Adenosine receptor
Adenosine receptors are a class of G protein-coupled receptors (GPCRs) that bind the endogenous purine nucleoside adenosine to mediate its regulatory effects on cellular signaling and physiological processes across multiple tissues. There are four distinct subtypes in mammals—A1, A2A, A2B, and A3—each encoded by separate genes and exhibiting unique affinities for adenosine, with A1 and A2A displaying high affinity (nanomolar range) and A2B and A3 showing lower affinity (micromolar range).[1] These receptors are characterized by a seven-transmembrane α-helical structure typical of GPCRs, with an extracellular N-terminus for ligand binding and an intracellular C-terminus involved in signal transduction and regulation.[2] The signaling pathways of adenosine receptors primarily involve modulation of adenylyl cyclase activity: A1 and A3 receptors couple to inhibitory G proteins (Gi/o), reducing cyclic AMP (cAMP) levels and inhibiting voltage-gated calcium channels while activating potassium channels, which contributes to presynaptic inhibition of neurotransmitter release.[3] In contrast, A2A and A2B receptors couple to stimulatory G proteins (Gs/olf), increasing cAMP production and activating protein kinase A, which promotes vasodilation, anti-inflammatory responses, and modulation of dopamine signaling in the brain.[4] Additional downstream effects include activation of phospholipase C and mitogen-activated protein kinases, enabling diverse roles in cytoprotection during stress, such as ischemia or hypoxia.[2] Adenosine receptors are widely distributed throughout the body, with A1 predominantly expressed in the brain (e.g., cortex, hippocampus) and heart, where they regulate sleep, seizure activity, and cardiac rate; A2A highly concentrated in the striatum and immune cells, influencing motor control and inflammation; A2B found in peripheral tissues like the lung and gastrointestinal tract, involved in bronchoconstriction and mast cell degranulation; and A3 present in the testis, lung, and immune system, contributing to immune modulation and fibrosis prevention.[3] Their expression and function are dynamically regulated by factors such as phosphorylation by G protein-coupled receptor kinases, desensitization via β-arrestin binding, and transcriptional control under conditions like oxidative stress or hypoxia, which upregulate A2A via NF-κB pathways.[2] Due to their central roles in neuromodulation, cardioprotection, and immunomodulation, adenosine receptors represent key therapeutic targets for conditions including Parkinson's disease, asthma, and ischemia-reperfusion injury.[4]Overview
Definition and general function
Adenosine receptors constitute a family of four subtypes—A1, A2A, A2B, and A3—classified within the P1 purinergic receptor group, which are G-protein-coupled receptors (GPCRs) that selectively bind adenosine as their endogenous ligand.[5] Unlike P2 purinergic receptors, which primarily respond to ATP and other nucleotides, P1 receptors are dedicated to adenosine signaling and play a central role in transducing its effects across various tissues.[6] These receptors were first formally identified in the late 1970s through investigations into adenosine's cardiac actions, such as inducing bradycardia and coronary vasodilation, marking a pivotal advancement in understanding purinergic signaling.[7][8] The primary function of adenosine receptors is to regulate adenosine-mediated signaling, which modulates key physiological processes including neurotransmission, vasodilation, immune responses, and energy homeostasis.[9] This regulation occurs mainly through their coupling to G proteins that either inhibit or stimulate adenylyl cyclase activity, thereby altering intracellular cyclic AMP (cAMP) levels and downstream signaling cascades.[2] For instance, A1 and A3 subtypes typically inhibit adenylyl cyclase via Gi/o proteins, promoting protective responses like reduced excitability, while A2A and A2B subtypes stimulate it via Gs or Golf proteins, enhancing processes such as anti-inflammatory actions.[10] As the endogenous ligand, adenosine is produced via enzymatic breakdown of extracellular ATP by ectonucleotidases or from intracellular cAMP hydrolysis, with its extracellular concentrations rising markedly during stress conditions like hypoxia, ischemia, or inflammation to activate these receptors and restore homeostasis.[9][11] This accumulation serves as a rapid distress signal, enabling adenosine receptors to fine-tune cellular responses in real time.[12]Physiological significance
