Membrane mimetic-dependence of GPCR energy landscapes.

We leveraged variable-temperature 19F-NMR spectroscopy to compare the conformational equilibria of the human A2A adenosine receptor (A2AAR), a class A G protein-coupled receptor (GPCR), across a range of temperatures ranging from lower temperatures typically employed in 19F-NMR experiments to physiological temperature. A2AAR complexes with partial agonists and full agonists showed large increases in the population of a fully active conformation with increasing temperature. NMR data measured at physiological temperature were more in line with functional data. This was pronounced for complexes with partial agonists, where the population of active A2AAR was nearly undetectable at lower temperature but became evident at physiological temperature. Temperature-dependent behavior of complexes with either full or partial agonists exhibited a pronounced sensitivity to the specific membrane mimetic employed. Cellular signaling experiments correlated with the temperature-dependent conformational equilibria of A2AAR in lipid nanodiscs but not in some detergents, underscoring the importance of the membrane environment in studies of GPCR function.

State-Targeting Stabilization of Adenosine A2A Receptor by Fusing a Custom-Made De Novo Designed α-Helical Protein.

G-protein coupled receptors (GPCRs) are known for their low stability and large conformational changes upon transitions between multiple states. A widely used method for stabilizing these receptors is to make chimeric receptors by fusing soluble proteins (i.e., fusion partner proteins) into the intracellular loop 3 (ICL3) connecting the transmembrane helices 5 and 6 (TM5 and TM6). However, this fusion approach requires experimental trial and error to identify appropriate soluble proteins, residue positions, and linker lengths for making the fusion. Moreover, this approach has not provided state-targeting stabilization of GPCRs. Here, to rationally stabilize a class A GPCR, adenosine A2A receptor (A2AR) in a target state, we carried out the custom-made de novo design of α-helical fusion partner proteins, which can fix the conformation of TM5 and TM6 to that in an inactive state of A2AR through straight helical connections without any kinks or intervening loops. The chimeric A2AR fused with one of the designs (FiX1) exhibited increased thermal stability. Moreover, compared with the wild type, the binding affinity of the chimera against the agonist NECA was significantly decreased, whereas that against the inverse agonist ZM241385 was similar, indicating that the inactive state was selectively stabilized. Our strategy contributes to the rational state-targeting stabilization of GPCRs.

Crystal Structure and Subsequent Ligand Design of a Nonriboside Partial Agonist Bound to the Adenosine A2A Receptor.

In this study, we determined the crystal structure of an engineered human adenosine A2A receptor bound to a partial agonist and compared it to structures cocrystallized with either a full agonist or an antagonist/inverse agonist. The interaction between the partial agonist, belonging to a class of dicyanopyridines, and amino acids in the ligand binding pocket inspired us to develop a small library of derivatives and assess their affinity in radioligand binding studies and potency and intrinsic activity in a functional, label-free, intact cell assay. It appeared that some of the derivatives retained the partial agonist profile, whereas other ligands turned into inverse agonists. We rationalized this remarkable behavior with additional computational docking studies.

Statistics for the analysis of molecular dynamics simulations: providing P values for agonist-dependent GPCR activation.

Molecular dynamics (MD) is the common computational technique for assessing efficacy of GPCR-bound ligands. Agonist efficacy measures the capability of the ligand-bound receptor of reaching the active state in comparison with the free receptor. In this respect, agonists, neutral antagonists and inverse agonists can be considered. A collection of MD simulations of both the ligand-bound and the free receptor are needed to provide reliable conclusions. Variability in the trajectories needs quantification and proper statistical tools for meaningful and non-subjective conclusions. Multiple-factor (time, ligand, lipid) ANOVA with repeated measurements on the time factor is proposed as a suitable statistical method for the analysis of agonist-dependent GPCR activation MD simulations. Inclusion of time factor in the ANOVA model is consistent with the time-dependent nature of MD. Ligand and lipid factors measure agonist and lipid influence on receptor activation. Previously reported MD simulations of adenosine A2a receptor (A2aR) are reanalyzed with this statistical method. TM6-TM3 and TM7-TM3 distances are selected as dependent variables in the ANOVA model. The ligand factor includes the presence or absence of adenosine whereas the lipid factor considers DOPC or DOPG lipids. Statistical analysis of MD simulations shows the efficacy of adenosine and the effect of the membrane lipid composition. Subsequent application of the statistical methodology to NECA A2aR agonist, with resulting P values in consistency with its pharmacological profile, suggests that the method is useful for ligand comparison and potentially for dynamic structure-based virtual screening.

