From the INSERM U 36 Collège de France Paris, 75005 Paris, France
Received for publication, September 3, 2002
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ABSTRACT |
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Endothelin-converting enzyme (ECE) is a membrane
metalloprotease that generates endothelin from its direct precursor big
endothelin. Four isoforms of ECE-1 are produced from a single
gene through the use of alternate promoters. These isoforms share the
same extracellular catalytic domain and contain unique cytosolic tails, which results in their specific subcellular targeting. We
investigated the distribution of ECE-1 isoforms in transfected AtT-20
neuroendocrine cells. Whereas ECE-1a and 1c were present at the plasma
membrane, ECE-1b and ECE-1d were retained inside the cells. We found
that both intracellular isoforms were concentrated in the endosomal system: ECE-1d in recycling endosomes, and ECE-1b in late
endosomes/multivesicular bodies. Leucine-based motifs were involved in
the intracellular retention of these isoforms, and the targeting of
ECE-1b to the degradation pathway required an additional signal in the
N terminus. The concentration of ECE-1 isoforms in the endosomal system
suggested new functions for these enzymes. Potential novel functions
include redistribution of other isoforms through direct interaction. We have showed that ECE-1 isoforms could heterodimerize, and that in such
heterodimers the ECE-1b targeting signal was dominant. Interaction of a
plasma membrane isoform with ECE-1b resulted in its intracellular
localization and decreased its extracellular activity. These
data demonstrated that the targeting signals specific for ECE-1b
constitute a regulatory domain per se that could modulate the localization and the activity of other isoforms.
Endothelins (ET1-1,
ET-2, and ET-3) are 21-residue peptides derived from three distinct
genes (1). They are pleiotropic factors that play an important role in
the regulation of the cardiovascular and endocrine systems (for review
see Refs. 2 and 3). Endothelins are also crucial developmental factors
as demonstrated by the targeted disruption of the genes coding for the
precursors of ET-1 and ET-3, and of the genes coding for their
receptors, ETA and ETB (4-7). In addition, the expression of
endothelin is associated with many pathological processes and with
tumor growth (2, 8-10). In order to fulfill such a wide spectrum of
physiological functions, endothelins act through autocrine and
paracrine mechanisms. Their biosynthesis thus requires tight local
control. Endothelins are synthesized in the endoplasmic reticulum as
precursors that undergo a two-step proteolytic maturation.
Pro-endothelins are first processed at conserved multibasic sites by
furin or a furin-like enzyme, in order to release an intermediate
called big endothelin (big-ET) (11, 12), which is devoid of biological
activity (13). Big-ET is then processed by endothelin converting enzyme (ECE) at a Trp-Val/Ile bond, which releases the biologically active peptide. This latter proteolytic step can occur in the extracellular medium and in the secretory pathway, so that cells secrete either big-ETs alone or together with endothelins (14, 15, 16). Endothelial
cells co-express the precursor, the converting enzyme and the receptor,
thus implying autocrine function of endothelin. On the other hand,
luteal cells (17) and neurons (18) express only the converting enzyme
and the receptor, and three different cell types express the precursor,
the converting enzyme and the receptor in seminiferous tubules (19),
demonstrating the paracrine function of endothelin through
extracellular processing of big-ET. The cellular distribution of ECE
thus plays a central role in controlling the biosynthetic pathway of endothelins.
ECE is a type II membrane protein of the neutral endopeptidase (NEP)
family, with an N-terminal cytosolic tail and a catalytic ectodomain.
Unlike other proteases of this family, ECE is expressed as covalent
dimers (20-22). Two genes coding for ECE-1 and ECE-2 have been cloned
(20, 23, 14, 24). ECE-1 has a broader tissue distribution and is always
expressed at higher levels than ECE-2. The targeted disruption of the
ECE-1 gene resulted in a lethal phenotype that combined both the
phenotypes of the inactivation of ET-1 or ETA and of ET-3 or ETB, thus
demonstrating the central role of ECE-1 in the endothelin system (25).
On the other hand, the inactivation of the ECE-2 gene did not result in
significant modification of the mouse phenotype unless it was combined
with the ECE-1 gene inactivation (26). ECE-1 is expressed in the endothelium of all organs as well as in nonvascular cells of many tissues including brain and neuroendocrine tissues (Refs. 14, 21, 18,
and 27; for review see Ref. 28). Four isoforms of ECE-1 have been
identified (Refs. 29 and 30; for review see Ref. 31), and a related
gene organization has recently been described for ECE-2 (32, 33). ECE-1
isoforms result from the use of alternate promoters located
upstream of specific exons (29, 30, 34). Thus, the 4 ECE-1 isoforms
share the same catalytic domain and only differ in their N terminus,
which codes for their cytosolic tails (Fig.1A). As these
domains are responsible for their targeting, the specificity of ECE
isoforms resides in their subcellular distribution. Considering the
importance of the intracellular and extracellular biosynthesis of
endothelins, the subcellular distribution of ECE isoforms may play a
central role in the regulation of the endothelin system and constitutes an important factor for the efficient inhibition of ECE in pathological conditions.
Extensive work aimed to define the intracellular localization of ECE-1
has been done in endothelial cells (21, 35, 36). Most of these studies
were however non relevant to isoform specificity due to the lack of
antibodies that can distinguish between ECE-1b, 1c, and 1d. More recent
work was based on the expression of ECE-1 isoforms in transfected
fibroblasts or epithelial cells. In these models, ECE-1a and ECE-1c
were targeted to the plasma membrane, whereas ECE-1b was localized to
intracellular organelles, and ECE-1d displayed an intermediate
distribution (30, 37-39). Neuroendocrine cells constitute another
major cell type expressing ECE-1 in vivo (28, 27). In these
cells, precursors and peptides, as well as processing enzymes, are
sorted between the constitutive and the regulated secretory pathways.
We used the neuroendocrine AtT-20 cells as a model. These are
corticotrope pituitary cells that are specialized in the synthesis and
regulated secretion of adrenocorticotropic hormone (ACTH). In these
cells, we investigated the subcellular distribution and the
heterodimerization of ECE-1 isoforms, as the function of intracellular
ECE-1 is a matter of debate, and the role of dimerization is still unknown.
Antibodies and Reagents--
The antiserum 29 was directed
against a peptide of human ECE-1 catalytic domain (40). The antisera
1206 specific for ECE-1a and 1208 specific for ECE-1b/c/d were directed
against peptides of the human ECE-1 cytosolic domain (56). The
monoclonal antibody ECE-6 directed against ECE-1 has already been
described (41). The antibodies directed against the following markers
were also used: TGN38, provided by Dr. S. L. Milgram (University of
North Carolina at Chapel Hill); ACTH, provided by the NIDDK, National Institutes of Health; transferrin receptor, from Zymed
Laboratories Inc.; Cation-independent mannose-6-phosphate
receptor, provided by Dr. W. J. Brown (Cornell University); lamp-1,
from the Developmental Study Hybridoma Bank (University of Iowa);
cathepsin D, from Santa Cruz Biotechnology (Santa Cruz, CA); M2
monoclonal antibody directed against the FLAG epitope, from Upstate
(Charlottesville, VA); the proprotein convertase furin, from Alexis
(San Diego, CA). Alexa Fluor-546 transferrin and Lysotracker Green were
from Molecular Probes (Eugene, OR). The rab7-GFP construct was kindly
provided by Dr. C. Bucci (Università di Napoli `Federico II',
Italy), and the dynamin-2-GFP wild type and K44A mutant by Dr. M. McNiven (Mayo Clinic, Rochester). Furin cDNA was a gift from Dr.
