Nedd4-2functionally interacts with ClC-5: involvement in constitutive albumin endocytosis in proximal tubule cells
Hryciw, Deanne, Ekberg, Jenny, Lee, Aven, Lensink, Ingrid, Kumar, Sharad, Guggino, William, Cook, David, Pollock, Carol, & Poronnik, Philip (2004) Nedd4-2functionally interacts with ClC-5: involvement in constitutive albumin endocytosis in proximal tubule cells. Journal of Biological Chemistry, 279, pp. 54996-55007.
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 μg/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 μg/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.
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|Item Type:||Journal Article|
|Additional Information:||Articles free to read on journal website after 12 months|
|Copyright Owner:||Copyright 2004 The American Society for Biochemistry and Molecular Biology|
|Deposited On:||12 Mar 2014 01:19|
|Last Modified:||04 Feb 2015 05:59|
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