The A3 adenosine receptor: An enigmatic player in cell biology

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Abstract

Adenosine is a primordial signaling molecule present in every cell of the human body that mediates its physiological functions by interacting with 4 subtypes of G-protein-coupled receptors, termed A1, A2A, A2B and A3. The A3 subtype is perhaps the most enigmatic among adenosine receptors since, although several studies have been performed in the years to elucidate its physiological function, it still presents in several cases a double nature in different pathophysiological conditions. The 2 personalities of A3 often come into direct conflict, e.g., in ischemia, inflammation and cancer, rendering this receptor as a single entity behaving in 2 different ways. This review focuses on the most relevant aspects of A3 adenosine subtype activation and summarizes the pharmacological evidence as the basis of the dichotomy of this receptor in different therapeutic fields. Although much is still to be learned about the function of the A3 receptor and in spite of its duality, at the present time it can be speculated that A3 receptor selective ligands might show utility in the treatment of ischemic conditions, glaucoma, asthma, arthritis, cancer and other disorders in which inflammation is a feature. The biggest and most intriguing challenge for the future is therefore to understand whether and where selective A3 agonists or antagonists are the best choice.

Introduction

The A3 adenosine receptor (A3AR) is the only adenosine subtype which was cloned before its pharmacological identification. It was originally isolated as an orphan receptor from rat testis, having 40% sequence homology with canine A1 and A2A subtypes (Meyerhof et al., 1991) and was identical with the A3AR later cloned from rat striatum (Zhou et al., 1992). Homologs of the rat striatal A3AR have been cloned from sheep and human, revealing large interspecies differences in A3AR structure. For example, the rat A3AR presents only 74% sequence homology with sheep and human A3AR, while there is 85% homology between sheep and human A3AR. This is reflected in the very different pharmacological profiles of the species homologs, especially in terms of antagonist binding that has made characterization of this adenosine subtype difficult. Recently equine A3AR has been cloned and pharmacologically characterized. Sequencing of the cDNA indicated that it has a high degree of sequence similarity with that of other mammalian A3AR transcripts, including human and sheep (Brandon et al., 2006).

The A3AR has been mapped on human chromosome 1p21-p13 (Atkinson et al., 1997) and consists of 318 aminoacid residues. Murrison et al. (1996) determined that the A3AR gene contains 2 exons separated by a single intron of about 2.2 kb. The upstream sequence does not contain a TATA-like motif, but it has a CCAAT sequence and consensus binding sites for SP1, NF-IL6, GATA1 and GATA3 transcription factors. Involvement of the latter in transcriptional control of this gene would be consistent with a role of the receptor in immune function. The A3AR is a G-protein-coupled receptor (GPCR) characterized by its C-terminal portion facing the intracellular compartment and 7 transmembrane spanning domains. In contrast to other adenosine receptors, the C-terminal region presents multiple serine and threonine residues, which may serve as potential sites of phosphorylation that are important for rapid receptor desensitization upon agonist application (Palmer & Stiles, 2000). Phosphorylation leads to a decrease of the number of receptors in the high-affinity state and a decrease of agonist potency to inhibit adenylyl cyclase activity. At the same time, the receptor is reversibly internalized in an agonist-dependent fashion (Trincavelli et al., 2002a).

The A3AR has widely distributed its mRNA being expressed in testis, lung, kidneys, placenta, heart, brain, spleen, liver, uterus, bladder, jejunum, proximal colon and eye of rat, sheep and humans (Zhou et al., 1992, Salvatore et al., 1993, Linden, 1994, Rivkees, 1994, Dixon et al., 1996). However, marked differences exist in expression levels within and among species. In particular rat testis and mast cells express high concentrations of A3 mRNA, while low levels have been detected in most other rat tissues (Linden et al., 1993, Salvatore et al., 1993). Lung and liver have been found as the organs expressing high levels of A3 mRNA in human, while low levels have been found in aorta and brain (Salvatore et al., 1993). Lung, spleen, pars tuberalis and pineal gland expressed the highest levels of A3 mRNA in sheep.

