Human Adenosine A2A Receptor: Molecular Mechanism of Ligand Binding and Activation.
ABSTRACT: Adenosine receptors (ARs) comprise the P1 class of purinergic receptors and belong to the largest family of integral membrane proteins in the human genome, the G protein-coupled receptors (GPCRs). ARs are classified into four subtypes, A1, A2A, A2B, and A3, which are all activated by extracellular adenosine, and play central roles in a broad range of physiological processes, including sleep regulation, angiogenesis and modulation of the immune system. ARs are potential therapeutic targets in a variety of pathophysiological conditions, including sleep disorders, cancer, and dementia, which has made them important targets for structural biology. Over a decade of research and innovation has culminated with the publication of more than 30 crystal structures of the human adenosine A2A receptor (A2AR), making it one of the best structurally characterized GPCRs at the atomic level. In this review we analyze the structural data reported for A2AR that described for the first time the binding of mode of antagonists, including newly developed drug candidates, synthetic and endogenous agonists, sodium ions and an engineered G protein. These structures have revealed the key conformational changes induced upon agonist and G protein binding that are central to signal transduction by A2AR, and have highlighted both similarities and differences in the activation mechanism of this receptor compared to other class A GPCRs. Finally, comparison of A2AR with the recently solved structures of A1R has provided the first structural insight into the molecular determinants of ligand binding specificity in different AR subtypes.
Project description:The purinergic type P1 (adenosine A1 and A2A) receptors and the type P2 (X7) receptor have been suggested to mediate physiological effects of adenosine and adenosine triphosphate on sleep. We aimed to determine gene expression of A1R (receptor), A2AR, and P2RX7 in leukocytes of healthy subjects during total sleep deprivation followed by sleep recovery. Expression of the pro-inflammatory cytokines IL-1? and TNF-? were also determined as they have been characterized as sleep regulatory substances, via P2RX7 activation. Blood sampling was performed on 14 young men (aged 31.9 ± 3.9) at baseline (B), after 24 h of sleep deprivation (24 h-SD), and after one night of sleep recovery (R). We compared gene expression levels after six nights of habitual (22.30-07.00) or extended (21.00-07.00) bedtimes. Using quantitative real-time PCR, the amount of mRNA for A1R, A2AR, P2RX7, TNF-?, and IL-1? was analyzed. After 24 h-SD compared to B, whatever prior sleep condition, a significant increase of A2AR expression was observed that returned to basal level after sleep recovery [day main effect, F(2, 26) = 10.8, p < 0.001]. In both sleep condition, a day main effect on P2RX7 mRNA was observed [F(2, 26) = 6.7, p = 0.005] with significant increases after R compared with 24 h-SD. TNF-? and IL-1? expressions were not significantly altered. Before 24 h-SD (baseline), the A2AR expression was negatively correlated with the latency of stage 3 sleep during the previous night, while that of the A1R positively. This was not observed after sleep recovery following 24 h-SD. This is the first study showing increased A2AR and not A1 gene expression after 24 h-SD in leukocytes of healthy subjects, and this even if bedtime was initially increased by 1.5 h per night for six nights. In conclusion, prolonged wakefulness induced an up-regulation of the A2A receptor gene expression in leukocytes from healthy subjects. Significant correlations between baseline expression of A1 and A2A receptors in peripheral cells and stage 3 sleep suggested their involvement in mediating the effects of adenosine on sleep.
Project description:The functional role of heteromers of G-protein-coupled receptors is a matter of debate. In the present study, we demonstrate that heteromerization of adenosine A1 receptors (A1Rs) and A2A receptors (A2ARs) allows adenosine to exert a fine-tuning modulation of glutamatergic neurotransmission. By means of coimmunoprecipitation, bioluminescence and time-resolved fluorescence resonance energy transfer techniques, we showed the existence of A1R-A2AR heteromers in the cell surface of cotransfected cells. Immunogold detection and coimmunoprecipitation experiments indicated that A1R and A2AR are colocalized in the same striatal glutamatergic nerve terminals. Radioligand-binding experiments in cotransfected cells and rat striatum showed that a main biochemical characteristic of the A1R-A2AR heteromer is the ability of A2AR activation to reduce the affinity of the A1R for agonists. This provides a switch mechanism by which low and high concentrations of adenosine inhibit and stimulate, respectively, glutamate release. Furthermore, it is also shown that A1R-A2AR heteromers constitute a unique target for caffeine and that chronic caffeine treatment leads to modifications in the function of the A1R-A2AR heteromer that could underlie the strong tolerance to the psychomotor effects of caffeine.
