© 1998 Wiley-Liss, Inc.

DRUG DEVELOPMENT RESEARCH 45:261–268 (1998)

Research Overview

Ecto-Adenosine Deaminase: An Ecto-Enzyme and aCostimulatory Protein Acting On a Variety of

Cell Surface ReceptorsRafael Franco,* Josefa Mallol, Vicent Casadó, Carmen Lluis, Enric I. Canela,

Carles Saura, Julià Blanco, and Francisco CiruelaDepartment of Biochemistry and Molecular Biology, University of Barcelona, Barcelona, Spain

ABSTRACT Adenosine deaminase (ADA, E.C. 3.5.4.4) is an enzyme of the purine metabolism that con-verts adenosine (Ado) and 2′deoxyadenosine (dAdo) into inosine and 2′deoxyinosine, respectively. Theenzyme has been the object of considerable interest because the congenital defect causes severe combinedimmunodeficiency (SCID). In the past 12 years, ADA, which was considered to be cytosolic, has been foundon the surface of many cells; therefore, it has to be also considered as an ecto-enzyme (ecto-ADA). There isrecent evidence about a specific role for ecto-ADA that is different from that of intracellular ADA. Apart fromdegrading extracellular Ado or dAdo, which are toxic for lymphocytes and other cells, ecto-ADA has got anextraenzymatic function by means of its interaction with cell surface proteins. The interactions in whichecto-ADA participates are summarized, and an overview of the known and suspected extraenzymatic rolesfor ecto-ADA is presented. Drug Dev. Res. 45:261–268, 1998. © 1998 Wiley-Liss, Inc.

Key words: A1 adenosine receptors; CD26; dipeptidylpeptidase IV; hsc73; signaling

Grant sponsor: Fondo de Investigaciones Sanitarias de laSeguridad Social; Grant number: 91/0272; Grant sponsor: CIRIT-CICYT; Grant number: QFN93/4423; Grant sponsor: CICYT; Grantnumber: PB94/0941; Grant number: SAF97/0066

*Correspondence to: Rafael Franco, Dept. Bioquímica iBiologia Molecular, Universitat de Barcelona, Martí i Franquès 1,08028 Barcelona, Spain. E-mail: r.franco@sun.bq.ub.es

Strategy, Management and Health Policy

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Clinical DevelopmentPhases I-IIIRegulatory, Quality,Manufacturing

PostmarketingPhase IV

ADA: HISTORICAL OVERVIEWADA and Severe Combined Immunodeficiency

In 1972, Giblett and coworkers identified a molecu-lar defect associated with severe combined immunodefi-ciency (SCID), which is characterized by an impairedfunction of the immune system, with reduced numbers ofcirculating T- and B-lymphocytes. The authors were study-ing a set of markers to be used in the assessment of bonemarrow transplants in the immunodeficient patients. Oneof those markers, adenosine deaminase (ADA), was reportedto be lacking in one of the candidates for transplantation.Shortly thereafter, the existence of another SCID patientwhose cells were ADA deficient was reported [reviewed inHershfield and Mitchell, 1995). This finding focussed theinterest in the understanding of how the lack of ADA couldlead to a nonfunctional immune system.

Chromosomal Location of ADA Gene

Upon the discovery of the relationship betweenADA an the development of the immune system, the ADA

gene was soon located in the long arm of chromosome20. The cDNA for the enzyme was one of the first to becloned; the sequence of the enzyme from Escherichia coli,mouse, and human is now available. A human genomicsequence for ADA is also known [reviewed in Hershfieldand Mitchell, 1995].

