From the Laboratories of Biochemical and Molecular
Neuroendocrinology, the Protein Engineering Network of Centres of
Excellence, Clinical Research Institute of Montreal, University of
Montreal, Montreal, Quebec H2W 1R7 Canada, the ¶ Laboratoire
de Biochimie Cellulaire CNRS-Unité Propre de Recherche de
l'Enseignement Supérieur Associe'e 6032, Faculté de
Pharmacie, 27 Blvd. Jean Moulin, 13385 Marseille, cedex 05, France,
and
INSERM U387 Hôpital Ste Marguerite, 270 Blvd. Ste
Marguerite, 13009 Marseille, cedex 29, France
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cellular trafficking of subtilisin/kexin-like
precursor convertases (PCs) may be regulated by a number of motifs,
some of which are present within the P-domain and in the C-terminal
sequence. Six of the seven known PCs contain a conserved RGD sequence
within the P domain. In order to investigate the functional importance of this motif, we generated mutants of PC1 that contain a Myc tag
epitope inserted between the prosegment and the catalytic subunit.
Cellular expression of vaccinia virus recombinants revealed that this
tag did not seem to influence the autocatalytic conversion of proPC1
into PC1 or its bioactivity. The two PC1 variants produced possess
either the wild type RGD sequence or its RGE mutant. Stable transfectants of these variants in AtT20 cells revealed that similar to
the wild type enzyme, PC1-RGD-Myc is sorted to secretory granules. In
contrast, PC1-RGE-Myc exits the cell via the constitutive secretory pathway. In vitro, a 14-mer peptide spanning the RGD
sequence of PC1, but not its RGE mutant, binds to cell surface
vitronectin-binding integrins of Chinese hamster ovary cells. However,
within the endoplasmic reticulum and in an RGD-independent fashion,
integrin Limited proteolysis of prohormones and proproteins is a general
mechanism generating bioactive peptides and proteins in a regulated
fashion. Often, this processing step involves specific cleavage(s) post
single basic or pairs of basic residues. Recently, seven mammalian
serine proteinases related to yeast kexin and bacterial subtilisins
were identified and shown to be responsible for such conversions. These
proprotein convertases (PCs)1
were called furin (also called PACE), PC1 (PC3), PC2, PACE4, PC4, PC5
(PC6), and PC7 (LPC, SPC7, or PC8) (for reviews and updates, see Refs.
1-5). Whereas furin, PACE4, PC5, and PC7 exhibit a widespread tissue
distribution, PC4 is predominantly found in testicular germ cells. The
expression of PC1 and PC2 is mostly restricted to endocrine and neural
cells. All of these PCs exhibit an N-terminal signal peptide, followed
by a prosegment, a catalytic domain, a P domain, and an enzyme-specific
C-terminal segment (4, 5). The most conserved catalytic domain of
eukaryotic precursor convertases exhibits the closest similarity to
bacterial subtilisins and contains the catalytic triad Asp, His, and
Ser, as well as the oxyanion hole Asn (4, 5). In contrast to the
relatively short bacterial subtilases, the catalytic domain of
eukaryotic convertases is extended at the C terminus by a 140-150 amino acid P domain, the function of which is less well understood.
The P domain has been shown to be critical for the zymogen cleavage of
prokexin (6), pro-PACE4 (7), proPC2 (8), and proPC1 (9); for the
substrate cleavage activity of furin (10, 11); and for the sorting of
PC2 (12) and PC1 (13). Based on molecular modeling, it has recently
been suggested that the P domain is an independently folded structure
and that in PC1 it consists of 8-stranded Within the P domain, a conserved RRGDL sequence is observed
in six (except for PC7) of the seven mammalian PCs, and its
RG dipeptide is always present in all PC orthologues (13).
For PC7, which exhibits an RRGSL sequence, the C-terminal
border is more extended (16). It has recently been suggested that the RGD sequence in PCs lies in between two In a previous report, we showed that the RGD sequence of PC1 is
critical for the efficient folding and zymogen processing of proPC1 (89 kDa) into PC1 (83 kDa) within the endoplasmic reticulum (ER) and for
its ability to be processed into the C-terminally truncated 66-kDa
PC1- In the present study, we investigated the functional importance of the
RGD sequence in the cellular sorting of PC1 in AtT20 cells that
normally express this enzyme endogenously. This was achieved using
stable transfectants of PC1-RGD and PC1-RGE containing a Myc epitope
tag. Biosynthetic and immunocytochemical data provided strong evidence
for the critical importance of the RGD motif in the sorting of PC1 to
secretory granules. Cell adhesion assays showed that in
vitro, a synthetic 14 amino acid (aa) peptide spanning the RGD
sequence of PC1 can bind to integrins. In contrast, co-expression studies in AtT20 cells provided evidence for an RGD-independent ER-localized binding of integrin mPC1-Myc Constructions--
The sequences of native mouse
(m)PC1 and its D520E (RGD/RGE) mutant described in Ref. 13 were
modified to include a 10-aa Myc antigen (underlined) inserted at the N
terminus of mature mPC1, resulting in the sequence
RSKR110EQKLISEEDL111SVQD
of full-length mPC1 (24). For this purpose, two complementary oligonucleotides containing the cohesive ends of the Psp1406-I restriction endonuclease (italics) were synthesized, annealed, and
phosphorylated. Their sequences were as follows: sense,
5'-CGTTCAGTTGAACAAAAGCTCATCTCAGAGGAAGATCTT-3', and
antisense, 5'-CGAAGATCTTCCTCTGAGATGAGCTTTTGTTCAACTGAA-3'. The double-stranded oligonucleotide was then ligated into the Psp1406-I
site of the cDNA of mPC1. The mutated mPC1-RGD-Myc and mPC1-RGE-Myc
were then subcloned either into the 5'
HindIII/SalI 3' sites of PMJ602 vector, and
vaccinia virus recombinants were isolated, as described Ref. 20, or
into the 5' HindIII/EcoRV 3' sites of pcDNA3 (Invitrogen).
