Characterization and Expression of the Human A2a Adenosine Receptor Gene

Abstract: The actions of the neurotransmitter adenosine are mediated by a family of high-affinity, G protein-coupled receptors. We have characterized the gene for the human A2a subtype of adenosine receptor (hA2aR) and determined levels of A2aR mRNA in human brain regions and nonneural tissues. Human genomic Southern blot analysis demonstrates the presence of a single gene encoding the hA2aR located on chromosome 22. Two overlapping cosmids containing the hA2aR gene were isolated from a chromosome 22 library and characterized. Southern blot and sequence analyses demonstrate that the hA2aR gene spans ∼9–10 kb with a single intron interrupting the coding sequence between the regions encoding transmembrane domains III and IV. The sequence of the hA2aR gene diverged from the reported cDNA structure in the 5' untranslated region. This divergence appears to result from an artifact in the construction of the original cDNA library. By northern blot analysis, high expression of the hA2aR gene was identified in the caudate nucleus with low levels of expression in other brain regions. High expression was also seen in immune tissues; lesser A2aR expression was detected in heart and lung. The gene for the A2a subtype of receptor for the neurotransmitter adenosine falls in the class of intron containing G protein-coupled receptor genes. Expression in the basal ganglia is consistent with a role for the hA2aR in motor control. Activation of the A2aR may also regulate immune responses and cardiopulmonary function.     

Multiple Human Taste Receptor Sites: A Molecular Modeling Approach

Numerous experimental data on the human peripheral taste systemsuggest the existence of multiple low-affinity and low-specificityreceptor sites which are responsible for the detection and thecomplete discrimination of a very large number of organic molecules.According to this hypothesis, a given molecule interacts withnumerous taste receptors and vice versa. Statistical analysisof taste intensities estimated by 58 human subjects for variousmolecules enables the calculation of taste intermolecular distances.For the present modeling study, we hypothesized that a shorttaste distance (i.e. taste similarity) between two distinctmolecules indicates that they bind with similar distributionsof affinities to the taste receptors, and hence display similarbinding motifs. In order to find common molecular binding motifsamong 14 selected organic tastants, hydrogen-bonding and hydrophobicinteraction properties were mapped onto their molecular surfaces.The 14 surfaces were then cut in 240 fragments, most of whichwere made up of 2–4 potentially interacting zones. A correspondenceindex was defined to measure the analogy between two optimallysuperimposed fragments. The 75 most representative fragmentswere all matched pairwise. Twelve distinct clusters of fragmentswere isolated from the 2775 calculated comparisons. These 12fragment types were used to calculate structural similaritydistances. We then performed a combinatorial analysis to identifywhich fragment combination best reconciled structural and tastedistances. We finally identified an optimal subset of sevenfragment types out of the 12, which significantly and best accountedfor the 91 pairwise taste distances between all 14 modeled tastants.These seven validated fragment types are therefore presentedas good candidates to be recognized by the same number of distincttaste receptor sites. Potential applications of these identifiedbinding motifs to tastant design are suggested. Chem. Senses21: 425–445, 1996.     

Human Adenosine A2A Receptor Binds Calmodulin with High Affinity in a Calcium-Dependent Manner

Understanding how ligands bind to G-protein-coupled receptors and how binding changes receptor structure to affect signaling is critical for developing a complete picture of the signal transduction process. The adenosine A2A receptor (A2AR) is a particularly interesting example, as it has an exceptionally long intracellular carboxyl terminus, which is predicted to be mainly disordered. Experimental data on the structure of the A2AR C-terminus is lacking, because published structures of A2AR do not include the C-terminus. Calmodulin has been reported to bind to the A2AR C-terminus, with a possible binding site on helix 8, next to the membrane. The biological meaning of the interaction as well as its calcium dependence, thermodynamic parameters, and organization of the proteins in the complex are unclear. Here, we characterized the structure of the A2AR C-terminus and the A2AR C-terminus-calmodulin complex using different biophysical methods, including native gel and analytical gel filtration, isothermal titration calorimetry, NMR spectroscopy, and small-angle X-ray scattering. We found that the C-terminus is disordered and flexible, and it binds with high affinity (K_d = 98 nM) to calmodulin without major conformational changes in the domain. Calmodulin binds to helix 8 of the A2AR in a calcium-dependent manner that can displace binding of A2AR to lipid vesicles. We also predicted and classified putative calmodulin-binding sites in a larger group of G-protein-coupled receptors.     

