From the Division of Genetics, International Center
for Medical Research, Kobe University School of Medicine, Kobe, 6500017 Japan and the ¶ Howard Hughes Medical Institute and Department of
Molecular Genetics, University of Texas Southwestern Medical Center at
Dallas, Dallas, Texas 75235-9050
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ABSTRACT |
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Endothelin-converting enzyme-1 (ECE-1) is a type
II membrane protein that catalyzes the proteolytic activation of big
endothelin-1 to endothelin-1 (ET-1). The subcellular distribution of
ECE-1, and hence the exact site of physiological activation of big
ET-1, remains controversial. Here, we demonstrate with several
complementary methods that the two alternatively spliced bovine ECE-1
isoforms, ECE-1a and ECE-1b, differing only in the first 30 amino acids of their N-terminal cytoplasmic tails, exhibit strikingly distinct intracellular sorting patterns. Bovine ECE-1a, which is responsible for
the intracellular cleavage of big ET-1 in endothelial cells, is
constitutively recruited into the lysosome, where it is rapidly degraded. In contrast, bovine ECE-1b, the isoform found in cultured smooth muscle cells, is transported to the plasma membrane by a default
pathway and functions as an ectoenzyme. Mutational analyses reveal that
the N-terminal tip of the cytoplasmic domain of bovine ECE-1a contains
novel proline-containing signals that mediate constitutive lysosomal
targeting. Analyses of chimeric ECE-1/transferrin receptors demonstrate
that the cytoplasmic tail of bovine ECE-1a is sufficient for the
lysosomal delivery and rapid degradation. Our results suggest that the
distinct intracellular targeting of bovine ECE-1 isoforms may provide
new insights into functional aspect of the endothelin system and that
the cell permeability of ECE inhibitor compounds should be carefully
considered during their pharmacological development.
The endothelins are a family of 21-amino acid peptides consisting
of three closely related isoforms termed endothelin-1
(ET-1),1 ET-2, and ET-3 (1,
2). They act on two molecularly distinct subtypes of seven
membrane-spanning G protein-coupled receptors, the endothelin A
(ETA) and endothelin B (ETB) receptors, to
mediate a wide variety of biological activities (3, 4). Recent studies with specific endothelin receptor antagonists have illustrated important roles of endothelins in a number of pathological conditions in humans including congestive heart failure, vascular restenosis, and
essential hypertension (5-9). Further, recent genetic studies in mice
demonstrated that the endothelin system is also required for
development of specific neural crest-derived tissues (10-13).
Biologically active endothelins are produced from prepropolypeptides
through two steps of proteolytic processing. The approximately 200-residue preproendothelins are first processed by a furin-like processing protease(s) into biologically inactive intermediates termed
big endothelins (big ETs). These are then proteolytically cleaved
between Trp21 and Val/Ile22 to produce active
endothelins. This proteolytic conversion is catalyzed by specific
endothelin-converting enzymes (ECEs). Two isozymes of ECE, ECE-1 and
ECE-2, have been molecularly identified (14-16). Both ECEs are type II
membrane proteins with highly conserved Zn2+
metalloprotease motifs and cleave big ET-1 to produce ET-1 in vitro as well as in transfected cells. The two enzymes work at different pH ranges; ECE-1 cleaves big endothelins at a neutral pH,
while ECE-2 functions in an acidic pH range. This implies that these
enzymes function in different subcellular locations: ECE-1 may act in
early components of the secretory pathway, presumably in the Golgi
apparatus, as well as on the cell surface. In contrast, ECE-2 probably
functions in highly acidified compartments of the secretory pathway,
including a portion of the trans-Golgi apparatus. Immunohistochemical analyses of ECE-1 suggest that it is responsible for the production of mature ET-1 in a variety of cells, including several kinds of endothelial cells (17, 18). These isozymes also appear
to have different functions during embryonic development. Targeted
disruption of the ECE-1 gene in mice revealed that ECE-1 is the major
enzyme involved in the activation of big ET-1 and big ET-3 at specific
developmental stages (19). Importantly, it appears that loss of ECE-1
protein cannot be compensated for by ECE-2.
However, the subcellular localization of ECE-1, as well as the exact
site of actual activation of big ET-1, remains controversial. ECE-1 has
been reported to be present in the Golgi apparatus (14, 20), in the
trans-Golgi network (21), in the intracellular vesicle (22,
23), and on the cell surface (17, 18, 21, 24, 25). More recent studies
using electron microscopy and a cell surface biotinylation method
demonstrated that ECE-1 in endothelial cells is present both on the
cell surface and in intracellular vesicles (23). In whole animal
preparations and isolated perfused tissues, exogenously administered
big endothelins induce vasopressor actions; these activities are
inhibited by the metalloprotease inhibitor, phosphoramidon (26). This
suggests that cleavage of exogenously supplied big ET-1 takes place on
the cell surface. In contrast, in vascular endothelial cells and
Chinese hamster ovary (CHO) cells transfected with the ECE-1 cDNA,
cleavage of endogenously synthesized big ET-1 occurred intracellularly
during transit through the Golgi apparatus (14, 27). Thus, there appear
to be different conversion sites of big ET-1. However, it is unclear
whether the same ECE molecule is responsible for these different
conversions. Clarifying the exact site at which ECE-1 functions is of
importance to understand its physiological roles and to design ECE
inhibitors for therapeutic purpose.
Recently, two subisoforms of ECE-1, termed ECE-1a and ECE-1b, which
differ from each other only in the N-terminal tip of their cytoplasmic
tail, were identified (14, 24, 28, 29). Structural studies of the human
ECE-1 gene indicate that these two isoforms are generated by
alternative splicing (29). To investigate the significance of these two
isoforms of ECE-1, we have studied the possibility that the N-terminal
cytoplasmic tails of bovine ECE-1a and ECE-1b determine subcellular
localization of the protein and hence the site at which big ET-1 is
converted to the mature peptide. We provide morphological, biochemical,
and pharmacological evidence showing that these subisoforms exhibit
distinct intracellular sorting patterns, both in native vascular cells
and in transfected CHO cells. Furthermore, we have uncovered signals in
the alternatively spliced cytoplasmic tail of bovine ECE-1a that are
responsible for constitutive lysosomal targeting of this integral
membrane protein.
