C-terminal splicing of NTPDase2 provides distinctive catalytic properties, cellular distribution and enzyme regulation.
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 NTPDase2beta isoform produced by alternative splicing of the rat NTPDase2 gene provides an extended intracellular C-terminus and distinguishes itself from NTPDase2alpha isoform in gaining several intracellular protein kinase CK2 (casein kinase 2) phosphorylation sites and losing the intracellular protein kinase C motif. The plasmids containing NTPDase2alpha or NTPDase2beta cDNA were used to stably transfect Chinese-hamster ovary-S cells. Imaging studies showed that NTPDase2alpha was predominantly membrane-bound, whereas NTPDase2beta had combined cell surface and intracellular localization. alpha and beta isoforms showed variations in divalent cation dependence and substrate specificity for nucleoside-5'-triphosphates and nucleoside-5'-diphosphates. NTPDase2beta 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. NTPDase2beta was up-regulated by intracellular protein kinase CK2 phosphorylation, whereas NTPDase2alpha 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.
Wang
Carol J H
CJ
Department of Physiology, Faculty of Medical and Health Sciences, The University of Auckland, Park Road, Grafton, Private Bag 92019, Auckland, New Zealand.
Vlajkovic
Srdjan M
SM
Housley
Gary D
GD
Braun
Norbert
N
Zimmermann
Herbert
H
Robson
Simon C
SC
Sévigny
Jean
J
Soeller
Christian
C
Thorne
Peter R
PR
D000249:Adenosine Monophosphate / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D017398:Alternative Splicing / Q000235:genetics*; D000818:Animals; D016466:CHO Cells; D002384:Catalysis; D002413:Cations, Divalent / Q000494:pharmacology; D006224:Cricetinae; D015870:Gene Expression; D006868:Hydrolysis / Q000187:drug effects; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011494:Protein Kinases / Q000378:metabolism; D021381:Protein Transport; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D017422:Sequence Analysis, DNA; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D014162:Transfection
D002413:Cations, Divalent; D007527:Isoenzymes; D047428:Protein Kinase Inhibitors; D012333:RNA, Messenger; D000249:Adenosine Monophosphate; D000255:Adenosine Triphosphate; D011494:Protein Kinases; D000251:Adenosine Triphosphatases; C027201:ectoATPase