Sequence and Three-Dimensional Structure

The product of human ADA gene consists of 363amino acids; despite the high degree of amino acid se-quence conservation among species, the human enzymehas a longer carboxy terminal region than the murine and

262 FRANCO ET AL.

bacterial homologues. The 41-kDa product of ADA genecan be easily studied in human erythrocytes. The isozymicpattern of the enzyme in erythrocytes from donors express-ing the most abundant phenotype (ADA1) consists of onemajor and two minor bands that result from posttranscrip-tional modification. It has been found that an Asp8/Asnsubstitution is responsible for genetic polymorphism. Infact, the substitution of a neutral asparagine for an anionicaspartic acid is consistent with the less anodal electro-phoretic migration of the protein band corresponding tothe ADA2 phenotype. Pedigree analysis has confirmed twoalleles (1 and 2) at an autosomal locus. From combinationof these two alleles (ADA1 and ADA2) three different phe-notypes can occur, i.e., ADA1, ADA2, and ADAD1-2. Phe-notype 2 is less common than phenotype 1 and appears atgene frequencies in the range 0.03–011 among differentpopulations [see Hershfield and Mitchell, 1995]. No simi-lar polymorphism has been reported in other mammals.

The structure of the enzyme complexed with aninhibitor resembling the tetrahedral intermediate of re-action was reported by Wilson et al. [1991]. The enzyme/inhibitor complex displays a parallel α/β-barrel motif witheight central β strands. Interestingly, the enzyme con-tains a Zn atom, which was not previously suspected andthat participates directly in the addition-elimination SN2

reaction mechanism.There are two kinds of ADA inhibitors, those re-

sembling the substrate, and those resembling the reac-tion tetrahedral intermediate; they have been named,respectively, ground-state and transition-state inhibitors.These inhibitors are useful in the therapy of viral infec-tions and of hairy-cell leukemia. They may also modu-late the immune response in human B-cell or T-cellmalignancies. The inhibitors are also useful for prevent-ing the deamination and subsequent inactivation of anti-tumor and cytotoxic agents that are nucleosides, forinstance ara-A and 3′deoxyadenosine or cordycepin [seeFranco et al., 1998].

Enzyme-Replacement andGene Therapies for SCID Patients

Several mechanisms whereby ADA may affect thedevelopment and function of lymphoid cells have beensuggested, but none can explain all the alterations foundin SCID [see Hershfield and Mitchell, 1995]. Before thediscovery of the molecular basis of SCID, bone-marrowtransplantation was the treatment of choice. However, inthe case of ADA-deficient patients, this treatment wasvery unsatisfactory. Fortunately, in ADA-deficient SCIDpatients the enzyme-replacement therapies have beenextremely helpful. A rather nonspecific form of enzymereplacement technique consists of transfusion of irradi-ated blood cells containing normal amounts of ADA. Dueto secondary effects a more specific protocol, which con-

sists of injecting a purified enzyme preparation of bovineorigin conjugated to polyethylene glycol is used [seeHershfield and Mitchell, 1995 for review]. Modification ofADA by polyethylene glycol extends the half-life of the cir-culating enzyme but interferes with uptake by the cells. Infact, a high proportion of ADA remains extracellular, thusindicating that (1) effective deamination of adenine nucleo-sides can occur outside the cell, and (2) interaction of ADAwith cell surface molecules can have a role in restoring theimmune function [Franco et al., 1998].

The therapy of ADA-deficient children has takenadvantage of the choice of the ADA gene as one of thefirst candidates for clinical trials of gene transfer. Thischoice is because ADA has been extensively character-ized, both from a biochemical and molecular biology pointof view and because ADA-related SCID is a broadly stud-ied genetic disease. The first clinical trials, developedsince 1992 in EEUU and Europe, are promising but fur-ther development is necessary [see Bordignon et al., 1995;Blaese et al., 1995].

