Short-Chain Ubiquitination Mediates the Regulated Endocytosis of the Aquaporin-2 Water Channel

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Title: Short-Chain Ubiquitination Mediates the Regulated Endocytosis of the Aquaporin-2 Water Channel
Authors: Kamsteeg, Erik-Jan; Hendriks, Giel; Boone, Michelle; Konings, Irene B. M.; Oorschot, Viola; van der Sluijs, Peter; Klumperman, Judith; Deen, Peter M.T.
Publisher: Proceedings of the National Academy of Sciences of the U.S.A
Date Published: November 28, 2006
Reference Number: 714
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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.
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This translation by the NDI Foundation is to assist the lay reader. To provide a clear, accessible interpretation of the original article, we eliminated or simplified some technical detail and complicated scientific language. We concentrated our translation on those aspects of the article dealing directly with NDI. The NDI Foundation thanks the researchers for their work toward understanding and more effectively treating this disorder.
© Copyright NDI Foundation 2007 (JC)

Aquaporin-2 (AQP2) is a protein that is located in the principal cells of the kidney collecting duct. When the hormone arginine vasopressin (AVP) links with its vasopressin-2 receptor (V2R) located on the membrane of the principal cell, it initiates a chemical sequence that stimulates AQP2 to travel from the cell interior to a specific section of the cell membrane called the apical membrane. This movement from the cell interior to the membrane is called exocytosis. Once AQP2 arrives at the membrane it inserts itself into the membrane and acts as a channel through which water can enter the cell.

When the body has absorbed enough water through the kidneys, AVP levels decrease and AQP2 travels from the membrane back to the cell interior, a process called endocytosis. Researchers know that the hormones prostaglandin E2 and dopamine cause AQP2 proteins to return to the cell interior by activating protein kinase C (PKC), yet the mechanics of AQP2 endocytosis are not fully understood. Erik-Jan Kamsteeg, et al., explored AQP2 process both in vitro (i.e., with specific laboratory cell line cultures) and in vivo (using cells taken from goat kidneys). The focus of their work was the function of ubiquitins and ubiquitination in AQP2 endocytocis.

An ubiquitin is a small protein comprised of 76 amino acids. They are found throughout all cells with a true nucleus (eukaryotes). One of their main functions is to mark other proteins for destruction. That is, to bring them to the process where they start being taken apart by cellular machinery, a process called proteolysis. However, ubiquitin can also mark proteins for removal from cellular membranes. Ubiquitination is the cellular process that marks a protein with ubiquitin.

The research team’s findings showed that a specific form of ubiquitination – short chain ubiquitination – is definitely involved in regulated AQP2 endocytosis. First, they determined that AQP2 is ubiquitinated both in vitro and in vivo by recording that AQP2 is modified with a limited number of ubiquitin molecules. Then they determined that AQP2 is preferentially ubiquinated if it is in the apical membrane. That is, the AQP2 residing in the apical membrane are more likely to be ubiquinated than those in the cell interior. Further, Kamsteeg, et al., determined that ubiquitination of AQP2 precedes its translocation to the cell interior. The team located the spot on the AQP2 that the ubiquitin connects to, which is a lysine amino acid that forms the 270th link in the chain of amino acids that comprises the AQP2.

Finding that AQP2 in the apical membrane is preferentially ubiquinated, and that ubiquination precedes AQP2 internalization, the researchers conducted further research which indicates that AQP2 ubiquitination enhances its endocytosis.