Nedd4-2 functionally interacts with ClC-5: involvement in constitutive albumin endocytosis in proximal tubule cells.
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.
School of Biomedical Sciences, The University of Queensland, Brisbane, Queensland 4072, Australia.
Deanne H
DH
Hryciw
Jenny
J
Ekberg
Aven
A
Lee
Ingrid L
IL
Lensink
Sharad
S
Kumar
William B
WB
Guggino
David I
DI
Cook
Carol A
CA
Pollock
Philip
P
Poronnik
D000418:Albumins / Q000378:metabolism*; D000818:Animals; D019921:Biotinylation; D015153:Blotting, Western; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D018118:Chloride Channels / Q000737:chemistry* / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004705:Endocytosis; D056827:Endosomal Sorting Complexes Required for Transport; D005982:Glutathione Transferase / Q000378:metabolism; D047468:Immunoprecipitation; D007684:Kidney Tubules / Q000166:cytology* / Q000378:metabolism; D008247:Lysosomes / Q000378:metabolism; D008954:Models, Biological; D000075702:Nedd4 Ubiquitin Protein Ligases; D009865:Oocytes / Q000378:metabolism; D009893:Opossums; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D011485:Protein Binding; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism; D025801:Ubiquitin / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000737:chemistry* / Q000378:metabolism; D014981:Xenopus; D029867:Xenopus Proteins
D000418:Albumins; C097503:CLC-5 chloride channel; D018118:Chloride Channels; D056827:Endosomal Sorting Complexes Required for Transport; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D025801:Ubiquitin; D029867:Xenopus Proteins; D000075702:Nedd4 Ubiquitin Protein Ligases; C000618482:Nedd4 protein, Xenopus; C000618460:nedd4l protein, Xenopus; D044767:Ubiquitin-Protein Ligases; D005982:Glutathione Transferase; D046988:Proteasome Endopeptidase Complex