The present study provides functional characterization of alternative splicing of the NTPDase2 (ecto-nucleoside triphosphate diphosphohydrolase-2) involved in the regulation of extracellular nucleotide concentrations in a range of organ systems. A novel NTPDase2β isoform produced by alternative splicing of the rat NTPDase2 gene provides an extended intracellular C-terminus and distinguishes itself from NTPDase2α isoform in gaining several intracellular protein kinase CK2 (casein kinase 2) phosphorylation sites and losing the intracellular protein kinase C motif. The plasmids containing NTPDase2α or NTPDase2β cDNA were used to stably transfect Chinese-hamster ovary-S cells. Imaging studies showed that NTPDase2α was predominantly membrane-bound, whereas NTPDase2β had combined cell surface and intracellular localization. α and β isoforms showed variations in divalent cation dependence and substrate specificity for nucleoside-5′-triphosphates and nucleoside-5′-diphosphates. NTPDase2β exhibited reduced ATPase activity and no apparent ADPase activity. NTPDase2 isoforms demonstrated similar sensitivity to inhibitors such as suramin and pyridoxal phosphate-6-azophenyl-2′,4′-disulphonic acid, and differential regulation by protein kinases. NTPDase2β was up-regulated by intracellular protein kinase CK2 phosphorylation, whereas NTPDase2α activity was down-regulated by protein kinase C phosphorylation. The results demonstrate that alternative coding of the intracellular C-terminal domain contributes distinctive phenotypic variation with respect to extracellular nucleotide specificity, hydrolysis kinetics, protein kinase-dependent intracellular regulation and protein trafficking. These findings advance the molecular physiology of this enzyme system by characterizing the contribution of the C-terminal domain to many of the enzyme's signature properties.
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February 2005
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Research Article|
January 24 2005
C-terminal splicing of NTPDase2 provides distinctive catalytic properties, cellular distribution and enzyme regulation
Carol J. H. WANG;
Carol J. H. WANG
*Department of Physiology, Faculty of Medical and Health Sciences, The University of Auckland, Park Road, Grafton, Private Bag 92019, Auckland, New Zealand
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Srdjan M. VLAJKOVIC;
Srdjan M. VLAJKOVIC
1
*Department of Physiology, Faculty of Medical and Health Sciences, The University of Auckland, Park Road, Grafton, Private Bag 92019, Auckland, New Zealand
1To whom correspondence should be addressed (email s.vlajkovic@auckland.ac.nz).
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Gary D. HOUSLEY;
Gary D. HOUSLEY
*Department of Physiology, Faculty of Medical and Health Sciences, The University of Auckland, Park Road, Grafton, Private Bag 92019, Auckland, New Zealand
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Norbert BRAUN;
Norbert BRAUN
†Biozentrum der J. W. Goethe-Universität, AK Neurochemie, Frankfurt am Main, Germany
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Herbert ZIMMERMANN;
Herbert ZIMMERMANN
†Biozentrum der J. W. Goethe-Universität, AK Neurochemie, Frankfurt am Main, Germany
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Simon C. ROBSON;
Simon C. ROBSON
‡Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, U.S.A.
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Jean SÉVIGNY;
Jean SÉVIGNY
§Centre de Recherche en Rhumatologie et Immunologie, CHUQ, Université Laval, Sainte-Foy, Québec, Canada
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Christian SOELLER;
Christian SOELLER
*Department of Physiology, Faculty of Medical and Health Sciences, The University of Auckland, Park Road, Grafton, Private Bag 92019, Auckland, New Zealand
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Peter R. THORNE
Peter R. THORNE
∥Discipline of Audiology, Faculty of Medical and Health Sciences, The University of Auckland, Park Road, Grafton, Private Bag 92019, Auckland, New Zealand
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Publisher: Portland Press Ltd
Received:
May 21 2004
Revision Received:
September 02 2004
Accepted:
September 14 2004
Accepted Manuscript online:
September 14 2004
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London
2005
Biochem J (2005) 385 (3): 729–736.
Article history
Received:
May 21 2004
Revision Received:
September 02 2004
Accepted:
September 14 2004
Accepted Manuscript online:
September 14 2004
Citation
Carol J. H. WANG, Srdjan M. VLAJKOVIC, Gary D. HOUSLEY, Norbert BRAUN, Herbert ZIMMERMANN, Simon C. ROBSON, Jean SÉVIGNY, Christian SOELLER, Peter R. THORNE; C-terminal splicing of NTPDase2 provides distinctive catalytic properties, cellular distribution and enzyme regulation. Biochem J 1 February 2005; 385 (3): 729–736. doi: https://doi.org/10.1042/BJ20040852
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