J. W. Creemers (University of Leuven, Belgium). Bafilomycin A1 was
from Kamiya (Seattle, WA). Clasto-lactacystin was from
Calbiochem (San Diego, CA).
Cell Culture--
AtT-20 cells were maintained at 37 °C in
5% CO2, in high glucose Dulbecco's modified Eagle's
medium (DMEM) (Invitrogen Life Technologies) containing
10% NuSerum IV (Becton Dickinson, Franklin Lakes, NJ), and 2.5%
fetal bovine serum (Invitrogen Life Technologies). AtT-20 cells were
provided by Dr. R. E. Mains (Johns Hopkins University School of
Medicine). Transfected cell lines were cultivated with either one or
both of the following selection agents: 200 µg/ml G418 (Invitrogen,
Carlsbad, CA); 50 µg/ml zeocin (Invitrogen Life Technologies). The
human microvascular dermal endothelial cell line HMEC-1 was cultivated
in MCDB-31 containing 20% fetal bovine serum, 10 ng/ml epidermal
growth factor, and 1 µg/ml hydrocortisone (42).
Site-directed Mutagenesis and Transfection--
Mutations were
generated by PCR-mediated methods. The PCR fragments were cloned into
pCDNA3.1 (Invitrogen Life Technologies) containing the human ECE-1
cDNA (39), at the NheI and HindIII restriction sites. All the cDNA fragments generated by PCR were verified by DNA sequencing after insertion in pCDNA3.1. The
sequence coding for the FLAG epitope was added at the 3'-end of ECE-1
cDNA by PCR. AtT-20 cells were transfected using LipofectAMINE 2000 according to the manufacturer's recommendations (Invitrogen Life Technologies). Clones were first screened by immunoblotting, and then
by immunofluorescence in order to select cells with a homogenous expression. 2-4 independent clones were analyzed for each construct.
For the double transfected cell lines, AtT-20 cells were first
transfected with pCDNA3.1 coding for ECE-1b with a C-terminal FLAG
epitope and for the G418 resistance. A stable clone was then transfected with pcDNA3.1 coding for ECE-1a and for the zeocin resistance.
For transient expression of rab7-GFP and dynamin-2-GFP, cells were
transfected on coverslips using LipofectAMINE 2000 for 3 h and
treated for immunofluorescence 16-20 h later.
Immunofluorescence--
Cells were seeded on 14-mm coverslips,
cultivated for 40-48 h, and fixed with cold methanol for 5 min. They
were then washed with PBS, and nonspecific binding was saturated with
either 1% BSA or 10% normal goat serum in PBS. They were then
incubated with the primary antibodies in 0.1% BSA or 1% normal goat
serum in PBS. Goat or donkey antibodies directed against rabbit IgG were coupled to Alexa Fluor-488 or Alexa Fluor-555 (Molecular Probes)
or to Cy5 (Amersham Biosciences). Goat antibodies directed against
mouse IgG and donkey antibodies directed against goat IgG were coupled
to tetramethylrhodamine (TRITC) (Jackson Immunoresearch) or Alexa
Fluor-546 (Molecular Probes). For antibody uptake experiments, cells
were preincubated for 30 min in DMEM containing 0.1% BSA. They were
then incubated with the 29 antiserum in the presence of 0.1% BSA and 5 mM dithiothreitol for 30 min at 37 °C as previously described (39). Cells were then fixed with methanol and incubated with
a secondary antibody coupled to Alexa Fluor-488. In some experiments,
cells were preincubated in DMEM containing 0.1% BSA and incubated for
20 min at 37 °C with 25 µg/ml Alexa Fluor-546 transferrin before fixation.
Coverslips were mounted with Mowiol and observed with a Leica TCS SP2
confocal microscope equipped with three external lasers (488, 543, and
633 nm) (Leica Microsystems). Images were acquired with a ×63/1.32 PL
APO objective lens. All the double labeling experiments were analyzed
in sequential scanning mode. The confocal sections always corresponded
to the medium level of the cells, except for the ACTH-labeled cells. In
these cells, sections corresponded to the basal level of the cells in
order to detect the secretory granules. The perinuclear zone thus
appeared more diffuse in these images. All the images correspond to
80 × 80 µm fields.
Pre-embedding Immunoperoxidase Staining--
Labeling was
performed directly on AtT-20 cells as previously described (43). Cells
were plated in 35-mm tissue culture dishes. They were fixed in
periodate-lysine-paraformaldehyde for 2 h at room
temperature, washed, preincubated in 50 mM NH4Cl, and
permeabilized with 0.005% saponin. After incubation with specific rabbit antibodies, cells were incubated with goat Fab anti-rabbit IgG
conjugated with peroxidase (Biosys; Compiègne, France). After washing, cells were postfixed in 1% glutaraldehyde, and the peroxidase activity was visualized using 3,3'-diaminobenzidine tetrahydrochloride. Cells were postfixed in 1% osmium tetroxide, dehydrated, and embedded in situ in Epon. Selected areas of immunoreactive cells were
sectioned as previously described. Ultrathin sections were examined at
the electron microscope without further staining.
Metabolic Labeling and Immunoprecipitation--
150,000 cells
were seeded in 12-well plates. 40 h later, cells were labeled for
30 min with 100 µCi/ml of [35S]methionine and cysteine
(ProMix, Amersham Biosciences, Piscataway, NJ). They were then directly
extracted or chased in DMEM containing 2% fetal bovine serum. Cells
were extracted on ice with 0.5% Nonidet P-40 and 0.5% deoxycholate in
25 mM Tris-HCl pH 7,4 containing 100 mM NaCl
and 5 mM EDTA. Before extraction, a protease inhibitor mixture (Sigma) was added to the lysis buffer. Lysates were spun for 10 min at 15,000 × g at 4 °C. The supernatant was
collected and ECE-1 was immunoprecipitated, using either the 29 or 1206 or 1208 antibodies. All steps were done at 4 °C. Briefly, extracts were precleared in the presence of protein A coupled to Sepharose CL-4B
(Amersham Biosciences). The supernatants were then incubated for 2 h in the presence of the antiserum, and the immune complexes were
precipitated using protein A coupled to Sepharose CL-4B, washed once
with the lysis buffer, once with PBS containing 0.5 M NaCl,
and twice with PBS. Immunoprecipitated material was analyzed by
SDS-PAGE. Gels were directly exposed to a Biomax film (Kodak). For the
measurement of the degradation rate of ECE-1, quantitative analysis of
radiolabeled material was done using an FX Molecular Imager and
QuantityOne software (Bio-Rad, Hercules, CA). In order to study the
degradation pathway of ECE-1b, cells were chased in the presence of 10 mM NH4Cl or 1 µM Bafilomycin A1
or 1 µM clasto-lactacystin.
