The long-term goals of this project are to determine the intrinsic sorting signals and necessary interacting proteins that enable differential trafficking of an essential transport enzyme, the Na,K-ATPase, to the apical and/or basolateral membranes of normal epithelial cells and those affected by disease. Reduced level of the -31-subunit and high abundance of the (32-subunit isoform in gastric and renal carcinoma and renal tubular cells of polycystic kidneys correlates with a switch from basolateral to apical localization of the pump and loss of normal cell-cell adhesion. We hypothesize that the N-glycans linked to the (31-subunit isoform of the Na,K-ATPase are important for stabilization of the pump on the lateral membranes and therefore for the maintenance of intercellular adhesion in epithelia. On the other hand, the N-glycans unique to the (32-isoform of the Na,K-ATPase are important for apical delivery of the pump.
The specific aims are: 1. Elucidate the role of individual N-glycans of the Na,K-ATPase (31- and (32-subunits in polarized sorting, trafficking, and stabilization of the pump in the apical and basolateral membranes of polarized renal cells in culture. 2. Investigate the role of the (31-subunit of the Na,K-ATPase and its glycosylation in intercellular adhesion of renal cells in normal conditions and upon reversible ischemic injury. We will study the effect of expression of glycosylation-deficient mutants of the two (3-subunit isoforms in MDCK cells on vesicular trafficking of the pump, its distribution between apical and basolateral membranes, association of the pump with the cytoskeleton, and in cell-cell adhesion. We will also determine the differences in the carbohydrate composition of the individual N-glycans in the (3-subunits and identify the lectin proteins that specifically recognize N-glycans and facilitate trafficking, sorting and membrane retention of the Na,K-ATPase (3-subunit isoforms. We will determine which type or which carbohydrate composition of N-glycans is preferable for cell contact formation. These observations will be extended to cells in culture under ischemic conditions to relate these observations to pathology of the kidney. Elucidation of the details of the Na,K-ATPase sorting and identification of the proteins that facilitate proper trafficking will give us a better understanding of the mechanism underlying disruption of cell-cell contacts in renal fibrosis, cancer, polycystic kidney disease and renal ischemia and could impact the development of novel therapeutics. ? ? ?
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