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J Biol Chem, Vol. 275, Issue 12, 8664-8671, March 24, 2000

Subtype-specific Trafficking of Endothelin Receptors^*

Yoichiro Abe, Kazuhisa Nakayama^§, Akihiro Yamanaka, Takeshi Sakurai, and Katsutoshi Goto^¶

From the Department of Pharmacology, Institute of Basic Medical Sciences and ^§ Institute of Biological Sciences and Gene Experiment Center, University of Tsukuba, Tsukuba, Ibaraki 305-8575, Japan

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

We investigated the subcellular localization of two endothelin receptors (ET_AR and ET_BR). To visualize these receptors directly, the C terminus of each receptor was fused to the N terminus of enhanced green fluorescent protein (designated as ETR-EGFP). When transiently expressed in various mammalian cell lines, ET_AR-EGFP was predominantly localized on the plasma membrane. By contrast, ET_BR-EGFP was, independent of ligand stimulation, predominantly localized on the intracellular vesicular structures containing Lamp-1. Immunoblot analyses revealed that at steady state ET_BR-EGFP was highly degraded, and its degradation was inhibited by bafilomycin A₁. Antibody uptake experiments suggested that the ET_BR-EGFP molecules were internalized from the plasma membrane. It is therefore likely that ET_BR is first transported to the plasma membrane and then internalized, irrespective of ligand stimulation, to lysosomes where it undergoes proteolytic degradation. Exchanging the C-terminal cytoplasmic tails of the two ETRs revealed that the cytoplasmic tail is responsible for both the intracellular localization and the degradation of the receptors. Deletion of the extreme C-terminal 35 amino acids from both receptors allowed the receptor proteins to localize predominantly in the intracellular vesicles and to degrade. These observations indicate that the cytoplasmic tail of ET_AR determines its plasma membrane localization. Stimulation with endothelin-1 increased the amount of intact ETR-EGFP fusion proteins without increasing their de novo synthesis, suggesting that binding of endothelin-1 stabilizes the ETRs.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Endothelins are a family of 21-amino acid peptides consisting of three isopeptides as follows: endothelin-1, endothelin-2, and endothelin-3 (ET-1,¹ ET-2, and ET-3, respectively) (1, 2). Endothelins have two receptor subtypes, termed endothelin-A receptor (ET_AR) and endothelin-B receptor (ET_BR), both of which belong to the G protein-coupled receptor (GPCR) superfamily (3, 4). ET_AR exhibits higher affinity for ET-1 and ET-2 than ET-3 (3), whereas ET_BR accepts all three endothelins with similar affinity (4). ET_AR is predominantly coupled to G_q and G_s, whereas ET_BR is predominantly coupled to G_q and G_i (5). Although these two receptors share approximately 55% overall amino acid sequence identity (Fig. 1A), several domains are less homologous to each other and contribute to the functional differences between these receptors. For example, transmembrane domains IV-VI of the ET_BR determine its specificity to certain ligands, such as ET-3 and IRL1620 (6). On the other hand, transmembrane domains I-III and VII determine the ability of ET_AR to bind its specific ligands, such as BQ 123 (6). Cytoplasmic loop II is essential for ET_AR to couple to G_s, whereas cytoplasmic loop III is necessary for ET_BR to couple to G_i (7).

In addition to the N-terminal region of the third cytoplasmic loop, the C-terminal cytoplasmic tail is the most divergent intracellular region between the two receptors (Fig. 1, A and B). It has been suggested that the C-terminal cytoplasmic tail is responsible for ligand-dependent internalization and/or desensitization of the GPCRs. In this regard, the most characterized GPCR is ₂-adrenergic receptor. When it is stimulated with its agonist, the serine and threonine residues in the cytoplasmic tail are phosphorylated by G protein-coupled receptor kinases and cAMP-dependent kinase, followed by its association with -arrestins, resulting in uncoupling to G proteins or desensitization. -Arrestins also play a crucial role in the ligand-dependent internalization of this receptor, mediated by clathrin-coated vesicles. The internalized receptors are dephosphorylated at endosomes, resulting in their resensitization and recycling back to the plasma membrane (8-10). Although several other GPCRs, such as protease-activated receptors, are also internalized in response to the cleavage in their N-terminal region and activation by proteases, they are sorted to lysosomes instead of recycling to the plasma membrane and undergo proteolytic degradation in order to terminate the irreversible activation of the signal transduction (11-14). It is the cytoplasmic tail that regulates the trafficking of these receptors (13).

