Originally published In Press as doi:10.1074/jbc.M903271199 on March 9, 2000
J. Biol. Chem., Vol. 275, Issue 21, 15741-15748, May 26, 2000
Cysteine-rich Fibroblast Growth Factor Receptor Alters Secretion
and Intracellular Routing of Fibroblast Growth Factor 3*
Roman
Köhl,
Marianne
Antoine,
Bradley B.
Olwin
,
Clive
Dickson§, and
Paul
Kiefer¶
From Ruhr-Universität Bochum, Medizinische Fakultät,
Abteilung für Virologie, Universitätsstrasse 150, Gebäuole MA 6/130, D-44780 Bochum, Germany, the
Department of Molecular, Cellular, and Developmental
Biology, University of Colorado, Boulder, Colorado 80308, and
§ Imperial Cancer Research Fund Laboratories, 44 Lincoln's
Inn Fields, London WC2A 3PX, United Kingdom
Received for publication, April 29, 1999, and in revised form, March 6, 2000
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ABSTRACT |
Expression of the cysteine-rich fibroblast growth
factor (FGF) receptor (CFR) in COS-1 cells strongly inhibits the
secretion of co-expressed FGF3. By using a column retention assay and
affinity chromatography, we demonstrate that at physiological salt
concentrations FGF3 binds with strong affinity to CFR in
vivo and in vitro. Furthermore, to show that FGF3
binds to CFR in vivo, truncation mutants of CFR with
changed subcellular distributions were shown to cause a similar
redistribution of FGF3. Although CFR is a 150-kDa integral membrane
glycoprotein that is primarily located in the Golgi apparatus, we show
here that in COS-1 cells a substantial proportion of CFR is secreted.
This is due to a carboxyl-terminal proteolytic cleavage that releases
the intraluminal portion of the protein for secretion. However, the
apparent size of the integral membrane and secreted CFR appears
similar, since the loss of protein mass is balanced by a gain of
complex carbohydrates. The released CFR is associated with the
extracellular matrix through its affinity for glycosaminoglycans. These findings show that CFR can modulate the secretion of FGF3 and may
control its biological activity by regulating its secretion.
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INTRODUCTION |
Fibroblast growth factors
(FGFs)1 are a family of at
least 18 structurally related polypeptides that can modulate the
growth, differentiation, migration, and survival of various cell types in culture (reviewed in Refs. 1-4). The properties of FGFs indicate they act primarily as intercellular signaling molecules, by binding to
and activating receptors on the surface of the same or adjacent cells.
Four high affinity FGF receptor genes (FGFR1-4) have been identified
that encode transmembrane tyrosine kinases (5, 6). Moreover, signal
transduction requires the presentation of FGFs by a second type of
receptor composed of heparan sulfate containing proteoglycan. In
addition to the transmembrane signaling receptor complex, a third class
of membrane-anchored FGF-binding protein has been identified as a
cysteine-rich FGF receptor (CFR) (7, 8).
CFR was purified as an FGF-binding protein from embryonic chick and
shown to be a 150-kDa integral membrane glycoprotein (9). It
subsequently emerged that the CFR is homologous to a rat protein cloned
as a Golgi-specific protein designated MG-160 (10-12). More recently,
the human homologue of the same protein was identified as an
E-selectin-binding protein (ESL-1) provided it was modified by
(1,3)-fucosylation (13, 14). A comparison of the derived amino acid
sequences of the chicken and human homologues shows more than 90%
sequence identity, demonstrating a high degree of conservation during
evolution (7, 9, 10). Chicken CFR and rat-MG160 were shown to localize
primarily to the Golgi apparatus, and ESL-1 was described as a cell
surface protein (11, 12, 16).
The sequence of CFR reveals 16 cysteine-rich repeats in the
intraluminal domain and a short cytoplasmic tail (7, 12). CFR binds
FGF1, FGF2, and FGF4, and its presence has been shown to alter the
levels of intracellular FGF1 and FGF2 suggesting a role in the
intracellular trafficking of the FGFs (7, 17). However, the CFR shows
no recognizable sequence homology to the FGF tyrosine kinase receptors,
nor to the proteoglycan co-receptors. The CFR failed to bind several
other growth factors tested (platelet-derived growth factor-BB,
epidermal growth factor, insulin, and insulin-like growth factor-II),
but forms a secreted complex with TGF-
(7, 18).
Previously we showed that mouse FGF3 was retained in the medial Golgi
complex of COS-1 cells and only slowly released into the culture
medium, raising the possibility that FGF3 export may be controlled
(19). Regulation at the level of secretion has been also described for
TGF-, and interestingly, this ligand is also retained in the Golgi
complex as an inactive precursor (18, 20). The coincident intracellular
localization of FGF3 and the CFR in the Golgi apparatus prompted us to
investigate whether there was a functional interaction between these
two proteins. We show here that FGF3 binds to the CFR and that the CFR
can alter FGF3 secretion and its subcellular localization.
