©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Endothelin-induced Endocytosis of Cell Surface ET Receptors
ENDOTHELIN REMAINS INTACT AND BOUND TO THE ET RECEPTOR (*)

Miyoung Chun (1)(§), Herbert Y. Lin (1) (2)(¶), Yoav I. Henis (1)(**), Harvey F. Lodish (1) (2)(§§)

From the (1) Whitehead Institute for Biomedical Research, Cambridge, Massachusetts 02142 and the (2) Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

We demonstrate unusual features of the intracellular processing of endothelin-1 (ET-1) and its receptor ET, the receptor subtype that mediates contraction of vascular smooth muscle cells. First, we show that in stably transfected CHO cells expressing ET, binding of an ET-1 ligand induces rapid endocytosis of cell surface ET. Receptor endocytosis was measured both by immunofluorescence and by radioiodinated antibodies specific for ET. Second, we demonstrate that ET-1 remains intact for up to 2 h after endocytosis and, as judged by co-immunoprecipitation, internalized I-ET-1 remains bound to ET receptors. We hypothesize that internalized ET-1, bound to ET receptors, continues to activate a signal-transducing G protein, thus accounting for the prolonged period of contraction induced in smooth muscle cells by a single administration of ET-1.


INTRODUCTION

Endothelin-1 (ET-1),() a 21-amino acid peptide with two intramolecular disulfide bonds, is the most potent vasopressor agent yet discovered (1) . Initially isolated from porcine aortic endothelial cells, it induces long-lasting hypertension; a single injection (1 nmol/kg) into the circulation of a rat increases blood pressure for several hours (2) . ET-1 and its isoforms ET-2 and ET-3 have numerous biological actions in vitro and in vivo, including hemodynamic, cardiac, renal, neuroendocrine, smooth muscle contraction, and protomitogenic effects (reviewed in Refs. 3 and 4).

These diverse responses are mediated by at least three distinct ET receptors which differ in their relative binding affinities to the three ET isoforms, ET, ET, and ET(5, 6, 7, 8, 9, 10, 11, 12, 13) . These receptors have seven putative membrane-spanning segments and are coupled to G proteins. ET receptors are widely expressed but distributed differently in tissues where they show pharmacological effects (6, 14, 15, 16) , such as the vascular system, brain, kidney, lung, and adrenal glands (5, 7, 8, 10, 11, 12, 13) . Importantly, ET but not ET mRNA is expressed in rat vascular smooth muscle A10 cells (5) and on rat aorta stripped of endothelial cells (10, 11) . Thus, the ET receptor mediates vascular constriction, although in some blood vessels, such as rabbit saphenous vein, ET appears to mediate vasoconstriction (17, 18) .

Agonist-induced receptor desensitization is a common feature of G-protein-coupled signal transduction systems (reviewed in Ref. 19). One type of receptor desensitization is characterized by phosphorylation of serine and threonine residues in the third cytosol-facing loop and the carboxyl-terminal segment of the receptor, reducing the ability of the receptor to interact with G proteins and thus causing a diminished responsiveness to agonist stimulation. The best-studied example is the -adrenergic receptor (20, 21) ; continued exposure of cells to -agonists causes an attenuation of signal transduction due to phosphorylation of the -adrenergic receptor by both the cAMP-dependent protein kinase (22) and the -adrenergic receptor kinase (23) . This desensitization is essentially complete within minutes. In contrast, activation of the ET receptor on smooth muscle cells causes contraction which lasts over 2 h, resulting in a sustained increase in blood pressure (24) . The cellular mechanisms which underlie the long-lasting vasopressor effects of endothelins, however, are not understood. In this paper, we suggest one mechanism for the protracted action of ET which relates to the unusual intracellular processing of the receptor following endocytosis.

