Role of anti-adhesin, podocalyxin and adaptor proteins in glomerular injury

我们已经在体内外证明了抗黏附素分子Podocalysin与NHERF2和Ezrin的第二个PDZ结构域结合，这种相互作用对于维持足细胞结构的独特组织是重要的。在这项研究项目中，我们研究了接头蛋白如何与Podocalyin在肾小球损伤中相互作用。结果：1.我们发现Podocalyin/NHERF2/Ezrin Comlex的磷酸化状态与汞汞损伤有关。用酵母双杂交系统鉴定了与NHERF2的第一个PDZ结构域相互作用的蛋白。令人惊讶的是，这就是NHERF2本身。GST下拉实验和免疫共沉淀证实了这种自我相互作用，在严重损伤的肾小球中这种自我相互作用被破坏。我们分析了Mundel Like博士建立的永生化小鼠肾小球上皮细胞的内吞功能。我们还发现，与Angioteins II浓度成正比，Podocalysin与上述细胞株的接头蛋白之间的相互作用减弱。当高极化MDCK细胞表达Podocalyin时，接头蛋白被重新定位到顶端膜下结构域和小G蛋白，Rho A被激活。这些结果表明，Podocalyx可能通过激活Rho A而导致接头蛋白重新定位到顶端区域。

We have previously demonstrated that anti-adhesin molecule, podocalyxin binds to the second PDZ domain of NHERF2 and ezrin in vitro and in vivo and that the interaction is important to maintain the unique organization of podocyte structure. In this research project, we investigated how the adaptor proteins interact with podocalyxin in glomerular injury.1. We found that phosphrylation status of podocalyxin/NHERF2/ezrin comlex linked to the golmerular injury.2. We identified the protein which interacted with first PDZ domain of NHERF2 by yeast two-hybrid system. Surprisingly, it was NHERF2 itself. This self-interaction was confirmed by GST pull-down assay and co-immunoprecipitation and was disrupted in severe injured glomeruli.3. We analyzed the endocytic function of mouse immortalized glomerular epithlial cells that was established by Dr. Mundel like as in vivo millue.4. We also found that the interaction between podocalyxin and adaptor proteins of above cell line was reduced in propotion to the angioteinsin II concentration.5. When highly polarized MDCK cells were transfected to express podocalyxin, the adaptor proteins were re-localized to the apical submembranous domain and small G-protein, Rho A was activated. These results suggest podocalyxin may lead the adaptor proteins re-locate to the apical domain through the activation of Rho A.