From the Institute for Cancer Research and Treatment (IRCC), University of Torino School of Medicine, 10060 Candiolo, Italy
Received for publication, October 3, 2002, and in revised form, January 9, 2003
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ABSTRACT |
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PLEXIN genes encode receptors for
secreted and membrane-bound semaphorins. It was proposed that the
extracellular domain of plexins acts as an inhibitory moiety,
preventing receptor activation. Here we show that plexin-B1 and
plexin-B2 undergo proteolytic processing in their extracellular
portion, thereby converting single-chain precursors into
non-disulfide-linked, heterodimeric receptors. We demonstrate
that plexin processing is mediated by subtilisin-like proprotein
convertases, by inhibition with Plexins function as cell surface receptors for all classes of
semaphorins, either alone or in complex with neuropilins (Refs. 1-4,
reviewed in Ref. 5). Semaphorins include secreted and transmembrane
proteins that act as repulsive cues for axon guidance and are
furthermore implicated in a variety of functions, spanning from immune
response to angiogenesis and tumor progression (reviewed in Refs. 5 and
6). The human plexin family contains at least nine members, classified
into four distinct subfamilies based on sequence similarity (1). In
addition, although A-subfamily plexins are predominantly expressed in
the developing nervous system, plexin-B1 (the B-subfamily prototype and
high-affinity receptor for semaphorin 4D) appears to be more
ubiquitously distributed (7).
The extracellular domain of all plexins has features in common with
scatter factor receptors (MET gene family) and semaphorins (3). In addition to mediating ligand binding, it can associate with
neuropilins (1, 4) and scatter factor receptors (8). It was reported
(9) that deleting part of the extracellular domain of plexins results
in a conformational change that activates receptor signaling. This
implies that, in the absence of the ligand, the extracellular
domain of plexins brings about a steric hindrance that inhibits
receptor function. Interestingly, the extracellular domains of
plexin-B1 and plexin-B2 contain a putative cleavage site for
subtilisin-like proprotein convertases, located in the proximity of the
transmembrane domain (1). Moreover, this site is phylogenetically
conserved, because it is also found in fly plexin B (1). The family of
subtilisin-like proprotein convertases (PCs)1 includes furin and
many other members (reviewed in Ref. 10). They are known to process a
variety of transmembrane and secreted proteins, including scatter
factor receptors and semaphorins, which are both phylogenetically
related to plexins. For class 3 semaphorins, proteolytic cleavage was
shown to regulate the axon-repelling activity (11). It has not yet been
shown whether plexins B actually undergo proteolytic processing in
cells. If this were true, they could either be converted into
heterodimeric receptors or their extracellular domain could be released
into the extracellular space.
The intracellular domain of plexins is extremely well conserved within
the family and across evolution and contains stretches that are
distantly related to GTPase activating proteins (GAPs, Ref. 12).
Furthermore, specific sequences in the cytoplasmic domains of plexins
of the B subfamily are responsible for binding activated Rac1 (13-15)
and PDZ domain-containing proteins (16-18).
In this study we have shown that plexin-B1 and plexin-B2 are found in
cells and tissues in a heterodimeric form because of proteolytic
cleavage by PCs. This event appears to require receptor localization at the cell surface, which in turn is regulated by sequences in the cytoplasmic domain. Finally, we have shown that the
proteolytic processing of plexins by PCs significantly increases ligand
binding and functional response.
cDNA Expression Constructs--
Plexin-B1 expression
construct (in pcDNA3 vector, Invitrogen) includes an in-frame VSV
tag immediately after the signal peptidase site. Plexin-B2 expression
construct (in pMT2 expression vector) includes a VSV tag at the COOH
terminus of the protein. The secreted form of plexin-B2 (B2-EC)
contains the whole extracellular domain of plexin-B2 (amino acids
1-1190) fused to glutathione S-transferase (GST) at the
COOH terminus by cloning into the expression vector pMT2-GST (a kind
gift from D. Shaap). Human PLEXIN-B1 and
PLEXIN-B2 cDNAs were mutated in their
subtilisin-like substrate-processing sites (R1302RRR
Plexin B1- Cells, Transfections, and Drugs--
Human HepG2, 293T, HT-29,
and SW-48 cells, canine kidney MDCK and murine NIH-3T3 cell lines were
purchased from ATCC. The human GTL-16 gastric carcinoma cell line and
the Suit2 pancreatic cell line were described in Refs. 20 and 21,
respectively. Cells were maintained in Dulbecco's modified Eagle's
medium, supplemented with 2 mM
L-glutamine and 10% fetal bovine serum
(Invitrogen). 293T cells were maintained in Iscove's modified
Dulbecco's medium (Sigma) supplemented with 10% fetal bovine serum.
NIH-3T3 cells were maintained in Dulbecco's modified Eagle's medium
with 10% heat-inactivated bovine serum. cDNA transfections in 293T
cells were carried out by calcium phosphate precipitation. MDCK cell clones stably expressing PLEXIN-B1 cDNA were obtained by
calcium phosphate transfection and selection with G-418 (Invitrogen). Pools of HepG2 and GTL-16 cells expressing
The inhibitor of PCs decanoyl-RVKR-chloromethylketone was purchased
from BACHEM (CH). Monensin and Brefeldin were from Sigma.
