### A. Overview

Extracellular purines (adenosine, ADP, and ATP) and pyrimidines (UDP and UTP) are important signaling molecules that mediate diverse biological effects via cell-surface receptors termed purine receptors. In this review particular emphasis is placed on the discrepancy between the

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Receptors for Purines and Pyrimidines

Four adenosine receptors have been cloned and characterized from several mammalian species. The receptors are named adenosine A(1), A(2A), A(2B), and A(3). The A(2A) and A(2B) receptors preferably interact with members of the G(s) family of G proteins and the A(1) and A(3) receptors with G(i/o) proteins. However, other G protein interactions have also been described. Adenosine is the preferred endogenous agonist at all these receptors, but inosine can also activate the A(3) receptor. The levels of adenosine seen under basal conditions are sufficient to cause some activation of all the receptors, at least where they are abundantly expressed. Adenosine levels during, e.g., ischemia can activate all receptors even when expressed in low abundance. Accordingly, experiments with receptor antagonists and mice with targeted disruption of adenosine A(1), A(2A), and A(3) expression reveal roles for these receptors under physiological and particularly pathophysiological conditions. There are pharmacological tools that can be used to classify A(1), A(2A), and A(3) receptors but few drugs that interact selectively with A(2B) receptors. Testable models of the interaction of these drugs with their receptors have been generated by site-directed mutagenesis and homology-based modelling. Both agonists and antagonists are being developed as potential drugs.

https://pharmrev.aspetjournals.org/content/pharmrev/53/4/527.full.pdf

International Union of Pharmacology. XXV. Nomenclature and Classification of Adenosine Receptors

“Receptors recognize a distinct chemical entity and translate information from that entity into a form that the cell can read to alter its state” (Kenakin et al., 1992). Even though the receptors are often pharmacologically defined on the basis of synthetic compounds, they are assumed to have developed to respond to endogenous molecules. Therefore, receptors are generally named on the basis of their natural ligands. Hence, it is appropriate to very briefly summarize the evidence that purine nucleotides and nucleosides are natural ligands for a wide class of receptors.

In a seminal paper, Drury and Szent-Gyorgyi (1929) showed that adenosine exerted a large number of biological effects, including bradycardia and vasodilation. A wider interest in the role of adenosine followed from the demonstration in 1963 that adenosine can be produced by the hypoxic heart. Two groups independently formulated the hypothesis that adenosine may be involved in the metabolic regulation of coronary blood flow (Berne, 1963; Gerlach et al., 1963). The observation by de Gubareff and Sleator (1965) that the actions of adenosine in heart tissue could be blocked by caffeine suggested the existence of an adenosine receptor. The potent cardiovascular effects of adenosine led to an interest in the synthesis of new adenosine analogs, and careful dose-response studies with a number of these drugs (Cobbin et al., 1974) strongly suggested the presence of a receptor for adenosine-like compounds. Sattin and Rall (1970) reported that adenosine increased cyclic AMP accumulation in slices of rodent brain and that this adenosine-induced second-messenger response was blocked by methylxanthines. Their findings suggested that adenosine receptors exist in the central nervous system. The essentially simultaneous findings by Mcilwain (1972), that such brain slices actually elaborate adenosine in concentrations that would be sufficient to elevate cyclic AMP, provided support that these putative receptors were physiologically occupied by adenosine. Thus, in the 1970s there was good evidence that there were receptors for adenosine at which methylxanthines acted as antagonists. Biochemical evidence for the existence of multiple adenosine receptors was subsequently provided by the demonstration that adenosine analogs increased cyclic AMP production in some preparations and decreased it in others. Because the relative agonist potency for a variety of adenosine analogs was different for these two types of effects, the presence of two classes of receptors, called A1 and A2 (van Calker et al., 1979) or Ri and Ra (Londos et al., 1980), was proposed. The A1/A2 nomenclature is now generally used.

The presence of receptors for ADP, particularly on blood platelets, was also recognized several decades ago. Studies of the factors in blood that induce platelet aggregation led to the identification of ADP as an active component present in red blood cell extracts (Gaarder et al., 1961). The evidence that ADP and adenosine (presumably A2) receptors exist on platelets was summarized by Haslam and Cusack (1981).

Four decades ago, ATP was shown to produce important cardiovascular effects (Green and Stoner, 1950) and to be released from sensory nerves (Holton and Holton, 1954; Holton, 1959), hinting at a role in neural transmission. In his landmark review of purinergic nerves, Burnstock (1972) postulated the existence of specific ATP receptors. Although evidence in support of this idea was not overwhelming at the time, many subsequent studies have supported the existence of receptors for extracellular ATP (Burnstock and Brown, 1981; Gordon, 1986; O’Connor et al., 1991). Similarly, the evidence is now compelling that ATP plays important physiological and/ or pathophysiological roles in a variety of biological systems, including that of a neurotransmitter in peripheral and central neurons. Finally, diadenosinetetraphosphate is a dinucleotide stored in synaptic vesicles and chromaffin granules (Flodgaard and Klenow, 1982; Rodriguez del Castillo et al., 1988) and released therefrom (Pintor et al., 1991a, 1992). The purine dinucleotide also binds with subnanomolar affinity to receptors (Pintor et al., 1991b, 1993) and exerts biological effects (Pintor et al., 1993), indicating that it is an endogenous purinoceptor ligand.