Vaccinia Virus--
The isolation of recombinant vaccinia
viruses (vvs) expressing mPC1 and mPC2, as well as their RGD mutants,
was previously reported (13, 20, 25). The recombinant vv:mPOMC was a
generous gift of Dr. Gary Thomas (Portland, OR). We have similarly
isolated vaccinia virus recombinants of the human (h) integrins
Stable and Transient Transfectants--
Full-length mPC1-RGD-Myc
and mPC1-RGE-Myc cDNAs cloned into the pcDNA3 vector
(Invitrogen) or an empty pcDNA3 vector were transfected into AtT20
cells using a DOSPER reagent as suggested by the manufacturer (Life
Technologies, Inc.). Six G418-resistant clones expressing each enzyme
were isolated. One of the clones expressing the highest amount of
mPC1-RGD-Myc was chosen for further analyses. All mPC1-RGE-Myc
G418-resistant clones expressed low levels of the enzyme, as estimated
by Western blots using an enhanced chemiluminescence kit (Boehringer
Mannheim) and the Myc-specific antibody at a dilution of 1:2000. Based
on immunoprecipitation of the radiolabeled protein, the highest
expressor (clone I) was selected for further investigation by
biosynthetic analyses and by immunocytochemistry.
Biosynthetic Analyses--
Seventeen hours postinfection with vv
recombinants, GH4C1 cells (3 × 106) were radiolabeled
with 200 µCi of [35S]Met for 3 h. The media and
cell extracts were immunoprecipitated with either an N-terminal
mPC1-antibody (20); an antiserum to Immunocytochemical Analysis of mPC1-RGD-Myc- and
mPC1-RGE-Myc-expressing Cells--
Because the monoclonal antibody to
Myc was not found to be suitable for immunofluorescence cytochemistry,
mPC1-Myc was localized in transfected AtT20 cells by using the
peroxidase/diamino benzamidine technique (28). The primary
Myc-1-9E10.2 antibody was used at a dilution of 1:100, and the
secondary antibody consisted of horseradish anti-mouse IgG coupled to
peroxidase used at a 1:75 dilution (Amersham Pharmacia Biotech).
Cell Adhesion Assays--
The synthetic peptides GRGDSP and
GRGESP were purchased from Bachem. The synthetic 14-mer mPC1 peptides,
TIEYSRRGD520LHVTL, representing aa 512-525
(24), and its D520E mutant TIEYSRRGE520LHVTL,
were prepared by solid phase synthesis using fast Fmoc (N-(9-fluorenyl)methoxycarbonyl) chemistry on a 431A
ABI-solid phase peptide synthesizer. Their quality and purity were
verified by reverse phase high pressure liquid chromatography and by
fast atom bombardment mass spectrometry, giving the expected molecular masses of 1658 and 1672 Da, respectively. Adhesion substrates were
prepared by coating flat-bottom 96-well microtiter plates for 1 h
at 37 °C with 50 µl of vitronectin (prepared as described in Ref.
29) at 1 µg/ml. Coated wells were then blocked with 1% bovine serum
albumin in phosphate-buffered saline for 1 h. CHO cells were
obtained in a single cell suspension by treatment of subconfluent cell
monolayers with 0.53 mM EDTA in phosphate-buffered saline,
washed twice with Dulbecco's modified Eagle's medium containing 0.2%
bovine serum albumin (adhesion buffer) and preincubated for 1 h at
4 °C with synthetic peptides. Cells (50,000 cells in 50 µl) were
added to each well and allowed to adhere to the substrata for 1 h
at 37 °C in a cell culture incubator. Unattached cells were removed
by gently washing three times with adhesion buffer and residual
attached cells were fixed by 1% glutaraldehyde. After staining by
0.1% crystal violet, cells were lysed with 1% SDS, and the absorbance
was measured at 600 nm by a microplate reader (model 960 from Metertech).
