From the Department of Biochemistry and Molecular
Biology, St. Louis University School of Medicine, St. Louis, Missouri
63104 and § Atherosclerosis Research, Monsanto Company,
St. Louis, Missouri 63167
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ABSTRACT |
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The cell surface retention sequence (CRS) binding
protein-1 (CRSBP-1) is a newly identified membrane glycoprotein which
is hypothesized to be responsible for cell surface retention of the oncogene v-sis and c-sis gene products and
other secretory proteins containing CRSs. In simian sarcoma
virus-transformed NIH 3T3 cells (SSV-NIH 3T3 cells), a fraction of
CRSBP-1 was demonstrated at the cell surface and underwent
internalization/recycling as revealed by cell surface 125I
labeling and its resistance/sensitivity to trypsin digestion. However,
the majority of CRSBP-1 was localized in intracellular compartments as
evidenced by the resistance of most of the
35S-metabolically labeled CRSBP-1 to trypsin digestion, and
by indirect immunofluorescent staining. CRSBP-1 appeared to form
complexes with proteolytically processed forms (generated at and/or
after the trans-Golgi network) of the v-sis gene product
and with a ~140-kDa proteolytically cleaved form of the
platelet-derived growth factor (PDGF) Many secretory proteins, including growth factors and cytokines,
have been demonstrated to undergo cell surface retention during
biosynthesis and secretion in cultured cells (1-8). These proteins are
secreted but remain tightly associated with the cell surface without
being released into the medium or extracellular compartment. The cell
surface-retained proteins may be released upon treatment with suramin,
heparin, or high salt or by protease digestion (1, 5, 6, 8). Upon
longer incubation (days), the cell surface-retained proteins may be
released (mainly as degraded forms) or diffuse slowly and become
associated with extracellular matrix (1, 8, 9). The proteins undergoing
cell surface retention possess cell surface retention sequence
(CRS)1 motifs which include a
stretch of basic amino acids; these motifs are responsible for their
cell surface retention (1-8). The cell surface retention of secretory
proteins is hypothesized to be mediated by high-affinity cell
surface-binding sites which have been recently identified by binding
and cross-linking of 125I-labeled peptides containing the
CRS motifs of platelet-derived growth factor-B (c-sis gene
product) and vascular endothelial cell growth factor (8). A major
CRS-binding protein, designated CRSBP-1, has been purified to near
homogeneity from plasma membranes of cultured cells and liver tissue
(8). CRSBP-1 is a major binding protein for CRS peptides which is
expressed in all cell types thus far examined including fibroblasts,
endothelial cells, epithelial cells, and smooth muscle cells
(8).2
The viral oncogene v-sis product of simian sarcoma virus
(SSV) was the first protein reported to undergo cell surface retention during secretion (1). The cell surface retention of the
v-sis gene product appeared to facilitate its binding to the
PDGF receptor at the cell surface of SSV-transformed cells (1). The
c-sis gene product (PDGF-B), the cellular homologue of the
v-sis gene product, was subsequently shown to undergo this
novel secretion process and its major structural determinant (CRS) was
localized near its C terminus (2, 6). Deletion of this major CRS in the
PDGF-B molecule diminished its cell surface retention and enhanced its
secretion but did not affect the transforming activity of the
c-sis gene product as measured by an in vitro
foci assay (6). This strongly argued that the major CRS was not
absolutely required for the transforming activity of the
c-sis gene product. However, the in vitro foci
assay used to determine the transforming activity of the
c-sis gene product did not yield information concerning its
tumorigenicity. Furthermore, the c-sis gene product may
contain other minor or cryptic CRSs which contribute to partial cell
surface retention even after deletion of the major CRS near its C
terminus (2, 6, 8). The CRSs of polypeptide growth factors are well
conserved in evolution (2, 6, 10-20), suggesting biological importance
of cell surface retention of these proteins.
v-sis- and c-sis-transformed fibroblasts have
been the best studied systems of autocrine transformation (1, 10, 11, 21-30). In v-sis- and c-sis-transformed cells,
the newly synthesized PDGF Materials--
Na125I (17 Ci/mg),
Tran35S-label (>1,000 Ci/mmol),
L-[35S]cysteine (>800 Ci/mmol), and
[ Metabolic Labeling and Immunoprecipitation--
Subconfluent
monolayers of SSV-NIH 3T3 (~3 ×106 cells/10-cm Petri
dish) or NIH 3T3 cells (~6 × 105 cells/10-cm Petri
dish) were starved in methionine-free DMEM for 3.5 h prior to
labeling with Tran35S-label or
L-[35S]cysteine (200 µCi/ml) for 3 h
(1, 22). For pulse and chase experiments, labeled cells were chased in
DMEM containing 10 mM unlabeled L-methionine or
L-cysteine for various times before harvesting (1, 22).
