1. Introduction

Adenosine Deaminase (ADA) is a cytosolic enzyme, which has been the object of considerable interest, mainly because in human a congenital defect in the enzyme causes severe combined immunodeficiency disease (SCID). ADA participates in the purine metabolism where it degrades either adenosine or 2’-deoxyadenosine producing inosine or 2’- deoxyinosine, respectively. Further metabolisation of these deaminated nucleosides leads to hypoxanthine, which can be either transformed into uric acid by xanthine oxidase or salvaged into mononucleotides by the action of hypoxanthine-guanine phosphoribosyl-transferase.
Different laboratories have shown that ADA may appear also on the cell surface (ecto-ADA, see review by (Franco et al., 1997)). Only one human gene for ADA has been detected which codes for a protein lacking both signal peptide and putative transmembrane domains. There probably exists some specific mechanism of ADA release. It should be noted that there are important growth factors (e.g. interleukin-1b) lacking signal peptides whose mechanism of secretion is also not known. No differences in catalytic activity between cytosolic ADA and ecto-ADA have been found.
In addition, ecto-ADA could have functions independent of its enzymatic activity. Ecto-ADA could bind directly to at least three different cell surface molecules, human CD26 (a lymphocytes activation marker), and A1 and A2B adenosine receptors. In such a context, ADA probably acts as a co stimulatory molecule of adenosine receptors and/or CD26 (Martin et al., 1995; Ciruela et al., 1996; Herrera et al., 2001)
 

 
2. Structure of Adenosine Deaminase

The product of human ADA gene consists of 363 amino acids (41 kDa) and there is a high degree of amino acids sequence conservation amongst species.
Click to see the amino acid sequence alignment comparison of human, murine, C. elegans and E. coli adenosine deaminases (Microsoft word .rtf format)

In 1991, the three-dimensional structure of recombinant murine ADA produced in E.coli was reported by Wilson and co-workers. The enzyme contains a parallel a/b-barrel motif with eight central b strands and eight peripheral a helices, which is common structure found in 1/10 of known enzymes (Farber and Petsko, 1990)); it also contains five additional helices. The oblong-shaped deep active site is lined  by the COOH-terminal segments and connecting loops of the b-barrel strands. The active site also contains a zinc atom, which participates directly in the deamination mechanism. There are several  hydrogen bonds between the substrate and the enzyme that stabilize the binding of substrate and the transition state.

Chang et al. (1991) proposed that catalytic functions are carried out by Cys 262, Asp 295, Asp 296, and His 214 of the mammalian adenosine deaminases. The zinc ion is coordinated by His 15, His 17, His 214 and Asp 295. From following studies (Wilson and Quiocho, 1993; Mohamedali et al., 1996; Sideraki et al., 1996), the mechanism for deamination was proposed, in which the zinc cofactor activates a liganded water molecule from which the nearby His 238 abstracts a proton, thus creating the attacking hydroxyl group. The incipient hydroxyl is orientated for attack on the C6 of the substrate through its interaction with Asp 295, His 238, and the zinc. Asp 295 is thought to hydrogen bond to the catalytic water and share a zinc ligand site with it. The protonated Glu 217 facilitates the reaction by donating a hydrogen bond to N1 of the purine, thus enabling the formation of tetrahedral C6. His 238 is possible candidate as a source of the proton added to the amino leaving group. Residues Asp 296 and Gly 184 participate in hydrogen bonds with N7 and N9 of adenosine.
In vitro mutagenesis of three residues coordinating the zinc atom (His 17, His 214, Asp 295) eliminated ADA activity (Bhaumik et al., 1993). Many of the point mutations thus far examined affect residues lodged in the b strands. Since the active site pocket, with a bound zinc, is lined by parts of the b strands, it is likely that any mutation that causes a misalignment of the b strands would have a deleterious effect on activity (Wilson et al., 1991).
 
