Role of the membrane-cytoskeleton in the spatial organization of the Na,K-ATPase in polarized epithelial cells.

Vectorial function of polarized transporting epithelia requires the establishment and maintenance of a nonrandom distribution of Na,K-ATPase on the cell surface. In many epithelia, the Na,K-ATPase is located at the basal-lateral domain of the plasma membrane. The mechanisms involved in the spatial organization of the Na,K-ATPase in these cells are poorly understood. We have been investigating the roles of regulated cell-cell contacts and assembly of the membrane-cytoskeleton in the development of the cell surface polarity of Na,K-ATPase. We have shown that the Na,K-ATPase colocalizes with distinct components of the membrane-cytoskeleton in polarized Madin-Darby canine kidney (MDCK) epithelial cells. Significantly, we showed directly that Na,K-ATPase is a high affinity binding site for the membrane-cytoskeletal proteins ankyrin and fodrin, and that all three proteins exist in a high molecular weight protein complex that also contains the cell adhesion molecule (CAM) uvomorulin. We have proposed that these interactions are important in the assembly at sites of cell-cell contact of the membrane-cytoskeleton, which in turn initiates the development of the nonrandom distribution of the Na,K-ATPase. To directly investigate the functional significance of these protein-protein interactions in the spatial organization of the Na,K-ATPase, we analyzed the distribution of the Na,K-ATPase in fibroblasts transfected with a cDNA encoding the epithelial CAM, uvomorulin. Our results showed that expression of uvomorulin is sufficient to induce a redistribution of Na,K-ATPase from an unrestricted distribution over the entire cell surface in nontransfected cells to a restricted distribution at sites of uvomorulin-mediated cell-cell contacts in the transfected cells; this distribution is similar to that in polarized epithelial cells. This restricted distribution of the Na,K-ATPase occurred in the absence of tight junctions, but coincided with the reorganization of the membrane-cytoskeleton. These results support a model in which the epithelial CAM uvomorulin functions as an inducer of cell surface polarity of Na,K-ATPase through cytoplasmic linkage to the membrane-cytoskeleton.