Cells were then extracted with 1.2 ml of RIPA buffer (1% Nonidet P-40,
0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris-HCl, pH
7.4, 0.15 M NaCl, 5 mM EDTA) and the cell extracts were immunoprecipitated using antiserum to CRSBP-1 (8) or the
v-sis gene product (31) and analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) as described previously (1, 22).
125I Labeling of Cell Surface Proteins--
Cell
surface proteins were labeled with 125I using the
lactoperoxidase method of Hubbard and Cohn (32). Briefly, confluent cells in 60-mm Petri dishes were placed on ice, rinsed with
phosphate-buffered saline (PBS), and then incubated for 20 min with 2.5 ml of PBS containing 1.5 mg of glucose, 120 µg of lactoperoxidase, 60 µg of glucose oxidase, and 250 µCi of Na125I. The
reaction was stopped by washing the cells twice with PBS containing 1 mM N-acetyltyrosine and 10 mM sodium
metabisulfite, and twice with PBS alone. The cell surface
125I-labeled cells were lysed and analyzed by
immunoprecipitation using antiserum to CRSBP-1 and SDS-PAGE.
CRSBP-1 and v-sis Gene Product
Co-immunoprecipitation--
Subconfluent cultures of SSV-NIH 3T3 cells
(~3 × 106 cells/10-cm Petri dish) or NIH 3T3 cells
(~6 × 105 cells/10-cm Petri dish) were incubated at
37 °C for 4 h in cysteine-free DMEM followed by labeling with
L-[35S]cysteine (200 µCi/ml) at 37 °C
for 1 h (1, 22). The metabolically labeled cells were washed twice
in PBS and collected in PBS. After centrifugation, the cells were
extracted in Triton X-100 buffer (20 mM Hepes/NaOH, pH 7.4, 1% Triton X-100, 50 mM NaCl, 5 mM EDTA) containing 5 µg/ml aprotinin, 1 µg/ml leupeptin, 1 µg/ml
pepstatin A, 5 mM iodoacetamide, and 5 mM
N-ethylmaleimide. The cell extracts were then precleared
with non-immune serum and incubated with antiserum to the
v-sis gene product, non-immune serum, or antiserum to
CRSBP-1 at 4 °C for a minimum of 4 h. The immunocomplexes were precipitated with protein A-Sepharose at 4 °C for 90 min. After washing with Triton X-100 buffer, the antigen-antibody complexes in the
immunoprecipitates were dissociated by heating the washed protein
A-Sepharose beads at 100 °C in SDS buffer (50 mM
Tris-HCl, pH 7.4, 1% SDS, 5 mM EDTA, 0.15 M
NaCl). After centrifugation, the supernatant was diluted 10-fold with
SDS-free RIPA buffer and re-immunoprecipitated with antiserum to the
v-sis gene product as described above. The final
immunoprecipitates were analyzed by SDS-PAGE.
Trypsin Digestion of Cell Surface CRSBP-1--
Cells were either
pulse labeled with Tran35S-label and chased for 2 h or
cell surface labeled with Na125I as described above.
Labeled cells were cooled on ice and washed twice with ice-cold
bicarbonate-free DMEM. Following the washes, the cells were treated
with trypsin (2 mg/ml) on ice for 2 h. STI was then added to a
final concentration of 6 mg/ml to stop the digestion reaction. In the
control in which the cells were treated without trypsin, STI was added
prior to the addition of trypsin. Cells treated with and without
trypsin were collected, washed twice with bicarbonate-free DMEM
containing 6 mg/ml STI, extracted with RIPA buffer, and
immunoprecipitated using antiserum to CRSBP-1.
Indirect Immunofluorescent Staining--
Cells were grown on
glass coverslips and fixed with methanol at Internalization and Recycling of Cell Surface
CRSBP-1--
Following cell surface iodination, cells were washed once
with bicarbonate-free DMEM (prewarmed to 37 °C). The cells were then
incubated at 37 °C for various time periods as indicated. After
incubation, cells were cooled to 0 °C on ice and treated with 2 mg/ml trypsin on ice for 2 h as described above. Control cells
were kept on ice after cell surface iodination and treated with trypsin
in the presence of STI (6 mg/ml). Finally, cells were solubilized with
RIPA buffer and the cell extracts were immunoprecipitated with
antiserum to CRSBP-1.