 
3. Expression of ADA in organism

In humans the highest ADA activity is found in thymus and other lymphoid tissues (~800 IU/mg), the lowest in erythrocytes (~1 IU/mg) (Hershfield and Mitchel, 1995).
Among nonlymphoid tissues in humans, relatively high levels of ADA are found in the villi of epithelial cells lining the duodenum; levels are lower in other portions of the gastrointestinal tract. The pattern of ADA expression differs among species. In mice, for example, ADA levels are more than tenfold higher in epithelial cells lining the proximal alimentary tract than in the thymus. The highest levels occur in the tongue, esophagus, forestomach, and proximal small intestine. Tissues, such as muscle, liver, kidney, brain and blood, have low activity in most species. The tissues with most consistent high activity are duodenum and spleen.
The activity of ADA enzyme is subject to changes depending upon the degree of activity of the cell, i.e. whether differentiation or proliferation occurs (Franco et al., 1998). Trotta and Balis (1977) have demonstrated a marked increase in specific ADA activity as the mitotically active crypt cells of rat intestinal jejenum differentiate. In experiments performed with thymocytes and peripheral T-lymphocyte subpopulations, it has been shown that the cellular content of ADA diminishes as maturation occurs (Ma et al., 1982; Massaia et al., 1982).
The ADA activity can be found at variable amounts in all cell types present in the nervous system. The embryonic development of the nervous system is mildly affected by the absence of ADA. Thus ADA-deficient patients exhibit some neurological abnormalities that improve after enzyme replacement therapy (Hirschhorn et al., 1980).
Meng et al., 1997 investigated the expression of a testis-specific ADA mRNA in the mouse and they demonstrated the presence of a major 1,350 bp testis-specific ADA mRNA and a weaker 1,200 bp ADA transcript whose developmental expression starts on day 28 of life. Sequence analysis of the testis-specific ADA cDNA indicated that exons 1 and 2, as well as the first 8 nucleotides of exon 3 of the somatic cell ADA cDNA were absent in the testicular ADA cDNA. This shorter version of ADA isn’t likely active adenosine deaminase due to the absence of His 15 and His 17 (however, it was not examined). Additionally, there exists one more cDNA in murine testis library, which is different from but similar to authentic murine ADA (unpublished data). This cDNA lacks 65 N-terminal amino acids in comparison to authentic ADA and thus also lacks with the highest probability deaminase enzymatic activity. This new gene seems to form special gene family with other homologs in Drosophila and Arabidopsis. The developmental onset of expression of the testis-specific ADA mRNA may be related to specific proliferation/differentiation events of spermatogenesis.
The levels of ADA activity and ADA protein correlate with steady state of ADA mRNA in tissues of mice as in cultured human lymphoblasts and fibroblasts, which suggests transcriptional control of ADA expression (Hershfield and Mitchel, 1995). Arrest of transcript elongation may play a role in tissue-specific control of ADA mRNA levels. Transcription of human and murine ADA genes constructs in frog oocytes gave rise to properly initiated but prematurely terminated transcripts (Chen et al., 1990; Ramamurthy et al., 1990). A 230-bp segment of the human ADA gene, consisting of the GC-rich promoter and the 5’ untranslated portion of the first exon, determines constitutive, or “housekeeping” ADA expression. However, other cis-acting elements are needed to specify very-high-level, tissue-specific ADA expression. Aronow et al., (1989; 1992) found the region 4.3-8.5 kb downstream of the first exon, within the large (15 kb) first intron of the human ADA gene, to have enhancer activity.
 

4. Physiological roles of ADA and adenosine

Additionally to the key role in the purine metabolism, i.e. degradation and/or conversion of adenosine/deoxyadenosine, ADA has also important physiological roles. These roles according to the present knowledge can be divided to
1. enzymatic activities both of cytosolic and ecto-ADA via the regulation of a concentration of both intracellular and extracellular adenosine
2. extraenzymatic activities of ecto-ADA via the binding to the cell surface molecules
 

4.1. Enzymatic activity of ADA – adenosine actions

First, physiological roles of ADA can be seen in connection with adenosine whose concentration can be modulated by enzymatic action of ADA. Adenosine is both a metabolic precursor for nucleic acids (intracellular adenosine) and a significant signalling molecule involved in regulation of various physiological processes. The physiological functions of adenosine are thought to be linked to its localized release (extracellular adenosine - Ado). Ado triggers the changes within the cells through its interaction with adenosine receptors. The responses to adenosine include coronary vasodilatation, reduction in heart rate and contractile force, inhibition of platelet aggregation, mast-cell degranulation, inactivation of eosinophil migration, renal vasoconstriction, regulation of ion channel activity, membrane potential and neurotransmitter and hormone release (Nyce, 1999). In some tissues the release of adenosine has been shown to be stimulated by hypoxia (Katori and Berne, 1966). Ado may also play a role in angiogenesis (Adair et al., 1990). Beside these effects, recent reports describe Ado as a novel modulator of cell proliferation and differentiation (see text further). These effects of Ado are again triggered through adenosine receptors.
 

4.1.1. Adenosine receptors

Adenosine receptors, as other G protein-linked receptors, contain seven transmembrane domains, with an intracellular carboxy terminus and an extracellular amino terminus.
There were identified 4 types of adenosine receptors in mammals. A1 and A3 receptors interact with G proteins Gi and A2a and A2b with Gs. They are typically coupled to the adenylate cyclase-cAMP signal-transduction pathway. A1 and A3 also signal via phospolipase C (PLC) and Ca2+.
The most widely recognized signaling pathway of A1 receptors is inhibition of adenylate cyclase causing a decrease in the second-messenger cAMP (Munshi et al., 1991) that in turn modulates the activity of cAMP-dependent protein kinase. Another signaling mechanism of A1 receptors is activation of PLC leading to membrane phosphoinositide metabolism and increased production of IP3 (and DAG) and Ca2+ mobilization (Iredale et al., 1994). Elevation of cytosolic Ca2+ by IP3 can stimulate a variety of signaling pathways, including a family of protein kinase C (PKC), phospolipase A2 (PLA2), Ca2+-dependent K+ channels, and nitric oxide synthase (NOS).
Schematic picture of transmembrane arrangement of adenosine receptor

The A3 receptor has also been shown to inhibit adenylate cyclase activity (Zhou et al., 1992). It is coupling to Gia2-, Gia3- and, to a lesser extent, to Gq/11 proteins and stimulates PLC and elevates IP3 levels and intracellular Ca2+.
The most commonly recognized signal transduction mechanism for A2A and A2B receptors is, on the other hand, activation of adenylate cyclase. This implies coupling with the G protein Gs (Palmer and Stiles, 1995).
 

