From the Institut de Pharmacologie Moléculaire et Cellulaire, CNRS, UPR 411, 660 Route des Lucioles, 06560 Valbonne, France
Received for publication, October 20, 2000, and in revised form, November 26, 2000
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ABSTRACT |
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The mechanisms by which prohormone precursors are
sorted to the regulated secretory pathway in neuroendocrine cells
remain poorly understood. Here, we investigated the presence of sorting signal(s) in proneurotensin/neuromedin N. The precursor sequence starts
with a long N-terminal domain followed by a Lys-Arg-(neuromedin N)-Lys-Arg-(neurotensin)-Lys-Arg- sequence and a short C-terminal tail.
An additional Arg-Arg dibasic is contained within the neurotensin sequence. Mutated precursors were expressed in endocrine insulinoma cells and analyzed for their regulated secretion. Deletion mutants revealed that the N-terminal domain and the Lys-Arg-(C-terminal tail)
sequence were not critical for precursor sorting to secretory granules.
In contrast, the Lys-Arg-(neuromedin N)-Lys-Arg-(neurotensin) sequence
contained essential sorting information. Point mutation of all three
dibasic sites within this sequence abolished regulated secretion.
However, keeping intact any one of the three dibasic sequences was
sufficient to maintain regulated secretion. Finally, fusing the
dibasic-containing C-terminal domain of the precursor to the C terminus
of Whereas all cells secrete proteins constitutively, neuroendocrine
and exocrine cells are also able to release a number of proteins and
peptides, including hormones and neuropeptides, prohormone convertases
(PCs)1 and digestive enzymes,
through a regulated secretory pathway (RSP) (1-3). Furthermore, in
neuroendocrine cells, neuropeptides and peptide hormones are generally
synthesized as part of large precursors in which they are flanked by
processing sites, usually pairs of basic residues. The precursors must
be cleaved at processing sites by PCs (for review, see Ref. 4) to yield
the biologically active peptides that will be secreted under
stimulation. Sorting between the constitutive and the regulated
secretory pathway is thought to occur in the trans-Golgi network (TGN)
and/or in immature secretory granules (IG) that originate from the TGN
(for review, see Ref. 5), ultimately leading to the packaging and
storage of regulated proteins and peptides into mature secretory granules.
Despite considerable advancement in our understanding of the regulated
secretion machinery in specialized cells, the sorting mechanisms into
the RSP remain unclear. Moore and Kelly (6) demonstrated that a
constitutive protein can be re-routed to the RSP when fused with a
regulated protein. This result led to the idea that the targeting of
proteins into the RSP is an active process, whereas constitutive
secretion occurs by default. Furthermore, it was recognized early that
aggregation of secreted proteins occurs in a late Golgi compartment
(1), and this led Kelly to suggest that specific aggregation of
regulated proteins could direct their sorting to the RSP (3). At least
two nonexclusive mechanisms have been proposed to account for the
sorting of proteins or protein aggregates to the RSP. The first
mechanism, designated sorting-for-entry (5), postulates that upon
reaching the TGN, regulated proteins or aggregates are actively
directed to IGs by a process that would involve their specific binding
to components of the TGN membrane. Thus, because of the homologies
between exocytosis and receptor-mediated endocytosis, Orci (7)
suggested the existence of TGN receptor(s) that would bind regulated
proteins and lead to their RSP sorting. The second mechanism, termed
sorting-by-retention (5), hypothesizes that both regulated and
constitutive proteins can enter IGs and that regulated proteins are
retained in the maturing granule while constitutive proteins are
eliminated (8-11). Two divergent hypotheses are currently put forward
to account for sorting in the IGs. The first one, recently reviewed
(5), proposes that constitutive proteins exit IGs via an active
mechanism that leads to their entry in constitutive-like vesicles
budding from IG membrane. In this model, regulated rather than
constitutive secretion would be the default pathway. It is not
excluded, however, that retention of regulated proteins in the granules
may be facilitated by their ability to aggregate or associate with
other proteins in the maturing secretory granules (5). The second
hypothesis, also recently reviewed and discussed (12), considers that
retention of regulated proteins in the IGs is achieved through
basically the same mechanism as that proposed to operate in the
sorting-for-entry model, i.e. aggregation and/or binding to
a sorting receptor in the granule membrane. In this case, constitutive
proteins could be passively excluded from the granules (12).
Whatever the sorting2
mechanism, interactions of regulated proteins with other proteins or
membrane components in the TGN or in the IGs are thought to play a
crucial role in sorting between the constitutive and the regulated
pathways. This has led to the search for sequences or structural
elements (sorting signal) in regulated proteins that would direct or
facilitate their storage in mature secretory granules. However, the
nature of these elements is still a matter of debate (recently reviewed
in Ref. 12). No consensus amino acid sequence was revealed by sequence
alignments of hormone precursors (13). Comparison between hydropathic
profiles of regulated proteins suggested that a N-terminal hydrophobic domain might represent a possible sorting signal for some regulated proteins (14). However, experimental evidence to support this hypothesis are lacking. In vitro studies demonstrated the
ability of granins to aggregate in the TGN (15). Further studies
indicated that a disulfide bond-stabilized loop in the N-terminal
region of chromogranin B (CgB) was necessary for its secretion through the RSP (16-18). A disulfide bond-delimited sequence in the N-terminal region of POMC was also found to be essential for addressing this precursor to the RSP (19). In this case, the disulfide-bonded sequence
was shown to bind to carboxypeptidase E (CPE), an enzyme involved in
late steps of precursor processing, and it was proposed that CPE might
be a sorting receptor in the TGN for several hormone precursors (20).
This view, however, is in contradiction with earlier studies showing
that the N-terminal domain of POMC was not required for its sorting to
the RSP but, rather, that cooperation between different internal POMC
domains might be involved (21). Furthermore, the proposal of CPE as a
sorting receptor for hormone precursors was recently challenged (22,
23). Attention was also paid to the dibasic sequences that are usually
numerous in prohormone precursors as possible sorting signals. Thus,
although initial studies suggested that the N-terminal domain of
prosomatostatin may play a role in precursor targeting to the RSP (24),
further work indicated that the somatostatin 28-containing C-terminal region might also be involved in sorting (25) and that a single mutation of the Arg-Lys dibasic that is normally processed to yield
somatostatin 14 prevented prosomatostatin from entering the RSP (26).
Similarly, following initial studies suggesting that in prorenin no
signal was present in the prodomain that may be involved in its routing
to the RSP (27-30), a more recent work showed that mutation of the
dibasic that is normally processed in prorenin to produce renin
prevented sorting of the protein to the RSP (31).
The aim of the present study was to look for structural elements that
could be responsible for the routing of proneurotensin/neuromedin N
(pro-NT/NN) into the RSP. This molecule is the common precursor of two
bioactive neuropeptides, neurotensin (NT) and neuromedin-N (NN) (32).
Pro-NT/NN is mainly expressed in the gut, brain, and adrenals. The
organization of the precursor is depicted in Fig. 1. NT and NN are
located near the C terminus of the precursor, where they are flanked
and separated by three dibasic sequences. A fourth dibasic site
preceding an NN-like sequence is present in the N-terminal region of
pro-NT/NN and a fifth dibasic site is found within the NT sequence.