Autophosphorylation of the PDGF CRSBP-1 Is Expressed at the Cell Surface of SSV-NIH 3T3 Cells and
Normal NIH 3T3 Cells--
The newly synthesized v-sis gene
product is secreted but retained at the cell surface of SSV-NIH 3T3
cells (1). If CRSBP-1 is the protein responsible for cell surface
retention of the v-sis gene product, SSV-NIH 3T3 cells
should express CRSBP-1 at its cell surface. To determine the cell
surface expression of CRSBP-1, proteins on the cell surface of SSV-NIH
3T3 cells were labeled with 125I at 0 °C using the
lactoperoxidase method (32). The 125I-labeled cell lysates
were then immunoprecipitated with antiserum to CRSBP-1 which reacts
with an N-terminal region in the cytoplasmic domain of CRSBP-1, a type
II membrane glycoprotein (8, 33). These were analyzed by SDS-PAGE and
autoradiography. As shown in Fig. 1, both
monomeric and dimeric forms (Fig. 1A, lane 1), and the
monomeric form (Fig. 1B, lane 1) of CRSBP-1 were identified in the cell lysates by analysis on SDS-PAGE under nonreducing and
reducing conditions, respectively. This result suggests that CRSBP-1 is
present at the cell surface and can form disulfide-linked dimers in
SSV-NIH 3T3 cells. Only the monomeric form of CRSBP-1 was detected in
normal NIH 3T3 cells by analysis on SDS-PAGE under either reducing or
nonreducing conditions (data not shown).
A Majority of CRSBP-1 Is Present in Intracellular
Compartments--
We hypothesized that CRSBP-1 forms complexes with
the newly synthesized v-sis gene product in the TGN before
the complex is transported to the cell surface (1, 8). To see if
CRSBP-1 is present in intracellular compartments, CRSBP-1 was
metabolically labeled with Tran35S-label in SSV-NIH 3T3
cells. After pulse and chase for 5 h to allow steady-state
cellular distribution of CRSBP-1, cells were treated with trypsin.
35S-Labeled cell lysates were then immunoprecipitated using
antiserum to CRSBP-1. The immunoprecipitates were analyzed by SDS-PAGE
and fluorography. As shown in Fig. 2, the
trypsin treatment of cells did not greatly affect the recovery of
35S-labeled CRSBP-1 in immunoprecipitates. Nearly all of
35S-labeled CRSBP-1 appeared to be recovered in the
immunoprecipitates of cells treated with trypsin when compared with the
amount of 35S-labeled CRSBP-1 in the immunoprecipitates of
cells treated without trypsin (Fig. 2A, lane 2 versus lane
1). In a control experiment, the trypsin treatment completely
abrogated the appearance of 125I-labeled cell surface
CRSBP-1 in the immunoprecipitates (Fig. 2B, lane 2 versus lane
1). These results suggest that a majority of metabolically labeled
cellular CRSBP-1 (>95%) is present in intracellular compartments,
explaining its resistance to trypsin digestion.
To define the subcellular localization of CRSBP-1, we performed
indirect immunofluorescent staining of SSV-NIH 3T3 cells using antiserum to CRSBP-1 and fluorescein isothiocyanate-conjugated goat
anti-rabbit IgG. As shown in Fig.
3A, CRSBP-1 was localized in
the vesicles near extranuclear membranes which may represent TGN and
endosomes/prelysosomal compartments. The immunofluorescent staining was
blocked in the presence of 2 mM peptide antigen (Fig. 3B).
CRSBP-1 Interacts with the v-sis Gene Product in SSV-NIH
3T3--
Since CRSBP-1 likely traffics between TGN and the cell
surface, CRSBP-1 could interact with the newly synthesized
v-sis gene product during routing of both proteins from TGN
to the cell surface. To test this possibility, we investigated the
interaction of CRSBP-1 with the newly synthesized v-sis gene
product using [35S]cysteine metabolic labeling followed
by sequential immunoprecipitation with antisera to CRSBP-1 and the
v-sis gene product. SSV-NIH 3T3 cells were metabolically
labeled with [35S]cysteine. The 35S-labeled
cells were then extracted with Triton X-100 or RIPA buffer (1, 22). The
Triton X-100 or RIPA buffer extracts were immunoprecipitated with
antiserum to the v-sis gene product, antiserum to CRSBP-1,
or non-immune serum. The antigen-antibody complexes in the
immunoprecipitates were dissociated by heating at 100 °C in RIPA
buffer and subjected to a second immunoprecipitation using antiserum to
the v-sis gene product or non-immune serum. The second
immunoprecipitates were analyzed by SDS-PAGE under nonreducing
conditions. As shown in Fig. 4, antiserum
to CRSBP-1 was able to co-immunoprecipitate the v-sis gene
products p44 and p36 in Triton X-100 buffer (Fig. 4, lane 2)
but not in RIPA buffer (Fig. 4, lane 5). RIPA buffer
contained 0.1% SDS which dissociates the complex of CRSBP-1 and the
v-sis gene product. In SSV-NIH 3T3 cells, the
v-sis gene product is known to be synthesized as a 72-kDa
(p72) homodimer and proteolytically processed to generate p68, p58,
p44, and p27 (1). Both p68 and p58 are generated at the ER and Golgi
complex. The production of p44 occurs at TGN and/or after exit from
TGN. p27 is a proteolytic product of the cell surface-bound p44 and
does not contain the major CRS (1). However, under mild conditions
(cell lysis in 0.1% Triton X-100), the various size forms of the
v-sis gene product (p58, p50, p44, p36, and p27) were
detected in SSV-NIH 3T3 cells in this study (Fig. 4, lane
1). p50 and p36 were not detected in the previous more restrictive
conditions of cell lysis (0.1% SDS) (1) and may represent degradation
products of p58 and p44, respectively. p27 was not detected on the
autoradiogram of the co-immunoprecipitates (even after longer time
exposure of the autoradiogram). These results suggest that CRSBP-1
interacts mainly with the v-sis gene products p44 and p36
which are produced at and/or after TGN (1).