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EXPERIMENTAL PROCEDURES |
Cell Culture--
COS-1 cells were maintained as described
previously (16). For transient DNA transfections, plasmid DNA as
indicated was introduced into 5 × 105 COS-1 cells by
electroporation (450 V/250 microfarads). Between 48 and 72 h after
transfection, the cells were harvested for immunoblot analysis or
processed for immunofluorescence.
Plasmid Constructions--
pCFR1.1 was constructed by inserting
anti-RGS(His)6 epitope downstream of the signal peptide
cleavage site of a chicken CFR cDNA (7). The amino-terminal 48 codons of chicken CFR were amplified by PCR using a 3' primer that
encodes the RGS-His epitope and included a BamHI site. A
second PCR fragment was generated using a 5' primer encoding the same
epitope and encompassing the BamHI site to amplify the
downstream CFR sequence beginning with codon 49. Both resulting PCR
fragments were fused via the newly created BamHI site
placing the RGS-His epitope 5 codons downstream from the signal peptide
cleavage site (17). The modified CFR cDNA was then inserted in the
expression vector pKC4 under control of the early SV40 promoter. To
obtain the plasmids pCFR2.1 and pCFR3.1, PCR was used to delete the
carboxyl-terminal 12 and 34 codons of CFR, respectively. The 3'
oligonucleotide primers were used to introduce a stop codon and an
EcoRI site. The resulting 3' fragments were used to replace
the 3' sequences of pCFR1.1 through a naturally occurring
HindIII site. PGEX-CFR was constructed by subcloning the
total CFR3.1 insert as a partial BamHI-EcoRI fragment into pGEX2T (Amersham Pharmacia Biotech).
Immunofluorescence--
COS-1 cells grown on glass coverslips
were transfected with the appropriate plasmids, and 48 h later the
cells were fixed and processed as described previously (19). For
surface immunostaining, the cells were incubated with antibodies
without permeabilizing; alternatively, cells were incubated with the
antibodies at 4 °C in the presence of 0.05% sodium azide prior to
fixation. After washing in PBS, the stained cells were mounted in 90%
glycerol containing p-phenylenediamine and viewed with
a 63× oil immersion lens on a Zeiss microscope equipped with
barrier filters for fluorescein or Texas Red. Rabbit antiserum directed
to the carboxyl terminus of mouse FGF3 was diluted 1:200 in PBS. CFR
was detected using a mouse monoclonal antibody, anti-LA epitope 15E9
against chicken CFR (8), or a mouse monoclonal antibody against the
RGS-His tag (Qiagen). The cis-Golgi compartment was visualized with a rabbit antibody against the cis-Golgi matrix protein GM130 (kindly provided by Dr. Graham Warren, ICRF (21)). For visualizing the endocytotic pathway, cells were incubated with 5 mg/ml lysine-fixable FITC-dextran (10.000 MG, Molecular Probes) in culture medium at 37 °C for 1 h before fixation.
Immunoblot Analysis--
The procedures used for preparing cell
lysates and ECM have been described in detail elsewhere (19, 22).
Samples from equivalent numbers of cells were fractionated by SDS-PAGE
in 12.5% gels, transferred to nitrocellulose membranes (Schleicher & Schuell), and then probed with rabbit antiserum to the carboxyl
terminus of FGF3 or monoclonal antibodies to detect CFR or the RGS-His epitope (Qiagen). Immunoreactive proteins were detected by enhanced chemiluminescence (Amersham Pharmacia Biotech).
Digestion of Carbohydrate Side Chains--
Medium and cell
extracts of CFR-transfected COS-1 cells were concentrated by acetone
precipitation, resuspended in denaturation buffer (0.5% SDS and 1%
-mercaptoethanol), and treated with neuraminidase and
N-glycanase as described by the manufacturer (Biolabs). For neuraminidase treatment, the acetone-precipitated proteins were resuspended in 50 mM sodium citrate, pH 4.5, as recommended
by the manufacturer (Biolabs). The digested products were analyzed by
SDS-PAGE and immunoblotting.
In Vivo CFR Column Retention Assay--
Cell extracts from FGF3
cDNA and pCFR1.1 co-transfected or from FGF3 cDNA alone
transfected COS-1 cells lysed by sonification in a 50 mM
phosphate buffer, pH 7.8, containing 300 mM sodium chloride
were incubated with 50 µl of Ni2+-nitrilotriacetic acid
resin (Qiagen) for 2.5 h at 4 °C in sonification buffer at a
final volume of 500 µl. After washing with a 200-fold volume of
sonification buffer containing 10 mM imidazole, the bound
proteins were eluted with sonification buffer containing increasing
concentrations of imidazole, and the eluates were analyzed by
immunoblotting using a polyclonal antibody against FGF3 or a monoclonal
antibody against CFR. The immune complexes were detected by the
enhanced chemiluminescence technique as described under "Experimental Procedures."