Thus, strong evidence for the presence of receptors for the endogenous ligands adenosine, ADP, ATP, and dia-denosinetetraphosphate had accumulated. This group of receptors is called the purinoceptors. If at some future time there is compelling evidence that UTP, or another pyrimidine nucleotide, is an endogenous ligand at receptors that respond poorly or not at all to ATP, then this terminology may need revision.

https://pharmrev.aspetjournals.org/content/pharmrev/46/2/143.full.pdf

Nomenclature and classification of purinoceptors

Adenosine receptors are major targets of caffeine, the most commonly consumed drug in the world There is growing evidence that they could also be promising therapeutic targets in a wide range of conditions, including cerebral and cardiac ischaemic diseases, sleep disorders, immune and inflammatory disorders and cancer After more than three decades of medicinal chemistry research, a considerable number of selective agonists and antagonists of adenosine receptors have been discovered, and some have been clinically evaluated, although none has yet received regulatory approval However, recent advances in the understanding of the roles of the various adenosine receptor subtypes, and in the development of selective and potent ligands, as discussed in this review, have brought the goal of therapeutic application of adenosine receptor modulators considerably closer

Adenosine receptors as therapeutic targets

In the 10 years since our previous International Union of Basic and Clinical Pharmacology report on the nomenclature and classification of adenosine receptors, no developments have led to major changes in the recommendations. However, there have been so many other developments that an update is needed. The fact that the structure of one of the adenosine receptors has recently been solved has already led to new ways of in silico screening of ligands. The evidence that adenosine receptors can form homo- and heteromultimers has accumulated, but the functional significance of such complexes remains unclear. The availability of mice with genetic modification of all the adenosine receptors has led to a clarification of the functional roles of adenosine, and to excellent means to study the specificity of drugs. There are also interesting associations between disease and structural variants in one or more of the adenosine receptors. Several new selective agonists and antagonists have become available. They provide improved possibilities for receptor classification. There are also developments hinting at the usefulness of allosteric modulators. Many drugs targeting adenosine receptors are in clinical trials, but the established therapeutic use is still very limited.

https://pharmrev.aspetjournals.org/content/pharmrev/63/1/1.full.pdf

International Union of Basic and Clinical Pharmacology. LXXXI. Nomenclature and Classification of Adenosine Receptors—An Update

The human A3 adenosine receptor was cloned from a striatal cDNA library using a probe derived from the homologous rat sequence. The cDNA encodes a protein of 318 amino acids and exhibits 72% and 85% overall identity with the rat and sheep A3 adenosine receptor sequences, respectively. Specific and saturable binding of the adenosine receptor agonist N6-(4-amino-3-[125I]iodobenzyl)adenosine [125I]ABA was measured on the human A3 receptor stably expressed in Chinese hamster ovary cells with a Kd = 10 nM. The potency order for adenosine receptor agonists was N-ethylcarboxamidoadenosine (NECA) > or = (R)-N6-phenyl-2-propyladenosine [(R)-PIA] > N6-cyclopentyladenosine (CPA) > (S)-N6-phenyl-2-propyladenosine [(S)-PIA]. The human receptor was blocked by xanthine antagonists, most potently by 3-(3-iodo-4-aminobenzyl)-8-(4-oxyacetate)phenyl-1-propylxanthine (I-ABOPX) with a potency order of I-ABOPX > 1,3-dipropyl-8-(4-acrylate)phenylxanthine > or = xanthine amino congener >> 1,3-dipropyl-8-cyclopentylxanthine. Adenosine, NECA, (R)- and (S)-PIA, and CPA inhibited forskolin-stimulated cAMP accumulation by 30-40% in stably transfected cells; I-ABA is a partial agonist. When measured in the presence of antagonists, the dose-response curves of NECA-induced inhibition of forskolin-stimulated cAMP accumulation were right-shifted. Antagonist potencies determined by Schild analyses correlated well with those established by competition for radioligand binding. The A3 adenosine receptor transcript is widespread and, in contrast to the A1, A2a, and A2b transcripts, the most abundant expression is found in the lung and liver. The tissue distribution of A3 mRNA is more similar to the widespread profile found in sheep than to the restricted profile found in the rat. This raises the possibility that numerous physiological effects of adenosine may be mediated by A3 adenosine receptors.