Adenosine serves as a key retaliatory metabolite, produced in response to cellular energy depletion from ATP breakdown during periods of high metabolic demand, thereby helping to restore energy homeostasis by modulating cellular activity and promoting protective adaptations.[13] This role is evident in its accumulation in brain regions active during wakefulness, where it signals the need for rest by inducing sleep through activation of adenosine receptors, particularly A1 and A2A subtypes, which enhance non-rapid eye movement sleep and slow-wave activity to facilitate energy recovery.[14] Additionally, adenosine exerts anti-inflammatory effects by suppressing pro-inflammatory cytokine release and immune cell activation via receptor-mediated signaling, thereby mitigating tissue damage during stress and supporting resolution of inflammatory responses.[15] The physiological importance of adenosine receptors is underscored by their evolutionary conservation across diverse species, including mammals and invertebrates such as Drosophila melanogaster and Caenorhabditis elegans, where homologs like AdoR regulate stress responses, neuronal activity, and metabolic adaptations, highlighting their fundamental role in cellular signaling and survival mechanisms.[16][17] Dysregulation of adenosine receptor signaling contributes to various pathological conditions, including epilepsy, where reduced adenosine tone exacerbates seizure susceptibility; asthma, characterized by heightened bronchoconstriction and inflammation due to altered receptor responsiveness; and ischemia, in which impaired adenosine-mediated protection amplifies tissue injury during oxygen deprivation.[18][19][20] A critical quantitative aspect of this signaling is the dramatic elevation of extracellular adenosine levels, which can increase more than 100-fold under hypoxic conditions, thereby intensifying receptor activation to elicit protective responses and maintain homeostasis.[21]Molecular structure
Protein architecture
Adenosine receptors belong to the class A subfamily of G protein-coupled receptors (GPCRs), characterized by a conserved topology consisting of seven transmembrane α-helices (7TM) that span the plasma membrane, an extracellular N-terminal domain, and an intracellular C-terminal tail.[2] This arrangement forms a central binding pocket within the transmembrane domain where the endogenous ligand adenosine interacts primarily through hydrogen bonding and hydrophobic contacts with residues in helices 3, 6, and 7.[22] The extracellular N-terminus is typically short and may include glycosylation sites, while the intracellular loops and C-terminus facilitate interactions with G proteins and regulatory proteins.[23] The orthosteric binding site, located deep within the helical bundle, is primarily formed by residues in transmembrane helices 6 and 7, along with contributions from helix 3 and the extracellular loop 2 (ECL2), enabling subtype-selective ligand recognition. Allosteric modulators, which fine-tune receptor activity without competing directly with orthosteric ligands, often bind to sites involving the extracellular loops, particularly ECL2 and ECL3, influencing ligand affinity and efficacy through conformational changes propagated across the receptor.[24] Subtle variations in helix lengths and loop flexibilities exist among subtypes, contributing to differences in ligand binding dynamics.[2] High-resolution crystal structures have elucidated the inactive and active conformations of adenosine receptors. The first structure of the human A2A adenosine receptor, solved in 2008 at 2.6 Å resolution in complex with the antagonist ZM241385, revealed an inactive state with a constricted binding pocket stabilized by ionic locks between helices 3 and 6.[22] Subsequent structures, including the A1 receptor in 2017 at 3.2 Å resolution bound to the covalent antagonist DU172, highlighted a more open orthosteric cavity in the inactive conformation compared to A2A, with active-state insights from agonist-bound A2A complexes showing outward movement of helix 6.[25] More recent advances include cryogenic electron microscopy structures of the full-length human A3 receptor bound to selective agonists in 2024, revealing Gi-coupled active states, and new crystal structures of the A2A receptor with nanomolar antagonists in 2025, further detailing allosteric modulation and subtype-specific conformations.[26][27] These structures underscore the conformational plasticity essential for receptor activation. Post-translational modifications play critical roles in receptor maturation and regulation. N-linked glycosylation at the extracellular N-terminus, typically at asparagine residues, promotes proper folding, trafficking to the cell surface, and stability, with deglycosylation leading to impaired function in some subtypes.[23] Phosphorylation of serine and threonine residues in the intracellular C-terminus and loops by G protein-coupled receptor kinases (GRKs) and second messenger-dependent kinases induces desensitization, β-arrestin recruitment, and internalization following prolonged agonist exposure.[28]Gene expression and distribution