The presence of A3AR protein has been evaluated through radioligand binding, immunoassay or functional assay in a variety of primary cells, tissues (Table 1) and cell lines (Table 2). In the mouse brain a widespread, relatively low level of A3AR binding sites, with a density (Bmax) of 15 fmol/mg of protein in cerebellum, was found (Jacobson et al., 1993). Similar data were obtained in the rat and in gerbil and rabbit brain (Ji et al., 1994). Due to this very low expression, other authors reported that from in situ hybridization experiments, it was not possible to detect either the A3 receptor gene or binding site in the central nervous system (CNS; Rivkees et al., 2000) and others described the expression of the A3AR in thalamus and hypothalamus (Yaar et al., 2002). However, electrophysiological and biochemical evidence suggested the presence of A3AR in the rat hippocampus (Dunwiddie et al., 1997, Macek et al., 1998, Lopes et al., 2003a) and cortex (Brand et al., 2001), and functional studies also indicated its presence in the brain (Jacobson et al., 1993, Von Lubitz et al., 1994, Haskó et al., 2005). In cardiomyocytes, there was no direct evidence of the presence of A3AR (Peart & Headrick, 2007) but a plethora of studies reported that it was responsible for cardioprotection in a variety of species and models, including isolated cardiomyocytes and isolated myocardial muscle preparations (Tracey et al., 1997, Shneyvays et al., 1998, Thourani et al., 1999a, Shneyvays et al., 2001, Cross et al., 2002, Harrison et al., 2002, Germack and Dickenson, 2004, Headrick and Peart, 2005, Xu et al., 2006). In the rat mast cell line RBL-2H3, binding experiments detected a density of about 1 pmol/mg of protein (Olah et al., 1994, Ramkumar et al., 1993) and several authors reported a role for A3AR in rat mast cell degranulation (Carruthers and Fozard, 1993, Fozard and Carruthers, 1993, Ramkumar et al., 1993, Hannon et al., 1995, El-Hashim et al., 1996, Fozard et al., 1996). In enteric neurons and epithelial cells, the A3AR was evidenced by immunohistochemical studies (Christofi et al., 2001), and subsequently, it was quantified in colonic mucosa by radioligand binding experiments (Gessi et al., 2004a). In lung parenchyma and in human lung type 2 alveolar-like cells (A549), the A3AR was detected through radioligand binding and immunohistochemical assays (Varani et al., 2006). Of note, the A3AR was detected in a variety of primary cells involved in inflammatory responses. Human eosinophils were the first cells in which native human A3AR was detected by using radioligand binding (Khono et al., 1996a), and then it was demonstrated in human neutrophils (Bouma et al., 1997, Gessi et al., 2002, Chen et al., 2006a), monocytes (Broussas et al., 1999, Broussas et al., 2002, Thiele et al., 2004), macrophages (McWhinney et al., 1996, Szabo et al., 1998), dendritic cells (Panther et al., 2001, Dickenson et al., 2003, Fossetta et al., 2003, Hofer et al., 2003) and lymphocytes (Gessi et al., 2004b). Finally, a very high expression of A3AR protein was observed in a variety of cancer cell lines (Merighi et al., 2001, Suh et al., 2001, Gessi et al., 2001, Gessi et al., 2007) and in cancer tissues, suggesting a role for this subtype as a tumoral marker (Gessi et al., 2004a, Madi et al., 2004).