Recruitment of a Cytoplasmic Chaperone Relay by the A2A Adenosine Receptor

The adenosine A_2A receptor is a prototypical rhodopsin-like G protein-coupled receptor but has several unique structural features, in particular a long C terminus (of >120 residues) devoid of a palmitoylation site. It is known to interact with several accessory proteins other than those canonically involved in signaling. However, it is evident that many more proteins must interact with the A_2A receptor, if the trafficking trajectory of the receptor is taken into account from its site of synthesis in the endoplasmic reticulum (ER) to its disposal by the lysosome. Affinity-tagged versions of the A_2A receptor were expressed in HEK293 cells to identify interacting partners residing in the ER by a proteomics approach based on tandem affinity purification. The receptor-protein complexes were purified in quantities sufficient for analysis by mass spectrometry. We identified molecular chaperones (heat-shock proteins HSP90α and HSP70-1A) that interact with and retain partially folded A_2A receptor prior to ER exit. Complex formation between the A_2A receptor and HSP90α (but not HSP90β) and HSP70-1A was confirmed by co-affinity precipitation. HSP90 inhibitors also enhanced surface expression of the receptor in PC12 cells, which endogenously express the A_2A receptor. Finally, proteins of the HSP relay machinery (e.g. HOP/HSC70-HSP90 organizing protein and P23/HSP90 co-chaperone) were recovered in complexes with the A_2A receptor. These observations are consistent with the proposed chaperone/coat protein complex II exchange model. This posits that cytosolic HSP proteins are sequentially recruited to folding intermediates of the A_2A receptor. Release of HSP90 is required prior to recruitment of coat protein complex II components. This prevents premature ER export of partially folded receptors.     

Molecular Modelling of the Antagonist Binding Site on the Adenosine A,Receptor

Abstract

With the aid of molecular modelling both adenosine and adenosine A, receptor antagonists belonging to various chemical classes were compared. A model for the antagonist binding site was developed. As a consequence 1H-imidazo[4, 5-c]-quinolin-4-amines were synthesized, constituting a novel class of potent non-xanthine adenosine receptor antagonists.     

Adenosine Stimulates Cone Photoreceptor Myoid Elongation via an Adenosine A2-Like Receptor

In several parts of the nervous system, adenosine has been shown to function as an extracellular neuromodulator binding to surface receptors on target cells. This study examines the possible role of adenosine in mediating light and circadian regulation of retinomotor movements in teleost cone photoreceptors. Teleost cones elongate in the dark and contract in the light. In continuous darkness, the cones continue to elongate and contract at subjective dusk and dawn in response to circadian signals. We report here that exogenous adenosine triggers elongation (the dark/night movement) in isolated cone inner segment-cone outer segment preparations (CIS-COS) in vitro. Agonist/antagonist potency profiles indicate that adenosine's effect on cone movement is mediated by an A2-like adenosine receptor, which like other A2 receptors enhances adenylate cyclase activity. Although closest to that expected for A2 receptors, the antagonist potency profile for CIS-COS does not correspond exactly to any known A2 receptor subtype, suggesting that the cone receptor may be a novel A2 subtype. Our findings are consistent with previous reports that retinal adenosine levels are higher in the dark, and further suggest that adenosine could act as a neuromodulatory "dark signal" influencing photoreceptor metabolism and function in the fish retina.     

A Functional Screening of Adenosine Analogues at the Adenosine A2B Receptor: A Search for Potent Agonists

Abstract

Various adenosine analogues were tested at the adenosine A_2B receptor. Agonist potencies were determined by measuring the cyclic AMP production in Chinese Hamster Ovary cells expressing human A_2B receptors. 5′-.N-Substituted carboxamidoadenosines were most potent. 5′-N-Ethylcarboxamidoadenosine (NECA) was most active with an ECso value of 3.1 μM. Other ribose modified derivatives displayed low to negligible activity. Potency was reduced by substitution on the exocyclic amino function (N⁶) of the purine ring system. The most active N⁶-substituted derivative N⁶-methyl-NECA was 5 fold less potent than NECA. C8-and most C2-substituted analogues were virtually inactive. 1-Deaza-analogues had a reduced potency, 3-and 7-deazaanalogues were not active.     