Reagents--
Synthetic human big ET-1-(1-38) and ET-1 were
obtained from American Peptides. Phosphoramidon and chloroquine were
obtained from Sigma. FR901533 (WS79089B) and FR139317 (WS79089B) were
gifts from Fujisawa Pharmaceutical Co., Ltd. Fura-2-AM was from
Molecular Probes, Inc. (Eugene, OR).
Enzyme Immunoassay of Endothelin Peptides--
The supernatant
of cultured cells was directly applied to a sandwich-type enzyme
immunoassay (EIA) that showed no cross-reactivity between big ET-1 and
ET-1 (14).
Cell Culture--
CHO cells were cultured as described (14). The
coding region of bovine ECE-1a or ECE-1b was subcloned into the pME18Sf
expression vector (14). Stable transfection of CHO cells and isolation of the transfectant clones (CHO/ECE-1a and CHO/ECE-1b) were performed as described (14). Clones that showed a similar level of expression by
Northern blotting were chosen for further analysis. Endothelial cells
were isolated from bovine coronary arteries by collagenase treatment as
described (14). Arterial smooth muscle cells were isolated from bovine
coronary artery by explantation. Bronchial smooth muscle cells were
isolated from bovine trachea by collagenase treatment or explantation
and were cultured in Dulbecco's modified Eagle's medium containing
10% (v/v) fetal bovine serum. Smooth muscle cells were identified by
staining of the smooth muscle cell-specific Reverse Transcription-PCR--
RNA was extracted from cells
using RNA STAT-60 (TEL-TEST "B", Inc.) as recommended by the
manufacturer. First-strand cDNA synthesis was carried out with 1 µg of total RNA and oligo(dT)12-18 primers by using
SuperScript reverse transcriptase II (Life Technologies, Inc.) as
recommended by the manufacturer. The PCR contained 20 mM
Tris-HCl (pH 8.5), 50 mM KCl, 1.5 mM
MgCl2, a 0.2 mM concentration of each dNTP, a
100 nM concentration of each amplification primer, 10 ng of
first strand cDNA, and 2.5 units of Taq DNA polymerase. The primers, 5'-ATGTCTCCCCGGGGGCAGGAT-3' (corresponding to amino acids
1-8 of bovine ECE-1a) and 5'-TTCACCTGCAGGGAAGGAGGCA-3' (amino acids
29-36 of ECE-1a) were used for bovine ECE-1a-specific amplification (108-base pair PCR product expected), and the primers,
5'-ATGTCTACCTACAAGCGGCCCA-3' (amino acids 1-8 of bovine ECE-1b) and
5'-TTCACCTGCAGGTGGTTGGGGT-3' (amino acids 24-31 of ECE-1b) were used
for bovine ECE-1b-specific amplification (93 base pairs expected).
Thirty cycles of PCR were performed at an annealing temperature of
60 °C, and the PCRs were separated on a 2% agarose gel. The PCR
products were verified by DNA sequencing.
Quantitative PCR--
Quantitative PCR was performed using real
time PCR detection technology and analyzed on a model 7700 Sequence
Detector (Applied Biosystems) (30, 31). The assay uses the 5'-nuclease
activity of Taq polymerase to cleave a nonextendible
hybridization probe during the extension phase of PCR. The fluorescent
reporter located on the 5'-end of the probe is released from a
quenching dye present on the 3'-end, and fluorescent emission is
measured in real time. Threshold values are calculated by determining
the point at which the fluorescence exceeds a threshold limit (usually
10 times the S.D. values of the base line). The primers
5'-ATGTCTACCTACAAGCGGGCCA-3' and 5'-TTGGTGGACGTCCACTTGAAGG-3' were used
for bovine ECE-1b-specific amplification, and the primers
5'-AAGAAGGCGTTTGAAGAGAGC-3' and 5'-TGGCCGATTTCCGAGTATC-3' were
used to amplify the common region of bovine ECE-1. The hybridization
probes were 5'-TACACGTCGCTCTCGGACAGCGAGT-3' (ECE-1b specific) and
5'-TCATCCATCCACTTCAGGGTGCTCA-3' (common region). These oligonucleotide
probes, which bind to the amplified PCR products, were labeled with a
reporter dye, FAM (6-carboxyfluorescein), on the 5' nucleotide and
a quenching dye, TAMRA (6-carboxytetramethylrhodamine), on the 3'
nucleotide. The PCRs were carried out using TaqMan PCR reagent (Applied
Biosystems) as recommended by the manufacturer. Each PCR amplification
was performed in quadruplicate, using the following conditions: 2 min
at 50 °C and 10 min at 95 °C, followed by a total of 40 two-temperature cycles (15 s at 95 °C and 1 min at 60 °C). For
the generation of standard curves, serial dilutions of a cDNA
sample made from CHO/ECE-1b cells were used. A normalization to ECE-1b
expressed in smooth muscle cell was performed for each sample. The
amount of ECE-1a was calculated by subtracting the amount of ECE-1b
from the total amount of ECE-1.
Fluorescent Immunocytochemistry--
Cells were seeded onto
coverslips and cultured for 2 days.
For intracellular staining, cells were fixed and permeabilized in
methanol for 5 min at
For cell surface staining, cells were fixed in PBS containing 4%
paraformaldehyde for 15 min at room temperature. Following two washes
in PBS, cells were incubated in PBS containing 10% (w/v) nonfat dry
milk (milk/PBS) for 1 h at 37 °C. Cells were incubated in
milk/PBS containing a polyclonal antibody (1:100) directed against
ECE-1 C-terminal peptides for 90 min at 37 °C. The cells were washed
six times with PBS for 10 min each and then incubated in milk/PBS
containing 7.5 µg/ml of fluorescein isothiocyanate-labeled goat
anti-rabbit IgG. After 45 min at 37 °C, the cells were washed nine
times with PBS for 10 min each. The coverslips were mounted on
microscope slides as described above. Three negative control conditions
were examined: staining with preimmune serum, antibody after
preabsorption, and omission of primary antibody. None of these
conditions resulted in cell staining.
Ca2+ Transient Bioassay--
The stable transfected
CHO cell lines, CHO/ECE-1a and ECE-1b, were transiently transfected
with the ETA endothelin receptor expression construct and
loaded with Fura 2-AM. Synthetic human ET-1 or big ET-1 was applied to
these cells, and intracellular calcium changes were monitored by a
Jasco CAM-110 intracellular ion analyzer as described (14). In some
experiments, cells were pretreated with FR139317, an ETA
receptor antagonist, or phosphoramidon.