ECTO-ADAEcto-ADA: Historical Overview

The first two lines of evidence indicating that ADAinteracts with membranes were given by Andy and Kornfeld[1982], who found that a protein able to interact with ADAwas a membrane protein, and by Trams and Lauter [1975],who found that ADA activity was enriched in the same sub-cellular fractions as the membrane-bound enzyme 5′-nucle-otidase, which produces adenosine from AMP. A similarenrichment was found in our laboratory in synaptosomesfrom rat brain [Franco et al., 1986]. Furthermore, we foundthat the topology of ADA activity was the same as that ofthe ecto-enzyme 5′-nucleotidase and different from that ofcytosolic enzymes such as lactate dehydrogenase. In fact,ADA and 5′-nucleotidase activities were similar beforeand after detergent disruption of synaptosomes. This find-ing suggested an ecto-enzymic pattern for ADA in syn-aptosomes. Subsequently, an extracellular orientation ofADA was also reported in blood and coronary arterialcells [Franco et al., 1997, 1998, and references therein].

ADA and Ecto-ADANo differences in catalytic activity or molecular

characteristics between cytosolic ADA and ecto-ADAhave been reported so far. Apart from biochemical datasupporting the homology between the two differentlylocated proteins, this is supported by the fact that onlyone gene for ADA has been detected, which codes for aprotein lacking both putative membrane domains andsignal peptide. Although it has not been possible to elu-cidate the mechanism by which cytosolic ADA appearson the cell surface as ecto-ADA, it has been possible toremove all intracellular ADA from mouse lymphocytes

ECTO-ADENOSINE DEAMINASE: ENZYME AND COSTIMULATORY MOLECULE 263

stabilized at low-pH acetate buffer [Senesi et al., 1988].The cells remain structurally intact, and other cellularenzymes such as lactate dehydrogenase are not released.This finding suggests the existence of specific mecha-nisms of ADA release. A portion, probably small, of ecto-ADA would also appear after release from dead cells andattachment to the surface of neighboring living cells.

Distribution of Ecto-ADA

Along the many years devoted to the study of ecto-ADA, we have rarely found a cell type lacking the ex-pression of the enzyme at the cell surface. Except in somefew cases such as in B-lymphocytes it is, however, rare tofind that ecto-ADA is present in all cells. This means that,in a given cell population, there is a mixture of ADA+

and ADA– cells. On the other hand, the number of ecto-ADA molecules, measured as intensity of the labelingwith an anti-ADA antibody, is very variable. Although wehave found ecto-ADA on the surface of platelets, fibro-blasts, lymphoid cells, epithelial cells, renal cells, etc.,ecto-ADA has been mainly characterized in peripheralnucleated blood cells. ADA is found as ecto-enzyme inthe majority of monocytes and B-cells and in some (10–20%) T-cells [Aran et al., 1991]. The expression of ecto-ADA is high in B-cell–derived lines (SKW6.4 cells, 95 ±5%), low in T-cell–derived lines (Jurkat cells, 10–20%),and moderate in cells derived from a chronic myelog-enous leukemia (K562 cells, 32 ± 8%) [Martín et al., 1995].Apart from the higher percentage of B-cells expressing ecto-ADA, the number of ADA molecules on the plasma mem-brane of ecto-ADA–expressing B-cells is much higher thanthat of ecto-ADA–expressing T-cells. This finding contrastswith the enzyme activity in whole cells, which is higher inT- than in B-cells [Aran et al., 1991]. These characteristicsindicate that expression of cell surface ADA is a well-regu-lated phenomenon. Because ecto-ADA anchors to differ-ent cell surface proteins, it should be noted also that thebinding of ecto-ADA to the cell surface can be sometimesweak; therefore, it can be lost in the washes performedduring the assays. Usually, this leads to underestimatingthe expression of the ecto-enzyme.

In some cells, the enzyme is homogenously distrib-uted along the cell membrane, but this is not always thecase. In collaboration with the group of M. Ros in CiudadReal (Spain), we have recently found a nonhomogenousecto-ADA distribution in primary cultures of neuronsfrom rat brain cortex (manuscript in preparation). More-over, in collaboration with the group of Dr. A. Minelli inPerugia (Italy), we are currently characterizing the ex-pression of ecto-ADA and of A1 adenosine receptor (A1R)in spermatozoa (data in preparation). These cells havepermitted the finding of a very strict distribution of ecto-ADA. Therefore, in spermatozoa it seems that ecto-ADAappears on the cell surface following specific routes,

which suggests that ecto-ADA has a very specific func-tion in the vital cycle of these cells.

