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Shank2 regulates NaPiIIa abundance and endocytosis in OK cells
Author(s) -
Dobrinskikh Evgenia,
Cranston DeeAnn,
Rachelson Joanna,
Moldovan Radu,
Lei Tim,
Doctor R. Brian
Publication year - 2012
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.26.1_supplement.1066.2
Subject(s) - internalization , endocytosis , microbiology and biotechnology , endosome , pdz domain , transport protein , live cell imaging , apical membrane , endocytic cycle , transfection , chemistry , biology , intracellular , cell , biochemistry , membrane , gene
Sodium phosphate co‐transporter IIa (NaPiIIa) is concentrated in the apical microvilli of renal proximal tubule (PT) cells under low serum phosphate (Pi) conditions but is internalized to and degraded within lysosomes under high Pi conditions. In vivo rat studies indicate that Shank2, a PDZ domain protein that binds the C‐terminal tail of NaPiIIa, undergoes a parallel pattern of internalization. The present study utilizes OK cells to determine the functional significance of Shank2 on NaPiIIa. Under low Pi (0.1 mM) conditions, KD of Shank2 expression resulted in a parallel decrease in NaPiIIa abundance. 3 hours after shifting to high Pi (2.0 mM) media, western blotting showed both NaPiIIa and Shank2 undergo degradation. After 1 hour in high Pi media, confocal imaging of fixed cells shows that both native and expressed NaPiIIa and Shank2 are entered the sub‐apical domain. Live‐cell imaging shows that GFP‐NaPiIIa and mRFP‐Shank2 traverse through the sub‐apical domain together as a dual‐labeled endosomes. Fluorescence cross‐correlation spectroscopy (FCCS) provided quantitative evidence at the molecular level of NaPiIIa and Shank2 co‐migration through the sub‐apical domain. Together these findings indicate that, under low Pi conditions, Shank2 contributes to the retention of NaPiIIa within PT cells and that, under high Pi conditions, Shank2 is positioned to impact the endocytosis and trafficking of NaPiIIa.