Insights into adenosine A2A receptor activation through cooperative modulation of agonist and allosteric lipid interactions.

The activation process of G protein-coupled receptors (GPCRs) has been extensively studied, both experimentally and computationally. In particular, Molecular Dynamics (MD) simulations have proven useful in exploring GPCR conformational space. The typical behaviour of class A GPCRs, when subjected to unbiased MD simulations from their crystallized inactive state, is to fluctuate between inactive and intermediate(s) conformations, even with bound agonist. Fully active conformation(s) are rarely stabilized unless a G protein is also bound. Despite several crystal structures of the adenosine A2a receptor (A2aR) having been resolved in complex with co-crystallized agonists and Gs protein, its agonist-mediated activation process is still not completely understood. In order to thoroughly examine the conformational landscape of A2aR activation, we performed unbiased microsecond-length MD simulations in quadruplicate, starting from the inactive conformation either in apo or with bound agonists: endogenous adenosine or synthetic NECA, embedded in two homogeneous phospholipid membranes: 1,2-dioleoyl-sn-glycerol-3-phosphoglycerol (DOPG) or 1,2-dioleoyl-sn-glycerol-3-phosphocholine (DOPC). In DOPC with bound adenosine or NECA, we observe transition to an intermediate receptor conformation consistent with the known adenosine-bound crystal state. In apo state in DOPG, two different intermediate conformations are obtained. One is similar to that observed with bound adenosine in DOPC, while the other is closer to the active state but not yet fully active. Exclusively, in DOPG with bound adenosine or NECA, we reproducibly identify receptor conformations with fully active features, which are able to dock Gs protein. These different receptor conformations can be attributed to the action/absence of agonist and phospholipid-mediated allosteric effects on the intracellular side of the receptor.

Polydeoxyribonucleotide ameliorates lipopolysaccharide-induced acute lung injury via modulation of the MAPK/NF-κB signaling pathway in rats.

Acute lung injury (ALI) is characterized by disruption of the alveolar-capillary membrane resulting in pulmonary edema and accumulation of associated proteinaceous alveolar exudate. Initiation of ALI upregulates tumor necrosis factor-α (TNF-α), which activates nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinases (MAPK) that induce various pro-inflammatory mediators. Polydexyribonucleotide (PDRN) is an adenosine A2A receptor agonist that exerts anti-inflammatory effects by suppressing the production of pro-inflammatory cytokines and apoptosis. We investigated the therapeutic efficiency of PDRN on ALI induced by lipopolysaccharide (LPS) in rats. ALI was induced by intratracheal instillation of LPS (5 mg/kg) in 200 µL saline. The PDRN treatment group received a single intraperitoneal injection of 500 µL saline including PDRN (8 mg/kg) 1 h after ALI induction. To confirm the involvement of the adenosine A2A receptor in PDRN, 8 mg/kg 7-dimethyl-1-propargylxanthine (DMPX) was applied with PDRN treatment. Rats were then sacrificed 12 h after PDRN and DMPX treatments. Intratracheal administration of LPS caused lung tissue damage and significantly increased the lung injury scores and levels of pro-inflammatory cytokines, and apoptotic factors. In addition, MAPK/NF-κB signaling factors were increased by ALI initiation. PDRN treatment potently suppressed expressions of MAPK/NF-κB signaling factors compared to the PDRN + DMPX co-treated group. These alterations led to a reduction of pro-inflammatory cytokines, apoptotic factors, and NF-κB and MAPK signaling, which promoted the recovery of damaged lung tissue. PDRN therapy demonstrated therapeutic effects for LPS-induced ALI compared to the non-treated and DMPX-treated groups. Therefore, PDRN may be used as a therapy for initial treatment of ALI.

Functional characterization of a novel adenosine A2B receptor agonist on short-term plasticity and synaptic inhibition during oxygen and glucose deprivation in the rat CA1 hippocampus.