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Molecular cloning and characterization of the human A3 adenosine receptor

Using the polymerase chain reaction, an A3 adenosine receptor has been cloned from the hypophysial par tuberalis of sheep. The clone encodes a 317-amino acid protein that is 72% identical to the rat A3 adenosine receptor. In contrast to rat, where abundant A3 mRNA transcript is found primarily in testis, the sheep transcript is most abundant in lung, spleen, and pineal gland and is present in moderate levels in brain, kidney, and testis. The agonist N6-amino[125I]iodobenzyladenosine binds with high affinity (Kd congruent to 6 nm) and specificity to recombinant A3 adenosine receptors expressed transiently in COS-1 cells or stably in CHO K1 cells. The potency order of agonists is N6-aminoiodobenzyladenosine > N-ethylcarboxamidoadenosine > or = (R)-phenylisopropyladenosine >> cyclopentyladenosine. Little or no binding of purine nucleotides was detected. The potency order of antagonists is 3-(3-iodo-4-aminobenzyl)-8-(4-oxyacetate)phenyl-1- propylxanthine (I-ABOPX) (Ki = 3 nM) > 1,3-dipropyl-8-(4-acrylate)phenylxanthine (BW-A1433) > 1,3-dipropyl-8-sulfophenylxanthine = xanthine amine cogener >> 8-cyclopentyl-1,3-dipropylxanthine. Enprofylline does not bind. These data indicate that, in contrast to A1 adenosine receptors, A3 adenosine receptors preferentially bind ligands with aryl rings in the N6-position of adenine and in the C8-position of xanthine. Among antagonists, the A3 adenosine receptor preferentially binds 8-phenylxanthines with acidic versus basic para-substituents (I-ABOPX > BW-A1433 > 1,3-dipropyl-8-sulfophenylxanthine = xanthine amine cogener). Agonists reduce forskolin-stimulated cAMP accumulation in Chinese hamster ovary cells stably transfected with recombinant sheep A3 adenosine receptors; the reduction is blocked by BW-A1433 but not by 8-cyclopentyl-1,3-dipropylxanthine. These data suggest that (i) A3 adenosine receptors display unusual structural diversity for species homologs, (ii) in contrast to rat, sheep A3 adenosine receptors have a broad tissue distribution, and (iii) some xanthines with acidic side chains bind with high affinity to A3 adenosine receptors.

Molecular cloning and functional expression of a sheep A3 adenosine receptor with widespread tissue distribution.

A1 adenosine receptors. Two amino acids are responsible for species differences in ligand recognition.

Double tagging recombinant A1- and A2A-adenosine receptors with hexahistidine and the FLAG epitope. Development of an efficient generic protein purification procedure.

The four human adenosine receptors (ARs), A 1 , A 2A , A 2B , and A 3 , have been stably expressed in mammalian cells. Radioligand binding properties were examined for all four subtypes, including, for the first time the A 2B AR. The A 1 AR-selective radioligand, [ 3 H]8-cyclopentyl-1,3-dipropylxanthine ([ 3 H]CPX; A 1 ; K D = 2 nM) also can be used to characterize recombinant A 2B AR (K D = 40 nM). Theophylline and enprofylline both bind to A 2B AR with K 1 of 7 μM, well within the therapeutic ranges of these compounds used to treat asthma. We have identified C B -(N-methylisopropyl)-amino-N 6 -(5'-endohydroxy)-endonorbornan-2-yl-9-methyladenine (WRC-0571) as the most selective antagonist of A 1 AR. Recombinant ARs have been extended with hexahistidine (H) and the FLAG (F) epitope to make H/F-A 1 and H/F-A 2A receptors that have been purified to near homogeneity under conditions that preserve radioligand binding. [ 3 H]Adenosine binds to purified H/F-A 1 AR-G protein complexes with a Ko of 0.95 ± 0.3 nM; binding is not affected by the adenosine deaminase inhibitor, erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA). Phosphoaminoacid analysis of H/F-A 1 AR and H/F-A 2A AR purified from mammalian cells incubated in [ 32 P]phosphate indicates that both receptors are phosphorylated on serine residues. Monoclonal antibodies were raised by using the purified H/F-A 2A AR as an antigen. Antibodies with higher affinity than antipeptide antisera were generated and used for Western blotting and rat brain immunocytochemistry.

Heidi E. Taylor

Papers

Molecular cloning and characterization of the human A3 adenosine receptor

Molecular cloning and functional expression of a sheep A3 adenosine receptor with widespread tissue distribution.

A1 adenosine receptors. Two amino acids are responsible for species differences in ligand recognition.

Double tagging recombinant A1- and A2A-adenosine receptors with hexahistidine and the FLAG epitope. Development of an efficient generic protein purification procedure.

Molecular characterization of recombinant human adenosine receptors