Processing studies demonstrated that PC1, PC2, and PC5-A differentially
process pro-NT/NN at the three dibasic sites that flank and separate NT
and NN with patterns that reproduce those observed in the gut, the
brain, and the adrenals, respectively (33, 34) (Fig. 1). In contrast,
the dibasic site upstream of the NN-like sequence and the one within NT
are not normally cleaved by any of these PCs.
As indicated above, disulfide bond-delimited domains were reported to
be essential for the sorting of both chromogranin B and POMC. As two
cysteine residues are present in the N-terminal region of pro-NT/NN, we
first demonstrated the existence of an intramolecular disulfide bridge
in the precursor and investigated its function by mutating the cysteine
residues or deleting the sequence in between (Fig. 2). Then, the role
of the long N-terminal region upstream of NN and of the short
C-terminal tail downstream of NT was studied with deletion. Finally,
deletions and mutations were also made in the region that contains the
dibasic sites and the active peptide sequences. All the constructs were
transiently transfected in beta TC7 cells, an insulinoma cell
line able to process endogenous insulin and to sort it to the RSP (35).
We then studied the intracellular processing pattern of wild type and
mutated pro-NT/NN and determined the secretion pathway of the
maturation products. Our results demonstrate that neither the disulfide
bridge nor the N- and C-terminal flanking domains are necessary for the
correct routing of the precursor to the RSP. In contrast, the
neuropeptide-encoding region of pro-NT/NN was shown to contain
structural elements that are critical for sorting to the RSP and those
elements were identified as the dibasic sequences within this region.
That the C-terminal domain of pro-NT/NN contains sorting motifs was
further demonstrated by fusing this domain to the C terminus of
Disulfide Bond Analysis--
Pro-NT/NN contains two cysteine
residues in positions 39 and 88. To see if they form a disulfide bond,
large E6I,3 the 1-117
pro-NT/NN fragment (Fig. 1), was
partially purified from rMTC 6-23 cells as reported previously (37)
and 70 pmol of this material were submitted to Arg-directed cleavage
using the citraconylation, trypsin digestion, and unblocking methods (CT procedure) described previously (37). The CT-treated material was
divided in two portions, one of which was reduced with 3% (v/v)
K6L and H10P Radioimmunoassays--
The 65-89 pro-NT/NN
fragment was assayed directly in each HPLC fraction using a previously
described K6L radioimmunoassay (RIA) (37) specific for the free N
terminus of the KLPLVL (K6L) peptide that corresponds to sequence
65-70 of pro-NT/NN. To detect the 9-56 pro-NT/NN fragment, a RIA
directed against the 19-28 sequence of pro-NT/NN (HASKVSKGSP, H10P)
was developed. For this purpose, a peptide corresponding to H10P with a
C-terminal Cys (H11C) was synthesized by Dr. Solange Lavielle
(Université Paris VII) and C-terminally coupled to keyhole limpet
hemocyanine according to previously published procedures (38).
Immunization of rabbits with the conjugate led to the production of an
antiserum that was used at a 1:65,000 dilution in a RIA that employed
125I-H11C as the tracer and unlabeled H11C as the standard.
H11C was iodinated on the His1 residue using the
lactoperoxidase method and the labeled peptide was purified on reverse
phase HPLC. The EC20, EC50, and
EC80 of the assay were (in fmol/tube) 24, 122, and 625, respectively. The RIA was specific for H11C, i.e. it did not
cross-react with a number of synthetic pro-NT/NN fragments unrelated to
H10P. In addition, large E6I was not recognized in the RIA, indicating that the H10P motif had to be exposed to react with the antiserum. The
H10P sequence in pro-NT/NN (and large E6I) is preceded by a Met residue
and can therefore be freed by CNBr cleavage. Portions of the HPLC
fractions (see above) were lyophilized and dissolved in 200 µl of
70% (v/v) formic acid to which were added 50 µl of 2% (w/v)
cyanogen bromide in 70% formic acid under argon, and the mixtures were
kept for 6 h in darkness. After addition of 1 ml of water and
lyophilization, the samples were reconstituted in H10P RIA buffer (50 mM Tris/HCl, pH 8.6) containing 0.1% (w/v) bovine serum
albumin and assayed for immunoreactive H10P.
Site-directed Mutagenesis of Pro-NT/NN--
Wild-type (WT)
prepro-NT/NN cDNA was kindly provided by Dr. P. R. Dobner
(University of Massachusetts, Worcester, MA). Deletions within the
N-terminal domain of pro-NT/NN ( Obtention of Cell Culture and Transfection--
Beta TC7 cells were cultured
in Dulbecco's modified Eagle's medium containing 10% fetal bovine
serum and 50 µg/ml gentamycin (Biomedia). Cells were maintained at
37 °C in 5% CO2 atmosphere. Culture media were changed
every other day, and cells were passed once a week. The day before
transfection, cells were plated in 35-mm dishes (1 × 106 cells/dish). Beta TC7 cells were transfected with
polyethyleneimine (800 kDa; Fluka) with few modifications to the
protocol described (39). For each plate, DNA (2 µg) was diluted in
100 µl of OptiMEM (Life Technologies, Inc.), and incubated for 30 min
at room temperature with 100 µl of OptiMEM containing 15 µl of
polyethyleneimine (0.9 mg/ml). Mix was then complemented with 800 µl
of OptiMEM and added to the cells. After 5 h, 1 ml of Dulbecco's
modified Eagle's medium containing 20% serum was added. 18-20 h
later, the supernatant was replaced with 2 ml of fresh complete culture
medium. Transiently transfected cells were analyzed 48 h after
transfection. To obtain stably transfected cell lines, beta TC7 were
passed into 150-mm dishes with the addition of 0.5 mg/ml G418 (Life
Technologies, Inc.) in the medium, 2 days after transfection. Medium
was changed every other day. After 2-3 weeks, clones were picked and
expanded in medium complemented with 0.25 mg/ml G418. Expression of the constructs was checked by RIA and Western blot assays. Mock-transfected clones (transfection with pcDNA3 alone) give no signal whatever the
technique. All clones were maintained in selection medium throughout,
until the begining of the experiments.
Beta TC7 Cell Incubation--
For regulated secretion studies,
the cells were incubated first with 500 µl of OptiMEM (30-min basal),
then with 500 µl of OptiMEM containing 46 mM KCl and 4.4 mM CaCl2 (30-min stimulation) and the
incubation media were kept frozen until further use. Finally, the cells
were washed with phosphate-buffered saline (PBS from Eurobio) and
extracted in 500 µl of cold 0.1 N HCl. The extracts were
incubated for 10 min at 95 °C, centrifuged, and the supernatants were kept frozen. Protein amounts were determined using the Bio-Rad protein assay reagent under the manufacturer's conditions.
