Identification of the amino acid region involved in the intercellular interaction between the β1 subunits of Na+/K+-ATPase

Epithelial junctions depend on intercellular interactions between beta(1) subunits of the Na+/K+-ATPase molecules of neighboring cells. The interaction between dog and rat subunits is less effective than the interaction between two dog b1 subunits, indicating the importance of species-specific regions for beta(1)-beta(1) binding. To identify these regions, the species-specific amino acid residues were mapped on a high-resolution structure of the Na+/K+-ATPase beta(1) subunit to select those exposed towards the beta(1) subunit of the neighboring cell. These exposed residues were mutated in both dog and rat YFP-linked beta(1) subunits (YFP-beta(1)) and also in the secreted extracellular domain of the dog beta(1) subunit. Five rat-like mutations in the amino acid region spanning residues 198-207 of the dog YFP-beta(1) expressed in Madin Darby canine kidney (MDCK) cells decreased co-precipitation of the endogenous dog beta(1) subunit with YFP-beta(1) to the level observed between dog beta(1) and rat YFP-beta(1). In parallel, these mutations impaired the recognition of YFP-beta(1) by the dog-specific antibody that inhibits cell adhesion between MDCK cells. Accordingly, dog-like mutations in rat YFP-beta(1) increased both the (YFP-beta(1))-beta(1) interaction in MDCK cells and recognition by the antibody. Conversely, rat-like mutations in the secreted extracellular domain of the dog beta(1) subunit increased its interaction with rat YFP-beta(1) in vitro. In addition, these mutations resulted in a reduction of intercellular adhesion between rat lung epithelial cells following addition of the secreted extracellular domain of the dog beta(1) subunit to a cell suspension. Therefore, the amino acid region 198-207 is crucial for both trans-dimerization of the Na+/K+-ATPase beta(1) subunits and cell-cell adhesion.