Association of paracellin-1 with ZO-1 augments the reabsorption of divalent cations in renal epithelial cells
Association of paracellin-1 with ZO-1 augments the reabsorption of divalent cations in renal epithelial cells
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DOI：
10.1074/jbc.m406331200
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发表时间：
2004-12-24
影响因子：
4.8
通讯作者：
Takagi, K
中科院分区：
文献类型：
--
作者：
Ikari, A;Hirai, N;Takagi, K
Paracellin-1 （PCLN-1）属于紧密连接蛋白claudin家族，可能在镁和钙的重吸收中起关键作用。到目前为止，PCLN-1的生理特性尚未明确。在本研究中，我们研究了PCLN-1是否与ZO-1相关。我们还研究了Ca-45（2+）通过细胞旁屏障的转运是否受到这种关联的影响。利用谷胱甘肽s -转移酶融合蛋白进行体外结合分析，发现PCLN-1的c端TRV序列，特别是Thr和Val残基与ZO-1相互作用。接下来，利用FLAG标记载体在Madin-Darby犬肾细胞中稳定表达PCLN-1。ZO-1与野生型PCLN-1和丙氨酸替代（TAV）突变体共同免疫沉淀。然而，缺失突变（DeltaTRV）和丙氨酸取代突变（ARV和TRA）抑制了PCLN-1与ZO-1的关联。共聚焦免疫荧光显示野生型PCLN-1和TAV突变体与ZO-1一起定位于紧密连接区，而DeltaTRV、ARV和TRA突变体广泛分布于包括紧密连接区在内的侧膜。有趣的是，与表达DeltaTRV、ARV和TRA突变体和模拟细胞相比，表达野生型PCLN-1和TAV突变体的单层细胞显示出更高的Ca-45（2+）从根尖到基底室的运输活性。Ca-45（2+）的转运被镁浓度的增加所抑制，这表明镁和钙是通过PCLN-1竞争性转运的。正电位梯度增强了Ca-45（2+）从根尖室向基底室的运输，但不影响相反方向的运输。因此，PCLN-1定位于紧密连接，随后与ZO-1和PCLN-1结合。ZO-1复合体可能在肾上皮细胞对二价阳离子的重吸收中起重要作用。
Paracellin-1 (PCLN-1) belongs to the claudin family of tight junction proteins and possibly plays a critical role in the reabsorption of magnesium and calcium. So far, the physiological properties of PCLN-1 have not been clarified. In the present study, we investigated whether PCLN-1 is associated with ZO-1. We also investigated whether Ca-45(2+) transport across the paracellular barrier is affected by this association. In vitro binding analysis using glutathione S-transferase fusion protein showed that the C-terminal TRV sequence, especially Thr and Val residues, of PCLN-1 interacts with ZO-1. Next, PCLN-1 was stably expressed in Madin-Darby canine kidney cells using a FLAG tagging vector. ZO-1 was co-immunoprecipitated with the wild-type PCLN-1 and the alanine substitution (TAV) mutant. However, mutants of the deletion (DeltaTRV) and the alanine substitution (ARV and TRA) inhibited the association of PCLN-1 with ZO-1. Confocal immunofluorescence demonstrated that the wild-type PCLN-1 and the TAV mutant localized in the tight junction along with ZO-1, but the DeltaTRV, ARV, and TRA mutants were widely distributed in the lateral membrane including the tight junction area. Interestingly, monolayers of cells expressing the wild-type PCLN-1 and the TAV mutant showed higher activities of Ca-45(2+) transport from apical to basal compartments, compared with those expressing the DeltaTRV, ARV, and TRA mutants and the mock cells. Ca-45(2+) transport was inhibited by increased magnesium concentration suggesting that magnesium and calcium were competitively transported by PCLN-1. It was noted that a positive electrical potential gradient enhanced Ca-45(2+) transport from apical to basal compartments without affecting the opposite direction of transport. Thus, PCLN-1 localizes to the tight junction followed by association with ZO-1, and the PCLN-1 . ZO-1 complex may play an essential role in the reabsorption of divalent cations in renal epithelial cells.