Activation-dependent trafficking of NTPDase2 in Chinese hamster ovary cells.
Activation-dependent trafficking of NTPDase2 in Chinese hamster ovary cells.
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中国仓鼠卵巢细胞中 NTPDase2 的激活依赖性运输
DOI：
10.1016/j.biocel.2007.01.003
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发表时间：
2007
期刊：
影响因子：
--
通讯作者：
Housley GD
中科院分区：
文献类型：
--
作者：
Vlajkovic SM;Wang CJ;Soeller C;Zimmermann H;Thorne PR;Housley GD
膜结合型NTPDase2是胞外核苷三磷酸二磷酸水解酶(E-NTPDase)家族的成员，参与P2受体信号的调节。NTPDase2对胞外核苷酸具有广泛的底物专一性，但对核苷5‘-三磷酸的水解性高于核苷5’-二磷酸。在这项研究中，我们试图确定作用于P2受体的酶底物如何影响NTPDase2的细胞内转运。为此，将绿色荧光蛋白(GFP)标记的大鼠特异性NTPDase2基因瞬时导入中国仓鼠卵巢(CHO)细胞，以直接观察NTPDase2的亚细胞定位和运输。将细胞与NTPDase2底物(三磷酸腺苷和三磷酸腺苷)及合成核苷酸类似物(三磷酸腺苷γS和二磷酸腺苷βS)进行超融合，每隔一定时间采集共聚焦图像。通过比较分析胞浆和细胞膜的荧光强度，确定NTPDase2掺入质膜。绿色荧光蛋白标记的NTPDase2具有完整的功能，三磷酸腺苷和三磷酸腺苷γβS诱导细胞内储存的绿色荧光蛋白-NTPDase2的膜掺入，而UTP和腺苷二磷酸γS则无效。ATP水解率的增加与NTPDase2转运到质膜的增加有关。ATP诱导的NTPDase2转运是通过激活内源性的涉及钙内流的P2X受体而不是通过P2Y受体诱导的细胞内钙释放来实现的。我们的结果表明，P2X受体的激活刺激了潜伏的NTPDase2插入质膜。表面定位的NTPDase2的增加可能反映了一种调节机制，抵消了P2受体的过度刺激和脱敏。
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 (ATPγS and ADPβS), 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 ATPγS induced membrane incorporation of GFP-NTPDase2 from putative intracellular stores, whilst UTP and ADPβS 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.
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小鼠耳蜗中的 NTPDase1 和 NTPDase2 免疫定位：对 P2 受体信号传导调节的影响
影响因子：
3.2
作者：
S. Vlajkovic;P. Thorne;J. Sévigny;S. Robson;G. Housley
通讯作者：
G. Housley
影响因子：
5.5
作者：
Gudipaty, L;Munetz, J;Dubyak, GR
通讯作者：
Dubyak, GR
外源 ATP 酶选择性剪接与嘌呤能传递终止相关的证据。
DOI：
--
发表时间：
1999
期刊：
Brain Research. Molecular Brain Research
影响因子：
--
作者：
S. Vlajkovic;G. Housley;D. Greenwood;P. Thorne
通讯作者：
P. Thorne
NTPDase2 的 C 端剪接提供了独特的催化特性、细胞分布和酶调节。
DOI：
10.1042/bj20040852
发表时间：
2005
期刊：
The Biochemical journal
影响因子：
--
作者：
Carol J. H. Wang;S. Vlajkovic;G. Housley;N. Braun;H. Zimmermann;S. Robson;J. Sévigny;C. Soeller;P. Thorne
通讯作者：
P. Thorne
人外源 ATP 酶 (E-NTPDase 2) 的羧基或氨基末端区域赋予鸡外源 ATP-二磷酸水解酶 (E-NTPDase 8) 洗涤剂和温度敏感性。
影响因子：
2.9
作者：
T. Mukasa;Yonghee Lee;A. Knowles
通讯作者：
A. Knowles