| 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 (Liu et al., 2002). This phenomenon is related to ouabain-induced endocytosis of plasmalemmal Na/K-ATPase in LLC-PK1 Cells by a clathrin-dependent mechanism (Liu et al., 2004). GRP78/BiP is a resident protein of the endoplasmic reticulum (ER) and acts as a molecular chaperone. Recently, several studies have shown 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 several up-regulated proteins and confirmed by Western Blot. Interestingly, GRP78/BiP was significantly upregulated in both early and late endosomes in response to ouabain treatment. By using a cell surface protein biotinylation technique to isolate the cell surface membrane proteins, we found that GRP/BiP is expressed on the cell surface of LLC-PK1 cells and down-regulated in a time-dependent manner in response to ouabain. By comparing the cellular redistribution, our data suggest that both the Na/K-ATPase a-1 subunit and GRP78/BiP follow the same pattern in response to ouabain. |