GST-CFR Fusion Protein Affinity Chromatography--
An overnight
30-ml Escherichia coli culture containing PGEX-CFR or a
control GST plasmid was diluted 10-fold into LB/ampicillin medium and
grown at 37 °C to OD 1.0 before induction with 0.5 mM
isopropyl-1-thio--D-galactopyranoside (BTS). Bacteria
were lysed by pulse sonication in lysis buffer (1% Triton X-100, 1.5% N-laurylsarcosine, 25 mM triethanolamine, 1 mM EDTA in PBS). 200 µl of a 50% slurry of GSH-agarose
beads (Molecular Probes) was added and incubated at 4 °C overnight.
After washing 6 times in an excess volume of PBS, 1% Triton X-100
(PBS-TX), agarose beads containing bound proteins were analyzed by
SDS-PAGE followed by Coomassie Blue staining and immunoblot analysis.
Interactions between GST-CFR and FGF3 cell-associated proteins were
analyzed using 0.5-ml aliquots of FGF3-transfected COS-1 cells (equal
to 2 × 105 cells) to which 50 µl of the prepared
GST- or GST-CFR-glutathione-agarose beads were added. The binding
reaction was incubated at 4 °C overnight. The beads were washed,
resuspended in PBS-TX buffer, and poured into a column. The columns
were extensively washed with an excess of PBS-TX, and the retained
proteins were analyzed by SDS-PAGE. FGF3-related proteins were detected
by immunoblotting.
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RESULTS |
CFR Is Secreted and Associated with the ECM--
As a prelude to
investigating the effect of CFR on FGF3 secretion, we have analyzed the
subcellular distribution of CFR using two expression plasmids, pCFR1
and pCFR1.1. pCFR1 contains a full-length chicken cDNA encoding
CFR, whereas pCFR1.1 additionally incorporates a histidine epitope at
the amino terminus of CFR (Fig.
1A). COS-1 cells transfected
with pCFR1 or pCFR1.1 contain products of the expected size of 150 kDa
that were detected by immunoblotting with a monoclonal antibody (LA
epitope) that maps toward the carboxyl terminus of CFR, or in the case
of pCFR1.1 protein was detected with an antibody to the histidine tag
(Fig. 1B).
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Fig. 1.
Presence of chicken CFR in the ECM and
culture medium. A, schematic depiction of pCFR1.1
showing the main features of the CFR: amino-terminal signal peptide
(heavy stippling), the transmembrane domain
(horizontal stripes), and the position of potential
N-linked glycosylation sites (Y). The
shaded box represents the RGS-His epitope
inserted carboxyl-terminal to the signal peptide cleavage site.
B, extracts from COS-1 cells transfected with pCFR1,
pCFR1.1, or the empty vector pKC4 were separated by SDS-PAGE, and the
CFR proteins were detected by immunoblotting with an antibody to the
CFR or the RGS-His epitope. C, COS-1 cells transfected with
pCFR1.1 were harvested after 48 h, and the culture fluid was
recovered. The cells were washed in PBS and removed from the culture
dish with 0.5% Triton X-100 in PBS. The material remaining on the dish
was operationally defined as ECM and recovered in dissociation buffer as described
under "Experimental Procedures." Samples of cell extract, ECM, and
culture medium were fractionated by SDS-PAGE on a 7.5% gel and
immunoblotted with the monoclonal antibody anti-RGS(His) against the
His tag of pCFR1.1. The + and  indicate whether the cells were
grown in the presence or absence of 10 µg/ml heparin. The
immunocomplexes were visualized by ECL technique using a specific
anti-mouse secondary antibody. D, cell extracts and culture
fluids were recovered from pCFR1.1 -transfected COS-1 cells grown in
the presence of heparin (10 µg/ml). Cell proteins were concentrated
by acetone precipitation and digested with neuraminidase
(Neu), or N-glycanase (N-gly) as
indicated. The products were then fractionated by SDS-PAGE, and CFR
products were detected by immunoblotting. Product sizes were calculated
relative to prestained protein standards.
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The identification of ESL-1 as a cell surface protein and MG-160 as
Golgi apparatus-specific, prompted a re-examination of the subcellular
location of CFR/ESL-1/MG-160. The ability of CFR to bind heparin also
suggested that a secreted product may remain associated with the cell
surface and extracellular matrix (ECM). To test these possibilities,
pCFR1 and pCFR1.1 were transfected into COS-1 cells and analyzed for
cell-associated and secreted products in the presence or absence of
heparin (Fig. 1C and data not shown). In the absence of
heparin, CFR was associated with the cell, ECM, and medium. However, in
the presence of heparin, more CFR was displaced into the medium from
the cell surface/ECM, indicating that the CFR is secreted from
transfected COS-1 cells.
Secreted CFR Is Truncated at the Carboxyl Terminus--
As
secretion of a transmembrane protein may involve proteolysis, the size
of the non-glycosylated CFR products was determined. Proteins were
prepared from pCFR1.1-transfected COS-1 cells and medium and treated
with glycosidases as described, and the sizes of the resultant CFR
proteins were determined by immunoblotting (Fig. 1D).