Antibodies--
IC2-specific polyclonal antibodies were raised
in rabbits against a GST fusion protein containing almost the entire
intracellular domain of human plexin-B1 (amino acids 1543-2135). EC6.9
monoclonal antibody, directed against the extracellular domain of
plexin-B1, was obtained by DNA and recombinant protein immunization of
mice.2 Neither of
these antibodies cross-reacts with plexin-B2. Monoclonal anti-VSV
antibodies (clone P5D4) were purchased from Sigma.
Immunoprecipitation and Western Blotting--
Cells were lysed
on ice in EB buffer (20 mM Tris-HCl, pH 7.4, 5 mM EDTA, 150 mM NaCl, 10% glycerol, 1% Triton
X-100) in the presence of a mixture of protease inhibitors
(phenylmethylsulfonyl fluoride, leupeptin, aprotinin, pepstatin).
Tissue samples were homogenized and lysed in EB buffer.
Immunoprecipitations and Western blotting were performed according to
standard protocols. Final detection was done with the ECL system
(Amersham Biosciences). Silver staining of polyacrylamide gels was
performed using the silver stain kit (Sigma).
Metabolic Labeling--
Cells were starved for 1 h in
serum-free medium (without cysteine and methionine) and pulsed from 20 min to 1 h in the presence of 200 µCi/ml
[35S]methionine/cysteine (Pro-mix, Amersham Biosciences),
then washed, and chased for different times in Dulbecco's modified
Eagle's medium containing 10% fetal bovine serum. Cells were lysed in EB buffer, and proteins were immunoprecipitated as described above and
submitted to SDS-PAGE. After fixation, gels were incubated with 1 M Na-salicylate for 30 min at room temperature.
After gel drying, detection was done by autofluorography or by a
PhosphorImager system (Amersham Biosciences).
Labeling of Cell Surface Proteins by Biotinylation--
Surface
proteins were labeled for 30 min at 4 °C using the ECL protein
biotinylation module (Amersham Biosciences). After labeling, cells were
rinsed twice with Dulbecco's modified Eagle's medium-1% bovine serum
albumin to quench unreacted biotin and then lysed in EB buffer. Cell
extracts were incubated with the appropriate antibodies or with
immobilized streptavidin-agarose (Pierce). Protein samples were
analyzed by SDS-PAGE and Western blotting; biotinylated proteins were
revealed by peroxidase-linked streptavidin (avidin-HRP) and the ECL
detection system.
Cell Surface Staining--
NIH-3T3 cells expressing plexin-B1,
plexin-B1-uncleav, or vector alone were seeded in 48-well
dishes coated with poly(L)-lysine. The next day, cells were
incubated with anti-VSV antibody diluted in culture medium for
1 h at 4 °C. After washing, cells were fixed with 3.7%
formaldehyde in phosphate-buffered saline for 20 min, and endogenous
phosphatases were inactivated by incubation in a water bath at 65 °C
for 15 min. Cell-bound antibodies were then detected with alkaline
phosphatase-conjugated anti-mouse antibodies (Molecular Probes) for 30 min at room temperature. Specifically bound alkaline phosphatase
activity was eventually revealed by p-nitrophenylphosphate
hydrolysis at 37 °C in a reaction buffer containing 1 M
Tris-HCl, pH 9.5, 1% bovine serum albumin, 1 mM MgCl2. Absorbance values were measured at a wavelength of
405 nm and corrected for background absorbance.
Semaphorin Binding and Collapse Assays--
The Sema4D binding
assay using NIH-3T3 cells expressing plexin-B1, B1-uncleav,
or vector alone was performed as described (1, 23). Briefly, cells
seeded in 48-well dishes were incubated for 30 min with different
concentrations of Sema4D fused to secreted alkaline phosphatase (SeAP).
Cells were then washed three times, fixed with acetone-formaldehyde,
and incubated at 65 °C for 15 min to inactivate endogenous
phosphatases. Cell-bound Sema4D was quantified by measuring SeAP
activity on p-nitrophenylphosphate, as above. Scatchard plot
analysis was performed using Equilibrate (by GertJan C. Veenstra).
The cell collapse assay was performed according to the protocol
described in Ref. 4, incubating NIH-3T3 cells with Sema4D-SeAP for 30 min at 37 °C, followed by incubation with NBT/BCIP (Promega). Stained cells were visualized with a Leica DMLB microscope, and images
were captured using a Leica DC300F camera.
Plexin-B1 Is Predominantly Found in a Cleaved Form in Cells and
Tissues--
PLEXIN-B1 encodes a large glycosylated
protein, functioning as receptor for semaphorin 4D (Sema4D/CD100) (1,
7). Previous experiments have shown that plexin-B1 mRNA, unlike
that of A subfamily members, is widely distributed in non-neuronal
tissues (7). To further analyze the expression of plexin-B1 receptor in
human cell lines and tissues, we raised specific polyclonal antibodies against its cytoplasmic sequence (IC2, described under "Experimental Procedures").
