Regulation of V2 vasopressin receptor degradation by agonist-promoted ubiquitination.

The seven-transmembrane-spanning vasopressin V2 receptor (V2R) is a Gs-coupled receptor that is rapidly phosphorylated and internalized following stimulation with the agonist, arginine-vasopressin. Herein, we show that the V2R is ubiquitinated following agonist stimulation. V2R-ubiquitination is not observed in a beta-arrestin1,2 deleted mouse fibroblast cell line and is restored following introduction of beta-arrestin2, thus indicating that beta-arrestin2 is required for the ubiquitination of V2R. A mutant V2R (K268R) that is not ubiquitinated still activates Gs and internalizes with similar kinetics as the wild type receptor. Unstimulated wild type and K268R mutant receptors degrade at similar rates and have comparable half-lives of 217 +/- 17 and 245 +/- 29 min as determined by pulse-chase experiments. However, following agonist stimulation, the rate of receptor degradation for the wild type is enhanced (half-life of 69 +/- 19 min), whereas that of the mutant is only minimally affected (half-life of 188 +/- 11 min). These data suggest that V2R levels are regulated through at least two processes. In the absence of agonist stimulation, a slow degradative pathway operates that is independent of receptor ubiquitination. However, receptor stimulation leads to rapid beta-arrestin2-dependent ubiquitination of the receptor and increased degradation.

The vasopressin V2 receptor (V2R) 1 is a seven-transmembrane-spanning receptor that is present primarily in the renal distal tubules and collecting ducts. Interaction with its ligand, arginine vasopressin (AVP), leads to activation of G s and consequent activation of adenylyl cyclase that increases cAMP levels in cells. A cascade of events following the elevated levels of cAMP in kidney cells leads to an increase in the water permeability of the collecting ducts and facilitates water reabsorption (1). Exposure of V2R to its ligand also triggers receptor phosphorylation, internalization, and desensitization resulting in a reduction of the cellular response to the agonist (2,3). The carboxyl terminus of the human V2R, similar to other G pro-tein-coupled receptors, contains multiple phosphorylation sites and is a major regulatory domain controlling receptor interaction with ␤-arrestins and their trafficking in the cell (2,4). Previous studies have shown that V2R interacts tightly with ␤-arrestin1 or -2 following phosphorylation (4). The receptor-␤-arrestin complex is then endocytosed via clathrin-coated pits and accumulated in large endocytic vesicles in the proximity of the nucleus. V2R undergoes rapid agonist-promoted endocytosis but appears to be poorly recycled. The intracellularly retained receptor is either slowly recycled to the surface or is degraded.
The 26 S proteasome and lysosomes are two major protein degradative pathways in cells. Lysosomes are vesicular organelles with decreased internal pH that can facilitate the breaking of peptide bonds. Proteasomes are multisubunit complexes consisting of a regulatory and a core particle that can recognize and degrade polyubiquitinated proteins (5). Proteins are targeted to the proteasome via covalent attachment of polyubiquitin chains consisting of at least four ubiquitin peptides attached at specific lysine residues (6). The regulatory particle of proteasome is responsible for the recognition and recycling of polyubiquitin chains back into the cytoplasm in addition to unfolding and threading the unfolded protein through the core particle of the proteasome. The core particle of proteasome cleaves proteins into small peptides ranging in size from 3 to 23 amino acids. Although the initial function assigned to ubiquitination was the targeting of proteins to proteasomes, recent studies indicate that ubiquitination of several membrane proteins serves as a targeting or sorting signal through the endocytic route leading either to the vacuole in yeast or to lysosomes in mammalian cells.
Agonist-promoted ubiquitination of several seven-transmembrane-spanning receptors has been shown to play a key role in either their endocytic trafficking or receptor degradation. For example, monoubiquitination of the yeast pheromone receptor promotes ligand-induced endocytosis and is essential for the targeting of the receptor to the vacuole for degradation (7). Several mammalian seven-transmembrane-spanning receptors such as CXCR4 and ␤2-adrenergic receptors are also ubiquitinated following agonist stimulation. Mutant versions of these receptors, which cannot undergo ubiquitin modification, internalize but are not degraded (8,9). In the case of the ␤2-adrenergic receptor, ubiquitination of ␤-arrestin is necessary for receptor internalization but apparently not for receptor degradation.