During receptor-mediated endocytosis, ligands bound to their receptors are internalized via clathrin-coated pits and vesicles (reviewed in Refs. 25 and 26). Most ligands are subsequently degraded in lysosomes (27, 28, 29, 30, 31) , whereas the fate of the internalized receptors varies. Some, such as epidermal growth factor receptors, are degraded together with their bound ligand (29) while others are recycled back to the cell surface after intracellular dissociation of their ligand (32, 33, 34, 35, 36) . Following binding of ligand, -adrenergic receptors are internalized by endocytosis (20, 37) . However, the fate of the ligand is unknown.

Here, we demonstrate unusual features of the intracellular processing of ET-1 and its receptor ET. First, we show that ET-1 binding induces rapid endocytosis of ET. Second, we demonstrate that ET-1 remains intact for up to 2 h after endocytosis, and that internalized ET-1 remains bound to an ET receptor. We hypothesize that internalized ET-1, bound to ET receptors, continues to activate a transducing G protein, thus accounting for the prolonged period of contraction induced in smooth muscle cells by a single administration of ET-1.


EXPERIMENTAL PROCEDURES

Materials

Rabbit anti-ET polyclonal antibody 291, an antipeptide antibody specific for the extracellular amino-terminal domain of ET, was described previously (38) .

Cell Culture and Transfection

The generation and maintenance of stably transfected CHO cell lines expressing ET were described previously (5) . Briefly, CHO cells were co-transfected with pcDNA-3 (39) and the ET cDNA in the vector pcDNA-1 (5) using the calcium phosphate precipitation technique. Two days after transfection, cells were plated in selective medium containing 500 µg/ml G418 (Life Technologies, Inc.). G418-resistant colonies were selected and amplified by subcloning single colonies. Two clones were selected for further studies. CHET-B (5) was used for the experiments in Figs. 1 and 2. The other cell line, CHO ET28, was used for the experiments in Figs. 3 and 4 and Tables I and II. CHET-B or CHO ET28 cells express 10 cell surface ET binding sites per cell (5) .()

Measurement of Internalization of Cell Surface ET Receptors by Immunofluorescence

Live CHO cells, either nontransfected or stably transfected with the ET cDNA, were grown on a coverslip, washed twice with ice cold phosphate-buffered saline (PBS), then once with HH/BSA buffer (Hank's balanced buffer also containing 10 mM Hepes, pH 7.4, and 2% bovine serum albumin (BSA)). The cells were then incubated successively on ice (1 h, with 3 washes between incubations) in HH/BSA with: (i) 20 µg/ml normal goat IgG (Sigma or The Jackson Laboratory); (ii) 20 µg/ml anti-ET 291 IgG; (iii) 50 µg/ml fluorescein isothiocyanate-conjugated goat anti-rabbit IgG (The Jackson Laboratory). The cells were then split into two sets. One set was further incubated with 50 pM (unlabeled) ET-1 at 4 °C for 2 h overnight, followed by washing with PBS to remove excess ET-1. The other set of cells was similarly treated except no ET-1 was added. The cells were then warmed to 37 °C with 1 ml of PBS containing 11 mM glucose for various times (0-180 min), fixed in PBS containing 2% paraformaldehyde for 20 min at room temperature, and washed extensively with PBS. The fixed cells were mounted with Slow-Fade antifade reagent (Molecular Probes) and viewed under a Bio-Rad MRC600 confocal fluorescence microscope.

Kinetics of Internalization of Cell Surface ET Receptors

A10 cells and CHO cells stably transfected with ET were used for this study; each data point was done in triplicate. Live cells, grown in 35-mm culture dishes, were incubated successively in 4 °C (1 h each incubation) with 20 µg/ml anti-ET IgG followed by I-labeled goat anti-rabbit IgG (0.2 µCi/ml in PBS, Amersham), then washed again three times with PBS. The cells were divided into two groups. One set was further incubated with 50 pM ET-1 at 4 °C for 2 h, and the other was similarly incubated without ET-1. The medium was removed, and both sets of cells were then incubated with 1 ml of PBS containing 11 mM glucose at 37 °C for various times (0-180 min) to allow endocytosis. After collecting the medium, the cells were incubated with 1 ml of 50 mM glycine, pH 2.5, three times for 10 min each at 4 °C, and the acid solutions were pooled; this fraction is referred to as surface-bound IgG. Consistently, in cells not incubated at 37 °C, more than 95% of surface-bound IgG was removed by this acid wash procedure. The cell pellet was dissolved in 1 ml of 1 N NaOH at room temperature; this fraction contains the internalized IgG which is resistant to the acid wash. The amount of I-IgG from the medium, cell surface, and cell pellet were measured using a liquid scintillation counter.