Co-immunoprecipitation of Integrin
Insertion of a Myc Tag at the N-terminal of Mature mPC1 Does Not
Affect Its Autoactivation or Its Ability to Process POMC--
In order
to differentiate the expression of exogenous PC1 from its endogenous
homologue in cells, we introduced a 10-aa Myc epitope tag between the
prosegment and the start of the catalytic subunit. This strategic site
was chosen because it was previously shown that insertion of a
FLAG-epitope tag at a similar site in furin did not affect its
enzymatic properties (30). As a test for the effect introducing the Myc
tag, we overexpressed vaccinia virus recombinants of PC1-RGD and
PC1-RGE in GH4C1 cells (devoid of endogenous PC1 (31)) and compared
their biosynthesis to those of their Myc-tagged derivatives PC1-RGD-Myc
and PC1-RGE-Myc. As shown in Fig. 1, the
insertion of a Myc epitope does not affect the autocatalytic conversion
(32) of proPC1 (89 kDa) into PC1 (83 kDa) for either the wild type
enzyme or its RGE mutant. Because the extent of processing of PC1-RGD
and PC1-RGD-Myc into the 66-kDa PC1-
As a functional test for the Myc-tagged PC1 activity, we compared the
ex vivo ability of PC1 and PC1-Myc enzymes to process POMC
into Stable Transfectants of PC1-RGD-Myc and PC1-RGE-Myc in AtT20
Cells--
In order to study the importance of the integrity of RGD
motif on the sorting of PC1 to secretory granules, we chose the widely used AtT20 cells (which endogenously synthesize large amounts of wild
type PC1) as the ideal cell type to perform this investigation. We thus
generated stable transfectants of both PC1-RGD-Myc and PC1-RGE-Myc and
selected the highest expressing clones for each, based on levels
estimated by Western blots using the Myc antibody (Fig.
3). Although at least three clones
exhibiting high expression levels of PC1-RGD-Myc were isolated (we only
show one of them), all positive PC1-RGE-Myc clones revealed relatively
much lower intracellular levels of Myc-tagged PC1. Again, no 66-kDa
form of PC1 was observed for the latter clones (Fig. 3). We thus
selected one of them (clone I) for biosynthetic analyses (Fig.
4) and immunocytochemical localization
(Fig. 5).
The stable transfectants were pulse-labeled with [35S]Met
for 15 min, followed by a chase of either 1 or 3 h in the presence or absence of 8-Br-cAMP (Fig. 4). Under these conditions, after 1 h of chase, the ratio of the levels of PC1-RGD-Myc (83 kDa) in cAMP
treated versus control cells was 1.8, as compared with 3.1 for the 66-kDa derivative. In contrast, the ratio of the levels of
secreted 83-kDa PC1-RGE-Myc in the presence versus absence of cAMP was 0.9. At the 3 h-chase time, the corresponding values for
the secreted 83- and 66-kDa RGD forms were 1.1 and 2.7, respectively. Concomitantly, the corresponding intracellular ratios of the 83- and
66-kDa forms of PC1-RGD-Myc were 0.9 and 0.5, respectively. Thus,
following the 3-h chase, practically all the 83-kDa form of PC1 is
converted into PC1-
As shown in Fig. 5A, immunostaining for PC1-RGD-Myc was
observed both paranuclearly and at the cellular tips (dark
triangles). This pattern is very similar to that previously
observed in AtT20 cells in our laboratory with the endocrine hormone
ACTH, which is localized both at the TGN (paranuclear) and in secretory
granules (cellular extension tips) (34). In contrast, in the case of PC1-RGE-Myc most of the Myc immunoreaction was found paranuclearly (Fig. 5B) and the staining at the cellular extension tips
was very weak (Fig. 5B, arrows). This pattern resembles that
of constitutively secreted proteins such as Interaction of PC1 with Cell Adhesion Integrins--
The
conserved RRGDL sequence in convertases (4, 13) suggested
that PCs might interact with cell surface integrins that recognize
RGD-containing sequences. In that context, phage display revealed that
integrins
In order to test the possible intracellular binding of full-length mPC1
with integrins and the dependence of this plausible interaction on the
RGD sequence, we selected
Fig. 8 depicts the results of
immunoprecipitation of cell extracts using an The results presented in this work represent a first attempt to
define the functional significance of the conserved RGD motif in
proprotein convertases. The available data clearly point out that the
integrity of this motif is crucial for the stability (13) and the
sorting of the convertase PC1 (see above). Thus, whereas the wild-type
PC1 enters secretory granules (Figs. 4 and 5), possibly assisted by its
C-terminal sorting domain Integrins are a major group of versatile adhesion receptors endowed
with both adhesive and signaling functions. They possess shared and
unique specificities both outside and inside the cell. Many of the
integrins share an affinity toward the RGD recognition sequence in
their extracellular matrix ligands but are still capable of
distinguishing different RGD-containing proteins (18). In attempt to
define the ability of the conserved RRGDL motif to bind
integrins extracellularly, we tested the in vitro integrin binding of a 14-aa synthetic peptide spanning this sequence (Fig. 6).
