DISTINGUISHING ROLES OF THE MEMBRANE-CYTOSKELETON AND CADHERIN MEDIATED CELL-CELL ADHESION IN GENERATING DIFFERENT NA-ATPASE DISTRIBUTIONS IN POLARIZED EPITHELIA(,K+)

In simple epithelia, the distribution of ion transporting proteins bet ween the apical or basal-lateral domains of the plasma membrane is imp ortant for determining directions of vectorial ion transport across th e epithelium. In the choroid plexus, Na+,K+-ATPase is localized to the apical plasma membrane domain where it regulates sodium secretion and production of cerebrospinal fluid; in contrast, Na+,K+-ATPase is loca lized to the basal-lateral membrane of cells in the kidney nephron whe re it regulates ion and solute reabsorption. The mechanisms involved i n restricting Na+,K+-ATPase distribution to different membrane domains in these simple epithelia are poorly understood. Previous studies hav e indicated a role for E-cadherin mediated cell-cell adhesion and memb rane-cytoskeleton (ankyrin and fodrin) assembly in regulating Na+,K+-A TPase distribution in absorptive kidney epithelial cells. Confocal imm unofluorescence microscopy reveals that in chicken and rat choroid ple xus epithelium, fodrin, and ankyrin colocalize with Na+,K+-ATPase at t he apical plasma membrane, but fodrin, ankyrin, and adducin also local ize at the lateral plasma membrane where Na+,K+-ATPase is absent. Bioc hemical analysis shows that fodrin, ankyrin, and Na+,K+-ATPase are rel atively resistant to extraction from cells in buffers containing Trito n X-100. The fractions of Na+,K+-ATPase, fodrin, and ankyrin that are extracted from cells cosediment in sucrose gradients at approximately 10.5 S. Further separation of the 10.5 S peak of proteins by electroph oresis in nondenaturing polyacrylamide gels revealed that fodrin, anky rin, and Na+,K+-ATPase comigrate, indicating that these proteins are i n a high molecular weight complex similar to that found previously in kidney epithelial cells. In contrast, the anion exchanger (AE2), a mar ker protein of the basal-lateral plasma membrane in the choroid plexus , did not cosediment in sucrose gradients or comigrate in nondenaturin g polyacrylamide gels with the complex of Na+,K+-ATPase, ankyrin, and fodrin. Ca++-dependent cell adhesion molecules (cadherins) were detect ed at lateral membranes of the choroid plexus epithelium and colocaliz ed with a distinct fraction of ankyrin, fodrin, and adducin. Cadherins did not colocalize with Na+,K+-ATPase and were absent from the apical membrane. The fraction of cadherins that was extracted with buffers c ontaining Triton X-100 cosedimented with ankyrin and fodrin in sucrose gradients and comigrated in nondenaturing gels with ankyrin and fodri n in a high molecular weight complex. Since a previous study showed th at E-cadherin is an instructive inducer of Na+,K+-ATPase distribution, we examined protein distributions in fibroblasts transfected with B-c adherin, a prominent cadherin expressed in the choroid plexus epitheli um. The results show that Na+,K+-ATPase and fodrin become concentrated at cell-cell contracts in cells expressing E-cadherin, but not in cel ls expressing B-cadherin, indicating differences in the potential of t hese cadherins to induce Na+,K+-ATPase accumulation at cell-cell conta cts. These results provide new insights into the roles of the membrane -cytoskeleton and cadherins in generating different distributions of t he same protein in simple epithelial cells.