Kinetics of Internalization of Cell Surface ET-1

Cells were incubated with 50 pMI-ET-1 in PBS at 4 °C for 2 h to overnight, washed with PBS, then incubated as above at 37 °C at various times (0-180 min). The medium was collected. As above, the cells were acid-washed and then dissolved in 1 N NaOH, yielding cell surface and internalized I-ET-1. The amount of I-ET-1 from the medium, cell surface, and cell pellet was measured.

Characterization of I-ET-1 by High Performance Liquid Chromatography (HPLC)

CHO cells transfected with ET, grown in 35-mm tissue culture dishes, were incubated with 50 pMI-ET-1 in PBS at 4 °C for 2 h, then washed extensively with PBS. As above, the cells were warmed to 37 °C for various times (0-180 min). Cell surface-bound I-ET-1 was removed by the acid-wash procedure. The cells were then dissolved in 1 ml of 5% acetic acid, and the cell supernatant, containing internalized I-ET-1, was lyophilized and redissolved in a 50 µl of 0.1% trifluoroacetic acid. I-ET-1 was then analyzed by HPLC using a C18 column and a linear gradient of 100% solvent A (0.1% trifluoroacetic acid) to 100% solvent B (90% CHCN in solvent A). Fractions were collected every 30 s of the 50-min run, and the radioactivity of each was measured.

Ammonium Sulfate Precipitation of Internalized I-ET-1

CHO cells either nontransfected or transfected with ET cDNA were labeled with 50 pMI-ET-1 in PBS at 4 °C for 2 h, followed by incubation at 37 °C (0-180 min) as above. Surface-bound I-ET-1 was removed by the acid wash procedure. The cells were solubilized in 50 mM Tris buffer, pH 7.4, containing 1% Nonidet P-40 (Sigma), 1 mg/ml BSA, and 1 nM unlabeled ET-1. After low-speed centrifugation, the cell supernatant was incubated with 60% ammonium sulfate for 30 min on ice. The precipitated I-ET-1 was collected on a Millipore filter (0.2 µm pore size) followed by extensive washing with PBS, and the radioactivity on the filters was measured.

Immunoprecipitation of Internalized I-ET-1 by an ET Antibody

CHO cells either nontransfected or transfected with ET cDNA were labeled with 50 pMI-ET-1 in PBS at 4 °C for 2 h and incubated 0-180 min at 37 °C, and the surface-associated ligand was removed by an acid wash as above. The cells were solubilized at 4 °C with 1 ml of TBST (10 mM Tris, pH 8.0, 0.15 M NaCl, 1% Triton X-100, 2 mM phenylmethylsulfonyl fluoride) containing 60 mM octyl glucoside. After low-speed centrifugation, the cell supernatant was incubated for 30 min at 4 °C with 50 µg/ml rabbit anti-ET IgG. This rabbit anti-ET IgG had been reacted with 5 µg/ml Protein A-Sepharose (Sigma) overnight before it was added to the cell supernatant. The Protein A-Sepharose was collected by centrifugation and washed four times with TBST. The radioactivity in the recovered I-ET-1ETET antibody complex was measured using a liquid scintillation counter.