ECTO-ADA ANCHORING PROTEINS

CD26, was the first cell surface protein identifiedas a “receptor” for ecto-ADA [Kameoka et al., 1993].Shortly afterward, A1 adenosine receptors (A1Rs) wereidentified in our laboratory as another cell surface mole-cule able to interact with ecto-ADA [Ciruela et al., 1996].

CD26

CD26 (dipeptidylpeptidase IV) is a type II membranesialoglycoprotein consisting of two identical subunits ofabout 100 kDa. The human protein is predominantly ex-tracellular with a 32-amino acid hydrophobic transmem-brane region and a cytoplasmic tail of only 8 amino acids.CD26, which has been mainly studied in lymphocytes, isidentical to dipeptidylpeptidase IV (DPPIV), which hasbeen extensively studied in cells derived from the gas-trointestinal tract. The protein cleaves dipeptides from theN-terminal region of polypeptides having Pro at thepenultimate position. The search for physiologic substrateshas finally been successful, because CD26/DPPIV seemsto cleave chemokines such as SDF-1 [Shioda et al., 1998].

Kameoka et al. [1993] demonstrated that CD26 wasan ecto-ADA anchoring protein. First, it was shown thata band of 41 kDa coprecipitated with CD26. A partialsequencing of the protein in the band indicated that itwas ADA. Furthermore, an excellent correlation betweenthe expression level of cell-surface ecto-ADA and that ofcell-surface CD26/DPPIV was found. It should be notedthat a good correlation between ecto-ADA and CD26/DPPIV expression in lymphocytes has been reportedfrom data in our laboratory [See Franco et al., 1998]. In agiven lymphocyte population, the percentage of cellsexpressing CD26/DPPIV is always higher or equal to thatof cells expressing ecto-ADA. In a given cell, CD26/DPPIV molecules can appear as either “empty” or “filled”with ecto-ADA. On the other hand, ADA in cell-free lym-phocyte extracts can be found free or bound to CD26giving rise to the existence of ADA activity in high-mo-lecular weight forms.

A1 adenosine receptors.

One of the substrates of ADA, Ado, is an autocoidthat exerts multiple physiologic actions in various sys-tems. Ado acts through specific receptors located on thesurface of cells; four of these receptors have been clonedand are known as A1, A2A, A2B, and A3. All of them belongto the superfamily of G protein-coupled receptors andpossess seven transmembrane domains. A1Rs are widelydistributed in different tissues. Lymphoid cells, however,usually lack this specific receptor subtype or express itat low levels.

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Working with nonlymphoid cells, we have accumu-lated enough evidence to be sure that ADA and A1R in-teract on the cell surface. First, in pig brain cortexmembranes whose Ado concentration was undetectable,we found that ADA was necessary for the identificationof the high-affinity component of the binding of potentagonists to A1Rs. In fact, in absence of ADA, a single low-affinity binding site is found [Ciruela et al., 1996]. Thiswould explain the controversy arising in the first bindingexperiments used to characterize the receptor in brainmembranes. Depending on the absence or presence ofexogenous ADA, one single (low affinity) [Wu et al., 1980;Wu and Phillis, 1982] or two binding sites (low and highaffinity) [Williams and Risley, 1980a,b] were found for[3H]2-chloroadenosine. The appearance of a high-affin-ity binding site in the presence of ADA was explained bythe disappearance of endogenous adenosine, or by as-suming that ADA had an extra-catalytic high-affinity bind-ing site for 2-chloroadenosine.