Analysis of Proneurotensin Processing Products--
HPLC and RIA
procedures have been described previously (37, 40). Briefly, the NT 29G
and NT 28H antisera (kind gifts of Jean-Claude Cuber, INSERM U 45, Lyon) react with the C terminus and N terminus of NT, respectively, the
NN antiserum with the N terminus of NN, and the K6L antiserum with the
N terminus of the K6L sequence (see above). These antisera recognize
their respective antigen only when the adjacent dibasic sequence in the
precursor has been cleaved. Both the NN and K6L sequence start with a
Lys residue which itself is preceded by a Lys-Arg doublet in pro-NT/NN. To detect cryptic NN and K6L sequences in large pro-NT/NN fragments, Arg-directed trypsin cleavage was performed following protection of Lys
residues by citraconylation (hence the abbreviation CT for
citraconylation-trypsin digestion). The CT procedure was applied to
media and cell extracts, and the CT-treated samples were assayed for
their immunoreactive NN and K6L contents. The intracellular CT
immunoreactive NN (CTiNN) contents thus measured represent the total
amount of precursor (processed + unprocessed) stored in the cells at
the time of cell extraction. As NN immunoreactivity corresponds to
processed precursor, we determine the amount of unprocessed large forms
by subtracting iNN (processed) from CTiNN (processed + unprocessed).
Pulse-Chase Experiments--
Beta TC7 cells clones were plated
in duplicate 60-mm dishes the day before. They were preincubated in
methionine- and cysteine-free Dulbecco's modified Eagle's medium for
2 h (Life Technologies, Inc.). Cells were then labeled for 30 min
in 1.0 ml of methionine/cysteine-depleted medium containing 500 µCi
of [35S]methionine and [35S]cysteine
Tran35S-label (ICN). After the pulse, cells in one dish
were harvested in 500 µl of RIPA (150 mM NaCl, 50 mM Na2HPO4, 1% SDS, 0.5% Nonidet P-40, 0.1% Triton X-100). The others were incubated in OptiMEM. Every
30 min, medium was collected and replaced with fresh OptiMEM. The
collected medium was centrifuged and kept frozen until use. After
2 h, cells were incubated in OptiMEM for 1 h, then for 30 min
(basal), and then for 30 min in stimulation medium (see above) (stimulated). Cells were harvested, and the cell extract was
centrifuged. The supernatant was collected, and 500 µl of PBS were
added. Immunoprecipitation was performed with 1 µl of anti-pro-NT/NN
antibody (kindly provided by P. R. Dobner) overnight at 4 °C
under agitation. Protein A-Sepharose 4B (Zymed Laboratories
Inc.) was added (40 µl/sample) followed by a 2-h incubation at
4 °C. After centrifugation, the pellet was rinsed five times in RIPA
buffer, and twice in PBS. The final pellet was resuspended in Laemmli
buffer, heated for 5 min at 95 °C, and loaded on a 15% acrylamide
gel. After electrophoresis, the gel was dried and analyzed by autoradiography.
Western Blot Analysis--
Immunoblot analysis of PC1 and PC2 in
beta TC7 cells were performed as described previously (33) with PC1 and
PC2 antisera generously provided by Iris Lindberg (Louisiana State
University, New Orleans, LA). Western blots with the Immunohistochemical Studies--
Cells were grown on
polylysine-coated coverslips. They were fixed in 4% paraformaldehyde
in phosphate buffer (15 min) at room temperature and permeabilized in
PBS containing 0.3% Tween 20 (5 min). After blocking for 20 min in PBS
containing 1% normal goat serum, cells were incubated for 2 h
with primary antibody diluted in PBS containing 1% normal goat serum.
The granule marker anti-secretogranin II (kindly provided by S. A. Tooze, Imperial Cancer Research Fund, London, United Kingdom) was used
at 1:300, and the anti- Characterization of Pro-NT/NN Processing in the Mouse Insulinoma
Beta TC7 Cell Line--
The beta TC7 cell line was derived from mouse
pancreatic beta cells and shown to retain the properties of
insulin-secreting cells (35, 41). Proinsulin processing into mature
insulin has been shown to result from the action of two prohormone
convertases, PC1 and PC2, that are normally expressed in pancreatic
beta cells (42, 43). Western blot analysis of beta TC7 cell extracts revealed that the cell line does express both PC1 and PC2 (Fig. 3A). Pro-NT/NN was transiently
transfected in beta TC7 cells and the transfected cells were analyzed
for their ability to process the precursor and to secrete the
maturation products in a regulated manner. Intracellular levels of
precursor (processed + unprocessed) as determined by CTiNN levels
amounted to 1 pmol/mg of protein (Table
I). Pro-NT/NN was efficiently processed
to yield similar intracellular amounts of immunoreactive NT and NN (iNT
and iNN) that represented 70-80% of synthesized precursor (Fig.
3B). HPLC analysis identified intracellular iNN and iNT as
authentic NN and NT, respectively (Fig. 3C). Both products
were secreted under stimulation (Fig. 3B). No large NT or
large NN was detectable, neither intracellularly nor in the secretion
medium (not shown). Altogether, these data show that beta TC7 cells
efficiently processed pro-NT/NN with a pattern that is consistent with
their expressing PC1 and PC2 (33). Furthermore, release experiments
demonstrate that the processing products were stored within secretory
granules. Thus, beta TC7 cells represent a good endocrine cell line
model for studying the processing and targeting to the RSP of pro-NT/NN and pro-NT/NN mutants following transient cell transfection.
The Disulfide Bridge Is Not Necessary for Pro-NT/NN Processing and
Sorting--
As the primary sequence of pro-NT/NN contains two
cysteine residues (Fig. 1), we checked whether they formed a disulfide
bridge. For this purpose, partially purified large E6I was cleaved by Arg-directed tryptic digestion between the two cysteines, thus generating several peptides, among them the 9-56 and the 65-89 fragments each containing one of the cysteines (Fig.
4A). The digested material was
then treated or not with
To assess the importance of the disulfide bridge and the
disulfide-bonded sequence for pro-NT/NN processing and sorting to the
RSP, two mutant precursors were constructed in which either both
cysteine residues were mutated to serine (C39S/C88S) or the 39-88
precursor sequence was deleted ( The N-terminal Region of Pro-NT/NN Is Not Involved in Sorting and
Processing--
We then investigated the role in sorting and
processing of the N-terminal region of pro-NT/NN that extends from
residue 14 after the signal peptide to residue 117 just prior to the
dibasic that flanks the N terminus of NN. A first
Unexpected results were obtained with the The KR-NT Sequence Contains Essential Information for
Sorting--
As deletions of the N-terminal region did not prevent
pro-NT/NN processing and sorting to the RSP, we surmised that the
C-terminal precursor region that contains the neuropeptide sequences
and the dibasic sequences that flank them might play a role in sorting. To test this hypothesis, C-terminal deletions were performed to yield
large NT ( Dibasic Sites Are Essential for Pro-NT/NN Sorting--
The
C-terminal neuropeptide-encoding region of pro-NT/NN contains four
dibasic sequences (Fig. 1): the three Lys-Arg dibasic sequences that
flank and separate NT and NN, and an Arg-Arg dibasic in position 8-9
of the NT sequence (position 135-136 in pro-NT/NN) that is not
normally processed in tissues. As three out of the four dibasic
sequences are removed in large NN, we hypothesized that these motifs
might alone or in combination play a role in pro-NT/NN sorting to the
RSP. Since large NT, which contains three of the four dibasic sequences
was efficiently processed and targeted to the RSP as shown above, it
appears that the dibasic site that flanks the C terminus of NT is not
alone necessary for pro-NT/NN sorting to the RSP. To assess the role of
the remaining three dibasic sequences in large NT, we constructed all
possible combinations of large NT in which either one, two, or all
three dibasic sequences were mutated on one of the basic residues (Fig.