The classical pathways associated with A3AR activation are the inhibition of adenylyl cyclase activity, through the coupling with Gi proteins, and the stimulation of phospholipase C (PLC), inositol triphosphate (IP3) and intracellular calcium (Ca2+), via Gq proteins (Ramkumar et al., 1993, Abbracchio et al., 1995, Palmer et al., 1995). However, more recently additional intracellular pathways have been described as relevant for A3AR signaling. For example, in the heart, A3AR mediates cardioprotective effects through ATP-sensitive potassium (KATP) channel activation. Moreover, it is coupled to activation of RhoA and a subsequent stimulation of phospholipase D (PLD), which in turn mediates protection of cardiac myocytes from ischemia (Mozzicato et al., 2004). In addition, in different recombinant and native cell lines, A3AR is involved, like the other adenosine subtypes, in the modulation of mitogen-activated protein kinase (MAPK) activity (Schulte & Fredholm, 2000). A3AR signaling in Chinese Hamster Ovary cells transfected with human A3AR (CHO-hA3) leads to stimulation of extracellular signal-regulated kinases (ERK1/2). In particular, A3AR signaling to ERK1/2 depends on βγ release from pertussis toxin (PTX)-sensitive G proteins, phosphoinositide 3-kinase (PI3K), Ras and mitogen-activated protein kinase kinase (MEK; Schulte & Fredholm, 2002). Functional A3AR activating ERK1/2 has also been described in microglia cells (Hammarberg et al., 2003). In this cell model, by selectively stimulating the A3AR in both primary mouse microglia cells and in the N13 microglia cell line with the agonist 2-chloro-N6-(3-iodobenzyl)-N-methyl-5′-carbamoyladenosine (Cl-IB-MECA), a biphasic, partly Gi-protein-dependent influence on the phosphorylation of the ERK1/2 has been found. In colon cancer cells, after adenosine deaminase (ADA) treatment, A3AR activation stimulates cell proliferation through ERK1/2 activation (Gessi et al., 2007). In contrast in melanoma cells it stimulates PI3K-dependent phosphorylation of protein kinase B (PKB/Akt) leading to the reduction of basal levels of ERK1/2 phosphorylation, which in turn inhibits cell proliferation (Merighi et al., 2005a). This mechanism may be selective for melanoma cells, having a misregulation of proliferative pathways. Furthermore, it has been shown that MAPK activation is involved in A3AR regulation, both contributing to direct phosphorylation and controlling GPCR kinase protein membrane translocation, which are involved in receptor phosphorylation. Thus, an active MAPK signaling pathway appears to be essential for A3AR phosphorylation, desensitization and internalization (Trincavelli et al., 2002b). ERK1/2 are also involved in cardiac hypertrophy and can play a protective role in ischaemic myocardium. Interestingly, A3AR activation in rat cardiomyocytes has been demonstrated to increase ERK1/2 phosphorylation by involving Gi/o proteins, protein kinase C (PKC) and tyrosine kinase-dependent and independent pathways. It has been found that Cl-IB-MECA produced a biphasic effect on cAMP accumulation with a stimulatory action starting at a concentration of 3 nM. This activity was triggered through PLC/PKC and not via direct Gs coupling (Germack & Dickenson, 2004). Another important pathway triggered by adenosine via A3AR is that of PI3K/Akt. There is evidence that A3AR activation mediates phosphorylation of PKB/Akt, protecting rat basophilic leukemia (RBL)-2H3 mast cells from apoptosis by involving the βγ subunits of Gi and PI3K-β. It is well known that protein kinase A (PKA) and PKB/Akt phosphorylate and inactivate glycogen synthase kinase 3β (GSK-3β), a serine/threonine kinase acting as a key element in the Wnt signaling pathway. In its active form, GSK-3β suppresses mammalian cell proliferation (Fishman et al., 2002). It has been reported that A3AR activation is able to decrease the levels of PKA, a downstream effector of cAMP, and of the phosphorylated form of PKB/Akt in melanoma cells. This implies the deregulation of the Wnt signaling pathway, generally active during embryogenesis and tumorigenesis to increase cell cycle progression and cell proliferation (Fishman et al., 2002). Involvement of the PI-3K/PKB pathway has been linked with preconditioning effects induced by A3AR activation in cardiomyocytes from newborn rats (Germack & Dickenson, 2005). An elegant study has recently documented a role of A3AR in cell survival signaling in resveratrol preconditioning of the heart. This study provides evidence that resveratrol preconditions the heart through the activation of adenosine A1 and A3AR, transmitting a survival signal through both the PI3K-Akt-Bcl2 and, only in the case of A3AR, cAMP response element-binding protein (CREB)-Bcl2 pathways (Das et al., 2005a). Subsequently it has been demonstrated that CREB phosphorylation occurs through both Akt-dependent and -independent signaling (Das et al., 2005b). Activation of PI3K-Akt-pBAD by A3AR has been observed recently in glioblastoma cells leading to cell survival in hypoxic conditions (Merighi et al., 2007a). Further studies indicate that A3AR activation by interfering with PKB/Akt pathways can decrease interleukin-12 (IL-12) production in human monocytes (Haskó et al., 1998, la Sala et al., 2005). Collectively, these findings demonstrate that several intracellular mechanisms are involved following A3AR stimulation, the understanding of which may be essential and crucial for explaining the different aspect of its activation.

