Distinct subcellular expression of endogenous polycystin-2 in the plasma membrane and Golgi apparatus of MDCK cells
Distinct subcellular expression of endogenous polycystin-2 in the plasma membrane and Golgi apparatus of MDCK cells
复制标题
DOI：
10.1093/hmg/11.1.59
复制
发表时间：
2002-01-01
影响因子：
3.5
通讯作者：
Peters, DJM
中科院分区：
文献类型：
--
作者：
Scheffers, MS;Le, H;Peters, DJM
多囊蛋白-2是一种具有非选择性阳离子通道活性的膜蛋白。该蛋白质由PKD 2基因编码，该基因在15%的常染色体显性多囊肾病（ADPKD）患者中发生突变。多囊蛋白-2可以与PKD 1基因的跨膜蛋白多囊蛋白-1相互作用。然而，内质网（ER）定位（异源表达）多囊蛋白-2在培养细胞和基底外侧定位已报告在肾组织。使用两个多克隆抗血清提出了对多囊蛋白-2，我们证明了不同的表达的内源性蛋白质的高尔基体和质膜的MDCK细胞。相反，大多数异源表达的多囊蛋白-2（PC 2-EGFP）保留在ER中，与ER标记物蛋白质二硫键异构酶（PDI）的染色模式基本重叠。仅在这些细胞的一小部分中观察到弱质膜信号。膜染色也建议通过免疫电子显微镜，并证实了亚细胞分级蔗糖密度梯度。用含有Triton X-100的缓冲液提取后，质膜染色消失，而多囊蛋白-1和E-钙粘蛋白的信号仍然可见，表明多囊蛋白-2既不与Triton X-100不溶性细胞骨架紧密结合，也不与这些蛋白质结合。我们得出结论，内源性多囊蛋白-2通过高尔基体运输到质膜，并具有更广泛的膜定位比多囊蛋白-1。这些数据表明，多囊蛋白-2可以在膜的某些区域自由移动，在那里它可能作为一个通道，激活，或与多囊蛋白-1复合。
Polycystin-2 is a predicted integral membrane protein with non-selective cation channel activity. The protein is encoded by the PKD2 gene, which is mutated in similar to15% of patients with autosomal dominant polycystic kidney disease (ADPKD). Polycystin-2 can interact with the transmembrane protein polycystin-1, the product of the PKD1 gene. However, endoplasmic reticulum (ER) localization was reported for (heterologously expressed) polycystin-2 in cultured cells and baso-lateral localization has been reported in renal tissues. Using two polyclonal antisera raised against polycystin-2 we demonstrated distinct expression of the endogenous protein in the Golgi apparatus and the plasma membrane of MDCK cells. In contrast, most of the heterologously expressed polycystin-2 (PC2-EGFP) remained in the ER, substantially overlapping with the staining pattern of protein-disulfide isomerase (PDI), a marker for the ER. Only in a small subset of these cells weak plasma membrane signals were observed. Membrane staining was also suggested by immunoelectron microscopy and was confirmed by subcellular fractionation on sucrose density gradients. The plasma membrane staining disappeared following extraction with a buffer containing Triton X-100, whereas signals for polycystin-1 and E-cadherin remained visible, suggesting that polycystin-2 is neither tightly bound to the Triton X-100 insoluble cytoskeleton, nor to these proteins. We conclude that endogenous polycystin-2 is transported via the Golgi apparatus to the plasma membrane and has a broader membrane localization than polycystin-1. These data suggest that polycystin-2 can move freely in certain regions of the membrane where it probably functions as a channel, activated by, or in complex with, polycystin-1.