In some experiments, immunoprecipitated material was analyzed by
immunoblotting. Proteins were separated by SDS-PAGE and transferred to
polyvinylidene difluoride membranes. Nonspecific binding was saturated
in 10 mM Tris, 150 mM NaCl, pH 7.4 containing
5% low fat milk, and membranes were then incubated with the antibodies in the same buffer containing 0.5% low fat milk and 0.2% Tween-20. Alkaline phosphatase activity was detected using Attophos (Promega, Madison, WI) as a substrate and analyzed with an FX Molecular Imager
and Quantity One software (Bio-Rad).
Determination of ECE-1 Activity--
ECE-1 activity was measured
using the BK2 peptide (aminomethylcoumarin-RPPGFSAFR-dinitrophenyl) as
a substrate. The proteolysis of this quenched peptide at the
Ala7-Phe8 bond by ECE-1 has already been
characterized (44). The BK2 peptide was synthesized by Sigma-Genosys
(The Woodlands, TX). 5000 cells were seeded per well of 96-well plates
40 h before activity measurement. Cells were preincubated 60 min
in serum-free medium. They were then washed twice, and the assay was
performed in Hank's Balanced Salt Solution containing 25 mM PIPES pH 6.5, in the presence of the following protease
inhibitors: 1 mM AEBSF, 800 nM aprotinin, 20 µM leupeptin, 36 µM bestatin, 15 µM pepstatin A, and 14 µM E-64. The
experiments were performed in the presence of 30 µM of
the BK2 peptide. Cells were preincubated in the reaction buffer, in the
presence or absence of 0.4% n-octyl-glucoside, at 37 °C
for 15 min prior to addition of the substrate. The activity was
assessed in triplicate wells for 30 min at 37 °C. Fluorescence was
measured using a Gemini XS microtiter plate reader and pro-Max software
(Molecular Device, Sunnyvale, CA) using 330 nm excitation and 395 nm
emission wavelengths. The protein content was determined in a duplicate
96-well plate using the BCA protein assay (Pierce). ECE-1 activity was
normalized to the expression level of the enzyme in each clone as
measured by the activity detected in the presence of 0.4% of the
detergent n-octyl-glucoside. Values are relative fluorescence units per min per µg of total proteins. They
corresponded to the mean activity determined in triplicate culture
wells. Experiments were reproduced two or three times and provided
similar ratios of extracellular per total activity.
Characterization of ECE Isoforms in AtT-20 Cells--
Mouse
pituitary corticotrope AtT-20 cells were used to analyze the function
of ECE-1 isoforms in neuroendocrine cells. The four ECE-1 isoforms were
stably expressed in AtT-20 cells at similar levels. We first analyzed
the maturation of ECE-1 by metabolic labeling (Fig.
1B). ECE-1 forms
disulfide-bound dimers (20-22). Immunoprecipitation demonstrated that
the 4 ECE-1 isoforms dimerized with similar kinetics as only the
dimeric form was detected after a 2-h chase. At the end of the labeling
period, a monomeric form could be detected together with a
diffuse band of higher molecular weight. This band
disappeared after the chase period, which suggested that it
corresponded to intermediate dimers of ECE-1 either incompletely folded
or incompletely glycosylated. These experiments also provided information concerning the degradation rate of ECE-1. Whereas the
half-life of ECE-1a, 1c, and 1d was ~7 h, ECE-1b was degraded much
more rapidly, with a half-life of ~3 h.
We then investigated the subcellular distribution of ECE-1 isoforms in
AtT-20 cells by immunofluorescence (Fig. 1C). No endogenous ECE-1 was detected in wild type cells. ECE-1a and 1c were mainly present at the plasma membrane, whereas ECE-1b was detected inside the
cells. A similar pattern of localization was observed in transfected CHO cells (37, 39). In AtT-20 cells, ECE-1d was faintly detected on the
plasma membrane and was mainly found inside the cells. We had observed
the prominent labeling of the plasma membrane for this isoform in CHO
cells (30). This difference could result from the higher level of
expression in CHO cells as a strong labeling of the plasma membrane was
also detected in AtT-20 cells expressing higher levels of ECE-1d. The
major intracellular localization of ECE-1d was also observed when a
GFP-ECE-1d chimera was expressed in AtT-20 cells and when ECE-1d was
expressed in the epithelial MDCK cells (data not shown). In addition,
the same subcellular distributions were observed using different
antisera directed against the catalytic domain of ECE-1 (29 and ECE-6)
or directed against the cytosolic domains of ECE-1 isoforms (1206 and
1208), as well as using ECE-1 isoforms tagged with a FLAG epitope.
However, there were some differences in the distribution of the
intracellular isoforms: ECE-1d labeled a unique diffuse area in the
perinuclear region, whereas ECE-1b was concentrated in numerous large
vesicles dispersed in the cytoplasm.
We further investigated the presence of ECE-1 on the plasma membrane by
measuring its extracellular activity. For this purpose, cells were
incubated in the presence of the fluorescent quenched peptide BK2. Some
endogenous processing of BK2 was detected with wild type AtT-20 cells
(Fig. 1D). It was inhibited by 100 µM
ortho-phenantrolin and by 1 mM EDTA, but not by 100 µM phosphoramidon, the common NEP/ECE-1 inhibitor. The
processing detected with cells expressing ECE-1a was ~3-fold higher
both in the extracellular medium and in the presence of the detergent
n-octyl-glucoside. It was not inhibited by 6 µM batimastat, a matrix metalloprotease inhibitor, nor by
100 µM thiorphan, an inhibitor of NEP, nor by 1 µM lysinopril, an inhibitor of angiotensine converting
enzyme. On the other hand, 1 µM phosphoramidon almost
completely inhibited BK2 processing by AtT-20 cells expressing ECE-1
(data not shown). Background activity detected with wild type AtT-20
cells was subtracted from the activity measured with AtT-20/ECE-1
cells. ECE-1a and 1c displayed a high extracellular activity, whereas
ECE-1d and ECE-1b extracellular activity was much lower, in agreement
with the immunofluorescence data (Fig. 1E).
Taken together, these experiments provide new data on the most recently
identified isoform, ECE-1d. In AtT-20 cells, this isoform was mainly
present in intracellular compartments although low levels could also be
detected at the plasma membrane. In addition, these experiments
demonstrate that ECE-1a, 1b and 1c displayed the same localization in
neuroendocrine cells, as in fibroblasts (37, 39).
ECE-1b and 1d Were Not Concentrated in the Secretory
Pathway--
The labeling observed with ECE-1b and ECE-1d suggested
that these isoforms could be targeted to different intracellular
compartments (Fig. 1C). Previous studies had proposed that
ECE-1b specifically functioned in the intracellular production of
endothelin. Such a hypothesis would require the concentration of ECE-1b
in the secretory pathway. Thus, we investigated the expression of
ECE-1b and 1d in the secretory pathway by double immunofluorescence. Big endothelin is first generated in the trans-Golgi network (TGN) by
the processing of pro-endothelin by furin or PC7/PC8/LPC
(11, 12), both resident enzymes of this compartment (Refs. 45 and 46
and see Fig. 5C). We thus investigated the presence of
ECE-1b and
These experiments demonstrated that ECE-1b and 1d were present in the
Golgi zone to a limited extent. Unlike the proprotein convertase furin,
which cleaves pro-endothelin in the TGN, ECE-1b, and 1d were
accumulated in a compartment that is distinct from the secretory pathway.