Here we sought to investigate the role of V2 vasopressin receptor ubiquitination in receptor trafficking and degradation. Our findings indicate that V2R is ubiquitinated in an agonist and ␤-arrestin-dependent manner at a single lysine residue (lysine 268) in the third intracellular loop and that this ubiquitination forces the V2R to a rapid degradative pathway. * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

EXPERIMENTAL PROCEDURES
Materials-LipofectAMINE was from Invitrogen. HA affinity agarose beads were purchased from Covance or Sigma. Arginine-vasopressin and MG132 were from Sigma. Ubiquitin antibody UbP4D1 and anti-HA rabbit polyclonal antibody were from Santa Cruz Biotechnology. Easy tag TM express protein labeling mix, 35 S, and [ 3 H]Arg-vasopressin were from PerkinElmer Life Sciences.
V2 receptor mutants (K268R and K367R) were constructed using the QuikChange site-directed mutagenesis kit (Stratagene). The sequence of oligos used for mutagenesis can be provided upon request. All constructs were verified by DNA sequencing (Howard Hughes Medical Institute sequencing facilities, Duke University).
Receptor Immunoprecipitation and Western Blotting-COS-7 cells were transiently transfected with HA-tagged V2R and FLAG-tagged ␤-arrestin2. Twenty-four hours after transfection, cells were split into appropriate plates for ubiquitination and binding assays. Forty-eight hours following transfection, cells were serum-starved for 2 h (with or without 10 M MG132 where appropriate) and then stimulated for the designated amount of time with 1 M AVP. Cells were lysed using a buffer containing 50 mM HEPES (pH 7.5), 0.5% Nonidet P-40, 250 mM NaCl, 2 mM EDTA, 10% (v/v) glycerol, 1 mM sodium orthovanadate, 1 mM sodium fluoride, 1 mM phenylmethylsulfonyl fluoride, leupeptin (5 g/ml), aprotonin (5 g/ml), pepstatin A (1 g/ml), and 100 M benzamidine. Cell lysates were incubated with HA beads overnight at 4°C. The next day, the cell lysate was removed, and the beads were washed four times with the lysis buffer to eliminate nonspecific binding. Laemmeli's sample buffer was used to elute the immunoprecipitated receptor, and the resulting solution was resolved on polyacrylamide gels. The proteins were transferred to Polyscreen® polyvinylidene difluoride hybridization transfer membrane for Western blotting and were detected by the chemiluminescent reagent SuperSignal® West Pico (Pierce).
Binding Assay-Twenty-four hours after transfection, cells were split into 12-well plates. Forty-eight hours following transfection, cells were washed twice with a wash buffer consisting of Dulbecco's modified Eagle's medium, 2% bovine serum albumin, and 1 mM HEPES (pH 7.5). Cells were incubated with radiolabel solution [ 3 H]Arg-vasopressin (or a mixture of radioligand and 10 M AVP) for 1 h at room temperature. All wells were washed thrice with the wash buffer to remove all nonspecific binding. Cells were lysed with a solution containing 0.1% SDS and 0.1 M NaOH. The amount of radioactivity retained in cells was measured and receptor concentration was calculated using GraphPad software.
cAMP Assay-A 3 H-labeled cAMP assay system (Amersham Biosciences) was used to determine the amount of accumulated cAMP response in transiently transfected cells following stimulation as per previously described protocol (10).
Internalization Assay-Twenty-four hours after transfection with HA-tagged V2R and FLAG-tagged ␤-arrestin2, COS-7 cells were split into appropriate plates. Forty-eight hours following transfection, cells were placed on ice and washed thrice with the wash buffer (Dulbecco's modified Eagle's medium, 1% bovine serum albumin, and 1 mM HEPES). Radioligand [ 3 H]vasopressin was added to cells, and cells were placed on a 37°C hot plate for indicated times to allow for receptor internalization. Cells were washed three times to remove unbound radioligand. Bound cell surface ligand was acid washed, collected, and quantified to determine the amount of receptor that remained on the surface. Following the acid wash, cells were lysed with solution containing 0.1% SDS and 0.1 M NaOH, and the amount of internalized receptor was determined. As a control, binding assays (as described above) were performed simultaneously to determine the total number of receptors on the cell surface prior to internalization assay.
Confocal Microscopy-HEK-293 cells were transfected with HAtagged V2R and GFP-tagged ␤-arrestin2 using FuGENE. Twenty-four hours following transfection, cells were split and plated on collagencoated 35-mm glass-bottom plates and serum-starved for several hours. After addition of an agonist (1 M AVP), images were obtained at different time points ranging from 0 to 60 min poststimulation on a Zeiss LSM510 laser scanning microscope. Receptor expression was confirmed by radioligand binding.
