Agonist-promoted ubiquitination differentially regulates receptor trafficking of endothelin type A and type B receptors

Agonist-stimulation induces different intracellular trafficking of endothelin receptors (ET A/B R) after internalization. The mechanism is unclear. Results: Stimulation induces ubiquitination, lysosomal targeting and decreased cell surface ET B R levels. Non-ubiquitinated ET A R and ET B R mutant recycled to plasma membrane with smaller changes in the levels. Conclusion: Ubiquitination determines intracellular trafficking of endothelin receptors. Significance: ET ubiquitination fine-tunes cellular responses to agonist, by regulating cellular receptor levels. ABSTRACT Two types of G protein-coupled receptors for

increase in the intracellular Ca 2+ concentration upon repetitive ET-1 stimulation were larger. A series of ETBR mutants (designated "4KR mutant") in which either one of 5 arginine residues of the 5KR mutant was reverted to lysine were normally ubiquitinated, internalized and degraded, with ERK phosphorylation being normalized.
These results demonstrate that agonist-induced ubiquitination at either lysine residue in C-tail of ETBR but not ETAR switches intracellular trafficking from recycling to plasma membrane to targeting to lysosome, causing decreases in cell surface level of ETBR and intracellular signaling.
Endothelin-1 (ET-1) is a vasoconstricting peptide of 21 amino acids, which is synthesized and released in endothelial cells (1). ET-1 is considered to play an important role in the physiological control of blood pressure and cardiac function and also in genesis and development of cardiovascular diseases such as atherosclerosis (2), cardiac remodeling accompanying chronic heart failure (3), and pulmonary arterial hypertension (4). There are two types of receptors for ET-1: endothelin type A receptor (ETAR) and ETBR, both of which are G protein-coupled receptors (GPCRs) (5,6). ETAR is coupled with Gq and Gs (7,8), while ETBR is coupled with Gq and Gi (7,9). Typically, ETAR is present on vascular smooth muscle cells and upon agonist stimulation, it induces contraction of the cells to cause vasoconstriction. On the other hand, ETBR is present on vascular endothelial cells and upon agonist stimulation, it induces nitric oxide production through activation of endothelial nitric oxide synthase to cause vascular relaxation (10). ETBR is also known to function as a clearance receptor for ET-1, which removes ET-1 from the extracellular fluid by binding ET-1 and rapidly internalizing into the inside of the cell (11,12).
Administration of a receptor agonist ET-1 to whole animals is reported to induce a transient decrease in the blood pressure resulting from vasodilatation via ETBR, followed by a long-lasting increase in the blood pressure resulting from vasoconstriction via ETAR. The biphasic change in the blood pressure is considered to result from rapid desensitization of ETBR-mediated response and negligible desensitization of ETAR-mediated response (1,12,13).
The different susceptibility to desensitization of ETAR-and ETBR-mediated responses could be explained mainly by different intracellular trafficking of both receptors after stimulation with their agonist ET-1 (14,15), although ETAR and ETBR closely resemble each other (≈55% overall; 77% within the putative transmembrane helices) (16,17). Upon agonist stimulation, both receptor subtypes are rapidly internalized to early endosome, but subsequently targeted to different intracellular destinations (14,18).
ETAR is recycled back to the plasma membrane through the pericentriolar recycling compartment, while ETBR is targeted to lysosomes for degradation through late endosome (14,18).
Several lines of evidence indicate that cytoplasmic carboxyl terminal tail (C-tail) determines the pathway of agonist-induced trafficking of several GPCRs including endothelin receptors (ETRs) (19,20). With respect to ETRs, this conclusion is based on the studies using chimeric mutants of ETRs where the C-tails of ETAR and ETBR were swapped (15,21). The chimeric ETBR with C-tail of ETAR was capable of recycling like wild-type ETAR, whereas the chimeric ETAR with C-tail of ETBR behaved like wild-type ETBR (15,21). However, the detailed mechanism for the different fates of ETAR and ETBR is at present totally unknown.
We have recently shown that ETBR is ubiquitinated more abundantly than ETAR in the absence of agonist stimulation (22). To get insights into the mechanisms for different fates of these two receptors, we decided to examine agonist-induced ubiquitination of ETAR and ETBR and to analyze the role of ubiquitination in receptor trafficking.
In this study, we show that 1) the different fates of ETAR and ETBR are mainly due to agonist-induced ubiquitination of ETBR but not ETAR, which occurs on the cell membrane, 2) ubiquitination of ETBR switches intracellular trafficking of the receptor after agonist-induced internalization, from recycling back to the plasma membrane to lysosomal targeting for degradation, causing a decrease in cell surface levels of the receptor, 3) ubiquitination of the receptor and consequent decrease in its cell surface levels induce quenching of ETBR-mediated responses such as ERK phosphorylation and an increase in the intracellular Ca 2+ concentration ([Ca 2+ ]i) to repetitive agonist stimulation, and 4) ubiquitination at either one of five lysine residues except lysine 411 in the distal end of C-tail of ETBR is sufficient for a switch of the intracellular trafficking.