ECE-1b and 1d Were Concentrated in Distinct Endosomal
Compartments--
The strong intracellular staining of ECE-1b and 1d
that was not localized to the secretory pathway suggested that these
isoforms could be concentrated in the endosomal system. We thus
investigated their localization using the transferrin receptor and
internalized transferrin as markers for recycling endosomes,
mannose-6-phosphate receptor and rab7 as markers for late
endosomes/multivesicular bodies, and cathepsin D and Lamp-1 as markers
for lysosomes. ECE-1d was completely co-localized with the transferrin
receptor, which was detected in the diffuse perinuclear compartment
described above (Fig. 3A).
Similar results were obtained using internalized transferrin as a
marker (data not shown). These data demonstrated that ECE-1d was
concentrated in the recycling endosomes. ECE-1b was also detected in
the recycling endosomes. However, the large vesicles that contained
most of the ECE-1b labeling were not labeled with the transferrin
receptor. On the other hand, ECE-1b was partially co-localized with a
rab7-GFP chimera, whereas ECE-1d was not (Fig. 3B). As
already demonstrated for other endosomal proteins (47), the
overexpression of rab7-GFP did not modify the distribution of ECE-1b
and 1d. In agreement with these results, ECE-1b was also partially
co-localized with the mannose-6-phosphate receptor, a late endosome
marker (data not shown). ECE-1b and 1d were not detected in lysosomes,
as demonstrated by cathepsin D (Fig. 3C) or Lamp-1 (data not
shown) labeling or with the lysosomal marker Lysotracker (data not
shown). The same pattern of co-localizations with endosome markers was
observed when ECE-1b and 1d were transiently expressed in HeLa cells
(data not shown).
In order to further identify the large vesicles labeled with ECE-1b, we
used electron microscope immunocytochemistry. These experiments
demonstrated the presence of ECE-1b in multivesicular bodies, as
identified by their morphology (Fig.
4A, arrowheads). We
had already described the expression of ECE-1b in these structures in
transfected CHO cells (39). In addition, ECE-1b was detected in a
central zone consisting of aggregated small vesicles (Fig. 4A, asterisk). These membrane structures likely
corresponded to the recycling endosomes that were co-labeled with the
transferrin receptor and ECE-1b (Fig. 3A). Degradation of
proteins is initiated in multivesicular bodies, the transport
intermediate to lysosomes. The presence of ECE-1b in these organelles
was thus in agreement with the rapid degradation of this isoform (Fig.
1B). We further investigated the degradation of ECE-1b with
inhibitors (Fig. 4B). After a 4-h chase period, ECE-1 was
completely glycosylated and most of the neosynthesized ECE-1 was
degraded (Fig. 4B, lane 2). The lysosomotropic agent
(NH4Cl) and the vacuolar ATPase inhibitor Bafilomycin A1,
which both prevent the luminal acidification of the pH, could block
ECE-1b degradation. On the other hand, the cell-permeant proteasome
inhibitor clasto-lactacystin had no effect on ECE-1b
degradation.
ECE-1b and 1d Were Transiently Expressed at the Cell
Surface--
The detection of extracellular enzymatic activity from
cells transfected with ECE-1b and ECE-1d suggested a higher level of expression on the plasma membrane than revealed by immunofluorescence. The transient presence of ECE-1b and ECE-1d at the cell surface could
account for this difference. In order to investigate such possibility,
we performed antibody uptake experiments. We used cells expressing
ECE-1c as a control since this isoform was detected both at the cell
surface and inside the cells (Fig. 1C). The cells were first
incubated in the presence of the 29 antiserum, directed against the
ectodomain, at 37 °C in order to detect internalized ECE-1. They
were then fixed with methanol and the total ECE-1 content was detected
with the monoclonal ECE-6 antibody. AtT-20 cells expressing ECE-1c
could uptake the 29 antiserum. The labeling of internalized ECE-1c
completely overlapped that of total ECE-1c. In addition, both labeling
provided a signal as intense, as shown by the yellow appearance on the
merged image. These results suggested a high rate of recycling for this
isoform. ECE-1b and ECE- 1d could also internalize the 29 antiserum
(Fig. 5A), but the level of
antibody uptake was much lower than for cells expressing ECE-1c. Antibody uptake was very faint in some cells expressing ECE-1b (asterisk in Fig. 5A). To confirm these results,
AtT-20 cells expressing ECE-1b or ECE-1d were transiently transfected
with either the wild type dynamin-2 or the dominant-negative K44A
mutant. Dynamin-2 is involved in the internalization of plasma membrane proteins (for review see Ref. 48). Transfection of the wild type
dynamin did not modify the distribution of ECE-1b and 1d. The K44A
mutant prevents the endocytosis of plasma membrane proteins. When
expressed with the dominant-negative mutant, ECE-1b and ECE-1d were
detected at the cell surface (Fig. 5B). As a control, we investigated the effect of the expression of the dynamin mutant on the
distribution of furin, the enzyme responsible for the first step in the
processing of pro-endothelin. Furin was completely co-localized with
TGN38 (Fig. 5C), as already described (45). The K44A dynamin
mutant did not modify the distribution of furin nor of TGN38 (Fig.
5C). These data were in agreement with the concentration of
ECE-1b and 1d in a compartment distinct from the TGN. They further
demonstrated that these ECE isoforms were concentrated in some
endosomal compartment cycling with the plasma membrane. Taken together,
these data were in agreement with the extracellular activity from
AtT-20 cells expressing ECE-1b and ECE-1d (Fig. 1D). They
suggested that these isoforms were targeted to the endosomal system,
both directly from the TGN and after transport to the plasma membrane.
Recycling of ECE-1b and 1d was however occurring at a much lower rate
than for the plasma membrane isoform ECE-1c.
Identification of the Signals Responsible for Targeting to the
Endosomal Pathway--
We have already shown that the intracellular
localization of ECE-1b is due to two leucine-based motifs: one common
to ECE-1b, 1c and 1d (Leu31-Val32 in ECE-1b),
and one specific for ECE-1b (Leu12-Leu13) (39).
Interestingly, ECE-1d also contains a specific leucine-based motif
(Val8-Leu9). In order to investigate the
relative role of these motifs in targeting to different endosomes, we
mutated these into alanines and exchanged them between ECE-1b and
ECE-1d. The mutation of ECE-1b Leu31-Val32 into
Ala-Ala (b LV/AA) did not affect its sorting to the degradation pathway
(Table I). On the other hand, the loss of
the ECE-1b Leu12-Leu13 (b LL/AA) motif reduced
the degradation rate of ECE-1b (Table I). In agreement with these
results, the distribution of the b LV/AA mutant was not modified (Fig.
