Mark E. Olah

The role of receptor structure in determining adenosine receptor activity.

Adenosine produces a wide variety of physiological effects through the activation of cell surface adenosine receptors (ARs). ARs are members of the G-protein-coupled receptor family, and currently, four subtypes, the A1AR, A2AAR, A2BAR, and A3AR, are recognized. This review focuses on the role of receptor structure in governing various facets of AR activity. Ligand-binding properties of ARs are primarily dictated by amino acids in the transmembrane domains of the receptors, although a role for extracellular domains of certain ARs has been suggested. Studies have identified certain amino acids conserved amongst AR subtypes that are critical for ligand recognition, as well as additional residues that may differentiate between agonist and antagonist ligands. Receptor regions responsible for activation of Gs have been identified for the A2AAR. The location of these intracellular sites is consistent with findings described for other G-protein-coupled receptors. Site-directed mutagenesis has been employed to analyze the structural basis for the differences in the kinetics of the desensitization response displayed by various AR subtypes. For the A2AAR and A3AR, agonist-stimulated phosphorylation of the AR, presumably via a G-protein receptor kinase, has been shown to occur. For these AR subtypes, intracellular regions or individual amino acids that may be targets for this phosphorylation have been identified. Finally, the role of A1AR gene structure in regulating the expression of this AR subtype is reviewed.

Pharmacological characterization of novel A3 adenosine receptor-selective antagonists

The effects of putative A3 adenosine receptor antagonists of three diverse chemical classes (the flavonoid MRS 1067, the 6-phenyl-1,4-dihydropyridines MRS 1097 and MRS 1191, and the triazoloquinazoline MRS 1220) were characterized in receptor binding and functional assays. MRS1067, MRS 1191 and MRS 1220 were found to be competitive in saturation binding studies using the agonist radioligand [125I]AB-MECA (N6-(4-amino-3-iodobenzyl)adenosine-5-N-methyluronamide) at cloned human brain A3 receptors expressed in HEK-293 cells. Antagonism was demonstrated in functional assays consisting of agonist-induced inhibition of adenylate cyclase and the stimulation of binding of [35S]guanosine 5-O-(3-thiotriphosphate) ([35S]GTP-gamma-S) to the associated G-proteins. MRS 1220 and MRS 1191, with KB values of 1.7 and 92 nM, respectively, proved to be highly selective for human A3 receptor vs human A1 receptor-mediated effects on adenylate cyclase. In addition, MRS 1220 reversed the effect of A3 agonist-elicited inhibition of tumor necrosis factor-alpha formation in the human macrophage U-937 cell line, with an IC50 value of 0.3 microM.

A role for central A3-adenosine receptors. Mediation of behavioral depressant effects.

The behavioral effects of a selective A3 adenosine receptor agonist 3‐IB‐MECA (N 6‐(3‐iodobenzyl)‐5‐N‐methylcarboxamidoadenosine) in mice and the localization of radioligand binding sites in mouse brain were examined. Low levels of A3 adenosine receptors were detected in various regions of the mouse brain (hippocampus, cortex, cerebellum, striatum), using a radioiodinated, high‐affinity A3‐agonist radioligand [125I]AB‐MECA (N 6‐(3‐iodo‐4‐aminobenzyl)‐5‐N‐methylcarboxamidoadenosine). Scatchard analysis in the cerebellum showed that the K d value for binding to A3 receptors was 1.39 ± 0.04 nM with a B max of 14.8 ±2.1 protein. 3‐IB‐MECA at 0.1 i.p. was a locomotor depressant with > 50% reduction in activity. Although selective A1 or A2a antagonists reversed locomotor depression elicited by selective A1 or A2a agonists, respectively, the behavioral depressant effects of 3‐IB‐MECA were unaffected. 3‐IB‐MECA also caused scratching in mice, which was prevented by coadministration of the histamine antagonist cyproheptadine. The demonstration of a marked behavioral effect of A3 receptor activation suggests that the A3 receptor represents a potential new therapeutic target.

A SURVEY OF NONXANTHINE DERIVATIVES AS ADENOSINE RECEPTOR LIGANDS

The binding affinities at rat A1, A2a, and A3 adenosine receptors of a wide range of heterocyclic derivatives have been determined. Mono-, bi-, tricyclic and macrocyclic compounds were screened in binding assays, using either [3H]PIA or [3H]CGS 21680 in rat brain membranes or [125I]AB-MECA in CHO cells stably transfected with rat A3 receptors. Several new classes of adenosine antagonists (e.g. 5-oxoimidazopyrimidines and a pyrazoloquinazoline) were identified. Various sulfonylpiperazines, 11-hydroxytetrahydrocarbazolenine, 4H-pyrido[1,2-a]pyrimidinone, folic acid, and cytochalasin H and J bound to A3 receptors selectively. Moreover, cytochalasin A, which bound to A1 adenosine receptors with Ki value of 1.9 μM, inhibited adenylyl cyclase in rat adipocytes, but not via reversible A1 receptor binding.