Expression of more than one type of adenosine receptor on the same cell may allow the common agonist of adenosine to activate multiple signaling pathways. Adenylate cyclase is a common effector, which is negatively coupled to A1 and A3 receptors and positively coupled to A2 receptors, affording the opportunity for reciprocal control and, therefore, fine tuning of this signaling pathway. The extracellular adenosine concentration may be a crucial determinant of the differential activation of coexisting adenosine receptors under pathophysiological as well as physiological conditions.

Overexpression of the A2 adenosine receptor in mice promoted gland hyperplasia. Catherine Ledent and colleagues generated transgenic mice expressing the canine A2 adenosine receptor under control of the bovine thyroglobulin gene promoter (Ledent et al., 1992). This promoter targets the expression of a reporter gene to the thyroid cells with a very tight specificity. High level of A2 receptor transcript and the presence of a functional receptor were detected in transgenic thyroids. This correlated with increased levels of cAMP in the thyroid of transgenic animals, demonstrating that the A2 adenosine receptor acts in vivo as a constitutive activator of adenylyl cyclase. Expression of the A2 adenosine receptor promoted gland hyperplasia and severe hyperthyroidism. The progressive heterogeneity of the tissue in older mice and the presence of dense tissue nodules suggested that the stimulation of adenylyl cyclase could eventually favor the development of thyroid cancer.

Extracellular adenosine inhibits DNA synthesis induced by TSH in FRTL-5 thyroid cells through the A1 adenosine receptor-Gi system (Sho et al., 1999). TSH promotes G1 cyclin expression by inducing cAMP production. This cyclin expression then triggers cell cycle progression from the G0/G1 to the S phase. PIA inhibited the TSH induction but not inhibited dibutyryl cAMP, a cell-permeable cAMP derivative, induction of cell cycle progression. The inhibitory actions of PIA on the TSH actions were prevented by the treatment of the cells by pertussis toxin, which is an inactivator of Gi. Therefore, PIA actions are performed by the activation of the A1 adenosine receptor and inactivation of adenylyl cyclase by Gi  coupled with the A1 receptor. Previously, these authors also showed that in FRTL-5 cells both adenosine and PIA inhibited the TSH-induced cAMP production; whereas, in the absence of TSH, the agonist stimulated cAMP production in the cells where the A1 receptor-mediated pathway was inactivated by the pertussis toxin (Nazarea et al., 1991). This suggests that, in FRTL-5 cells, both A1 and A2 adenosine receptors coexist; the A1 receptor mediates an inhibitory signal for adenylyl cyclase through Gi, and the A2 receptor is responsible for activating adenylyl cyclase through Gs.
 

Additionally to expression of different adenosine receptor on the cell surface, any of previously described actions of adenosine can be potentially influenced by two mechanisms regulating the concentration of adenosine:
1. transportation of adenosine across the cell membrane
2. enzymatic regulation of adenosine concentration by ADA.
 

4.1.2. Adenosine transportation

The termination of adenosine actions with its receptor involves its transport across the plasma membrane and subsequent metabolism (Thorn and Jarvis, 1996). Adenosine is hydrophilic and specialized transport systems are required for its movement across the cell membrane. There exist two types of nucleoside transport systems in mammalian cells – facilitated-diffusion nucleoside transport (equilibrative adenosine carriers) and active transport driven by an inwardly directed transmembrane sodium gradient (Na+/adenosine cotransporters). Inhibition of these transporters potentiates the actions of adenosine. Because of the relatively high activity of intracellular adenosine
kinase, adenosine concentrations inside cells are normally low, so the net flux through the equilibrative transporters is inwardly directed. However, under conditions where intracellular adenosine concentrations rise, these transporters can release adenosine.
In the heart, hypoxia produces a profound inhibition of adenosine kinase activity (to as low as 6% of normal activity) (Decking et al., 1997). Inhibition of adenosine kinase probably has little effect on ATP levels, but it generates large amounts of adenosine and profoundly increases adenosine release.
 

4.1.3. Regulation of adenosine concentration by ADA and consequences

It is clear that ADA may potentially influence any previously described actions of adenosine through its enzymatic regulation of adenosine concentration.
For example, Lelievre et al., (1998a; 1998b) showed that ADA can modulate cell growth in colon cancer cell lines. Additionally, Dexter et al., 1981 showed that agents that trigger colon cancer cell line HT29 differentiation also trigger modifications in the activity of ADA. Thus, adenosine deaminase-induced extracellular adenosine deprivation may result in the selection of differentiated cells. Lelievre et al. (2000) demonstrated that a concomitant down-regulation of adenosine A1 receptors and up regulation of adenosine A2 receptors occurred in cloned cells of colon cancer cell line with a strong reduction in proliferation. Colquhoun and Newsholme, 1997 showed that removal of exogenous adenosine by growth in the presence of adenosine deaminase also inhibited the proliferation of other human tumor cells.

However, in contrast to previous paragraphs adenosine can stimulate the proliferation of other cells types. For example, Ethier et al. (1993) found that addition of physiological concentration of Ado to human umbilical vein endothelial cell cultures stimulated proliferation. Or, the release of adenosine under certain conditions in the brain can stimulate in presence of both adenosine A1 and A2  receptors the proliferation of microglial cells (Gebicke-Haerter et al., 1996). MacLaughlin et al., 1997 demonstrated that Ado induced mesengial cell proliferation by a mechanism that seems to involve both adenosine A1 and A2 receptor type activation and increase in both second messengers, cytosolic free calcium and cAMP.