CRSBP-1 Undergoes Rapid Turnover in SSV-NIH 3T3 Cells but Not in
Normal NIH 3T3 Cells--
The v-sis gene product undergoes
rapid turnover in SSV-transformed cells (1). If CRSBP-1 forms complexes
with the v-sis gene product in SSV-NIH 3T3 cells, CRSBP-1
should undergo rapid turnover as the v-sis product does (1).
We, therefore, determined the turnover rate of CRSBP-1 in SSV-NIH 3T3
cells and normal NIH 3T3 cells using pulse and chase experiments. As
shown in Fig. 5, CRSBP-1 underwent rapid
turnover with a t1/2 of ~2.5 h in SSV-NIH 3T3
cells (Fig. 5, A and C). In contrast, CRSBP-1 was
relatively stable even after a 4-h chase in normal NIH 3T3 cells (Fig.
5, B and C). The rapid turnover of CRSBP-1 in
SSV-NIH 3T3 cells was completely blocked by suramin, which is also
known to block the intracellular turnover of the v-sis gene
product and PDGF Cell Surface CRSBP-1 Undergoes Internalization and Recycling in
SSV-NIH 3T3 Cells but Not in Normal NIH 3T3 Cells--
Since the
majority of CRSBP-1 is present intracellularly, the turnover of CRSBP-1
should occur intracellularly as well (Fig. 5, A and
C). It was therefore of interest to see if cell surface CRSBP-1 undergoes ligand-dependent turnover. To examine the
stability of cell surface CRSBP-1, CRSBP-1 on the cell surfaces of
SSV-NIH 3T3 cells and normal NIH 3T3 cells was labeled with
125I at 0 °C using the lactoperoxidase procedure (32).
The 125I-labeled cells were warmed to 37 °C for various
time periods to allow internalization of 125I-labeled cell
surface CRSBP-1. Following these warming time periods, cells were
cooled to 0 °C and subjected to trypsin digestion. The cell lysates
were then analyzed by SDS-PAGE and autoradiography under reducing
conditions. As shown in Fig.
6A, 125I-labeled
cell surface CRSBP-1 in SSV-NIH 3T3 cells was sensitive to trypsin
digestion prior to warming at 37 °C and became almost completely
resistant to trypsin digestion after a 6-min incubation at 37 °C
(Fig. 6A, lane 4), indicating that internalization of cell
surface CRSBP-1 had occurred. After further incubation at 37 °C, the
internalized 125I-CRSBP-1 gradually recycled back to the
cell surface and became sensitive to trypsin digestion again. After an
18-min incubation at 37 °C, 125I-labeled cell surface
CRSBP-1 achieved a second full internalization (Fig. 6A, lane
8). This result indicates that the cell surface CRSBP-1 undergoes
rapid internalization and recycling with a cycling time of ~12 min. A
third peak of cell surface CRSBP-1 internalization occurred after a
30-min incubation at 37 °C (Fig. 6A, lane 10). Quantitative analysis by densitometric scanning revealed that approximately ~30% of the internalized CRSBP-1 was recycled and that
the remaining ~70% underwent turnover. In normal NIH 3T3 cells, the
125I-labeled cell surface CRSBP-1 did not undergo
internalization under similar conditions and was sensitive to trypsin
digestion even after incubation up to 30 min at 37 °C (Fig.