Adenosine is an endogenous neuromodulator exerting its biological functions via four receptor subtypes, A1, A2A, A2B, and A3. A2B receptors (A2BRs) are expressed at hippocampal level where they are known to inhibit paired pulse facilitation (PPF), whose reduction reflects an increase in presynaptic glutamate release. The effect of A2BRs on PPF is known to be sensitive not only to A2BR blockade but also to the A1R antagonist DPCPX, indicating that it involves A1R activation. In this study we provide the first functional characterization of the newly synthesized non-nucleoside like A2BR agonist P453, belonging to the amino-3,5-dicyanopyridine series. By extracellular electrophysiological recordings, we demonstrated that P453 mimicked the effect of the prototypical A2BR agonist BAY60-6583 in decreasing PPF at Schaffer collateral-CA1 synapses in rat acute hippocampal slices. This effect was prevented by two different A2BR antagonists, PSB603 and MRS1754, and by the A1R antagonist DPCPX. We also investigated the functional role of A2BR during a 2 min of oxygen and glucose deprivation (OGD) insult, known to produce a reversible fEPSP inhibition due to adenosine A1R activation. We found that P453 and BAY60-6583 significantly delayed the onset of fEPSP reduction induced by OGD and the effect was blocked by PSB603. We conclude that P453 is a functional A2BR agonist whose activation decreases PPF by increasing glutamate release at presynaptic terminals and delays A1R-mediated fEPSP inhibition during a 2-minute OGD insult.

Dynamic Role of the G Protein in Stabilizing the Active State of the Adenosine A2A Receptor.

Agonist binding in the extracellular region of the G protein-coupled adenosine A2A receptor increases its affinity to the G proteins in the intracellular region, and vice versa. The structural basis for this effect is not evident from the crystal structures of A2AR in various conformational states since it stems from the receptor dynamics. Using atomistic molecular dynamics simulations on four different conformational states of the adenosine A2A receptor, we observed that the agonists show decreased ligand mobility, lower entropy of the extracellular loops in the active-intermediate state compared with the inactive state. In contrast, the entropy of the intracellular region increases to prime the receptor for coupling the G protein. Coupling of the G protein to A2AR shrinks the agonist binding site, making tighter receptor agonist contacts with an increase in the strength of allosteric communication compared with the active-intermediate state. These insights provide a strong basis for structure-based ligand design studies.

A2A Adenosine Receptor Agonists and their Potential Therapeutic Applications. An Update.

BACKGROUND: Adenosine is an endogenous purine nucleoside, which mediates a variety of important biological processes and diseases, such as vasodilation, inflammation, cancer, wound healing, ischemia reperfusion injury, Parkinson disease, infectious diseases, and other CNS disorders. Particularly important are the A2A receptors that have been expressed in the lung, liver, heart, cardiovascular tissues, leukocytes, neutrophils, and endothelial cells. This review provides an update of the latest A2A receptor agonists developed in the period 2005-2017, their selectivity regarding other adenosine receptors and their potential therapeutic applications. METHODS: I have conducted an extensive search from the most common bibliographic databases for critically review the most recent works on the A2A receptor agonists and their therapeutic applications in inflammation, asthma and chronic obstructive pulmonary disease, myocardial perfusion imaging, sepsis, rheumatoid arthritis, and wound healing, among others. RESULTS: In the last decade, a great deal of effort has been devoted to develop adenosine receptor agonists and antagonists for treatment of a number of diseases. Thus, for A2A receptor agonists more than 130 papers and reviews have been found, many of them highlighting the usefulness of these compounds in the field. CONCLUSIONS: Although so far many of the A2A receptor agonists have failed in clinical trials due to their side effects, some of them have been approved for protection against cardiac ischemia-reperfusion injury and anemia. The recently reported crystal structure of the human A2A receptor in complex with the agonist UK-432097 is a fundamental keystone for the development of new and selective A2A ligands with new therapeutic applications.

Mapping the allosteric sites of the A2A adenosine receptor.