Generation of Stable Cell Lines-HEK293T cells stably expressing wild type or mutant N-terminally HA-tagged ETRs, and HEK293T cells stably expressing myc-ubiquitin were generated by retroviral gene transfer as described previously (23,24). The positive cells were selected in DMEM containing 2 µg/ml puromycin for a week, and individual lines were tested by Western blot using an anti-HA antibody. Mutagenesis was performed using KOD-Plus-Mutagenesis Kit (Toyobo) according to the manufacturer's instructions.
All of the constructs were verified by DNA sequencing.
Western Blot-The proteins were separated by SDS-PAGE and electrotransferred to a polyvinylidene fluoride membrane (Millipore) with an electroblotter.
After transfer, the membranes were washed three times with TBST (10 mM Tris-HCl (pH 8.0), 100 mM NaCl, and 0.1% Tween-20) followed by blocking (2% non-fat dry milk in TBST) of nonspecific binding for 1 h at room temperature. The membranes were incubated with specific antibodies as a primary antibody at 4°C overnight. The primary antibody was detected with a secondary antibody conjugated with HRP.
The protein-antibody complex was detected by enhanced chemiluminescence Western blot reagent (Thermo Fisher Scientific). The membranes were exposed to Amersham Hyperfilm TM ECL (GE Healthcare) and the signals were quantified with Image J1.37 software (National Institutes of Health).
The immunocomplexes were washed four times with washing buffer, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) and then analyzed for ubiquitination of receptors by Western blot using rabbit polyclonal anti-myc antibody and goat polyclonal anti-rabbit IgG antibody conjugated to HRP as primary and secondary antibodies, respectively. Reprobing for immunoprecipitated HA-ETRs was performed using anti-HA antibody and goat polyclonal anti-mouse IgG antibody conjugated to HRP as primary and secondary antibodies.
Ubiquitination assay for endogenous ETBR in HPASMC was performed in a manner essentially similar to that for exogenously expressed HA-ETBR in HEK293T cells, except for the following points. For immunoprecipitation of ETBR, rabbit monoclonal anti-ETBR antibody instead of anti-HA antibody was added to the supernatants of cell lysates, while Western blot analysis of ubiquitinated ETBR was performed using mouse monoclonal anti-ubiquitin antibody and goat polyclonal anti-mouse IgG antibody conjugated to HRP as primary and secondary antibodies, respectively.
Reprobing for immunoprecipitated endogenous ETBR was performed using rabbit monoclonal anti-ETBR antibody and light chain specific mouse monoclonal anti-rabbit IgG antibody conjugated to HRP as primary and secondary antibodies, respectively.
Cell Surface HA-ETR Assay-The cells (2 × 10 6 ) expressing wild type or mutant HA-ETRs were incubated with 30 nM ET-1 for the indicated times at 37°C. After washing three times with ice-cold PBS (pH 7.4) and resuspension in PBS, the cells were incubated with rotation with 0.5 mg/ml EZ-Link TM Sulfo-NHS-SS-Biotin at 4°C for 1 h to biotinylate cell surface proteins, and the reaction was quenched by adding 50 mM Tris-HCl (pH 8.0)/PBS followed by two washes with ice-cold PBS. The cells were lysed with lysis buffer and centrifuged at 20,000 × g for 20 min at 4ºC. The supernatants were incubated with streptavidin agarose resin at 4°C for 1.5 h to collect biotinylated proteins.
The precipitates were washed four times with washing buffer and biotinylated proteins on the streptavidin agarose resin were eluted by adding SDS sample buffer (62.5 mM Tris-HCl (pH 6.8), 10% glycerol, 5% 2-mercaptoethanol, 2.5% SDS, 0.1% bromophenol blue). The resulting supernatant was subjected to Western blot analysis to detect HA-ETRs, which had been on the cell surface after ET-1 stimulation.
Analysis of intracellular trafficking by confocal microscopy-To determine intracellular trafficking pathways for ETRs, we analyzed co-localization of ETRs with either Rab7 or Rab11 as a marker for late endosome/lysosome or recycling endosome, respectively.