Project description:BACKGROUND:G-protein-coupled receptor (GPCR) heteromeric complexes have distinct properties from homomeric GPCRs, giving rise to new receptor functionalities. Adenosine receptors (A1R or A2AR) can form A1R-A2AR heteromers (A1-A2AHet), and their activation leads to canonical G-protein-dependent (adenylate cyclase mediated) and -independent (?-arrestin mediated) signaling. Adenosine has different affinities for A1R and A2AR, allowing the heteromeric receptor to detect its concentration by integrating the downstream Gi- and Gs-dependent signals. cAMP accumulation and ?-arrestin recruitment assays have shown that, within the complex, activation of A2AR impedes signaling via A1R. RESULTS:We examined the mechanism by which A1-A2AHet integrates Gi- and Gs-dependent signals. A1R blockade by A2AR in the A1-A2AHet is not observed in the absence of A2AR activation by agonists, in the absence of the C-terminal domain of A2AR, or in the presence of synthetic peptides that disrupt the heteromer interface of A1-A2AHet, indicating that signaling mediated by A1R and A2AR is controlled by both Gi and Gs proteins. CONCLUSIONS:We identified a new mechanism of signal transduction that implies a cross-communication between Gi and Gs proteins guided by the C-terminal tail of the A2AR. This mechanism provides the molecular basis for the operation of the A1-A2AHet as an adenosine concentration-sensing device that modulates the signals originating at both A1R and A2AR.
Project description:Adenosine is a widespread neuromodulator within the CNS and its extracellular level is increased during hypoxia or intense synaptic activity, modulating pre- and postsynaptic sites. We studied the neuromodulatory action of adenosine on glutamatergic currents in the hippocampus, showing that activation of multiple adenosine receptors (ARs) by basal adenosine impacts postsynaptic site. Specifically, the stimulation of both A1R and A3R reduces AMPA currents, while A2AR has an opposite potentiating effect. The effect of ARs stimulation on glutamatergic currents in hippocampal cultures was investigated using pharmacological and genetic approaches. A3R inhibition by MRS1523 increased GluR1-Ser845 phosphorylation and potentiated AMPA current amplitude, increasing the apparent affinity for the agonist. A similar effect was observed blocking A1R with DPCPX or by genetic deletion of either A3R or A1R. Conversely, impairment of A2AR reduced AMPA currents, and decreased agonist sensitivity. Consistently, in hippocampal slices, ARs activation by AR agonist NECA modulated glutamatergic current amplitude evoked by AMPA application or afferent fiber stimulation. Opposite effects of AR subtypes stimulation are likely associated to changes in GluR1 phosphorylation and represent a novel mechanism of physiological modulation of glutamatergic transmission by adenosine, likely acting in normal conditions in the brain, depending on the level of extracellular adenosine and the distribution of AR subtypes.
Project description:The inhibitory adenosine A1 receptor (A1R) and excitatory A2A receptor (A2AR) are predominantly expressed in the brain. Whereas the A2AR has been implicated in normal aging and enhancing neurotoxicity in multiple neurodegenerative diseases, the inhibitory A1R has traditionally been ascribed to have a neuroprotective function in various brain insults. This review provides a summary of the emerging role of prolonged A1R signaling and its potential cross-talk with A2AR in the cellular basis for increased neurotoxicity in neurodegenerative disorders. This A1R signaling enhances A2AR-mediated neurodegeneration, and provides a platform for future development of neuroprotective agents in stroke, Parkinson's disease and epilepsy.
Project description:Background: Caffeine is the most widely consumed psychoactive substance in the world, even during pregnancy. Its stimulatory effects are mainly due to antagonism of adenosine actions by blocking adenosine A1 and A2A receptors. Previous studies have shown that caffeine can cross the placenta and therefore modulate these receptors not only in the fetal brain but also in the heart. Methods: In the present work, the effect of caffeine chronically consumed during pregnancy on A1 and A2A receptors in Wistar rat heart, from both mothers and their fetuses, were studied using radioligand binding, Western-blotting, and adenylyl cyclase activity assays, as well as reverse transcription polymerase chain reaction. Results: Caffeine did not significantly alter A1R neither at protein nor at gene expression level in both the maternal and fetal heart. On the contrary, A2AR significantly decreased in the maternal heart, although mRNA was not affected. Gi and Gs proteins were also preserved. Finally, A1R-mediated inhibition of adenylyl cyclase activity did not change in the maternal heart, but A2AR mediated stimulation of this enzymatic activity significantly decreased according to the detected loss of this receptor. Conclusions: Opposite to the downregulation and desensitization of the A1R/AC pathway previously reported in the brain, these results show that this pathway is not affected in rat heart after caffeine exposure during pregnancy. In addition, A2AR is downregulated and desensitized in the maternal heart, suggesting a differential modulation of these receptor-mediated pathways by caffeine.