6C), whereas the loss of the
ECE-1b-specific LL motif (b LL/AA) resulted in an intracellular distribution resembling that of ECE-1d with increased presence of the
enzyme at the cell surface (Fig. 6A). Thus, only the
b-specific di-leucine motif is involved in the targeting to the
degradation pathway. However, the nature of the residues in
the di-leucine motif could not alone account for the rapid
degradation of ECE-1b. First, the ECE-1b LL/VL mutant was degraded as
rapidly as wild type ECE-1b (Table I) and displayed the same
distribution as wild type ECE-1b (Fig. 6B). Second, the
ECE-1d VL/LL mutant (d VL/LL) was not degraded as rapidly as ECE-1b
(Table I). The distribution of this mutant however was slightly
modified, and ECE-1d VL/LL was detected both in the central recycling
endosomes and in surrounding large vesicles (Fig. 6E). In
addition, the VL motif in ECE-1d was involved in the intracellular
localization of this isoform, as its mutation into Ala-Ala could
increase its plasma membrane targeting (Fig. 6D).
Taken together, these results suggested that ECE-1b contained
additional information responsible for its rapid degradation. In order
to test this hypothesis, we constructed a chimera consisting of the 10 N-terminal residues of ECE-1b followed by the
Val8-Leu9 motif of ECE-1d (b-d). This chimera
was degraded as rapidly as ECE-1b, suggesting that another determinant
important for ECE-1b degradation was present at the N terminus of the
di-leucine motif (Table I). In order to identify a second signal
involved, we mutated each residue between Gly3 and
Ser10 into Ala (data not shown). This alanine scan could
not identify another signal responsible for the rapid degradation of
ECE-1b. These experiments suggested that, in addition to the di-leucine motif, the 10 N-terminal residues of ECE-1b contain some sorting determinants involved in the rapid degradation of this isoform but not
encoded by the primary sequence.
Heterodimerization of ECE Isoforms--
ECE-1 forms dimers bound
by a disulfide bond at Cys412 in the extracellular domain
(22). Our metabolic labeling experiments demonstrated that 100% of
ECE-1 is expressed as dimers within 2 h after synthesis (Fig.
1B). We investigated the possibility that ECE-1 forms
heterodimers in cells that express several isoforms. We used antiserum
1206 directed against the cytosolic domain of ECE-1a, and antiserum
1208 directed against a peptide common to the cytosolic domains of
ECE-1b, 1c, and 1d. The specificity of these antibodies was
demonstrated by immunofluorescence and immunoprecipitation of extracts
from AtT-20 cells overexpressing ECE-1a or ECE-1b (Fig.
7, A and B). We
investigated ECE-1 heterodimerization in endothelial cells that
co-express endogenous ECE-1 isoforms. For this purpose, we used
endothelial cells from the HMEC-1 cell line. These dermal microvascular
cells express many endothelial markers (42). After metabolic labeling,
the cell extracts were immunoprecipitated with the three antisera. We
could detect ECE-1 isoforms in HMEC-1 cell extracts (Fig.
7C, lanes 1-3). Antiserum 1206 could detect very
little ECE-1a. In order to analyze the presence of heterodimers, the
1206 immune complexes were denatured, and ECE-1b/c/d was
immunoprecipitated with antiserum 1208 (Fig. 7C, lane
4). These experiments allowed the detection of ECE-1 dimers that
contained ECE-1a, from the first immunoprecipitation, and either ECE-1b
or 1c or 1d, as demonstrated by the second immunoprecipitation. The low
efficacy of the 1206 antiserum in immunoprecipitation experiments, as
compared with that of antiserum 29 directed against the catalytic
domain of ECE-1 (Fig. 7B) prevented the quantitation of
heterodimerization.
AtT-20 cell lines that co-expressed ECE-1a and 1b were generated to
further investigate heterodimerization of ECE-1 isoforms (Fig.
8, total lysate). The presence
of heterodimers was first investigated. ECE-1b was immunoprecipitated
from the lysates with the 1208 antiserum and the immune complexes were
analyzed by immunoblotting with either the 1206 or the 1208 antisera.
These experiments demonstrated the presence of heterodimers containing
ECE-1a and ECE-1b in the double transfected cells (Fig. 8, 1208 immunoprecipitate).
We then investigated whether ECE-1 heterodimerization affected the
subcellular distribution of these isoforms. For this purpose, we used
an ECE-1b construct that contained a FLAG epitope at the C terminus, in
order to allow for the double immunolabeling using a monoclonal
antibody directed against FLAG and antiserum 1206. The introduction of
the FLAG epitope did not modify the intracellular distribution of
ECE-1b (compare Figs. 1C and
9A). Upon co-expression of
ECE-1bFLAG with ECE-1a, the localization of ECE-1a was partly shifted
from the plasma membrane to intracellular vesicles. These vesicles also
contained ECE-1b and displayed the pattern characteristic for this
isoform (Fig. 9A). It is important to notice that, whereas the distribution of ECE-1a was modified, that of ECE-1b was not: no
ECE-1b was detected at the plasma membrane upon co-expression with
ECE-1a, even though some ECE-1a was still targeted to the plasma
membrane.
To identify the intracellular compartment that contained both ECE-1a
and ECE-1b, cells expressing either ECE-1a alone or together with
ECE-1b were transiently transfected with the rab7-GFP chimera. Double
immunofluorescence in these cells demonstrated that ECE-1a/ECE-1b dimers were partly present in late endosomes/vesicular bodies, as both
isoforms were detected in some rab7 containing vesicles (Fig.
9B, arrowheads and white in merged
inset). Some intracellular ECE-1a was not co-localized with
rab7-GFP, even though it was co-localized with ECE-1b (Fig.
9B, arrows and pink in merged
inset). This corresponded to the observation that ECE-1b
alone was only partially present in late endosomes/multivesicular
bodies (Figs. 3B and 4A). Control experiments
demonstrated that ECE-1a was not present in rab7-containing vesicles
when expressed alone (Fig. 9B).
To investigate the function of ECE-1 heterodimerization, we compared
the extracellular activity of ECE-1a when expressed alone or in
combination with ECE-1b. The insertion of the FLAG epitope at the C
terminus of ECE-1b resulted in the loss of enzyme activity of this
construct (Fig. 9C). Similar data were obtained by measuring ET-1 by ELISA after addition of big-ET-1 in the medium of cells expressing ECE-1bFLAG.2 We
could not measure the effect of the FLAG epitope on the activity of
ECE-1a/ECE-1bFLAG heterodimers. In such heterodimers, the FLAG epitope
could either not alter or decrease the ECE-1a activity detected. Thus,
our results could overestimate the ratio of extracellular per total
ECE-1a activity in the double transfected cell lines. Upon
co-expression of the two isoforms, the extracellular activity of ECE-1a
per total activity was decreased (Fig. 9D). The activity was
measured in three independent clones expressing ECE-1a alone and three
independent clones expressing ECE-1a and ECE-1bFLAG. The mean value of
the ratio of extracellular activity per total activity was 90.4, 59.7, and 80,6 for ECE-1a-expressing cells, and 60.5, 51.7, and 59.3 for
double-transfected cells. Values are the mean of two or three
independent experiments in which activity was determined in triplicate
culture wells. The ratio of ECE-1a and ECE-1bFLAG in these clones was
similar as estimated by Western blotting with the 1206 and 1208 antisera.