These interpretations have proved to be incorrectbecause the high-affinity binding is only found when ADAis coupled to A1R. Further proof that this is the case hascome from confocal microscopy, affinity chromatographydata, and coprecipitation by using DDT1MF-2 cells,which are a model of A1R-expressing cell. Some of theexperiments were possible by the development of anti-bodies against A1R, which work beautifully in immuno-cytochemical, immunoprecipitation, and immunoblottingassays. A1R was retained in a matrix of ADA-Sepharose,and both ADA and A1R colocalized on the surface of thecells [Ciruela et al., 1996]. In these cells, the degree ofcolocalization between ADA and CD26 was lower thanthe colocalization between ADA and A1R. By using cellextracts both proteins coimmunoprecipitated. Althoughthe existence of a third protein acting as a bridge be-tween ecto-ADA and A1R cannot be ruled out, our dataobtained by using DDT1MF-2 cells clearly indicate thatthis protein would not be CD26. These and other datagiven below constitute the first evidence demonstratinga functional interaction between a degradative enzymeand the receptor whose ligand is the enzyme substrate.

Other partners for ecto-ADA?

ADA is very heterogeneous in terms of distribu-tion of hydrophilic and hydrophobic regions [Franco etal., 1998]. Therefore, the possibility of multiple interac-tions mapping to different zones of the molecule is veryattractive. Actually, there is evidence for the existence offurther ecto-ADA anchoring proteins. One possible novelinteraction for ecto-ADA has been investigated in Chi-nese ovary hamster (CHO) cells. In these cells, as in othercells from rodents, CD26/DPPIV does not interact withecto-ADA. Besides, CHO cells do not express significantamounts of A1Rs. Interestingly, transfection of another

subtype of adenosine receptor in CHO cells leads to theappearance of ecto-ADA on the cell surface. Althoughthis ecto-ADA interacts with these adenosine receptorson the surface of CHO cells, the physiologic relevance ofthe interaction is under investigation.

A new and recent finding demonstrates that thereis still room for more interactions involving ADA. Thus,a specific interaction has been also described for intrac-ellular ADA [Ramos-Morales et al., 1997]. This interac-tion involves Grb3-3 and must have a precise significantrole, because the larger homologue of Grb3-3, i.e., Grb2,does not establish molecular contacts with ADA. BecauseGrb3-3 is an intracellular adaptor molecule, it is possiblethat this interaction has no relationship with ecto-ADA.A role for Grb3-3 in the mechanism of transport of ADAto the cell surface, however, cannot be ruled out.

ROLES FOR ECTO-ADAEnzymatic Behavior

After the correlation between ADA and SCID wasestablished, experiments aimed to assess whether Adoand/or dAdo are toxic for lymphocytes were widely per-formed. dAdo at 1 µM inhibits the function of human T-lymphocytes; at higher concentrations, dAdo blocksinterleukin-2 production and expression of the inter-leukin-2 receptor. Ado and dAdo induce apoptosis in cul-tured murine thymocytes treated with ADA inhibitors.Apoptosis can also be triggered by synthetic Ado analogsthat are resistant to deamination by ADA [see Hershfieldand Mitchell, 1995].

Until recently, the contribution of intracellular ADAto maintain nontoxic levels of Ado and dAdo was thoughtto be major. However, there is new evidence that indi-cates that ecto-ADA has an important role in controllingthe concentration of extracellular Ado. Thus, Dong et al.[1996] have elegantly shown that CD26-transfected JurkatT-lymphocytes, which express high quantities of ecto-ADA, are more resistant than untransfected cells to theinhibitory action of adenine nucleosides.

There is also indirect evidence for extracellulardeamination of adenosine in the central nervous system.Actually relatively high levels of inosine have been foundin all body fluids assayed. In rat striatum, inosine con-centration measured by microdialysis is much higher (110nM) than adenosine concentration (40 nM) [Ballarin etal., 1991], thus suggesting that ecto-ADA deaminates ex-tracellular Ado in the CNS.