RESULTS

ET-1 Induces Internalization of Cell Surface ET

To determine whether cell surface ET undergoes endocytosis in CHO cells stably transfected with ET cDNA, we used polyclonal anti-peptide antibody 291 specific for the extracellular amino-terminal domain of ET. Antibody 291 is an excellent probe for receptor internalization because it binds to receptors on live cells and does not inhibit binding of radioiodinated ET-1 (data not shown; see below). We characterized extensively the specificity of this antibody: as judged by immunofluorescence it detected ET receptors in ET-transfected, but not untransfected live, nonpermeabilized, CHO cells, consistent with the proposed ectodomain topology of the amino terminus. Moreover, the binding of Ab 291 to ET transfected cells was inhibited by 1 µg/ml (100) concentration of the immunizing peptide but not of irrelevant peptides (data not shown).

Internalization of cell surface ET was first monitored by immunofluorescence. To assess internalization without ET-1 ligand, cells were incubated with Ab 291 for 1 h at 4 °C, then with fluorescein isothiocyanate-conjugated goat anti-rabbit IgG. Then the cells were warmed to 37 °C for various times (0 to 180 min) to allow endocytosis, fixed with paraformaldehyde, and visualized by confocal fluorescence microscopy. Fig. 1 a shows the distribution of ET before incubation at 37 °C. The bright staining of the cell periphery ( arrows) is indicative of cell surface labeling. This pattern did not change up to 3 h at 37 °C, suggesting that the labeled antibodies remain at or near the cell surface (Fig. 1 b and data not shown). These data indicate that, in the absence of ET-1, the ET receptor is not internalized.


Figure 1: ET-1 induced internalization of cell surface ET. Live CHO cells, stably transfected with ET cDNA, were incubated at 4 °C with anti-ET IgG followed by fluorescein isothiocyanate-conjugated goat anti-rabbit IgG (see ``Experimental Procedures''). The cells were further incubated either without ET-1 ( a and b) or with 50 pM ET-1 ( c and d) at 4 °C for 2 h, followed by extensive washing to remove excess ET-1. Then the cells were warmed to 37 °C for 0 min ( a and c), 30 min ( b), and 10 min ( d) and then fixed and visualized by confocal fluorescence microscopy. Shown here are the optical scans taken 4 µm above the surface of the slide. Bar represents 5 µm.



Endocytosis of ET does occur following addition of ET-1, as illustrated in Fig. 1 d. After incubation of CHO cells, stably transfected with ET with Ab 291 and fluorescein isothiocyanate-conjugated secondary Ab, ET-1 was added for 2 h at 4 °C. Subsequently, the cells were warmed to 37 °C for 0-180 min, fixed, and visualized by confocal microscopy. Without incubation at 37 °C, the labeled antibody remained on the cell surface (Fig. 1 c). However, after a 10-min incubation at 37 °C, a marked change in receptor distribution was observed (Fig. 1 d). Cell surface staining was dramatically reduced ( closed arrows), and punctate staining of antigen appeared throughout the cytoplasm ( open arrows). These data indicate that ET-1 induces internalization of cell surface ET receptors.

To confirm the immunofluorescence results quantitatively and to obtain actual rates of endocytosis, we directly determined the kinetics of endocytosis of ET-1 and ET using I-labeled goat anti-rabbit IgG (to monitor ET) or I-labeled ET-1 (to monitor ET-1) and an acid-stripping procedure to distinguish surface-bound antibody or ET-1 from internalized ones (see ``Experimental Procedures''). As shown in Fig. 2 a, in the absence of ET-1, less than 10% of I-labeled IgG bound to cell surface ET receptors are internalized even after a 3-h incubation at 37 °C. Most of the I-labeled IgG is recovered in the medium, but we have not investigated the properties of this material. On the other hand, I-ET-1 is internalized rapidly (Fig. 2 b). After 10 min of incubation at 37 °C, 35% of surface-bound I-ET-1 has become internalized rising to 80% by 1 h at 37 °C. We were concerned that overexpression of ET in stably transfected CHO cells might somehow cause endocytosis of bound ET-1. However, Fig. 2 d shows the same rate and extent of endocytosis of surface-bound I-ET-1 by vascular smooth muscle A10 cells, which express the ET receptor endogenously.


