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ARF6-GTP recruits Nm23-H1 to facilitate dynamin-mediated endocytosis during adherens junctions disassembly

Abstract

ARF6-regulated endocytosis of E-cadherin is essential during the disassembly of adherens junctions in epithelial cells. Here, we show that activation of ARF6 promotes clathrin-dependent internalization of E-cadherin and caveolae at the basolateral cell surface. Furthermore, we demonstrate that ARF6-GTP, a constitutively activate form of ARF6, interacts with and recruits Nm23-H1, a nucleoside diphosphate (NDP) kinase that provides a source of GTP for dynamin-dependent fission of coated vesicles during endocytosis. Finally, we show that ARF6-mediated recruitment of Nm-23-H1 to cell junctions is accompanied by a decrease in the cellular levels of Rac1-GTP, consistent with previous findings that Nm23-H1 down-regulates activation of Rac1. These studies provide a molecular basis for ARF6 function in polarized epithelia during adherens junction disassembly.

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Figure 1: ARF6 regulates ligand internalization rate at the basolateral surface of MDCK cells.
Figure 2: ARF6 regulates dynamin-dependent endocytosis of E-cadherin, Tf and caveolae.
Figure 3: ARF6 recruits Nm23-H1 to promote vesicle fission.
Figure 4: ARF6 modulates Rac1-GTP levels by recruitment of Nm23-H1.
Figure 5: Role of ARF6 in adherens junction disassembly and migratory potential.

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Acknowledgements

We thank L. van Aelst for the plasmid encoding the CRIB domain of PAK fused to GST, the T cell library used in the yeast two hybrid screen and for helpful discussions on the screening procedure, M. McNiven for the plasmids encoding dynamin 2, P. Chavrier for critical reading of the manuscript, and S. Kerbawy and K. Kruger-Passig for excellent technical assistance. F.P. and J.S. are recipients of predoctoral and post-doctoral fellowships, respectively, from the Walther Cancer Institute. This work was supported in part by grants from the American Heart Association and the US Department of Defense to C.D.-S.

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Palacios, F., Schweitzer, J., Boshans, R. et al. ARF6-GTP recruits Nm23-H1 to facilitate dynamin-mediated endocytosis during adherens junctions disassembly. Nat Cell Biol 4, 929–936 (2002). https://doi.org/10.1038/ncb881

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