ARNO through its coiled-coil domain regulates endocytosis at the apical surface of polarized epithelial cells.
ARNO is a guanine-nucleotide exchange protein for the ARF family of GTPases. Here we show that in polarized epithelial cells, ARNO is localized exclusively to the apical plasma membrane, where it regulates endocytosis. Expression of ARNO stimulates apical endocytosis of the polymeric immunoglobulin receptor, and coexpression of ARF6 with ARNO leads to a synergistic stimulation of apical endocytosis. Expression of a dominant negative ARF6 mutant, ARF6-T27N, antagonizes this stimulatory effect. Deletion of the N-terminal coiled-coil (CC) domain of ARNO causes the mutant ARNO to localize to both the apical and basolateral plasma membranes. Expression of the CC domain alone abolishes ARNO-induced apical endocytosis as well as co-localization of IgA-receptor complexes with ARNO and clathrin. These results suggest that the CC domain contributes to the specificity of apical localization of ARNO through association with components of the apical plasma membrane. We conclude that ARNO acts together with ARF6 to regulate apical endocytosis.
Department of Pharmacology, School of Pharmacy, Faculty of Medicine, Hebrew University of Jerusalem, Jerusalem 91120, Israel.
Miriam
M
Shmuel
Department of Pharmacology, School of Pharmacy, Faculty of Medicine, Hebrew University of Jerusalem, Jerusalem 91120, Israel.
Lorraine C
LC
Santy
Department of Pharmacology, School of Pharmacy, Faculty of Medicine, Hebrew University of Jerusalem, Jerusalem 91120, Israel.
Scott
S
Frank
Department of Pharmacology, School of Pharmacy, Faculty of Medicine, Hebrew University of Jerusalem, Jerusalem 91120, Israel.
Dana
D
Avrahami
Department of Cell Biology, University of Virginia Health Sciences Center, Charlottesville, Virginia 22908.
James E
JE
Casanova
Department of Pharmacology, School of Pharmacy, Faculty of Medicine, Hebrew University of Jerusalem, Jerusalem 91120, Israel. Electronic address: yoram11@md.huji.ac.il.
Yoram
Y
Altschuler
D000089982:ADP-Ribosylation Factor 6; D020727:ADP-Ribosylation Factors / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D004285:Dogs; D004705:Endocytosis / Q000502:physiology*; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism*; D020690:GTPase-Activating Proteins / Q000235:genetics / Q000378:metabolism*; D005786:Gene Expression Regulation; D006801:Humans; D017434:Protein Structure, Tertiary; D021381:Protein Transport
D000089982:ADP-Ribosylation Factor 6; D020690:GTPase-Activating Proteins; C102474:cytohesin-2; D020727:ADP-Ribosylation Factors; C000715348:ARF6 protein, human