Adenosine deaminase has also been shown to impair the insulin sensitivity for glucose transport and antilipolysis by inactivating extracellular adenosine, which adipocytes release spontaneously (Green, 1987). Takasuga and coworkers showed that adenosine enhanced both the insulin-induced accumulation of PIP3 and the insulin-induced activation of PKB by a mechanism independent of its inhibitory action on adenylyl cyclase (Takasuga et al., 1999):
1. Insulin induces in rat adipocytes the production of PIP3 by PI 3-kinase. Treatment of cells with adenosine deaminase suppresses the insulin-induced production of PIP3. Rat adipocytes spontaneously release adenosine, which in turn binds to the A1 receptors on the cells. Degradation of this adenosine by addition of adenosine deaminase modulates the insulin action on glucose uptake. Inhibitors of the adenosine A1 receptors also inhibit the insulin-induced PIP3 production, which supports that adenosine deaminase modulates the insulin action by eliminating the adenosine action on the A1 receptors.
2. When the spontaneously released adenosine was inactivated by adenosine deaminase, PIA, a poorly hydrolysable analogue of adenosine, effectively enhanced the insulin-induced PIP3 production. PGE2, which activates the GTP-binding proteins in the cells, also increases the accumulation of the insulin-induced PIP3, even if inhibitors of the adenosine A1 receptors are present.
3. Insulin is known to activate rapidly a serine/threonine kinase PKB in rat adipocytes (Wijkander et al., 1997). The activity of PKB is increased by lipid products of PI 3-kinase (Franke et al., 1997). Incubation of the cells with adenosine deaminase attenuates the insulin-induced activation of PKB. Addition of PIA reverses the PKB activity to the level of insulin alone and PGE2 also increased the PKB activity in the presence of both insulin and adenosine deaminase.
4. Direct inhibition of adenylyl cyclase by DDA did not affect the insulin actions on PIP3 and PKB.
 

Pak et al. (1994) showed that adenosine deaminase inhibitors have little or no influence on the concentration of extracellular adenosine in nervous system. However, adenosine usually comprises <10% of the total purine efflux in hippocampus, whereas the remainder appears as the adenosine metabolites inosine, hypoxanthine, or xanthine (Lloyd et al., 1993), which imply that adenosine deaminase is relatively important in clearing the extracellular adenosine. The resolution of these seemingly paradoxical observations according to Dunwiddie and Masino (2001) is that the majority of adenosine in the extracellular space is cleared via reuptake; however, any metabolites that are formed are much more likely than adenosine to diffuse out of the slice without being recaptured and, hence, make a disproportionate contribution to purine efflux.
 

4.1.4. Severe Combined Immunodeficiency Disease (SCID)

A lot of knowledg about how ADA can influence different adenosine actions arose from the examination of severe combined immunodeficiency disease (SCID) pathogenesis. ADA deficiency is the cause of one form of SCID, in which there is dysfunction of both B and T lymphocytes with impaired cellular immunity and decreased production of immunoglobulins. ADA deficiency accounts for about one-half of cases of autosomal recessive SCID (OMIM *102700).
Although ADA enzyme activity is missing in every cell in the body, only the immune system is significantly affected. Approx. 50% of ADA-deficient patients have pathological changes in chondro-osseous tissues with few proliferating, some hypertrophic and some necrotic chondrocytes (Cederbaum et al., 1976).  In addition, some patients have neurological abnormalities that may be due to ADA deficiency. Mesangial sclerosis in renal tissues, pulmonary insufficiency and liver abnormalities have been also seen in ADA-deficient patients (Hershfield and Mitchell, 1995).
Immunodeficiency is likely the consequence of the particular sensitivity of immature lymphoid cells to the toxic effects of the ADA substrates adenosine and/or deoxyadenosine. However, the exact molecular mechanisms of lymphotoxicity remain obscure, and the relative contribution of intracellular lymphotoxic versus signaling properties of extracellular adenosine on ADA SCID have not been sufficiently explored.
ADA deficiency may provoke a variety of consequences either through metabolic disturbances caused by elevated 2-deoxyadenosine or cell signaling disturbances caused by elevated adenosine.
2‘-deoxyadenosine may act as a cytotoxic metabolite that can mediate its effects directly at the nucleoside level or after conversion to dATP (reviewed by Aldrich et al., 2000). 2‘-deoxyadenosine functions as a suicide inhibitor of the enzyme S-adenosylhomocysteine (AdoHcy) hydrolase, an enzyme critical to cellular transmethylation reactions. The resulting accumulation of AdoHcy could also modulate APO-1/Fas-mediated cell death. 2‘-deoxyadenosine, which can be phosphorylated to dATP, can cause DNA nicking and induction of apoptosis. Elevated dATP levels can also cause ribonucleotide reductase inhibition, leading to inhibition of deoxynucleotide synthesis and the reduced ability to repair DNA.
Elevated adenosine levels could trigger aberrant adenosine receptor signaling. Adenosine level in thymus might normally be controlled by the relative activities of the adenosine-producing enzyme ecto-5’-nucleotidase (CD73) and adenosine-degrading enzyme ADA. Blackburn et al., 1992 found that adenosine concentration is proportional to the dynamic ratio of CD73 mRNA to ADA mRNA. Madara et al., 1993 described functional associations between CD73-generated adenosine and signaling via adenosine receptors in the gut epithelium. The cell-type-specific pattern of CD73 and ADA expression in the thymus suggests that regulated adenosine signaling may also be important at this site. The massive apoptotic cell death that occurs in the thymus provides a source of extracellular nucleotides for CD73 action (Resta et al., 1997).
Huang et al., 1997 demonstrated that extracellular adenosine strongly inhibits T-Cell Receptor (TCR)-triggered proliferation of peripheral T-cells and up regulation of their IL-2 receptor a chain molecules (CD25). Proper TCR signaling plays a critical role in survival and development of thymocytes in vivo. They suggested that these effects are likely to be mediated by A2a receptor-mediated signaling because the increases in cAMP mimicked the adenosine-induced inhibition of TCR-triggered CD25 up regulation and splenocyte proliferation. Apasov et al. (2000) showed that A2a receptor is the mostly responsible receptor for external adenosine-triggered signaling and thus cAMP accumulation in murine thymocytes. In their experiments, extAdo induces the cell loss of thymocytes from wild type (+/+) mice, but apoptosis was not detected in thymocytes from A2a receptor deficient (-/-) mice, providing genetic evidence of the necessity of A2a receptor for the transmission of extAdo-initiated apoptosis of thymocytes. However, only about 10-15% of thymocytes (represented by transient stage of thymocytes) express A2aR-induced apoptosis. Moreover, the adenosine was still able to block the TCR-triggered activation even in thymocytes that lack expressed A2a receptors.
The same authors used the advantage of availability of ADA-deficient mouse and showed in vivo that early events of TCR-mediated signaling in thymocytes (i.e. TCR z chain phosphorylation and Ca2+ accumulation) are inhibited in an ADA-deficient environment (Apasov et al., 2001). Interestingly, increased apoptosis was not noticed in histological studies and lymphocytes collected from ADA –/– mice spleens or lymph nodes, suggesting that mature lymphocytes are less sensitive to the cytotoxic properties of adenosine or 2‘-deoxyadenosine.
 