We first screened a number of cell lines derived from human
tumors by Western blotting and detected plexin-B1 expression in GTL-16
(derived from gastric carcinoma), Suit2 (pancreatic adenocarcinoma), HepG2 (liver hepatoma), and HT-29 (colon adenocarcinoma) cell lines
(Fig. 1A). In these cells, the
IC2 antibody specifically recognized low amounts of a protein with a
molecular mass of 300 kDa (most likely corresponding to the full-length
receptor) together with a predominant smaller protein of molecular mass
of 100 kDa that might correspond to a truncated form of
plexin-B1 containing the cytoplasmic domain and a portion of the
extracellular moiety. Control experiments with pre-immune serum did not
reveal any bands (data not shown). We then analyzed normal human tissue
samples (Fig. 1B) and confirmed that the predominant protein
species recognized by the IC2 antibody is a 100-kDa polypeptide,
whereas the p300 precursor protein is barely detectable. We ruled out
that the 100-kDa fragment was generated because of post-lytic protein
degradation, because it was also detected in protein extracts obtained
by cell lysis in boiling 2.5% SDS (data not shown). We concluded that in vivo endogenous plexin-B1 is predominantly expressed as a
truncated protein with a molecular mass of 100 kDa.
To confirm that the two proteins detected by IC2 were derived from
plexin-B1, we analyzed the receptor in transfected cells. When lysates
from 293T, MDCK, and NIH-3T3 cells expressing exogenous plexin-B1 were
analyzed with IC2, we again detected two protein species (p300 and
p100, Fig. 1C), identical to those found in cells and
tissues. Moreover, results obtained with in vitro
transcription/translation experiments from the same cDNA used in
transfections ruled out the possibility that downstream ATG codons
might be used for alternative translation initiation (data not shown).
Therefore, we concluded that the truncated fragment of plexin-B1 likely
derives from proteolytic processing.
Plexin-B1 and Plexin-B2 Are Synthesized as Single-chain Precursors
and Proteolytically Processed into Non-disulfide-linked
Heterodimers--
We have previously reported (1) that the
extracellular domain of plexin-B1 contains a tetrabasic amino acid
sequence located in the proximity of the transmembrane domain
(i.e. R1302RRR), which fits well with the
consensus for cleavage by subtilisin-like PCs. Proteolytic processing
in this site could therefore explain the consistent finding of cleaved
plexin-B1 in cells and tissues. By analogy with scatter factor
receptors, the proteolytic processing of plexin-B1 by PCs might lead to
the conversion of a single-chain precursor into a heterodimeric
receptor. This would result in an extracellular moiety (with a
molecular mass of ~200 kDa, subunit
Plexin-B2, another member of the B subfamily of plexin receptors, also
contains a polybasic sequence (R1161QKR) fitting with the
consensus for PC-specific cleavage. Because an antibody directed against plexin-B2 was not available, we immunopurified an
epitope-tagged form of plexin-B2 expressed in 293T cells. By Western
blotting experiments using anti-VSV antibody (recognizing the COOH
terminus of plexin-B2), we could detect two proteins with molecular
mass of 240 and 80 kDa (Fig.
3A). Reminiscent of plexin-B1
precursor and Plexin Processing Is Caused by Site-specific Cleavage by Proprotein
Convertases--
Considering the presence of a specific consensus
substrate-cleavage site for PCs in the extracellular domain of plexins
B, we verified whether receptor processing could be inhibited by treating cells with convertase inhibitors. We first tested the PC-specific inhibitor decanoyl-RVKR-chloromethylketone (25). As shown
in Fig. 4A, this inhibitor
completely blocked the processing of both plexin-B1 and plexin-B2.
Furthermore, we treated plexin-expressing cells with the PC-inhibitor
It is known that PCs require two basic charged residues in positions
Plexin-B2 Processing Depends on Protein Delivery at the Cell
Surface--
PC-dependent processing generally takes place
during transit through the Golgi complex or within secretory vesicles
en route to the cell membrane. However, proprotein convertases are also exposed on the plasma membrane and can be shed in the extracellular space or recycled into the cell (reviewed in Ref. 28). To determine whether the proteolytic cleavage of plexins-B takes place before or
after transit through the Golgi apparatus, we treated
plexin-B2-expressing cells with protein sorting inhibitors. Monensin A,
which inhibits vesicular trafficking between Golgi apparatus and the
plasma membrane, efficiently blocked the proteolytic processing of the
receptor (Fig. 6A). Brefeldin
A, which inhibits the trafficking between the endoplasmic reticulum and
the Golgi apparatus, was also effective.
As a complementary approach, we expressed a secretable form of
plexin-B2 extracellular domain, including its substrate-cleavage site,
fused to glutathione S-transferase at the COOH terminus (B2-EC) (Fig. 6B). The molecule was efficiently processed
and secreted, and two associated subunits accumulated in the
conditioned medium, as demonstrated by silver staining and anti-GST
Western blotting (Fig. 6B, left panels). Protein
p170 (indicated by b) corresponds to the plexin-B2 Sequences in the Cytoplasmic Domain of Plexin-B1 Regulate Cell
Surface Localization and Proteolytic Processing of the
Receptor--
It was reported recently that the COOH-terminal sequence
of plexin-B1 associates with a PDZ-Rho-GEF (16-18). Here we show that a deleted receptor unable to interact with PDZ-Rho-GEF (B1
Moreover, it has been shown that the cytoplasmic domain of plexin-B1
binds activated GTP-bound Rac1 (13-15). The interaction depends on the
sequence located between the two conserved regions of the SP domain.