It was recently shown that both ET_AR and ET_BR are phosphorylated by G protein-coupled receptor kinases and rapidly desensitized in an identical manner (15). However, truncation of the cytoplasmic tails of both ET_AR and ET_BR (lacking the last 36 and 40 amino acids, respectively) does not affect ligand-induced desensitization (16-18), suggesting that the cytoplasmic tail of both endothelin receptors is dispensable in ligand-induced desensitization. In addition, although ligand-induced association of -arrestins and internalization were observed in the case of ET_AR (19, 20), the role of the cytoplasmic tail in internalization is still unknown. In the present study, we therefore investigated the role of the C-terminal cytoplasmic tail of these receptors in intracellular trafficking. For this purpose, the C terminus of each ETR was fused with the N terminus of enhanced green fluorescent protein (EGFP) and transiently transfected in cell lines. We found that ET_BR is sorted to lysosomes and undergoes proteolytic degradation independent of ligand stimulation, probably due to the absence of the sequence necessary for its anchoring to the plasma membrane. We also found that prolonged stimulation with ET-1 allows for stabilization of both receptors.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Plasmid Construction-- The restriction maps of rat ET_AR and ET_BR cDNAs are shown in Fig. 1C. All mutations (Fig. 1C, underlined), as well as truncation of the C-terminal region of the receptors, were introduced by polymerase chain reaction using cDNAs for rat ET_AR (21) and ET_BR (4) as templates. The polymerase chain reaction products were subcloned into pCR2.1 TOPO TA cloning vector (Invitrogen) and sequenced by the dideoxy termination method using an autosequencer (Lichor). A BamHI site was introduced just before the termination codon of each clone and was used to insert it into pEGFP-N3 (CLONTECH) in frame. To exchange the C-terminal cytoplasmic tail, an AsnI site was introduced at Ile³⁶⁴-Asn³⁶⁵ of ET_AR corresponding to Ile³⁸⁰-Asn³⁸¹ of ET_BR. To exchange the third cytoplasmic loop, an RcaI site was introduced at Leu²⁹⁴-Met²⁹⁵-Thr²⁹⁶ of ET_BR, which corresponds to Leu²⁷⁷-Met²⁷⁸-Thr²⁷⁹ of ET_AR, and an Mlu NI site was introduced at Val³⁰⁴-Ala³⁰⁵-Lys³⁰⁶ of ET_AR, which corresponds to Val³²⁰-Ala³²¹-Lys³²² of ET_BR. All cDNAs of each ETR-EGFP fusion protein were subcloned between XhoI and NotI sites of pME 18Sf() vector.

Cell Culture and Transfection-- L tk cells obtained from Riken Cell Bank (Tsukuba, Japan) were maintained in Dulbecco's modified Eagle's medium (DMEM) (Life Technologies, Inc.) supplemented with 10% fetal calf serum. Transfection was performed by the DEAE-dextran method. HeLa cells were maintained in DMEM supplemented with 10% fetal calf serum. Transfection was performed using FuGENE 6 transfection reagent (Roche Molecular Biochemicals). Rat clone 9 cells were maintained in DMEM/F-12 medium (Life Technologies, Inc.) supplemented with 10% fetal calf serum. Transfection was performed using FuGENE 6 transfection reagent.

Immunofluorescence Analysis-- L tk cells seeded on a 6-cm dish (7.5 × 10⁵ cells) were transfected with each plasmid. Six hours after transfection, the cells were reseeded onto 8-well Lab-Tek II chamber slides (Nunc). The cells were cultured for 48 h and then fixed with 4% paraformaldehyde in PBS for 1 h at room temperature.

For lysosomal staining, rat clone 9 cells or HeLa cells seeded directly on Lab-Tek II chamber slides (5.0 × 10³ cell/well for clone 9 and 1.0 × 10⁴ cells/well for HeLa cells) were transfected with each plasmid. Forty-eight hours after transfection, cells were fixed with cold methanol, permeabilized with 0.1% Triton X-100 in PBS, and incubated with rabbit polyclonal anti-rat Lamp-1 antibody (kindly provided by Dr. Kenji Akasaki, Fukuyama University, Japan) for clone 9 or mouse monoclonal anti-human Lamp-1 antibody (PharMingen) for HeLa cells for 1 h at room temperature, followed by labeling with Cy3-conjugated goat antibodies against rabbit or mouse IgG, respectively (Jackson ImmunoResearch Laboratories) for 1 h at room temperature.

For analysis of endocytosis of ET_BR, clone 9 cells or HeLa cells transfected with the ET_BR-EGFP plasmid were incubated with 5 µg/ml rabbit anti-peptide antibody against the N-terminal region of human ET_BR (amino acids 27-42) (IBL) for 24 h simultaneously with transfection. After washing, fixing with 4% paraformaldehyde in PBS, and permeabilization with 0.1% Triton X-100 in PBS, cells were stained with Cy3-conjugated goat antibody against rabbit IgG.

All cells were observed with a TCS4D laser-scanning confocal microscope (Leica).

