Allosteric interactions between agonists and antagonists within the adenosine A2A receptor-dopamine D2 receptor heterotetramer

Adenosine A_2A receptor (A_2AR)-dopamine D₂ receptor (D₂R) heteromers are key modulators of striatal neuronal function. It has been suggested that the psychostimulant effects of caffeine depend on its ability to block an allosteric modulation within the A_2AR-D₂R heteromer, by which adenosine decreases the affinity and intrinsic efficacy of dopamine at the D₂R. We describe novel unsuspected allosteric mechanisms within the heteromer by which not only A_2AR agonists, but also A_2AR antagonists, decrease the affinity and intrinsic efficacy of D₂R agonists and the affinity of D₂R antagonists. Strikingly, these allosteric modulations disappear on agonist and antagonist coadministration. This can be explained by a model that considers A_2AR-D₂R heteromers as heterotetramers, constituted by A_2AR and D₂R homodimers, as demonstrated by experiments with bioluminescence resonance energy transfer and bimolecular fluorescence and bioluminescence complementation. As predicted by the model, high concentrations of A_2AR antagonists behaved as A_2AR agonists and decreased D₂R function in the brain.Most evidence indicates that G protein-coupled receptors (GPCRs) form homodimers and heteromers. Homodimers seem to be a predominant species, and oligomeric entities can be viewed as multiples of dimers (1). It has been proposed that GPCR heteromers are constituted mainly by heteromers of homodimers (1, 2). Allosteric mechanisms determine a multiplicity of unique pharmacologic properties of GPCR homodimers and heteromers (1, 3). First, binding of a ligand to one of the receptors in the heteromer can modify the affinity of ligands for the other receptor (1, 3, 4). The most widely reproduced allosteric modulation of ligand-binding properties in a GPCR heteromer is the ability of adenosine A_2A receptor (A_2AR) agonists to decrease the affinity of dopamine D₂ receptor (D₂R) agonists in the A_2AR-D₂R heteromer (5). A_2AR-D₂R heteromers have been revealed both in transfected cells (6, 7), striatal neurons in culture (6, 8) and in situ, in mammalian striatum (9, 10), where they play an important role in the modulation of GABAergic striatopallidal neuronal function (9, 11).In addition to ligand-binding properties, unique properties for each GPCR oligomer emerge in relation to the varying intrinsic efficacy of ligands for different signaling pathways (1–3). Intrinsic efficacy refers to the power of the agonist to induce a functional response, independent of its affinity for the receptor. Thus, allosteric modulation of an agonist can potentially involve changes in affinity and/or intrinsic efficacy (1, 3). This principle can be observed in the A_2AR-D₂R heteromer, where a decrease in D₂R agonist affinity cannot alone explain the ability of an A_2AR agonist to abolish the decreased excitability of GABAergic striatopallidal neurons induced by high concentration of a D₂R agonist (9), which should overcome the decrease in affinity. Furthermore, a differential effect of allosteric modulations of different agonist-mediated signaling responses (i.e., functional selectivity) can occur within GPCR heteromers (1, 2, 8). Again, the A_2AR-D₂R heteromer provides a valuable example. A recent study has shown that different levels of intracellular Ca²⁺ exert different modulations of A_2AR-D₂R heteromer signaling (8). This depends on the ability of low and high Ca²⁺ to promote a selective interaction of the heteromer with different Ca²⁺-binding proteins, which differentially modulate allosteric interactions in the heteromer (8).It has been hypothesized that the allosteric interactions between A_2AR and D₂R agonists within the A_2AR-D₂R heteromer provide a mechanism responsible not only for the depressant effects of A_2AR agonists, but also for the psychostimulant effects of adenosine A_2AR antagonists and the nonselective adenosine receptor antagonist caffeine (9, 11, 12), with implications for several neuropsychiatric disorders (13). In fact, the same mechanism has provided the rationale for the use of A_2AR antagonists in patients with Parkinson’s disease (13, 14). The initial aim of the present study was to study in detail the ability of caffeine to counteract allosteric modulations between A_2AR and D₂R agonists (affinity and intrinsic efficacy) within the A_2AR-D₂R heteromer. Unexpectedly, when performing control radioligand-binding experiments, not only an A_2AR agonist, but also caffeine, significantly decreased D₂R agonist binding. However, when coadministered, the A_2AR agonist and caffeine co-counteracted their ability to modulate D₂R agonist binding. By exploring the molecular mechanisms behind these apparent inconsistencies, the present study provides new insight into the quaternary structure and function of A_2AR-D₂R heteromers.