Section snippets

Neuroprotection versus neurodegeneration

Considerable interest has been shown in understanding the involvement of A3AR in normal and pathological conditions of the CNS despite its low expression in the brain (Rivkees et al., 2000). Even though the function of A3AR in the CNS has been controversial in terms of protective versus toxic actions, actually several data point towards a neuroprotective effect. Firstly, a dual role of A3AR was described in a model of global ischemia in gerbils where acute preischemic administration of the

Cardioprotection versus cardiotoxicity

To date several pieces of evidence support the conclusion that activation of A3AR is crucial for cardioprotection during and following ischemia–reperfusion and it has been suggested that a consistent part of the cardioprotective effects exerted by adenosine, once largely attributed to the A1 receptor, may now be in part ascribed to A3AR activation (Headrick & Peart, 2005). Even though there is a low expression of A3AR in myocardial tissue, a number of studies have demonstrated that acute

Anti-inflammatory versus proinflammatory effects

The interest in the elucidation of A3AR involvement in inflammation is attested by the large amount of experimental work carried out in cells of the immune system and in a variety of inflammatory conditions (Fig. 3). However, as in the SNC or in the cardiovascular system the A3AR subtype appears to have a complex or “enigmatic” role, as both proinflammatory and antiinflammatory effects have been demonstrated. One of the first evidence for a role of A3AR in increasing inflammation derived by

Antitumor versus tumorigen effects

A very interesting area of potential application of A3 ligands concerns cancer therapies. The possibility that A3 adenosine receptor plays a role in the development of cancer has aroused considerable interest in recent years (Fishman et al., 2002, Merighi et al., 2003). The A3 subtype has been described in the regulation of the cell cycle and both pro- and antiapoptotic effects have been reported depending on the level of receptor activation (Jacobson, 1998, Yao et al., 1997, Gao et al., 2001,

Immunosuppressive versus immunostimulating effects

The ability of immune cells to fight tumor cells is fundamental for successful host defense against cancer. Adenosine, whose concentration increases within hypoxic regions of solid tumors, may interfere with the recognition of tumor cells by cytolytic effector cells of the immune system (Blay et al., 1997, Merighi et al., 2003). Adoptive immunotherapy with lymphokine-activated killer (LAK) cells has shown some promise in the treatment of certain cancers that are unresponsive to conventional

Outlook

The study of A3AR and its ligands is a rapidly growing and intense area of research in drug discovery. There is now extensive evidence for the involvement of A3AR in many disease pathways, and therefore continued research to discover agonists and antagonists for this target is warranted. Our knowledge of the structure and function of A3AR has evolved dramatically in the last decade, but still this receptor, that we classically like to imagine similar to the double-personality of the character

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