Metabolic Labeling and Immunoprecipitation--
Cells were
plated onto a 60-mm dish to obtain 70-80% confluency and grown
overnight. Cells were washed twice with starvation medium (methionine
and cysteine-free Dulbecco's modified Eagle's medium supplemented
with 1% (v/v) fetal bovine serum), preincubated in starvation medium
for 1 h, and incubated for 1 h in 1.5 ml of the same medium
containing 100 µCi/ml Trans35S-label (ICN Biomedical).
Pulse-labeled cells were chased for the designated times in complete
medium. In some experiments, cells were preincubated for 2-6 h in the
medium containing 100 µM phosphoramidon, 30 mM ammonium chloride, or 100 µM chloroquine and chased for 0, 1, 2, and 4 h in the same medium. At each time point, labeled cells were placed on ice and solubilized with lysis buffer (50 mM Tris-HCl, pH 8.0, 50 mM NaCl, 1%
Zwittergent). ECE-1a and ECE-1b were immunoprecipitated from
postnuclear supernatants using the polyclonal antibody directed against
the C-terminal 16 amino acids of bovine ECE-1. Immunoprecipitates were
analyzed on 7.0% polyacrylamide gels. Quantitation of radioactivity
was performed on a PhosphorImager (Molecular Dynamics, Inc., Sunnyvale, CA).
Immunoblotting--
Postnuclear lysate was subjected to
SDS-polyacrylamide gel electrophoresis, and filters were probed with an
antibody against the C terminus of bovine ECE-1 (16) and developed
using the ECL kit (Amersham Pharmacia Biotech) as recommended by the manufacturer.
Mutagenesis of Bovine ECE-1a or ECE-1b--
Deletion constructs
and alanine-substituted constructs were made by site-directed
mutagenesis (32). All mutants were verified by DNA sequencing of the
entire cytoplasmic domain. Stable transfection of CHO cells and
isolation of the transfectant clones were performed as described (14).
At least 12 independent stable transfectant clones from each mutant
were analyzed by indirect immunofluorescence and immunoblotting as well
as in some cases, by pulse-chase immunoprecipitation.
ECE-1-Transferrin Receptor Chimera Analysis--
The
cytoplasmic domain of bovine ECE-1a (amino acids 1-56) or ECE-1b
(amino acids 1-51) was fused in frame to the transmembrane helix
(residues 62-89) and the entire extracellular domain (residues 90-761) of the human transferrin receptor (TfR) as follows. Initially, a 683-base pair PCR product, containing the transmembrane helix and
part of extracellular domain of the human transferrin receptor, was
generated using the human transferrin receptor cDNA as a template. The sense primer,
5'-TGTAGTGGATCCATCTGCTATGGG-3', corresponds to
the first eight amino acids of the transmembrane helix of the human
transferrin receptor with the substitution of AGT for TCC (italic type)
to create a BamHI site (underlined) as a silent mutation.
The antisense primer, 5'-GGGAAATTTAGTCTGGTCCATGTAATA-3', represents
about 30 base pairs downstream of the unique HindIII site
(position 912) of the human transferrin receptor. The amplified fragment was digested with BamHI and HindIII.
Next, the cytoplasmic tails of bovine ECE-1a and ECE-1b were generated
by PCR. The sense primers
(5'-TTGAATTCCTGATGTCTCCCCGGGGGCAGGAT-3' for ECE-1a,
5'-TTGAATTCGGGATGTCTACCTACAAGCGGGCC-3' for ECE-1b)
represent the first seven amino acids of the cytoplasmic tail of bovine
ECE-1a or ECE-1b including an EcoRI site (underlined) in the
5'-end. The antisense primer,
5'-ATGGATCCACTACACCGCTTCTCCACCGGGGT-3', represents the
3'-end of the cytoplasmic tail of bovine ECE-1 (common for ECE-1a and
ECE-1b) and four amino acids of the human transferrin receptor
including the BamHI site (underlined), which allows the
fusion of the cytoplasmic tail of bovine ECE-1a or ECE-1b to occur in
the correct reading frame with respect to the human transferrin
receptor. The amplified PCR products were digested with
EcoRI and BamHI. All of the PCR products were
verified by DNA sequencing. After fusing these PCR products, we ligated
the resulting fragment into the rest of the C-terminal portion of the
human transferrin receptor cDNA. The chimeric cDNAs were
inserted into the expression vector, pME18Sf-(designated ECE-1a-TfR
or ECE-1b-TfR). The expression constructs were stably transfected into
CHO-K1 cells (CHO/ECE-1a-TfR or CHO/ECE-1b-TfR), and three independent clones from each stable transfected cell line were further
analyzed. The cell lines, which display similar levels of chimeric
protein expression between ECE-1a-TfR and ECE-1b-TfR, were compared
in parallel by immunostaining and immunoprecipitation as described
above. For pulse-chase experiments, we used a 20-µl mixture of
transferrin receptor monoclonal antibodies, which recognize the
extracellular portion of the human transferrin receptor (Ab-1 from
Calbiochem and CD71 (clone MEM-75) from Accurate Chemical & Scientific
Corp.). Protein G-Sepharose 4 Fast Flow (Amersham Pharmacia Biotech)
was used for immunoprecipitation. For immunostaining, CD71 (at a
dilution of 1:10) and fluorescein isothiocyanate-goat anti mouse IgG
(7.5 µg/ml, Zymed Laboratories Inc.) were used.
Expression of the Two Alternative Spliceoforms of Bovine
ECE-1--
Recent studies have shown that the ECE-1 gene contains two
alternative promoters and first exons, generating ECE-1a and ECE-1b polypeptides that differ from each other in the N-terminal half of
their cytoplasmic tails (24, 29) (see Fig. 7). We found that bovine
arterial and bronchial smooth muscle cells, both possessing endothelin
receptors (3), exclusively express ECE-1b mRNA (Fig. 1A). In contrast, both ECE-1a
and ECE-1b mRNAs were detectable in cultured vascular endothelial
cells by nonquantitative reverse transcription-PCR (Fig.
1A). Therefore, we determined the relative quantity of the
two isoforms in both cell types by real time quantitative PCR using a
fluorogenic 5' nuclease assay (30, 31). As shown in Fig. 1B,
95% of ECE-1 mRNA expressed in endothelial cells was ECE-1a
mRNA, indicating that endothelial cells predominantly express ECE-1a.