Extra-Enzymatic BehaviorCatania et al. [1991] were the first to demonstrate

in cerebellar neurons that, in a catalytically independentmanner, exogenous ADA leads to large increases in theinflux of 45Ca2+ and hydrolysis of polyphosphoinositides.Activation of A1Rs was not involved in these events be-

ECTO-ADENOSINE DEAMINASE: ENZYME AND COSTIMULATORY MOLECULE 265

cause the effect, which might be secondary to a releaseof excitatory amino acids, was antagonized by glutamatereceptor antagonists but not by agonists or antagonists ofadenosine receptors. These results pointed out anextraenzymatic behavior of ADA, but no action mecha-nism was suspected. Recently, we have been able to dem-onstrate that ecto-ADA has a costimulatory role eitherby means of its interaction with CD26/DPPIV or bymeans of its interaction with A1Rs.

Costimulation by means of CD26

After the first results proving an up-regulation in theexpression of both CD26/DPPIV and ecto-ADA upon acti-vation of T-lymphocytes [Martín et al., 1995], we have sub-sequently demonstrated that the interaction ADA/CD26on the cell surface of T-cells has a costimulatory effect[Franco et al., 1998 and references therein]. Thus, additionof ADA to activated T-cells increases the rate of cell prolif-eration, whereas the blocking of the ADA/CD26 interac-tion by means of an antibody completely prevents it. In thepresence of this anti-ADA antibody, which does not modifythe enzymatic activity, T-cells become unresponsive. Thus,a molecular interaction between ADA and CD26 is neededfor the activation of peripheral blood T-cells. These resultsidentify the ecto-ADA/CD26 complex as a costimulatorymodule in T-cell activation (Fig. 1A).

It should be noted that the ecto-ADA/CD26 interac-tion is disrupted by the human immunodeficiency virus(HIV), either by intact infectious particles or by the solubleenvelope glycoprotein gp120 [Valenzuela et al., 1997]. Thisabolishes the costimulatory role of ecto-ADA in T-cell acti-vation. We believe that this is one of the mechanisms ofimmunodeficiency happening in AIDS patients even be-fore any decline in CD4+ T-cell counts occurs (Fig. 1B)[Franco et al., 1998, and manuscript submitted].

Costimulation by means of A1 adenosine receptors.

Another evidence that ecto-ADA was not merelyan ecto-enzyme has come from investigating the role ofthe interaction ADA/A1R in the smooth muscle cell lineDDT1MF-2.

Regulation of ligand binding. A1Rs present twoaffinities for ligands. For the agonist R-PIA, there is alow affinity form of A1R found in cell membranes with aKd in the range 1–2 nM and a high affinity binding sitewith a Kd of about 0.1 nM. The low affinity state is be-lieved to represent free receptor molecules, whereas thehigh affinity site may represent G protein-coupled A1Rs.As mentioned above the high affinity state is only foundin the presence of ADA. Actually, in all the ligand bind-ing assays reported in the literature, ADA is always added.By doing this it is assumed that ADA deaminates endog-enous adenosine, which would compete with theradioligand for the binding to A1R. Although adenosine

may be present is some preparations, the level of thenucleoside in exhaustively washed cell membranes is verylow. On the other hand, in terms of catalytic activity, theamount of exogenous ADA required for the finding oftwo affinity states is relatively high. Actually the sameresults are obtained when ADA is completely inhibitedby Hg2+ ions. Therefore, what happens with ADA is thatit interacts with A1R in such a way that conformationalchanges in the receptor leads to the appearance of the highaffinity binding site. Under the classic assumption that thetwo forms of A1Rs correspond to receptors coupled or un-coupled to G proteins, this would indicate that the cou-pling to G proteins is only possible when ADA interactswith the receptor. It should be noted, however, that thecluster-arranged cooperative model, which accounts for the

Fig. 1. Role of ecto-ADA in the activation of T-cells. A: T-cells need theestablishment of the ADA/CD26 interaction to activate. There is a needfor at least three signals in T-cell activation. Apart from the recognition ofthe antigen by the T-cell receptor and CD4 modules the ADA/CD26module provides a third signal which is required for activation and subse-quent proliferation of T-lymphocytes. B: Alteration of T-cell activation byhuman immunodeficiency virus (HIV). The envelope glycoprotein pro-tein (gp120) of the virus blocks the ADA/CD26 interaction, thus leadingto a defective T-cell activation [see Franco et al, 1998 for review].

