Abstract
Regulation of our water homeostasis is fine-tuned by dynamic translocation of Aquaporin-2 (AQP2)-bearing vesicles to and from the plasma membrane of renal principal cells. Whereas binding of vasopressin to its type-2 receptor initiates a cAMP-protein kinase A cascade and AQP2 translocation to the apical membrane, this is counteracted by protein kinase C-activating hormones, resulting in ubiquitination-dependent internalization of AQP2. The proteins targeting AQP2 for ubiquitin-mediated degradation are unknown. In collecting duct mpkCCD cells, siRNA knockdown of NEDD4 and NEDD4L E3 ligases yielded increased AQP2 abundance, but they did not bind AQP2. Membrane Yeast Two-Hybrid assays using full-length AQP2 as bait, identified NEDD4 family interacting protein 2 (NDFIP2) to bind AQP2. NDFIP2 and its homologue NDFIP1 have PY motifs by which they bind NEDD4 family members and bring them close to target proteins. In HEK293 cells, NDFIP1 and NDFIP2 bound AQP2 and were essential for NEDD4/NEDD4L-mediated ubiquitination and degradation of AQP2, an effect not observed with PY-lacking NDFIP1/2 proteins. In mpkCCD cells, downregulation of NDFIP1, NEDD4 and NEDD4L, but not NDFIP2, increased AQP2 abundance. In mouse kidney, Ndfip1 and Ndfip2 mRNA distribution was similar and high in proximal tubules and collecting ducts, which was also found for NDFIP1 proteins. Our results reveal that NEDD4/NEDD4L mediate ubiquitination and degradation of AQP2, but that NDFIP proteins are needed to connect NEDD4/NEDD4L to AQP2. As NDFIP1/2 bind many NEDD4 family E3 ligases, which are implicated in several cellular processes, NDFIP1/2 may be the missing link for AQP2 ubiquitination and degradation from different subcellular locations.
Highlights
Hypernatremia or hypovolemia lead to an increased releases of vasopressin (AVP) from the pituitary
Instead, using a Membrane Yeast Two-Hybrid (MYTH) assay to identify proteins interacting with full-length AQP2, we found that NEDD4 family interacting protein (NDFIP) 1 and 2 interact with AQP2, are expressed in renal collecting ducts, and are essential for ubiquitination and degradation of AQP2 by NEDD4 and NEDD4L
To test the potential role of these E3 ligases in AQP2 ubiquitination, internalization and degradation regulation, we transfected Nedd4/Nedd4L siRNAs into dDAVP-stimulated mpkCCD cells to test their effects on AQP2 abundance
Summary
Hypernatremia or hypovolemia lead to an increased releases of vasopressin (AVP) from the pituitary. Driven by an osmotic gradient, water enters the cells through AQP2 and exits the cell via AQP3 and AQP4, which corrects blood tonicity and volume and results in concentrated urine [1] These corrected osmo and volume balances normalize blood AVP levels, which subsequently induces the internalization of AQP2 to storage vesicles and its lysosomal degradation, coinciding with a reduced water reabsorption. AQP2 binding and functional evidence of involvement of any of these E3 ligases in AQP2 ubiquitination and degradation has not been reported. Instead, using a Membrane Yeast Two-Hybrid (MYTH) assay to identify proteins interacting with full-length AQP2, we found that NEDD4 family interacting protein (NDFIP) 1 and 2 interact with AQP2, are expressed in renal collecting ducts, and are essential for ubiquitination and degradation of AQP2 by NEDD4 and NEDD4L
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