Project description:Compounds designed to display polypharmacology may have utility in treating complex diseases, where activity at multiple targets is required to produce a clinical effect. In particular, suitable compounds may be useful in treating neurodegenerative diseases by promoting neuronal survival in a synergistic manner via their multi-target activity at the adenosine A1 and A2A receptors (A1R and A2AR) and phosphodiesterase 10A (PDE10A), which modulate intracellular cAMP levels. Hence, in this work we describe a computational method for the design of synthetically feasible ligands that bind to A1 and A2A receptors and inhibit phosphodiesterase 10A (PDE10A), involving a retrosynthetic approach employing in silico target prediction and docking, which may be generally applicable to multi-target compound design at several target classes. This approach has identified 2-aminopyridine-3-carbonitriles as the first multi-target ligands at A1R, A2AR and PDE10A, by showing agreement between the ligand and structure based predictions at these targets. The series were synthesized via an efficient one-pot scheme and validated pharmacologically as A1R/A2AR-PDE10A ligands, with IC50 values of 2.4-10.0 ?M at PDE10A and Ki values of 34-294 nM at A1R and/or A2AR. Furthermore, selectivity profiling of the synthesized 2-amino-pyridin-3-carbonitriles against other subtypes of both protein families showed that the multi-target ligand 8 exhibited a minimum of twofold selectivity over all tested off-targets. In addition, both compounds 8 and 16 exhibited the desired multi-target profile, which could be considered for further functional efficacy assessment, analog modification for the improvement of selectivity towards A1R, A2AR and PDE10A collectively, and evaluation of their potential synergy in modulating cAMP levels.
Project description:Cholesterol is a key component of cell membranes with a proven modulatory role on the function and ligand-binding properties of G-protein-coupled receptors (GPCRs). Crystal structures of prototypical GPCRs such as the adenosine A2A receptor (A2AR) have confirmed that cholesterol finds stable binding sites at the receptor surface suggesting an allosteric role of this lipid. Here we combine experimental and computational approaches to show that cholesterol can spontaneously enter the A2AR-binding pocket from the membrane milieu using the same portal gate previously suggested for opsin ligands. We confirm the presence of cholesterol inside the receptor by chemical modification of the A2AR interior in a biotinylation assay. Overall, we show that cholesterol's impact on A2AR-binding affinity goes beyond pure allosteric modulation and unveils a new interaction mode between cholesterol and the A2AR that could potentially apply to other GPCRs.
Project description:To investigate the involvement of peripheral adenosine receptors in the effect of electroacupuncture (EA) on visceral pain in mice with inflammatory bowel disease (IBD). 2,4,6-Trinitrobenzene sulfonic acid (TNBS) was used to induce the visceral pain model. EA (1 mA, 2 Hz, 30 min) treatment was applied to bilateral acupoints "Dachangshu" (BL25) 1 day after TNBS injection once daily for 7 consecutive days. Von Frey filaments were used to measure the mechanical pain threshold. Western blot was used to detect the protein expression levels of adenosine 1 receptor (A1R), adenosine 2a receptor (A2aR), adenosine 2b receptor (A2bR), adenosine 3 receptor (A3R), substance P (SP), and interleukin 1 beta (IL-1?) in colon tissue. EA significantly ameliorated the disease-related indices and reduced the expression of SP and IL-1? in the colon tissues of mice with IBD. EA increased the expression of A1R, A2aR, and A3R and decreased the expression of A2bR in the colon tissue. Furthermore, the administration of adenosine receptor antagonists influenced the effect of EA. EA can inhibit the expression of the inflammatory factors SP and IL-1? by regulating peripheral A1, A2a, A2b, and A3 receptors, thus inhibiting visceral pain in IBD mice.
Project description:Adenosine is an endogenous nucleoside in the central nervous system that acts on adenosine receptors. These are G protein-coupled receptors that have four known subtypes: A1, A2A, A2B, and A3 receptors. In the present study, we aimed to map the location of the adenosine receptor subtypes in adult wild-type zebrafish retina using in situ hybridization and immunohistochemistry. A1R, A2AR, and A2BR mRNA were detected in the ganglion cell layer (GCL), the inner nuclear layer (INL), the outer nuclear layer (ONL), and the outer segment (OS). A3R mRNA was detected in the GCL, ONL, and OS. A1R-immunoreactivity was expressed as puncta in the INL and in the outer plexiform layer (OPL). A1Rs were located within the cone pedicle and contiguous to horizontal cell tips in the OPL. A2AR-immunoreactivity was expressed as puncta in the GCL, inner plexiform layer (IPL), INL, and outer retina. A2AR puncta in the outer retina were situated around the ellipsoids and nuclei of cones, and weakly around the rod nuclei. A1Rs and A2ARs were clustered around ON cone bipolar cell terminals and present in the OFF lamina of the INL but were not expressed on mixed rod/cone response bipolar cell terminals. A2BR-immunoreactivity was mainly localized to the Müller cells, while A3Rs were found to be expressed in retinal ganglion cells of the GCL, INL, ONL, and OS. In summary, all four adenosine receptor subtypes were localized in the zebrafish retina and are in agreement with expression patterns shown in retinas from other species.