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

Abstract

Epithelial junctions depend on intercellular interactions between the Na,K-ATPase β1 subunits of neighboring cells. The interaction between dog and rat subunits is less effective than the interaction between two dog β1 subunits, indicating the importance of species-specific regions for β1-β1 binding. To identify these regions, the species-specific amino-acid residues were mapped onto a high resolution structure of the Na,K-ATPase β1 subunit to select those exposed towards the β1 subunit of the neighboring cell. These exposed residues were mutated in both dog and rat YFP linked β1 subunits (YFP-β1) and also in the secreted extracellular domain of the dog β1 subunit. Five rat-like mutations in the 198–207 amino-acid region of the dog YFP-β1 expressed in Madin Darby canine kidney (MDCK) cells decreased co-precipitation of the endogenous dog β1 subunit with YFP-β1 to the level observed between dog β1 and rat YFP-β1. In parallel, these mutations impaired the recognition of YFP-β1 by the dog-specific antibody that inhibits cell adhesion between MDCK cells. Accordingly, dog-like mutations in rat YFP-β1 increased both (YFP-β1)-β1 interaction in MDCK cells and recognition by the antibody. Conversely, rat-like mutations in the secreted extracellular domain of the dog β1 subunit increased its interaction with rat YFP-β1 in vitro. In addition, these mutations resulted in reduction of intercellular adhesion between rat lung epithelial cells following addition of the secreted extracellular domain of the dog β1 subunit to a cell suspension. Therefore, the 198–207 amino-acid region is critical for both trans-dimerization of the Na,K-ATPase β1 subunits and cell-cell adhesion.