 
4.2. Extraenzymatic activities of Ecto-ADA

Ecto-ADA could have functions independent of its enzymatic activity. These extraenzymatic functions have been linked to the binding of ecto-ADA directly to at least three different cell surface molecules: CD26 (a lymphocytes activation marker), A1 and A2B adenosine receptors. In such a context, ADA probably acts as a co stimulatory molecule of adenosine receptors and/or CD26 (Martin et al., 1995; Ciruela et al., 1996; Herrera et al., 2001).
 

4.2.1. Ecto-ADA interaction with CD26

The first cell surface protein able to bind ADA was identified in 1993 as human CD26 by (Kameoka et al., 1993).  The CD26 protein has been extensively studied in T-lymphocytes (for review see De Meester et al., 1999), where its physiological role seems to be related to T-cell activation (when cells become activated, the level of CD26 expression increases markedly). But CD26 is found in many cell types, even in resting T cells (Franco et al., 1997). CD26 has an enzyme activity, which consists of the cleavage of dipeptides from N-terminus of polypeptides having Pro at the penultimate position (therefore CD26 is also called dipeptidyl peptidase IV – DPPIV). But neither the physiological substrate nor the exact physiological role of the enzymatic activity is known.
Dong et al. (1996) demonstrated that neither the protease activity nor the deaminase activity are required for the association between CD26 and ecto-ADA. They also showed that ADA on the cell surface could be derived from the intracellular ADA of cells.  They found that the human CD26-transfected murine pre-B cell line did not co-express ADA on the cell surface, as shown by anti-ADA Ab (human ADA specific; murine ADA doesn’t bind to the human CD26), although human CD26 was clearly expressed on the cell surface. The overnight co culture of human CD26 transfectants with CD26-negative parental Jurkat cells resulted in a high expression of human ADA on the cell surface of human CD26 transfectants by two-color immunofluorescence analysis. This result showed that ADA inside the cells could be released into the medium, either actively or passively, and could bind to CD26 on the cell surface of other cells.
Martin et al. (1995) demonstrated that ecto-ADA could act as a co-stimulatory molecule. Cell proliferation is accelerated when peripheral T cells are activated in the presence of exogenous ADA. In contrast, T cells became anergic in the presence of anti-ADA antibodies, which do not modify the enzymatic activity. Thus, a molecular interaction between ADA and CD26 was needed for the activation of peripheral blood T cells.
Despite the high similarity between murine and human ADA, neither does murine ADA interact with human or murine CD26 nor does murine CD26 interact with human or murine ADA (Franco et al., 1998). Thus it seems that interaction between ADA and CD26 is probably restricted to human.
 

4.2.2. Ecto-ADA binding to adenosine receptors

Rafael Franco’s group found that ADA interacts with at least two types of adenosine receptors. First, they found that in pig brain cortex membranes ADA interacts with A1R adenosine receptor (Saura et al., 1996). Recently, they demonstrated that in transfected Chinese hamster ovary cells and Jurkat J32 T lymphocytes ADA anchors to adenosine receptors of the A2B subtype (Herrera et al., 2001). These data constitute the first evidence demonstrating an interaction between a degradative ecto-enzyme (i.e. ecto-ADA) and the receptor whose ligand (i.e. adenosine) is the enzyme substrate.
Franco et al. (1998) suggest that the interaction ecto-ADA/A1R is necessary for high-affinity binding of Ado and subsequently for allowing efficient signal transduction (high-affinity binding component of the A1 receptor was identified only in the presence of exogenous ADA). They also found that low concentration of Hg2+ completely abolished ADA activity without affecting the ADA-induced enhancement of second messengers via A1R. Thus it seems that enzymatic activity of ADA is not required for this type of ADA action and it can probably acts as a co stimulatory molecule.
 
