Purinergic Signalling (2006) 2:409–430 DOI 10.1007/s11302-006-9003-5 ORIGINAL ARTICLE The E-NTPDase family of ectonucleotidases: Structure function relationships and pathophysiological significance Simon C. Robson & Jean Se´vigny & Herbert Zimmermann Received: 21 December 2005 /Accepted: 23 January 2006 / Published online: 30 May 2006 # Springer Science + Business Media B.V. 2006 Abstract Ectonucleotidases are ectoenzymes that hy- patho-physiological processes. Examples include acute drolyze extracellular nucleotides to the respective effects on cellular metabolism, adhesion, activation and nucleosides. Within the past decade, ectonucleotidases migration with other protracted impacts upon develop- belonging to several enzyme families have been mental responses, inclusive of cellular proliferation, discovered, cloned and characterized. In this article, differentiation and apoptosis, as seen with atheroscle- we specifically address the cell surface-located mem- rosis, degenerative neurological diseases and immune bers of the ecto-nucleoside triphosphate diphosphohy- rejection of transplanted organs and cells. Future drolase (E-NTPDase/CD39) family (NTPDase1,2,3, clinical applications are expected to involve the devel- and 8). The molecular identification of individual opment of new therapeutic strategies for transplanta- NTPDase subtypes, genetic engineering, mutational tion and various inflammatory cardiovascular, analyses, and the generation of subtype-specific anti- gastrointestinal and neurological diseases. bodies have resulted in considerable insights into enzyme structure and function. These advances also Keywords apyrase . brain . CD39 . ecto-ATPase . allow definition of physiological and patho-physiologi- immunology . ischemia . kidney . liver . nervous tissue . cal implications of NTPDases in a considerable variety NTPDase . platelet . vasculature of tissues. Biological actions of NTPDases are a consequence (at least in part) of the regulated phos- Abbreviations phohydrolytic activity on extracellular nucleotides and ACR apyrase conserved regions consequent effects on P2-receptor signaling. It further ADA adenosine deaminase appears that the spatial and temporal expression of ATPDase ATP diphosphohydrolase NTPDases by various cell types within the vasculature, bFGF basic fibroblast growth factor the nervous tissues and other tissues impacts on several CCK-8 cholecystokinin octapeptide-8 EC endothelial cell E-NTPDase ecto-nucleoside triphosphate S. C. Robson diphosphohydrolase Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, ERK extracellular regulated kinase Boston, Massachusetts, USA FAK focal adhesion kinase MAP mitogen-activated protein J. Se´vigny MAPK MAP kinase Centre de Recherche en Rhumatologie et Immunologie, Universite´ Laval, Que´bec, Que´bec, Canada NO nitric oxide NPP nucleotide pyrophosphatase/ H. Zimmermann ()) phosphodiesterase Institut fuer Zellbiologie und Neurowissenschaft, NTPDase nucleoside triphosphate Biozentrum der J.W. Goethe-Universitaet, Marie-Curie-Str. 9, D-60439, Frankfurt am Main, Germany diphosphohydrolase e-mail: [email protected] PDGF platelet derived growth factor 410 Purinergic Signalling (2006) 2:409–430 PI3K phosphatidylinositol 3-kinase org/index.html and http://www.geocities.com/bioinfor- RanBPM ran binding protein maticsweb/speciesspecificdatabases.htm), and, 3) select TCDD tetrachlorodibenzo-p dioxin ectonucleotidases that dictate the cellular responses by the stepwise degradation of extracellular nucleotides to nucleosides [17–20]. Introduction Ensembles of ectonucleotidases, associated Extracellular nucleotides modulate a multiplicity of receptors and signaling molecules tissue functions including development, blood flow, secretion, inflammation and immune reactions. Indeed, Within the past decade, ectonucleotidases belonging to signaling via extracellular nucleotides has been recog- several enzyme families have been discovered, cloned nized for over a decade as one of the most ubiquitous and functionally characterized by pharmacological intercellular signaling mechanisms [1, 2]. Essentially means. Specifically, we refer here to members of the every cell in a mammalian organism leaks or releases ecto-nucleoside triphosphate diphosphohydrolase (E- these mediators, and carries receptors for nucleotides NTPDase) family (EC 3.6.1.5) as ectoenzymes that of which seven ionotropic (P2X) and at least eight hydrolyze extracellular nucleoside tri- and diphos- metabotropic (P2Y) receptor subtypes have been iden- phates and have a defined pharmacological profile. tified and characterized to date. Whereas P2X recep- Most notably, in many tissues and cells, NTPDases tors respond to ATP, P2Y receptors can be activated comprise dominant parts of a complex cell