Immunoblotting-- L tk cells on 10-cm dishes (1.5 × 10⁶ cells) were transfected with each plasmid. Forty-eight hours after transfection, cells were collected and lysed with SDS-PAGE sample buffer (10⁷ cells/ml), then subjected to SDS-PAGE, followed by electrical transfer to a PVDF membrane. EGFP or EGFP fusion proteins were detected with polyclonal anti-GFP antibody (CLONTECH), horseradish peroxidase-labeled goat anti-rabbit IgG, and enhanced chemiluminescence reagents (Amersham Pharmacia Biotech).

Northern Blot Analysis-- Transfected L tk cells on 10-cm dishes (1.5 × 10⁶ cells/dish) were cultured for 48 h, and cellular total RNA was extracted with ISOGEN (Nippon Gene). The extracted RNA (10 µg) was separated by formaldehyde/agarose gel electrophoresis, transferred to a hybond N Nylon membrane (Amersham Pharmacia Biotech), and hybridized with rat ET_AR, rat ET_BR, or EGFP cDNA fragment as probes. Each of the probes was labeled with [³²P]dCTP (Amersham Pharmacia Biotech) by the random priming method.

Binding Experiments-- Cells were subjected to competitive radioligand binding assay as described (21). All assays were started at 48 h after transfection. Transfected cells were seeded in 24-well plates at a density of 1.0 × 10⁵ cells/well and further cultured until the cells adhered (about 3 h). The cells were then washed twice and incubated with 20 pM ¹²⁵I-ET-1 (Amersham Pharmacia Biotech) in the presence of various concentrations of non-labeled ET-1 or ET-3 for 2 h at 22 °C. After extensive washing, cells were lysed by the addition of 0.1 N NaOH, and cell-bound radioactivities were determined by -counter (Aloka).

Intracellular Ca²⁺ Mobilization-- L tk cells on 10-cm dishes (1.5 × 10⁶ cells/dish) were transfected with each ETR cDNA together with the _1B adrenergic receptor cDNA (pcDV1R_1B; kindly provided by Dr. Robert J. Lefkowitz, Duke University, North Carolina). Forty-eight hours after transfection, cells were dispersed and loaded with Fura 2-AM (Dojin) in solution A (140 mM NaCl, 4 mM KCl, 1 mM Na₂HPO₄, 1 mM MgCl₂, 1.25 mM CaCl₂, 11 mM glucose, 5 mM HEPES, 0.2% bovine serum albumin) for 30 min at 37 °C. The Fura 2-loaded cells were washed, resuspended in solution A, and stimulated with ET-1 (100 nM) (Peptide Institute, Inc.) or noradrenaline (10 µM) (Wako). The fluorescence of the cells was measured with excitation at 340 and 380 nm and emission at 500 nm with a CAF 110 ion analyzer (Japan Spectroscopic Co.). Values were represented as percent of maximal response of noradrenaline (10 µM) stimulation.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Difference in the Subcellular Localization between ET_AR and ET_BR-- To visualize the intracellular localization of two subtypes of the endothelin receptor, ET_AR and ET_BR, the C terminus of each receptor was fused to the N terminus of EGFP (designated as ET_AR-EGFP and ET_BR-EGFP). When expressed in L tk cells, both ET_AR-EGFP and ET_BR-EGFP were transcribed at comparable levels to wild-type ET_AR and ET_BR, respectively (Fig. 2, A and B, lower panels, lane 1, 2, 7, and 8), and had virtually the same binding characteristics as ET_AR and ET_BR, respectively (Fig. 2, C and D, and Table I). In addition, measurement of intracellular Ca²⁺ mobilization in the transfected L tk cells revealed that ET_AR-EGFP and ET_BR-EGFP could activate the intracellular signal transduction system to essentially the same extent as ET_AR and ET_BR, respectively (Fig. 2, E and F). To normalize transfection efficiency, _1B adrenergic receptor was cotransfected, and the effects of ET-1 (100 nM) were estimated as percent of maximal responses obtained by stimulation with noradrenaline (10 µM). The values of intracellular Ca²⁺ increment in ET_AR, ET_AR-EGFP, ET_BR, and ET_BR-EGFP were 148 ± 22, 145 ± 35, 81 ± 2, and 89 ± 7%, respectively. These data confirm that the fusion of EGFP does not affect either ligand binding or signal transduction of the endothelin receptors. Therefore, we performed the following experiments using these EGFP fusion constructs in the transient transfection system.

View this table: [in this window] [in a new window]   Table I Ligand binding properties of the endothelin receptors and their derivatives transiently expressed in L tk cells Values are means ± S.E. of four independent experiments performed in duplicate.