For this purpose, HEK293T cells were plated on a collagen-coated 35-mm diameter glass base dish (Iwaki, Japan) at a density of 3 × 10 5 cells per dish. The cells were transiently transfected with either of expression vectors for C-terminally GFP-tagged WT ETAR (ETAR-GFP), ETBR WT-GFP and ETBR 5KR-GFP, along with either C-terminally tdTomato-tagged Rab7 (Rab7-tdTomato) or Rab11-tdTomato. Twenty four hours after transfection, the cells were incubated with or without ET-1 for 30 min, and fixed in 4% paraformaldehyde for 15 minutes at room temperature. Images were captured by confocal laser microscopy (FV10i, Olympus) and analyzed quantitatively using MetaMorph software (Universal Imaging, West Chester, PA). Namely, vesicles positive for GFP signal or tdTomato signal within each cell were defined based on their intensity and diameter, and subsequently, the number of vesicles within each cell which showed signals for either GFP, tdTomato or both was counted. The extent of co-localization of receptors with Rab proteins was represented as a percentage of the number of vesicles showing both signals to total number of vesicles showing GFP signal alone. Results were obtained from three independent experiments, with 10-13 cells being analyzed in each experiment.

Analysis of internalization of ETRs by confocal microscopy-HA-ETBR-expressing cells were
washed and incubated with Alexa488-conjugated anti-HA antibody for 1 h at 4ºC in serum free DMEM. After washing twice with PBS, the cells were incubated with vehicle or 30 nM ET-1 for 30 min at 37ºC, washed with PBS and fixed with 4% paraformaldehyde. Images were captured by confocal laser microscopy (FV10i, Olympus). Using MetaMorph software, measurements were made in single cells by selecting a region encompassing the entire plasma membrane (defined as total cell region) and then selecting a region just inside the plasma membrane (1.6 µm inside the total cell region; defined as cell inside region).
The difference between these two regions was defined as cell membrane region. Fluorescence intensity in total cell region and cell inside region was measured, and fluorescence intensity in cell membrane region was calculated based on fluorescence intensity in these two regions. For estimation of the amount of the internalized receptors, the ratio of the fluorescence intensity in cell membrane region to that in total cell region was determined. 2+ ]i was measured as described previously (25,26). HEK293T cells expressing wild type or mutant HA-ETBRs were incubated in culture medium containing with 4 µM fura-2/AM, 2.5 mM probenecid and 0.04% pluronic F-127 at 37°C for 60 min under reduced light. After washing, the cells were suspended in Ca 2+ -free Krebs-HEPES solution (140 mM NaCl, 3 mM KCl, 1 mM MgCl2, 11 mM D-(+)-glucose, 10 mM HEPES; adjusted to pH 7.3 with NaOH) at 4 × 10 5 cells/ml, and stored at 4°C under reduced light. Immediately before [Ca 2+ ]i measurement, CaCl2 was added to 0.5-ml aliquot of the cell suspension at the final concentration of 2 mM. [Ca 2+ ]i was measured at 30°C using a CAF-110 spectrophotometer (JASCO) with excitation wavelengths of 340 and 380 nm and an emission wavelength of 500 nm. Data were collected and analyzed using MacLab/8s and Chart (v. 3.5) software (ADInstruments Japan).

Measurement of the intracellular Ca 2+ concentration ([Ca 2+ ]i)-[Ca
Data Analysis-All data were presented as mean ± S.E.M.
The significance of the difference between mean values was evaluated with GraphPad PRISM™ (version 3.00, GraphPad Software Inc) by Student's unpaired t-test. A P value less than 0.05 was considered to indicate statistically significant differences.

RESULTS
ETBR but not ETAR is ubiquitinated in C-tail in response to ET-1 stimulation-To evaluate possible ubiquitin modification of the ETRs, we generated HEK293T cells stably expressing either N-terminally HA-tagged ETAR or ETBR (referred to as HA-ETAR or HA-ETBR, respectively). The cells were transiently transfected with expression vector encoding myc-ubiquitin (myc-ub), stimulated with ET-1 for the indicated times and lysed.
HA-ETAR or HA-ETBR was immunoprecipitated, and the extent of receptor ubiquitination was determined by Western blot (Fig. 1A). Before stimulation with ET-1 (at 0 min), a broad band larger than 80 kDa representing ubiquitinated receptor was observed for HA-ETBR, but essentially no signal was observed for HA-ETAR. After stimulation with 30 nM ET-1, no detectable ubiquitination of HA-ETAR was observed. In sharp contrast, ubiquitination of HA-ETBR was augmented within 5 min after ET-1 stimulation and lasted, at least, for 20 min (Fig. 1, A and B). To confirm the relevance of our findings on exogenously expressed receptors in HEK293T cells, we investigated ET-1-induced ubiquitination of endogenously expressed ETBR in human pulmonary artery smooth muscle cells (HPASMC), and obtained essentially similar results ( Fig. 1, C and D). β-Arrestins have been shown to function as adaptors for ubiquitination of GPCRs such as β2-adrenergic receptor (β2-AR) (27,28), while both ETRs are known to recruit β-arrestins (18,21). Therefore, we examined a role of β-arrestins in ubiquitination of ETBR. Knockdown of β-arrestin1 and β-arrestin2 was without effect on basal and ET-1-induced ubiquitination of ETBR, demonstrating that β-arrestins play no significant role in ubiquitination of ETBR (Fig. 2).