Figure 2: Kinetics of internalization of I-anti-ET IgG and I-ET-1. The cells used were CHO cells transfected with ET ( a, b, and c) or A10 cells ( d). They were labeled at 4 °C with anti-ET IgG followed by I-goat anti-rabbit IgG ( a and c) or with 50 pMI-ET-1 ( b and d), as described under ``Experimental Procedures.'' In a and c, an additional incubation (2 h, 4 °C) was performed with ( c) or without ( a) 50 pM unlabeled ET-1. The cells were then incubated at 37 °C up to 180 min. For each time point, the percentage of I-labeled IgG or ET-1 in the medium ( open circles), at the cell surface ( open triangles), and inside the cells ( closed squares) was measured, using the acid-stripping procedure (see ``Experimental Procedures'') to differentiate surface-bound from internalized radioactivity. Each point is the average of triplicate measurements of two ( a, b, and c) or three ( d) independent experiments. There was no more than 5-10% variation among experiments. a, internalization of I-IgG bound to cell surface ET in CHO cells stably transfected with ET. b, internalization of I-ET-1 in CHO cells expressing ET. c, internalization of I-IgG bound to cell surface ET in CHO cells expressing ET, ET-1 was present during incubation at 37 °C. d, internalization of I-ET-1 in A10 cells.



A comparison of the experiments in a and c of Fig. 2shows directly that addition of unlabeled ET-1 causes the internalization of cell surface ET. In the experiment in c, live CHO cells transfected with ET were incubated with Ab 291 at 4 °C for 1 h and then, as with those used in a, with I-IgG at 4 °C for 1 h. Unbound I-IgG was removed, and the cells were incubated with ET-1 (50 pM) for 2 h at 4 °C. After excess unbound ET-1 was removed, the cells were warmed to 37 °C. In contrast to the results in a, endocytosis of I-IgG complexes to cell surface ET was rapid and efficient. Importantly, the rate and the maximal extent of internalization of I-IgG was almost identical with the rate and the maximal extent of internalization of I-ET-1 (compare b and c of Fig. 2 ). A comparison of a and c of Fig. 2 establishes that ET-1 induces the internalization of cell surface ET. These experiments confirm the results of Fig. 1 and demonstrate that ET-1 induces a rapid internalization of cell surface ET receptors. Over 80% of both cell surface ET receptors and surface-bound ET-1 become internalized within a 1-h incubation at 37 °C.

ET-1 Remains Intact 2 h after Endocytosis

To determine whether ET-1 remains intact following receptor-mediated endocytosis, I-ET-1 was incubated with ET-expressing CHO cells, and internalized I-ET-1 was analyzed by HPLC (Fig. 3). Specifically, I-ET-1 was allowed to bind to the cells at 4 °C; after removal of unbound ligand, the cells were then warmed to 37 °C for various times. Surface-bound I-ET-1 was removed by an acid wash, then the cells were dissolved in 5% acetic acid. The cell supernatant, containing internalized radiolabeled ET-1, was lyophilized, redissolved in 0.1% trifluoroacetic acid, and analyzed using a C18 HPLC column.


Figure 3: Analysis of internalized I-ET-1 by high performance liquid chromatography. a, I-ET-1 standard. b, I-ET-1 bound to the surface of CHO cells transfected with ET. The cells were incubated with 50 pMI-ET-1, dissolved, and analyzed by HPLC as described under ``Experimental Procedures.'' c, internalized I-ET-1 after 20 min at 37 °C. ET-expressing CHO cells were labeled as in b and warmed to 37 °C for 20 min. After acid-stripping to remove the surface-associated ligand, they were dissolved and analyzed by HPLC. d, internalized I-ET-1 after 120 min at 37 °C. The cells were treated as in c, except that the incubation at 37 °C was for 120 min.



