Abstract
We report that the small GTPase, ADP-ribosylation factor 6 (ARF6), is present only on the apical surface of polarized MDCK epithelial cells. Overexpression of a mutant of ARF6, ARF6-Q67L, which is predicted to be in the GTP-bound form, stimulates endocytosis exclusively at this surface. Surprisingly, overexpression of the mutant ARF6-T27N, which is predicted to be in the GDP-bound form, also stimulated apical endocytosis, though to a lesser extent. ARF6-stimulated endocytosis is inhibited by a dominant-negative form of dynamin, or a dominant-negative hub fragment of clathrin heavy chain, indicating that it is mediated by clathrin. Correspondingly, overexpression of either mutant of ARF6 leads to an increase in the number of clathrin-coated pits at the apical plasma membrane. When ARF6-Q67L is overexpressed in the presence of the dominant-negative dynamin, the ARF6-Q67L colocalizes with clathrin and with IgA bound to its receptor. We conclude that ARF6 is an important modulator of clathrin-mediated endocytosis at the apical surface of epithelial cells.
Publication types
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Research Support, U.S. Gov't, Non-P.H.S.
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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ADP-Ribosylation Factor 6
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ADP-Ribosylation Factors / genetics
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ADP-Ribosylation Factors / metabolism*
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Animals
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Cell Line
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Cell Polarity*
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Clathrin / genetics
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Clathrin / metabolism*
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Clathrin Heavy Chains
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Coated Pits, Cell-Membrane / metabolism
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Coated Pits, Cell-Membrane / ultrastructure
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Dogs
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Dynamins
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Endocytosis*
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Epithelial Cells / cytology*
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Epithelial Cells / metabolism
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Epithelial Cells / ultrastructure
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GTP Phosphohydrolases / genetics
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GTP Phosphohydrolases / metabolism
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Genes, Dominant / genetics
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Guanosine Diphosphate / metabolism
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Guanosine Triphosphate / metabolism
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Immunoglobulin A / immunology
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Kidney
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Microscopy, Electron
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Mutation
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Peptide Fragments / genetics
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Peptide Fragments / metabolism
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Recombinant Proteins / genetics
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Recombinant Proteins / metabolism
Substances
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ADP-Ribosylation Factor 6
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Clathrin
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Immunoglobulin A
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Peptide Fragments
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Recombinant Proteins
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Clathrin Heavy Chains
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Guanosine Diphosphate
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Guanosine Triphosphate
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GTP Phosphohydrolases
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ADP-Ribosylation Factors
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Dynamins