The data demonstrated that this peptide behaved similarly to a
fibronectin-related hexapeptide, GRGDSP. Importantly, the RGE mutants
of either the mPC1- or the fibronectin-derived peptide did not bind CHO
cells, as expected from RGD-dependent integrin binding.
These data suggested that secreted PC1, or a fragment thereof, could in
principle bind to surface integrins such as those found on CHO cells.
However, we also wanted to investigate the possible intracellular
interaction of integrins with PC1 and PC2, because it is now known that
the processing of the Earlier data suggested that residues within the P domain of PC1
influence the sorting of this enzyme to the regulated secretory pathway
(13). Based on the above data, we can conclude that integrin
5
1 associates primarily with the
zymogens proPC1, proPC1-
C (missing the C-terminal 137 residues), as
well as proPC2. Thus, the observed discrimination between the secretion
routes of PC1-RGD and PC1-RGE does not implicate integrins such as
5
1.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-barrels with a well
organized inner hydrophobic core (14). Furthermore, because it is
different from subtilisins, the dibasic cleavage specificity of PCs may require the presence of a large number of acidic (Asp and Glu) residues
within the substrate binding region. It was suggested that the P
domain, unique to PCs, contributes to the stabilization of their
catalytic subunit (14). Studies of various chimeras implied that the P
domain is necessary to both fold and maintain the catalytic pocket in
an active form and to regulate the calcium and pH dependence of PCs
(15).
-strands (
3 and
4), which contain 6 of the predicted 22 residues forming the inner hydrophobic core of the P domain (14). Because an RGD sequence is found
in a number of proteins that bind cell adhesion integrins, such as
fibronectin and vitronectin, it was originally suggested that this
motif might be important for the interaction of the PCs with integrins
(17). Binding of fibronectin to its integrin receptor was shown to be
dependent on the RGD sequence and to be abrogated when replaced by RGE
(18). The conservation of the RGD motif in mammalian PCs suggested a
structural and/or functional role, which may be common to all of them.
C (13). Furthermore, previous studies demonstrated that the
66-kDa form of PC1 is formed inside granules (19-22). Accordingly,
data derived from vaccinia virus expression of the RGE mutant of PC1
suggested that the RGD motif is important for the sorting of PC1 into
the regulated secretory pathway (13).
5
1 to
proPC1, proPC1-
C (missing the 137-aa C-terminal segment
C but
still containing the RGD sequence (23)) and proPC2.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
5 and
1, kindly provided by Dr. E. Ruoslahti (La Jolla Cancer Research Foundation, La Jolla, CA). GH4C1 or
AtT20 cells were infected with the specified plaque-forming units
(pfu)/cell vv:mPC1-RGD, vv:mPC2-RGD, vv:mPC1-RGE, vv:mPC2-RGE,
vv:mPC1-RGD-Myc, vv:mPC1-RGE-Myc, vv:mPOMC, vv:h
5, and
vv:h
1.
-endorphin (26), or a
Myc-specific monoclonal antibody (Myc-1-9E10.2) obtained from ATCC
(clone CRL-1729). The immunoprecipitates were resolved by SDS-PAGE on
either 8% or 15% polyacrylamide gels, and the dried gels were
autoradiographed (20, 26). All biosynthesis experiments were performed
at least twice. For stable transfectants, AtT20 cells were pulsed for
15 min with [35S]Met followed by a chase of 1 or 3 h
in the presence or absence of 5 mM of the secretagogue
8-Br-cAMP (27).
5
1 with PC1 and PC2--
AtT-20 cells
were solubilized with 50 mM Tris-HCl, pH 8, 200 mM NaCl, 1 mM EDTA, and 1% Triton X-100
containing 0.5% bovine serum albumin and a mixture of proteinase
inhibitors (1 mM phenylmethylsulfonyl fluoride, 500 units/ml aprotinin, 1 µg/ml leupeptin, 1 µM pepstatin, 1 mM iodoacetamide, and 1 mM
o-phenanthroline). Samples were incubated for 5 h at
4 °C with 10 µg/ml of purified rabbit polyclonal antibodies against the C-terminal portion of the
5-integrin
subunit. Control experiments included a preincubation of the
anti-
5 antibody with excess of the C-terminal peptide
immunogen. Immunoprecipitated material was submitted to SDS-PAGE on a
7% polyacrylamide gel and blotted onto nitrocellulose. For Western
blot analyses, polyclonal antibodies against the N terminus of PC1 and
the C terminus of PC2 were used. The labeled proteins were detected
using the above enhanced chemiluminescence system.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
C is quite similar (Fig.