An I-ET-1 standard was run on the same HPLC column; fractions 61 to 65 contained virtually all of the radioactivity (Fig. 3 a, arrow) and therefore represent intact I-ET-1. I-ET-1 bound to cell surface receptors at 4 °C (without incubation at 37 °C or an acid wash) migrated identically with the ET-1 standard (Fig. 3 b, arrow). After a 20-min incubation at 37 °C, 90% of the internalized ( i.e. resistant to the acid wash) I radioactivity co-migrated with the ET-1 standard (Fig. 3 c, arrow). A small peak that eluted earlier contained the remaining 10% of the radioactivity (Fig. 3 c, open arrow). Two hours after warming to 37 °C, more than 30% of the internalized I-ET-1 co-migrated with the I-ET-1 standard, indicating that 30% of internalized ET-1 remained intact (Fig. 3 d, arrow). Several peaks of radioactive material eluted earlier from the column (Fig. 3 d, open arrows), indicating that some degradation had occurred. Therefore, internalized ET-1 remained almost fully intact after 20 min at 37 °C (Fig. 3 c), and, even after 2 h of internalization, more than 30% of ET-1 was intact (Fig. 3 d).

ET-1 Remains Bound to ET 2 h after Endocytosis

We utilized two techniques to show that the majority of internalized ET-1 is still bound to ET: ammonium sulfate precipitation of receptor-ligand complexes (), and immunoprecipitation of internalized I-ET-1 by an antibody specific to ET (). In both experiments, nontransfected (control) and ET-transfected CHO cells were incubated with 50 pMI-ET-1 at 4 °C for 2 h, warmed to 37 °C for various times, then surface-bound I-ET-1 was removed by an acid wash. The cells were solubilized in a detergent solution, and, after clarification by low-speed centrifugation, receptor-ligand complexes were quantified.

In the first study, the cell supernatant was brought to 60% ammonium sulfate and incubated on ice for 30 min before filtration. Unbound I-ET-1 does not precipitate under these conditions. However, as shown in , 20 min after endocytosis, 50% of the total radioactivity from internalized I-ET-1 is recovered in the precipitate, and, even 2 h after internalization, more than 30% of the internalized I-ET-1 is precipitated. Thus, a significant fraction of internalized ET-1 remains bound to some protein, presumably to the ET receptor, even 2 h after internalization. Note that, as an additional control, no significant amount of I-ET-1 is recovered in extracts of control, nontransfected, CHO cells.

To confirm the binding of ET-1 to the ET receptor following endocytosis, immunoprecipitation of the ligand-receptor complexes was performed (). The cell supernatant was incubated with antibody 291, specific for the ET receptor, and immunocomplexes were recovered using Protein A-Sepharose beads. shows that 20 min after endocytosis 55% of the total radioactivity from internalized I-ET-1 is recovered in the anti-ET immunoprecipitate, and more than 25% of the internalized I-ET-1 is immunoprecipitated after 120 min at 37 °C. At 120 min of endocytosis, 30% of internalized I-ET-1 is intact (Fig. 3), and 25-30% of the radioactivity from internalized I-ET-1 is bound to ET receptors (Tables I and II). We conclude that, at this time, all of the internalized, intact I-ET-1 is bound to an ET receptor. It is possible that the intracellular ET-1ET complexes continue to activate G proteins.


DISCUSSION

Contraction and relaxation of smooth muscle cells that line the arteries is one of the major ways by which blood pressure is regulated, and these cells are affected by a number of competing vasodilator and vasoconstrictor hormones. The long term vasopressor effect of ET-1 (24) is one important aspect of this regulatory system, and, therefore, it is necessary to have a molecular and cellular understanding of how a single small injection of ET-1 into the circulation of a rat increases its blood pressure for several hours. This effect is likely to be mediated via ET, the ET receptor subtype expressed in vascular smooth muscle cells and thought to play critical roles in regulating vascular resistance and the distribution of blood flow (5, 10, 11) . For these reasons, we have studied the intracellular fate of ET receptors, its dependence on ligand (ET-1), and the state of the ET-1ET complexes after internalization.

