Gp135/podocalyxin and NHERF-2 participate in the formation of a preapical domain during polarization of MDCK cells.
Epithelial polarization involves the segregation of apical and basolateral membrane domains, which are stabilized and maintained by tight junctions and membrane traffic. We report that unlike most apical and basolateral proteins in MDCK cells, which separate only after junctions have formed, the apical marker gp135 signifies an early level of polarized membrane organization established already in single cells. We identified gp135 as the dog orthologue of podocalyxin. With a series of domain mutants we show that the COOH-terminal PSD-95/Dlg/ZO-1 (PDZ)-binding motif is targeting podocalyxin to the free surface of single cells as well as to a subdomain of the terminally polarized apical membrane. This special localization of podocalyxin is shared by the cytoplasmic PDZ-protein Na+/H+ exchanger regulatory factor (NHERF)-2. Depleting podocalyxin by RNA interference caused defects in epithelial polarization. Together, our data suggest that podocalyxin and NHERF-2 function in epithelial polarization by contributing to an early apical scaffold based on PDZ domain-mediated interactions.
Meder
Doris
D
Max Planck Institute of Molecular Cell Biology and Genetics, D-01307 Dresden, Germany.
Shevchenko
Anna
A
Simons
Kai
K
Füllekrug
Joachim
J
D000469:Alkaline Phosphatase; D000818:Animals; D015820:Cadherins / Q000378:metabolism; D002460:Cell Line; D016764:Cell Polarity / Q000235:genetics / Q000502:physiology*; D049109:Cell Proliferation; D003598:Cytoskeletal Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D018076:DNA, Complementary / Q000235:genetics; D004285:Dogs; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism / Q000502:physiology; D058851:GPI-Linked Proteins; D015870:Gene Expression / Q000235:genetics; D006023:Glycoproteins / Q000378:metabolism; D006031:Glycosylation; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D047468:Immunoprecipitation; D019012:Integrin beta1 / Q000378:metabolism; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D008856:Microscopy, Fluorescence; D009154:Mutation; D010957:Plasmids / Q000235:genetics; D011485:Protein Binding; D034622:RNA Interference; D012795:Sialoglycoproteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D014162:Transfection
D015820:Cadherins; D003598:Cytoskeletal Proteins; D018076:DNA, Complementary; D058851:GPI-Linked Proteins; D006023:Glycoproteins; D019012:Integrin beta1; D007527:Isoenzymes; D008565:Membrane Proteins; D012795:Sialoglycoproteins; C001643:podocalyxin; D049452:Green Fluorescent Proteins; D000469:Alkaline Phosphatase; C019839:alkaline phosphatase, placental