 

Adair,T.H., Gay,W.J., and Montani,J.P. (1990). Growth regulation of the vascular system: evidence for a metabolic hypothesis. Am. J. Physiol 259, R393-R404.

Aldrich,M.B., Blackburn,M.R., and Kellems,R.E. (2000). The importance of adenosine deaminase for lymphocyte development and function. Biochem. Biophys. Res. Commun. 272, 311-315.

Apasov,S., Chen,J.F., Smith,P., and Sitkovsky,M. (2000). A(2A) receptor dependent and A(2A) receptor independent effects of extracellular adenosine on murine thymocytes in conditions of adenosine deaminase deficiency. Blood 95, 3859-3867.

Apasov,S.G., Blackburn,M.R., Kellems,R.E., Smith,P.T., and Sitkovsky,M.V. (2001). Adenosine deaminase deficiency increases thymic apoptosis and causes defective T cell receptor signaling. J. Clin. Invest 108, 131-141.

Aronow,B., Lattier,D., Silbiger,R., Dusing,M., Hutton,J., Jones,G., Stock,J., McNeish,J., Potter,S., Witte,D., and . (1989). Evidence for a complex regulatory array in the first intron of the human adenosine deaminase gene. Genes Dev. 3, 1384-1400.

Aronow,B.J., Silbiger,R.N., Dusing,M.R., Stock,J.L., Yager,K.L., Potter,S.S., Hutton,J.J., and Wiginton,D.A. (1992). Functional analysis of the human adenosine deaminase gene thymic regulatory region and its ability to generate position-independent transgene expression. Mol. Cell Biol. 12, 4170-4185.

Bhaumik,D., Medin,J., Gathy,K., and Coleman,M.S. (1993). Mutational analysis of active site residues of human adenosine deaminase. J. Biol. Chem. 268, 5464-5470.

Blackburn,M.R., Gao,X., Airhart,M.J., Skalko,R.G., Thompson,L.F., and Knudsen,T.B. (1992). Adenosine levels in the postimplantation mouse uterus: quantitation by HPLC-fluorometric detection and spatiotemporal regulation by 5'- nucleotidase and adenosine deaminase. Dev. Dyn. 194, 155-168.

Cederbaum,S.D., Kaitila,I., Rimoin,D.L., and Stiehm,E.R. (1976). The chondro-osseous dysplasia of adenosine deaminase deficiency with severe combined immunodeficiency. J. Pediatr. 89, 737-742.

Chang,Z.Y., Nygaard,P., Chinault,A.C., and Kellems,R.E. (1991). Deduced amino acid sequence of Escherichia coli adenosine deaminase reveals evolutionarily conserved amino acid residues: implications for catalytic function. Biochemistry 30, 2273-2280.

Chen,Z., Harless,M.L., Wright,D.A., and Kellems,R.E. (1990). Identification and characterization of transcriptional arrest sites in exon 1 of the human adenosine deaminase gene. Mol. Cell Biol. 10, 4555-4564.

Ciruela,F., Saura,C., Canela,E.I., Mallol,J., Lluis,C., and Franco,R. (1996). Adenosine deaminase affects ligand-induced signalling by interacting with cell surface adenosine receptors. FEBS Lett. 380, 219-223.

Colquhoun,A. and Newsholme,E.A. (1997). Inhibition of human tumour cell proliferation by analogues of adenosine. Cell Biochem. Funct. 15, 135-139.

De,M., I, Korom,S., Van Damme,J., and Scharpe,S. (1999). CD26, let it cut or cut it down. Immunol. Today 20, 367-375.

Decking,U.K., Schlieper,G., Kroll,K., and Schrader,J. (1997). Hypoxia-induced inhibition of adenosine kinase potentiates cardiac adenosine release. Circ. Res. 81, 154-164.

Dexter,D.L., Crabtree,G.W., Stoeckler,J.D., Savarese,T.M., Ghoda,L.Y., Rogler-Brown,T.L., Parks,R.E., Jr., and Calabresi,P. (1981). N,N-dimethylformamide and sodium butyrate modulation of the activities of purine-metabolizing enzymes in cultured human colon carcinoma cells. Cancer Res. 41, 808-812.

Dong,R.P., Kameoka,J., Hegen,M., Tanaka,T., Xu,Y., Schlossman,S.F., and Morimoto,C. (1996). Characterization of adenosine deaminase binding to human CD26 on T cells and its biologic role in immune response. J. Immunol. 156, 1349-1355.

Dunwiddie,T.V. and Masino,S.A. (2001). The role and regulation of adenosine in the central nervous system. Annu. Rev. Neurosci. 24, 31-55.

Ethier,M.F., Chander,V., and Dobson,J.G., Jr. (1993). Adenosine stimulates proliferation of human endothelial cells in culture. Am. J. Physiol 265, H131-H138.