1A), this demonstrates that the Myc tag does not
significantly affect the late processing (19-22) of the 83-kDa form of
the wild type PC1 into its 66-kDa derivative. In addition, our data
show that the Myc monoclonal antibody allows the immunoprecipitation of
radiolabeled Myc-tagged PC1s (Fig. 1B). Finally, it is clear
that no 66-kDa form of PC1-RGE-Myc is secreted into the medium (Fig. 1,
A and B) and that both PC1-RGE and PC1-RGE-Myc
exhibit lower levels in the medium as compared with their RGD parents
(Fig. 1A) (13). Furthermore, overexposure of the
autoradiogram did not reveal the presence of any trace of 66-kDa
PC1-RGE in either media (not shown). Intracellularly, because both the N-terminal PC1 antibody (Fig. 1A, CTL) and
the monoclonal Myc antibody (Fig. 1B, RGD and
RGE) detect either a nonspecific protein or the endogenous
(nonsecretable) c-Myc protein migrating closely to the 66-kDa position
(33) (Fig. 1B), it is difficult to assess the level of
intracellular 66-kDa PC1.
View larger version (39K):
[in a new window]
Fig. 1.
Vaccinia virus expression of mPC1-RGD-Myc and
its RGE mutant in GH4C1 cells. GH4C1 (3 × 106)
cells were infected with either vv:wild type (control
(CTL)), vv:mPC1 (RGD), vv:mPC1-Myc
(RGD/myc), vv:mPC1-RGE, or vv:mPC1-RGE-Myc
(RGE/myc), each at 2 pfu/cell for native mPC1
(RGD) and 3 pfu/cell for the RGE mutants. Seventeen hours
postinfection, the cells were pulse-labeled with [35S]Met
for 3 h. The cell extracts and media were immunoprecipitated with
either a PC1 N-terminal antibody recognizing the segment 84-100 of
mPC1 (20,24) or a Myc mouse monoclonal antibody. The immunoprecipitates
were separated on an 8% SDS-polyacrylamide gel and fluorographed as
described (13). The resulting autoradiogram also depicts the migration
position of proPC1 (89 kDa), PC1 (83 kDa), and the C-terminally
truncated PC1- C (66 kDa).
-lipotrophic hormone (26). As shown in Fig.
2, the levels of secreted
-lipotrophic
hormone are similar in the presence or absence of the Myc epitope tag.
This demonstrates that the introduction of the Myc tag at the selected
position did not affect the intracellular enzymatic activity or
cleavage selectivity of PC1. Finally, and in accordance with the lower
overall levels of PC1-RGE and PC1-RGE-Myc as compared with their RGD
parents (Fig. 1), we note that the former produces less
-lipotrophic hormone than the latter, as was previously observed for the RGE mutant
of PC1 (13).
View larger version (51K):
[in a new window]
Fig. 2.
Comparative POMC processing by mPC1 and
mPC1-Myc. GH4C1 (3 × 106) cells were infected
with 2 pfu/cell vv:mPOMC together with either 2 pfu/cell vv:wild type
(control (CTL)), vv:mPC1 (RGD), vv:mPC1-Myc
(RGD/myc), 3 pfu/cell vv:mPC1-RGE, or vv:mPC1-RGE-Myc
(RGE/myc). Seventeen hours postinfection, the cells were
pulse-labeled with [35S]Met for 3 h. Cell lysates
and media were immunoprecipitated with an antibody specific to
-endorphin (26). The immunoprecipitates were separated on a 15%
SDS-PAGE gel, and the dried gel was autoradiographed. The migration
positions of unprocessed POMC and of
-LPH are emphasized.
View larger version (40K):
[in a new window]
Fig. 3.
Western blot of stable transfectants of
mPC1-RGD-Myc and mPC1-RGE-Myc in AtT20 cells. Cell extracts of
stable transfectants of mPC1-RGD-Myc (one clone) and mPC1-RGE-Myc
(clones I-IV) in AtT20 cells (50 µg of total protein/lane, as
estimated by a Bio-Rad assay) were resolved by SDS-PAGE on an 8%
acrylamide gel. As a control, we also obtained a stable transfectant of
the pcDNA3 vector alone. The separated proteins were probed with a
Myc monoclonal antibody at a dilution of 1:2000. The proteins were
visualized by enhanced chemiluminescence. The migration positions of
PC1 and PC1- C and the positions of the molecular mass markers are
shown.
View larger version (65K):
[in a new window]
Fig. 4.
Biosynthetic analysis of stable transfectants
of mPC1-Myc in AtT20 cells. AtT20 cells (3 × 106) stably transfected with either mPC1-Myc-RGD or
mPC1-Myc-RGE in pcDNA3 were pulse-labeled for 15 min with
[35S]Met and chased for either 1 or 3 h in the
presence (+) or absence ( ) of 5 mM 8-Br-cAMP. The
mPC1-Myc proteins in the cells and media were analyzed under the same
conditions as in Fig. 1 using the Myc monoclonal antibody. The
resulting autoradiogram also depicts the migration positions of proPC1
(89 kDa), PC1 (83 kDa), and the C-terminally truncated PC1-
C (66 kDa).
View larger version (82K):
[in a new window]
Fig. 5.