6B, lanes 2-10). In SSV-NIH 3T3 cells, the internalization
and recycling of 125I-labeled cell surface CRSBP-1 appeared
to be blocked by 100 µM suramin. In the presence of
suramin, 125I-labeled cell surface CRSBP-1 remained at the
cell surface and was sensitive to trypsin digestion (Fig. 6A,
lanes 12-20). Suramin did not affect the status of the cell
surface CRSBP-1 in normal NIH 3T3 cells (Fig. 6B, lanes
12-20). The cell surface CRSBP-1 was sensitive to trypsin
digestion during incubation at 37 °C either in the presence or
absence of suramin (Fig. 6B). These results suggest that
cell surface CRSBP-1 undergoes ligand (v-sis gene
product)-dependent internalization and recycling. We have also examined the internalization and recycling of cell surface CRSBP-1
in other cell types. Only cells expressing the v- or
c-sis gene product (SSV-NIH 3T3, SSV-NRK, and
c-sis-NIH 3T3 cells) exhibited cell surface CRSBP-1
internalization and recycling while, in other cell types which did not
express the v- or c-sis gene product (neu/erbB2-transformed NIH 3T3 cells, ras-transformed NIH
3T3 cells, normal NRK cells, myc-transformed NIH 3T3 cells),
CRSBP-1 did not undergo detectable internalization and recycling (data not shown). These results indicate that the internalization and recycling of CRSBP-1 is not a general feature associated with transformation.
CRSBP-1 Interacts with the PDGF CRSBP-1 is expressed in all cell types thus far examined and
appears to be a major cell surface binding protein for
125I-labeled CRS peptides (8). The intracellular and cell
surface localization and broad pH ligand binding activity of CRSBP-1
allow CRSBP-1 to function as a ligand-specific transport protein
trafficking between intracellular organelles (TGN) and the plasma
membrane. Interestingly, in contrast to other transport proteins (low
density lypoprotein receptor, transferrin receptor, and mannose
6-phosphate/insulin-like growth factor II receptor) (34-36), CRSBP-1
undergoes ligand-dependent internalization and recycling at
the cell surface. This ligand-dependent internalization and
recycling is supported by the following evidence: 1) the
internalization and recycling of CRSBP-1 occurs in cells expressing the
ligand v-sis gene product (SSV-NIH 3T3 cells and SSV-NRK
cells) but not in other cell types, including transformed cells, and 2)
suramin, a potent inhibitor of the ligand binding activity of CRSBP-1
(8), abolishes the internalization and recycling of CRSBP-1 in
v-sis-transformed cells. The mechanism of this
ligand-dependent internalization and recycling is not known. We hypothesize that the ligand v-sis gene product
stimulates internalization and recycling of CRSBP-1 by inducing
dimerization or oligomerization of CRSBP-1 through its homodimer
structure. This is supported by the observation that a disulfide-linked
dimer of CRSBP-1 was detected only in cells expressing the
v-sis gene product but not in normal NIH 3T3 cells.
Several lines of evidence suggest that the v-sis gene
product interacts with CRSBP-1 in SSV-NIH 3T3 cells. These include: 1)
the v-sis gene products p44 and p36, which are generated by proteolysis at and/or after TGN were co-immunoprecipitated with antiserum to CRSBP-1. This is consistent with the observation that the
majority of CRSBP-1 is localized in TGN and endosomes/prelysosomal compartments. 2) CRSBP-1 only underwent rapid turnover in cells expressing the v-sis gene product, and 3) suramin, which is
known to enter and accumulate in TGN and endosomes/prelysosomal
compartments (37, 38), blocked the intracellular turnover of CRSBP-1 as it blocked the intracellular turnover of the v-sis gene
product and PDGF Autocrine transformation by the v-sis gene product involves
the activation of the PDGF -type receptor, as
demonstrated by metabolic labeling and co-immunoprecipitation. CRSBP-1,
like the v-sis gene product and PDGF
-type receptor,
underwent rapid turnover which was blocked in the presence of 100 µM suramin. In normal and other transformed NIH 3T3
cells, CRSBP-1 was relatively stable and did not undergo rapid turnover
and internalization/recycling at the cell surface. These results
suggest that in SSV-NIH 3T3 cells, CRSBP-1 interacts with and forms
ternary and binary complexes with the newly synthesized
v-sis gene product and PDGF
-type receptor at the
trans-Golgi network and that the stable binary (CRSBP-1·v-sis gene product) complex is transported to
the cell surface where it presents the v-sis gene product
to unoccupied PDGF
-type receptors during
internalization/recycling.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-type receptor is activated by the newly
synthesized v-sis or c-sis gene product during
cellular routing of both proteins from the endoplasmic reticulum (ER)
to plasma membranes or extracellular compartment (1, 8, 22, 23,
28-30). The ER and Golgi complex interactions of the PDGF
-type
receptor and v-sis or c-sis gene product result
in the rapid turnover of both proteins (1, 22, 23). A fraction of the
newly synthesized v-sis or c-sis gene product,
which does not interact with the PDGF
-type receptor in these
intracellular compartments, is secreted and retained at the cell
surface (1). The cell surface-retained v-sis or c-sis gene product is then efficiently transferred to
unoccupied cell surface PDGF
-type receptors (1). To test the
hypothesis that CRSBP-1 is responsible for cell surface retention of
the v-sis gene product, we investigated the interactions of
CRSBP-1 with the v-sis gene product and PDGF
-type
receptor in SSV-transformed cells. If CRSBP-1 is able to present the
v-sis gene product to the PDGF
-type receptor at the cell
surface and/or in intracellular compartments (22), CRSBP-1 should have
the potential to form complexes with the v-sis gene product
and PDGF
-type receptor in SSV-transformed cells. In this article,
we demonstrate that the majority of CRSBP-1 is present in intracellular
compartments (endosomes/prelysosomal compartments and TGN) of SSV-NIH
3T3 cells and normal NIH 3T3 cells. CRSBP-1 forms complexes with the
v-sis product in SSV-NIH 3T3 cells as demonstrated by
metabolic labeling and co-immunoprecipitation. CRSBP-1 also forms
complexes specifically with a ~140-kDa PDGF
-type receptor which
represents an activated (phosphorylated) and proteolytically cleaved
form of the PDGF
-type receptor as demonstrated by
co-immunoprecipitation/immunocomplex phosphorylation analysis.