ECE-1 belongs to the family of NEP metalloproteases. Among these
enzymes, ECE-1 has two unique characteristics: first, it is present and
active both at the cell surface and in intracellular organelles;
second, it exists as covalent dimers. In the present paper, we have
investigated these two aspects of ECE-1 cell biology. Our experiments
provided the first extensive analysis of the intracellular distribution
of the 4 ECE-1 isoforms in the secretory pathway and in the endosomal
system. The two isoforms localized in intracellular compartments,
ECE-1b and 1d, displayed distinct but complementary patterns of
distribution in the endosomal system: ECE-1d was present in the
recycling endosomes and ECE-1b in the late endosomes/multivesicular bodies. Using biochemical methods, we could show that ECE-1 isoforms exist as heterodimers. The discovery of such heterodimers suggested a
novel role for the isoform-specific targeting of ECE-1. We showed that
the targeting signal responsible for the intracellular distribution of
ECE-1b could redirect the plasma membrane ECE-1a to the endosomal system. These data demonstrated that the specific N terminus of ECE-1b
constitutes a regulatory domain that is able to modify the distribution
of other ECE-1 isoforms. Such a regulatory mechanism could be
physiologically relevant as many cell types, including endothelial
cells, co-express endogenously several ECE-1 isoforms (37, 30).
Our study was undertaken in the AtT-20 cell line due to the important
function of endothelins in regulating the activity of neuroendocrine
cells through autocrine and paracrine mechanisms (Ref. 49; for review
see Ref. 3). Endothelins have been detected in the secretory granules
of neuroendocrine cells (50), thus raising the possibility that ECE-1
was targeted to the secretory granules, like common neuroendocrine
endopeptidases. Our results rejected the possibility that big-ET was
processed in secretory granules, as none of the intracellular isoforms
were detected in the secretory granules. Our data thus suggested that
the mature endothelin detected in the secretory granules might result
from processing in the TGN. Processing of pro-endothelin by furin in this compartment is the first step that is required for ECE-1 to
release endothelin from its precursor (51). Even though we did not find
that ECE-1 was concentrated in the TGN, like furin is (45), ECE-1 is
transported through this compartment during biosynthesis. The
intracellular processing of big-ET in the TGN would thus not be a
specialized function for any ECE-1 isoform. Indeed, we have already
shown that ECE-1a, a plasma membrane isoform, could generate endothelin
in the secretory pathway as well as in the extracellular medium (16).
Whereas ECE-1b and ECE-1d could generate endothelin in the secretory
pathway, these isoforms should not be considered more specific than
ECE-1a or 1c for this processing. In support of this, big-ET is the
main species secreted from cells that co-express pre-pro-endothelin and
an intracellular isoform of ECE-1, thus indicating the low efficiency
of intracellular processing of big-ET by ECE-1 (14). On the other hand,
ECE-2 could specifically fulfill this role as it displays the acidic optimum pH required for efficient conversion of precursors in the lumen
of the TGN and the secretory vesicles (24), and as 2 of the 4 recently
identified isoforms of ECE-2 were found in intracellular compartments
(33).
Previous studies aimed at identifying the intracellular compartments
containing ECE-1b have produced controversial results. ECE-1b was
detected in the Golgi apparatus, where it was co-localized with the
Golgi marker wheat germ agglutinin in endothelial cells (35) and with
TGN38 in transfected fibroblasts (37, 39). In addition, the presence of
ECE-1b in late endosomes/multivesicular bodies has been described in
transfected epithelial cells and fibroblasts (38, 39). Our results were
in agreement with these latter studies. In addition, we demonstrated
that ECE-1d was also present in the endosomal system, but concentrated
in the recycling endosomes. We showed that ECE-1b and 1d were cycling
between the plasma membrane and endosomes using three different lines
of evidence: first, extracellular activity was detected from cells
expressing ECE-1b and 1d; second, cells expressing these isoforms were
able to capture antibodies directed against the ectodomain; third, expression of a dominant negative mutant of dynamin, which blocks internalization of proteins, retained these ECE-1 isoforms at the
plasma membrane, but had no effect on furin and on TGN38. In agreement
with these results, ECE-1b was co-localized with rab5, a marker for
early endosomes, which is the first intracellular compartment where
endocytic vesicles fuse (38).
The ECE-1b-specific di-leucine motif was responsible for the
intracellular retention of this isoform (52, 39). In the present study,
we demonstrated that the intracellular localization of ECE-1d also
relies on the presence of a specific leucine-based motif
(Val8-Leu9). We had shown that a second
leucine-based motif (Leu31-Val32), common to
ECE-1b, 1c and 1d, was also involved in ECE-1b intracellular retention
(39). Here we found that this signal is not involved in ECE-1b
degradation. On the other hand, the ECE-1b specific di-leucine motif
(Leu12-Leu13) was responsible for the rapid
degradation of this isoform. This motif alone was however not
sufficient. Even though leucine-based motifs have been implicated at
multiple steps of intracellular sorting in the TGN, the endosomes and
the plasma membrane (for review see Ref. 53), the determinants
responsible for the specificity of the transport steps involved remain
to be identified (54). Several lines of evidence suggested that the
sorting of ECE-1b relied on additional signal to the LL motif: first,
the mutation of the ECE-1b LL motif only slightly increased the plasma
membrane localization, compared with the mutation of ECE-1d VL motif;
second, exchanging the VL and LL motifs modified ECE-1d degradation and localization, but not that of ECE-1b; third, the chimera containing the
N-terminal residues of ECE-1b upstream of the VL motif of ECE-1d
resulted in the rapid degradation of this construct. Our results
suggested that ECE-1b N terminus encodes another complementary signal
that does not reside in the primary structure. The additional signal
could correspond to a secondary structure or to a post-translational modification that would increase the affinity of the di-leucine motif
for an associated protein responsible for sorting to the degradation
pathway. In the case of bovine isoforms of ECE-1, Emoto et
al. (55) have provided evidence for the targeting of bovine ECE-1b
to the plasma membrane, and for the sorting of bovine ECE-1a to
lysosomes (55). It should be noted that bovine ECE-1b corresponds to
human ECE-1c, which we also found targeted to the plasma membrane, and
that bovine ECE-1a is poorly conserved with human ECE-1a. Indeed, the
targeting signals responsible for bovine ECE-1a sorting are not present
in human ECE-1.
The detection of ECE-1 isoforms in the endosomal system could
correspond to some novel physiological function of this enzyme, as
big-ET is not present in these compartments. Endosomal ECE-1 could
constitute an intracellular pool that could be rapidly translocated to
the plasma membrane upon stimulation of the cells, as suggested by the
cycling of ECE-1b and ECE-1d between endosomes and plasma membrane.
Alternatively, the intracellular retention of some ECE isoform could
regulate the distribution of other isoforms through direct protein
interaction. ECE-1 is expressed as disulfide-linked dimers, but the
role of ECE-1 dimerization has not yet been found (20-22). We have
already showed that ECE-1 is present only as dimers on the plasma
membrane of endothelial cells (56). The present analysis of ECE-1
biosynthesis demonstrated that the complete dimerization of ECE-1 is a
feature common to the 4 isoforms. Dimerization occurs rapidly after
synthesis, most probably in the endoplasmic reticulum as we could
observe the transient appearance of dimer intermediates that most
likely corresponded to incompletely glycosylated or unfolded ECE-1. The
Cys412 residue, present in the catalytic domain, is
responsible for the dimerization (22). The role of ECE-1 dimerization
has been investigated using site-directed mutagenesis of this cysteine. Mutation into a serine did not modify the glycosylation and the subcellular distribution of ECE-1. However, this mutation slightly modified the enzymatic characteristics of ECE-1 (22, 57).