Adenosine and 2-Chloroadenosine Deaza-Analogues as Adenosine Receptor Agonists1

Abstract

Several deaza-analogues of adenosine and 2-chloro-adenosine have been examined for their adenosine receptor affinity. It was found that the relative contribution of the nitrogen atoms of the purine moiety to binding at A₁ rat brain adenosine receptor, follows the order N⁷ > N³ > N¹. The affinity of the adenosine analogues for the adenosine rat brain receptor was besides compared with their activity as inhibitors of platelet aggregation. A synthesis of 2-chloro-1-deazaadenosine by two alternative routes starting from 7-nitroimidazo[4,5-b]pyridine-4-oxide is also reported.     

Platelet Aggregation Inhibitory Activity of Selective A2 Adenosine Receptor Agonists

Abstract

A series of new 2-alkynyl, 2-cycloalkynyl, and 2-aralkynyl derivatives of adenosine-5′-ethyluronamide (NECA) were synthesized and evaluated in binding studies and functional assays to assess their potency and selectivity at A₂ vs A₁ receptors. The new derivatives were also tested as inhibitors of rabbit platelet aggregation induced by ADP. While the presence of an aromatic or heteroaromatic ring conjugated to the triple bond decreased antiplatelet activity, the introduction of a hydroxyl group or a heterocyclic ring on the alkynyl side chain increased the antiaggregatory activity in comparison with NECA, resulting in the most potent inhibitors of platelet aggregation so far known in the nucleoside series. However, the presence of an α-quaternary carbon markedly reduced the antiaggregatory potency without affecting the A₂ binding affinity, suggesting that the platelet receptor is not a typical A_2a site.     

A2B Adenosine Receptor Induces Protective Antihelminth Type 2 Immune Responses

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Caffeine Neuroprotection Decreases A2A Adenosine Receptor Content in Aged Mice

Neurochemical Research - Caffeine is a bioactive compound worldwide consumed with effect into the brain. Part of its action in reducing incidence or delaying Alzheimer’s and Parkinson’s...     

The Synthesis and Biological activity of a Highly Selective Adenosine A2a. Receptor Agonist

Abstract

Three novel nucleosides 1, 2, and 3 were prepared that contained side chains at the 2-position of adenosine. Compound 1 was shown to be the most selective A_2a receptor agonist reported to date having an A₁/A₂ ratio of 2400. In addition, compound 1 was shown to reduce blood pressure in rats and dogs with only minimal effects on heart rate.     

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 A_2A adenosine receptor (A_2AAR) 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 2¹⁰ 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.     

Allosteric Coupling of Drug Binding and Intracellular Signaling in the A2A Adenosine Receptor

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Protein Kinase C-Mediated Suppression of the Presynaptic Adenosine A1 Receptor by a Facilitatory Metabotropic Glutamate Receptor

Abstract: KCl-evoked glutamate exocytosis from cerebrocortical synaptosomes can be inhibited by the adenosine A1 receptor agonist cyclohexyladenosine (CHA). Inhibition is associated with a decreased KCl-evoked Ca²⁺ level elevation, and the effect of the agonist is occluded by prior incubation with the Agelenopsis aperta neurotoxin ω-agatoxin-IVA at 250 n M . The inhibition is suppressed in the presence of 3 n M phorbol dibutyrate (PDBu) or by activation of the protein kinase C (PKC)-coupled metabotropic glutamate receptor by 100 µ M (1 S ,3 R )-1-aminocyclopentane-1,3-dicarboxylate [(1 S ,3 R )ACPD]. A tonic inhibition of release by leaked exogenous adenosine can be reversed by adenosine deaminase or by PDBu addition. The CHA-induced inhibition can be enhanced by the PKC inhibitor Ro 31-8220. The mechanism for the suppression of the adenosine A1 receptor-mediated inhibition is distinct from that previously described for the (1 S ,3 R )ACPD-evoked, PKC-mediated, facilitatory pathway, which enhances phosphorylation of the MARCKS protein, 4-aminopyridine-induced action potentials, and release of glutamate because the latter requires at least 100 n M PDBu [or the combination of (1 S ,3 R )ACPD and arachidonic acid] and is not seen following KCl depolarization. Both PKC-mediated pathways may be involved in the presynaptic events associated with the establishment of synaptic plasticity.