When expressed in L tk cells, the ET_AR-EGFP fusion protein was localized predominantly on the plasma membrane (Fig. 3A). By contrast, the ET_BR-EGFP fusion protein was localized not only on the plasma membrane but also on intracellular vesicular structures (Fig. 3B), even in the absence of ligand stimulation. Essentially the same localization pattern of each fusion protein was observed using rat Clone 9 cells or human HeLa cells as the host (Fig. 3, I-L), excluding the possibility that the observations are cell line-specific. We previously observed that human ET_BR-EGFP is localized on the intracellular vesicles, as well as on the plasma membrane,² confirming that the observation is a general phenomenon for ET_BR across species.

We then analyzed the molecular forms of the fusion proteins by immunoblotting of the lysates from L tk cells transfected with these constructs using polyclonal anti-GFP antibody. No band was detected with the anti-GFP antibody in the lysate from cells transfected with either ET_AR or ET_BR (Fig. 4, lane 1 and lane 8, respectively), and only one band at a molecular mass of 27 kDa was detected in the lysate from cells transfected with EGFP (Fig. 4, lane 7), demonstrating the specificity of the anti-GFP antibody. Under these experimental conditions, heterogeneous bands of ET_AR-EGFP were detected between 75 and 100 kDa (Fig. 4, lane 2). The bands probably resulted from the heterogeneity in the protein glycosylation. On the other hand, ET_BR-EGFP was highly degraded to smaller molecular forms (Fig. 4, lane 9). The most intense band detected at a molecular mass of 27 kDa may be the EGFP portion itself, which was generated by intracellular cleavage of the fusion protein and accumulated due to its unusually long half-life (approximately 24 h).

Cytoplasmic Tail of ET_AR Is Responsible for Its Localization to the Plasma Membrane-- ET_AR and ET_BR are highly conserved in the intracellular regions, except for the N terminus of the third cytoplasmic loop and the C-terminal cytoplasmic tail (Fig. 1, A and B). In the case of other GPCRs, corresponding regions have been shown to be implicated in their desensitization and internalization. Therefore, it is reasonable to speculate that the sequences that determine the difference between the two receptors in the subcellular localization are within these regions. We therefore made chimeric constructs of ETR-EGFP having either the third cytoplasmic loop or the cytoplasmic tail, or both regions, derived from the other ETR subtype. When expressed in L tk cells, the levels of mRNA of these ET_AR-EGFP and ET_BR-EGFP derivatives were approximately the same as those of wild-type ETR-EGFP (Fig. 2, A and B, respectively). In addition, as summarized in Table I and Fig. 2, E and F, the binding property and signal transduction ability of these chimeric receptors were essentially the same as those of the wild-type receptors. These data confirm that these substitutions do not affect the function of ET_AR and ET_BR. Exchange of the third cytoplasmic loop of one receptor subtype with that of the other (ET_AR (B_CL3)-EGFP and ET_BR (A_CL3)-EGFP) did not alter either their subcellular localization (Fig. 3, E and F) or stability (Fig. 4 lanes 4 and 11) as compared with the wild-type receptors (Fig. 3, A and B, and Fig. 4, lanes 2 and 9). By contrast, exchange of the cytoplasmic tail (ET_AR (B_CT)-EGFP and ET_BR (A_CT)-EGFP) led the chimeric receptors to characteristics that are completely different from the wild-type receptors as follows: ET_AR (B_CT)-EGFP was localized on both the plasma membrane and vesicular structures (Fig. 3C) and was highly degraded (Fig. 4, lane 3), whereas ET_BR (A_CT)-EGFP was localized predominantly on the plasma membrane (Fig. 3D) and not so degraded (Fig. 4, lane 10). We obtained essentially the same results by exchanging both the third intracellular loop and the cytoplasmic tail (Fig. 4, lanes 5 and 12 and data not shown).

View larger version (20K): [in this window] [in a new window]   Fig. 1.   Schematic representation of the rat endothelin receptors. A, comparison of the structure of the two endothelin receptors, ETAR and ETBR. Amino acids that are identical in both receptors (open circles), different in both receptors (closed circles), similar to each other (shaded circles), only in ETAR (doubled circles), and only in ETBR (circles with balck dots), are indicated. The sites exchanging the third cytoplasmic loop (CL3) and the cytoplasmic tail (C tail) are indicated by arrowheads. The site with the truncated cytoplasmic tail in its receptors is indicated (CT). B, comparison of the amino acid sequences of the third cytoplasmic loop and cytoplasmic tail of ETAR and ETBR. The amino acids that are identical in the two receptors are shaded. The truncation site of the cytoplasmic tail is indicated by an arrow. C, restriction maps for cDNAs encoding rat ETAR and ETBR. Restriction sites introduced by polymerase chain reaction are underlined. The regions corresponding to the transmembrane domains are represented as solid bar.

