title: Distinguishing roles of the membrane-cytoskeleton and cadherin mediated cell-cell adhesion in generating different Na+,K(+)-ATPase distributions in polarized epithelia.
abstract: In simple epithelia, the distribution of ion transporting proteins between the apical or basal-lateral domains of the plasma membrane is important for determining directions of vectorial ion transport across the 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 localized to the basal-lateral membrane of cells in the kidney nephron where it regulates ion and solute reabsorption. The mechanisms involved in restricting Na+,K(+)-ATPase distribution to different membrane domains in these simple epithelia are poorly understood. Previous studies have indicated a role for E-cadherin mediated cell-cell adhesion and membrane-cytoskeleton (ankyrin and fodrin) assembly in regulating Na+,K(+)-ATPase distribution in absorptive kidney epithelial cells. Confocal immunofluorescence microscopy reveals that in chicken and rat choroid plexus epithelium, fodrin, and ankyrin colocalize with Na+,K(+)-ATPase at the apical plasma membrane, but fodrin, ankyrin, and adducin also localize at the lateral plasma membrane where Na+,K(+)-ATPase is absent. Biochemical analysis shows that fodrin, ankyrin, and Na+,K(+)-ATPase are relatively resistant to extraction from cells in buffers containing Triton 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 electrophoresis in nondenaturing polyacrylamide gels revealed that fodrin, ankyrin, and Na+,K(+)-ATPase comigrate, indicating that these proteins are in a high molecular weight complex similar to that found previously in kidney epithelial cells. In contrast, the anion exchanger (AE2), a marker protein of the basal-lateral plasma membrane in the choroid plexus, did not cosediment in sucrose gradients or comigrate in nondenaturing polyacrylamide gels with the complex of Na+,K(+)-ATPase, ankyrin, and fodrin. Ca(++)-dependent cell adhesion molecules (cadherins) were detected at lateral membranes of the choroid plexus epithelium and colocalized 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 containing Triton X-100 cosedimented with ankyrin and fodrin in sucrose gradients and comigrated in nondenaturing gels with ankyrin and fodrin in a high molecular weight complex. Since a previous study showed that E-cadherin is an instructive inducer of Na+,K(+)-ATPase distribution, we examined protein distributions in fibroblasts transfected with B-cadherin, a prominent cadherin expressed in the choroid plexus epithelium.(ABSTRACT TRUNCATED AT 400 WORDS)
authors.affiliation: Department of Molecular and Cellular Physiology, Beckman Center for Molecular and Genetic Medicine, Stanford University School of Medicine, California 94305-5426.
authors.forename: J A
authors.initials: JA
authors.lastname: Marrs
authors.forename: E W
authors.initials: EW
authors.lastname: Napolitano
authors.forename: C
authors.initials: C
authors.lastname: Murphy-Erdosh
authors.forename: R W
authors.initials: RW
authors.lastname: Mays
authors.forename: L F
authors.initials: LF
authors.lastname: Reichardt
authors.forename: W J
authors.initials: WJ
authors.lastname: Nelson
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D027321:Anion Transport Proteins; D017487:Ankyrins / Q000302:isolation & purification; D017920:Antiporters; D001483:Base Sequence; D015820:Cadherins / Q000302:isolation & purification; D002352:Carrier Proteins / Q000302:isolation & purification; D002448:Cell Adhesion / Q000502:physiology*; D002451:Cell Compartmentation; D002462:Cell Membrane / Q000378:metabolism; D016764:Cell Polarity; D002478:Cells, Cultured; D002499:Centrifugation, Density Gradient; D002645:Chickens; D002831:Choroid Plexus / Q000378:metabolism* / Q000648:ultrastructure; D003598:Cytoskeletal Proteins / Q000302:isolation & purification; D003599:Cytoskeleton / Q000378:metabolism; D004285:Dogs; D004847:Epithelial Cells; D004848:Epithelium / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D046911:Macromolecular Substances; D008565:Membrane Proteins / Q000302:isolation & purification; D008840:Microfilament Proteins / Q000302:isolation & purification; D008969:Molecular Sequence Data; D051381:Rats; D064606:SLC4A Proteins; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism; D014162:Transfection; D013049:Spectrin
chemical_list: D027321:Anion Transport Proteins; D017487:Ankyrins; D017920:Antiporters; D015820:Cadherins; D002352:Carrier Proteins; D003598:Cytoskeletal Proteins; D046911:Macromolecular Substances; D008565:Membrane Proteins; D008840:Microfilament Proteins; D064606:SLC4A Proteins; D000254:Sodium-Potassium-Exchanging ATPase; D013049:Spectrin