PTH transiently increases the percent mobile fraction of Npt2a in OK cells as determined by FRAP.
PTH transiently increases the percent mobile fraction of Npt2a in OK cells as determined by FRAP.
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根据 FRAP 测定，PTH 暂时增加 OK 细胞中 Npt2a 的移动分数百分比。
DOI：
10.1152/ajprenal.90657.2008
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发表时间：
2009
期刊：
影响因子：
--
通讯作者：
Donowitz,Mark
中科院分区：
文献类型：
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作者：
Weinman,EdwardJ;Steplock,Deborah;Cha,Boyoung;Kovbasnjuk,Olga;Frost,NicholasA;Cunningham,Rochelle;Shenolikar,Shirish;Blanpied,ThomasA;Donowitz,Mark
肾钠依赖性磷酸盐转运蛋白2a （Npt2a）结合多种PDZ接头蛋白，包括钠氢交换调节因子-1 (NHERF-1)，其调节其在肾近端小管细胞顶膜中的滞留和对甲状旁腺激素（PTH）的反应。本实验旨在利用光漂白后荧光恢复（FRAP）研究增强型绿色荧光蛋白(EGFP)-Npt2a在近端小管样负鼠肾（OK）细胞中的横向迁移，并确定PDZ结合蛋白在介导PTH作用中的作用。野生型Npt2a （EGFP-Npt2a-TRL）在基础条件下的迁移率为~ 17%。用双（磺基琥珀酰亚基）硫酸酯（一种水溶性交联剂）处理细胞，几乎完全消除了恢复，表明恢复代表了质膜的横向扩散，而不是胞外分泌或未漂白转运体的合成。用AAA取代c端氨基酸PDZ结合序列TRL （EGFP-Npt2a-AAA）导致Npt2a的移动百分率增加近两倍。用甲状旁腺激素治疗的细胞导致可移动百分数迅速增加到bb0 - 30%，随后随时间变化下降到基线或以下。PTH对天然OK细胞中表达的EGFP-Npt2a-AAA的移动性和缺乏NHERF-1的OK- h细胞中表达的野生型EGFP-Npt2a-TRL的移动性没有影响。这些发现表明，Npt2a与PDZ结合蛋白的结合限制了肾近端小管细胞顶膜转运蛋白的横向迁移。PTH治疗，可能是通过解离NHERF-1/Npt2a复合物，短暂地增加了Npt2a的流动性，这表明从细胞骨架中释放Npt2a先于PTH介导的内吞噬作用。
Renal sodium-dependent phosphate transporter 2a (Npt2a) binds to a number of PDZ adaptor proteins including sodium-hydrogen exchanger regulatory factor-1 (NHERF-1), which regulates its retention in the apical membrane of renal proximal tubule cells and the response to parathyroid hormone (PTH). The present experiments were designed to study the lateral mobility of enhanced green fluorescent protein (EGFP)-Npt2a in proximal tubule-like opossum kidney (OK) cells using fluorescence recovery after photobleaching (FRAP) and to determine the role of PDZ binding proteins in mediating the effects of PTH. The mobile fraction of wild-type Npt2a (EGFP-Npt2a-TRL) under basal conditions was ∼17%. Treatment of the cells with Bis(sulfosuccinimidyl) suberate, a water-soluble cross-linker, abolished recovery nearly completely, indicating that recovery represented lateral diffusion in the plasma membrane and not the exocytosis or synthesis of unbleached transporter. Substitution of the C-terminal amino acid PDZ binding sequence TRL with AAA (EGFP-Npt2a-AAA) resulted in a nearly twofold increase in percent mobile fraction of Npt2a. Treatment of cells with PTH resulted in a rapid increase in the percent mobile fraction to >30% followed by a time-dependent decrease to baseline or below. PTH had no effect on the mobility of EGFP-Npt2a-AAA expressed in native OK cells or on wild-type EGFP-Npt2a-TRL expressed in OK-H cells deficient in NHERF-1. These findings indicate that the association of Npt2a with PDZ binding proteins limits the lateral mobility of the transporter in the apical membrane of renal proximal tubule cells. Treatment with PTH, presumably by dissociating NHERF-1/Npt2a complexes, transiently increases the mobility of Npt2a, suggesting that freeing of Npt2a from the cytoskeleton precedes PTH-mediated endocytosis.