Farber,G.K. and Petsko,G.A. (1990). The evolution of alpha/beta barrel enzymes. Trends Biochem. Sci. 15, 228-234.

Franco,R., Casado,V., Ciruela,F., Saura,C., Mallol,J., Canela,E.I., and Lluis,C. (1997). Cell surface adenosine deaminase: much more than an ectoenzyme. Prog. Neurobiol. 52, 283-294.

Franco,R., Valenzuela,A., Lluis,C., and Blanco,J. (1998). Enzymatic and extraenzymatic role of ecto-adenosine deaminase in lymphocytes. Immunol. Rev. 161, 27-42.

Franke,T.F., Kaplan,D.R., Cantley,L.C., and Toker,A. (1997). Direct regulation of the Akt proto-oncogene product by phosphatidylinositol-3,4-bisphosphate [see comments]. Science 275, 665-668.

Gebicke-Haerter,P.J., Christoffel,F., Timmer,J., Northoff,H., Berger,M., and van Calker,D. (1996). Both adenosine A1- and A2-receptors are required to stimulate microglial proliferation. Neurochem. Int. 29, 37-42.

Green,A. (1987). Adenosine receptor down-regulation and insulin resistance following prolonged incubation of adipocytes with an A1 adenosine receptor agonist. J. Biol Chem. 262, 15702-15707.

Herrera,C., Casado,V., Ciruela,F., Schofield,P., Mallol,J., Lluis,C., and Franco,R. (2001). Adenosine A2B receptors behave as an alternative anchoring protein for cell surface adenosine deaminase in lymphocytes and cultured cells. Mol. Pharmacol. 59, 127-134.

Hershfield, M. S.; Mitchell, B. S. (1995). Immunodeficiency diseases caused by adenosine deaminase deficiency and purine nucleoside phosphorylase deficiency. In: Scriver, C. R.; Beaudet, A. L.; Sly, W. S.; Valle, D. (eds.): The Metabolic and Molecular Bases of Inherited Disease. Vol. 2. New York: McGraw-Hill (7th ed.). 1725-1768.

Hirschhorn,R., Paageorgiou,P.S., Kesarwala,H.H., and Taft,L.T. (1980). Amerioration of neurologic abnormalities after "enzyme replacement" in adenosine deaminase deficiency. N. Engl. J. Med. 303, 377-380.

Huang,S., Apasov,S., Koshiba,M., and Sitkovsky,M. (1997). Role of A2a extracellular adenosine receptor-mediated signaling in adenosine-mediated inhibition of T-cell activation and expansion. Blood 90, 1600-1610.

Iredale,P.A., Alexander,S.P., and Hill,S.J. (1994). Coupling of a transfected human brain A1 adenosine receptor in CHO-K1 cells to calcium mobilisation via a pertussis toxin-sensitive mechanism. Br. J. Pharmacol. 111, 1252-1256.

Kameoka,J., Tanaka,T., Nojima,Y., Schlossman,S.F., and Morimoto,C. (1993). Direct association of adenosine deaminase with a T cell activation antigen, CD26. Science 261, 466-469.

Katori,M. and Berne,R.M. (1966). Release of adenosine from anoxic hearts. Relationship to coronary flow. Circ. Res. 19, 420-425.

Ledent,C., Dumont,J.E., Vassart,G., and Parmentier,M. (1992). Thyroid expression of an A2 adenosine receptor transgene induces thyroid hyperplasia and hyperthyroidism. EMBO J. 11, 537-542.

Lelievre,V., Caigneaux,E., Muller,J.M., and Falcon,J. (2000). Extracellular adenosine deprivation induces epithelial differentiation of HT29 cells: evidence for a concomitant adenosine A(1)/A(2) receptor balance regulation. Eur. J. Pharmacol. 391, 21-29.

Lelievre,V., Muller,J.M., and Falcon,J. (1998a). Adenosine modulates cell proliferation in human colonic adenocarcinoma. I. Possible involvement of adenosine A1 receptor subtypes in HT29 cells. Eur. J. Pharmacol. 341, 289-297.

Lelievre,V., Muller,J.M., and Falcon,J. (1998b). Adenosine modulates cell proliferation in human colonic carcinoma. II. Differential behavior of HT29, DLD-1, Caco-2 and SW403 cell lines. Eur. J. Pharmacol. 341, 299-308.

Lloyd,H.G., Lindstrom,K., and Fredholm,B.B. (1993). Intracellular formation and release of adenosine from rat hippocampal slices evoked by electrical stimulation or energy depletion. Neurochem. Int. 23, 173-185.

Ma,D.D., Sylwestrowicz,T.A., Granger,S., Massaia,M., Franks,R., Janossy,G., and Hoffbrand,A.V. (1982). Distribution of terminal deoxynucleotidyl transferase and purine degradative and synthetic enzymes in subpopulations of human thymocytes. J. Immunol. 129, 1430-1435.

MacLaughlin,M., Martinez-Salgado,C., Eleno,N., Olivera,A., and Lopez-Novoa,J.M. (1997). Adenosine activates mesangial cell proliferation. Cell Signal. 9, 59-63.

Madara,J.L., Patapoff,T.W., Gillece-Castro,B., Colgan,S.P., Parkos,C.A., Delp,C., and Mrsny,R.J. (1993). 5'-adenosine monophosphate is the neutrophil-derived paracrine factor that elicits chloride secretion from T84 intestinal epithelial cell monolayers. J. Clin. Invest 91, 2320-2325.