Here, we provided evidence for the formation of heterodimers in
endothelial cells endogenously co-expressing several isoforms. The
investigation of the formation of heterodimers in these cells was
however limited due to the low level of expression of ECE-1a, and to
the lack of antibodies specific for either ECE-1b or 1c or 1d.
Therefore, we generated AtT-20 cell lines that co-expressed ECE-1a, the
isoform with the higher plasma membrane expression, and ECE-1b, which
displays the more stable intracellular localization. In double
transfected cells we could confirm the presence of heterodimers. Both
isoforms were colocalized in intracellular vesicles and the extracellular activity of ECE-1a was decreased. In addition, triple labeling experiments demonstrated that ECE-1a was translocated to the
late endosomes/multivesicular bodies that contained ECE-1b. Importantly, heterodimerization of these isoforms did not increase the
expression of ECE-1b at the plasma membrane, thus demonstrating the
dominant role of ECE-1b sorting signals. This was in agreement with the
active sorting of ECE-1b to endosomes through a di-leucine motif,
whereas ECE-1a might be transported to, and maintained at, the plasma
membrane by the lack of a sorting signal rather than by active sorting.
Taken together, these data suggested that heterodimerization could
regulate ECE-1 extracellular activity through the translocation of the
plasma membrane isoform to intracellular compartments. Such a mechanism
could be physiologically relevant at two levels. First at the
expression level: heterodimerization could be directly regulated by the
expression of each isoform. In fact, our experiments suggested that
ECE-1 dimerization occurred early in the secretory pathway. The
expression of ECE-1 isoforms is controlled by the presence of alternate
promoters in the gene structure (29, 30, 34). Recent studies have
provided evidence for the isoform-specific regulation of expression
(58-61). Heterodimerization could thus modulate the shift from plasma
membrane activity to intracellular activity generated at the level of
translation. In support of this hypothesis, the ECE-1 activity measured
during liver wound healing was unchanged whereas the expression of
ECE-1 isoforms was differentially regulated (58). An additionnal level
of regulation could correspond to the exchange of the dimerized
isoforms. Recent studies have provided evidence for the presence of
protein disulfide isomerase activities at the plasma membrane (62-65).
In this regard, the cycling of ECE-1b and ECE-1d between the plasma
membrane and endosomes could provide a mean for internalizing the
plasma membrane ECE-1a and ECE-1c through disulfide exchange mediated
by a plasma membrane disulfide isomerase.
Processing enzymes are regulated through different pathways, like
activation of proenzymes or regulation of activity by endogenous inhibitors. ECE-1 is not synthesized as a proenzyme, and no associated protein or endogenous inhibitor has been documented so far. In the
present study, we propose that heterodimerization of ECE-1 isoforms
could constitute a mean for regulating their distribution and,
subsequently, their extracellular activity. Heterodimerization could
also be important for other metalloproteases like aminopeptidases and
matrix metalloproteases, which are known to form disulfide-linked dimers.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Expression of ECE-1 isoforms in AtT-20
cells. A, schematic representation of the structure of
ECE-1 isoforms. The peptides used for generating the polyclonal
antibodies 29, 1206, and 1208 are indicated by the bars. B,
biosynthesis of ECE-1 isoforms. AtT-20 cells expressing ECE-1a, ECE-1b,
ECE-1c, ECE-1d were metabolically labeled for 30 min and chased for the
indicated time periods. ECE-1 was immunoprecipitated with antiserum 29. Immunoprecipitates were separated by SDS-PAGE under nonreducing
conditions and radiolabeled proteins were detected by autoradiography.
Monomeric and dimeric ECE-1 are indicated by arrows. The
asterisk corresponds to intermediates in the formation of
dimers. C, subcellular localization of ECE-1 isoforms.
AtT-20 cells (WT) and stable clones expressing ECE-1a,
ECE-1b, ECE-1c, and ECE-1d were fixed with cold methanol and ECE-1
immunoreactivity was detected using the monoclonal antibody ECE-6.
D and E, catalytic activity of the ECE-1
isoforms. D, cells were incubated in the presence of BK2 as
described under "Materials and Methods." Wild type (open
bars) and ECE-1a-expressing cells (closed bars) were
incubated in the absence or presence of n-octyl-glucoside in
order to measure the extracellular and the total activity,
respectively. E, catalytic activity was measured from cells
expressing each ECE-1 isoform. Activity of wild type AtT-20 cells was
subtracted and extracellular activity was normalized to ECE-1
expression as measured by total ECE-1 activity in the presence of
n-octyl-glucoside. Results are expressed as relative
fluorescence unit per µg of total protein per min.
1d in the TGN, using TGN38 as a marker. These isoforms
were not concentrated in this compartment (Fig.
2A). We also investigated the
presence of ECE-1b and 1d in the regulated secretory pathway using ACTH
as a marker. Proopiomelanocortin, the precursor of ACTH, is the
secretory product specific for AtT-20 cells. The antibody directed
against ACTH labeled the juxtanuclear zone, corresponding to the Golgi
apparatus, and the secretory granules accumulated in the cell processes
(arrowhead in Fig. 2B). ECE-1b and 1d
colocalization with ACTH was faint in the Golgi zone, and absent in the
secretory granules (Fig. 2B). We could further demonstrate that these ECE-1 isoforms were not concentrated in the secretory pathway, as they were not redistributed to the endoplasmic reticulum by
brefeldin A as ACTH was (data not shown).
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Fig. 2.
ECE-1b and 1d are not concentrated in the
secretory pathway. AtT-20 cells stably expressing either ECE-1b or
ECE-1d were fixed with cold methanol. ECE-1 co-immunolocalization was
performed using polyclonal antibodies directed against TGN38
(A), a marker for the TGN, or ACTH (B), a marker
for the regulated secretory pathway, and the monoclonal antibody ECE-6.
ACTH was present in the Golgi zone and in the secretory granules
accumulated in the cell processes
(arrowheads).
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Fig. 3.
ECE-1b and 1d are concentrated in endosomal
compartments. AtT-20 cells expressing either ECE-1b or ECE-1d were
fixed with cold methanol. ECE-1 immunoreactivity was detected using
antiserum 29 (A, B, and C). The
transferrin receptor, a marker for the recycling endosomes, was
immunolocalized with a monoclonal antibody (A) and cathepsin
D, a marker for lysosomes, with a goat antibody (C).
Alternatively, AtT-20 cells stably expressing either ECE-1b or ECE-1d
were transiently transfected with a rab7-GFP chimera (B), a
marker for the late endosomes/multivesicular bodies, and ECE-1
immunoreactivity was detected 16 h after transfection.
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Fig. 4.
ECE-1b is present in multivesicular
bodies. A, ECE-1b was detected by immunoelectron
microscopy using the 29 antiserum. Immunoperoxidase activity was
present both in aggregated small vesicles that corresponded to the
recycling endosomes (asterisk) and in the large
multivesicular bodies that surrounds them (arrowheads).