Recently, it has been proposed that association with Rac-GTP regulates
cell surface expression of plexin-B1 (29). Here we show that a mutated
receptor unable to bind Rac-GTP (B1-GGA, Ref. 13) was virtually absent
from the cell surface and accumulated intracellularly (Fig.
6C). Interestingly, the proteolytic cleavage of
plexin-B1-GGA is totally abrogated, indicating that GTPase association
is required for the physiological processing of plexin-B1 occurring on
the plasma membrane.
Together these data indicate that the proteolytic cleavage of plexin-B1
by proprotein convertases is dependent on cell surface localization. We
also show that specific sequences in the cytoplasmic domain of
plexin-B1 are responsible for regulating cell surface targeting of the
receptor, probably via interaction with activated Rac-1 and PDZ
domain-containing proteins.
The Proteolytic Processing of Plexin-B1 Results in Increased Ligand
Binding and Functional Response--
To analyze the functional role of
receptor processing, we expressed wild-type and uncleavable
(B1-uncleav) forms of plexin-B1 in NIH-3T3 fibroblasts and
tested their ability to interact with the specific ligand Sema4D. As
shown in Fig. 7A, the
wild-type receptor is largely present as a heterodimer, whereas the
uncleavable plexin-B1 is found as single-chain precursor. The two
receptor forms are expressed at comparable levels at the plasma
membrane, as quantified by surface staining with antibodies (Fig.
7B) and cell surface biotinylation (data not shown).
However, when tested in a binding assay, the cells expressing the
uncleavable receptor were significantly less efficient in binding
Sema4D (Fig. 7C). By analyzing these data in a Scatchard
plot, we calculated comparable affinity constants for wild-type and
uncleavable receptors and an average 3-fold difference in
Bmax values (at concentrations of ligand binding
plateau). This indicates that the different binding ability of the
uncleaved receptors should be attributed to a reduced number of
functional binding sites at the cell surface.
Moreover, we observed a reduced ability of Sema4D to elicit a
functional response in cells expressing the uncleavable receptor. This
was determined by studying Sema-dependent cellular collapse of fibroblasts, by analogy to what has previously been described in
other cell types (4, 30). As shown in Fig. 7D, cells
expressing processed plexin-B1 underwent cell contraction and rounding
up upon treatment with Sema4D; in contrast, the single-chain receptor could not trigger an analogous response. In conclusion, we implicate the proteolytic processing of plexin-B1 in the formation of functional Sema4D binding sites at the cell surface.
We have previously reported that the human genome contains at
least nine different plexin genes (1). Plexins belonging to the A
subfamily are known to be predominantly expressed in the nervous system
(7) and have been implicated in axon guidance. However we found that
plexin-B1 is widely expressed in a variety of epithelial tissues and
cell lines, suggesting that plexin-B1, and possibly other plexins, may
have a general role in regulating cell migration and cell
clustering/dissociation (reviewed in Ref. 31).
We found that endogenous plexin-B1 is mainly found as a heterodimer,
because of proteolytic processing by PCs. Moreover, we showed that
another member of the plexin-B subfamily, plexin-B2, is converted into
a heterodimer by proprotein convertases. The cleavage is directed to
specific sites in the extracellular domain of the receptors, fitting
the consensus for PCs. In the extracellular domain of fly plexin B
(i.e. R1196KKR) is also found a potential PC-specific processing site, suggesting phylogenetic conservation. The
extracellular domains of other plexins do not contain bona fide cleavage sites for PCs. In fact, we did not observe
proteolytic processing of other plexins in our experimental conditions
(data not shown). Members of the PC family identified to date include SPC-1/furin, PACE-4, PC2, PC1/3, PC4, PC5/6A-PC5/6B, and PC7. We
demonstrate that plexin processing is inhibited by Several PCs are transmembrane proteins found in vesicles cycling
between the Golgi apparatus and the plasma membrane (for a review, see
Ref. 34). PCs localize transiently on the cell surface, from where they
are recycled to endosomes and the trans-Golgi network. In
addition, an active form of the enzyme is shed extracellularly (reviewed in Ref. 28). Here we report a new family of receptors that
appear to be processed by PCs in a post-Golgi compartment and likely at
the cell surface, suggesting that the activity of certain convertases
may be regulated by their subcellular localization. This conclusion is
based on the following evidence. First, an inhibitor that blocks
vesicular trafficking from post-Golgi to the plasma membrane abrogates
the processing. Second, the uncleaved precursor of the extracellular
domain of plexin-B2 (B2-EC) can be found inside the cells and at the
cell surface, whereas its processed form is only found in the
conditioned medium. Third, mutated plexins that are impaired in
subcellular trafficking and are not localized at the cell surface do
not undergo proteolytic processing. Our results could also be explained
by a processing step immediately preceding plexin localization at the
cell surface. Notably, using uncleavable mutants we ruled out that the
cleavage is required for surface localization.