The genes encoding the four subtypes of adenosine receptors in humans are designated ADORA1, ADORA2A, ADORA2B, and ADORA3. The ADORA1 gene is located on chromosome 1q32.1.[29] The ADORA2A gene resides on chromosome 22q12.[30] The ADORA2B gene is situated on chromosome 17p12.[31] The ADORA3 gene maps to chromosome 1p13.2.[32] Expression of adenosine receptor genes exhibits tissue-specific patterns that underlie their physiological roles. The ADORA1 gene is ubiquitously expressed but shows particularly high levels in the brain (including cortex, hippocampus, and cerebellum) and heart, with notable presence in adipose tissue and kidney.[2] ADORA2A expression is prominent in the striatum and basal ganglia of the brain, as well as in immune cells such as leukocytes and platelets.[2] ADORA2B is expressed at moderate to high levels in peripheral tissues like the lung and gastrointestinal tract, with lower abundance in the central nervous system.[2] ADORA3 displays restricted expression, with elevated levels in the testes and immune tissues including mast cells and eosinophils.[2] Regulation of adenosine receptor gene expression involves environmental and genetic factors. Hypoxia-inducible factors (HIFs), particularly HIF-1α, upregulate ADORA2B transcription through a functional HIF-binding site in its promoter, enhancing receptor expression under hypoxic conditions to amplify adenosine signaling.[33] Genetic polymorphisms also influence expression; for instance, the ADORA2A rs5751876 variant (1976T>C) significantly modulates ADORA2A mRNA levels in multiple tissues, including the brain, thereby affecting receptor density and functional responses.[34] Evolutionarily, the adenosine receptor family (P1 purinergic receptors) arose through gene duplications from an ancestral purinergic receptor precursor, with a key whole-genome duplication event in early vertebrates leading to the divergence of subtypes like ADORA2B into paralogs such as adorb1 and adorb2.[35]Signaling mechanisms
G-protein coupling
Adenosine receptors, as members of the class A G protein-coupled receptor (GPCR) family, initiate signaling by coupling to heterotrimeric G proteins upon ligand binding. The subtypes exhibit distinct coupling preferences: A1 and A3 receptors primarily associate with inhibitory Gi/o proteins, which suppress adenylyl cyclase activity; A2A receptors couple to stimulatory Gs proteins (and Golf in the striatum), enhancing adenylyl cyclase function, and recent structural studies have shown they can also couple to Go proteins, albeit with lower efficacy due to distinct conformational dynamics; and A2B receptors mainly engage Gs but can also interact with Gq proteins under certain conditions, leading to phospholipase C activation.[2][36] These couplings determine the overall inhibitory or stimulatory nature of adenosine signaling in target cells.[37] The activation process begins with adenosine or an agonist binding to the orthosteric site in the receptor's transmembrane domain, inducing a conformational shift from inactive to active states. This change exposes key intracellular residues, particularly in transmembrane helix 6 (TM6) and the third intracellular loop (ICL3), which interact with the G protein heterotrimer (Gαβγ). The receptor acts as a guanine nucleotide exchange factor (GEF), catalyzing the release of GDP from the Gα subunit and its replacement with GTP. GTP binding triggers dissociation of the Gα-GTP subunit from the Gβγ dimer, allowing both to engage downstream effectors; the intrinsic GTPase activity of Gα eventually hydrolyzes GTP to GDP, reforming the inactive heterotrimer and terminating signaling until re-engagement.[38][39] Interactions between receptor subunits and G proteins are mediated primarily by the receptor's intracellular domains. The C-terminal tail and ICLs contact the α5 helix of the Gα subunit's C-terminus, stabilizing the active complex and facilitating nucleotide exchange; for instance, in A2A receptors, residues in the C-tail contribute to Gs selectivity. Uncoupling occurs via regulatory mechanisms involving β-arrestins: agonist-bound receptors are phosphorylated on serine/threonine residues in the C-terminal tail by G protein-coupled receptor kinases (GRKs), recruiting β-arrestins that sterically block G protein rebinding and promote receptor internalization.[40][2] Specificity in G protein coupling is influenced by factors such as membrane lipid rafts, cholesterol-rich microdomains that cluster receptors and G proteins to optimize interaction efficiency and signaling fidelity; disruption of rafts alters adenosine receptor-mediated responses in neurons and immune cells. Additionally, experiments with chimeric receptors—swapping intracellular loops or tails between subtypes—reveal interchangeability of coupling preferences, underscoring the modular role of these domains in dictating Gi/o versus Gs/Gq selectivity without altering ligand binding.[41]Downstream effectors