In order to determine the role of ubiquitination in the regulation of ETBR trafficking, we constructed mutant ETBR, which would not be ubiqutinated. ETBR has 16 lysine residues in the cytoplasmic region; 8 lysine residues in C-tail (Fig. 3A) and 8 other lysine residues in the cytoplasmic loops.
Because recent studies have indicated that C-tail might be a major site of ubiquitination in GPCRs (29-32), we first replaced 8 lysine residues in C-tail with arginine (designated HA-ETBR 8KR).
In contrast with wild-type HA-ETBR, the mutant receptor was found to be not ubiquitinated in an agonist-dependent manner (Fig. 3B), suggesting that lysine residues in C-tail are major sites of ubiquitination in ETBR.
Previously, we demonstrated that ETBR is palmitoylated at the cysteine cluster of C-tail, and anchored to the plasma membrane ( Fig. 3A) (9), generating a small loop structure between the 7th transmembrane domain and the palmitoylation sites (Fig. 3A). The amino acid sequences of the loop structure are very similar between ETAR and ETBR (84% identical) (15), whereas the sequence homology of the region distal to palmitoylation sites is relatively low (26% identical), suggesting the possibility that lysine residues in the region distal to palmitoylation sites in C-tail of ETBR are the targets for ubiquitination. Therefore, we generated another ETBR mutant in which 5 lysine residues (K411, K417, K422, K424 and K438) distal to the palmitoylation sites in C-tail were replaced with arginine (designated HA-ETBR 5KR). As shown in Fig. 3C, HA-ETBR 5KR was not ubiquitinated in an agonist-dependent manner, suggesting that lysine residues distal to the palmitoylation sites are major targets for ubiquitination of ETBR.
Receptor ubiquitination is involved in ET-1-induced ETBR internalization-To get insights into the functional significance of ETR ubiquitination, receptor internalization was examined using biotin-streptavidin system to measure the amount of cell surface receptor. The level of cell surface HA-ETBR rapidly decreased following ET-1 stimulation with a half-life of about 15 min (Fig. 4, A and B). The level of cell surface HA-ETAR decreased similarly with HA-ETBR up to 10 min, but thereafter, it remained almost unchanged (Fig. 4, A and B). Notably, HA-ETBR 5KR, the mutant which was not ubiquitinated, disappeared from the cell surface at the rate similar to that of HA-ETBR WT up to 10 min, but thereafter, at a far slower rate (Fig. 4, C and D): the time course of disappearance was similar to that of HA-ETAR (Fig. 4, B and D).
Next, we attempted to confirm slower internalization of HA-ETBR 5KR in comparison with that of HA-ETBR WT using confocal microscopy. Before stimulation with ET-1, major part of HA-ETBR WT and HA-ETBR 5KR was present on the cell membrane (Fig. 5, A, upper panels; Fig. 5, B, left panel). Thirty minutes after stimulation with ET-1, most of both receptors were internalized, and the number of the receptors remaining on the cell membrane became smaller. Quantitative analysis showed that before ET-1 stimulation, the ratio of cell membrane fluorescence intensity to total cell fluorescence intensity was comparable between cells expressing HA-ETBR WT and HA-ETBR 5KR, but that after 30 min-stimulation with ET-1, it was twice as large in cells expressing HA-ETBR 5KR as that in cells expressing HA-ETBR WT (Fig. 5, A, lower panels; Fig. 5

, B, right panel).
Receptor ubiquitination is involved in ET-1-induced ETBR degradation-We next investigated the role of ubiquitination in ET-1-induced receptor degradation.
For this purpose, we determined the disappearance rate of total ETRs in the whole cells after ET-1 stimulation in the presence of a protein synthesis inhibitor, cycloheximide (CHX). In the presence of CHX, the level of total HA-ETBR decreased with time after ET-1 stimulation, whereas that of total HA-ETAR was almost unchanged, at least, for 30 min (Fig. 6, A and B). The disappearance rate of non-ubiquitinated HA-ETBR 5KR was slower than HA-ETBR WT (Fig. 6, C and D) and comparable to that of HA-ETAR (Fig. 6, B and D). These results suggest that receptor ubiquitination accelerates ETBR degradation.
Time course for recovery of cell surface levels of HA-ETBR WT and HA-ETBR 5KR following ET-1 stimulation-Toward further understanding of cellular dynamics of HA-ETBR and HA-ETBR 5KR, we examined the time course for recovery of cell surface levels of these receptors following ET-1 stimulation. For this purpose, after 30-min stimulation with ET-1, the cells stably expressing HA-ETBR WT and HA-ETBR 5KR were washed two times with ET-1-free medium, cultured for the indicated times and lysed for quantification of cell surface levels of HA-ETBR WT and HA-ETBR 5KR by biotinylation assay.