2), transiently transfected the mutants in beta TC7 cells, and analyzed
precursor processing and regulated secretion (Fig.
6).
All the large NT mutants were well expressed in beta TC7 cells, as
evaluated by CTiNN measurements (Table I). Extracts from all of the
large NT mutant-expressing cells (Fig. 6) reacted positively with the
C-terminal NT antiserum, consistent with the fact that the mutants
possess a free C-terminal NT sequence. In all cases, the dibasic
sequences that flank NN were processed to yield N-terminal iNN and
N-terminal iNT when they were left intact, whereas their mutation
totally prevented cleavage. N-terminal iNN levels ranged from 75% to
90% of intracellular CTiNN levels, indicating that cleavage at the
dibasic that precedes NN proceeded near completion. N-terminal iNT
concentrations amounted only to 30% of CTiNN, suggesting that
processing at the dibasic that separates NN and NT was somewhat hindered by mutations of either of the adjacent dibasic. All the single
and double mutants yielded immunoreactive products that could be
released in a regulated manner (Fig. 6). In contrast, the large NT
construct with the triple mutation was not secreted upon cell
stimulation. These results indicate that the integrity of at least one
of any of the three dibasic sequences in large NT is necessary and
sufficient for its storage into secretory granules.
To see if adding the C-terminal KR-tail sequence to large NT bearing
the triple mutation would restore regulated secretion, a triple
pro-NT/NN mutant was constructed and transiently transfected in beta
TC7 cells. The mutant was well expressed as indicated by intracellular
CTiNN levels (Table I). Interestingly, although the dibasic that
follows NT was efficiently processed to yield C-terminal iNT (69.7 ± 11%, n = 4), the immunoreactive product generated
by the cleavage was not released upon cell stimulation (Fig. 6). This
suggests that cleavage of the Lys141-Arg142
sequence occurred before sorting could take place, possibly through the
action of PC1 since this enzyme has been shown to be activated in the
endoplasmic reticulum of neuroendocrine cells, in contrast to PC2,
which is activated much later in secretory granules (45-47).
Precursor Mutants That Lack Regulated Secretion Are Efficiently
Secreted in a Constitutive Manner--
To further investigate the
cellular fate of those pro-NT/NN mutants that lacked regulated
secretion, i.e. the large NN deletion mutant and the triple
large NT mutant, the constructs were stably transfected in beta TC7
cells and metabolic studies were performed. The K118Q large NT mutant
was also stably transfected in beta TC7 cells. The latter was chosen as
a control in this series of experiments because, as shown above, it is
processed at the Lys126-Arg127 dibasic to yield
a large precursor form that presumably is identical to large NN except
for the K118Q substitution and, unlike large NN, undergoes regulated
secretion. Three clones, each expressing high levels of one of the
mutants, were pulse-labeled for 30 min and chased for 4 h. The
medium was collected at varying time intervals and replaced with fresh
normal medium for the first 210 min of chase and with depolarizing
medium for the last 30-min chase interval. Cell lysates and media were
immunoprecipitated with the pro-NT/NN antiserum and analyzed by
SDS-polyacrylamide gel electrophoresis and autoradiography (Fig.
7). For the K118Q large NT mutant (Fig. 7A), a 30-min pulse without chase led to the intracellular
labeling of a 17-kDa protein corresponding in size to the whole
pro-NT/NN mutant sequence minus the signal peptide (lane
i). After a 4-h chase, the 17-kDa band was no longer
observed in the cell lysate but a 15-kDa protein corresponding in size
to the K118Q large NN processing product was detected in amounts that
represented 40% of the 17-kDa protein present after the pulse period
(lane h). A fraction of the 17-kDa protein was
secreted during the first 60 min of chase (lanes
a and b). The protein was barely detectable at
longer chase times (lanes c-f) and could not be
released upon depolarization (lane h). Similarly,
a fraction of the 15-kDa protein was secreted during the chase period
with a peak at 60 min and a progressive decline thereafter
(lanes a-f). However, unlike the 17-kDa protein,
it was readily released upon cell depolarization with a 4-fold
stimulation (compare lane g with lane
f) in amounts that represented 15% of the material labeled
during the pulse. Altogether, these data are consistent with the above
data derived from RIA measurements. They show that the K118Q large NT
mutant is processed intracellularly to yield a K118Q large NN product that is largely stored in secretory granules and released upon cell
stimulation.
With the The C-terminal Pro-NT/NN Domain Is a Transferable Sorting
Signal--
The above results demonstrate the critical importance of
the C-terminal encoding-peptide domain of pro-NT/NN for its addressing to secretory granules. We then sought to investigate whether this domain could by itself reroute a constitutive protein to the RSP. The
chosen constitutive protein was In this work, we provide strong evidence that basic residues
within the neuropeptide-containing region of pro-NT/NN are essential for precursor entry and/or retention into the RSP, whereas the large
N-terminal domain and the disulfide bond it contains are not required
for products from pro-NT/NN to reach the RSP.
As recalled in the Introduction, a disulfide bond-stabilized loop in
the N-terminal region of CgB was shown to be necessary for its sorting
to the RSP (16, 17). A disulfide bond-delimited sequence in the
N-terminal region of POMC was also proposed to be essential for the
sorting of this precursor to the RSP (19) through binding to a receptor
identified as CPE (20). Because pro-NT/NN contains two cysteine
residues, we investigated the presence of a disulfide bond in the
precursor and its role as a sorting signal. Our data clearly
established the existence of a disulfide bridge within the N-terminal
domain of pro-NT/NN and demonstrated that the disulfide bond and the
loop it delimits were not involved in precursor sorting and processing
into the RSP. In fact, virtually all of the large N-terminal region
upstream of the neuropeptide-containing sequence in pro-NT/NN could be removed without affecting the regulated secretion of processing products. Thus, the N-terminal region of pro-NT/NN comprising more than
three-quarters of the precursor sequence is clearly not involved in
sorting. Furthermore, since deleting large portions of the disulfide
bridge-containing N-terminal domain of pro-NT/NN is likely to affect
the secondary and tertiary structures of the precursor, it would appear
that pro-NT/NN sorting to the RSP is independent of its global
conformation and does not involve N-terminal conformational elements,
contrary to what was reported for CgB (16) and POMC (19).
This led us to investigate the possibility that the
neuropeptide-encoding C-terminal region of pro-NT/NN contains discrete motifs that would be involved in its sorting to the RSP. Sequential C-terminal deletions of the precursor revealed that the large NT
construct was processed and addressed to the RSP as efficiently as wild
type pro-NT/NN, indicating that the C-terminal tail played no essential
role in sorting. In contrast, the large NN fragment was neither
processed nor targeted to the RSP. The latter finding supports the
conclusion that the N-terminal domain of pro-NT/NN is not involved in
targeting the precursor to the RSP and shows that essential sorting
elements are contained within the KR-NT sequence of pro-NT/NN. Further
mutagenesis studies showed that the three dibasic sequences in the
C-terminal region of large NT played an important role in sorting as
mutating all of them prevented large NT from reaching mature secretory
granules. Interestingly, mutating any combinations of two of the three
dibasics yielded large NT constructs that were sorted to the RSP,
indicating that only one intact dibasic site in the C-terminal domain
of large NT is required for sorting. As the large NN deletion mutant
that contains the Lys118-Arg119 dibasic site
but lacks the 126-140 region of large NT was not sorted to the RSP, we
conclude that in pro-NT/NN the minimal sorting signal consists of a
dibasic motif located in the neuropeptide-containing region extending
from residue 118 to 140 of the precursor and that, within this
sequence, any one of the three 118-119, 126-127, and 135-136 dibasic
sites can function as the sorting signal.