Upon activation by adenosine, adenosine receptors initiate diverse intracellular signaling cascades through their coupled G proteins, primarily modulating adenylyl cyclase (AC) activity to alter cyclic AMP (cAMP) levels. The A1 and A3 subtypes, coupled to Gi/o proteins, inhibit AC, leading to decreased cAMP production and subsequent reduction in protein kinase A (PKA) activity, which dampens downstream processes like gene transcription and ion channel regulation.[9] In contrast, the A2A subtype, coupled to Gs proteins, stimulates AC, resulting in elevated cAMP levels and PKA activation, which promotes cellular responses such as vasodilation and anti-inflammatory effects.[9] The A2B subtype primarily couples to Gs for cAMP increase but can also engage Gq proteins to activate phospholipase C (PLC), particularly at higher agonist concentrations.[9] Beyond AC modulation, adenosine receptors influence other key effectors. For A2B and A3 receptors, Gq-mediated PLC activation hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP2) to produce inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG), elevating intracellular calcium via IP3 receptors and activating protein kinase C (PKC) through DAG.[9] The A1 receptor, via Gi/o βγ subunits, directly opens G protein-coupled inwardly rectifying potassium (GIRK) channels, causing membrane hyperpolarization that inhibits neuronal excitability and cardiac action potentials.[42] Adenosine receptor signaling integrates with broader pathways, including mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) cascades, which mediate cell proliferation, survival, and differentiation; for instance, A2A and A2B activation enhances ERK phosphorylation in immune cells.[9] Similarly, phosphoinositide 3-kinase (PI3K)/Akt pathways are engaged, particularly by A3 receptors, to promote cell survival and anti-apoptotic effects in stressed tissues.[9] Prolonged receptor activation leads to desensitization through phosphorylation by G protein-coupled receptor kinases (GRKs), which recruits β-arrestins to uncouple the receptor from G proteins and facilitate internalization.[28] Quantitative assessments of these effectors reveal subtype-specific potencies; for example, adenosine induces cAMP accumulation via A2A receptors with an EC50 of approximately 0.7 μM in cellular assays, reflecting its high-affinity coupling to Gs-AC signaling.[43] These dose-response characteristics underscore the receptors' roles in fine-tuning physiological responses across tissues.[9]Subtypes
Comparative properties
The four subtypes of adenosine receptors—A1, A2A, A2B, and A3—share a common G protein-coupled receptor architecture but exhibit distinct pharmacological and physiological profiles that enable differential responses to adenosine. These differences arise from variations in G protein coupling, ligand affinity, tissue distribution, and signaling outcomes, allowing the system to fine-tune cellular responses across physiological conditions. A comparative overview is presented in the table below, drawing from established pharmacological characterizations.| Subtype | G-protein coupling | Affinity for adenosine (approximate Ki) | Primary tissues | Key functions |
|---|---|---|---|---|
| A1 | Gi/o (inhibitory) | High (1–10 nM) | Brain (cortex, hippocampus, cerebellum), heart, kidney | Neuroprotection, modulation of neurotransmitter release, negative chronotropic effects in heart |
| A2A | Gs (stimulatory) | High (20–50 nM) | Striatum, immune cells (e.g., leukocytes), blood vessels, olfactory tubercle | Vasodilation, anti-inflammatory effects, regulation of motor function and dopamine signaling |
| A2B | Gs (stimulatory; Gq/11 at high expression) | Low (1–5 μM) | Lung, gastrointestinal tract, immune cells, bladder | Promotion of angiogenesis, modulation of inflammation and fibrosis, cytokine release under hypoxia |
| A3 | Gi/o (inhibitory; Gq/11 in some contexts) | Intermediate (50–100 nM) | Brain (thalamus, hippocampus), immune cells, lung, mast cells | Cardioprotection, immune modulation, induction of apoptosis in certain cell types |
A1 adenosine receptor