Distinct Subcellular Localization of ECE-1a and ECE-1b--
Since
the only sequence difference between bovine ECE-1a and ECE-1b was found
in the tip of the cytoplasmic tails, we investigated the possibility
that these isoenzymes exhibit different subcellular localizations. We
immunostained both endothelial cells and smooth muscle cells with
antibodies that recognize the common C-terminal ectodomain of bovine
ECE-1. Without prior permeabilization, endothelial cells stained only
faintly (Fig. 1C). After permeabilization, endothelial cells
showed strong staining in intracellular vesicles, the majority of which
overlapped with Golgi staining, visualized using lentil lectin (Fig.
1C). In contrast, smooth muscle cells exhibited a robust
cell surface staining without permeabilization (Fig. 1C).
With permeabilization, smooth muscle cells exhibited both intracellular
and cell surface staining (data not shown). These findings indicate
that the endothelial cells predominantly express intracellular ECE-1,
whereas the smooth muscle cells express ECE-1 on the cell surface. To
examine where the actual cleavage of endogenously produced big ET-1
occurs in these endothelial cells, we cultured the cells in the
presence of phosphoramidon, a cell-permeable ECE-1 inhibitor (27), or
FR901533, a non-cell-permeable ECE-1 inhibitor (14). We then assayed
the levels of big and mature ET-1 peptides secreted into the medium.
Secretion of mature ET-1 from these cells was significantly inhibited
by phosphoramidon, with a concomitant increase in big ET-1 secretion
(Fig. 1D). In contrast, FR901533, at concentrations
sufficient to inhibit extracellular cleavage of big ET-1 (see Fig.
3A), could not inhibit the processing of big ET-1 in
endothelial cells, presumably because the compound did not have access
to the site of big ET-1 cleavage. These findings support the notion
that cleavage of endogenously produced big ET-1 occurs intracellularly
in endothelial cells.
To examine whether the distinct subcellular localization of bovine
ECE-1a and ECE-1b could be reproduced in heterologous cell lines, we
stably transfected CHO cells (which do not express detectable levels of
ECE-1) with either bovine ECE-1a or ECE-1b cDNAs and immunostained
multiple transfected clones with an anti-ECE-1 antibody. Most cells
from monoclonal CHO/ECE-1a transfectant cell lines were not stained
without prior permeabilization (Fig.
2A). With permeabilization, a
robust staining of CHO/ECE-1a cells was observed in Golgi-like areas,
where we also observed strong lectin staining (Fig. 2B). In
contrast, CHO/ECE-1b cells exhibited strong cell surface staining
without prior permeabilization (Fig. 2C).
To functionally confirm the intracellular versus cell
surface localization of bovine ECE-1a and ECE-1b proteins in
transfected cells, we co-cultured CHO/prepro-ET-1 cells, which secrete
big ET-1, with the same number of either CHO/ECE-1a or CHO/ECE-1b cells
and assessed the generation of mature ET-1 in the medium. CHO/ECE-1b
cells generated large amounts of mature ET-1 when cocultured with
CHO/prepro-ET-1 cells (Fig.
3A). The production of mature ET-1 was efficiently inhibited by FR901533, with a reciprocal increase
in big ET-1 levels, indicating that cleavage occurred in the
extracellular space where FR901533 had access. In contrast, co-culture
of CHO/ECE-1a and CHO/prepro-ET-1 cells yielded only low levels of
mature peptide, indicating that CHO/ECE-1a cells express little
functional ECE-1 on the cell surface (Fig. 3A). This is not
due to an absence of functional ECE-1 in CHO/ECE-1a cells, since we
previously found that these same cell lines, when further transfected
with prepro-ET-1 cDNA, could cleave 50-90% of the endogenously
synthesized big ET-1 intracellularly (14). Furthermore, we assessed the
functional localization using CHO cell lines that express both ECE-1
and ETA receptor as reporter cells. When CHO/ECE-1b cells
were transiently transfected with an ETA receptor cDNA,
these cells became responsive to exogenous big ET-1 as assessed by
intracellular Ca2+ transients (Fig. 3B). The
action of big ET-1 was completely abolished by an ECE inhibitor (Fig.
3B), indicating that exogenous big ET-1 could be efficiently
cleaved into active peptide by ECE-1b present on the cell surface. In
contrast, co-expression of ECE-1a and the ETA receptor did
not render CHO cells responsive to exogenous big ET-1, while ET-1
produced a response of similar amplitude to that observed in CHO/ECE-1b
cells, indicating that little functional ECE-1 is expressed on the cell
surface in CHO/ECE-1a cells (Fig. 3B).
Selective Lysosomal Sorting and Rapid Degradation of
ECE-1a--
In the process of further dissecting the cellular
mechanisms that may lead to the distinct steady-state subcellular
localization of bovine ECE-1a and ECE-1b, we found that the turnover
rate of ECE-1a protein was much higher than that of ECE-1b. CHO/ECE-1a and CHO/ECE-1b cells were metabolically labeled with
35S-amino acids for 60 min and then rinsed and further
cultured in medium without tracers. At specific time intervals, cell
extracts were prepared and immunoprecipitated with an anti-ECE-1
antibody. These pulse-chase experiments demonstrated that, although
ECE-1a and ECE-1b mRNAs are translated at similar rates (compare
the initial lanes in Fig.
4), the half-life of ECE-1a protein
(~1.5 h) is much shorter than that of ECE-1b (~20 h).
A previous study reported that treatment of vascular endothelial cells
with phosphoramidon caused a marked increase in the cellular amount of
ECE-1 protein, although the mechanism responsible for this was unknown
(18, 28). We were able to reproduce these observations: when
the endothelial cells were cultured in the presence of phosphoramidon
for 24 h, they contained significantly larger amounts of ECE-1
protein as compared with untreated cells (Fig.
5A). The level of ECE-1
mRNA was unaffected by phosphoramidon treatment (data not shown).
Importantly, however, this phenomenon was not observed in the smooth
muscle cells (Fig. 5A). Similar experiments in transfected
CHO cells confirmed that this "pseudoinduction" of the ECE-1
protein occurs only in CHO/ECE-1a cells and not in CHO/ECE-1b cells
(Fig. 5A). Taken together, these findings led us to
hypothesize that phosphoramidon may somehow protect bovine ECE-1a from
its rapid degradation, thereby causing an accumulation of the
isoenzyme. Indeed, we found that, in both endothelial and CHO/ECE-1a
cells, phosphoramidon treatment markedly prolonged the half-life of
ECE-1a protein as judged by pulse-chase immunoprecipitation (Figs. 4
and 5B).