It could be argued that the pro-NT/NN mutants that lacked regulated
secretion, i.e. large NN and the triple large NT mutant, might, as a consequence of the mutations, be unable to reach the TGN
and be trapped as misfolded proteins in the ER where they would either
remain or be degraded. Metabolic labeling studies performed with these
mutants clearly rule out such a possibility. Thus, following their
biosynthesis, both mutants were readily secreted in a constitutive
manner from beta TC7 cells and totally lacked regulated secretion,
indicating that they reached the TGN but could not enter or remain
within secretory granules. In these experiments, the K118Q large NT
mutant was used as a control. As shown both by RIA and metabolic
labeling experiments, the mutant was processed to yield a K118Q large
NN product that is identical to large NN except for the K118Q mutation.
Interestingly, unlike large NN, this product was very efficiently
stored in secretory granules wherefrom it could be released in a
regulated manner. Therefore, the sorting signal defined above,
i.e. the C-terminal precursor domain and the dibasic
sequences it contains, represent a strong and efficient (all or none)
structural requirement for allowing storage of pro-NT/NN in mature
secretory granules.
This point was further demonstrated by fusing the C-terminal domain of
pro-NT/NN to the C terminus of Our data show that not all dibasic sites in pro-NT/NN may serve as
processing site and/or sorting signal. Thus, in the It was further shown in this study (31) that mutation of the dibasic
sequences in the fusion protein abolished both processing and regulated
secretion, leading to the interesting suggestion that targeting to the
RSP might be linked to processing. In this context, a recent study on
insulin and proinsulin sorting suggested that dibasic cleavage may
increase storage of the hormone in secretory granules (48). In the
present study, virtually all of the pro-NT/NN mutants that were sorted
to the RSP were processed at at least one of the dibasic sequences
proposed to be involved in sorting pro-NT/NN to the RSP. The only
exception is the K118Q,R127Q large NT mutant in which the only intact
dibasic site is Arg135-Arg136 within the NT
sequence, a site that is not processed in normal tissues that express
pro-NT/NN. This mutant was sorted to the RSP. However, we were not able
to assess whether the Arg135-Arg136 dibasic
site remained intact in beta TC7 cells that express the K118Q,R127Q
large NT mutant due to the lack of adequate antibodies. Therefore, we
cannot definitively conclude that processing is linked to sorting for
pro-NT/NN. We can only suggest that dibasic sequences in the C-terminal
domain of pro-NT/NN might interact with other regulated proteins, among
which the PCs represent only a possibility, and that this interaction
might play a role in the sorting of the precursor. Should this be the
case, it would represent a crucial sorting step since our data show
that deleting or mutating the dibasic sequences abolishes regulated secretion.
As already mentioned, dibasic sequences have been proposed to act as
sorting signals for prosomatostatin and prorenin (26). Sorting motifs
have also been investigated in POMC and conflicting results were
obtained. One group reported that POMC sorting involved N-terminal
conformational elements delimited by a disulfide bridge (19). Others
reported that the disulfide bond-containing domain of POMC was not
involved in RSP sorting (21) and further showed that deletion of
internal precursor sequences abolished POMC targeting to secretory
granules (49). As these sequences contain a number of dibasic
sequences, it cannot be excluded that such motifs were necessary for
POMC sorting. CgB was shown to be addressed to the RSP through a
disulfide bond-delimited loop in its N-terminal domain (17, 50).
However, CgB appears to play a role by itself in assisting the
packaging of certain hormone and neuropeptide precursors into secretory
granules (51). It is therefore conceivable that the sorting mechanism
of CgB might differ from that of classical prohormones and
proneuropeptides. Clearly, more work is needed to answer the question
as to whether sorting of hormone and neuropeptide precursors to the RSP
through dibasic sequences is a general mechanism. It is, however, an
appealing suggestion since sorting to the RSP is a property shared by
all neuroendocrine cells and although hormone and neuropeptide
precursors exhibit widely different structures they have in common the
presence of dibasic motifs in their sequence. Thus, in addition to
serving as processing sites for the limited set of prohormone
convertases present in neuroendocrine cells, dibasic sequences could
also function as recognition sequence for the sorting machinery.
As mentioned in the Introduction, two models termed sorting-for-entry
and sorting-by-retention are currently viewed as possible mechanisms
for segregating regulated proteins in secretory granules. The latter
model can be further subdivided in two schemes: one in which retention
of regulated proteins in IGs is an active process and the other in
which retention would be passive while exclusion of constitutive
proteins from the IGs is the active process (5, 12). Our present data
do not allow to conclude as to which mechanism might operate for the
sorting of pro-NT/NN. However, because of the all-or-none effect of the
C-terminal domain of pro-NT/NN on the sorting of the precursor and the
high efficiency of this domain in routing a constitutive protein
(-lactamase, a bacterial enzyme that is constitutively secreted
when expressed in neuroendocrine cells, resulted in efficient sorting
of the fusion protein to secretory granules in insulinoma cells. We
conclude that dibasic motifs within the neuropeptide domain of
proneurotensin/neuromedin N constitute a necessary and sufficient
signal for sorting proteins to the regulated secretory pathway.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-lactamase, a bacterial protein that is constitutively secreted when
expressed in eukaryotic cells (36), and showing that the fusion protein
was redirected to the RSP in beta TC7 cells.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-mercaptoethanol. This generates several peptides, among them the
9-56 and the 65-89 pro-NT/NN fragments, each of which contains one of
the Cys residues. If a disulfide bond links the Cys, both fragments
should migrate as a single entity on reverse phase HPLC prior to
reduction, whereas they should elute as two peaks after reduction.
Unreduced or reduced CT-treated large E6I (30 pmol) was injected onto a
4.6 × 250-mm reverse phase C-18 column (5 µm, 100 Å). Elution
was carried out in 0.1% trifluoroacetic acid, 0.05% triethylamine.
The column was equilibrated in 10% acetonitrile, and 10 min after
sample injection three linear gradients were run sequentially from 10%
to 40%, 40% to 50%, and 50% to 90% acetonitrile in 42, 30, and 10 min, respectively, at a flow rate of 1 ml/min. One-ml fractions were
collected and assayed for the presence of the 9-56 and 65-89
pro-NT/NN fragments.
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Fig. 1.
Diagrammatic representation of rat
pro-NT/NN. Rat prepro-NT/NN is 169 amino acids long and starts
with a 22-residue signal peptide not shown here. Amino acid residues in
pro-NT/NN are numbered from 1, starting with the first
residue of the prosequence. The Cys residues in positions 39 and 88 in
pro-NT/NN are shown linked by a disulfide bridge. The positions of the
dibasic sites are indicated by vertical thick
lines. The main processing products generated by PC1 (NT and
large NN), PC2 (NN, NT, and large E6I) and PC5-A (NT, large NT, and
large NN) are represented (33, 34).