Bar, 1 µm. B, degradation of ECE-1b. AtT-20
cells expressing ECE-1b were metabolically labeled for 30 min and
directly extracted (lane 1) or chased for 4 h under
control conditions (lane 2) or in the presence of 1 µM clasto-lactacystin (lane 3) or
10 mM NH4Cl (lane 4) or 1 µM
Bafilomycin A1 (lane 5). ECE-1 was immunoprecipitated with
antiserum 29. Immunoprecipitates were separated by SDS-PAGE under
reducing conditions and radiolabeled proteins were detected with a
Molecular Imager. Arrowhead indicates the position of
core-glycosylated ECE-1. Arrow indicates the position of
mature ECE-1.
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Fig. 5.
ECE-1b and ECE-1d are transiently expressed
at the cell surface. A, AtT-20 cells expressing ECE-1b,
ECE-1c, or ECE-1d were incubated with antiserum 29 at 37 °C in order
to detect the plasma membrane and internalized ECE-1. Cells were then
fixed with cold methanol and incubated with the monoclonal antibody
ECE-6 in order to detect the total ECE-1. Confocal analysis was
performed using the same settings for internalized ECE-1 and for total
ECE-1 in the three cell lines in order to provide semiquantitative
information. A cell expressing ECE-1b unable to uptake antibody is
shown (asterisk). B, AtT-20 cells expressing
ECE-1b or ECE-1d were transiently transfected with the GFP chimera of
either the wild type or the K44A mutant of dynamin-2
(green). ECE-1 immunoreactivity was detected with the 29 antiserum (red). Double transfected cells are identified on
the overlay image, and the effect of the transfected protein on the
distribution of ECE-1 is shown on the right image. C, wild
type AtT-20 cells were transiently transfected either with furin alone
or co-transfected with furin and the GFP chimera of the K44A mutant of
dynamin-2. TGN38, a marker for the TGN, was detected with a polyclonal
antibody and furin with the MON-139 monoclonal antibody. The left
panel shows the GFP fluorescence in black and white.
The merged image shows the co-localization of furin and TGN38
only.
The half life of wild type and mutant ECE-1b and 1d was calculated
after performing pulse chase experiments as described under
"Materials and Methods." Experiments were reproduced three times on
2-4 clones per mutation.
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Fig. 6.
Leucine-based motifs are involved in the
intracellular retention of ECE-1b and ECE-1d. ECE-1
immunoreactivity was detected with the 29 antiserum in AtT-20 cells
expressing the following mutants: A, 1b LL/AA; B,
1b LL/VL; C, 1b LV/AA; D, 1d VL/AA; E,
1d VL/LL.
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Fig. 7.
ECE-1 forms heterodimers in endothelial
cells. A, specificity of the antibodies directed
against the cytosolic domain of ECE-1 isoforms. AtT-20 cells expressing
ECE-1a or ECE-1b were fixed with methanol and labeled with either
antiserum 29, directed against the ectodomain common to all isoforms,
or antiserum 1206, directed against ECE-1a, or antiserum 1208, directed
against ECE-1b/c/d. B, AtT-20 cells expressing ECE-1a or
ECE-1b were metabolically labeled for 30 min and chased for 2 h.
ECE-1 was immunoprecipitated with either the 29 or the 1206 or the 1208 antisera. Immunoprecipitates were separated by SDS-PAGE under reducing
conditions. C, HMEC-1 cells were metabolically labeled for
1 h and chased for 2 h. ECE-1 was immunoprecipitated with
either the 29 (lane 1) or the 1206 (lane 2) or
the 1208 (lane 3) antiserum. Immunoprecipitates were
separated by SDS-PAGE under reducing conditions. Alternatively, the
1208 immune complexes (IP1) were denatured and immunoprecipitated again
using the 1206 antiserum (IP2). The IP2 immunoprecipitate was then
separated by SDS-PAGE under reducing conditions (lane 4).
Radiolabeled proteins were detected using a Molecular Imager. The
material present in lane 4 was detected using a higher
sensitivity setting of the Molecular Imager.
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Fig. 8.
ECE-1 forms heterodimers in double
transfected AtT-20 cells. Cells expressing ECE-1a or ECE-1b/FLAG
either alone or together were extracted. Total proteins were separated
by SDS-PAGE under reducing conditions. Alternatively, ECE-1b was
immunoprecipitated using the 1208 antiserum before reducing SDS-PAGE of
the immune complexes. ECE-1 was detected using either the 1206 (left panels) or the 1208 antisera (right
panels).
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Fig. 9.
ECE-1b redistributes ECE-1a to intracellular
vesicles. A, AtT-20 cells expressing ECE-1a either
alone or together with ECE-1bFLAG were fixed with methanol. ECE-1a was
detected using the 1206 antiserum. ECE-1bFLAG was detected using the
monoclonal M2 antibody. B, AtT-20 cells expressing ECE-1a
either alone or together with ECE-1bFLAG were transiently transfected
with a rab7-GFP chimera. ECE-1bFLAG was detected using the monoclonal
M2 antibody and a secondary antibody coupled to Alexa Fluor-546. ECE-1a
was detected using the 1206 antiserum and a secondary antibody coupled
to Cy5. Arrows indicate double labeling of ECE-1a and
ECE-1bFLAG. Arrowheads indicate triple labeling of vesicles.
C, total activity from wild type AtT-20 cells or from cells
expressing ECE-1b or ECE-1bFLAG was determined in the presence of 0.4%
n-octyl-glucoside. Results are expressed as relative
fluorescence unit per µg of total protein per min. D,
catalytic activity was measured from cells expressing either ECE-1a
alone or together with ECE-1bFLAG. Activity of wild type AtT-20 cells
was subtracted, and the ratio of extracellular per total activity was
calculated. Values are the mean of three independent experiments in
which activity was measured in triplicate culture wells.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We thank V. Neiveyans, M. Bignon, and C. Soundaramourty for technical assistance. Electron microscopy was done with the help of F. Mongiat. We thank Dr. K. Frenzel for reading the article. We are grateful to Drs. S. L. Milgram (University of North Carolina at Chapel Hill), W. Brown (Cornell University), C. Bucci (Università di Napoli `Federico II', Italy), M. McNiven (Mayo Clinic, Rochester), and J. W. Creemers (University of Leuven, Belgium) for the gift of antibodies and cDNAs.
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FOOTNOTES |
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* 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: INSERM U36,
Collège de France, 11 Place M.Berthelot, 75005 Paris, France.
Tel: 33-1-44-27-14-29; Fax: 33-1-44-27-16-91; E-mail:
laurent.muller@college-de-france.fr.
§ Present address: Dept. of Animal Science, Faculty of Agricultural, Food and Environmental Quality Sciences, The Hebrew University of Jerusalem, Rehovot 76100, Israel.
¶ Present address: AXOVAN AG, Gewerbestrasse 16, 4123 Allschwil, Switzerland.
Published, JBC Papers in Press, October 21, 2002, DOI 10.1074/jbc.M208949200
2 R. Meidan and L. Muller, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are: ET, endothelin; ACTH, adrenocorticotropic hormone; Big-ET, big endothelin; ECE, endothelin-converting enzyme; NEP, neutral endopeptidase; TGN, trans-Golgi network; PBS, phosphate-buffered saline; DMEM, Dulbecco's modified Eagle's medium; PIPES, 1,4-piperazinediethanesulfonic acid.
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