These findings prompted us to speculate that ECE-1a protein is
constitutively recruited into a lysosomal compartment, where it is
rapidly degraded. To test this hypothesis, we treated CHO/ECE-1a cells
with inhibitors of lysosomal function. Both NH4Cl and
chloroquine markedly prolonged the half-life of ECE-1a protein in these
cells (Fig. 5C). Furthermore, after the NH4Cl or
chloroquine treatment, strong ECE-1 immunoreactivity was detected in
coarse granular compartments more distal to the nuclei, which we did
not previously see in untreated CHO/ECE-1a cells (compare Figs. 2 and
6). Double staining of CHO/ECE-1a cells
in the presence of lysosomal inhibitors with an anti-ECE-1 antibody and
a monoclonal antibody that recognizes lysosomal membrane glycoprotein B
confirmed that ECE-1-immunoreactive granular structures were also
lysosomal membrane glycoprotein B-positive (Figs. 6, G and
H). These results indicate that bovine ECE-1a is
constitutively targeted to lysosomes. We observed similar ECE-1-immunoreactive granular compartments in phosphoramidon-treated CHO/ECE-1a cells and endothelial cells (Fig. 6, B and
F). In contrast, the addition of FR901533 up to 100 µM did not cause the accumulation of bovine ECE-1 protein
in either CHO/ECE-1a cells or endothelial cells (data not shown).
The Cytoplasmic Tail of Bovine ECE-1a Contains Signals That Mediate
the Constitutive Lysosomal Targeting--
The cDNA-predicted
bovine ECE-1a and ECE-1b polypeptides have a N-terminal putative
cytoplasmic tail of 56 residues and 51 residues, respectively (Fig.
7). The last (C-terminal) 24 amino acids
of these cytoplasmic tails as well as the entire transmembrane domains
and ectodomains are identical between the two isoenzymes. Therefore,
the structural determinants that cause the striking difference in
subcellular localization and trafficking of bovine ECE-1a and ECE-1b
proteins must be embedded within the first ~30 amino acid residues of
the cytoplasmic tails. To further elucidate the nature of these
presumptive signal(s), we performed a series of mutagenesis studies
within the cytoplasmic tails. The mutant constructs were stably
transfected into CHO cells, and multiple clones from each transfection
were assessed by immunofluorescence staining as well as by pulse-chase
immunoprecipitations. A deletion of all of the alternatively spliced
portion of the tail resulted in a robust cell surface expression of the
mutant protein, which was indistinguishable from the distribution of
wild-type bovine ECE-1b (Fig. 7). This indicates that, in the absence
of the putative signal(s), the enzyme goes to the cell surface by
default. In fact, a deletion of the first 5 amino acids at the
N-terminal tip of the bovine ECE-1a tail is sufficient to cause a full
cell surface expression (Fig. 7, ECE-1a
Next, we examined whether the cytoplasmic tail of bovine ECE-1a is
sufficient to mediate the lysosomal targeting of a heterologous membrane protein. Chimeric constructs were created by attaching the
cytoplasmic tail of bovine ECE-1a or ECE-1b to the transmembrane and
ectodomain of the TfR, another type II membrane protein (Fig. 8A). Pulse-chase experiments
with stable transfectant CHO cells constitutively expressing these
constructs demonstrated that the half-life of the ECE-1a-TfR chimeric
molecule (~1 h) was similar to that of ECE-1a and was much shorter
than that of ECE-1b-TfR (~7 h) and wild-type TfR (~10 h, data not
shown) (Fig. 8B). These findings indicate that ECE-1a-TfR
protein is constitutively recruited to the lysosomal compartment and
rapidly degraded. Immunostaining of these cells using antibodies that
recognize the extracellular domain of the human transferrin receptor
revealed that the ECE-1a-TfR chimeric protein exhibited juxtanuclear
staining, which is similar to the pattern observed for wild type bovine
ECE-1a protein (data not shown). Taken together, these results indicate
that the cytoplasmic tail of bovine ECE-1a contains signals necessary
and sufficient for lysosomal targeting of this membrane protein.
We have shown that the alternative splicing of the ECE-1 gene
first exon generates two distinct integral membrane isoenzymes, which
exhibit strikingly different subcellular localizations. ECE-1a, the
predominant isoform found in cultured vascular endothelial cells,
resides in an intracellular compartment that largely overlaps with the
Golgi apparatus. Our experiments have shown that this isoenzyme is
responsible for the intracellular, co-secretory cleavage of
endogenously produced big ET-1 in endothelial cells. In contrast, ECE-1b is expressed on the cell surface as an ectoenzyme in the endothelin receptor-containing smooth muscle cells. We have shown that
this isoenzyme can catalyze the cell surface activation of extracellularly supplied big ET-1. Obviously, a more careful
examination on the expression pattern of ECE-1a versus
ECE-1b will be required to establish these relationships. Nevertheless,
based on our present findings, we propose a model of
endothelin-mediated cell-cell communications depicted in Fig.
9. This scheme dictates that cleavage of
big ET-1 by the "generator" cells takes place primarily inside the
cell, provided that those cells express sufficient levels of ECE-1, as
in the case of vascular endothelial cells. ET-1 secreted as mature
peptide probably works locally in a paracrine or autocrine manner. In
addition to the intracellular cleavage of big ET-1 at the level of the
generator cells, our model dictates that extracellular big ET-1 will be
further cleaved by cell surface ECE-1 expressed by the "target"
cells, with the resultant mature peptide readily acting on ET
receptors. Extracellular big ET-1 may not only come from generator
cells that do not express high levels of ECE-1; it may also be able to
travel a considerable distance. In this context, it is interesting to
note that the circulating plasma half-life of big ET-1 is significantly
longer than that of mature ET-1 (33). The absolute concentration of
circulating big ET-1 is also higher than that of mature peptide
(34).
INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References
EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References
-actin and also by their
characteristic spindle shape. For co-culture experiments, approximately
1 × 105 cells of CHO/prepro-ET-1, a CHO stable cell
line that expresses big ET-1 (14), and 1 × 105 cells
of either CHO/ECE-1a or CHO/ECE-1b were plated onto a 12-well plate and
grown overnight. After washing twice with medium, cells were fed fresh
complete medium in the presence or absence of FR901533 at the
designated concentrations and incubated for 12 h. The supernatant was directly used for enzyme immunoassay to measure big ET-1 and ET-1.
20 °C. After washing in phosphate-buffered saline (PBS), PBS containing 10% (v/v) normal goat serum (NGS/PBS) was
added. Following a 1-h incubation at 37 °C, the NGS/PBS was replaced
with buffer containing polyclonal antibody (1:100) directed against
bovine ECE-1 C-terminal peptides (16). After incubation for 90 min at
37 °C, the cells were washed six times with PBS for 10 min each and
then incubated in NGS/PBS containing 7.5 µg/ml of fluorescein
isothiocyanate-goat anti-rabbit IgG (Zymed Laboratories Inc.). After 45 min at 37 °C, the cells were washed nine times with PBS for 10 min each. The coverslips were mounted on microscope slides with 90% (v/v) glycerol, 50 mM Tris-HCl (pH 9.0),
and 2.5% (w/v) 1,4-diazabicyclo[2.2.2]octane. The
rhodamine-lentil lectin (Vector Laboratories), used to counterstain the
Golgi apparatus, was incubated at 2.5 µg/ml. Monoclonal antibody UH3,
which recognizes hamster lysosomal membrane glycoprotein B, was
obtained from The Developmental Studies Hybridoma Bank (University of
Iowa, Iowa City, IA).
RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References
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Fig. 1.
Expression and subcellular localization of
bovine ECE-1a and ECE-1b in native cells. A, expression
of ECE-1a and ECE-1b mRNA in endothelial cells, arterial smooth
muscle cells (SMC), and bronchial smooth muscle cells. RNA
from each cell type was reverse-transcribed and amplified using
specific primer pairs for bovine ECE-1a (a) or ECE-1b
(b). CHO/ECE-1a and CHO/ECE-1b are the CHO stable
transfectant cell lines that constitutively express bovine ECE-1a and
ECE-1b, respectively. The amplified products were subjected to
electrophoresis on agarose gels and were visualized by staining with
ethidium bromide. B, comparison between mRNA levels of
ECE-1a and ECE-1b by quantitative real time PCR. The relative amount of
ECE-1a and ECE-1b mRNA expressed in endothelial cells was
normalized against ECE-1b expressed in arterial smooth muscle cells.
ND, not detected. C, fluorescence
immunocytochemistry of endothelial cells and smooth muscle cells. Cells
were stained for cell surface (surface; a and
b) or intracellular (intra.; c and
d) staining as described under "Experimental
Procedures." Double staining with an ECE-1 antibody
(ECE-1; c) and lentil lectin (LL;
d), as a Golgi marker, was performed for intracellular
staining. Without permeabilization, endothelial cells stained only
faintly (a), whereas smooth muscle cells exhibited a robust
cell surface staining (b). The intense immunofluorescence
for ECE-1 (c) colocalizes with the lectin Golgi staining
(d). D, effects of phosphoramidon and FR901533 on
the production of big and mature ET-1 from cultured endothelial cells.
Endothelial cells were cultured in medium containing designated
concentrations of phosphoramidon (Phos.) or FR901533
(FR) for 24 h, and the concentration of big ET-1
(open circle) and mature ET-1 (closed
circle) in the medium was then determined by EIA.
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Fig. 2.
Fluorescence immunocytochemistry of CHO
stable transfected cell lines. CHO cell lines were stained for
cell surface (surface; A, C, and
I) or intracellular (intra.; B,
D, E, F, G, H,
and J) staining as described under "Experimental
Procedures." CHO/ECE-1a cell lines were not stained without prior
permeabilization (A). With permeabilization, a robust
staining of CHO/ECE-1a cells is observed in Golgi-like areas
(B). CHO/ECE-1b cells exhibit strong cell surface staining
without permeabilization (C). Double staining of ECE-1
(E and G) and wheat germ agglutinin
(WGA; F) or lentil lectin (LL;
H) demonstrates that the intracellular ECE-1 staining
(E and G) observed in CHO/ECE-1a cells is
colocalized with lectin Golgi staining (F and H).
The staining of CHO cells expressing ECE-1a R4A, an alanine
substitution mutant shown in Fig. 7, shows intense cell surface
staining for ECE-1 (I). CHO-K1 cells, the parental CHO cell
line, exhibits no staining (J).
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Fig. 3.
Functional subcellular localization of bovine
ECE-1a and ECE-1b. A, effects of FR901533 on the
production of big and mature ET-1 by 1:1 cocultures of CHO/prepro-ET-1
and either CHO/ECE-1a or CHO/ECE-1b cells. Cells were cocultured for
12 h in the absence or presence of the designated concentrations
of FR901533, and big and mature ET-1 levels in the medium were
determined by EIA. CHO/ECE-1b cells, but not CHO/ECE-1a cells, cleave
extracellularly supplied big ET-1 on the cell surface. B,
bioassay of the subcellular localization of ECE-1 using human
ETA-transfected CHO/ECE-1 cells as reporter cells.
Synthetic ET-1 (20 pM) or big ET-1 (2 nM) was
applied to CHO/ECE-1 cells transiently transfected with a human
ETA cDNA, and intracellular Ca2+ was
monitored as described under "Experimental Procedures."
Co-expression of ECE-1a and ETA receptor renders CHO cells
responsive to exogenous big ET-1. FR139317, an ETA receptor
antagonist, or phosphoramidon completely abolished the action of big
ET-1.
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Fig. 4.
Degradation of bovine ECE-1a and ECE-1b
protein expressed in CHO cells. Pulse-chase and
immunoprecipitation experiments of CHO/ECE-1 cells are shown.
Equivalent numbers of CHO cells constitutively expressing ECE-1a
(open circle) or ECE-1b (closed
circle) were pulse-labeled with 35S label for
1 h and then chased for the designated time in the complete
medium. ECE-1 protein was immunoprecipitated and analyzed on
SDS-polyacrylamide gels as described under "Experimental
Procedures." The relative amount of immunoprecipitate at each time
point was calculated as a percentage of the amount labeled at 0 h.
An autoradiograph from a representative experiment is shown, and the
quantitative data shown represent the mean of results from three
independent experiments in which three different clones of CHO cells
were analyzed for ECE-1a and ECE-1b.