The A1 adenosine receptor (A1AR) is a subtype of G protein-coupled receptor that predominantly couples to Gi/o proteins, thereby inhibiting adenylyl cyclase activity and reducing intracellular cyclic AMP (cAMP) levels.[2] This coupling also facilitates the activation of G protein-gated inward rectifier potassium (GIRK) channels, promoting membrane hyperpolarization, while simultaneously inhibiting voltage-gated calcium (Ca²⁺) channels, which decreases neuronal excitability.[44] Presynaptically, A1AR activation suppresses neurotransmitter release, particularly of excitatory transmitters like glutamate, by limiting Ca²⁺ influx and modulating vesicular release machinery in various brain regions.[45] Pharmacologically, A1AR exhibits high affinity for selective agonists such as N⁶-cyclopentyladenosine (CPA), which potently activates the receptor at nanomolar concentrations and mimics endogenous adenosine effects.[46] Non-selective antagonists like caffeine block A1AR at low doses (around 10-50 μM), contributing to its stimulant properties by preventing adenosine-mediated inhibition, though higher doses also affect other subtypes.[47] In cardioprotection, A1AR activation during ischemic preconditioning—brief episodes of ischemia prior to prolonged occlusion—triggers downstream survival pathways that limit infarct size and improve post-ischemic recovery in myocardial tissue.[48] Within the brain, A1AR stimulation exerts anticonvulsant effects by dampening hyperexcitability and halting seizure propagation in experimental models of temporal lobe epilepsy.[18] In the renal system, A1AR mediates vasoconstriction of afferent arterioles, reducing glomerular filtration rate and contributing to tubuloglomerular feedback regulation of blood flow.[49] Preclinical evidence also highlights A1AR's potential in migraine prophylaxis, where agonists inhibit trigeminovascular nociceptive transmission without inducing vasoconstriction, suggesting therapeutic utility.[50]A2A adenosine receptor
The A2A adenosine receptor (A2AR) is a G protein-coupled receptor primarily coupled to the stimulatory G protein (Gs), which upon activation by adenosine or agonists stimulates adenylyl cyclase to increase intracellular cyclic AMP (cAMP) levels, subsequently activating protein kinase A (PKA) and downstream effectors such as CREB for transcriptional regulation.[51][52] This Gs-mediated pathway contrasts with the Gi/o coupling of other adenosine receptor subtypes, enabling A2AR to promote stimulatory signaling in various cell types. In the striatum, A2AR co-localizes with dopamine D2 receptors, forming functional heterodimers that facilitate allosteric interactions, where A2AR activation can modulate D2 receptor signaling and influence striatal medium spiny neuron activity.[53][54] A2AR plays key roles in anti-inflammatory responses by suppressing macrophage activation and cytokine release, predominantly through its high expression on immune cells like monocytes and macrophages, thereby dampening excessive inflammation in conditions such as tissue injury.[55] In the cardiovascular system, A2AR activation induces potent vasodilation in coronary arteries, enhancing blood flow and providing cardioprotection during ischemia by mediating hyperemia without significant effects on heart rate or contractility.[56][57] In the central nervous system, particularly in Parkinson's disease, A2AR antagonism relieves inhibitory effects on dopamine D2 receptor signaling in the basal ganglia, improving motor symptoms by enhancing striatal output and reducing levodopa-induced dyskinesia.[58] Selective pharmacological modulation of A2AR includes the agonist CGS-21680, which exhibits high affinity (Ki ≈ 27 nM) for A2AR and is used in preclinical models to mimic adenosine's effects on cAMP elevation and vasodilation.[59] The antagonist istradefylline (Nourianz), a selective A2AR blocker, was approved by the FDA in 2019 as an adjunct to levodopa/carbidopa for treating "off" episodes in Parkinson's disease patients, demonstrating efficacy in extending "on" time without exacerbating dyskinesia.[60] Crystal structures of A2AR, first resolved in 2008 with antagonists like ZM241385 and later with diverse ligands, have been instrumental in structure-based drug design, revealing key binding pocket residues (e.g., Asn253^{6.55}) that guide the development of subtype-selective modulators for neurological and inflammatory disorders.[61][62] Recent insights highlight A2AR's role in COVID-19-related immunosuppression, where agonists like regadenoson reduced viral burden and inflammation in preclinical models by enhancing immune clearance.[63]A2B adenosine receptor
