Final Report Summary - AQUASHUTTLE (Regulation of endocytosis of the aquaporin-2 water channel: an interplay of phosphorylation and ubiquitination)
Executive Summary:
The Kidney is the main organ for the regulation of water homeostasis. Of the 180 liters of pro-urine produced daily, 90% of the water is constitutively reabsorbed in the renal proximal tubules and descending thin limbs of Henle. This massive reabsorption has been attributed to the aquaporin-1 (AQP1) water channel, which is expressed in the apical and basolateral membranes of the epithelial cells that line these nephron segments. The remaining 10% can be reabsorbed in collecting ducts via aquaporin-2 (AQP2). This is under control of the antidiuretic hormone arginine vasopressin (AVP), which is released from the pituitary after sensing hypernatremia. In the principal cells of collecting ducts, binding of AVP to its type 2 receptor (V2R) initiates a cAMP signalling cascade that results in the activation of protein kinase A (PKA). PKA phosphorylates AQP2, which is then redistributed from intracellular vesicles to the apical membrane, thereby rendering this membrane water permeable. Removal of AVP initiates endocytosis of AQP2 and restores the water-impermeable state of the apical membrane. In this respect, the plasma membrane expression of AQP2 appeared key to the extent of water reabsorption in health and disease, because in disorders with excessive loss of water, such as congenital or acquired nephrogenic diabetes insipidus (NDI), AQP2 plasma membrane expression is severely reduced. In disorders with excessive water reabsorption, such as in congestive heart failure, liver cirrhosis and preeclampsia, plasma membrane AQP2 expression is increased. Plasma membrane expression of channels is often a balance between exocytosis and endocytosis (shuttling). The shuttling of AQP2 to and from the plasma membrane is regulated by a dynamic interplay of two posttranslational modification processes, being (de)phosphorylation and (de)ubiquitination. Vasopressin-induced PKA activation phosphorylates AQP2 at serine 256 (pS256) in the C-tail, which is essential for its plasma membrane expression. Besides S256, vasopressin also triggers the phosphorylation of S264 and S269 in AQP2. In contrast, AQP2 is highly phosphorylated at S261 when unstimulated, but is dephosphorylated at this site upon treatment with vasopressin. In addition AQP2 plasma membrane expression is also regulated by ubiquitination at lysine 270 (K270).
The scientific focus of the project was therefore to get more insight in the regulation of the endocytosis of AQP2 and to identify novel players which are involved in the phosphorylation and ubiquitination of AQP2 and to understand the interplay of the two modification processes.
To shed more light on the role of pS261 in the regulation of AQP2 translocation in relation to AQP2 phosphorylation at S256 and K270 ubiquitination, we studied the localization and regulation of several AQP2 proteins mutated at these sites in the Madin-Darby canine kidney (MDCK) cell model. Our data suggest that pS261 of AQP2 occurs after ubiquitin-mediated endocytosis and thus that pS261 does not induce AQP2 ubiquitination itself. Instead, pS261 may stabilize ubiquitinated AQP2. Together with the plasma membrane targeting signal of S256 and S269 phosphorylation, K270 ubiquitination finetunes the subcellular distribution of AQP2. The role of AQP2 phosphorylation at S264 in this process remains unclear.
Interestingly, a changed regulation of AQP2 phosphorylation/ubiquitination of a certain AQP2 mutation may underlie its missorting in patients suffering from recessive NDI. While most AQP2 mutants in recessive NDI are misfolded and retained in the endoplasmic reticulum, AQP2-P262L in NDI was impaired in its vasopressin-dependent translocation from vesicles to the plasma membrane Our data reveal that vasopressin induces instead of reduces phosphorylation of S261 in AQP2-P262L, but it remains to be established whether the changed phosphorylation causes its missorting in NDI.
AQP2 plasma membrane expression is also regulated by ubiquitination. Ubiquitination is a posttranslational modification in which the 76 amino acid protein ubiquitin is covalently coupled via its C-terminal glycine to the ε-amine of an internal lysine residue in substrate proteins. The reaction is carried out by three enzymes and their sequential action: ubiquitin-activating enzyme (E1) charges ubiquitin with ATP, followed by transfer to a ubiquitin-conjugating enzyme (E2). A ubiquitin protein ligase (E3) binds E2 and the substrate protein, and catalyses the transfer of the activated ubiquitin to the substrate. However, the E3 ligase responsible for coupling the ubiquitin to AQP2 remains to be identified. Here, by using a Membrane Yeast Two-Hybrid (MYTH) assay to identify protein interactors of full-length integral membrane proteins, we detect an interaction between NEDD4 interacting protein (NDFIP) 1 and 2 with AQP2. Moreover, we find that NDFIP1, but not NDFIP2, is expressed in principal cells and that it is essential for ubiquitination and degradation of AQP2 by the E3 ubiquitin ligases NEDD4 and NEDD4L.
AQP2 is known to be available for recycling to the plasma membrane at least six times. This suggests that, like in many other membrane proteins, also deubiquitination is involved. By using siRNA in a cell model that endogenously expresses AQP2 we screened possible candidate enzymes. Our data suggest, that USP8, USP4 and UCHL3 are involved in the regulation of AQP2. Further studies are necessary to pinpoint the direct or indirect involvement of these DUBs on the deubiquitination of AQP2.
The abundance of AQP2 expression in the plasma membrane is a determining factor in the extent of renal water reabsorption and is fundamental to common disorders characterized by excessive water loss (such as NDI), and reabsorption, as found with the syndrome of inappropriate release of AVP (SIADH), congestive heart failure, liver cirrhosis and preeclampsia. For example, 0.01% of our population uses lithium as a therapy for (mainly) bipolar disorders of which 20% develops irreversible NDI in time. To unravel how and which proteins regulate the AQP2 membrane expression is anticipated to reveal new targets for therapeutic treatment of these diseases of impaired water homeostasis.