surface- by ATP, ADP, UTP, UDP, ITP, and nucleotide sug- located nucleotide hydrolyzing and interconverting ars, albeit agonist specificity varies between subtypes machinery. This ensemble includes the ecto-nucleotide and the multiple animal species [3]. Depending on the pyrophosphatase phosphodiesterases (E-NPPs), NAD- P2 receptor subtype and signaling pathways involved, glycohydrolases, CD38/NADase, alkaline phospha- these receptors trigger and mediate short-term (acute) tases, dinucleoside polyphosphate hydrolases, adeny- processes that affect cellular metabolism, adhesion, late kinase, nucleoside diphosphate kinase, and activation or migration. In addition, purinergic signal- potentially ecto-F1–Fo ATP synthases [21–25] that ing also has profound impacts upon other more may interact in various tissues and cellular systems. protracted responses, including cell proliferation, dif- The ectonucleotidase chain or cascade, as initiated ferentiation and apoptosis, such as seen in atheroscle- by NTPDases can be terminated by ecto-50-nucleotid- rosis, degenerative neurological diseases and in several ase (CD73; EC 3.1.3.5) with hydrolysis of nucleoside inflammatory conditions [2, 4, 5]. monophosphates [26]. Together, ecto-50-nucleotidase The effects of extracellular nucleotides appear to and adenosine deaminase (ADA; EC 3.5.4.4), another overlap, at least in part, with those of vascular growth ectoenzyme that is involved in purine salvage path- factors, cytokines (inflammatory), adhesion molecules ways and converts adenosine to inosine, closely and nitric oxide (NO). Nucleotide-mediated activation regulate local and pericellular extracellular and plasma may be also synergistic with polypeptide growth concentrations of adenosine [10, 27]. factors (PDGF, bFGF) and insulin, the signaling being Several of these ectonucleotidase families and mediated via phospholipase C and D, diacylglycerol, additional functions of NTPDases [28–30]are protein kinase C, ERKs, phosphatidylinositol 3-kinases addressed elsewhere in this issue in detail. This review (PI3K), MAP kinases (MAPK) and Rho [6–8]. The focuses on the surface-located mammalian members of situation concerning extracellular nucleotide-signaling the E-NTPDase protein family. It starts with a brief can be suitably contrasted with the unique specificity introduction of molecular structure and functional of peptide hormones or vasoactive factors for often properties, followed by an analysis of the physiological single, defined receptors [9, 10]. Within purinergic/ and pathophysiological roles at various sites with an pyrimidinergic signaling events specificity is dictated emphasis on vasculature and neural tissues. by three essential modulatory components: 1) The derivation or source of the extracellular nucleotides [1, 11, 12]; 2) the expression of specific receptors for these Molecular identities unraveled molecular transmitters (and for the nucleotide and nucleoside derivatives) [13–16] (See also Molecular The literature on the molecular and functional char- Recognition Section of National Institutes of Health, acterization of the E-NTPDase family has been http://mgddk1.niddk.nih.gov/ also http://www.ensembl. intensively reviewed [18–22, 31–36] and will not be Purinergic Signalling (2006) 2:409–430 411 repeated here in detail. Our intent is to summarize came from three independent approaches. Handa and principal properties of the enzymes that will be of use Guidotti [42] purified and cloned a soluble ATP for the reader new to this field. diphosphohydrolase (apyrase) from potato tubers and Eight different ENTPD genes (Table 1 and Fig. 1) noted that this protein was related not only to similar encode members of the NTPDase protein family. Four enzymes of some protozoans, plants and yeast but of the NTPDases are typical cell surface-located also to human CD39. They also recognized conserved enzymes with an extracellularly facing catalytic site sequence domains and the relation to members of the (NTPDase1, 2, 3, 8). NTPDases 5 and 6 exhibit intracel- actin-hsp70-hexokinase superfamily. This was then lular localization and undergo secretion after heterolo- followed by the functional expression of human gous expression. NTPDases 4 and 7 are entirely CD39 and the demonstration that this protein was in intracellularly located, facing the lumen of cytoplasmic fact an ecto-apyrase [43]. In parallel, ectonucleoti- organelles (Fig. 1). The molecular identification of dases (termed ATP diphosphohydrolases) from por- individual NTPDase subtypes, genetic engineering, cine pancreas and bovine aorta were purified. The mutational analyses, and the generation of subtype- partial amino acid sequences for both ATP diphos- specific antibodies have not only led to considerable phohydrolases