After 30-min stimulation with ET-1, cell surface levels of HA-ETBR WT and HA-ETBR 5KR were reduced to approximately 30% and 70%, respectively, of control values before stimulation, remained constant up to 2 h and thereafter, began to increase: the level of HA-ETBR 5KR was recovered to the control level around 4 h, while recovery for the level of HA-ETBR WT was not complete even 8 h after stimulation (Fig. 7).
ETBR is ubiquitinated on the cell membrane-We asked whether the ET-1-induced ETBR ubiquitination occurs, before or after internalization.
For that purpose, we overexpressed FLAG-tagged dominant-negative mutant of rat dynamin (DN-dynamin), to inhibit ETBR internalization (21), and examined its effect on ETBR ubiquitination.
In control cells transfected with empty vector alone, cell surface HA-ETBR rapidly decreased following ET-1 stimulation (Fig. 8A, top panel). In contrast, in cells transfected with DN-dynamin, the disappearance rate of cell surface HA-ETBR became markedly slower (Fig. 8A, top panel), indicating that internalization of cell surface HA-ETBR was inhibited by DN-dynamin.
Notably, inhibition of internalization by DN-dynamin did not affect ETBR ubiquitination induced by ET-1 stimulation (Fig. 8, B and C), indicating that ETBR is ubiquitinated before internalization, probably on the cell membrane.
Confocal microscopic study of intracellular trafficking of ETAR, ETBR or ETBR 5KR-The apparent rate of receptor internalization in the present study reflects the sum of the rate of receptor endocytosis and the rate of receptor recycling to plasma membrane. Therefore, the slower disappearance of HA-ETBR 5KR from cell surface following ET-1 stimulation could result from either a decrease in the rate of receptor endocytosis process itself or an increase in receptor recycling to plasma membrane. To differentiate these two possibilities, we analyzed intracellular trafficking pathways of HA-ETAR, HA-ETBR WT and HA-ETBR 5KR using confocal microscopy. For this purpose, we examined co-localization of these receptors with Rab7, a marker for late endosome/lysosome, and Rab11, a marker for recycling endosome (33,34). In one series of experiment, either one of ETAR-GFP, ETBR-GFP and ETBR 5KR-GFP was transiently transfected into HEK293T cells along with Rab7-tdTomato (Fig. 9, A and C), while in the other series, either one of the receptors was transiently transfected with Rab11-tdTomato ( Fig.  9, B and D). In the absence of ET-1, most of these receptors were localized to the plasma membrane. After 30-min stimulation with ET-1, most of ETAR-GFP, ETBR-GFP and ETBR 5KR-GFP were internalized to the intracellular vesicular structures.
ETAR-GFP was rarely co-localized with Rab7-tdTomato, whereas ETBR WT-GFP was frequently co-localized with Rab7-tdTomato (Fig. 9, A and C). Notably, ETBR 5KR-GFP showed rare co-localization with Rab7-tdTomato, like ETAR-GFP (Fig. 9, A and C). Conversely, ETAR-GFP and ETBR 5KR-GFP were frequently co-localized with Rab11-tdTomato ( Fig.  9, B and D), whereas ETBR WT-GFP was rarely co-localized with it ( Fig. 9, B and D). These data clearly demonstrate that after agonist stimulation, ETAR and non-ubiquitinated ETBR mutant (5KR) are mainly on the recycling pathway, whereas ETBR is mainly targeted to lysosome for degradation, and hence, that the slower rate of apparent internalization for ETBR 5KR at the later phase after ET-1 stimulation is mainly due to recycling to plasma membrane of the receptor.
Functional significance of ETBR ubiquitination in cellular signaling-To clarify how receptor ubiquitination affects ETBR-mediated intracellular signaling, we examined ERK phosphorylation and an increase in [Ca 2+ ]i in cells stably expressing HA-ETBR WT or HA-ETBR 5KR in response to two successive stimulation with ET-1. For this purpose, the cells were first stimulated for 30 min with 30 nM ET-1, and they were washed two times with culture medium to remove ET-1: after incubation in fresh medium for 15 min for equilibration, the cells were again stimulated with 30 nM ET-1. In response to the 1st stimulation, ERK phosphorylation levels were increased in cells expressing HA-ETBR WT or HA-ETBR 5KR, and the increase was comparable to each other (Fig. 10, A and B).
