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

Abstract

Epithelial junctions depend on intercellular interactions between β1 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 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 on 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 amino acid region spanning residues 198-207 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 the (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 a 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 amino acid region 198-207 is crucial for both trans-dimerization of the Na+/K+-ATPase β1 subunits and cell-cell adhesion. © 2012.