Like the v-sis gene product and PDGF
-type receptor,
CRSBP-1 undergoes rapid turnover which can be blocked by suramin. The
cell surface CRSBP-1 exhibits ligand-dependent internalization and recycling. These results raise the possibility that
CRSBP-1 may play an important role in the autocrine growth of
SSV-transformed cells. The finding of a proteolytically cleaved form
(identified as a ~140-kDa phosphorylated protein) of the PDGF
-type receptor specifically complexed with CRSBP-1 suggests a novel
mechanism of signal transduction in autocrine growth.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]ATP (4,500 Ci/mmol) were purchased from ICN
Biochemicals (Irvine, CA). Suramin was obtained from FBA
Pharmaceuticals (West Haven, CT). Iodoacetamide,
N-ethylmaleimide, glucose oxidase, lactoperoxidase, trypsin,
soybean trypsin inhibitor (STI), Triton X-100, phenylmethanesulfonyl fluoride, bovine serum albumin, chloramine T, high molecular mass protein standards (myosin, 205 kDa;
-galactosidase, 116 kDa; phosphorylase b, 97 kDa; bovine serum albumin, 66 kDa;
ovalbumin, 45 kDa; carbonic anhydrase, 29 kDa), and low molecular
weight standards (bovine serum albumin, 66 kDa; ovalbumin, 45 kDa;
pepsin, 34.7 kDa; trypsinogen, 24 kDa;
-lactoglobulin, 18.4 kDa;
lysozyme, 14.3 kDa) were obtained from Sigma. IgGsorb was obtained from the Enzyme Center (Malden, MA), and protein A-Sepharose beads were
purchased from Life Technologies, Inc. (Grand Island, NY). Antiserum to
PDGF-B/v-sis gene product was raised in rabbits as described
previously (1, 31). Antiserum to the PDGF
-type receptor was raised
in rabbits according to our published procedure (22) and was used for
most experiments, or purchased from Oncogene Research Products
(Cambridge, MA). For preparation of antiserum to CRSBP-1, a C-terminal
cysteinated peptide whose amino acid sequence was derived from the
N-terminal amino acid sequence of CRSBP-1 was synthesized and
conjugated to bovine thyroglobulin (8). CRSBP-1 N-terminal
peptide-thyroglobulin conjugate and Freund's adjuvant were injected
subcutaneously into rabbits for generation of antiserum (8). The
specificity of the antiserum to CRSBP-1 was verified by Western blot
analysis and enzyme-linked immunoabsorbent assay (8). NRK cells and NIH
3T3 cells were obtained from American Type Culture Collection
(Rockville, MD). SSV-transformed NIH 3T3 cells (SSV-NIH 3T3 cells) and
SSV-transformed NRK cells (SSV-NRK cells) were provided by Drs. Stuart
A. Aaronson and Kenneth C. Robbins (National Institutes of Health, NCI,
Bethesda, MD). neu/erbB2-transformed NIH 3T3 cells (B104
cells) and ras-transformed NIH 3T3 cells were obtained from
Dr. Ming-chi Hung (M.D. Anderson Cancer Center, Houston, TX). All cell
lines were cultured and maintained in Dulbecco's modified Eagle's
medium (DMEM) containing 10% fetal calf serum.
20 °C for 15 min.