Comparative immunocytochemistry of
mPC1-RGD-Myc and mPC1-RGE-Myc in AtT20 cells. Distribution pattern
of mPC1-RGD-Myc (A) and mPC1-RGE-Myc (B) revealed
with a Myc monoclonal antibody using the diamino
benzamidine-immunoperoxidase technique (28). Immunostaining to
mPC1-RGD-Myc was found paranuclearly and in some tips of the cellular
extensions (A, dark triangles). This pattern resembles that
of ACTH in AtT20 cells (34). In contrast, for the mPC1-RGE-Myc,
immunostaining was predominantly found paranuclearly (B),
suggesting a Golgi-like localization similar to that reported for the
TGN marker TGN38 (34). Furthermore, within cellular extensions, very
little Myc immunoreactivity was found (B, arrows).
Magnification, × 500; scale bar, 25 µm.
C (which lacks the C-terminal 137 aa but still
contains the RGD sequence), and cAMP can only stimulate the secretion
of the latter form. These results are in agreement with the fact that
the 66-kDa form is primarily found in granules, whereas the 83-kDa form
is mostly in the trans-Golgi Network (TGN) (19-22). At the
3-h chase period, the level of secreted 83-kDa PC1-RGE-Myc is also
slightly lower in the presence of cAMP. These data strongly suggest
that the majority of PC1-RGE-Myc exit the cells via the unregulated,
constitutive secretory pathway. Finally, it seems that the major
intracellular form of PC1-RGE-Myc exhibits a lower molecular mass than
the secreted form and that its levels did not appreciably diminish
after 3 h chase, suggesting a low turnover of this likely immature
ER-associated form.
1-PDX in AtT20 cells and
of resident Golgi proteins, such as TGN38 (34). These data agree with
those in Fig. 4, whereby PC1-RGD-Myc can enter the regulated secretory pathway, whereas PC1-RGE-Myc exits the cell predominantly via the
constitutive pathway.
5
1 and
v
3 recognize an RRGDL sequence (35).
Thus, in order to test the possibility that PC1 can interact with
integrins, we synthesized a 14-aa peptide spanning the RRGDL sequence
of structure: TIEYSRRGD520LHVTL (24), as well
as its D520E (RGE) mutant. For cell adhesion assays, we chose CHO
cells, which bind vitronectin in an RGD-dependent fashion
(36). We thus compared the ability of the synthetic mPC1-RGD and RGE
peptides, as well as the model hexapeptides GRGDSP and
GRGESP to compete with vitronectin in the binding of CHO
cells. Thus, the data in Fig. 6 show that
the peptide mPC1-RGD is about 2.5-fold more potent in competing with
vitronectin for CHO cells as compared with the synthetic GRGDSP
peptide. As expected, both RGE-containing peptides do not prevent
vitronectin from binding CHO cells. These data suggested that in
vitro mPC1 could interact with integrins, such as those found on
the cell surface of CHO cells.
View larger version (17K):
[in a new window]
Fig. 6.
Attachment of CHO cells to vitronectin in the
presence of increasing amounts of competing peptides. CHO cells
(50,000 cells/50 µl) were added to each well and allowed to adhere to
a substrata of vitronectin (1 h at 37 °C). Attached cells were fixed
with 1% glutaraldehyde, stained with 0.1% crystal violet, and then
lysed with 1% SDS. The amount of adherent cells was estimated from the
absorbance at 600 nm. The mean of triplicate measurements and the S.E.
(less than 10%) are shown. RGD, ; RGE,
; RRGDL,
; RRGEL,
.
5
1, because it binds preferentially RRGDL sequences as compared with
v
3 (35). AtT20 cells were triply infected
with vv:
5, vv:
1, and the vaccinia recombinants of either mPC1-RGD, mPC1-RGE, mPC1-
C, mPC2-RGD, or
mPC2-RGE, in the presence or absence of brefeldin A (BFA). The fungal
metabolite BFA is known to disassemble the Golgi complex and produce
fusion of the cis-, medial-, and trans-Golgi (but not the TGN) with the ER (37). We first assessed the levels of
intracellular expression of each protein by Western blots and did not
observe significant differences between each infection (Fig.
7). Notice that either in the presence or
absence of BFA more than 50% of proPC1 is converted into PC1 and that
in the case of the RGE mutant it remains mostly as proPC1-RGE. In
contrast, and in accordance with the known slower proPC2 to PC2
conversion late along the secretory pathway (20), we observed a
preponderance of proPC2 in the cells with a minor fraction being
converted into PC2 (66 kDa) only in the absence of BFA. The production
of the 71-kDa proPC2, in which the prosegment is cleaved at an internal site, has been previously reported (20, 21).
View larger version (55K):
[in a new window]
Fig. 7.
Expression levels of vaccinia virus-expressed
mPC1 and mPC2 and their RGD/RGE mutants in AtT20 cells co-expressing
human integrin
5
1.