Purification and characterization of bovine cerebral cortex A1 adenosine receptor.

A1 adenosine receptors (A1AR) acting via the inhibitory guanine nucleotide binding protein inhibit adenylate cyclase activity in brain, cardiac, and adipose tissue. We now report the purification of the A1AR from bovine cerebral cortex. This A1AR is distinct from other A1ARs in that it displays an agonist potency series of N6-R-phenylisopropyladenosine (R-PIA) greater than N6-S-phenylisopropyladenosine greater than (S-PIA) greater than 5-N-ethylcarboxamidoadenosine (NECA) compared to the traditional potency series of R-PIA greater than NECA greater than S-PIA. The A1AR was solubilized in 1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (Chaps) and then purified by chromatography on an antagonist [xanthine amine congener (XAC)]-coupled Affi-Gel 10 followed by hydroxylapatite chromatography. Following purification, sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single protein of Mr 36,000 by silver staining, Na125I iodination with chloramine T and photoaffinity labeling with [125I]8-[4-[[[[2-(4-aminophenyl acetylamino) ethyl] carbonyl] methyl] oxy]-phenyl]-1,3- dipropylxanthine. This single protein displayed all the characteristics of the A1AR, including binding an antagonist radioligand [( 3H]XAC) with high affinity (Kd = 0.7 nM) and in a saturable manner (Bmax greater than 4500 pmol/mg). Agonist competition curves demonstrated the expected bovine brain A1AR pharmacology: R-PIA greater than S-PIA greater than NECA. The overall yield from soluble preparation was 7%. The glycoprotein nature of the purified A1AR was determined with endo- and exoglycosidases. Deglycosylation with endoglycosidase F increased the mobility of the A1AR from Mr 36,000 to Mr 32,000 in a single step. The A1AR was sensitive to neuraminidase but resistant to alpha-mannosidase, suggesting the single carbohydrate chain was of the complex type. This makes the bovine brain A1AR similar to rat brain and fat A1AR in terms of its carbohydrate chains yet the purified A1AR retains its unique agonist potency series observed in membranes.

Agonists and Antagonists Recognize Different but Overlapping Populations of A1 Adenosine Receptors: Modulation of Receptor Number by MgCl2, Solubilization, and Guanine Nucleotides

Abstract: A1 selective agonist and antagonist radioligands bind to the same A1 adenosine receptor binding subunit, as documented by photoaffinity labelling and partial peptide maps. In this study we document that although these radioligands recognize the same A, adenosine receptor (A1AR), they recognize different numbers of A1ARs in bovine brain membranes, with agonist number being greater than antagonist number. Neither addition of guanine nucleotides nor removal of Mg2+ ions enhanced antagonist binding in membranes. On solubilization, agonists still recognized a greater number of A1ARs but addition of guanine nucleotides or removal of Mg2+ substantially increased the number of receptors detected with antagonist radioligands. The effects of Mg2+ and guanine nucleotides were not additive, suggesting that formation of a “low agonist‐receptor‐G protein state” by either modulating agent was sufficient to alter the receptor conformation such that it could be recognized by antagonist. These studies suggest that a proportion of the “precoupled A1AR‐G protein complex” in membranes are in a conformation that cannot be recognized by antagonists and that membrane constraints are such that ions or guanine nucleotides cannot sufficiently modulate the conformation to allow it to recognize antagonists. On removal of membrane structure by solubilization, these constraints are removed.

Affinity chromatography of the bovine cerebral cortex A1 adenosine receptor

An approximate 140‐fold purification of the A1 adenosine receptor of bovine cerebral cortex has been obtained via affinity chromatography. The affinity column consists of Affi‐Gel 10 coupled through an amide linkage to XAC, a high‐affinity A1 adenosine receptor antagonist. As assessed by [3h]XAC binding, bovine brain membranes solubilized with the detergent CHAPS had a specific binding activity of 1.1 protein. Interaction of solubilized A1 adenosine receptors with the XAC‐Affi‐Gel was biospecific and 30% of the receptor activity was bound by the gel. Demonstration of [3h]XAC binding in the material eluted from the column with R‐PIA required insertion of receptor into phospholipid vesicles. The specific activity of the affinity column purified receptor was 146 ± 22 protein with typically 5–15% of the bound receptor recovered. The purified receptor displayed high‐affinity antagonist binding and bound agonists with the potency order expected of the bovine brain A1 adenosine receptor: R‐PIA > S‐PIA >62; NECA. In purified preparations, the photoaffinity probe [1251]PAPAXAC‐SANPAH specifically labelled a protein of molecular mass 38000 which has previously been shown to be the A1 adenosine receptor binding subunit.