266 FRANCO ET AL.

kinetics of ligand binding to A1R [Franco et al., 1996], showsthat high- and low-affinity sites are a consequence of nega-tive cooperativity of agonist binding and may not be re-lated to the content of free and G-protein–coupledreceptors. Therefore, ADA would affect cooperativity with-out affecting the A1R-G protein coupling.

Regulation of signaling. Regulation of ligand bind-ing by ADA is an indication that the enzyme would alsomodulate signaling events. Again, in all assays of signal trans-duction reported in the literature, ADA always is added. Incontrast to what happens in most ligand binding assays thatare performed in cell membranes, experiments of signal-ing by means of A1R are usually performed in intact cells.In such conditions, endogenous adenosine is present, andthis can indeed lead to understimulation of the signal trans-duction pathways elicited by means of A1Rs. We have in-vestigated in detail the role of ADA on signaling by meansof A1R in DDT1MF-2 cells, and the results obtained indi-cate that the interaction between ADA and A1R is requiredfor an efficient phosphorylatium of the receptor, mobiliza-tion of intracellular Ca2+ and inositol phosphate produc-tion. Actually, in the absence of the enzyme, the Ca2+ signaland the inositol phosphate production are almost undetect-able. Similar results are obtained by using active ADA orADA inhibited by Hg2+, which again indicates that the ef-fect is not caused by the enzymatic activity of the protein(Ciruela et al., 1996, 1997)

On the basis of results accumulated over the years,we have proposed a model that assumes that the role ofecto-ADA differs depending upon the adenosine concen-tration. It is possible that at low concentrations, adenos-ine interacts with high affinity A1R sites, thus makingpossible the delivery of signals; at low concentrationsadenosine would not be significantly degraded becausethe Km for deamination is around 50 µM. In contrast,when adenosine accumulates in the extracellular medium,the nucleoside would prevent the ecto-ADA/A1R inter-action and would lead to a very inefficient signaling. Thisis supported by the finding that inhibitors interacting withthe active site of the enzyme prevent its binding to thereceptor. Furthermore, the high concentration of thenucleoside would facilitate its degradation by theectoenzyme to prevent, minimize, or both, the down-regulation of the receptor. If this model is close to whathappens in vivo, the interaction ADA/A1R would havethe effect of facilitate signal transduction at low adenos-ine concentrations and would constitute an automaticpathway for short-term desensitization when the autocoidaccumulates in the extracellular space (Fig. 2).

A possible role in regulating A1R desensitizationand traffic. Efforts to identify proteins interacting withthe third intracellular loop of A1R have been performedin our laboratory. By using affinity chromatography,coimmunoprecipitation and biosensor technology it has

been shown that the heat shock cognate protein hsc73 isable to bind to A1R [Sarrió et al., manuscript submitted].The interaction between this protein and the A1R resultsin reduced binding of ligands, thus suggesting a role forhsc73 in the regulation of the receptor function. The ef-fect of hsc73 upon purified A1Rs was even stronger thanthat exerted by Gpp(NH)p, which uncouples the recep-tor from G proteins. It is noteworthy that there is a block-ing by ADA of this structural change induced by hsc73in A1Rs. The blocking of this hsc73-induced structuralmodification by ADA is not due to competition for thebinding to A1R.