Pulse-Chase Assay-COS-7 cells were transiently transfected with HA-tagged V2R and FLAG-tagged ␤-arrestin2. Forty-eight hours after transfection, cells were washed twice with phosphate-buffered saline MEF-␤arr1 Ϫ /␤arr2 Ϫ , ␤arr1 ϩ , and ␤arr2 ϩ were transiently transfected with HA-tagged vasopressin receptor and stimulated for 1 h with 1 M AVP in the presence of 10 M MG132. All blots were stripped and reprobed for the presence of the receptor. Binding assays revealed receptor expression levels of 220, 370, and 160 fmol/mg of protein in MEF-␤arr1 Ϫ /␤arr2 Ϫ , ␤arr1 ϩ , and ␤arr2 ϩ , respectively. Each experiment was reproduced three times yielding similar results. IP, immunoprecipitate; IB, immunoblot; KO, knock-out. and starved in serum-free medium lacking Met and Cys. 100 Ci of Easy tag TM express protein labeling mix, 35 S, per milliliter of serumfree buffer (2 ml/10-cm plate), was used to metabolically label the cells for 90 min. Cells were washed with phosphate-buffered saline three times to remove unbound label. The medium was replaced with serumfree Dulbecco's modified Eagle's medium, and cells were stimulated with 1 M AVP for the indicated amount of time. Cells were lysed, and the HA-tagged receptors were immunoprecipitated and run on SDSpolyacrylamide gels as described above. Dried gels were exposed to x-ray film. The bands on the autoradiograph were quantitated using a Bio-Rad Flour-S imager.

RESULTS AND DISCUSSION
To investigate whether human V2R is ubiquitinated following agonist stimulation, COS-7 cells were transiently transfected with HA-tagged V2R and stimulated for 1 h with the agonist, AVP. As shown in Fig. 1a (upper panels), V2R is ubiquitinated following agonist stimulation. The ubiquitinated receptor is detected as a smear on Western blots because of the variable number of conjugated ubiquitin moieties and could only be detected in the presence of the proteasomal inhibitor MG132 that prevents the rapid degradation of the polyubiquitin-conjugated proteins. A time course study determined that after agonist stimulation, V2R ubiquitination was apparent as early as 60 min (Fig. 1b, upper panel) and was prominent at 120 min. For each agonist-stimulated sample, the amount of V2R in the immunoprecipitates was determined by blotting for the HA tag on the receptor (Fig. 1b, lower panel). No changes in the receptor levels were detected after stimulation with AVP.
Detection of V2R ubiquitination requires preincubation of cells with MG132 unlike the ␤2-adrenergic receptor (8). The known role of MG132 is the inhibition of proteasomal activity. This is suggestive of a degradative role for the proteasomes in the case of V2R apart from the previously implied role in endosomal sorting of cell surface receptor (16). Alternatively, ubiquitination of the V2R may require stabilization of an unknown protein that is rapidly targeted to the proteasomes for degradation.
Next, we determined the site of ubiquitination in the V2R. Ubiquitin is covalently attached to lysine residues on proteins. Human V2R contains four lysine residues (Fig. 3a). Based on the predicted structure of human V2R, Lys-100 and Lys-116 do not face the cytosol and are thus unlikely to be ubiquitinated. We suspected that the site of ubiquitination of the receptor was either on residue Lys-268, located in the third intracellular loop, or residue Lys-367, located in the cytoplasmic tail of the receptor. Two V2R mutants, K268R and K367R, were created to determine the possible site(s) of receptor ubiquitination. Mutants and the wild type receptor were transiently transfected into COS-7 cells and assayed for agonist-stimulated ubiquitination. Both wild type and K367R receptors were ubiquitinated. However, no ubiquitinated species were detected in the immunoprecipitated K268R receptor mutant (Fig. 3b). Ligand binding data confirmed that K268R was indeed expressed in sufficient amounts for the detection of ubiquitinated species. These findings thus implicate residue Lys-268 as the primary site of receptor ubiquitination.