View larger version (25K): [in this window] [in a new window]   Fig. 2.   Expression, ligand binding, and signal transduction of ETAR, ETBR, and their derivatives. A, Northern blot analysis of L tk cells transfected with cDNAs encoding ETAR and their derivatives as indicated using probes synthesized with cDNAs encoding EGFP (upper panel) or XhoI/SfuI fragment (Fig. 1C) of rat ETAR (lower panel). B, Northern blot analysis of L tk cells transfected with cDNAs encoding ETBR and their derivatives as indicated using probes synthesized with cDNAs encoding EGFP (upper panel) or XhoI/EcoRV fragment (Fig. 1C) of rat ETBR (lower panel). C, ligand binding property of ETAR and ETAR-EGFP. L tk cells were transfected with cDNA encoding ETAR (closed symbols) or ETAR-EGFP (open symbols). Transfected cells were incubated with 125I-ET-1 in the presence of various concentrations of cold ET-1 (circles) or ET-3 (squares). Bound radioactivity was measured and estimated as percent of radioactivity obtained from binding of 125I-ET-1 without cold ligand. Values are means ± S.E. of four independent experiments performed in duplicate. D, ligand binding property of ETBR and ETBR-EGFP. L tk cells were transfected with cDNA encoding ETBR (closed symbols) or ETBR-EGFP (open symbols). Transfected cells were incubated with 125I-ET-1 in the presence of various concentrations of cold ET-1 (circles) or ET-3 (squares). Bound radioactivity was measured and estimated as percnet of radioactivity obtained from binding of 125I-ET-1 without cold ligand. Values are means ± S.E. of four independent experiments performed in duplicate. E, intracellular Ca2+ mobilization mediated by ETAR and its derivatives. L tk cells were transfected cDNAs as indicated, together with 1B adrenergic receptor, and stimulated with ET-1 (100 nM). The increment of intracellular Ca2+ concentration is estimated as percent of maximal response stimulated with noradrenaline (10 µM). Values are means ± S.E. of four independent experiments. F, intracellular Ca2+ mobilization mediated by ETBR and its derivatives. L tk cells were transfected cDNAs as indicated, together with 1B adrenergic receptor, and stimulated with ET-1 (100 nM). The increment of intracellular Ca2+ concentration is estimated as percent of maximal response stimulated with noradrenaline (10 µM). Values are means ± S.E. of four independent experiments.

View larger version (57K): [in this window] [in a new window]   Fig. 3.   Subcellular localization of ETAR-EGFP, ETBR-EGFP, and their derivatives. L tk cells were transfected with ETAR-EGFP or its derivatives (A, C, E, and G) or ETBR-EGFP or its derivatives (B, D, F, and H). Each ETR-EGFP, in which the cytoplasmic tail is exchanged with each of the others, is represented as CT (C and D). Each ETR-EGFP, in which the third cytoplasmic loop is exchanged with each of the others, is represented as CL3 (E and F). Each ETR-EGFP, in which the cytoplasmic tail is truncated, is represented as CT (G and H). Rat clone 9 cells (I and J) or HeLa cells (K and L) were also transfected with ETAR-EGFP (I and K) or ETBR-EGFP (J and L). Forty-eight hours after transfection, all cells were fixed with 4% paraformaldehyde in PBS for 1 h at room temperature, and fluorescence of EGFP was detected by laser-scanning confocal microscopy. Each of the results was reproduced at least three times.

View larger version (85K): [in this window] [in a new window]   Fig. 4.   Immunoblotting of lysates from L tk cells transfected with ETAR, ETBR, or their derivatives. Lysates from L tk cells transfected with ETAR (lane 1); ETAR-EGFP (lane 2); ETAR-EGFP, in which the cytoplasmic tail (ETAR (BCT)-EGFP, lane 3), the third cytoplasmic loop (ETAR (BCL3)-EGFP, lane 4), or both (ETAR (BCL3/BCT)-EGFP, lane 5) are exchanged with those of ETBR; ETAR-EGFP, in which the cytoplasmic tail is truncated (ETAR (CT)-EGFP, lane 6); EGFP (lane 7); ETBR (lane 8); ETBR-EGFP (lane 9); ETBR-EGFP, in which the cytoplasmic tail (ETBR (ACT)-EGFP, lane 10), the third cytoplasmic loop (ETBR (ACL3)-EGFP, lane 11), or both (ETBR (ACL3/ACT)-EGFP, lane 12) are exchanged with those of ETAR or ETBR-EGFP, in which the cytoplasmic tail is truncated (ETBR (CT)-EGFP, lane 13), were extracted and subjected to SDS-PAGE followed by electrical transfer to polyvinylidene difluoride membrane and detected with polyclonal rabbit anti-GFP antibody. Each of the results was reproduced at least three times.