The A2A adenosine receptor (A2A AR) is a G protein-coupled receptor that is pharmacologically targeted for the treatment of inflammation, sepsis, cancer, neurodegeneration, and Parkinson's disease. Recently, we applied long-timescale molecular dynamics simulations on two ligand-free receptor conformations, starting from the agonist-bound (PDB ID: 3QAK) and antagonist-bound (PDB ID: 3EML) X-ray structures. This analysis revealed four distinct conformers of the A2A AR: the active, intermediate 1, intermediate 2, and inactive. In this study, we apply the fragment-based mapping algorithm, FTMap, on these receptor conformations to uncover five non-orthosteric sites on the A2A AR. Two sites that are identified in the active conformation are located in the intracellular region of the transmembrane helices (TM) 3/TM4 and the G protein-binding site in the intracellular region between TM2/TM3/TM6/TM7. Three sites are identified in the intermediate 1 and intermediate 2 conformations, annexing a site in the lipid interface of TM5/TM6. Five sites are identified in the inactive conformation, comprising a site in the intracellular region of TM1/TM7 and in the extracellular region of TM3/TM4 of the A2A AR. We postulate that these sites on the A2A AR be screened for allosteric modulators for the treatment of inflammatory and neurological diseases.

Role and Function of A2A and A3 Adenosine Receptors in Patients with Ankylosing Spondylitis, Psoriatic Arthritis and Rheumatoid Arthritis.

Rheumatoid arthritis (RA), ankylosing spondylitis (AS) and psoriatic arthritis (PsA) are chronic inflammatory rheumatic diseases that affect joints, causing debilitating pain and disability. Adenosine receptors (ARs) play a key role in the mechanism of inflammation, and the activation of A2A and A3AR subtypes is often associated with a reduction of the inflammatory status. The aim of this study was to investigate the involvement of ARs in patients suffering from early-RA (ERA), RA, AS and PsA. Messenger RNA (mRNA) analysis and saturation binding experiments indicated an upregulation of A2A and A3ARs in lymphocytes obtained from patients when compared with healthy subjects. A2A and A3AR agonists inhibited nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) activation and reduced inflammatory cytokines release, such as tumor necrosis factor-α (TNF-α), interleukin (IL)-1ß and IL-6. Moreover, A2A and A3AR activation mediated a reduction of metalloproteinases (MMP)-1 and MMP-3. The effect of the agonists was abrogated by selective antagonists demonstrating the direct involvement of these receptor subtypes. Taken together, these data confirmed the involvement of ARs in chronic autoimmune rheumatic diseases highlighting the possibility to exploit A2A and A3ARs as therapeutic targets, with the aim to limit the inflammatory responses usually associated with RA, AS and PsA.

Validation of a Na+-shift binding assay for estimation of the intrinsic efficacy of ligands at the A2A adenosine receptor.

INTRODUCTION: Determination of the intrinsic efficacy of ligands at the A2A receptor is important for selecting drug candidates, e.g. in the case of inflammatory diseases where agonists are searched for or in Parkinson disease (antagonists). METHODS: Three functional binding assays were compared with up to seven ligands with different efficacies: the GTP-shift method based on the decrease of affinity observed with agonists when GTP is added to the competition binding assay; the Ki ratio method based on the different affinity states of the receptor when using an agonist or antagonist radioligand and the Na+-shift assay based on the difference of affinity of agonists when tested in a medium containing a divalent cation (50mM MgCl2) favoring the G protein coupled agonist-receptor complex or sodium (100mM NaCl) as negative allosteric modulator. RESULTS: The Na+-shift assay proposed herein successfully discriminated the full agonists CGS21680, NECA and adenosine (IC50 ratio=13-14) from the weak inverse agonists ZM241385 and IBMX (IC50 ratio=0.85) and the partial agonists LUF5834 and regadenoson (IC50 ratios equal to 3 and 10, respectively). DISCUSSION: We conclude that the Na+-shift assay proposed herein for the A2A receptors has been validated and represents a rapid, economic and efficient functional binding assay to be used in a drug development program for early estimation of the intrinsic efficacy of hits.

The hybrid molecule, VCP746, is a potent adenosine A2B receptor agonist that stimulates anti-fibrotic signalling.