The most important results of this paper are that binding of ET-1 to cell surface ET receptors induces endocytosis of ET-1 ET complexes, and that 30% of internalized ET-1 remains intact and tightly bound to ET receptors even 2 h after endocytosis. We hypothesize that internalized ET-1ET complexes might continue to activate a transducing G protein, thus inducing prolonged contraction in smooth muscle cells. First, we showed in transfected CHO cells that binding of an ET-1 ligand induces rapid endocytosis of cell surface ET. We measured receptor endocytosis both by immunofluorescence and by radioiodinated antibodies specific for ET. Second, we demonstrated that 30% of internalized I-ET-1 remains intact for up to 2 h after endocytosis, as judged by HPLC. Third, as shown by co-immunoprecipitation, internalized, intact I-ET-1 remains bound to an ET receptor. A potential limitation of transfected cells expressing a high number of receptors is that they may not regulate receptors in the same way as smooth muscle cells. Thus, we repeated these experiments in A10 cells which endogenously express ET. There was no difference between CHO and A10 cell lines in the kinetics of ET-1-induced ET endocytosis (Fig. 2 d). These experiments examine redistribution of only cell surface receptors; thus, these findings suggest that redistribution is mediated by an existing pool of cellular proteins, not by newly synthesized receptors.

Interestingly, 90% of internalized ET-1 remains intact 20 min following endocytosis (Fig. 3). At this time, however, only 50% of the radioactivity from internalized I-ET-1 is bound to the ET receptor, and, thus, a significant amount of internalized ET-1 is free. Two hours following internalization, however, about 30% of internalized ET-1 remains intact, and 25-30% of I radioactivity from internalized ET-1 is bound to ET receptors (Tables I and II). Thus, at 2 h after internalization, all intact ET-1 is bound to ET receptors, and all unbound ET-1 has been degraded. The slow dissociation of internalized ET-1 from ET receptors is consistent with our kinetic studies of the binding of ET-1 to ET. At pH 7, the dissociation of I-ET-1 from cell surface ET receptors, in transfected CHO cells, was extremely slow, with a half-time of about 2 h at 4 °C. Significantly, the rate of dissociation of I-ET-1 from ET was the same at pH values as low as 4.0 (data not shown). The internal pH of endosomes is thought to be between 5.5 and 6.0, and that of lysosomes about 4.5 to 5.5 (reviewed in Ref. 40). Thus, at any pH value likely to be found in internal endosomes, ET-1 should remain bound to ET receptors.

The finding that a significant amount of intact ET-1 is still bound to ET even hours after endocytosis gives rise to the hypothesis that it may transduce signals even after internalization. In such a case, internalized ET-1 ET complexes should be able to interact with downstream components in the ET-1 signal transduction pathway. This pathway involves activation of phospholipase C, protein kinase C, Phospholipase A, opening of non-selective membrane cation channels, L-type Ca channels, and cAMP-dependent protein kinase-dependent Cl channels (41, 42, 43, 44, 45, 46) . It is not known which, if any, of these proteins co-localize to endosomes containing internalized ET-1 ET complexes. Some insight into this issue derives from our recent demonstration that plasma membrane ET receptors, together with bound ET-1 ligand, are localized to caveolae (38) .

Caveolae are non-clathrin-coated invaginations of the plasma membrane. In different cell types, caveolae contain different proteins that participate in signal transduction by G protein-coupled and other receptors (47) . We showed directly that ET endothelin receptors, together with its bound ligand, are found in plasma membrane caveolin-containing complexes (38) . In capillary endothelial cells and visceral smooth muscle cells, two signal-transducing proteins activated by binding of ET-1 to the ET receptor, a calcium channel and an IP receptor, are enriched in plasma membrane caveolae (48, 49) . Caveolae isolated from tissues and cultured cells are enriched in both G and G subunits of heterotrimeric GTP-binding proteins (50) . In particular, G, the subunit of the heterotrimeric G protein that is coupled to ET, is also present in caveolae (47) . Taken together, these results strongly indicate that the endothelin receptor binds its ligand and generates an intracellular signal while localized in plasma membrane caveolae.

