title: Activation-dependent trafficking of NTPDase2 in Chinese hamster ovary cells.
abstract: Membrane-bound NTPDase2 is a member of the ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDase) enzyme family involved in the regulation of P2 receptor signaling. NTPDase2 has broad substrate specificity for extracellular nucleotides, but hydrolyses nucleoside 5'-triphosphates with high preference over nucleoside 5'-diphosphates. In this study, we have sought to determine how enzyme substrates acting on P2 receptors affect intracellular NTPDase2 trafficking. To achieve this, Chinese hamster ovary (CHO) cells were transiently transfected with rat-specific NTPDase2 cDNA tagged with green fluorescent protein (GFP), to allow direct visualisation of subcellular localisation and trafficking of NTPDase2. Cells were superfused with NTPDase2 substrates (ATP and UTP) and synthetic nucleotide analogues (ATPgammaS and ADPbetaS), and confocal image stacks were acquired at regular time intervals. NTPDase2 incorporation into the plasma membrane was determined by comparative analysis of fluorescence intensity in the cytosolic and membrane compartments. GFP-tagged NTPDase2 was fully functional and ATP and ATPgammaS induced membrane incorporation of GFP-NTPDase2 from putative intracellular stores, whilst UTP and ADPbetaS were ineffective. The increased ATP hydrolysis rate correlated with increased NTPDase2 trafficking to the plasma membrane. ATP-induced NTPDase2 trafficking was mediated by activation of endogenous P2X receptors involving Ca2+ entry rather than by P2Y receptor-induced release of Ca2+ from intracellular stores. Our results suggest that P2X receptor activation stimulates insertion of latent NTPDase2 into the plasma membrane. The increase in surface-located NTPDase2 may reflect a regulatory mechanism counteracting excessive stimulation and desensitisation of P2 receptors.
authors.affiliation: Department of Physiology, Faculty of Medical and Health Sciences, University of Auckland, Auckland, New Zealand. s.vlajkovic@auckland.ac.nz
authors.forename: Srdjan M
authors.initials: SM
authors.lastname: Vlajkovic
authors.forename: Carol J H
authors.initials: CJ
authors.lastname: Wang
authors.forename: Christian
authors.initials: C
authors.lastname: Soeller
authors.forename: Herbert
authors.initials: H
authors.lastname: Zimmermann
authors.forename: Peter R
authors.initials: PR
authors.lastname: Thorne
authors.forename: Gary D
authors.initials: GD
authors.lastname: Housley
mesh_terms: D000244:Adenosine Diphosphate / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism / Q000494:pharmacology; D000818:Animals; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D002462:Cell Membrane / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006868:Hydrolysis; D007424:Intracellular Fluid / Q000378:metabolism; D018613:Microscopy, Confocal; D021381:Protein Transport / Q000187:drug effects; D058910:Purinergic P2 Receptor Agonists; D058919:Purinergic P2 Receptor Antagonists; D011732:Pyridoxal Phosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D051381:Rats; D018048:Receptors, Purinergic P2 / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D013498:Suramin / Q000494:pharmacology; D013997:Time Factors; D014162:Transfection; D014530:Uridine Diphosphate / Q000378:metabolism; D014544:Uridine Triphosphate / Q000378:metabolism
chemical_list: D058910:Purinergic P2 Receptor Agonists; D058919:Purinergic P2 Receptor Antagonists; D018048:Receptors, Purinergic P2; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; C077792:pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid; D014530:Uridine Diphosphate; D011732:Pyridoxal Phosphate; D013498:Suramin; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; C027201:ectoATPase; D002118:Calcium; D014544:Uridine Triphosphate