Digestion of the cell extract with neuraminidase alone did not result
in a recognizable decrease in the size of the CFR suggesting that the
majority of the intracellular CFR forms have not yet gained sialic acid
modifications. However, when the same cell extract was treated with
neuraminidase and N-glycanase, the 150-kDa form was almost
completely reduced to approximately 135 kDa, presumably corresponding
to the non-glycosylated translation product after signal peptide
cleavage (Fig. 1D). By contrast, the majority of secreted
CFR proteins reduced their estimated molecular mass to approximately
112-124 kDa following digestion with neuraminidase alone, consistent
with sialic acid modifications, and their mass was further decreased to
100-110 kDa in the presence of additional N-glycanase (Fig.
1D). The faint 150-kDa band still visible after digestion
with neuraminidase presumably represents residual undigested secreted
CFR. Also the weak 135-140-kDa band present in the
N-glycanase and neuraminidase digestion is probably due to
partial digestion. Since the double digestion conditions have been
optimized for N-glycanase but not for neuraminidase, it is
quite likely that some N-linked carbohydrates are less
accessible and therefore less efficiently cleaved (23). In addition,
concentration of the proteins by acetone precipitation prior
neuraminidase and N-glycanase treatment may have affected
the efficiency of the digestions. However, the majority of the secreted
protein is sensitive to N-glycanase and neuraminidase, and
the decrease in mass is consistent with loss of carbohydrate from the
five predicted Asn-linked glycosylation sites. This demonstrates that
the core protein(s) of secreted CFR are considerably smaller than the
intracellular precursor, although they appear to have similar mass
because additional sialylation compensates for the reduction of protein
content. As the CFR proteins were recognized by a monoclonal antibody
against the amino-terminal histidine epitope, differences in mass
between the non-glycosylated extracellular and intracellular forms of the CFRs must occur at their respective carboxyl termini. This would
suggest the presence of proteolytic cleavage sites located amino-terminal to the transmembrane domain, which would also provide an
explanation for CFR secretion (see Fig. 1A for potential
position of cleavage sites).
CFR Co-expression Alters FGF3 Secretion--
To examine the effect
of CFR on the subcellular distribution and trafficking of FGF3, COS-1
cells were co-transfected with pCFR1.1 and a plasmid (pKC3.2) designed
for the efficient expression of secreted FGF3 (19). Four intracellular
isoforms of FGF3 have been identified as follows: two major
glycosylated species of 31.5 and 30.5 kDa (gp31.5 and gp30.5) and two
less abundant non-glycosylated forms, 28.5 and 27.5 kDa (Fig.
2B). The higher molecular
weight species of each pair of proteins was previously shown to differ from the lower molecular mass members by retention of the signal peptide. These four protein isoforms of FGF3 reside in the secretory pathway, predominantly associated with the medial Golgi compartment (19). A form of FGF3 (gp32.5), which is endoglycosidase H-resistant, is
secreted and can associate with the ECM (Fig. 2A). As CFR
and FGF3 are both retained in the Golgi apparatus, there is a
possibility that the presence of an FGF-binding protein in the same
compartment is directly influencing the intracellular trafficking of
FGF3. To test this possibility, a cDNA encoding FGF3 was
co-transfected into COS-1 cells with increasing amounts of pCFR1.1
(Fig. 2A). The results show that transfection of even a
small amount of CFR cDNA causes a diminished release of FGF3 into
the culture medium. This was not due to a reduction of intracellular
FGF3 synthesis as the total amount of FGF3 detected in COS-1 cells was
not detectably diminished, even at higher concentrations of CFR
cDNA (Fig. 2B). Under the conditions used, the amount of
CFR protein detected was proportional to the amount of input cDNA
(data not shown). Thus it would appear that FGF3 is sequestered in the
secretory pathway, possibly through an association with CFR. This
finding also suggests that the amount of CFR that is secreted would not be sufficient to compensate for the amount retained by the bulk of CFR
that resides in the Golgi.
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Fig. 2.
CFR reduces the secretion efficiency of
FGF3. COS-1 cells were co-transfected with the indicated amount of
pCFR1.1 plasmid DNA, together with 10 µg of DNA of pKC3.2 or empty
vector DNA. 12 h after transfection the cell culture medium was
replaced by medium containing 0.1% FCS. After 48 h cell extracts
and culture medium were harvested as described under "Experimental
Procedures." Cell extracts and the culture medium proteins were
analyzed by SDS-PAGE and immunoblotted with a rabbit polyclonal
antibody against the carboxyl-terminal peptide of FGF3. The
immunocomplexes were visualized by ECL technique. The sizes of the
various FGF3 related proteins are indicated in kilodaltons and were
calculated relative to prestained protein standards.
