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Sleep control is ascribed to a two-process model, a widely accepted concept that posits homoeostatic drive and a circadian process as the major sleep-regulating factors. Cognitive and emotional factors also influence sleep–wake behaviour; however, the precise circuit mechanisms underlying their effects on sleep control are unknown. Previous studies suggest that adenosine has a role affecting behavioural arousal in the nucleus accumbens (NAc), a brain area critical for reinforcement and reward. Here, we show that chemogenetic or optogenetic activation of excitatory adenosine A2A receptor-expressing indirect pathway neurons in the core region of the NAc strongly induces slow-wave sleep. Chemogenetic inhibition of the NAc indirect pathway neurons prevents the sleep induction, but does not affect the homoeostatic sleep rebound. In addition, motivational stimuli inhibit the activity of ventral pallidum-projecting NAc indirect pathway neurons and suppress sleep. Our findings reveal a prominent contribution of this indirect pathway to sleep control associated with motivation.

Determination of the crystal structure of the human A 2A adenosine receptor in complex with an inverse-agonist antibody shows that the allosteric site of the receptor inhibits agonist binding and that the antibody locks the receptor in an inactive conformation. GPCR modulation by an antibody G-protein-coupled receptors are the largest class of cell-surface receptors, and many of them are considered drug targets. The crystal structure of the human A2A adenosine receptor in complex with a mouse antibody fragment has now been determined. This structure includes the intracellular loop 3, critical for G-protein binding. In addition, the use of an antibody fragment to stabilize the receptor in an inactive conformation suggests a new strategy to modulate the activity of G-protein-coupled receptors. G-protein-coupled receptors are the largest class of cell-surface receptors, and these membrane proteins exist in equilibrium between inactive and active states 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 . Conformational changes induced by extracellular ligands binding to G-protein-coupled receptors result in a cellular response through the activation of G proteins. The A 2A adenosine receptor (A 2A AR) is responsible for regulating blood flow to the cardiac muscle and is important in the regulation of glutamate and dopamine release in the brain 14 . Here we report the raising of a mouse monoclonal antibody against human A 2A AR that prevents agonist but not antagonist binding to the extracellular ligand-binding pocket, and describe the structure of A 2A AR in complex with the antibody Fab fragment (Fab2838). This structure reveals that Fab2838 recognizes the intracellular surface of A 2A AR and that its complementarity-determining region, CDR-H3, penetrates into the receptor. CDR-H3 is located in a similar position to the G-protein carboxy-terminal fragment in the active opsin structure 1 and to CDR-3 of the nanobody in the active β 2 -adrenergic receptor structure 2 , but locks A 2A AR in an inactive conformation. These results suggest a new strategy to modulate the activity of G-protein-coupled receptors.

G-protein coupled receptors (GPCRs) constitute the largest class of membrane proteins and are a major drug target. A serious obstacle to studying GPCR structure/function characteristics is the requirement to extract the receptors from their native environment in the plasma membrane, coupled with the inherent instability of GPCRs in the detergents required for their solubilization. In the present study, we report the first solubilization and purification of a functional GPCR [human adenosine A2A receptor (A2AR)], in the total absence of detergent at any stage, by exploiting spontaneous encapsulation by styrene maleic acid (SMA) co-polymer direct from the membrane into a nanoscale SMA lipid particle (SMALP). Furthermore, the A2AR–SMALP, generated from yeast (Pichia pastoris) or mammalian cells, exhibited increased thermostability (∼5°C) compared with detergent [DDM (n-dodecyl-β-D-maltopyranoside)]-solubilized A2AR controls. The A2AR–SMALP was also stable when stored for prolonged periods at 4°C and was resistant to multiple freeze-thaw cycles, in marked contrast with the detergent-solubilized receptor. These properties establish the potential for using GPCR–SMALP in receptor-based drug discovery assays. Moreover, in contrast with nanodiscs stabilized by scaffold proteins, the non-proteinaceous nature of the SMA polymer allowed unobscured biophysical characterization of the embedded receptor. Consequently, CD spectroscopy was used to relate changes in secondary structure to loss of ligand binding ([3H]ZM241385) capability. SMALP-solubilization of GPCRs, retaining the annular lipid environment, will enable a wide range of therapeutic targets to be prepared in native-like state to aid drug discovery and understanding of GPCR molecular mechanisms.

