding to A2AARs and displayed strongly reduced potency in cAMP accumulation and dynamic mass redistribution (DMR) assays. These results have major implications for the use of A2AAR ligands as drugs as they will fail to modulate the receptor in an A2A-A2B heteromer context. Accordingly, A2A-A2BAR heteromers represent novel pharmacological targets.
Highlights
Adenosine receptors (ARs) are G protein-coupled receptors (GPCRs) activated by the nucleoside adenosine
To investigate a possible A2BAR subtypes are established (A2A)-A2BAR interaction, Förster resonance energy transfer (FRET) experiments were performed in Chinese hamster ovary (CHO-K1) cells transiently transfected with fusion proteins of green fluorescent protein variant 2 (GFP2) and enhanced yellow fluorescent protein (EYFP) attached to the C-terminus of the receptors [36] (Figure 1A–1C)
The results indicated that the A2AAR that was lacking the C-terminal domain was still fully able to form heteromers with the A2BAR (FRET efficiency 0.24, Figure 1B) suggesting that different receptor domains, possibly helical domains, have to be involved in heteromer formation
Summary
Adenosine receptors (ARs) are G protein-coupled receptors (GPCRs) activated by the nucleoside adenosine. Four subtypes designated A1, A2A, A2B and A3ARs exist. A2B and A3ARs were shown to couple to Gq proteins which results in phospholipase C activation followed by a rise in inositol trisphosphate levels mediating intracellular calcium release [2,3]. The A2AAR is expressed in high density in the caudate-putamen, and at low levels in most other brain regions. The A2AAR is highly expressed in cells of the immune system and blood platelets, and at lower levels in many other cells and organs [4]. The A2BAR is broadly expressed but mostly at moderate to low levels. A2A and A2BARs are the most closely related AR subtypes with an overall sequence identity of 58% and a similarity of 73% [5]
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