Alpha-actinin associates with polycystin-2 and regulates its channel activity
Alpha-actinin associates with polycystin-2 and regulates its channel activity
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DOI：
10.1093/hmg/ddi167
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发表时间：
2005-06-15
影响因子：
3.5
通讯作者：
Chen, XZ
中科院分区：
文献类型：
--
作者：
Li, Q;Montalbetti, N;Chen, XZ
多囊蛋白-2（PC 2）是PKD 2基因的产物，其在10-15%的常染色体显性多囊肾病（ADPKD）患者中发生突变。PC 2是一种完整的跨膜蛋白，并充当钙渗透性阳离子通道。其他蛋白质伴侣对该通道的功能调节在很大程度上仍然未知。在本研究中，使用酵母双杂交方法，我们发现，细胞内的N-和C-末端的PC 2与α-辅肌动蛋白，肌动蛋白结合和肌动蛋白捆绑蛋白在细胞骨架组织，细胞粘附，增殖和迁移的重要。通过体外谷胱甘肽S-转移酶下拉和斑点印迹重叠测定证实了PC 2-α-辅肌动蛋白的结合。此外，在人胚肾293和Madin-Darby犬肾（MDCK）细胞，大鼠肾脏和心脏组织和人合胞体滋养层（hST）顶膜囊泡中，通过免疫共沉淀证明了内源性PC 2和α-辅肌动蛋白之间的体内相互作用。免疫荧光实验表明，PC 2和α-辅肌动蛋白部分共定位于上皮MDCK和内髓集合管细胞，NIH 3 T3成纤维细胞和hST囊泡。我们研究了在脂质双层电生理系统中使用体外翻译的PC 2的α-辅肌动蛋白的PC 2的功能调制，发现α-辅肌动蛋白实质上刺激重建的PC 2的通道活性。当hST囊泡在脂质双层中重构时，也观察到α-辅肌动蛋白对PC 2的类似刺激作用。因此，PC 2和α-辅肌动蛋白之间的物理和功能相互作用可能在ADPKD中观察到的异常细胞粘附，增殖和迁移中发挥重要作用。
Polycystin-2 (PC2) is the product of the PKD2 gene, which is mutated in 10-15% patients of autosomal dominant polycystic kidney disease (ADPKD). PC2 is an integral transmembrane protein and acts as a calcium-permeable cation channel. The functional modulation of this channel by other protein partners remains largely unknown. In the present study, using a yeast two-hybrid approach, we discovered that both intracellular N- and C-termini of PC2 associate with alpha-actinins, actin-binding and actin-bundling proteins important in cytoskeleton organization, cell adhesion, proliferation and migration. The PC2-alpha-actinin association was confirmed by in vitro glutathione S-transferase pull-down and dot blot overlay assays. In addition, the in vivo interaction between endogenous PC2 and alpha-actinins was demonstrated by co-immunoprecipitation in human embryonic kidney 293 and Madin-Darby canine kidney (MDCK) cells, rat kidney and heart tissues and human syncytiotrophoblast (hST) apical membrane vesicles. Immunofluorescence experiments showed that PC2 and alpha-actinin were partially co-localized in epithelial MDCK and inner medullary collecting duct cells, NIH 3T3 fibroblasts and hST vesicles. We studied the functional modulation of PC2 by alpha-actinin in a lipid bilayer electrophysiology system using in vitro translated PC2 and found that alpha-actinin substantially stimulated the channel activity of reconstituted PC2. A similar stimulatory effect of alpha-actinin on PC2 was also observed when hST vesicles were reconstituted in lipid bilayer. Thus, physical and functional interactions between PC2 and alpha-actinin may play an important role in abnormal cell adhesion, proliferation and migration observed in ADPKD.