We demonstrate that specific sequences in the cytoplasmic domain of
plexin-B1 are responsible for regulating cell surface targeting of the
receptor and subsequent processing. The COOH-terminal sequence (likely
through association with PDZ domain-containing proteins) greatly
enhances cell surface localization of the receptor but is not directly
required to regulate processing by PCs. The sequence mediating
interaction with activated Rac1, instead, both regulates cell surface
localization and is absolutely required for proteolytic processing of
the receptor. This may be consistent with its role in localizing the
receptor in specific membrane microdomains. GTPase- and PDZ
domain-interacting sequences have been identified in plexin-B2 and in
plexin-B3 as well; this suggests the existence of common regulatory
mechanisms for this semaphorin receptor subfamily.
We showed that PC-mediated processing converts plexins B into
heterodimers containing an The specific functional role of plexins B processing by PCs is
therefore a challenging question. PCs are implicated in the proteolytic
processing of a number of proteins, including plexin ligands, the
semaphorins (11), and plasma membrane receptors, such as scatter factor
receptors, notably homologous to plexins (35). Importantly,
PC-deficient animals display major developmental defects (36, 37; for a
review see Ref. 10). However, in most cases, the mechanisms by which
proteolytic cleavage regulates receptor function are unclear. The Notch
receptor is a notable exception because its cleavage by PCs is required
for efficient ligand binding (38). We addressed the issue of the
specific function of plexin proteolytic processing in a number of ways. First, we demonstrated that this is a physiological event, consistently occurring in a variety of tissues and cell types. Second, we expressed wild-type and uncleavable plexin-B1 at comparable levels at the cell
surface and found that higher amounts of Sema4D bind to cells expressing heterodimeric receptors, as compared with single-chain precursors. The affinity constant for the ligand does not seem to
change with receptor cleavage. We thus speculate that the proteolytic processing of plexins B exposes additional ligand binding sites or
promotes the formation of multimeric receptor complexes required for
semaphorin binding. Rohm et al. (39) have previously
reported another example of regulation of the number of semaphorin
binding sites because of plexin-neuropilin interactions. Finally, we
found that the functional response to the ligand is significantly
increased in cells expressing plexin-B1 heterodimeric receptors, as
compared with their uncleavable forms. Notably, cells expressing
single-chain receptors did not collapse in response to low
concentrations of Sema4D that were still effective in the presence of
heterodimeric receptors.
In this study we started to address the issue of plexin diversity in
mammalians. Whereas in the genome of invertebrates only two plexins are
present, humans express at least nine different family members. We have
previously shown that plexin-B1, the prototype of receptor subfamily B,
is clearly distinguished by the ability to bind its semaphorin ligand
in the absence of neuropilins, in contrast to plexins A. Now we show
that plexins B are also distinguished by a heterodimeric structure due
to proteolytic processing. It has been suggested that plexins of the A
subfamily are activated by a conformational change because of
neuropilin engagement by secreted semaphorins (9). We have not
observed a similar behavior for
plexin-B1.3 This raises the
possibility that different plexins follow distinct activation
mechanisms. For instance, a conformational change induced by
proteolytic processing of plexins B may allow for a more efficient ligand binding and functional response to transmembrane semaphorins, which directly interact with plexins without the need for neuropilins. The diversity of plexin receptors in vertebrates may thus reflect multiple functions and signaling modes deserving further investigation.
1-antitrypsin Portland, and by
mutagenesis of the substrate-cleavage sites. We provide evidence
indicating that proprotein convertases cleave plexins in a
post-Golgi compartment and, likely, at the cell surface. In addition,
we find that both cell surface targeting and proteolytic processing of
plexin-B1 depend on protein-protein interaction motifs in the
cytoplasmic domain of the receptor. We then show that proteolytic
conversion of plexin-B1 into a heterodimeric receptor greatly increases
the binding and the functional response to its specific ligand
semaphorin 4D/CD100. Thus, we conclude that cleavage by
proprotein convertases is a novel regulatory step for semaphorin
receptors localized at the cell surface.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
AAAA and R1161QKR
AQKA, respectively) to generate uncleavable forms by virtue of a recombinant PCR-based approach (19).
The mutated sequences were verified by DNA sequencing. For gene
transfer experiments, PLEXIN-B1 and PLEXIN-B1-uncleav cDNAs were subcloned in the lentiviral transfer vector
pRRLsin.cPPT.hCMV.Wpre (kindly provided by L. Naldini, University of Torino).
10 and B1-GGA mutants were kindly provided by J.M.
Swiercz (University of Heidelberg) and H.G. Vikis (University of
Michigan Medical School), respectively.
1-antitrypsin
Portland cDNA cloned in pcDNA3.1Zeo (a generous gift from G. Thomas, Oregon University) was also subcloned into lentiviral
transfer vector pRRLsin.cPPT.hCMV.Wpre.
1-antitrypsin Portland and NIH-3T3 cells expressing plexin-B1 or B1-uncleav were
obtained by transduction with lentiviral vectors according to published protocols (22).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Plexin-B1 is cleaved in cell lines and
tissues. Immunoprecipitation and Western blot analysis of
plexin-B1 expression in a variety of human tumor cell lines
(A) and tissue samples (B) using a specific
anti-plexin-B1 antibody raised against the cytoplasmic domain of the
protein (IC2). Besides a barely detectable protein with a molecular
mass of 300 kDa (p300) corresponding to full-length plexin-B1, we could
detect a smaller protein with a molecular mass of 100 kDa (p100)
corresponding to a truncated form of the receptor. C, human
plexin-B1 was transfected into 293T, MDCK, and NIH-3T3 cells and
analyzed by immunoblotting with IC2, as above.