The A2B adenosine receptor (A2BR), encoded by the ADORA2B gene, is a low-affinity G protein-coupled receptor subtype that binds adenosine with a dissociation constant in the micromolar range, distinguishing it from high-affinity subtypes like A1 and A2A. Unlike other adenosine receptors, A2BR is primarily activated under conditions of elevated extracellular adenosine levels, such as those occurring during pathological states including inflammation, hypoxia, and tissue injury. This context-specific activation positions A2BR as a sensor for stress signals, contributing to adaptive responses in various tissues.[64] A2BR signaling exhibits promiscuity in G protein coupling, primarily associating with Gs proteins at low receptor expression levels to stimulate adenylyl cyclase and elevate cyclic AMP (cAMP) levels, thereby activating protein kinase A (PKA). At higher expression levels or in specific cellular contexts, such as in immune cells or under inflammatory conditions, A2BR couples to Gq/11 proteins, activating phospholipase C (PLC) and leading to inositol trisphosphate (IP3) production, intracellular calcium mobilization, and protein kinase C (PKC) activation. This dual coupling enables diverse downstream effects, with Gs-mediated pathways often promoting anti-inflammatory actions and Gq/PLC pathways facilitating pro-inflammatory mediator release during adenosine surges in pathologies like inflammation.[65][66] In the brain, A2BR modulates glial cell activation, particularly in astrocytes and microglia, contributing to neuroprotection following ischemic events by attenuating neuroinflammation and supporting neuronal survival. Post-ischemia, A2BR stimulation reduces pro-inflammatory cytokine release from glia and promotes tissue repair mechanisms. Additionally, A2BR enhances angiogenesis in hypoxic environments by upregulating vascular endothelial growth factor (VEGF) expression in endothelial cells, aiding oxygen delivery in ischemic tissues. A 2024 study highlighted A2BR's role in astrocytic signaling, where adenosine activation coordinates brain glucose metabolism and lactate release to support neuronal function, underscoring its importance in glial-neuronal metabolic coupling.[67][68][69][70] Pharmacologically, A2BR is targeted by selective agonists like BAY-60-6583, a partial agonist with high potency (EC50 ≈ 3 nM) and selectivity over other adenosine receptor subtypes, used in preclinical models to mimic pathological adenosine effects. Antagonist development remains challenging due to structural homology with the A2A subtype, resulting in limited highly selective A2BR antagonists; compounds like PSB-603 offer some selectivity but often exhibit cross-reactivity. In asthma, A2BR expression is upregulated in airway epithelial and smooth muscle cells, exacerbating inflammation and bronchoconstriction through IL-6 and IL-19 induction, making it a potential therapeutic target.[71][72][73]A3 adenosine receptor
The A3 adenosine receptor (A3AR) is a G protein-coupled receptor primarily coupled to Gi/o proteins, leading to inhibition of adenylyl cyclase and a subsequent decrease in intracellular cyclic AMP levels. This coupling is pertussis toxin-sensitive and mediates many of the receptor's inhibitory effects on cellular processes. Additionally, A3AR exhibits cross-talk with Gq proteins, which can activate phospholipase C and increase inositol trisphosphate production, contributing to calcium mobilization in certain cell types. In cancer cells, A3AR activation modulates the Wnt/β-catenin signaling pathway by downregulating β-catenin levels and inhibiting downstream gene transcription, thereby promoting apoptosis and suppressing proliferation.[74][9][75] A3AR activation confers cytoprotective effects during ischemia, particularly in myocardial and cerebral tissues, by preconditioning cells to reduce infarct size and oxidative stress through mechanisms involving protein kinase C and mitogen-activated protein kinase pathways. In fibroblasts, A3AR stimulation exerts anti-proliferative effects, limiting collagen synthesis and extracellular matrix remodeling, which is relevant in fibrotic conditions. In rheumatoid arthritis, A3AR plays a key role in modulating immune responses via stabilization of mast cells, reducing degranulation and histamine release to attenuate joint inflammation. Unlike the A2B receptor, which promotes acute inflammatory responses through Gq-mediated stimulation under stress, A3AR's Gi/o-dominant signaling favors chronic anti-inflammatory inhibition.[76][9][77][78] Key synthetic ligands for A3AR include the highly selective agonist IB-MECA (also known as Cl-IB-MECA), which mimics adenosine's effects at nanomolar concentrations, and the antagonist MRS-1191, which blocks receptor activation with high potency and specificity. A3AR exhibits lower baseline expression in most normal tissues compared to inflammatory or neoplastic cells, which restricts off-target effects but also limits its broad therapeutic applicability due to the need for targeted delivery in low-expressing contexts. A 2021 review highlighted A3AR's overexpression in hepatocellular carcinoma, where agonists like namodenoson target the receptor to induce tumor regression via Wnt/β-catenin deregulation, with ongoing clinical trials evaluating its efficacy. Species differences are notable, as rodent A3AR shows higher affinity for certain agonists and faster desensitization compared to the human ortholog, complicating preclinical-to-clinical translation.[79][80][74][75][81]Pharmacology
