Nedd4-2 functionally interacts with ClC-5: Involvement in constitutive albumin endocytosis in proximal tubule cells

Deanne H. Hryciw, Jenny Ekberg, Aven Lee, Ingrid L. Lensink, Sharad Kumar, William B Guggino, David I. Cook, Carol A. Pollock, Philip Poronnik

Research output: Contribution to journalArticle

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 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.

Original languageEnglish (US)
Pages (from-to)54996-55007
Number of pages12
JournalJournal of Biological Chemistry
Volume279
Issue number53
DOIs
StatePublished - Dec 31 2004

Fingerprint

Endocytosis
Albumins
Proteasome Endopeptidase Complex
Ligases
Ubiquitination
Ubiquitin
Opossums
Proximal Kidney Tubule
Xenopus
Lysosomes
Glutathione Transferase
Immunoprecipitation
Small Interfering RNA
Oocytes
Anti-Idiotypic Antibodies
Antibodies

ASJC Scopus subject areas

  • Biochemistry

Cite this

Nedd4-2 functionally interacts with ClC-5 : Involvement in constitutive albumin endocytosis in proximal tubule cells. / Hryciw, Deanne H.; Ekberg, Jenny; Lee, Aven; Lensink, Ingrid L.; Kumar, Sharad; Guggino, William B; Cook, David I.; Pollock, Carol A.; Poronnik, Philip.

In: Journal of Biological Chemistry, Vol. 279, No. 53, 31.12.2004, p. 54996-55007.

Research output: Contribution to journalArticle

Hryciw, DH, Ekberg, J, Lee, A, Lensink, IL, Kumar, S, Guggino, WB, Cook, DI, Pollock, CA & Poronnik, P 2004, 'Nedd4-2 functionally interacts with ClC-5: Involvement in constitutive albumin endocytosis in proximal tubule cells', Journal of Biological Chemistry, vol. 279, no. 53, pp. 54996-55007. https://doi.org/10.1074/jbc.M411491200
Hryciw, Deanne H. ; Ekberg, Jenny ; Lee, Aven ; Lensink, Ingrid L. ; Kumar, Sharad ; Guggino, William B ; Cook, David I. ; Pollock, Carol A. ; Poronnik, Philip. / Nedd4-2 functionally interacts with ClC-5 : Involvement in constitutive albumin endocytosis in proximal tubule cells. In: Journal of Biological Chemistry. 2004 ; Vol. 279, No. 53. pp. 54996-55007.
@article{6de7bc4d2bdf44ee85bd2a4bcc38262b,
title = "Nedd4-2 functionally interacts with ClC-5: Involvement in constitutive albumin endocytosis in proximal tubule cells",
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 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.",
author = "Hryciw, {Deanne H.} and Jenny Ekberg and Aven Lee and Lensink, {Ingrid L.} and Sharad Kumar and Guggino, {William B} and Cook, {David I.} and Pollock, {Carol A.} and Philip Poronnik",
year = "2004",
month = "12",
day = "31",
doi = "10.1074/jbc.M411491200",
language = "English (US)",
volume = "279",
pages = "54996--55007",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "53",

}

TY - JOUR

T1 - Nedd4-2 functionally interacts with ClC-5

T2 - Involvement in constitutive albumin endocytosis in proximal tubule cells

AU - Hryciw, Deanne H.

AU - Ekberg, Jenny

AU - Lee, Aven

AU - Lensink, Ingrid L.

AU - Kumar, Sharad

AU - Guggino, William B

AU - Cook, David I.

AU - Pollock, Carol A.

AU - Poronnik, Philip

PY - 2004/12/31

Y1 - 2004/12/31

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=11244331526&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=11244331526&partnerID=8YFLogxK

U2 - 10.1074/jbc.M411491200

DO - 10.1074/jbc.M411491200

M3 - Article

C2 - 15489223

AN - SCOPUS:11244331526

VL - 279

SP - 54996

EP - 55007

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 53

ER -