39-88,
14-88,
14-117,
71-117) (Fig. 2) were performed using the Ex-site method
(Stratagene, La Jolla, CA). Point mutations, apart from C39S and C88S
performed with the USE mutagenesis kit (Amersham Pharmacia Biotech),
were done with the Quickchange kit from Stratagene (C39S/C88S, K118Q, R127Q, R136Q, and combination of dibasic mutations) (Fig.
2). This kit was also used to obtain the
C-terminal truncations (
118-147,
126-147,
141-147) by
turning a codon into a stop codon. All kits were used according to the
manufacturer's recommendations. PCR products were transfected into BMH
bacteria (USE) or into XL1 bacteria (Quickchange). Bacteria were grown
in LB + ampicillin (50 µg/ml). Plasmids were purified using the
Wizard protocol (Promega, Madison, WI), and pro-NT/NN mutants were
subcloned into the EcoRl restriction site of the eukaryotic
expression pcDNA3 (Invitrogen, Leeks, The Netherlands). The correct
insertion of pro-NT/NN DNA fragment was checked by restriction mapping
with XbaI. Plasmids were used to transform TOP 10F bacteria.
The mutations were checked by sequencing the pro-NT/NN cDNAs using
a dideoxy method (Prism Dye Deoxy terminator kit; PerkinElmer Life
Sciences) and primers, either outside the pro-NT/NN sequence (T7, Sp6
primers) or inside.
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Fig. 2.
Schematic representation of the pro-NT/NN
mutants. The upper part represents the
mutations performed in the wild type precursor, and the
lower part represents the mutations in large NT.
Internal deletions are indicated by dashed lines.
Punctual mutations are represented by open
circles.
-Lactamase-Pro-NT/NN Chimeric
Proteins--
-Lactamase sequence (ampicillinase) was obtained by
PCR amplification of the ampicillin resistance gene (TEM-1) from
pcDNA3. We used a 36-mer oligonucleotide (oligo 1), the first 24 5'
nucleotides identical with the 3' end sequence of
-lactamase, and
the other 12 identical with the sequence encoding for the 109-112
domain of pro-NT/NN. Another 36-mer oligonucleotide was used (oligo 2), the first 24 5' nucleotides of which are reverse complementary to the
sequence encoding for the 109-116 domain of pro-NT/NN and the other 12 reverse complementary to the 3' end of the
-lactamase nucleotide
sequence. The
-lactamase sequence was PCR-amplified with oligo 1 and
an oligonucleotide identical with the 5' end of the
-lactamase
sequence (apart from a punctual mutation giving rise to a
HindIII restriction site). The 109-147 sequence of
pro-NT/NN was amplified with Sp6 oligo (3' end of the polylinker) and
oligo 2 from the WT pro-NT/NN-encoding plasmid. The 109-125-encoding sequence was obtained with the same oligos on the previously obtained
126-147-encoding plasmid as matrix. The
-lactamase fragment and
the C-term pro-NT/NN segment were purified, mixed at a 1:1 molecular
ratio, and a PCR amplification (with the Sp6 oligo and the
oligonucleotide specific for the 5' of the
-lactamase sequence) allowed the obtention of the
-lactamase/pro-NT/NN sequence. This product was purified and subcloned in
HindIII/NotI-digested pcDNA3.
-lactamase were
performed similarly, except that proteins (50 µg) were separated by
electrophoresis on 15% polyacrylamide minigels. The
-lactamase
antibody was purchased from 5 Prime
3 Prime, Inc.
(Eppendorf-5Prime; Boulder, CO) and used at a 1:1000 dilution.
-lactamase purchased from 5 Prime
3 Prime, Inc. was used at a dilution of 1:500. Labeling was revealed with
a Texas Red-conjugated goat anti-rabbit IgG (Jackson Immunoresearch) used at a dilution of 1:100 for 45 min. Coverslips were then mounted with glycerol and examined with a Leica TSC SP confocal imaging spectrophotometer equipped with a argon/krypton laser. Texas Red signals were imaged by exciting samples at 568 nm.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 3.
Processing and regulated release of pro-NT/NN
in beta TC7 cells. A, Western blot of analysis of PC1
and PC2 in beta TC7 cells. Intracellular proteins (50 µg) were
analyzed by Western blotting as described under "Experimental
Procedures." PC12 cells do not express PC1 nor PC2 and were used as a
negative control. B, processing and release of WT pro-NT/NN
in beta TC7 cells. Cells transiently transfected with WT pro-NT/NN were
stimulated and extracted as described under "Experimental
Procedures." The left part of the
graph shows intracellular amounts of immunoreactive NN
(filled bar), N-terminal NT (shaded
bar), and C-terminal NT (open bar)
expressed as the ratio of immunoreactive material over intracellular
CTiNN. The right part represents the stimulated
release of immunoreactive NN (filled bar) and
C-terminal iNT (open bar) expressed as the ratio
of released material under stimulated conditions over that in basal
conditions. Values are the mean ± S.E. from 14 experiments.
C, reverse phase HPLC analysis of intracellular
immunoreactive NN (open circles), N-terminal NT
(filled squares), and C-terminal NT
(open squares). Arrows indicate the
elution position of synthetic NN and NT.
Expression of pro-NT/NN mutants in transiently transfected beta TC7
cells
-mercaptoethanol and the reduced and
nonreduced products were submitted to HPLC. The fractions were analyzed
by RIA using two antibodies, each specific for one of the tryptic
fragments (Fig. 4A). Fig. 4B shows that
immunoreactivities coeluted under nonreducing conditions, whereas they
separated under reducing conditions. These results are consistent with
the existence of a disulfide bridge between the fragments. To
demonstrate that it is an intramolecular bond, pro-NT/NN was
transiently expressed in COS M6 cells and cell extracts were
electrophoresed under reducing and nonreducing conditions and
immunoblotted with a pro-NT/NN antiserum. In both cases, a 17-kDa band
was observed (data not shown), which corresponds to the expected
molecular mass of pro-NT/NN deduced from its amino acid sequence.
Hence, pro-NT/NN was synthesized as a monomer. Taken together, the data
demonstrate the existence of an intramolecular disulfide bridge in the
precursor.
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Fig. 4.
Evidence of an intracellular disulfide bridge
in the proregion of pro-NT/NN. A, schematic
representation of the products obtained after successive Arg-directed
(CT procedure) and Met-directed (CNBr) cleavages of large E6I, leading
to the exposure of the H10P and K6L sequences. B, HPLC
analysis of immunoreactive H10P (circles) and K6L
(squares) prior to (open symbols) and
after (closed symbols) reduction of cleaved large
E6I. K6L immunoreactivity was directly assayed in aliquot portions of
each HPLC fraction, whereas H10P immunoreactivity was assayed after
CNBr treatment of portions of the fractions.
39-88) (Fig. 2). The mutants were
transiently transfected in beta TC7 cells. Intracellular CTiNN levels
of both mutants were comparable to those of wild type pro-NT/NN (Table
I). The C39S/C88S mutant behaved quite similarly to wild type pro-NT/NN
with regard to both the extent of processing and the regulated
secretion (Fig. 5). The
39-88 mutant
exhibited a 10-30% reduction in processing efficiency, as compared
with wild type pro-NT/NN (Fig. 5). However, the regulated secretion
pattern of processing products was similar to that of the wild type
precursor (Fig. 5). These data show that the disulfide bond and the
sequence it delimits in pro-NT/NN are not necessary for precursor
sorting and processing into the RSP.