We have recently described the rationally-designed adenosine receptor agonist, 4-(5-amino-4-benzoyl-3-(3-(trifluoromethyl)phenyl)thiophen-2-yl)-N-(6-(9-((2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxylmethyl)tetrahydro-furan-2-yl)-9H-purin-6-ylamino)hexyl)benzamide (VCP746), a hybrid molecule consisting of an adenosine moiety linked to an adenosine A1 receptor (A1AR) allosteric modulator moiety. At the A1AR, VCP746 mediated cardioprotection in the absence of haemodynamic side effects such as bradycardia. The current study has now identified VCP746 as an important pharmacological tool for the adenosine A2B receptor (A2BAR). The binding and function of VCP746 at the A2BAR was rigorously characterised in a heterologous expression system, in addition to examination of its anti-fibrotic signalling in cardiac- and renal-derived cells. In FlpInCHO cells stably expressing the human A2BAR, VCP746 was a high affinity, high potency A2BAR agonist that stimulated Gs- and Gq-mediated signal transduction, with an apparent lack of system bias relative to prototypical A2BAR agonists. The distinct agonist profile may result from an atypical binding mode of VCP746 at the A2BAR, which was consistent with a bivalent mechanism of receptor interaction. In isolated neonatal rat cardiac fibroblasts (NCF), VCP746 stimulated potent inhibition of both TGF-ß1- and angiotensin II-mediated collagen synthesis. Similar attenuation of TGF-ß1-mediated collagen synthesis was observed in renal mesangial cells (RMC). The anti-fibrotic signalling mediated by VCP746 in NCF and RMC was selectively reversed in the presence of an A2BAR antagonist. Thus, we believe, VCP746 represents an important tool to further investigate the role of the A2BAR in cardiac (patho)physiology.

Structure of the adenosine A(2A) receptor bound to an engineered G protein.

G-protein-coupled receptors (GPCRs) are essential components of the signalling network throughout the body. To understand the molecular mechanism of G-protein-mediated signalling, solved structures of receptors in inactive conformations and in the active conformation coupled to a G protein are necessary. Here we present the structure of the adenosine A(2A) receptor (A(2A)R) bound to an engineered G protein, mini-Gs, at 3.4 Å resolution. Mini-Gs binds to A(2A)R through an extensive interface (1,048 Å2) that is similar, but not identical, to the interface between Gs and the ß2-adrenergic receptor. The transition of the receptor from an agonist-bound active-intermediate state to an active G-protein-bound state is characterized by a 14 Å shift of the cytoplasmic end of transmembrane helix 6 (H6) away from the receptor core, slight changes in the positions of the cytoplasmic ends of H5 and H7 and rotamer changes of the amino acid side chains Arg3.50, Tyr5.58 and Tyr7.53. There are no substantial differences in the extracellular half of the receptor around the ligand binding pocket. The A(2A)R-mini-Gs structure highlights both the diversity and similarity in G-protein coupling to GPCRs and hints at the potential complexity of the molecular basis for G-protein specificity.

[Adenosine A2A receptor as a drug target for treatment of sepsis].

Sepsis is a generalized infection accompanied by response of the body that manifests in a clinical and laboratory syndrome, namely, in the systemic inflammatory response syndrome (SIRS) from the organism to the infection. Although sepsis is a widespread and life-threatening disease, the assortment of drugs for its treatment is mostly limited by antibiotics. Therefore, the search for new cellular targets for drug therapy of sepsis is an urgent task of modern medicine and pharmacology. One of the most promising targets is the adenosine A(2A) receptor (A(2A)AR). The activation of this receptor, which is mediated by extracellular adenosine, manifests in almost all types of immune cells (lymphocytes, monocytes, macrophages, and dendritic cells) and results in reducing the severity of inflammation and reperfusion injury in various tissues. The activation of adenosine A(2A) receptor inhibits the proliferation of T cells and production of proinflammatory cytokines, which contributes to the activation of the synthesis of anti-inflammatory cytokines, thereby suppressing the systemic response. For this reason, various selective A(2A)AR agonists and antagonists may be considered to be drug candidates for sepsis pharmacotherapy. Nevertheless, they remain only efficient ligands and objects of pre-clinical and clinical trials. This review examines the molecular mechanisms of inflammatory response in sepsis and the structure and functions of A(2A)AR and its role in the pathogenesis of sepsis, as well as examples of using agonists and antagonists of this receptor for the treatment of SIRS and sepsis.

Purification and Crystallization of a Thermostabilized Agonist-Bound Conformation of the Human Adenosine A(2A) Receptor.

Crystallization of G protein-coupled receptors (GPCRs) is successful due to the development of generic protein engineering strategies, which has resulted in the structure determination of more than 25 GPCRs, including representatives from class A, B, C, and F. Most of the X-ray structures available correspond to an inactive conformation of the receptor bound to an antagonist. Only a few high-resolution structures of agonist-bound conformations of GPCRs have been determined over the last 6 years. Here, we describe the purification and crystallization protocols of a thermostabilized agonist-bound conformation of the human adenosine A2A receptor.

