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The regulatory mechanism of phosphate transport activity mediated by sodium-dependent phosphate transporter

钠依赖性磷酸盐转运蛋白介导的磷酸盐转运活性的调节机制

基本信息

批准号：
11671038
负责人：
TAKEDA Eiji
金额：
$ 2.3万
依托单位：
The University of Tokushima
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

项目摘要

Inorganic phosphate (phosphate) is an essential nutrient in the processes of glycolysis, gluconeogenesis, energy metabolism and skeletal mineralization. Type II sodium-dependent phosphate transporter (NPT2) expressed on renal brush border membrane (BBM) serves to physiologically and pathophysiologically regulate phosphate homeostasis. The NPT2 expression is regulated by transcriptional level, intracellular translocation of NPT2, and post-transcriptional level. The present study was performed to investigate the molecular mechanism of intracellular translocation of NPT2.PTH inhibits renal phosphate transport at the cellular level involves selective endocytic removal of NPT2 from the BBM of the renal proximal tubule mediated by the activation of both protein kinases A and C activated. PTH also stimulates phosphorylation of 100 or 77 kDa proteins, but not NPT2. Similar observation was found in calcitonin. The endocytosed NPT2 are redistributed to the cytoplasm but they appear to be targeted for rapid lysosomal degradation rather than recycling to the BBM if PTH treatment is prolonged.In another mechanism, the involvement of unique NPT2-related proteins (NPT2α, NPT2β, and NPT2γ) isolated from a rat kidney were investigated. NPT2α and NPT2γ were glycosylated and revealed to be 45- and 35-kDa proteins, respectively. In isolated BBM vesicles, an N-terminal antibody was reacted with the 45- and 40-kDa proteins, and a C-terminal antibody was reacted with the 37-kDa protein. The sizes of these proteins corresponded to those in glycosylated forms. A functional analysis demonstrated that NPT2γ markedly inhibited NPT2 activity in Xenopus oocytes. The findings suggest that this short isoform may function as a dominant negative inhibitor of the full-length transporter.Therefore, it is concluded PTH and short isoform of NPT2 stimulate rapid endocytic internalization of NPT2 at the brush border membrane, leads to an inhibition of phosphate reabsorption in the proximal tubules.

无机磷酸盐（磷酸盐）是糖酵解、代谢、能量代谢和骨骼矿化过程中的必需营养素。II型钠依赖性磷酸盐转运蛋白（NPT 2）表达于肾刷状缘膜（BBM）上，在生理和病理生理上调节磷酸盐稳态。NPT 2的表达受转录水平、NPT 2的胞内转运和转录后水平的调控。本研究旨在探讨NPT 2胞内转运的分子机制。PTH在细胞水平上抑制肾磷酸盐转运，其机制是通过激活蛋白激酶A和蛋白激酶C介导NPT 2从肾近曲小管BBM中被选择性内吞清除。PTH还刺激100或77 kDa蛋白的磷酸化，但不刺激NPT 2。在降钙素中发现了类似的观察结果。内吞的NPT 2被重新分配到细胞质中，但如果PTH处理时间延长，它们似乎被快速溶酶体降解而不是再循环到BBM。在另一种机制中，研究了从大鼠肾脏分离的独特NPT 2相关蛋白（NPT 2 α，NPT 2 β和NPT 2 γ）的参与。NPT 2 α和NPT 2 γ被糖基化，分别为45和35 kDa蛋白。在分离的BBM囊泡中，N-末端抗体与45-和40-kDa蛋白反应，C-末端抗体与37-kDa蛋白反应。这些蛋白质的大小对应于糖基化形式的那些。功能分析表明，NPT 2 γ显著抑制爪蟾卵母细胞中的NPT 2活性。结果表明，短型PTH和短型NPT 2可能是全长转运蛋白的显性负性抑制剂，因此，PTH和短型NPT 2可刺激NPT 2在刷状缘膜上的快速内吞内化，从而抑制近端小管对磷酸盐的重吸收。