These observations suggest two alternative possibilities for the role of the cytoplasmic tail of the ETRs. One is that ET_BR has a determinant for internalization and degradation in its cytoplasmic tail. The other is that the cytoplasmic tail of ET_AR has a sequence responsible for its anchoring to the plasma membrane. To discriminate between these possibilities, we deleted the cytoplasmic tail of each receptor (ET_AR (CT)-EGFP and ET_BR (CT)-EGFP). Because recent reports had demonstrated that several Cys residues proximal to the membrane within the cytoplasmic tail (amino acids 385-388 in ET_AR and 401-404 in ET_BR) are palmitoylated and necessary for signal transduction (16, 22-24), we truncated ET_AR and ET_BR from the C terminus up to Ser³⁹¹ and Thr⁴⁰⁶, respectively (Fig. 1, A and B). The deletion of the cytoplasmic tail led to localization to intracellular vesicular structures (Fig. 3, G and H) and complete degradation (Fig. 4, lanes 6 and 13) of both receptors. These data indicate that ET_AR has a signal responsible for its anchoring to the plasma membrane within its cytoplasmic tail.

ET_BR-EGFP Is Delivered to Lysosomes and Degraded-- Because ET_BR-EGFP was localized in intracellular vesicular structures and was highly degraded, it is reasonable to speculate that the structures to which ET_BR-EGFP is delivered are lysosomes. To confirm this speculation, rat Clone 9 cells and HeLa cells transiently expressing ET_BR-EGFP were stained with antibodies against a lysosomal marker, lysosome-associated membrane protein-1 (Lamp-1). As shown in Fig. 5, the signal of ET_BR-EGFP was superimposed almost completely on the staining for Lamp-1 in both cell lines. To corroborate this observation, L tk cells transfected with ET_BR-EGFP were treated with bafilomycin A₁, which is known to inhibit specifically vacuolar type H⁺-ATPase and thereby inhibit lysosomal proteases by raising the pH of intracellular acidic compartments, including lysosomes (25). As shown in Fig. 6, the degradation of ET_BR-EGFP was significantly suppressed by the treatment of the transfected L tk cells with bafilomycin A₁, whereas ET_AR-EGFP was not affected by the treatment (Fig. 6, lanes 1 and 2). These results indicate that ET_BR is sorted to lysosomes and undergoes proteolytic degradation.

View larger version (19K): [in this window] [in a new window]   Fig. 5.   Colocalization of ETBR-EGFP with Lamp-1 in rat clone 9 and HeLa cells. Rat clone 9 cells (left panels) or HeLa cells (right panels) were transfected with ETBR-EGFP. Forty-eight hours after transfection, clone 9 cells and HeLa cells were fixed, permeabilized, and stained with rabbit anti-rat Lamp-1 antibody and mouse anti-human Lamp-1, respectively. ETBR-EGFP is shown in green. Lamp-1 labeled with Cy3-conjugated goat antibodies against rabbit (for rat clone 9 cells) or mouse (for HeLa cells) IgG are shown in red. Colocalization of ETBR EGFP and Lamp-1 in lysosomes appears as yellow. Each of the results was reproduced at least three times.

View larger version (51K): [in this window] [in a new window]   Fig. 6.   The effect of bafilomycin A1 on proteolytic degradation of ETBR-EGFP in L tk cells. L tk cells were transfected with ETAR-EGFP (lanes 1 and 2) or ETBR-EGFP (lanes 3 and 4). Twenty four hours after transfection, cells were treated with (lanes 2 and 4) or without (lanes 1 and 3) bafilomycin A1 (100 nM) for 12 h, and the cell lysate was subjected to SDS-PAGE followed by immunoblotting using polyclonal anti-GFP antibody. Each of the results was reproduced at least three times.

We needed to determine whether ET_BR-EGFP is delivered directly from the Golgi to lysosomes or whether there is a mechanism whereby this receptor, once delivered to the plasma membrane, is then internalized to lysosomes. To find out, we examined uptake of an antibody against the 16 amino acids in the extracellular N-terminal domain of ET_BR in ET_BR-EGFP-transfected Clone 9 or HeLa cells. Only the cells with the fluorescence of ET_BR-EGFP were recognized by the antibody, confirming its specificity.³ As shown in Fig. 7, the antibody was internalized to intracellular vesicular structures that overlapped, although not completely, with the labeling of ET_BR-EGFP. These observations indicate that at least some fraction of the ET_BR molecules are constitutively internalized from the plasma membrane to lysosomes.

View larger version (40K): [in this window] [in a new window]   Fig. 7.   Endocytosis of ETBR-EGFP. Rat clone 9 cells (left panels) and HeLa cells (right panels) were transfected with ETBR-EGFP for 24 h. Five µg/ml of rabbit anti-peptide antibody against the N terminus of human ETBR (amino acids 27-42) was added during the transfection. Cell-bound and internalized antibody was labeled with Cy3-conjugated goat anti-rabbit IgG. ETBR-EGFP is shown in green. Antibody against the N terminus of human ETBR labeled with Cy3-conjugated goat antibody against rabbit IgG is shown in red. Colocalization of ETBR-EGFP and anti-N terminus antibody appears as yellow. Each of the results was reproduced at least three times.