) and a transmembrane moiety
(with a molecular mass of ~100 kDa, subunit
) that contains a
small extracellular region and the plexin cytoplasmic domain (Fig.
2A). In fact, we found that
cells transfected with plexin-B1 express both the full-length receptor (p300) and two cleaved subunits (p200 and p100) at the cell surface (Fig. 2B). The identity of p100 as
subunit was confirmed
by detection with IC2 antibodies, whereas p200
subunit was
identified by anti-VSV immunoblotting. In addition, we demonstrated the
presence of plexin-B1 heterodimers on the surface of cells expressing
endogenous receptors (Fig. 2C), using a monoclonal antibody
directed against its extracellular domain (EC6.9). To confirm that the
two subunits of plexin-B1 are associated in a complex, we show that the
extracellular subunit is efficiently purified with an antibody (IC2)
directed against the cytoplasmic domain of the receptor (Fig.
2D). In contrast to the plexin-B1 p300 precursor, the
cleaved subunits were easily detectable at the cell surface (as
demonstrated by surface biotinylation).
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Fig. 2.
Plexin-B1 cleaved subunits are associated in
a complex. A, schematic representation of plexin-B1.
The predicted processing site is indicated by an arrowhead.
Epitopes recognized by antibodies EC6.9 and IC2, and the predicted
molecular mass of the two subunits released by the cleavage ( and
), are also indicated. B, 293T cells transfected with
plexin-B1 or control vector were surface-biotinylated. Proteins
purified with anti-VSV antibodies were separated by SDS-PAGE,
transferred to nitrocellulose, and decorated with avidin-HRP, followed
by antibodies directed against the intracellular (IC2) or extracellular
(anti-VSV) domains of the receptor. p300 precursor appears as a
doublet; the upper band most likely corresponds to a
post-translationally modified form of the receptor and is enriched at
the cell surface. C, GTL-16, HepG2, and Suit2 cells
expressing endogenous plexin-B1 and NIH-3T3 cells wild type or
expressing the receptor were surface-biotinylated. Cell lysates were
immunopurified using a monoclonal antibody raised against the
extracellular domain of plexin-B1 (EC6.9) and further analyzed by
avidin-HRP detection and IC2 immunoblotting. D, NIH-3T3
cells expressing plexin-B1 were labeled by surface biotinylation. Cell
lysates were immunoprecipitated with IC2 antibodies recognizing the
cytoplasmic domain of the receptor and analyzed as in panel
C.
subunit, p240 has the predicted size of a
single-chain plexin-B2 precursor, and p80 may represent the plexin-B2
subunit. This would also imply the existence of an
subunit of plexin-B2 at the cell surface. Indeed, by cell surface
biotinylation, we could easily detect a protein with a molecular mass
of ~170 kDa, likely corresponding to the cleaved
subunit of
plexin-B2, containing almost the entire extracellular domain (Fig.
3A). A large amount of p80
subunit is detected with
anti-VSV antibodies among biotinylated cell membrane proteins, whereas
only a minor fraction of uncleaved plexin-B2 is exposed at the cell
surface (Fig. 3A, right panel). Similar to
plexin-B1, we conclude that plexin-B2 is predominantly expressed at the
cell surface as a heterodimer, whereas p240 probably represents the
full-length precursor. Furthermore, to confirm that both p170 and p80
are derived from the proteolytic cleavage of p240 precursor, we carried
out pulse-chase experiments. Fig. 3B shows that the
plexin-B2 p240 precursor decreased during the chase, whereas p80 and
p170 subunits accumulated with the same kinetics and could be
co-purified. We concluded that p170 represents the
subunit of
plexin-B2, released by cleavage and yet associated with p80
subunit. In addition, we found that the receptor subunits are not
covalently linked by disulfide bonds, as demonstrated by SDS-PAGE under
non-reducing conditions of immunopurified plexin-B1 and plexin-B2 (Fig.
3C). The disulfide-linked heterodimeric receptor Met was
included as a control (24).
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Fig. 3.
Plexin-B2 is proteolytically cleaved and
expressed at the cell surface as a heterodimeric complex.
A, surface-biotinylated 293T cells expressing VSV-tagged
plexin-B2 were analyzed by immunoblotting. Anti-tag antibodies detected
a p240, corresponding to the full-length precursor of plexin-B2, and a
smaller protein p80, corresponding to the receptor subunit.
Avidin-HRP detected a minor fraction of the plexin-B2 precursor p240,
together with p170 and p80 (the latter contains a very short
extracellular sequence). In the third panel, cell surface
proteins purified with avidin-agarose and immunoblotted with anti-VSV
antibody are shown. B, pulse-chase analysis of plexin-B2
expressed in 293T cells. Cells were pulsed with
[35S]methionine/cysteine for 20 min and then chased for
the times indicated; cell lysates were immunoprecipitated with anti-tag
antibody, separated by SDS-PAGE, and submitted to autofluorography.
