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https://hdl.handle.net/2440/9549
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Type: | Journal article |
Title: | Nedd4-2 functionally interacts with CIC-5: Involvement in constitutive albumin endocytosis in proximal tubule cells |
Author: | Hryciw, D. Ekberg, J. Lee, A. Lensink, I. Kumar, S. Guggino, W. Cook, D. Pollock, C. Poronnik, P. |
Citation: | Journal of Biological Chemistry, 2004; 279(53):54996-55007 |
Publisher: | Amer Soc Biochemistry Molecular Biology Inc |
Issue Date: | 2004 |
ISSN: | 0021-9258 1083-351X |
Statement of Responsibility: | Deanne H. Hryciw, Jenny Ekberg, Aven Lee, Ingrid L. Lensink, Sharad Kumar, William B. Guggino, David I. Cook, Carol A. Pollock and Philip Poronnik |
Abstract: | Constitutive albumin uptake by the proximal tubule is achieved by a receptor-mediated process in which the Cl(-) channel, ClC-5, plays an obligate role. Here we investigated the functional interaction between ClC-5 and ubiquitin ligases Nedd4 and Nedd4-2 and their role in albumin uptake in opossum kidney proximal tubule (OK) cells. In vivo immunoprecipitation using an anti-HECT antibody demonstrated that ClC-5 bound to ubiquitin ligases, whereas glutathione S-transferase pull-downs confirmed that the C terminus of ClC-5 bound both Nedd4 and Nedd4-2. Nedd4-2 alone was able to alter ClC-5 currents in Xenopus oocytes by decreasing cell surface expression of ClC-5. In OK cells, a physiological concentration of albumin (10 mug/ml) rapidly increased cell surface expression of ClC-5, which was also accompanied by the ubiquitination of ClC-5. Albumin uptake was reduced by inhibiting either the lysosome or proteasome. Total levels of Nedd4-2 and proteasome activity also increased rapidly in response to albumin. Overexpression of ligase defective Nedd4-2 or knockdown of endogenous Nedd4-2 with small interfering RNA resulted in significant decreases in albumin uptake. In contrast, pathophysiological concentrations of albumin (100 and 1000 mug/ml) reduced the levels of ClC-5 and Nedd4-2 and the activity of the proteasome to the levels seen in the absence of albumin. These data demonstrate that normal constitutive uptake of albumin by the proximal tubule requires Nedd4-2, which may act via ubiquitination to shunt ClC-5 into the endocytic pathway. |
Keywords: | Oocytes Kidney Tubules Cell Line Cell Membrane Lysosomes Animals Xenopus Opossums Proteasome Endopeptidase Complex Ubiquitin-Protein Ligases Glutathione Transferase Albumins Xenopus Proteins Chloride Channels Ubiquitin RNA, Small Interfering RNA, Messenger Blotting, Western Biotinylation Immunoprecipitation Endocytosis Protein Binding Dose-Response Relationship, Drug Models, Biological Endosomal Sorting Complexes Required for Transport Nedd4 Ubiquitin Protein Ligases |
DOI: | 10.1074/jbc.M411491200 |
Published version: | http://dx.doi.org/10.1074/jbc.m411491200 |
Appears in Collections: | Aurora harvest 4 Medicine publications |
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