Notably, ERK phosphorylation levels in response to the 2nd stimulation became smaller compared to the 1st responses in both types of cells, but the levels were 2-3 times larger in cells expressing HA-ETBR 5KR than those in cells expressing HA-ETBR WT (Fig. 10, A and B). The increase in [Ca 2+ ]i induced by the first ET-1 stimulation in HA-ETBR 5KR-expressing cells was similar to that in HA-ETBR WT-expressing cells (Fig. 10C, left panel), but the increase by the second stimulation was about 60% larger than that in HA-ETBR WT-expressing cells (Fig. 10C, right panel). Taking into consideration that the 2nd stimulation was given during the time when changes in cell surface levels of HA-ETBR WT and HA-ETBR 5KR following the 1st stimulation are maintained at about 30% and 70% of the values before the 1st stimulation, respectively, these results strongly demonstrate that larger responses to the 2nd stimulation for HA-ETBR 5KR are due mainly to higher levels of cell surface HA-ETBR 5KR following the 1st stimulation, resulting from a switch of intracellular trafficking from lysosomal targeting to recycling to plasma membrane. However, considering that ERK phosphorylation and an increase in [Ca 2+ ]i induced by the second ET-1 stimulation are significantly smaller than the responses expected from cell surface levels of the receptors, it is likely that desensitization mechanisms other than reduced levels of receptors are involved in the reduced responses to the second ET-1 stimulation.

Ubiquitination of either one lysine residue is sufficient
for ET-1-induced ETBR internalization-We asked which lysine residue(s) in C-tail was responsible for ET-1-induced ubiquitination and lysosomal targeting of ETBR. To clarify this point, we generated a series of ETBR mutants in which either one of 5 arginine residues (R411, R417, R422, R424 and R438) in C-tail of HA-ETBR 5KR was reverted to the original lysine (referred to as HA-ETBR 4KR-411K, 417K, 422K, 424K or 438K, respectively), and examined the ET-1-induced ubiquitination of these mutants. In response to ET-1 stimulation, all of these mutant ETBRs were ubiquitinated to the level comparable to that of HA-ETBR WT (Fig. 11, A and B), suggesting that either one of 5 lysine residues in C-tail of ETBR WT can be a site of ubiquitination, and also that ETBR WT is ubiquitinated at only one lysine residue.
We next examined ET-1-induced internalization of these HA-ETBR 4KR mutants. The disappearance rate of all these mutants except HA-ETBR 4KR-411K from the cell surface was recovered to that of HA-ETBR WT: the disappearance rate of HA-ETBR 4KR-411K remained low, and was similar to that of non-ubiquitinated mutant, HA-ETBR 5KR (Fig.  11C). We wondered whether the dissociation between ubiquitination and internalization of HA-ETBR 4KR 411K could be due to difference of assay conditions, i.e., the absence or presence of overexpression of myc-ub.
Therefore, internalization of the receptor was analyzed after overexpression of myc-ub. Overexpression of myc-ub did not affect the disappearance rate of HA-ETBR 4KR 411K from the cell surface compared to the rate in the absence of overexpression of myc-ub (data not shown). These data suggest that ubiquitination of any one lysine residue in C-tail, except for lysine 411 is sufficient for ET-1-induced ETBR internalization.
We also examined the ET-1-induced degradation rates of HA-ETBR 4KR mutants, which were indexed by the ET-1-induced decrease in whole cell levels of wild type or mutant HA-ETBRs during 30 min after treatment with CHX, a protein synthesis inhibitor. As described in Fig. 6, B and D, in the presence of CHX, ET-1 stimulation for 30 min decreased the whole cell level of wild type HA-ETBR to 65% of the control value without the stimulation, whereas it had little effect on the whole cell level of non-ubiquitinated mutant, HA-ETBR 5KR (Fig. 11D). In response to ET-1, the whole cell levels of all HA-ETBR 4KR mutants except for 4KR-411K decreased to the extent comparable to those of wild type HA-ETBR, whereas the level of HA-ETBR 4KR-411K was virtually unchanged (Fig. 11D). Overexpression of myc-ub did not affect the ET-1-induced degradation rate of HA-ETBR 4KR 411K (data not shown). These results indicate that ubiquitination of either one of 5 lysine residues except for lysine 411 in C-tail of ETBR is sufficient for ET-1-induced ETBR degradation.
Finally, we examined ERK phosphorylation in cells stably expressing HA-ETBR 4KR in response to two successive stimulation with ET-1. An increase in ERK phosphorylation in response to the 1st stimulation was similar between HA-ETBR WT and HA-ETBR 4KR mutant (Fig. 12, A and  data not shown).
In accordance with the recovery of receptor internalization and degradation rates, ERK phosphorylation response to the 2nd stimulation was recovered in cells expressing either one of HA-ETBR 4KR mutants except for HA-ETBR 4KR-411K to the level in cells expressing HA-ETBR WT (Fig. 12, B and, data not shown), whereas the response in cells expressing HA-ETBR 4KR-411K remained larger than that in cells expressing HA-ETBR WT.