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In Vivo and in Vitro Detection of CFR-FGF3 Binding--
By using a
column retention assay, we tested the capacity of CFR to bind FGF3
in vivo. Cell extracts were prepared from COS-1 cells
transfected with FGF3 or made from cells that have been co-transfected
with the FGF3 cDNA and the histidine tag containing pCFR1.1
plasmid. The extracts were applied to a Ni2+-chelate column
to test the ability of FGF3 to bind CFR that is attached to the column
via its His tag. After washing the columns, the bound proteins were
eluted with increasing concentrations of imidazole to compete off the
His-tagged CFR protein from the affinity column. The eluates were
separated by SDS-PAGE and analyzed by immunoblotting using FGF3 and
CFR-specific antibody, respectively. As shown in Fig.
3A, FGF3 without CFR is
completely eluted from the column with 20 mM imidazole,
whereas FGF3 co-expressed with CFR elutes at much higher concentrations
of imidazole and at the same concentration as the CFR (Fig.
3B). To determine whether cell-associated FGF3 interacts
with recombinant CFR in vitro, COS-1 cells expressing FGF3
were used to prepare a total cell extract, and the ability of
immobilized CFR to bind FGF3 in the extract was assessed. E. coli containing an expression plasmid encoding GST or a GST-CFR
fusion protein was used to prepare extracts as described (Fig.
4A). After incubating the
bacterial extracts with GSH-agarose beads, they were washed and
incubated with cell extracts containing FGF3 as described. After
extensive washing the presence of FGF3 retained by the beads was
assessed by immunoblotting (Fig. 4B). The GST-CFR fusion
protein was clearly able to retain FGF3, whereas even an excess (about
5 times over GST-CFR fusion protein) of the control GST protein did
not, demonstrating a strong affinity of FGF3 for CFR.
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Fig. 3.
Binding of FGF3 to CFR in
vivo. COS-1 cells were transfected with FGF3 cDNA
(A) or co-transfected with FGF3 cDNA and pCFR1.1
containing the His6 cassette (B). The cell
extracts were incubated for 2.5 h at 4 °C with the
Ni2+-nitrilotriacetic acid resin. The bound proteins were
eluted with increasing concentrations of imidazole as indicated and
analyzed by immunoblotting using a polyclonal antibody against FGF3 and
a monoclonal antibody against the His tag.
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Fig. 4.
CFR binds FGF3 in
vitro. A, left panel, a Coomassie
Blue-stained SDS-PAGE analysis of bacterial cultures uninduced or
induced (3 h) to express a GST-CFR fusion proteins as indicated and
described under "Experimental Procedures." The right-hand
tracks show the proteins recovered from an aliquot (5 µl) of the
washed GSH-agarose resin after binding of the bacterial extracts.
Right panel, an immunoblot analysis of the bacterially
expressed GST-CFR fusion proteins and the CFR proteins recovered from
an aliquot (5 µl) of the washed GSH-agarose beads after binding
GST-CFR. B, an immunoblot of the FGF3 proteins retained by
the loaded resins after incubation with an extract of FGF3-expressing
COS-1 cells (right two lanes) or incubation of the GST-CFR
resin with a cell extract from COS-1 cells containing the empty
expression vector pKC3.
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FGF3 Secretion Is Modulated by CFR--
Targeting signals that
affect the trafficking and secretion of integral membrane proteins
often reside on the intra-luminal domain or close to the transmembrane
domain. Assuming that deletion of the carboxyl-terminal domains may
alter the localization or secretion of CFR, a concomitant change of the
FGF3 secretion efficiency and subcellular localization would provide
evidence that CFR binds FGF3 in vivo. Two truncation mutants
of pCFR1.1 were generated; one contained a deletion encompassing the
intra-luminal region (pCFR2.1), and for the other the deletion extended
to a position amino-terminal to the transmembrane domain (pCFR3.1)
(Fig. 5A). The expression and
secretion of the truncated CFRs were analyzed by immunoblotting the
culture medium, cell extracts, and ECM in comparison with the
full-length protein (Fig. 5B). The ratio of the relative
amount of cell extract/medium/ECM analyzed was 1:4:8. As expected, the
truncated protein encoded by pCFR3.1 and lacking a transmembrane domain
was efficiently secreted as a soluble protein. Cells expressing pCFR2.1
also produced a predominantly secreted CFR protein, despite retaining
the sequences encoding the transmembrane domain. Although 80-90% of
the wild-type CFR protein remains cell-associated, 30-50% of the
truncated CFR proteins (CFR2.1 and CFR3.1, respectively) were found in
the extracellular space. However, all extracellular CFR-related
products had the same apparent size, indicating that the secreted
proteins derived from full-length and truncated forms are similar,
presumably through proteolytic cleavage at sites amino-terminally
located to the transmembrane domain as described above.
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Fig. 5.