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Fig. 5.
Inhibition of rapid degradation of bovine
ECE-1a protein in endothelial cells and CHO/ECE-1a cells.
A, immunoblot analysis of endothelial cells, arterial smooth
muscle cells, CHO/ECE-1a, and CHO/ECE-1b cells treated with
phosphoramidon. A confluent monolayer of cells was incubated in the
usual medium in the absence ( ) or presence (+) of 10 µM
phosphoramidon for 16 h. The postnuclear lysate (20 µg of BCAEC,
80 µg of smooth muscle cells, 20 µg of CHO/ECE-1a, and 2 µg of
CHO/ECE-1b) was subjected to immunoblotting using an ECE-1 antibody.
"Pseudoinduction" of ECE-1 protein by phosphoramidon occurs in
endothelial cells and CHO/ECE-1a cells. B, pulse-chase and
immunoprecipitation experiments of cultured endothelial cells in the
absence (
) or presence (+) of phosphoramidon. Endothelial cells were
pulsed with 35S label and chased for the indicated times in
the absence or presence of 10 µM phosphoramidon. The
postnuclear lysates were immunoprecipitated and analyzed as described
in the legend to Fig. 4. Phosphoramidon prolongs the half-life of ECE-1
protein in endothelial cells. C, pulse-chase and
immunoprecipitation experiments of CHO/ECE-1a cells treated with
phosphoramidon, ammonium chloride, or chloroquine. CHO/ECE-1a cells
were preincubated with each inhibitor, pulsed with 35S
label, and chased for the indicated times described under
"Experimental Procedures." These inhibitors prolong the half-life
of ECE-1a protein expressed in CHO cells.
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Fig. 6.
Fluorescence immunocytochemistry of CHO/ECE-1
cells and endothelial cells treated with phosphoramidon or
inhibitors. Fluorescence immunocytochemistry is shown of CHO/ECE-1
cells (A-E, G, and H) and endothelial
cells (F) treated with phosphoramidon (B,
D, and F), chloroquine (E), or
ammonium chloride (G and H). Cells were treated
with each inhibitor as described under "Experimental Procedures"
and stained for intracellular staining using an ECE-1 antibody. Double
staining of CHO/ECE-1a cells with an ECE-1 antibody and UH3, a
lysosomal marker, was also performed (G and H).
Strong ECE-1 immunoreactivity was detected in coarse granular
compartments in CHO/ECE-1a cells (B, E, and
G) and endothelial cells (F) treated with the
inhibitors.
2-6), indicating that this portion of bovine ECE-1a tail contains at least a part of the essential
signal that prevents cell surface expression. Alanine scan mutagenesis
studies in the critical N-terminal portion of bovine ECE-1a tail
revealed that there are two appreciable clusters of indispensable amino
acid residues: one at Pro3-Arg4 and the other
at Pro12-Leu13-Leu14 (Fig. 7). In
all cases, the results from pulse-chase immunoprecipitation experiments
were in accordance with the immunofluorescence assessments. We observed
a rapid turnover of the mutant protein whenever the protein exhibited
intracellular localization; in contrast, mutant proteins that exhibited
cell surface localization were all long lived (data not shown).
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Fig. 7.
Mutational analyses of alternatively spliced
N-terminal cytoplasmic tail of bovine ECE-1a and ECE-1b. The amino
acid sequences of the cytoplasmic tails of wild-type bovine ECE-1a and
ECE-1b are shown in one-letter codes. Alanine substitutions are shown
as A. Dashes represent unchanged residues. The mutant
constructs were stably transfected into CHO cells, and multiple clones
from each transfection were assessed by immunofluorescence staining as
well as by pulse-chase immunoprecipitations for intracellular
(closed circle) or cell surface (open
circle) localization. Mutational analyses reveal that there
are two proline-containing clusters of indispensable amino acid
residues: Pro3-Arg4 and
Pro12-Leu13-Leu14.
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Fig. 8.
Analysis of
ECE-1-transferrin receptor chimeras.
A, schematic illustration of ECE-1-TfR constructs.
Chimeric constructs were created by attaching the cytoplasmic
tail of bovine ECE-1a (amino acids 1-56) or ECE-1b (amino acids 1-51)
to the transmembrane (closed box) and
extracellular domain of the TfR. ECE-1a- and ECE-1b-specific sequences
are marked by striped boxes. These chimeric
cDNAs were stably transfected into CHO cells, and multiple clones
were analyzed in the following studies. B, rapid degradation
of ECE-1a-TfR chimera protein expressed in CHO cells. Equivalent
numbers of CHO cells constitutively expressing ECE-1a-TfR
(open circle) or ECE-1b-TfR (closed
circle) were pulse-labeled with 35S label for
1 h, and chased for the various periods of time (h) as indicated.
ECE-1-TfR chimeras were immunoprecipitated and analyzed as described
in the legend to Fig. 4. An autoradiograph from a representative
experiment is shown, and the quantitative data represent the mean of
three experiments on two different clones of CHO cells expressing each
chimera.
DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References
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Fig. 9.
A working model of endothelin-mediated
cell-cell communications. ECE-1a, the predominant isoform found in
cultured vascular endothelial cells, resides in an intracellular
compartment that largely overlaps with the Golgi apparatus and is
responsible for the intracellular cleavage of endogenously produced big
ET-1 in endothelial cells. ECE-1a is constitutively recruited into a
lysosomal compartment and rapidly degraded. ECE-1b is expressed on the
cell surface as an ectoenzyme in the endothelin receptor-containing
smooth muscle cells. ECE-1b can catalyze the cell surface activation of
extracellularly supplied big ET-1.
Our model is also supported by the observation obtained from a recent
series of endothelin-related gene disruption studies in mice.
ECE-1/
mice (19) reproduced the phenotype
observed in ET-1-deficient embryos (10): craniofacial and
cardiovascular abnormalities. However, a large amount of mature ET-1
peptide was still present in near term
ECE-1
/
embryos. This suggests that mature
ET-1 must be produced at the exact sites where it functions, since the
mature peptide present could not rescue the developmental phenotype of
ECE-1
/
mice. This demonstrates that
endothelin secreted as mature peptide acts in a highly local fashion.