AtT20 (1 × 107) cells were infected for 2 h at
27 °C with 1 pfu/cell each: vv:
5 and
vv:
1 together with either vv:wild type (WT),
vv:mPC1 (RGD (top)), vv:mPC1-RGE (RGE
(top)), vv:mPC2 (RGD
(bottom)), or vv:mPC2-RGE (RGE
(bottom)). The cells were then incubated for 17 h in
the presence or absence of 5 µg/ml of BFA. Cell lysates were then
separated by SDS-PAGE on a 7% polyacrylamide gel and then analyzed by
Western blot using an N-terminal PC1 and a C-terminal PC2 antiserum
(20). Notice the endogenous 89-kDa PC1 and 66-kDa PC1-
C in the cells
infected with the wild type (WT) virus control. The
arrows at the right on the upper and
lower panels depict the positions of the 72-kDa proPC1-
C
and the 71-kDa proPC2 intermediate (20, 22).
5-specific
antibody followed by Western blotting analysis of the
immunoprecipitates by SDS-PAGE using either an N-terminal PC1 antiserum
or a C-terminal PC2-antibody. Control experiments in which the
anti-
5 antibody was preincubated with an excess (10 µg) of the C-terminal peptide immunogen confirmed the specificity of
the immunoprecipitations (not shown). Unexpectedly, proPC1-
C (72 kDa) and both RGD and RGE forms of both proPC1 (89 kDa) and proPC2 (75 kDa) co-immunoprecipitate with
5
1 in the presence of BFA, suggesting that at the level of the ER, these zymogens
can interact with
5
1 in an
RGD-independent manner. Furthermore, proPC1 or proPC1-
C, neither of
which can exit the ER (20, 22), bind
5
1
primarily in the presence of BFA. The low level of these zymogens
observed in the absence of BFA may be related to their short residence
time in the ER. Only a relatively minor amount of PC1-
C (66 kDa) and
less so of PC1 (83 kDa) (seen upon overexposure of the gel) bind
5
1, even though their levels are not
negligible (Fig. 7). Thus, these data suggest that the C-terminal
137-aa
C segment of PC1 is not critical for this interaction which
occurs in the ER. The data also show that proPC2 binds
5
1 in both the presence and absence of
BFA, in accordance with its long residence time in the ER (20). It is
difficult at the moment to eliminate the possibility that at the level
of the TGN proPC2 remains bound to
5
1,
because some of the carbohydrate chains of PC2 always remain sensitive
to endoglycosidase H (20). The above data demonstrated that the
co-immunoprecipitation of
5 and the PCs is not simply
due to their overexpression, as we do not see any immunoprecipitates
for PC1-RGD and PC1-
C in absence of BFA. An additional control
included the co-expression of rPC7 (16) with
5
1, which showed no
co-immunoprecipitation even in the presence of BFA (not shown).
Finally, when immunoprecipitations were performed with PC-specific
antisera, Western blots showed that
5 is detected (not
shown). In conclusion, although the specific binding of
5
1 to the neural and endocrine
convertases occurs in the ER, neither the RGD sequence of proPC1 and
proPC2 nor the
C polypeptide of proPC1 is critical for the
interaction of these zymogens with
5
1.
View larger version (23K):
[in a new window]
Fig. 8.
Co-immunoprecipitation of human integrin
5
1
with mPC1 and mPC2 and their RGD/RGE mutants in AtT20 cells. AtT20
(1 × 107) cells were infected for 2 h at
27 °C with 1 pfu/cell each: vv:
5 and
vv:
1, together with either vv:wild type control
(WT), vv:mPC1 (RGD (left)),
vv:mPC1-RGE (RGE (left)), vv:mPC2 (RGD
(right)), or vv:mPC2-RGE (RGE
(right)). The cells were then incubated for 17 h in the
presence or absence of 5 µg/ml of BFA. Cell lysates were then
immunoprecipitated with an integrin
5 polyclonal
antibody. The immunoprecipitates were separated by SDS-PAGE on a 7%
polyacrylamide gel, followed by Western blotting of the gel using
either an N-terminal mPC1 or C-terminal mPC2 antibodies (20). The
migration of purified PC1 (83 kDa), proPC2 (75 kDa), and PC2 (66 kDa),
as well as the positions of proPC1 (89 kDa), proPC1-
C (72 kDa), and
PC1-
C (66 kDa), are indicated.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
C (23, 38), the PC1-RGE mutant is rapidly
degraded early along the secretory pathway (13), and the little that
does exit the ER is secreted via the constitutive secretory pathway
(Figs. 4 and 5). In agreement, we observed that all stable
transfectants of PC1-RGE-Myc exhibit much lower levels of expression
than the corresponding PC1-RGD-Myc clones (Fig. 3). This result is in
accord with our earlier observation that the steady-state levels of
vaccinia virus expressed recombinants of PC1-RGE are much lower than
those of their corresponding wild type enzyme (13). We surmise that it is likely that the RGD motif plays an important role in the folding of
PC1 within the ER. The structural alterations imposed by the RGD/RGE
mutation are such that PC1-RGE can no longer enter secretory granules.
