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 prese nt only on the apical surface of polarized MDCK epithelial cells. Overexpre ssion of a mutant of ARF6, ARF6-Q67L, which is predicted to be in the GTP-b ound form, stimulates endocytosis exclusively at this surface. Surprisingly , overexpression of the mutant ARF6-T27N, which is predicted to be in the G DP-bound form, also stimulated apical endocytosis, though to a lesser exten t. ARF6-stimulated endocytosis is inhibited by a dominant-negative form of dynamin, or a dominant-negative hub fragment of clathrin heavy chain, indic ating that it is mediated by clathrin. Correspondingly, overexpression of e ither 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 c lathrin and with IgA bound to its receptor. We conclude that ARF6 is an imp ortant modulator of clathrin-mediated endocytosis at the apical surface of epithelial cells.