CD73 inhibits antitumor immunity through the activation of adenosine receptors expressed on multiple immune subsets. CD73 also enhances tumor metastasis, although the nature of the immune subsets and adenosine receptor subtypes involved in this process are largely unknown. In this study, we revealed that A ₂A/A ₂B receptor antagonists were effective in reducing the metastasis of tumors expressing CD73 endogenously (4T1.2 breast tumors) and when CD73 was ectopically expressed (B16F10 melanoma). A ₂A⁻/⁻ mice were strongly protected against tumor metastasis, indicating that host A ₂A receptors enhanced tumor metastasis. A ₂A blockade enhanced natural killer (NK) cell maturation and cytotoxic function in vitro, reduced metastasis in a perforin-dependent manner, and enhanced NK cell expression of granzyme B in vivo, strongly suggesting that the antimetastatic effect of A ₂A blockade was due to enhanced NK cell function. Interestingly, A ₂B blockade had no effect on NK cell cytotoxicity, indicating that an NK cell-independent mechanism also contributed to the increased metastasis of CD73 ⁺ tumors. Our results thus revealed that CD73 promotes tumor metastasis through multiple mechanisms, including suppression of NK cell function. Furthermore, our data strongly suggest that A ₂A or A ₂B antagonists may be useful for the treatment of metastatic disease. Overall, our study has potential therapeutic implications given that A ₂A/A ₂B receptor antagonists have already entered clinical trials in other therapeutic settings.

Background Cancer immunotherapy offers a promising approach in cancer treatment. The adenosine A2A receptor (A2AR) could protect cancerous tissues from immune clearance via inhibiting T cells response. To date, the role of A2AR in head and neck squamous cell carcinoma (HNSCC) has not been investigated. Here, we sought to explore the expression and immunotherapeutic value of A2AR blockade in HNSCC. Methods The expression of A2AR was evaluated by immunostaining in 43 normal mucosae, 48 dysplasia and 165 primary HNSCC tissues. The immunotherapeutic value of A2AR blockade was assessed in vivo in genetically defined immunocompetent HNSCC mouse model. Results Immunostaining of HNSCC tissue samples revealed that increased expression of A2AR on tumor infiltrating immune cells correlated with advanced pathological grade, larger tumor size and positive lymph node status. Elevated A2AR expression was also detected in recurrent HNSCC and HNSCC tissues with induction chemotherapy. The expression of A2AR was found to be significantly correlated with HIF-1α, CD73, CD8 and Foxp3. Furthermore, the increased population of CD4 + Foxp3 + regulatory T cells (Tregs), which partially expressed A2AR, was observed in an immunocompetent mouse model that spontaneously develops HNSCC. Pharmacological blockade of A2AR by SCH58261 delayed the tumor growth in the HNSCC mouse model. Meanwhile, A2AR blockade significantly reduced the population of CD4 + Foxp3 + Tregs and enhanced the anti-tumor response of CD8 + T cells. Conclusions These results offer a preclinical proof for the administration of A2AR inhibitor on prophylactic experimental therapy of HNSCC and suggest that A2AR blockade can be a potential novel strategy for HNSCC immunotherapy.

Caffeine produces mild psychostimulant and sometimes anxiogenic effects by antagonizing adenosine at A 1 and A 2A receptors, and perhaps through interactions with other transmitter systems. Adenosine receptors are colocalized and functionally interact with dopamine receptors in the brain. Thus, functional polymorphisms in the genes for either adenosine or dopamine receptors may affect responses to caffeine. In this study, we examined associations between self-reported anxiogenic effects of caffeine and variation in the genes for A 2A ( ADORA2A ) and DRD 2 ( DRD2 ) receptors. Healthy male and female individuals ( n =102), who consumed less than 300 mg caffeine per week, ingested capsules containing 0, 50, 150, and 450 mg caffeine under double-blind conditions in four separate experimental sessions. Subjective anxiety was measured before and at repeated times after capsules were consumed. At the 150 mg dose of caffeine, we found a significant association between caffeine-induced anxiety (Visual Analog Scales, VAS) and ADORA2A rs5751876 (1976C/T), rs2298383 (intron 1a) and rs4822492 (3′-flank), and DRD2 rs1110976 (intron 6). Caffeine-induced anxiety (VAS) was also associated with two-loci interactions of selected ADORA2A and DRD2 polymorphisms. The lowest dose of caffeine did not increase ratings of anxiety while the highest dose increased anxiety in the majority of subjects. These findings provide support for an association between an ADORA2A polymorphism and self-reported anxiety after a moderate dose of caffeine. It is likely that other ADORA2A and DRD2 polymorphisms also contribute to responses to caffeine.

G protein-coupled receptors (GPCRs) are the largest family of plasma membrane-embedded signaling proteins. These receptors are involved in a wide array of physiological processes, marking them as attractive targets for drug development. Bitopic ligands, which are comprised of a pharmacophore that targets the receptor orthosteric site and a linked moiety that binds to a separate site, have considerable potential for addressing GPCR function. Here, we report the synthesis and evaluation of novel bitopic conjugates consisting of a small molecule pharmacophore that activates the adenosine A2A receptor (A2AR) linked to antibody fragments (nanobodies, Nbs). This approach leverages the high-affinity and specificity binding of Nbs to non orthosteric sites on engineered A2AR variants to provide bitopic Nb-ligand conjugates that stimulate strong and enduring signaling responses. We further demonstrate that such bitopic conjugates can induce activation by spanning two distinct receptor protomers. This property enables the selective targeting of receptor pairs over either individual receptor, as a form of “logic-gated” activity. We showcase the broad applicability of bitopic conjugates in this context by demonstrating their activity in targeting several pairs of co-expressed receptors, including GPCR monomers from different classes. Furthermore, we demonstrate that this dual-targeting strategy initiates signaling responses that diverge from those induced by monovalent ligands. The ability to target receptor pairs using Nb-ligand conjugates offers a powerful strategy with potential for cell type-selective signaling and implications for GPCR drug discovery efforts more broadly.

