Kesiry Riad, Liu Jiang
Department of Medicine, Medical College of Ohio, 3120 Glendale Avenue, Toledo, Ohio 43614-5089, USA.
Front Biosci. 2005 Sep 1;10:2045-55. doi: 10.2741/1680.
We have demonstrated that ouabain causes dose- and time-dependent decreases both in 86Rb+ uptake and plasmalemmal Na/K-ATPase content of LLC-PK1 cells, which is related to ouabain-induced endocytosis of plasmalemmal Na/K-ATPase in LLC-PK1 cells through a clathrin-dependent mechanism. GRP78/BiP is a resident protein of the endoplasmic reticulum (ER) and acts as a molecular chaperone. Recently, several studies have shown that GRP78/BiP is also expressed on the cell surface and forms heterogeneous, high molecular weight complexes with other proteins. To identify the proteins that are possibly involved in ouabain-induced endocytosis of the Na/K-ATPase in LLC-PK1 cells, we separated and identified endosomal proteins by 2D gel electrophoresis and MS/MS from both control and ouabain-treated LLC-PK1 cells. GRP78/BiP was identified by MS/MS as one of the several up-regulated proteins and confirmed by Western Blot. By using a cell surface protein biotinylation technique to isolate the cell surface membrane proteins, we found that GRP78/BiP is also expressed on the cell surface of LLC-PK1 cells, and surface-expressed GRP78/BiP is down regulated in a time-dependent manner in response to ouabain. By comparing the cellular redistributions, our data suggest that both the Na/K-ATPase alpha-1 subunit and GRP78/BiP follow the same redistribution pattern in response to ouabain.
我们已经证明，哇巴因会导致LLC-PK1细胞中86Rb+摄取量和质膜Na/K-ATP酶含量呈剂量和时间依赖性降低，这与哇巴因通过网格蛋白依赖性机制诱导LLC-PK1细胞质膜Na/K-ATP酶的内吞作用有关。GRP78/BiP是内质网（ER）的驻留蛋白，起分子伴侣的作用。最近，多项研究表明，GRP78/BiP也在细胞表面表达，并与其他蛋白质形成异质性高分子量复合物。为了鉴定可能参与哇巴因诱导的LLC-PK1细胞中Na/K-ATP酶内吞作用的蛋白质，我们通过二维凝胶电泳和串联质谱（MS/MS）从对照和哇巴因处理的LLC-PK1细胞中分离并鉴定了内体蛋白。通过串联质谱（MS/MS）鉴定出GRP78/BiP是几种上调蛋白之一，并通过蛋白质免疫印迹法进行了确认。通过使用细胞表面蛋白生物素化技术分离细胞表面膜蛋白，我们发现GRP78/BiP也在LLC-PK1细胞的细胞表面表达，并且响应哇巴因，表面表达的GRP78/BiP会以时间依赖性方式下调。通过比较细胞内的重新分布，我们的数据表明，Na/K-ATP酶α-1亚基和GRP78/BiP在响应哇巴因时遵循相同的重新分布模式。
