Short-chain ubiquitination mediates the regulated endocytosis of the aquaporin-2 water channel

Abstract

To regulate mammalian water homeostasis, arginine-vasopressin (AVP) induces phosphorylation and thereby redistribution of renal aquaporin-2 (AQP2) water channels from vesicles to the apical membrane. Vice versa, AVP (or forskolin) removal and hormones activating PKC cause AQP2 internalization, but the mechanism is unknown. Here, we show that a fraction of AQP2 is modified with two to three ubiquitin moieties in vitro and in vivo. Mutagenesis revealed that AQP2 is ubiquitinated with one K63-linked chain at K270 only. In Madin-Darby canine kidney cells, AQP2 ubiquitination occurs preferentially when present in the apical membrane, is transiently increased with forskolin removal or PKC activation, and precedes its internalization. Internalization kinetics assays with wild type (wt) and ubiquitination-deficient (K270R) AQP2 revealed that ubiquitination enhances AQP2 endocytosis. Electron microscopy showed that a translational fusion of AQP2 with ubiquitin (AQP2-Ub) localized particularly to internal vesicles of multivesicular bodies (MVBs), whereas AQP2-K270R largely localized to the apical membrane, early endosomes, and the limiting membrane of MVBs. Consistent with this distribution pattern, lysosomal degradation was extensive for AQP2-Ub, low for AQP2-K270R, and intermediate for wt-AQP2. Our data show that short-chain ubiquitination is involved in the regulated endocytosis, MVB sorting, and degradation of AQP2 and may be the mechanism used by AVP removal and PKC-activating hormones to reduce renal water reabsorption. Moreover, because several other channels are also (short-chain) ubiquitinated, our data suggest that ubiquitination may be a general mediator for the regulated endocytosis and degradation of channels in higher eukaryotes.homeostasis ͉ internalization ͉ signal transduction ͉ sorting ͉ transmembrane protein A quaporin (AQP) water channels are important for rapid and selective osmotic water transport across cell membranes. Most AQPs have a constitutive open pore, and regulation of transmembrane water transport is thus controlled by channel insertion into and retrieval from the cell surface (1-3). AQP2 is one of the best-characterized AQPs and confers water permeability to the kidney-collecting duct to serve body water homeostasis. This process is of pathophysiological importance, because inadequate cell-surface expression of AQP2 results in nephrogenic diabetes insipidus (4, 5), whereas increased cell surface expression and excessive water reabsorption is observed in congestive heart failure, preecclampsia, and the syndrome of inappropriate release of the hormone arginine-vasopressin (AVP) (6).Hypernatremia and hypovolemia induce the release of AVP, which regulates the cell-surface expression of AQP2. AVP occupation of its renal type 2 receptors initiates a signaling cascade resulting in phosphorylation of AQP2 at S256. Phosphorylation of AQP2 is necessary for the fusion of AQP2-containing vesicles with the apical membrane, where AQP2 facilitates water reabsorption (2, 7-11). Moreover, AVP also regulate...

Proteome-Wide Measurement of Protein Half-Lives and Translation Rates in Vasopressin-Sensitive Collecting Duct Cells

“…In that study, a significant increase was seen within 0.5 hours, which implies that the protein half-life of AQP2 must have been much shorter in the cells studied by Nedvestsky et al 12 versus our study. The data from both studies correlate well with the observation that a reduction in vasopressin signaling, mimicked by washout of forskolin, results in an increase in AQP2 ubiquitylation, 19 a well known signal for protein degradation. Figure 5 also shows several additional proteins with translation rates that change in response to dDAVP that are potentially involved in vasopressin signaling and/or AQP2 regulation: Mal2, Akap12, gelsolin, myosin light chain kinase, annexin-2, and Hsp70 (Supplemental Dataset 8).…”

Acute regulation of aquaporin-2 phosphorylation at Ser-264 by vasopressin

“…Indeed, it has been shown recently that pS256 is important for a direct interaction of AQP-2 with 70-kDa heat shock proteins and, ultimately, the AQP-2 shuttle (16). Moreover, recent evidence suggests that ubiquitination of the C-terminal tail of AQP2 at K270 is important for internalization (17), and it would be interesting to determine whether S264 phosphorylation can influence the level of AQP2 ubiquitination. Once ubiquitinated, AQP2 can be readily internalized, where it is transported to multivesicular bodies and targeted for proteasomal degradation.…”

Ubc13 and COOH Terminus of Hsp70-interacting Protein (CHIP) Are Required for Growth Hormone Receptor Endocytosis

“…The yeast monocarboxylate transporter, Jen1, requires HECT-ubiquitin ligase Rsp5-dependent Lys 63 ubiquitination for endocytosis (12). Short chain Lys 63 ubiquitination mediates the regulated endocytosis of the aquaporin-2 water channel (13), and forced expression of ubiquitin mutants indicates that Lys 63 ubiquitination of the prolactin receptor is important for its degradation (14). Chain assembly on substrates is an orchestrated interplay between an ubiquitin activating enzyme (E1), an conjugating enzyme (E2), and an ubiquitin ligase (E3).…”