Linking of NEDD4 or NEDD4L to AQP2 by NDFIP1 or NDFIP2 mediates AQP2 ubiquitination.

<p>(A) <i>NDFIP1/2 link binding of NEDD4/NEDD4L to AQP2</i>. HEK293 cells transiently-expressing AQP2 and NEDD4 or AQP2 and NEDD4L with or without wildtype-NDFIP1/NDFIP2 (1, 2) or their PY-mutants (1-PY*, 2-PY*) were lysed and subjected to AQP2-immunoprecipitation (IP: α-AQP2). The AQP2 IP-fractions (upper panel) and lysates (lysate) were subjected to immunoblotting for NEDD4 or NEDD4L (IB: α-NEDD4/NEDD4L), NDFIP1/2/1-PY*/2-PY* (IB: α-myc), and AQP2 (IB: α-AQP2). Only upon co-expression with AQP2 and wild type NDFIP1/NDFIP2, NEDD4 or NEDD4L were detected in the immunoprecipitate. (B/C) <i>NEDD4/NEDD4L binding to NDFIP1-2 is needed for AQP2 ubiquitination and degradation</i>. HEK293 cells transiently expressed AQP2 and NEDD4 (B) or NEDD4L (C) with or without NDFIP1/NDFIP2 (1, 2) or their PY-mutants (1-PY*, 2-PY*). AQP2 was immunoprecipitated (IP: α-AQP2) and subjected to immunoblotting for ubiquitin (IB: α-Ub), revealing AQP2 coupled to one (AQP2-Ub₁), two (AQP2-Ub₂) or three (AQP2-Ub₃) ubiquitin molecules. Semi-quantification of the ubiquitinated AQP2 signals is given (control is set to 100%). Co-transfection of AQP2 with NEDD4/NEDD4L and NDFIP1/NDFIP2 increased AQP2 ubiquitination, whereas with NDFIP1/2-PY mutants no increase was detected. Co-transfection of AQP2 with NEDD4 or NEDD4L alone does not increase the abundance of ubiquitinated AQP2. Immuno-precipitating IgGs ({IgG}) are indicated. Coomassie staining of the blots confirmed loading of protein equivalents. Molecular masses of proteins are indicated on the left (in kDa).</p

Expression of NDFIP1, NDFIP2, NEDD4 and NEDD4L in the mouse kidney.

<p>(A) <i>Ndfip1</i>, <i>Ndfip2</i>, <i>Nedd4 and Nedd4L mRNA expression along the nephron</i>. mRNA was isolated from different mouse nephron segments, and subjected to RT-qPCR to determine Ndfip1, Ndfip2, Nedd4 and Nedd4L mRNA levels. The determined mRNA levels were normalized to that of the housekeeping gene Rpl26 and presented as fraction of the segment with the highest level of expression, which was set to 1. Values are means ± SE from 6 mice. Segments indicated are proximal convoluted (S1) and straight (S3) tubule (PCT, PST), medullary (mTAL) and cortical (cTAL) thick ascending limb of Henle’s loop, distal convoluted tubule (DCT), connecting tubule (CNT), and the cortical (CCD) and outer medullary (OMCD) collecting duct. (B) <i>NDFIP1</i>, <i>but not NDFIP2</i>, <i>co-localizes with AQP2</i>. Cryo-sections of mouse kidneys were subjected to immunohistochemistry for NDFIP1 or NDFIP2 (green), AQP2 (red), and the nuclear dye DAPI (blue). Especially in the cortex and inner medulla, NDFIP1 colocalized with AQP2 in collecting duct principal cells. For NDFIP2, co-localization with AQP2 was never observed. Scale bars represent 25 μm.</p

NDFIP2, a NEDD4 family interacting protein, binds to AQP2 in a MYTH assay.

<p>(A) <i>Specific interaction of NDFIP2 with AQP2</i>. Colony growth on interaction-selection media of yeast cells expressing NubG-NDFIP2 together with AQP2 indicates interaction (left two panels). No colony growth is detected with the artificial bait (CD4-Cub) a human integral membrane protein (right two panels). NubG-OST and NubI-OST serve as negative and positive controls for interaction in the ER-membrane, respectively, whereas NubG-FUR4 and NubI-FUR4 serve as negative and positive controls for interaction in the plasma membrane, respectively. These data confirm expression of AQP2 in the ER and plasma membrane of yeast cells and reveal the absence of self-activation. (B) <i>Topology and interaction elements of NDFIP2</i>. The part of NDFIP2 found to interact with AQP2 (amino acid 203–336, indicated with grey marking) covers part of the N-tail, the transmembrane domains (indicated with dashed line) and a part of the luminal/extracellular C-terminus. The PY elements which interact with the NEDD4/4L ww domains are indicated with a closed line. (C) <i>Alignment of the sequences of human NDFIP2 and NDFIP1</i>. Of the sequence of NDFIP2 (isoform 1; NP_061953.2) found to interact with AQP2 in the MYTH assay (aa 203–336, grey), 68% is identical (*) and 86% is similar (. or:) to that of NDFIP1 (NP_085048.1). The PPY-motifs (black rectangle) known to bind to NEDD4 and NEDD4L are not, but the transmembrane domains (dashed rectangle) are within the AQP2 binding region. (D) <i>NDFIP1 and NDFIP2 interact with AQP2</i>. HEK cells were transiently-transfected with an empty construct (-), or constructs encoding myc-tagged NDFIP1, NDFIP2 or AQP2 separately or combined (indicated on top), grown for 2 days, lysed and subjected to AQP2-immunoprecipitation (IP: α-AQP2). The IP-fractions (upper panel) and total lysates (indicated) were immunoblotted for NDFIP1 or -2 (IB: α-myc), or AQP2 (IB: α-AQP2). Only when co-expressed with AQP2, NDFIP1 and NDFIP2 were detected in the immunoprecipitate. Coomassie staining of the blots confirmed loading of protein equivalents. Molecular masses of proteins are indicated on the left (in kDa).</p