Fixed cells were washed and rehydrated in PBS and blocked with 6%
bovine serum albumin and 3% normal goat serum for 45 min at room
temperature. For the first antibody reaction, cells were incubated with
either normal rabbit serum or antiserum to CRSBP-1 (1:200 dilution) in
the presence and absence of 2 mM peptide antigen (8) for
2 h at room temperature. Cells were washed and incubated with
fluorescein isothiocyanate-conjugated goat anti-rabbit IgG (Cappel, NC)
for 1.5 h at room temperature. After additional washes, the
coverslips were mounted in VECTASHIELD (Vector Laboratories, CA) and
viewed under an Olympus AHBT3 microscope using interference blue filters.
-Type Receptor in the
Immunoprecipitates--
SSV-NIH 3T3 cells were lysed in 1% Triton
X-100 buffer, diluted 10-fold with 20 mM Hepes-NaOH, pH
7.4, 50 mM NaCl and immunoprecipitated with antiserum to
the PDGF
-type receptor or antiserum to CRSBP-1 in the presence of
protein A-Sepharose. The immunoprecipitates were washed with 0.1%
Triton X-100 buffer. The autophosphorylation (tyrosine-specific
phosphorylation) reaction of the PDGF
-type receptor was started by
addition of 5 µCi/1 µM [
-32P]ATP and
3.2 mM MnCl2. After 10 min at 0 °C, the
immunoprecipitates were washed three times with 0.1% Triton X-100
buffer. The antibody-antigen complexes in the immunoprecipitates were
then dissociated in the presence of 1% SDS and re-immunoprecipitated
with antiserum to the PDGF
-type receptor as described above.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
125I Labeling of the cell surface
CRSBP-1 in SSV-NIH 3T3 cells. Cell surface proteins of SSV-NIH 3T3
cells were labeled with 125I on ice using the
lactoperoxidase method. 125I-Labeled cell lysates were
immunoprecipitated with antiserum to CRSBP-1 or non-immune serum
(N.S.). The immunoprecipitates were analyzed by 6 and 8%
SDS-PAGE under nonreducing (A) and reducing (B)
conditions, respectively. 125I-Labeled CRSBP-1 on the gel
was visualized by autoradiography. The arrow and
arrowhead indicate the locations of CRSBP-1 (72 kDa) and a
~135-kDa protein which is presumed to be the dimer of CRSBP-1,
respectively. The exposure time of the autoradiograms was 3 days.
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Fig. 2.
Trypsin sensitivity of
Tran35S-label metabolically labeled and cell surface
125I-labeled CRSBP-1 in SSV-NIH 3T3 cells. SSV-NIH 3T3
cells, which had been either pulse labeled with
Tran35S-label for 3 h and chased in 10 mM
unlabeled L-methionine for 2 h (A), or
labeled with 125I and lactoperoxidase at the cell surface
(B), were treated with (+) or without ( ) trypsin for
2 h on ice. The cell lysates were analyzed by immunoprecipitation
using antiserum to CRSBP-1 followed by 10% (A) or 8%
(B) SDS-PAGE under reducing conditions and fluorography or
autoradiography. The exposure times of the fluorogram and autoradiogram
were 3 weeks and 25 days, respectively.
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Fig. 3.
Cellular localization of CRSBP-1 in SSV-NIH
3T3 cells by indirect immunofluorescent staining. Cells were fixed
and incubated with antiserum to CRSBP-1 (A) or with
antiserum to CRSBP-1 plus 2 mM peptide antigen
(B). CRSBP-1 was then visualized using fluorescein
isothiocyanate-conjugated goat anti-rabbit IgG.
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Fig. 4.
Co-immunoprecipitation of the
v-sis gene product and CRSBP-1 in SSV-NIH 3T3
cells. Cells were incubated in cysteine-free DMEM for 4 h
followed by metabolic labeling with [35S]cysteine for
2.5 h. The 35S-labeled cells were lysed and the cell
lysates were immunoprecipitated with antiserum to the v-sis
gene product (lanes 1 and 4), antiserum to
CRSBP-1 (lanes 2 and 5), or non-immune serum
(N.S.)(lane 3) in the presence of either Triton
X-100 buffer (lanes 1-3) or RIPA buffer (lanes 4 and 5). The immunoprecipitates were heated at 100 °C in
the presence of 1% SDS. After dilution to a final SDS concentration of
0.1%, all samples were re-immunoprecipitated with antiserum to the
v-sis gene product or CRSBP-1. The immunoprecipitates were
analyzed by 10% SDS-PAGE under nonreducing conditions. The
35S-labeled v-sis gene products in the
immunoprecipitates were visualized by fluorography. The
arrows indicate the locations of the v-sis gene
products. The exposure time of the fluorogram was 22 days.
-type receptor in SSV-transformed cells (1, 22).