Endothelin Receptors Are Stabilized by Prolonged Stimulation with ET-1-- We also investigated the effects of prolonged stimulation with the ligand ET-1 on the internalization and degradation of ETRs. Expression levels of mRNA for ET_AR-EGFP or ET_BR-EGFP were not altered by the stimulation with ET-1 for 24 h (Fig. 8B). However, such prolonged stimulation led to a significant increase in the total amount of both ET_AR-EGFP and ET_BR-EGFP proteins (49 and 131%, respectively) (Fig. 8A, lanes 1 and 2 and 5 and 6, respectively), although ET_AR-EGFP as well as ET_BR-EGFP was delivered to lysosomes under these conditions (Fig. 8C). Especially in the case of ET_BR-EGFP, the amount of ~90- and ~65-kDa species increased markedly. In the absence of ET-1, treatment of cells with cycloheximide (10 µM) for 24 h reduced the amount of both ET_AR-EGFP and ET_BR-EGFP (55 and 58%, respectively) (Fig. 8A, lanes 1 and 3 and 5 and 7, respectively). However, ET-1 increased the amount of both receptors, even in these cycloheximide-treated cells (110 and 64%, respectively) (Fig. 8A, lane 3 and 4, and 7 and 8). These results suggest that prolonged stimulation with ET-1 increases the intact ET_AR and ET_BR not by enhancing the expression of these proteins but by stabilizing them.

View larger version (26K): [in this window] [in a new window]   Fig. 8.   Effects of endothelin-1 on localization and stability of endothelin receptors. A, immunoblotting of the lysate from the cells expressing each ETR-EGFP. L tk cells were transiently transfected with ETAR-EGFP (lanes 1-4) or ETBR-EGFP (lanes 5-8). Twenty four hours after transfection, cells were treated with (lanes 2, 4, 6, and 8) or without (lanes 1, 3, 5, and 7) ET-1 (100 nM) for 24 h. Cycloheximide (10 µM) was treated (lanes 3, 4, 7, and 8) simultaneously with ET-1. Each of the results was reproduced at least three times. B, Northern blot analysis of the cells expressing each endothelin receptor. Total RNA extracted from L tk cells transiently transfected with expression constructs of ETAR-EGFP (lanes 1 and 2) or ETBR EGFP (lanes 3 and 4). Twenty four hours after transfection, cells were stimulated with (lanes 2 and 4) or without (lanes 1 and 3) ET-1 (100 nM) for a further 24 h. Each of the results was reproduced at least three times. C, lysosomal localization of endothelin receptors in rat Clone 9 cells stimulated with ET-1. Cells were transiently transfected with ETAR-EGFP (left panels) or ETBR-EGFP (right panels). Twenty four hours after transfection, cells were stimulated with ET-1 (100 nM) for a further 24 h, followed by immunofluorescent staining as described for Fig. 5.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

In the present study, we expressed EGFP-tagged ETRs in various cell lines and examined their subcellular localization. We found that, at steady state, ET_AR was mainly localized on the plasma membrane, whereas ET_BR was localized not only on the plasma membrane but also on lysosomes, where it appeared to be degraded by lysosomal proteases. Furthermore, an antibody uptake experiment suggested that at least some fraction of ET_BR molecules are internalized from the plasma membrane to lysosomes. These observations indicate that ET_BR, once delivered to the plasma membrane, then constitutively internalizes to lysosomes for degradation, although we cannot exclude the possibility that some fractions of ET_BR molecules are directly sorted from the Golgi to lysosomes. Nevertheless, we favor the former possibility because experiments using chimeric and truncated constructs suggest the presence of a specific signal for anchoring of ET_AR to the plasma membrane but not for direct sorting of ET_BR from the Golgi to lysosomes; ET_BR bearing the cytoplasmic tail of ET_AR was mainly localized on the plasma membrane, and not only ET_AR with the ET_BR cytoplasmic tail but also both receptors lacking the cytoplasmic tail are mainly localized on lysosomes. Although the physiological significance of the unique behavior of ET_BR is currently unknown, its fast turnover is in line with previous reports consistently showing that expression of ET_BR mRNA is up- or down-regulated by various stimuli in many cell types and tissues (26-35).