C, 293T cells expressing plexin-B1 and plexin-B2 were
surface-biotinylated. Cell lysates were immunopurified with anti-VSV
antibody and fractionated by SDS-PAGE in the presence or absence of
reducing agents. Avidin-HRP detected plexin-B1 p300 precursor and
p200/p100 cleaved subunits, as well as (barely detectable) plexin-B2
p240 precursor and p170 subunit, in both conditions. As a control, the
disulfide-linked heterodimeric scatter factor receptor Met was
immunoprecipitated from GTL-16 cells and analyzed under identical
conditions. Met
subunit, with a molecular mass of 145 kDa, was
separated in the presence of reducing agents, whereas covalently linked
and
subunits run with a molecular mass of 190 kDa under
non-reducing conditions.
1-antitrypsin Portland (
1-PDX, Refs. 26 and 27). As shown in Fig.
4B, we observed a dramatic impairment of the proteolytic
processing of both plexins upon co-expression with
1-PDX. We also
observed increased amounts of the uncleaved precursors on the cell
surface (data not shown). Analogously,
1-antitrypsin
Portland inhibited receptor processing and formation of p100 in cells
expressing endogenous plexin-B1 (Fig. 4C).
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Fig. 4.
The processing of plexin-B1 and plexin-B2 is
blocked by specific inhibitors of proprotein convertases.
A, MDCK cells stably expressing plexin-B1 and 293T cells
transfected with plexin-B2 were seeded in the presence of 50 µM decanoyl-RVKR-chloromethylketone
(Dec-RVKR-CMK) and cultured for 36 h. Cell lysates were
immunoprecipitated with anti-tag antibody and analyzed with IC2 or
anti-VSV antibodies, respectively. Plexin-B1 and plexin-B2 processing
(as indicated by the presence of p100 and p80 subunits,
respectively) is suppressed by inhibition of PCs. B,
Plexin-B1 and plexin-B2 co-transfected with
1-antitrypsin Portland
(
1-PDX) were analyzed by immunoblotting, as above.
C, the processing of endogenous plexin-B1 was analyzed in
HepG2 and GTL-16 cells transduced with
1-PDX or a control vector.
Cell extracts were analyzed by immunoblotting with IC2
antibodies.
1 and
4 with respect to the actual substrate-cleavage site, leading
to the identification of the minimal consensus sequence RXXR
(reviewed in Ref. 10). We have identified sequences in the
extracellular domain of human plexin-B1 and plexin-B2 that fit this
consensus and described a proteolytic processing consistent with the
use of these sites. To confirm that plexin processing is caused by
cleavage by PCs in the indicated positions, we mutated the specific
consensus sequences in the extracellular domain of plexin-B1 and
plexin-B2. As shown in Fig. 5, these
mutated receptors, although normally targeted to the cell surface,
cannot be proteolytically cleaved, indicating that PC-specific cleavage
sites at the identified positions are absolutely required for
processing.
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Fig. 5.
Mutation of the PC-specific
substrate-cleavage site prevents plexin-B1 and plexin-B2
processing. Wild-type plexin-B1 (A) and plexin-B2
(B) and the respective uncleavable forms mutated in
PC-specific sites (R1302RRR AAAA,
B1-uncleav; and R1161QKR
AQKA,
B2-uncleav) were expressed in 293T cells. Cells were
surface-biotinylated, and lysates were immunoprecipitated with anti-VSV
antibodies and analyzed by Western blotting.
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Fig. 6.
The extracellular domain of plexin-B1 and
plexin-B2 is cleaved upon cell surface localization. A,
293T cells expressing plexin-B2 were labeled with
[35S]methionine/cysteine for 1 h and chased for
3 h in the presence or absence of 5 µM Monensin
(Mon) or 10 µg/ml Brefeldin A (BFA). Cell
lysates were immunoprecipitated with anti-VSV antibodies and analyzed
by SDS-PAGE, followed by autofluorography. B, the
extracellular domain of plexin-B2 (amino acids 1-1190) was fused to
glutathione S-transferase (GST) to generate the
secretable protein B2-EC. In the drawing, the predicted molecular mass
of cleaved B2-EC fragments are indicated. The larger fragment
(b, 170 kDa) corresponds to the subunit of
plexin-B2, whereas the smaller protein (c, 30 kDa) includes GST and the short extracellular sequence downstream
the substrate-cleavage site (indicated by an arrowhead).
Plexin-B2-EC construct and its mutant in the PC site (uncleavable) were
expressed in 293T cells, and the conditioned media were collected after
72 h from transfection. Secreted proteins were purified with
glutathione-Sepharose and analyzed by anti-GST immunoblotting and
silver staining. Lysates of surface-biotinylated cells were equally
purified and analyzed with anti-GST antibodies and avidin-HRP.