DISCUSSION
Two types of receptors for ET-1 such as ETAR and ETBR closely resemble each other, but after stimulation with their agonist, they are well-known to follow totally different intracellular trafficking pathways (14,15).
That is, after agonist-induced endocytosis, ETAR is mainly recycled back to the plasma membrane, whereas ETBR is targeted to lysosome for degradation (14,18).
In the present study, we have demonstrated that 1) the different fates of ETAR and ETBR are mainly due to agonist-induced ubiquitination of ETBR but not ETAR, which occurs on the cell membrane, 2) ubiquitination of ETBR switches intracellular trafficking of the receptor after agonist-induced internalization, from recycling back to the plasma membrane to targeting to lysosome for degradation, causing a decrease in cell surface levels of the receptor, 3) ubiquitination of the receptor and consequent decrease in its cell surface levels induce quenching of ETBR-mediated responses such as ERK phosphorylation and an increase in [Ca 2+ ]i to repetitive agonist stimulation, and 4) ubiquitination at either one of five lysine residues except lysine 411 in the distal end of C-tail of ETBR is sufficient for a switch of the intracellular trafficking.
ETBR but not ETAR, expressed endogenously in cell lines (HPASMC) and exogenously in HEK293T cells, was specifically ubiquitinated in an agonist-dependent manner, although both receptors possessed several lysine residues as potential ubiquitination sites. Wild-type ETBR has total 16 lysine residues in the cytoplasmic region as potential ubiquitination sites; 8 lysine residues in the cytoplasmic loops and 8 lysine residues in C-tail. In C-tail, 3 lysine residues are present between the 7th transmembrane domain and palmitoylation sites, while 5 lysine residues are present between the palmitoylation sites and C-tail end (distal C-tail) (Fig. 3A). Using mutant receptors named HA-ETBR 8KR and HA-ETBR 5KR (in which lysine residues in the whole C-tail and distal C-tail were replaced with arginine, respectively), it was clearly shown that among these 16 potential ubiquitination sites of ETBR, it is the 5 lysine residues in distal C-tail that are critical for agonist-induced ubiquitination of ETBR. Conversely, using another series of mutant receptors named ETBR 4KR in which either one of 5 arginine residues in HA-ETBR 5KR was reverted to the original lysine, it was shown that wild type ETBR was ubiquitinated at either one of 5 lysine residues in its C-tail, and that any single lysine residue in distal C-tail of ETBR except lysine 411 can function as a ubiquitination site. However, it is unknown whether a preferable ubiquitination site is present and, if present, which lysine residue is the most preferable for ubiquitination. Furthermore, the ubiquitination of ETBR was found to occur on the cell membrane, according to the experiments with dominant-negative dynamin. This result is consistent with the previous report on µ-opioid receptor (MOR) (35). The reason for lack of ETAR ubiquitination is at present unknown, although ETAR has 3 lysine residues as potential ubiquitination sites in the distal C-tail. This difference could be due to that ETAR is not a good substrate for any E3 ligase.
It is well-known that β-arrestins function as adaptors for ubiquitination of GPCRs such as β2-AR (27,28) and also that both ETAR and ETBR recruit β-arrestins upon agonist stimulation (18,21). However, it is unlikely that β-arrestins are involved in ubiquitination of ETBR, based on the result that knockdown of both β-arrestin-1 and β-arrestin-2 has little effect on ET-1-induced ubiquitination of the receptor. These results demonstrate that E3 ligase for ETBR may not require adaptor proteins for its recruitment to the receptor, or that it may use other adaptor proteins than β-arrestins.
Ubiquitination of ETBR switches intracellular trafficking of the receptor after agonist-induced endocytosis, from recycling back to the plasma membrane to targeting to lysosome for degradation, causing a decrease in cell surface levels of ETBR. That is, ubiquitinated ETBR WT was targeted to lysosome for degradation, judging from ET-1-induced decrease in total amount of the receptor following protein synthesis inhibition by CHX and by dominant localization of ETBR WT to late endosomes/lysosomes following ET-1 stimulation. In contrast, non-ubiquitinated ETBR mutant (ETBR 5 KR) was found to be recycled back to plasma membrane, as evidenced by negligible ET-1-induced decrease in total amount of the receptor following protein synthesis inhibition and by dominant localization of the receptor to recycling endosomes following ET-1 stimulation.
This pattern of intracellular trafficking for non-ubiquitinated ETBR mutant is similar to that for ETAR, which is not ubiquitinated. Furthermore, restoration of any one arginine residue of ETBR 5KR C-tail to lysine (ETBR 4KR) normalized receptor ubiquitination and intracellular trafficking to those of ETBR WT.