Effect of carboxyl-terminal truncations on
the secretion of CFR. A, schematic depiction of the
parental CFR cDNA present in plasmid vector pCFR1.1, pCFR2.1, and
pCFR3.1 containing carboxyl-terminal truncations. Most of the marked
features are described in the legend to Fig. 1. B,
immunoblot analysis of cell extracts, ECM, and culture medium from
COS-1 cells transfected with pCFR1.1, pCFR2.1, pCFR3.1, or the control
vector pKC4 (see "Experimental Procedures" and Fig. 1 for
procedures). The CFR proteins were detected with a monoclonal antibody
(LA) against CFR.
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To determine the effect truncated CFR proteins might have on FGF3
secretion, they were co-expressed in COS-1 cells, and the distribution
of FGF3 between cell and extracellular compartments was assessed (Fig.
6A). Although co-expression of
the truncated CFRs substantially reduced the amount of FGF3 secreted
into the culture medium, more FGF3 was found under conditions where
more CFR was exported. To investigate further the influence of CFR and
the CFR mutants on the intracellular distribution of FGF3, the
subcellular location of FGF3 and CFR was examined by immunofluorescence staining. Expression of FGF3 alone showed the typical dense
juxtanuclear staining expected from a protein mainly localized in the
medial Golgi as previously reported (Fig. 6B). However,
co-expression of FGF3 with normal or truncated CFRs revealed additional
staining of FGF3 in reticular structures, as well as peripheral
vesicular structures (Fig. 6, C-E). This latter staining
was more pronounced when FGF3 was co-expressed with truncated CFRs,
indicating a change in FGF3 trafficking.
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Fig. 6.
Effect of carboxyl-terminal truncated CFRs on
the secretion and subcellular localization of FGF3. A,
an immunoblot analysis of culture medium from COS-1 cells
co-transfected with 10 µg of pKC3.2, together with 5 µg of pCFR1.1,
pCFR2.1, pCFR3.1, or the empty vector pKC4. FGF3 proteins were detected
with a rabbit antiserum directed to FGF3. The immunocomplexes were
visualized by ECL. The subcellular localization of FGF3 in COS-1 cells
transiently co-transfected with 10 µg of pKC3.2 together with 5 µg
of DNA of the empty vector pKC4 (B) or 5 µg of each of the
CFR cDNAs pCFR1.1 (C), pCFR2.1 (D), and
pCFR3.1 (E). The FGF3-related proteins were detected by
immunostaining with the rabbit antiserum and visualized with Texas
Red-conjugated secondary antibodies. When co-transfected with CFR, FGF3
proteins are localized in additional vesicular compartments.
Representative examples are shown.
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Subcellular Localization of Normal and Truncated CFR Proteins and
Co-localization with FGF3--
To determine whether this routing of
FGF3 was due to its association with CFR, the subcellular localization
of both wild-type and truncated CFR was examined by immunofluorescence
staining. In agreement with previous reports, wild-type CFR was
primarily concentrated in areas adjacent to the nucleus in a position
characteristic of the Golgi complex (Fig.
7A) (10, 11, 15, 16). To
establish the juxtanuclear staining as Golgi, the COS-1 cells were
stained with two antibodies, one against the His tag and a second
against another cis-medial Golgi matrix protein GM130 (21). A
comparison of the two staining patterns showed a substantial overlap,
confirming the Golgi apparatus as the main intracellular location for
CFR (Fig. 7, C and D). The cell surface/ECM
location of the CFR predicted from the cell fraction study was not
apparent in the staining pattern with fixed and permeabilized cells,
probably due to the strong intracellular signal. Therefore, to
investigate the predicted cell surface staining, transfected COS-1
cells were fixed in 4% paraformaldehyde but not permeabilized prior to
antibody staining. As can be seen in Fig. 7B, cell surface
staining, particularly of the microvilli, was now clearly revealed by
this procedure. In presence of heparin, cell surface staining for CFR
was significantly diminished but not abolished (data not shown). In
addition to the juxtanuclear and plasma membrane staining, CFR
was also present in numerous cytoplasmic vesicles seen before in the
FGF3 co-transfection experiment (Fig. 5, B-E). These
structures are reminiscent of the endosomal/lysosomal system suggesting
a dynamic pattern of intracellular trafficking (Fig.
7A).
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Fig. 7.