Selectivity is species-dependent: Characterization of standard agonists and antagonists at human, rat, and mouse adenosine receptors.

Adenosine receptors (ARs) have emerged as new drug targets. The majority of data on affinity/potency and selectivity of AR ligands described in the literature has been obtained for the human species. However, preclinical studies are mostly performed in mouse or rat, and standard AR agonists and antagonists are frequently used for studies in rodents without knowing their selectivity in the investigated species. In the present study, we selected a set of frequently used standard AR ligands, 8 agonists and 16 antagonists, and investigated them in radioligand binding studies at all four AR subtypes, A1, A2A, A2B, and A3, of three species, human, rat, and mouse. Recommended, selective agonists include CCPA (for A1AR of rat and mouse), CGS-21680 (for A2A AR of rat), and Cl-IB-MECA (for A3AR of all three species). The functionally selective partial A2B agonist BAY60-6583 was found to additionally bind to A1 and A3AR and act as an antagonist at both receptor subtypes. The antagonists PSB-36 (A1), preladenant (A2A), and PSB-603 (A2B) displayed high selectivity in all three investigated species. MRS-1523 acts as a selective A3AR antagonist in human and rat, but is only moderately selective in mouse. The comprehensive data presented herein provide a solid basis for selecting suitable AR ligands for biological studies.

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

Adenosine A2A receptor (A2AR)-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 A2AR-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 A2AR agonists, but also A2AR 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 A2AR-D2R heteromers as heterotetramers, constituted by A2AR 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 A2AR antagonists behaved as A2AR agonists and decreased D2R function in the brain.

Design, synthesis, and biological evaluation of novel 2-((2-(4-(substituted)phenylpiperazin-1-yl)ethyl)amino)-5'-N-ethylcarboxamidoadenosines as potent and selective agonists of the A2A adenosine receptor.

Stimulation of A2A adenosine receptors (AR) promotes anti-inflammatory responses in animal models of allergic rhinitis, asthma, chronic obstructive pulmonary disease, and rheumatic diseases. Herein we describe the results of a research program aimed at identifying potent and selective agonists of the A2AAR as potential anti-inflammatory agents. The recent crystallographic analysis of A2AAR agonists and antagonists in complex with the receptor provided key information on the structural determinants leading to receptor activation or blocking. In light of this, we designed a new series of 2-((4-aryl(alkyl)piperazin-1-yl)alkylamino)-5'-N-ethylcarboxamidoadenosines with high A2AAR affinity, activation potency and selectivity obtained by merging distinctive structural elements of known agonists and antagonists of the investigated target. Docking-based SAR optimization allowed us to identify compound 42 as one of the most potent and selective A2A agonist discovered so far (Ki hA2AAR = 4.8 nM, EC50 hA2AAR = 4.9 nM, Ki hA1AR > 10 000 nM, Ki hA3AR = 1487 nM, EC50 hA2BAR > 10 000 nM).

Communication over the network of binary switches regulates the activation of A2A adenosine receptor.

Dynamics and functions of G-protein coupled receptors (GPCRs) are accurately regulated by the type of ligands that bind to the orthosteric or allosteric binding sites. To glean the structural and dynamical origin of ligand-dependent modulation of GPCR activity, we performed total ~ 5 µsec molecular dynamics simulations of A2A adenosine receptor (A2AAR) in its apo, antagonist-bound, and agonist-bound forms in an explicit water and membrane environment, and examined the corresponding dynamics and correlation between the 10 key structural motifs that serve as the allosteric hotspots in intramolecular signaling network. We dubbed these 10 structural motifs "binary switches" as they display molecular interactions that switch between two distinct states. By projecting the receptor dynamics on these binary switches that yield 2(10) microstates, we show that (i) the receptors in apo, antagonist-bound, and agonist-bound states explore vastly different conformational space; (ii) among the three receptor states the apo state explores the broadest range of microstates; (iii) in the presence of the agonist, the active conformation is maintained through coherent couplings among the binary switches; and (iv) to be most specific, our analysis shows that W246, located deep inside the binding cleft, can serve as both an agonist sensor and actuator of ensuing intramolecular signaling for the receptor activation. Finally, our analysis of multiple trajectories generated by inserting an agonist to the apo state underscores that the transition of the receptor from inactive to active form requires the disruption of ionic-lock in the DRY motif.