On the other hand, ET-1
/
embryos showed an
incomplete penetrance of cardiovascular abnormalities, which are
observed in all ECE-1
/
and
ETA
/
embryos (13, 19). This
suggests that the cardiovascular phenotype in
ET-1
/
embryos is partially rescued by
maternally or placentally produced big ET-1, but not mature ET-1, which
is delivered to the embryo and locally processed to ET-1 by ECE-1. This
establishes that big ET-1 can work as a long distance carrier of the
biological signals of ET-1. Taken together, these genetic studies
strongly suggest that ET-1 functions only locally, whereas big ET-1 can act as a distant carrier of ET-1 signals.
In the results presented in this paper, it does not appear that ECE-1a is actively retained within Golgi in the same fashion as glycosyltransferases and other Golgi-resident membrane proteins (35). Instead, the Golgi-like localization of ECE-1a protein in the steady state appears to be due to its constitutive targeting into a lysosomal compartment, where the protein is rapidly degraded by an acidification-dependent mechanism. This scheme is highly analogous to the one for the constitutive lysosomal sorting and rapid degradation of P-selectin in endothelial cells (36, 37) (see below). Interestingly, treatment of ECE-1a-expressing cells with the cell-permeable ECE-1 inhibitor phosphoramidon prevents the rapid turnover of the isoenzyme, resulting in a direct accumulation of the protein in the lysosomal compartments. Phosphoramidon, a competitive inhibitor of ECE-1, presumably causes a conformational change in ECE-1a by binding its active pocket, rendering the isoenzyme immune to the lysosomal degradation machinery. Alternatively, phosphoramidon may inhibit other lysosomal metalloprotease(s) that are essential for initiating the rapid degradation of ECE-1a. In this case, we expect that lysosomal degradation of a number of other proteins unrelated to ECE-1 may possibly be prevented by phosphoramidon.
Alanine scan mutagenesis studies demonstrated that the cytoplasmic tail of ECE-1a contains two clusters of indispensable amino acid residues, Pro3-Arg4 and Pro12-Leu13-Leu14, both of which are essential for specifying the steady-state intracellular localization and rapid lysosomal turnover of ECE-1a protein. To specifically determine the influence of these residues on the intracellular targeting of ECE-1a, it will be necessary to assess the relative amount of ECE-1a localized on the cell surface of these mutants by quantitative assay methods including cell surface biotinylation or iodination. Nevertheless, these findings suggest that the sorting determinant mediating lysosomal targeting of bovine ECE-1a is located within the two proline-containing signals. It has been previously demonstrated that constitutive, direct lysosomal targeting of P-selectin at the trans-Golgi network can be abrogated by deleting a 10-amino acid stretch, DGKCPLNPHS, from the C-terminal cytoplasmic tail (36). Interestingly, this stretch of P-selectin sequence contains two proline residues, in the contexts PLN and PH. More recently, the proline residue within the sequence KCPL was shown to make a major contribution to the efficiency of lysosomal targeting of P-selectin without affecting internalization (37). It is tempting to speculate that ECE-1a and P-selectin are constitutively delivered to lysosomal compartments through proline-containing signals by similar molecular mechanisms. In this regard, it has also been shown that P-selectin reaches lysosomes in CHO cells via the plasma membrane (38). We cannot establish from the results presented in this paper whether ECE-1a is delivered to lysosomes directly from the trans-Golgi network, or indirectly via the plasma membrane. However, our live cell-based assay using ECE inhibitors as pharmacological probes, coupled to our preliminary internalization experiments using ECE-1a-TfR chimera2 suggests that ECE-1a is delivered to lysosomes without appearing on the plasma membrane.
Studies on cultured cells transfected with ECE cDNA have demonstrated that ECE-1a is localized to the plasma membrane by immunofluorescence microscopy analysis (17, 21, 24). In fact, we observed cell surface expression of ECE-1 in a small subset of CHO/ECE-1a cells by immunocytochemistry, although the majority of cells showed intracellular localization. However, our biochemical and pharmacological studies demonstrated that all CHO/ECE-1a stable transfectant cell lines exhibit lysosomal targeting. In this regard, we observed that ECE-1a protein does "leak" to the cell surface when it is massively overexpressed, for example, under a strong promoter after transient transfection into the episomal replication-competent COS cells (data not shown; see Ref. 17). We feel that cell surface localization of ECE-1 observed in a few CHO/ECE-1a cells is largely due to massive overexpression. This suggests that the lysosomal targeting of ECE-1a occurs through a saturable mechanism. It is tempting to hypothesize that proline-containing sequences unique to the ECE-1a tail may interact with other regulatory proteins involved in lysosomal trafficking at the trans-Golgi network. A differential protein interaction screen using cytoplasmic tails of ECE-1a and ECE-1b as positive and negative "baits" may prove a feasible strategy to identify such proteins.
Subcellular localization of ECE-1 has remained controversial. A number
of apparently contradicting observations have been reported in recent
years using a variety of cultured cells (14, 18, 21, 23, 39). Our
present study suggests that some of the controversies may have arisen
due to the presence of two distinct spliceoforms of ECE-1 that exhibit
completely different localizations. ECE-1 provides an attractive target
for pharmacological intervention to reduce the formation of active
endothelin in pathological states involving a deregulation of
endothelin production. However, the results presented here indicate
that a careful consideration on cell permeability of inhibitor
compounds is essential in the future development of ECE-1 inhibitors.
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ACKNOWLEDGEMENTS |
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We thank Sumio Kiyoto for a sample of FR901533, Nobuhiro Suzuki and Hirokazu Matsumoto for the EIA antibodies, and Damiane deWit for technical assistance.
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FOOTNOTES |
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* This work was supported by grants from the Ministry of Education, Science and Culture of Japan and a Japan Heart Foundation Research Grant.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.
§ To whom correspondence should be addressed: Division of Genetics, International Center for Medical Research, Kobe University School of Medicine, 7-5-1 Kusunoki, Chuo, Kobe, 6500017 Japan. Tel.: 81-78-341-7451 (ext. 3562); Fax: 81-78-362-6064; E-mail: emoto{at}med.kobe-u.ac.jp.
The abbreviations used are: ET, endothelin; ECE, endothelin-converting enzyme; CHO, Chinese hamster ovary; EC, endothelial cells; EIA, enzyme immunoassay; PCR, polymerase chain reaction; TfR, human transferrin receptor; NGS, normal goat serum; PBS, phosphate-buffered saline.
2 N. Emoto and M. Matsuo, unpublished observations.
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