C, 293T cells were transfected with wild-type plexin-B1 or
with the mutated receptors B1-
10 and B1-GGA and
surface-biotinylated. Cell lysates were immunoprecipitated with
anti-VSV and analyzed by immunoblotting with IC2 antibodies. Receptors
exposed on the plasma membrane were revealed by avidin-HRP. Note that
the
subunit of B1-
10 runs slightly faster because of the
deletion of its C-terminal tail.
subunit, and protein p30 (c) to the short extracellular sequence of the
subunit fused to GST. The cleavage efficiency of B2-EC was
comparable with that of the full-length plexin, inasmuch as some of it
was found in the cell lysate as precursor (Fig. 6B,
right panels, a), probably because of protein
overexpression. In contrast, we could not find any processed B2-EC
protein intracellularly or associated with the cell surface, suggesting
that the cleavage occurs after plexin delivery at the plasma membrane
or immediately preceding secretion. Intriguingly, the presence of some
uncleaved precursor bound to cell surface proteins suggests that
proteolytic cleavage might induce a conformational change, leading to
the secretion of the associated subunits into the medium. To
demonstrate this assumption, we produced an uncleavable form of B2-EC
by mutating the conserved substrate-cleavage site, analogously to what
is shown in Fig. 5 for the full-size receptor. This uncleavable mutant
remained attached to the cell surface and was absent from the
medium (Fig. 6B, far right lanes).
10, Ref.
16) is inefficiently targeted to the cell surface and is mostly found
as a precursor protein (Fig. 6C). Notably, we observed that
the deletion of the essential COOH-terminal leucin residue in the
plexin-B1 PDZ-binding consensus sequence, or the introduction of an
unrelated COOH-terminal sequence, is not sufficient to block cell
surface targeting of the receptor and consequently its proteolytic processing (data not shown).
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Fig. 7.
Uncleavable plexin-B1 displays reduced ligand
binding ability and functional response. NIH-3T3 cells were
transduced with plexin-B1, mutant plexin-B1 uncleav, or
control vector and analyzed as follows. A, receptor
expression was confirmed by Western blotting with IC2 antibody.
B, cell surface localization of plexin-B1 and plexin-B1
uncleav was determined by immunostaining with anti-VSV,
followed by alkaline phosphatase (AP)-coupled secondary antibody. AP
activity was measured using p-nitrophenylphosphate as
colorimetric substrate as described under "Experimental
Procedures." Data are representative of three independent
experiments. C, cells were incubated with different
concentrations of Sema4D-AP for 30 min at 37 °C, and specifically
bound AP activity was quantified by colorimetric analysis, as above.
Results shown are representative of three experiments performed in
duplicate; error bars indicate the S.D. Scatchard plots are
shown, and linear regression analysis was used to determine the line of
best fit. Calculated KD values are indicated.
D, cells incubated with Sema4D-AP as described in
(C) were stained with AP substrate NBT/BCIP. The cellular
collapse triggered by plexin-B1 activation is significantly reduced in
cells expressing plexin-B1 uncleav.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1-antitrypsin Portland (
1-PDX), known to specifically block a number of PCs, including SPC1/furin. LoVo colon adenocarcinoma cells lack expression of furin and have been used to discriminate proteins that are exclusively processed by this convertase from those that can be substrates of other family members (32, 33). When plexin-B1 and
plexin-B2 were expressed into LoVo cells, receptor processing was
unaffected (data not shown), implicating the function of other PCs.
and a
subunit. The
subunit is
inserted in the plasma membrane and contains a short extracellular sequence, plus the specific cytoplasmic domain of plexins. The
subunit, including most of the extracellular domain, remains associated
in the complex by force of weak bonds. The absence of disulfide bonds
between the two subunits was confirmed by analysis under non-reducing
conditions; in the case of plexin-B2, this is also consistent with the
fact that no cysteine residues are found in the short extracellular
sequence of the
subunit. This leaves open the possibility that
receptor heterodimers may dissociate spontaneously or under specific
conditions. However, we did not find receptor fragments shed in the
conditioned media of cells expressing plexin-B1 or plexin-B2, either
basally or upon semaphorin stimulation (data not shown). Moreover, we
did not observe changes in plexin-B1 processing upon ligand stimulation.
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ACKNOWLEDGEMENTS |
---|
We thank B. Neel (Harvard Medical School) and Prof. W. Grigioni (University of Bologna) for help and advice. We thank G. Thomas, J. M. Swiercz, and H. G. Vikis for providing constructs. We are grateful to Laura Palmas and Raffaella Albano for technical assistance and Elaine Wright for reviewing this manuscript. We thank Silvia Giordano and our colleagues in the laboratory for advice and encouragement.
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FOOTNOTES |
---|
* This work was supported by Grant E-1129 from the Telethon Foundation and from the European Molecular Biology Organization (EMBO) Young Investigator Program (to L. T.) and by funds from the Armenise-Harvard Foundation for Advanced Scientific Research and the Italian Association for Cancer Research (AIRC) (to P. M. C.).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.
Recipient of a fellowship from the Italian Foundation for Cancer
Research (FIRC).
§ To whom correspondence should be addressed: University of Torino, Institute for Cancer Research and Treatment, SP 142, 10060 Candiolo, Italy. Tel.: 39-011-9933-204; Fax: 39-011-9933-225; E-mail: luca.tamagnone@ircc.it.
Published, JBC Papers in Press, January 17, 2003, DOI 10.1074/jbc.M210156200
2 L. Tamagnone, D. Barberis, S. Artigiani, and P. M. Comoglio, manuscript in preparation.
3 L. Tamagnone and S. Artigiani, unpublished observations.
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ABBREVIATIONS |
---|
The abbreviations used are: PC, proprotein convertase; VSV, vesicular stomatitis virus; GST, glutathione S-transferase; MDCK, Madin-Darby canine kidney cells; HRP, horseradish peroxidase; NBT/BCIP, nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate.
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