The liver enzyme cytochrome P450 1A2 (CYP1A2) is responsible for 90% of caffeine metabolism, while caffeine exerts many of its effects via antagonist binding to adenosine A2a receptors (ADORA2A). This study aimed to examine whether functional single nucleotide polymorphisms (SNPs) in 1976T > C (ADORA2A; rs5751876) and −163C > A (CYP1A2; rs762551) influence the effect of caffeine on the postprandial glucose (GLU) response to a carbohydrate meal. We report that individuals with the 1976T > C CC, but not CT/TT genotypes display elevated GLU levels after consuming caffeine and carbohydrate (CHO + CAFF) versus carbohydrate only (CHO). The GLU area under the curve (AUC) was also greater during the CHO + CAFF condition compared to the CHO condition in CC, but not the CT/TT genotypes. The −163C > A AC/CC, but not AA, genotypes displayed greater GLU concentrations 60-min post meal during CHO + CAFF versus CHO. Our data suggest that caffeine-induced impairments in postprandial glycaemia are related to 1976T > C and −163C > A SNPs.

Vascular inflammation and endothelial dysfunction secondary to unchecked activation of endothelium are key mechanisms underlying sepsis and organ failure. However, the intrinsic processes that mitigate excessive endothelial cell activation remain incompletely understood. To determine the central role of adenosine A2a receptor (A2aR) on macrophages in modulating lipopolysaccharide (LPS)‐induced vascular endothelial dysfunction, we constructed macrophage A2aR‐conditional knockout (Mac‐A2aR KO) mice, and stimulated the mice and macrophages with LPS. A2aR agonist, CGS21680, was administered to these models to further explore its impact. Results showed that knockout of Macrophage A2aR exacerbated LPS‐induced vascular permeability, oedema, inflammatory cardiac damage and upregulated expression of intercellular adhesion molecule‐1 (ICAM‐1) and E‐selectin in cardiopulmonary vascular endothelium. Moreover, deletion of A2aR on macrophages also markedly aggravated LPS‐induced increases in reactive oxygen species (ROS) and declines in antioxidant enzyme gene mRNA and protein expression levels related to oxidative stress (OS). Furthermore, deficiency of A2aR in bone marrow‐derived macrophages (BMDMs) promotes LPS‐induced macrophage M1 polarisation and secretion of inflammatory cytokines, especially tumour necrosis factor‐alpha (TNF‐α). Conversely, the pretreatment with CGS21680 in vivo and in vitro showed corresponding improvement in functions of vascular endothelial dysfunction. These data demonstrate that A2aR in macrophages represents a promising novel therapeutic target for LPS‐induced uncontrolled vascular endothelial injury and inflammation potentially through reducing macrophage M1 polarisation and OS and inhibiting the production and release of TNF‐α production.

Ischemia-reperfusion (IR) injury after lung transplantation, which affects both short- and long-term allograft survival, involves activation of NADPH oxidase 2 (NOX2) and activation of invariant natural killer T (iNKT) cells to produce IL-17. Adenosine A2A receptor (A2AR) agonists are known to potently attenuate lung IR injury and IL-17 production. However, mechanisms for iNKT cell activation after IR and A2AR agonist-mediated protection remain unclear. We tested the hypothesis that NOX2 mediates IL-17 production by iNKT cells after IR and that A2AR agonism prevents IR injury by blocking NOX2 activation in iNKT cells. An in vivo murine hilar ligation model of IR injury was used, in which left lungs underwent 1 hour of ischemia and 2 hours of reperfusion. Adoptive transfer of iNKT cells from p47(phox-/-) or NOX2(-/-) mice to Jα18(-/-) (iNKT cell-deficient) mice significantly attenuated lung IR injury and IL-17 production. Treatment with an A2AR agonist attenuated IR injury and IL-17 production in wild-type (WT) mice and in Jα18(-/-) mice reconstituted with WT, but not A2AR(-/-), iNKT cells. Furthermore, the A2AR agonist prevented IL-17 production by murine and human iNKT cells after acute hypoxia-reoxygenation by blocking p47(phox) phosphorylation, a critical step for NOX2 activation. NOX2 plays a key role in inducing iNKT cell-mediated IL-17 production and subsequent lung injury after IR. A primary mechanism for A2AR agonist-mediated protection entails inhibition of NOX2 in iNKT cells. Therefore, agonism of A2ARs on iNKT cells may be a novel therapeutic strategy to prevent primary graft dysfunction after lung transplantation.