This suggests that the RGD sequence influences either its degree of
calcium-dependent aggregation and/or its possible interaction with a putative sorting receptor in the TGN/immature secretory granules, both processes proposed as likely secretory granule
sorting mechanisms (39-41). Based on the predicted structure of the P
domain of PC1 (14), the RGD sequence lies between the diagonally
opposed third and fourth
-strands and is likely on the hydrophilic
side (13) of the predicted 8-stranded-
-barrel structure exhibiting a
Greek key motif (14). Because it is proposed that the P domain consists
of two
-sheets formed by
-strands 1, 2, 4, and 7 and
-strands
3, 8, 5, and 6, respectively (14), we envisage that the RGD sequence
would sit in between the two
-sheets.
-chain of certain integrins is performed
within the TGN by furin-like enzymes (42). We opted to co-express
5
1 with PC1 and another neuroendocrine
convertase PC2, because phage display revealed that this integrin binds
preferentially to an RRGDL-containing sequence (17, 35) found in these
PCs (13). Unexpectedly, co-immunoprecipitation results demonstrated
that
5
1 binds to both proPC1 and proPC2
within the ER in an RGD-independent fashion (Fig. 8). Furthermore, our
data showed that the C-terminal
C segment of PC1 is not implicated
in this interaction. It is intriguing that primarily the zymogens
proPC1 and proPC2 bind to
5
1 in the ER,
even though PC1 is formed in this organelle and remains so in the
presence of BFA (Fig. 8). This might implicate the participation, at
least in part, of the prosegments of PC1 and/or PC2 in this interaction. Interestingly, no RGD sequence is found in the prosegments of any PC. Recently, it was reported that motifs other than
RGD can be recognized by
5
1,
e.g. an RHD sequence found in the Alzheimer's disease, causing peptide A
40 and A
42 (43). In that context, an
RYD sequence is found in the catalytic subunit of mPC1 (aa 199-201), mPC5 (aa 205-207), and hPACE4 (aa 237-240). Thus, more work is necessary to define the critical motif(s) responsible for the
binding of proPC1 and proPC2 to
5
1 in the
ER. Nevertheless, it is noteworthy that
5
1 intracellularly binds to these
zymogens even though it represents one of the most discriminating of
the RGD-recognizing integrins with regard to extracellular ligand specificity (18). However, we cannot eliminate the possibility that the
ER interaction of
5
1 with proPC1 and/or
proPC2 is indirect and could involve one or more intermediary proteins,
such as chaperones for example. In that context, the presence of
multicomponent complexes in the ER involved in the folding of proteins
has recently been reported (44). Finally, similar results were also
obtained in GH4C1 cells (not shown) that do not express either PC1 or
PC2 (31), thereby eliminating the possibility that the endogenous enzymes interfered with our assays in AtT20 cells.
5
1 is likely not involved in the
discriminative sorting of PC1-RGD toward secretory granules and of
PC1-RGE to the constitutive secretory pathway. In addition, the present
data demonstrate that although the conserved RGD sequence in PC1 is
required for its stability and sorting toward secretory granules, this
does not seem to implicate an intracellular RGD-dependent
interaction with integrin
5
1. Rather,
this integrin seems to selectively bind proPC1 and proPC2 within the
ER. The understanding of the functional implication of this interaction
will surely lead to a more profound definition of the cellular biology
of PCs and integrins.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank S. Benjannet, A. M. Mamarbachi, J. Hamelin, J. Rochemont, J. Marcinkiewicz, A. Lemieux, and F. Parat for technical help throughout this study. Many thanks are due to A. Chen for the monoclonal Myc antibody preparation. We also thank Dr. M. Marcinkiewicz for help on the immunocytochemistry of the PC1-transfectants. The secretarial assistance of Sylvie Emond is appreciated.
![]() |
FOOTNOTES |
---|
* This work was supported by Medical Research Council program Grant PG-11474 and by the Government of Canada's Network of Centers of Excellence program supported by the Medical Research Council and Natural Sciences and Engineering Research Council of Canada through the Protein Engineering Network of Centers of Excellence.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Present address: Institut de Pharmacologie du CNRS, 660 route des Lucioles, 06560 Valbonne, Sophia-Antipolis, France. Supported in part by the Fondation Fyssen (Paris, France) and by the Fonds de la Recherche en Sante du Québec (Montreal, Quebec, Canada).
** Present address: Loeb Health Research Institute, Ottawa Civic Hospital, 725 Parkdale Ave., Ottawa, Ontario K1Y 4K9, Canada.
To whom correspondence should be addressed: Clinical Research
Institute of Montreal, 110 Pine Ave. West, Montreal, Quebec H2W 1R7,
Canada. Tel.: 514-987-5609; Fax: 514-987-5542; E-mail: seidahn{at}ircm.qc.ca.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: PC, precursor convertase; vv, vaccinia virus; POMC, pro-opiomelanocortin; ER, endoplasmic reticulum; TGN, trans-Golgi network; PAGE, polyacrylamide gel electrophoresis; CHO, Chinese hamster ovary; BFA, brefeldin A; mPC1, mouse PC1; pfu, plaque-forming unit(s); aa, amino acid(s).
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|