PDZ domain-mediated interaction of rabbit podocalyxin and Na+/H+ exchange regulatory factor-2
PDZ domain-mediated interaction of rabbit podocalyxin and Na+/H+ exchange regulatory factor-2
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DOI：
10.1152/ajprenal.00131.2001
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发表时间：
2002-06-01
影响因子：
4.2
通讯作者：
Kershaw, DB
中科院分区：
文献类型：
--
作者：
Li, Y;Li, J;Kershaw, DB
跨膜唾液酸糖蛋白足萼蛋白被认为是足细胞的精细交错足突结构中必不可少的。足糖萼蛋白的胞内COOH末端氨基酸Asp-Thr-His-Leu（DTHL）包含I型PSD 95-Dlg-Coccludens-1（PDZ）结构域的推定配体。用含有该基序的20个氨基酸的合成肽筛选cDNA文库，获得兔Na+/H+交换调节因子-2（NHERF-2）的克隆。体外分析表明，NHERF-2的每个PDZ结构域可以独立地结合足糖萼蛋白。肾小球提取物中的NHERF-2与足糖萼蛋白共沉淀，足糖萼蛋白和NHERF-2共定位于肾小球毛细血管袢中，表明足糖萼蛋白和NHERF-2可能在体内相互作用。缺失末端亮氨酸（-DTHL）的足糖萼蛋白肽在体外不能与NHERF-2相互作用。足糖萼蛋白定位于转染的Madin-Darby犬肾（MDCK）细胞的顶膜。然而，突变体足糖萼蛋白（缺失功能DTHL COOH-末端基序）在转染细胞中显示细胞质和顶端膜定位，并且在顶端膜处也不太稳定，如通过共聚焦显微镜和生物素化研究所评估的。突变体足糖萼蛋白确实降低了MDCK细胞单层的跨上皮电阻，尽管程度低于全长足糖萼蛋白。我们得出结论，足糖萼蛋白可以与NHERF-2的两个PDZ结构域相互作用，这种相互作用需要足糖萼蛋白的完整COOH末端，这也是负责转染MDCK细胞中足糖萼蛋白的有效顶端定位。这些结果表明，足细胞标记蛋白与NHERF-2的相互作用可能有效地将足细胞标记蛋白保留在足细胞的顶端表面，并提供了将足细胞标记蛋白与肌动蛋白细胞骨架连接的机制。
The transmembrane sialoglycoprotein podocalyxin is thought to be essential in the fine interdigitating foot process structure of the podocyte. The intracellular COOH-terminal amino acids Asp-Thr-His-Leu (DTHL) of podocalyxin comprise a putative ligand for a type I PSD95-Dlg-zona occludens-1 (PDZ) domain. A 20-amino acid synthetic peptide containing this motif was used to screen a cDNA library, and clones of rabbit Na+/H+ exchange regulatory factor-2 (NHERF-2) were obtained. In vitro analysis demonstrated that each PDZ domain of NHERF-2 could bind podocalyxin independently. NHERF-2 coprecipitated from glomerular extracts with podocalyxin, and podocalyxin and NHERF-2 colocalized in the glomerular capillary loops, indicating that podocalyxin and NHERF-2 may interact in vivo. Podocalyxin peptide missing the terminal leucine (-DTHL) failed to interact with NHERF-2 in vitro. Podocalyxin localized to the apical membrane of transfected Madin-Darby canine kidney (MDCK) cells. However, mutant podocalyxin (missing a functional DTHL COOH-terminal motif) showed cytoplasmic and apical membrane localization in transfected cells and was also less stable at the apical membrane, as assessed by confocal microscopy and biotinylation studies. Mutant podocalyxin did lower the transepithelial resistance of MDCK cell monolayers, albeit to a lesser extent than full-length podocalyxin. We conclude that podocalyxin can interact with both PDZ domains of NHERF-2 and that this interaction requires the intact COOH terminus of podocalyxin, which is also responsible for the efficient apical localization of podocalyxin in transfected MDCK cells. These results suggest that the interaction of podocalyxin with NHERF-2 may function to efficiently retain podocalyxin at the apical surface of the podocyte and provide a mechanism linking podocalyxin to the actin cytoskeleton.