ADP-ribosylation factor 6 and endocytosis at the apical surface of Madin-Darby canine kidney cells.
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.
Altschuler
Y
Y
Department of Anatomy, University of California, San Francisco, California 94143-0452, USA. yoram@itsa.ucsf.edu
Liu
S
S
Katz
L
L
Tang
K
K
Hardy
S
S
Brodsky
F
F
Apodaca
G
G
Mostov
K
K
D000089982:ADP-Ribosylation Factor 6; D020727:ADP-Ribosylation Factors / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D002460:Cell Line; D016764:Cell Polarity; D002966:Clathrin / Q000235:genetics / Q000378:metabolism*; D033922:Clathrin Heavy Chains; D003034:Coated Pits, Cell-Membrane / Q000378:metabolism / Q000648:ultrastructure; D004285:Dogs; D034281:Dynamins; D004705:Endocytosis; D004847:Epithelial Cells / Q000166:cytology* / Q000378:metabolism / Q000648:ultrastructure; D020558:GTP Phosphohydrolases / Q000235:genetics / Q000378:metabolism; D005799:Genes, Dominant / Q000235:genetics; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D007070:Immunoglobulin A / Q000276:immunology; D007668:Kidney; D008854:Microscopy, Electron; D009154:Mutation; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
D000089982:ADP-Ribosylation Factor 6; D002966:Clathrin; D007070:Immunoglobulin A; D010446:Peptide Fragments; D011994:Recombinant Proteins; D033922:Clathrin Heavy Chains; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; D020558:GTP Phosphohydrolases; D020727:ADP-Ribosylation Factors; D034281:Dynamins