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Fig. 5.
Processing and regulated release of disulfide
bond, N-terminally deleted, and C-terminally deleted pro-NT/NN
mutants. The various constructs depicted in the figure were
transiently transfected in beta TC7 cells. 48 h later, cells were
incubated for 30 min first in normal medium and then for another 30 min
in depolarizing medium. Cell extracts were also collected. Processing
at the level of the indicated dibasic sequences is expressed as the
ratio of immunoreactive peptide generated after cleavage at the level
of this doublet over intracellular CTiNN. The right
column represents the stimulated release of immunoreactive
CTiNN expressed as the ratio of released material under stimulated
conditions over that in basal conditions. The values are the mean ± S.E. from the number of experiments given in Table I. ns,
not significantly different from wild type pro-NT/NN; *,
p < 0.05 when compared with wild type pro-NT/NN; °,
not significantly different from 1.
14-117 deletion
mutant was constructed and transfected in beta TC7 cells. However, this mutant was not expressed to any detectable level in the cell line (data
not shown), likely because its short size did not allow signal peptide
removal (44), which possibly led to the degradation of the abnormal
protein. Two other deletion mutants,
14-88 and
71-117, were
then constructed (Fig. 2) and transiently transfected in beta TC7
cells. Both mutant precursors were expressed (Table I) and efficiently
processed to give iNN and iNT, and the maturation products were
secreted under stimulation, relevant to their sorting to the RSP (Fig.
5). Hence, no sorting signal was present in the N-terminal region of
pro-NT/NN.
71-117 mutant. First,
this mutant exhibited regulated secretion levels higher than the other
mutants (Fig. 5). Second, it generated substantial amounts of
immunoreactive K6L material (80 ± 3% of CTiNN, n = 4), indicating that the first Lys63-Arg64
dibasic site was cleaved as efficiently as the other dibasic sequences.
This cleavage was not observed with wild type pro-NT/NN nor with any of
the mutants that contained the KR-K6L sequence (data not shown).
141-147 mutant) and large NN (
126-147 mutant) (Fig.
2). Both deletion mutants were transiently transfected in beta TC7
cells (Table I), and their processing and regulated release were
analyzed (Fig. 5). Large NT was processed to yield iNT and iNN in
proportions similar to those obtained with wild type pro-NT/NN and
secretion was enhanced upon stimulation (Fig. 5). Therefore, the
KR-tail sequence is not by itself necessary for pro-NT/NN processing
and sorting into the RSP. In contrast, large NN was not processed,
i.e. cleavage of the Lys118-Arg119
dibasic sequence that normally proceeds to greater than 80% in wild-type pro-NT/NN was markedly reduced to less than 10% in the deletion mutant (Fig. 5). HPLC analysis confirmed that large NN was the
main intracellular product (data not shown). Furthermore, large NN was
not secreted in a regulated manner by beta TC7 cells (Fig. 5). Thus,
large NN was not stored in the RSP compartments where processing takes
place. Altogether, the data obtained with large NT and large NN show
that the C-terminal KR-NT sequence of large NT contains elements that
are essential for storage in the RSP.
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Fig. 6.
Processing and regulated release of dibasic
mutants. Each construct is schematically depicted with the
indication of its mutation(s). All possible mutations of the 118-119,
126-127, and 135-136 dibasic sequences were performed in large NT
( 141-147) as depicted in the top part of the
figure. In the bottom line is depicted the triple
pro-NT/NN mutant in which the 118-119, 126-127, and 135-136 dibasic
sequences were mutated. Processing and release were analyzed as
described in Fig. 5. The values are the mean ± S.E. the number of
experiments given in Table I. ns, not significantly
different from large NT (
141-147); *, p < 0.05 when compared with large NT (
141-147); °, not significantly
different from 1.
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Fig. 7.
Release of newly synthesized pro-NT/NN
mutants. Beta TC7 cells were stably transfected with the K118Q
large NT (A), 126-147 (large NN) (B), or
triple large NT mutants (C). Cells were pulse-labeled with
[35S]methionine/cysteine for 30 min. After the pulse, one
dish for each mutant was harvested for determining the intracellular
content of labeled proteins after immunoprecipitation with the
pro-NT/NN antiserum, SDS-polyacrylamide gel electrophoresis, and
autoradiography (lane i). In another dish, the
cells were washed with fresh medium and chased for varying periods of
time. At the end of each chase period, the medium was collected and
replaced with fresh medium and labeled products were analyzed as above.
Chase times were 30, 60, 90, 120, 180, and 210 min (lanes
a-f). After the last chase period, regulated secretion was
assayed by exposing the cells to depolarizing medium for 30 min. The
medium (lane g) and the cells (lane
h) were then collected and analyzed. The autoradiograms are
from one representative experiment with each pro-NT/NN mutant. Below,
the autoradiograms were quantitated by densitometry. The data in
lanes a-h are expressed as the percentage of
material present in the cells after the pulse (lane
i) and are the means from four separate experiments in
A and B, and two separate experiments in
C.
126-147 pro-NT/NN mutant (large NN) (Fig. 7B),
a unique intracellular 15-kDa protein, corresponding in size to
unprocessed large NN, was labeled during the 30-min pulse
(lane i) and was still present in cell lysates
after 4 h of chase (lane h), although at
much reduced levels (15%). Most of the 15-kDa protein (>75%) was
secreted from the cells during the first 210 min of chase (lanes a-f) and no stimulated release could be
observed (lane g). Similar results were obtained
with the triple large NT mutant (Fig. 7C). A single 17-kDa
intracellular protein with the expected size of the unprocessed mutant
was labeled during the 30-min pulse (lane i) and
was still present at low levels (10%) after 4 h of chase
(lane h). The protein was progressively secreted
from the cell for the first 210 min of chase lane (lanes
a-f), and no stimulated release was observed during the
last 30-min chase interval (lane g). These
results are in full agreement with the above RIA data showing that both
mutants were not processed and lacked regulated secretion. They further
show that the mutants were not stored intracellularly but were readily
secreted in a constitutive manner, thus indicating that they reached
the TGN and were not blocked in the endoplasmic reticulum as misfolded proteins.
-lactamase (also called
ampicillinase), a bacterial protein that possesses a signal peptide and
has been shown to be constitutively secreted when expressed in
neuroendocrine cells (36). Two chimeric proteins depicted in Fig.
8A were constructed by fusing
to the C terminus of
-lactamase either the C-terminal 109-147
pro-NT/NN domain (
-Lac-109-147) or the 109-125 pro-NT/NN fragment
with a C-terminal NN moiety (
-Lac-109-125). These constructs were
stably transfected in beta TC7 cells. Clones that express high levels
of the constructs were selected and tested for their ability to process
and release the chimeric proteins. The results clearly show that
processing and regulated secretion readily occurred for
-Lac-109-147 and, in contrast, were markedly impaired for
-Lac-109-125. Thus, iNN measurements in cell extracts indicated that cleavage of the Lys118-Arg119 dibasic
sequence that precedes NN proceeded to greater than 50% with
-Lac-109-147, whereas it was less than 10% for
-Lac-109-125 (Fig. 8A). Western blot analysis using a
-lactamase
antiserum (Fig. 8B) revealed that in
-Lac-109-147-expressing cells, both the intact 34-kDa fusion
protein and a 29-kDa processing product corresponding in size to
-lactamase minus its pro-NT/NN-derived C-terminal extension were
present. Furthermore, the 29-kDa product was released upon cell
depolarization, whereas the 34-kDa precursor was not found in the
medium of either unstimulated or stimulated cells. In contrast, the
-Lac-109-125 chimera was expressed as a single 31-kDa band
corresponding in size to the full sequence of the fusion protein (Fig.