Martin,M., Huguet,J., Centelles,J.J., and Franco,R. (1995). Expression of ecto-adenosine deaminase and CD26 in human T cells triggered by the TCR-CD3 complex. Possible role of adenosine deaminase as costimulatory molecule. J. Immunol. 155, 4630-4643.

Massaia,M., Ma,D.D., Sylwestrowicz,T.A., Tidman,N., Price,G., Janossy,G., and Hoffbrand,A.V. (1982). Enzymes of purine metabolism in human peripheral lymphocyte subpopulations. Clin. Exp. Immunol. 50, 148-154.

Meng,J.P., Zhang,F.P., Huhtaniemi,I., and Pakarinen,P. (1997). Characterization and developmental expression of a testis-specific adenosine deaminase mRNA in the mouse. J. Androl 18, 88-95.

Mohamedali,K.A., Kurz,L.C., and Rudolph,F.B. (1996). Site-directed mutagenesis of active site glutamate-217 in mouse adenosine deaminase. Biochemistry 35, 1672-1680.

Munshi,R., Pang,I.H., Sternweis,P.C., and Linden,J. (1991). A1 adenosine receptors of bovine brain couple to guanine nucleotide- binding proteins Gi1, Gi2, and Go. J. Biol Chem. 266, 22285-22289.

Nazarea,M., Okajima,F., and Kondo,Y. (1991). P2-purinergic activation of phosphoinositide turnover is potentiated by A1-receptor stimulation in thyroid cells. Eur. J. Pharmacol. 206, 47-52.

Nyce,J.W. (1999). Insight into adenosine receptor function using antisense and gene- knockout approaches. Trends Pharmacol. Sci. 20, 79-83.

Pak,M.A., Haas,H.L., Decking,U.K., and Schrader,J. (1994). Inhibition of adenosine kinase increases endogenous adenosine and depresses neuronal activity in hippocampal slices. Neuropharmacology 33, 1049-1053.

Palmer,T.M. and Stiles,G.L. (1995). Adenosine receptors. Neuropharmacology 34, 683-694.

Ramamurthy,V., Maa,M.C., Harless,M.L., Wright,D.A., and Kellems,R.E. (1990). Sequence requirements for transcriptional arrest in exon 1 of the murine adenosine deaminase gene. Mol. Cell Biol. 10, 1484-1491.

Resta,R., Hooker,S.W., Laurent,A.B., Jamshedur Rahman,S.M., Franklin,M., Knudsen,T.B., Nadon,N.L., and Thompson,L.F. (1997). Insights into thymic purine metabolism and adenosine deaminase deficiency revealed by transgenic mice overexpressing ecto-5'- nucleotidase (CD73). J. Clin. Invest 99, 676-683.

Saura,C., Ciruela,F., Casado,V., Canela,E.I., Mallol,J., Lluis,C., and Franco,R. (1996). Adenosine deaminase interacts with A1 adenosine receptors in pig brain cortical membranes. J. Neurochem. 66, 1675-1682.

Sho,K., Narita,T., Okajima,F., and Kondo,Y. (1999). An adenosine receptor agonist-induced modulation of TSH-dependent cell growth in FRTL-5 thyroid cells mediated by inhibitory G protein, Gi. Biochimie 81 , 341-346.

Sideraki,V., Mohamedali,K.A., Wilson,D.K., Chang,Z., Kellems,R.E., Quiocho,F.A., and Rudolph,F.B. (1996). Probing the functional role of two conserved active site aspartates in mouse adenosine deaminase. Biochemistry 35, 7862-7872.

Takasuga,S., Katada,T., Ui,M., and Hazeki,O. (1999). Enhancement by adenosine of insulin-induced activation of phosphoinositide 3-kinase and protein kinase B in rat adipocytes. J. Biol Chem. 274, 19545-19550.

Thorn,J.A. and Jarvis,S.M. (1996). Adenosine transporters. Gen. Pharmacol. 27, 613-620.

Trotta,P.P. and Balis,M.E. (1977). Structural and kinetic alterations in adenosine deaminase associated with the differentiation of rat intestinal cells. Cancer Res. 37, 2297-2305.

Wijkander,J., Holst,L.S., Rahn,T., Resjo,S., Castan,I., Manganiello,V., Belfrage,P., and Degerman,E. (1997). Regulation of protein kinase B in rat adipocytes by insulin, vanadate, and peroxovanadate. Membrane translocation in response to peroxovanadate. J. Biol Chem. 272, 21520-21526.

Wilson,D.K. and Quiocho,F.A. (1993). A pre-transition-state mimic of an enzyme: X-ray structure of adenosine deaminase with bound 1-deazaadenosine and zinc-activated water. Biochemistry 32, 1689-1694.

Wilson,D.K., Rudolph,F.B., and Quiocho,F.A. (1991). Atomic structure of adenosine deaminase complexed with a transition- state analog: understanding catalysis and immunodeficiency mutations. Science 252, 1278-1284.

Zhou,Q.Y., Li,C., Olah,M.E., Johnson,R.A., Stiles,G.L., and Civelli,O. (1992). Molecular cloning and characterization of an adenosine receptor: the A3 adenosine receptor. Proc. Natl. Acad. Sci. U. S. A 89 , 7432-7436.

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