GRP78/BiP is involved in ouabain-induced endocytosis of the Na/K-ATPase in LLC-PK1 cells

We have demonstrated that ouabain causes dose- and time-dependent decreases both in Rb-86(+) 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.