Many GPCRs, such as AT_1A, TSH, CCKBR, NK-1, neurotensin, GRP-R, -opioid, SSTR3, SSTR5, H₂, TXA₂R, and CCR2B receptors (36-47), are known to be impaired in their internalization by truncation of the cytoplasmic tail, although the consensus sequence for the internalization remains to be identified. By contrast, as is the case for µ-opioid receptor (48) and SSTR2 (49), both ETRs lacking the cytoplasmic tail undergo constitutive internalization. Therefore, the determinant for internalization appears to be within a region/regions other than C-terminal 35 amino acids. In the case of muscarinic acetylcholine receptors, the third cytoplasmic loop has been shown to contribute to their internalization (50, 51). Moreover, in the case of ₂-adrenergic receptor, its internalization depends not only on appropriate interactions of multiple molecular determinants within the cytoplasmic regions, including the first and second cytoplasmic loops and the cytoplasmic tail, but also on conformational determinants that may influence their orientation (52). However, our experiments showed that neither the third cytoplasmic loop nor the cytoplasmic tail contributes to the ETR internalization. Further experiments will be required to determine the region(s) responsible for the internalization of the ETRs.

In contrast to ET_BR that constitutively internalized to lysosomes, ET_AR is internalized to lysosomes in an agonist-dependent manner. It is therefore likely that ET_AR has a sequence within its cytoplasmic tail that prevents the receptor from constitutive internalization. However, we cannot rule out the possibility that the cytoplasmic tail of ET_BR also contains a sequence that decelerates the internalization. Many GPCRs are known to recycle between the plasma membrane and intracellular compartments (8-10). In the case of V₂ vasopressin receptor, the signal responsible for the recycling has been shown to be within its cytoplasmic tail (53). Receptors without such a motif once internalized seem to be sorted to lysosome (13). Neither ET_AR nor ET_BR seem to possess such a recycling motif, because both stay in lysosomes, once internalized.

When continuously exposed to ET-1, both ET_AR and ET_BR were stabilized although they were localized to lysosomes. Especially in the case of ET_BR, which is highly degraded at steady state, the amount of ~90- and ~65-kDa species was significantly increased by agonist stimulation. Previous studies have reported that, in various tissues, ET_BR exists not only as a 52-kDa intact species but also as a 34-kDa species that is generated through cleavage in the N-terminal extracellular domain by a metalloprotease(s) (54-56). These two species have been shown to be able to bind ligands with high affinity (54). Taking into account the molecular mass of the EGFP portion (27 kDa) and heterogeneity in glycosylation, it is likely that the 90- and 65-kDa species of ET_BR-EGFP detected in our experiments correspond to the 52- and 34-kDa species, respectively, detected in tissues. These ET_BR species have been shown to form a very stable complex with ET in vitro, even in the presence of high concentrations (up to 2%) of SDS (56). Chun et al. (19) reported that cell surface ET_AR that binds ET-1 undergoes internalization and also forms a stable complex with ET-1 for at least 2 h and that ET_AR and bound ET-1 are localized in caveolae (57). These may be implicated in the production of the long lasting responses evoked by ET in vivo, such as contraction of smooth muscles and elevation of blood pressure (1). Although we did not show the localization of endothelin receptors in caveolae, our observations on the agonist-dependent stabilization of both ET_AR and ET_BR make it tempting to speculate that ET evokes such long lasting cellular responses, at least in part, through stabilizing its receptors.

	ACKNOWLEDGEMENTS

We thank Dr. Kenji Akasaki for providing anti-Lamp-1 antibody and Dr. Robert J. Lefkowitz for providing pcDV1R_1B; Dr. Ikuo Nishimoto for critical reading of this manuscript; Dr. Dovie Wylie for help with preparation of the manuscript; and Miki Kiuchi for excellent technical assistance.

	FOOTNOTES

^* This work was supported in part by research grants from the Ministry of Education, Science and Culture of Japan, from the University of Tsukuba Research Project, and from the Japan Society for the Promotion of Science (JSPS-RFTF96I00202).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Present address: Dept. of Pharmacology and Neuroscience, KEIO University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan.

^¶ To whom correspondence should be addressed: Dept. of Pharmacology, Institute of Basic Medical Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8575, Japan. Tel.: 81-298-53-3277; Fax: 81-298-53-3039; E-mail: kgoto@md.tsukuba.ac.jp.

² Y. Abe, T. Sakurai, T. Yamada, T. Nakamura, M. Yanagisawa, and K. Goto, submitted for publication.

³ Y. Abe, K. Nakayama, A. Yamanaka, T. Sakurai, and K. Goto, unpublished observations.

	ABBREVIATIONS

The abbreviations used are: ET-1, endothelin-1; ET-2, endothelin-2; ET-3, endothelin-3; ET_AR, endothelin-A receptor; ET_BR, endothelin-B receptor; GPCR, G protein-coupled receptor; EGFP, enhanced green fluorescent protein; DMEM, Dulbecco's modified Eagle medium; Lamp-1, lysosome-associated membrane protein-1; CT, cytoplasmic tail; CL, cytoplasmic loop; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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