It is reported that some membrane proteins including β2AR and low-density lipoprotein receptor-related protein are recycled to the plasma membrane by trans-acting proteins such as sorting nexin 27 (SNX27) and SNX17, respectively, which interact with C-tail of those membrane proteins (36,37). Therefore, it is possible that such trans-acting proteins are involved in recycling of ETAR and probably ETBR 5KR which shows the same receptor kinetics as ETAR. In the case of wild type ETBR, lysosomal targeting signal by receptor ubiquitination might overwhelm recycling signal by trans-acting proteins or ubiquitination of ETBR might simply inhibit the binding of trans-acting proteins to the receptor. The previous report that a C-tail-truncated mutant of ETAR is neither internalized nor recycled to the plasma membrane (15,21) may support the presence of such trans-acting proteins. Such trans-acting proteins of ETAR and ETBR remain to be identified.
The present study clearly shows that ubiquitination at either one of five lysine residue in the distal C-tail of ETBR except lysine 411 is sufficient for switching intracellular trafficking of ETBR from recycling to plasma membrane to lysosomal targeting. However, the reason for which intracellular trafficking of HA-ETBR 4KR-411K is not recovered in spite of its ability to be ubiquitinated is at present unknown. It might be due to that ubiquitination of HA-ETBR 4KR at 411K is not an effective signal either for lysosomal targeting or for inhibiting the binding of recycling machinery such as the above-mentioned trans-acting proteins to the receptor. The present result that ubiquitination promotes degradation of ETBR is consistent with previous reports for β2AR, CXCR4, vasopressin V2 receptor (V2R), protease-activated receptor 2 (PAR2), neurokinin-1 receptor (NK1R), κ-opioid receptor (KOR) and δ-opioid receptor (DOR) (28-31, [38][39][40]. On the other hand, there are few reports showing the involvement of ubiquitination in apparent receptor internalization (or the rate of disappearance of cell surface receptors). That is, in the case of β2AR, CXCR4, V2R, KOR and DOR, ubiquitination has little effect on the rate of apparent receptor internalization, whereas it accelerates internalization of MOR without effect on receptor recycling to plasma membrane. In this context, ubiquitination of ETBR is unique in that it accelerates the apparent receptor internalization by switching intracellular trafficking from recycling to lysosomal targeting, presumably without effect on internalization process itself.
However, it remains to be determined whether ubiquitination of ETBR directly affects internalization process itself. The reason for different roles of ubiquitination in receptor internalization among ETBR, MOR and the other GPCRs is at present unknown, but it might depend on difference of E3 ligases to be involved and the consequent properties/sites of ubiquitination. Indeed, a recent study on β2AR demonstrates that following binding of different ligands (one is isoprenaline [βAR agonist], while the other is carvedilol [βAR antagonist]) to the receptor, different E3 ligases (such as Nedd4 and MARCH2, respectively) are recruited to ubiquitinate different sites of the receptor, causing different regulation of receptor trafficking: that is, ubiquitination of β2AR by Nedd4 accelerates only lysosomal targeting of the receptor, whereas that by MARCH2 accelerates both receptor internalization and lysosomal targeting (41).
Regarding the functional significance of agonist-induced ubiquitination of ETBR, the present study demonstrates that a decrease in cell surface levels of ETBR, resulting from agonist-induced acceleration of degradation of ubiquitinated ETBR, is mainly responsible for quenching of ETBR-mediated intracellular responses such as ERK phosphorylation and an increase in [Ca 2+ ]i to repetitive agonist stimulation. However, considering that ERK phosphorylation and [Ca 2+ ]i response induced by the second ET-1 stimulation are significantly smaller than the responses expected from the extent of a decrease in cell surface levels of the receptors, it is likely that receptor desensitization as well as reduced levels of receptors are involved in the reduced responses to the second ET-1 stimulation. The mechanisms for desensitization remain to be determined.
The ETBR expression level is reported to be elevated in several pathologic conditions such as atherosclerosis, chronic heart failure and pulmonary hypertension (42)(43)(44)(45), and it is probable that the elevated ETBR levels play a critical role in the genesis and/or development of these pathological conditions. In this context, it is important to investigate the molecular mechanism for the elevated receptor levels, especially in terms of disturbed ubiquitination or enhanced deubiquitination in those pathological conditions. Such study might lead to discovery of novel mechanism for genesis and/or development of these pathological conditions and also of novel therapeutic strategy for those pathological conditions.    for 30 min at 37°C. Subsequently, they were fixed with 4% paraformaldehyde and subjected to image analysis by confocal microscopy (A). Quantitative analysis of confocal images was performed using MetaMorph software (B). Namely, total cell region and cell membrane region were selected in single cells as described in "Experimental Procedures", fluorescence intensity in these regions was determined and the ratio of the fluorescence intensity in total cell region to that in cell membrane region was calculated. Each bar graph represents mean ± S.E.M. n = 15-17. **, P < 0.01 versus HA-ETBR WT. Scale bar, 10 µm.