Lys-268 is located in the C-terminal side of the third intracellular loop of the receptor, which is a predicted region for FIG. 4. Characterization of the K268R mutant. a, functional assays were performed on transiently transfected COS-7 cells as described previously. Accumulated cAMP levels, following 5 min of stimulation with 1 M AVP, were normalized against forskolin stimulation for each receptor in three independent experiments (ϮS.E.). Wild type, K268R, and K367R receptor expressions were determined to be at 150 -450 fmol of receptor/mg of protein by binding assays. b, HEK-293 transfected with GFP-␤-arrestin2 and either wild type or the K268R mutant receptor were stimulated with AVP. Distribution of GFP-␤-arrestin2 was determined by confocal microscopy. Prior to stimulation, GFP-␤-arrestin2 was uniformly distributed throughout the cytoplasm. After receptor stimulation, GFP-␤-arrestin2 was recruited into endocytic vesicles signifying its interaction with the activated receptor. Both wild type and K268R were capable of changing GFP-␤-arrestin2 distribution in the cell to form cytosolic rings following stimulation. Two independently performed experiments conferred similar results. c, internalization assays were performed on transiently transfected COS-7 cells as described under "Experimental Procedures." Graphed data are representative of four independent experiments. Error bars depict Ϯ S.E. WT, wild type. receptor and G protein interaction. Severely impaired receptor signaling or improper internalization might influence K268R receptor ubiquitination. We therefore investigated the signaling and internalization of the receptor mutant. Binding and functional assays show that when receptor mutants and the wild type receptor are expressed at comparable levels, both mutants are capable of activating G s protein in response to the agonist AVP. As predicted by the location of the mutation K268R, this receptor mutant is partially impaired in activating G s and elevating cAMP levels in cells (Fig. 4a). Our data also indicate that the K268R mutant resides at the plasma membrane because the agonist AVP, being hydrophilic, is not capable of permeating through the cell membrane.
␤-Arrestin2 interaction with the V2R is crucial for receptor ubiquitination (Fig. 2). Therefore, impaired interaction of K268R with ␤-arrestin2 might explain the lack of ubiquitination of the receptor mutant. To investigate this possibility, HEK-293 cells were transiently transfected with GFP-tagged ␤-arrestin2 and either wild type or K268R mutant receptor. Intact cells were stimulated for 40 min with AVP, and the recruitment of GFP-␤-arrestin2 to the endocytic vesicles was observed using confocal microscopy. GFP-␤-arrestin2 was uniformly distributed throughout the cytoplasm prior to agonist stimulation (Fig. 4b). Following stimulation, GFP-␤-arrestin2 was recruited to the plasma membrane and eventually accumulated within the cell in endocytic vesicles (Fig. 4b). This phenomenon has been described for several G protein-coupled receptors (11). Both wild type and K268R mutant receptors were capable of this interaction with GFP-␤-arrestin2 as shown in Fig. 4b. According to previous reports ␤-arrestin receptor interaction requires phosphorylation of serine clusters in the V2R tail region (4,14). Because the K268R mutant receptor is capable of recruiting ␤-arrestin2, its phosphorylation state is not likely to be affected. Moreover, the nonubiquitinated V2R is internalized similar to the wild type receptor. As shown in Fig. 4c, ϳ80% of the surface receptors (K268R and the wild type) are internalized after 60 min. Therefore, lack of ubiquitination in K268R had no effect on receptor internalization.
To determine whether ubiquitination could regulate the life cycle of V2R, the half-lives of the wild type and the mutant K268R receptor were determined by pulse-chase experiments.
FIG. 5. Effects of mutation of the V2R ubiquitination site on receptor degradation in the absence of stimulation. Pulse-chase experiments were performed on transiently transfected COS-7 cells with either wild type (wt) or K268R mutant receptor to determine the rate of receptor degradation and receptor half-lives. Following metabolic labeling, HA-tagged receptors were immunoprecipitated at different time points and analyzed as described under "Pulse-Chase Assay" under "Experimental Procedures." a, receptor levels were determined up to 13 h following metabolic labeling. The density of bands corresponding to each receptor for different time points was normalized against the band for the receptor levels at time point 0 (immunoprecipitated immediately following metabolic labeling). Wild type and mu-tant vasopressin receptor degradation data were fitted with exponential decay curves and analyzed using GraphPad software. No significant differences between the degradation rates of the wild type and the mutant vasopressin receptor were detected (assessed by performing two-way analysis of variance on the graph data). Each graph represents the result of three independent experiments. b, receptor levels were determined up to 6 h following stimulation with AVP. Data were fitted with exponential decay curves. The degradation rate of the wild type vasopressin receptor is considerably increased. Differences in the wild type and the mutant receptor (K268R) degradation are especially profound for the first 2 h following stimulation. Analysis of graph data with two-way analysis of variance confirmed the differences between the two graphs (p Ͻ 0.005). Five independent experiments yielded similar results, which are reflected on the graphs. c, GraphPad software was used to analyze the data and fit curves to include all the experimental points. The generated curve for each independent experiment was used to determine the time point at which 50% of receptor remains. The halflives from individual experiments were then used to generate the bar graphs. Half-lives of receptors were compared using a two-tailed t test. No significant differences between the half-lives of the wild type and the mutant were detected in unstimulated cells. The calculated half-life of the wild type receptor was lowered from 217 Ϯ 17 to 69 Ϯ 19 min following AVP treatment. On the other hand, modest reduction in the half-life of the nonubiquitinated mutant receptor, from 245 Ϯ 29 to 188 Ϯ 11 min, was observed. The half-lives of the wild type and the K268R mutant are significantly different after stimulation with AVP (**, p Ͻ 0.005). NS, nonstimulated.