Intracellular localization of wild-type and
mutant CFR by immunofluorescence microscopy. COS-1 cells were
transfected with pCFR1.1, fixed, and permeabilized for intracellular
staining (A) or left unpermeabilized for selective cell
surface staining (B). CFR proteins were detected with the
monoclonal antibody anti-RGS(His) against the His tag. C and
D, partial co-localization of CFR and the cis-Golgi matrix
protein GM130 by immunofluorescence microscopy. COS-1 cells were
transfected with pCFR1.1, fixed, and permeabilized. The cells were
stained with the monoclonal antibody anti-RGS(His) against the His tag
and a rabbit antiserum against GM130. C shows detection of
anti-RGS(His) antibody with a FITC-coupled secondary antibody, and
D shows staining with anti-GM130 with a Texas Red-coupled
secondary antibody. E and F, intracellular
localization of mutant CFR by immunofluorescence microscopy. COS-1
cells expressing pCFR2.1 (E) or pCFR3.1 (F) were
stained with anti-RGS(His) and visualized with a Texas Red-tagged
secondary antibody (E and F). CFR-related
proteins are present in the endosomal compartment (G and
H). To localize the endosomal/lysosomal compartment, COS-1
cells transfected with pCFR3.1 were also incubated with FITC-dextran
prior to fixation. The CFR protein was detected with a Texas
Red-labeled antibody (G), and the fluorescence from the
FITC-dextran is shown in H. To facilitate the comparison of
the staining pattern, some of the vesicular structures were highlighted
by arrows or boxed. I and J
show co-staining of COS-1 cells transfected with pKC3.2 and pCFR3.1
demonstrating co-localization of FGF3 and CFR proteins in the endosomal
compartment using the mouse monoclonal anti-RGS(His) to detect CFR and
a rabbit polyclonal antiserum against FGF3.
|
|
A similar analysis using the deletion mutants pCFR2.1 and pCFR3.1
showed the intracellular distribution of the truncated CFR mutants was
changed. Surprisingly, both deletion mutants still showed a
juxtanuclear staining as observed for wild-type CFR, despite the loss
of the transmembrane domain and cytoplasmic tail for the product
encoded by pCFR3.1 (Fig. 7, E and F). Moreover, staining of peripheral vesicular structures was considerably more pronounced with the truncated CFRs. The identity of these structures as
endocytotic vesicles was suggested by co-staining pCFR3.1-transfected cells for CFR and showing partial coincidence with the uptake of
lysine-fixable FITC-labeled dextran added 1 h prior to fixation (Fig. 7, G and H). The staining pattern of FGF3
in the co-transfected experiments was very similar to the pattern
observed for normal or truncated CFR proteins, respectively.
Co-staining of pCFR3.1- and pKC3.2-transfected COS-1 cells with a mouse
monoclonal antibody against the His tag and a rabbit antiserum against
FGF3, respectively, and detection of the immunocomplexes with
species-specific secondary antibodies obtained an almost coincident
staining pattern in agreement with a new co-localization of CFR and
FGF3 in the peripheral vesicles (Fig. 7, I and
J). Thus the reduced secretion of FGF3 probably reflects its
sequestration by CFRs in the medial Golgi complex, and then it would be
either secreted or routed to the endocytic pathway in association
with CFR. The staining pattern and the co-expression experiments imply
that FGF3 is captured and can be redistributed by CFR proteins to
intracellular compartments normally not entered by FGF3.
| |
DISCUSSION |
In this study we show that FGF3 can associate with the CFR, and
this could account for its poor secretion and retention in the Golgi
apparatus (19). CFR protein (MG160) is primarily located in the Golgi
apparatus, consistent with previous reports, but we also detect a
soluble form of the protein that associates with the cell surface and
ECM (11, 12, 16, 17). From the size of the core protein after
carbohydrate removal, the secreted CFR is cleaved at the carboxyl
terminus, which removes the intra-luminal and transmembrane domain
allowing the protein to be secreted. Since we could never detect
extracellular or cell-associated CFR forms with a significantly higher
molecular mass than 170 kDa, which might correspond to a CFR protein
with a sialylated intact protein backbone, the residual bands present
after treatment of CFR medium proteins with glycosidases derive most
likely from partial digestion. The extracellular and soluble CFR
appears to be associated with the ECM and/or cell surface through an
affinity with glycosaminoglycans, since soluble heparin is efficient at competing CFR from the cell surface and ECM. CFR/MG160 appears to be
homologous to ESL-1, a high affinity binding protein for E-selectin.
ESL-1 was identified as a cell surface glycoprotein expressed on
myeloid and some lymphoid cells (11, 12, 15, 16). However, in the light
of the results presented here, it would be interesting to know whether
cell surface ESL-1 is present as an integral membrane protein or
associated with the cell surface by its affinity to glycosaminoglycans.
Although the bulk of the intracellular CFR is Golgi complex-associated,
it was also found in endosomal vesicles that were identified by
co-staining with FITC-labeled dextran. Interestingly, the CFR
carboxyl-terminal deletion mutants that lack either the cytoplasmic
domain or both cytoplasmic and transmembrane domain showed a more
pronounced staining of endosomal compartment. This suggests that the
deletion mutations have weakened but not destroyed the Golgi retention and have allowed a greater amount of CFR to enter endosomes. Moreover, a spontaneous carboxyl-terminal truncation mutant of CFR which lacks
the cytoplasmic, transmembrane, and juxtamembrane regions appeared to
be widely distributed in the cell suggesting that the intraluminal
juxtamembrane domain is important for targeting and retention of CFR to
the medial Golgi (17).