Plasma membrane caveolae are thought to pinch off (51) and form endosome-like structures (52) . The ET receptor with its bound ET-1 ligand is likely be incorporated in these vesicles. Other caveolar-localized proteins that participate in signal transduction, such as G, might also be incorporated into these vesicles. G proteins are associated with a variety of intracellular membranes (reviewed in Refs. 53 and 54). In polarized epithelial LLC-PK-1 cells, the G subunit is localized to the cytoplasmic face of Golgi cisternae; it was distributed across the entire Golgi stack (55, 56) . In polarized Madin-Darby canine kidney cells, both the and subunits of G function to control vesicular transport (57, 58) . Although evidence is lacking, G might also be found in internalized caveolae. G could be a resident protein in these internal organelles or it could move to them from the plasma membrane along with ET receptors. Little is known of the nature of internal membranes that contain caveolin and other proteins characteristic of plasma membrane caveolae, and, at this time, we are unsure of the nature of the internal organelles that contain the ET-1ET complexes. Nonetheless, it is reasonable to suppose that signal transduction by the ET receptor occurs in internalized caveolae, accounting for the long-lasting elevation of cytosolic Ca and contraction of smooth muscle cells that occurs following a single addition of ET-1.

  
Table: Ammonium sulfate precipitation of I-ET-1

CHO cells, either nontransfected or transfected with ET cDNA (CHO ET), were incubated with I-ET-1 (2 h, 4 °C) and warmed to 37 °C for 20 or 120 min. After stripping surface ET-1 by an acid wash, the cells were dissolved in detergent. The cell extract were centrifuged at low speed, then the radioactivity in a portion of the cell supernatant was measured (before precipitation). The remaining cell supernatant was incubated with 60% ammonium sulfate for 30 min on ice, and the precipitated I-ET-1 was collected and counted (after precipitation).


  
Table: Immunoprecipitation of I-ET-1ET complex with anti-ET Ab

CHO cells, either nontransfected or transfected with ET cDNA (CHO ET), were incubated with I-ET-1 (2 h, 4 °C) and warmed to 37 °C for 20 min or 120 min. After stripping surface ET-1 by the acid wash procedure (see ``Experimental Procedures''), the cells were dissolved in a buffer containing 1% Triton X-100 and 60 mM octyl glucoside. ET was precipitated by anti-ET antibody and Protein A-Sepharose (see ``Experimental Procedures''). The immunocomplexes were recovered by centrifugation, washed, and counted. In control experiments, less than 1% of I-ET-1 was recovered in the ET immunoprecipitates.



FOOTNOTES

*
This work was funded in part by National Institutes of Health Grant HL-41484 (to H. F. L.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported by a fellowship from the Life Science Research Foundation.

Present address: Program in Membrane Biology, Renal Unit, Massachusetts General Hospital, Boston, MA 02114 and the Dept. of Medicine, Harvard Medical School, Boston, MA 02115.

**
Permanent address: Dept. of Biochemistry, the George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel.

§§
To whom correspondence and reprint requests should be addressed: Whitehead Institute for Biomedical Research, 9 Cambridge Center, Cambridge, MA 02142. Tel.: 617-258-5216; Fax: 617-258-9872. E-mail: lodish@wi.mit.edu.

The abbreviations used are: ET, endothelin; ET, endothelin receptor subtype A; ET, endothelin receptor subtype B; ET, endothelin receptor subtype C; G proteins, heterotrimeric GTP-binding signal-transducing proteins; HPLC, high performance liquid chromatography; PBS, phosphate-buffered saline; BSA, bovine serum albumin; Ab, antibody.

M. Chun and H. F. Lodish, unpublished data.


ACKNOWLEDGEMENTS

We thank Drs. H. E. Ives, E. Lobo, D. Neumann, and U. Liyanage for their invaluable discussions and N. Cohen for preparation of plasmids.


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