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Phosphorylation regulates clathrin‐mediated endocytosis of the thiazide‐sensitive cotransporter NCC (1181.12)
Author(s) -
Rosenbaek Lena,
Kortenoeven Marleen,
Aroankins Takwa,
Fenton Robert
Publication year - 2014
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.28.1_supplement.1181.12
Subject(s) - endocytosis , internalization , phosphorylation , clathrin , microbiology and biotechnology , chemistry , biology , biochemistry , cell
Our recent work suggests that AQP2 endocytosis rates can be regulated via phosphorylation. As NCC is phosphorylated at conserved residues in its intracellular NH2 terminus, we hypothesized that a similar mechanism may regulate NCC plasma membrane abundance. Novel MDCKI cell line models stably expressing NCC were generated to examine trafficking pathways of NCC and the role of phosphorylation in regulation of NCC internalization. In MDCKI‐NCC cells, NCC was observed as a highly glycosylated monomer and dimer. NCC constitutively recycled to/from the apical plasma membrane of MDCKI‐NCC cells, with 20‐30% of the apical membrane pool of NCC being internalized within 30 min. Using specific inhibitors of either clathrin‐dependent or ‐independent endocytosis we determined that NCC is internalized via a clathrin‐mediated pathway and reduction of endocytosis results in greater steady‐state NCC levels in the plasma membrane. Inducing phosphorylation of NCC via low chloride treatment or mimicking phosphorylation by replacing Thr53, Thr58, and Ser71 residues with Asp, resulted in increased apical membrane abundance and reduced rates of NCC internalization. Our data support a model where NCC is constitutively cycled to the plasma membrane, where upon stimulation it can be phosphorylated resulting in increased NCC activity and decreased NCC endocytosis, which together increase NaCl transport in the DCT. Grant Funding Source : Supported by Lundbeck Foundation and Danish Medical Research Foundation