Also of interest it the fact that ligand-induced de-sensitization of A1R leads to a subsequent internalization

Fig. 2. Interaction between A1R, ADA, and the heat-shock cognate pro-tein hsc73. A: A model for the functional coupling between ADA andA1R Two conditions are assumed: high extracellular adenosine concen-tration (poor signaling and rapid deamination) and low extracellular ad-enosine concentration (effective signaling and slow deamination). B: Apartfrom G proteins (not shown), A1Rs interact with ecto-ADA and cytosolichsc73. These two proteins regulate the ligand-induced internalization ofthe receptor. In the scheme, two internalization vesicles are shown in-side the cell. For clarity, G proteins are omitted in the schemes

ECTO-ADENOSINE DEAMINASE: ENZYME AND COSTIMULATORY MOLECULE 267

of both ADA and A1R [Saura et al., 1998]. A1R found ininternalization vesicles can be associated or not to hsc73.These findings open the possibility that cell surface A1R/ADA complexes and A1Rs not bound to ADA would fol-low distinct internalization pathways; A1R/ADA com-plexes would not likely traffic associated to hsc73, whereasA1R not bound to ADA would be internalized in hsc73-containing vesicles (see Fig. 2). Other interpretations forthese results (manuscripts submitted and in preparation),however, are possible.

Other possible roles for ecto-ADA.

The occurrence of ADA-related SCID, and bio-chemical and immunomorphologic studies of ADA dis-tribution in T- and B-lymphocytes and in lymphoid tissuessuggest that ADA is associated with differentiation of bothT- and B-lymphocytes. In fact, there is a different levelof enzyme activity in cells at different stages of matura-tion. It should be also noted that the ADA staining foundin the different cells is heterogeneous.

To what extent ecto-ADA is involved in the orga-nogenesis of the immune or other anatomical systemsand in maturation of T- and B-cells is an open question.In support of a specific role for ecto-ADA in these events,there is the fact that ADA replacement therapies, as in-fusion of ADA coupled to polyethylene glycol, used inADA-deficient children, have proved useful despite theenzyme remains extracellularly located. On the basis ofthe existence of signaling pathways in lymphocytes bymeans of the ADA/CD26 interaction, we have postulateda role for ecto-ADA in cell-to-cell contacts, which wouldbe important for the development of lymphoid tissuesand for maturation of lymphocytes. An indicated in Fig-ure 3, ADA would act as bridge between two cells ex-pressing CD26, A1Rs, or both. Because cells in thenervous system express ecto-ADA, CD26, and A1R, itwould be possible that similar ADA-mediated cell-to-cell contacts would have a role in some specific neuralfunctions.

In our laboratory as well as in a collaborative workestablished with Dr. A. Minelli from the University ofPerugia, we have some evidence for the participation ofADA in lymphocyte-to-epithelial cell contacts or semi-nal vesicle-to-sperm cell contacts. By using natural andtransfected T-lymphocytes expressing different amountsof ecto-ADA, we have been able to demonstrate that thereis a correlation between expression of CD26 and the ad-hesion of T-cells to a monolayer of epithelial cells. ThisCD26-mediated adhesion requires the presence of ADAon the surface of epithelial cells (manuscript in prepara-tion). We are currently interested in knowing whetherthis “trans” interaction leads to signaling in the T-cells,in the epithelial cells or in both. A cartoon depicting thisscenario is presented in Figure 3.

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Fig. 3. Ecto-ADA in cell-to-cell communication. A: Scheme represent-ing the possible role of ecto-ADA in differentiation and in maturation oflymphocytes. B: Scheme representing the summary of our results con-cerning the interaction between T-cells and epithelial cells (data in prepa-ration). T-cell-to-epithelial-cell contacts require “empty” CD26 moleculesexpressed on T-lymphocytes contacting ecto-ADA expressed on epithe-lial cells. Although not shown in the scheme, ecto-ADA in epithelial cellsmay be anchored to CD26 on the epithelial cell.

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