Transiently transfected COS-7 cells were metabolically labeled with 35 S-labeled Met and 35 S-labeled Cys. Tagged receptors were then immunoprecipitated, resolved on gels, and analyzed. In unstimulated cells, wild type and the K268R V2 receptors displayed comparable (p ϭ not significant) half-lives of 217 Ϯ 17 and 245 Ϯ 29 min, respectively (Fig. 5, a and c). Agonist stimulation reduced the half-life of the wild type receptor to 69 Ϯ 19 min while having little effect on the K268R mutant receptor half-life of 188 Ϯ 11 min (p Ͻ 0.005, Fig. 5, a and c). Three hours following the metabolic labeling, the nonubiquitinated mutant and the wild type vasopressin receptor levels were depleted in agonist-stimulated cells. Our data demonstrate a striking difference in the pattern of degradation for the receptors. The delay in K268R degradation reflects a possible lag in receptor sorting following stimulation. The alternate pathway of degradation for the nonubiquitinated receptor leads to elevated levels of receptor for up to 3 h. The agonist-dependent degradation of the K268R receptor mutant appears to be linear, whereas agonist-dependent degradation of the wild type receptor is non-linear and asymptotic. However, if one looks at the data presented in Fig. 5c, it becomes apparent that there is no significant difference in the half-life of the mutant receptor in the presence or absence of agonist. This suggests that the difference in the shape of the time curves for the mutant receptor degradation in the presence or absence of agonist may be more apparent than real.
Our data suggest that two different pathways degrade V2R. A slow degradative pathway in unstimulated cells is responsible for maintaining a balance between the synthesis and destruction of the V2 receptor. This slow pathway continuously degrades the V2 receptor using a ubiquitin-independent mechanism, because the mutant K268R that does not get ubiquitinated is still degraded by this pathway. Following agonist stimulation and subsequent ubiquitination of the receptor, a rapid ubiquitin-dependent mechanism speeds up the receptor degradation. Because of the lack of ubiquitination, the K268R mutant receptor degrades at a slower rate in the presence of agonist stimulation (Fig. 5c).
It has been reported that in-frame fusion of a single ubiquitin moiety to an otherwise stable plasma membrane ATPase protein in yeast leads to rapid endocytosis and vacuolar degradation of the chimeric protein (14). Furthermore, recent studies implicate postendocytic sorting to be mediated via ubiquitindependent mechanisms via the yeast Vps proteins and their mammalian homologs such as TSG101 and Hrs (15,16). Such recognition signals are single ubiquitin moieties on the endocytic cargo and the sorting proteins that are involved, such as Eps15 (17). On the other hand, the cell surface protein GapI (general amino acid permease) becomes polyubiquitinated before being degraded in the yeast vacuole (18).
In general, variety in the number of ubiquitin moieties that could be covalently attached to a protein can modulate the trafficking signal generated by this post-translational modification. For many proteins, single ubiquitin attachment signals internalization, whereas polyubiquitination tags them for destruction. In the case of V2R, the presence of an ubiquitinated receptor as a smear on Western blots is indicative of receptor polyubiquitination, and our studies of wild type and K268R receptor half-lives confirm that ubiquitination is the signal for rapid receptor degradation.
Our data are consistent with a number of mechanisms for V2 receptor degradation. Previous studies have suggested that V2 receptor can be degraded by processes that involve lysosomes, proteasomes, and cell membrane-bound metalloproteinases (19,22). The rapid agonist-stimulated receptor degradation likely involves lysosomes, because like many seven-transmembrane-spanning receptors, it has been demonstrated that V2 receptor traffics to lysosomes upon agonist stimulation (19). Our data, however, indicate that polyubiquitination of the receptor and the proteasomal machinery are also likely to be involved in the agonist-stimulated degradation because the proteasomal inhibitor MG132 is necessary to observe V2 receptor ubiquitination. Proteasomal involvement in endosomal sorting to the lysosomes has been described for several plasma membrane proteins (19 -21). On the other hand, it is possible that the slow constitutive ubiquitin-independent degradation of the receptors might be carried out by other mechanisms such as the previously described cell membrane-bound metalloproteinases (22).