The effect of CFR on the biological properties of FGFs remains
puzzling. A recent study looking at the effect on exogenously applied
FGF1 or FGF2 to Chinese hamster ovary cells expressing elevated levels
of CFR demonstrated a significant reduction in the intracellular levels
of these FGFs (17). As CFR did not appear to block entry of these FGFs,
or reduce their intracellular stability, the effect was ascribed to an
enhanced trafficking of the FGFs from endocytic compartments to the
cell exterior, possibly via the Golgi apparatus (17). The fate of FGF3
represents a very different situation; in this case FGF3 is synthesized
as a naturally secreted ligand that would be expected to encounter CFR
in the Golgi apparatus. Furthermore, we show here that FGF3 binds CFR
in vivo and in vitro (Figs. 3 and 4). In cell
culture, increased intracellular levels of CFR result in a progressive reduction in the secretion of FGF3 without a noticeable effect on FGF3
synthesis (Fig. 2B). In COS-1 cells, the gp30.5 and gp31.5 products associated with the medial Golgi have a half-life time in
excess of 4 h and are continuously sensitive to endoglycosidase H
digestion and represent the majority of the steady state level of
FGF3-related protein (19). The secreted 32.5-kDa form of FGF3 is
derived from the smaller products by secondary modification in a Golgi
or post-Golgi compartment (19). In the presence of heparin to displace
the already secreted form from its binding to cell surface
heparan-sulfate proteoglycans, no cell-associated gp32.5-kDa product is
detectable, suggesting that as soon as FGF3 leaves the medial Golgi the
further steps of secretion and modification appear to happen very fast
(19, 22). Since we have been unable to detect a cell-associated
gp32.5-kDa product as a consequence of co-expression of CFR (Fig.
2B), we concluded that CFR must interact with the FGF3 forms
when associated with the medial Golgi before they had undergone further
modification. However, since the overall amount of intracellular FGF3
protein is not changed within the limits of our assay, the half-life of
the FGF3 products routed via CFR in the endosomal-lysosomal compartment
is presumably considerably lower than those in the medial Golgi.
The sequestration of FGF3 by CFR was further supported by the behavior
of the carboxyl-terminal deletion mutants of CFR. The degree to which
the CFR proteins change the amount of extracellular FGF3 positively
correlates to their own Golgi retention, with the CFR3.1 mutant having
the smallest effect on the FGF3 release.
In essence, the redistribution of truncated CFR to intracellular
vesicles was reflected by an analogous distribution of FGF3 when
present in co-transfected COS-1 cells. The CFR potentially contains a
Golgi retention and endosomal targeting signal that might shuttle
internalized CFR·FGF complex between the endosomal compartment and
Golgi via the trans-Golgi network and thereby also provide an interface
with the cell surface via endosomes. A possible retrieval of FGF3 by
the CFR from the endosomal compartment back to the trans-Golgi network
could also account for the significant retardation of FGF3 secretion.
As the deleted CFRs may be less efficiently retrieved from the plasma
membrane, this would explain their accumulation in the
endosomal/lysosomal compartment and also their diminished effect on the
FGF secretion.
Recently, CFR was also identified as part of a secreted but latent
TGF- complex and was termed latent TGF--complexed protein-1 (LTCP-1) (18). It is interesting that TGF- as well as FGF3 are
retained in the Golgi apparatus as a pre-secreted store. Thus, the
presence of CFR/MG-160 in the latent transforming growth factor- complex and its localization to the medial cysternae of the Golgi complex suggest a possible function in the processing and secretion of
a member of another growth factor family (19, 20). The CFR/MG160/ESL-1/LTCP-1 appears to be a multifunctional protein, although its role within the organism is not clear. It is interesting that ESL-1, a ligand for E-selectin, is a cytokine modulated by cell
adhesion molecule that causes the binding of neutrophils to the
endothelium. Both FGFs and TGF- have been implicated as having an
involvement in tissue repair and inflammatory responses, albeit with
opposing effects. Therefore, we would speculate that maybe this
multi-functional protein may help to balance or buffer the effects of
these powerful cell signaling molecules in a developmental or tissue
repair context.
| |
ACKNOWLEDGEMENT |
We thank Graham Warren for the kind gift of
the antibody against GM130 and the useful comments and suggestions
regarding this manuscript.
| |
FOOTNOTES |
*
This work was supported by a grant from the National
Institutes of Health (to B. B. O.) and by a grant from the German
Research Society (to P. K.).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.:
49-234-322-6465; Fax.: 49-234-321-4352; E-mail:
paul.kiefer@ruhr-uni-bochum.de.
Published, JBC Papers in Press, March 9, 2000, DOI 10.1074/jbc.M903271199
| |
ABBREVIATIONS |
The abbreviations used are:
FGF, fibroblast
growth factor;
ECM, extracellular matrix;
CFR, cysteine-rich FGF
receptor;
PCR, polymerase chain reaction;
PBS, phosphate-buffered
saline;
FITC, fluorescein isothiocyanate;
PAGE, polyacrylamide gel
electrophoresis;
GST, glutathione S-transferase;
TGF-, transforming growth factor-.
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