8B). The protein was constitutively released in the medium
but lacked regulated secretion. Immunofluorescence studies coupled to
confocal microscopy were performed using the
-lactamase antiserum
and an antiserum directed against secretogranin II, a secretory granule
marker (Fig. 9). Secretogranin II
labeling in beta TC7 cells exhibited a highly punctate distribution and was heavily concentrated in a cytoplasmic region extending from the
nucleus to the plasma membrane (Fig. 9A).
-Lactamase
staining in
-Lac-109-147-expressing cells exhibited the same
punctiform and subcellular distribution as secretogranin II (Fig.
9B). In marked contrast,
-lactamase labeling in
-Lac-109-125-expressing cells was diffuse throughout the cytoplasm
of the cells (Fig. 9C).
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Fig. 8.
The pro-NT/NN C-terminal domain can act as a
sorting domain. Cell lines were developed that stably express
chimeric proteins in which either the 109-147 or the 109-125 domain
of pro-NT/NN was fused to the C terminus of -lactamase, leading to
the
-Lac-109-147 and the
-Lac-109-125 constructs, respectively.
A, schematic representation of the chimeras. Processing at
the level of the KR dibasic site upstream of NN was assayed in RIA as
described previously. B, regulated release of
-lactamase-containing products was investigated by incubating cells
for 30 min in normal medium (ns) and then in depolarizing
medium (s). Intracellular content was also collected
(intra). Intra- and extracellular media were analyzed by
Western blot with a
-lactamase antibody.
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Fig. 9.
Immunocytochemical localization of the
-lactamase fusion proteins. A,
immunostaining of beta TC7 cells with secretogranin II antibody.
B and C, immunostaining with an
anti-
-lactamase antibody of stably transfected beta TC7 cells
expressing
-Lac-109-147 (B) or
-Lac-109-125
(C). Same experiments with nontransfected cells give no
staining with the anti-
-lactamase antibody (scale
bar, 10 µ m).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-lactamase, a prokaryotic protein
that has been shown to be constitutively secreted when expressed in
neuroendocrine cells (36). The fusion protein expressed in beta TC7
cells clearly exhibited a subcellular localization and a pattern of
secretion consistent with it being efficiently sorted to the RSP. In
contrast, fusion of the C-terminal motif of large NN (KR-NN) to
-lactamase yielded a protein that displayed a diffuse cellular
distribution, was constitutively secreted, and lacked regulated
secretion. Therefore, it is clear that the C-terminal
neuropeptide-encoding domain of pro-NT/NN contains elements that are
necessary and sufficient to direct a protein to the RSP or to prevent
it from exiting IGs, independently of the nature of the protein to
which it is attached.
126-147 deletion mutant (large NN) and the triple large NT and pro-NT/NN mutants, the Lys63-Arg64 dibasic site in the
middle region of the proteins did not function as an alternative
processing site nor did it target the mutants to the RSP.
Interestingly, bringing this site closer to the C-terminal domain of
pro-NT/NN as in the
71-117 deletion mutant led to its efficient
processing, indicating that the C-terminal neuropeptide-containing region of pro-NT/NN is more accessible to processing enzymes than the
N-terminal region of the precursor. In any case, it may be suggested
that dibasic sequences have to be in a proper structural environment to
function as sorting signals. A similar conclusion was reached
concerning prorenin (31). This precursor contains, in addition to the
Lys-Arg processing site adjacent to renin, two other Lys-Arg sequences
in the N-terminal part of the proregion. Mutating the processing site
prevented prorenin processing and regulated secretion. However, fusing
the N-terminal 16 amino acid sequence of prorenin that contains the
silent dibasic sequences to a constitutive protein yielded a fusion
protein that was processed and targeted to the RSP. It was concluded
that the two N-terminal dibasic sequences could not be used as
alternative processing sites and sorting signals in prorenin because
they were not in a proper environment.
-lactamase) to the RSP, our data are difficult to reconcile with a
passive mechanism for retaining regulated proteins in IGs. Rather, we
would favor the hypothesis that the C-terminal region of pro-NT/NN and
the dibasic sequences it contains play an active role in precursor
sorting through protein-protein interactions. Further work will be
needed to determine the type of interactions involved: binding to a
putative sorting receptor(s), formation of protein aggregates or
cleavage-condensation assisted by PCs. The beta TC7 cell lines
developed here that stably express various pro-NT/NN constructs or the
-lactamase fusion proteins should provide useful tools in this notice.
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ACKNOWLEDGEMENTS |
---|
We thank P. R. Dobner for pro-NT/NN cDNA and antiserum, J. C. Cuber for NT antisera, and I. Lindberg for the PC1 and PC2 antibodies. We are grateful to C. Rovère for the PC1 and PC2 immunoblots in beta TC7 cells and for help in the development of the H10P RIA, and to S. Lavielle for providing synthetic peptides.
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FOOTNOTES |
---|
* 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. Tel.:
33-4-93-95-77-64; Fax: 33-4-93-95-77-08; E-mail:
kitabgi@ipmc.cnrs.fr.
§ Present address: Centre de Biochimie, INSERM, U470, 06108 Nice Cedex 2, France.
Published, JBC Papers in Press, December 4, 2000, DOI 10.1074/jbc.M009613200
2 Throughout this paper, the term "sorting" is used in a broad sense and is intended to mean that, at some point in the cell, regulated proteins are separated from constitutive proteins and ultimately stored in mature secretory granules. No assumption, unless explicitly indicated, is made as to the mechanisms underlying the different fate of regulated and constitutive proteins. In this line, a "sorting signal" could be simply defined as any sequence, motif or conformational element whose deletion from a regulated protein leads the protein to the constitutive pathway and, conversely, whose fusion to a constitutive protein leads the fusion protein to the regulated pathway, whatever the underlying sorting mechanism.
3 Precursor peptide fragments are designated as K6L, E6I, and H10P, in which the first letter corresponds to the single-letter code of the first amino acid, the middle number corresponds to the number of amino acids residues in the peptide, and the final letter corresponds to the single-letter code of the last amino acid.
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ABBREVIATIONS |
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The abbreviations used are: PC, prohormone convertase; CPE, carboxypeptidase E; CT, citraconylation/trypsin/unblocking procedure; HPLC, high pressure liquid chromatography; iNN, iNT, iK6L, iE6I, and iH10P, immunoreactive NN, NT, K6L, E6I, and H10P, respectively; NN, neuromedin N; NT, neurotensin; POMC, proopiomelanocortin; RIA, radioimmunoassay; RSP, regulated secretory pathway; TGN, trans-Golgi network; WT, wild type; PCR, polymerase chain reaction; CgB, chromogranin B; IG, immature secretory granule; PBS, phosphate-buffered saline.
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