Endogenous and exogenous ligands
Adenosine serves as the primary endogenous ligand for all four subtypes of adenosine receptors (A1, A2A, A2B, and A3), acting as a purine nucleoside that modulates cellular signaling in response to physiological stress such as hypoxia or inflammation.[9] Other endogenous nucleosides, including inosine and guanosine, function as weak partial agonists at these receptors, with inosine exhibiting biased agonism particularly at the A2A subtype to promote ERK1/2 signaling over cAMP pathways.[82] Guanosine enhances extracellular adenosine levels by slowing its cellular disposition through an unidentified mechanism, independent of nucleoside transporters or direct agonism at adenosine receptors.[83] The extracellular availability of these ligands is tightly regulated by nucleoside transporters, such as equilibrative nucleoside transporters (ENTs) and concentrative nucleoside transporters (CNTs), which control uptake and efflux to maintain signaling homeostasis during conditions like ischemia.[84] Exogenous ligands for adenosine receptors encompass a range of synthetic and natural compounds designed to mimic or block endogenous signaling. The xanthine class, including caffeine and theophylline, represents classic non-selective antagonists that competitively inhibit adenosine binding across multiple subtypes, contributing to their stimulant and bronchodilatory effects.[85] For greater subtype specificity, pyrimidine-based derivatives have been synthesized as selective A2B antagonists, such as 3,4-dihydropyrimidin-2(1H)-ones, which exhibit high potency and selectivity to target A2B-mediated pathways without broadly affecting other receptors.[86] Ligand interactions with adenosine receptors occur primarily at the orthosteric site within the receptor's transmembrane helical bundle, where endogenous agonists like adenosine bind to initiate G-protein coupling.[87] Distinct allosteric sites, often located extracellularly or within the intracellular loops, enable modulation of binding kinetics and efficacy by allosteric enhancers or inhibitors, allowing fine-tuning of receptor activation without direct competition at the orthosteric pocket.[87] Adenosine demonstrates moderate affinity across subtypes, with pKi values generally ranging from 6 to 8, reflecting nanomolar binding strengths that vary slightly by receptor (e.g., higher affinity at A1 and A3 compared to A2A and A2B).[9] The history of adenosine receptor ligand development traces back to early 20th-century investigations of xanthines, where caffeine's physiological antagonism was explored in studies from the 1920s onward, laying the groundwork for understanding receptor blockade.[88] By the 1990s, advances in high-throughput screening accelerated the discovery of selective ligands, enabling the identification of subtype-specific agonists and antagonists through large-scale compound libraries and radioligand binding assays.[89]Agonist and antagonist selectivity
Agonist selectivity for adenosine receptors is determined by binding affinities, often measured as inhibition constants (Ki) in radioligand binding assays, which reflect the potency of ligands in competing for receptor sites. Non-selective agonists like 5'-N-ethylcarboxamidoadenosine (NECA) exhibit similar high affinities across subtypes, with Ki values typically in the low nanomolar range for A1, A2A, and A3 receptors but reduced potency at A2B. In contrast, subtype-selective agonists are engineered through structural modifications to favor specific receptors, enabling targeted pharmacological studies and therapeutic applications.[90] The following table summarizes representative Ki values (in nM) for NECA and selected selective agonists at human adenosine receptors, derived from binding assays using recombinant or native tissues:| Ligand | A1 | A2A | A2B | A3 | Selectivity Profile |
|---|---|---|---|---|---|
| NECA (non-selective agonist) | 100 | 310 | 15000 | 290 | Broad potency, lowest at A2B |
| CPA (A1-selective) | 2.3 | 794 | 18600 | 72 | ~300-fold A1 over A2A, ~30-fold over A3 |
| CGS 21680 (A2A-selective) | 289 | 27 | >10000 | 67 | ~10-fold A2A over A1, ~2-fold over A3 |
| BAY 60-6583 (A2B-selective) | >10000 | >10000 | 6.5 | >10000 | >1500-fold A2B over others |
| Cl-IB-MECA (A3-selective) | 220 | 5360 | >10000 | 1.4 | ~150-fold A3 over A1, >3000-fold over A2A |