Interestingly, suramin not only blocked the turnover of CRSBP-1 but
also increased the accumulation of CRSBP-1 (Fig. 5, A and
C). Suramin did not affect the stability of CRSBP-1 in normal NIH 3T3 cells which do not express the v-sis gene
product (Fig. 5, B and C). The suramin blocking
of CRSBP-1 turnover appeared to be specific since it did not alter the
stability of overall cellular proteins as determined by measuring total
trichloroacetic acid precipitable 35S-labeled proteins
(data not shown). Since suramin is also an inhibitor for the ligand
binding activity of CRSBP-1 (8), these results suggest that suramin may
abolish the turnover of CRSBP-1 by blocking the complex formation of
CRSBP-1 and the v-sis gene product in SSV-NIH 3T3 cells. The
slower turnover rate of CRSBP-1 compared with those of the
v-sis gene product and PDGF
-type receptor (1, 22) may be
due to the large size of its pool.
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Fig. 5.
Effect of suramin on the turnover of CRSBP-1
in SSV-NIH 3T3 and normal NIH 3T3 cells. SSV-NIH 3T3
(A) and normal NIH 3T3 (B) cells were
pulse-labeled with Tran35S-label for 3 h and chased in
10 mM unlabeled methionine for 0, 2, and 4 h. The
cells were pulse-labeled and chased in the presence and absence of 0.1 mM suramin. After pulse and chase, cells were lysed and the
cell lysates were immunoprecipitated with antiserum to CRSBP-1 and
analyzed by 8% SDS-PAGE and fluorography. The exposure times were 10 days (SSV-NIH 3T3 cells without suramin), 7 days (SSV-NIH 3T3 cells
with suramin), 14 days (NIH 3T3 cells without suramin), and 16 days
(NIH 3T3 cells with suramin), respectively. The relative level of
35S-labeled CRSBP-1 was quantitated by densitometric
scanning. The quantitative analysis is shown in C, with the
relative level of 35S-labeled CRSBP-1 plotted against the
length of chase. The relative level of 35S-labeled CRSBP-1
at 0 h chase time was taken as 100%.
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Fig. 6.
Effect of suramin on the internalization and
recycling of 125I-labeled cell surface CRSBP-1 in SSV-NIH
3T3 and normal NIH 3T3 cells. Cell surface proteins of SSV-NIH 3T3
cells (A) and normal NIH 3T3 cells (B) were
labeled with 125I and lactoperoxidase on ice and then
warmed to 37 °C for 0, 3, 6, 9, 12, 15, 18, 21, or 30 min. Cells
were then treated with trypsin for 2 h on ice. For suramin
treatment, cells were incubated in the presence and absence of 0.1 mM suramin for 30 min at 37 °C prior to cell surface
labeling with 125I. After trypsin digestion, cells were
lysed and the cell lysates were immunoprecipitated with antiserum to
CRSBP-1 and analyzed by 8% SDS-PAGE and autoradiography. The exposure
time of the autoradiograms was 3 days.
-Type Receptor in SSV-NIH 3T3
Cells--
In SSV-transformed cells, the v-sis gene product
interacts with the PDGF
-type receptor in the lumen of the ER and
Golgi complex and at the cell surface (1, 22, 23, 29). Since CRSBP-1
was found to interact with the v-sis gene product, we believed it was possible that CRSBP-1 may form a transient ternary complex with the v-sis gene product and PDGF
-type
receptor. To test this possibility, we investigated the interaction of
CRSBP-1 and the PDGF
-type receptor by performing sequential
immunoprecipitation using antisera to CRSBP-1 and the PDGF
-type
receptor. SSV-NIH 3T3 cells were lysed with 1% Triton X-100 buffer.
The Triton X-100 extracts were then immunoprecipitated with antiserum
to CRSBP-1, antiserum to PDGF
-type receptor or non-immune serum.
The immunoprecipitates were incubated with
[
-32P]ATP to label the PDGF
-type receptor
(through autophosphorylation), heated at 100 °C in 0.1% SDS, and
subjected to a second immunoprecipitation using antiserum to the PDGF
-type receptor or non-immune serum. The immunoprecipitates were
analyzed by SDS-PAGE and autoradiography. As shown in Fig.
7, the 32P-labeled 180-kDa
(mature form), 160-kDa (precursor form), and ~140-kDa forms of the
PDGF
-type receptor were identified in the immunoprecipitates of
SSV-NIH 3T3 cells using antiserum to the PDGF
-type receptor
(lane 1). The ~140-kDa PDGF
-type receptor represents
an activated (phosphorylated) and proteolytically processed form of the
PDGF
-type receptor and was previously identified in SSV-NIH 3T3
cells (22). Antiserum to CRSBP-1 co-immunoprecipitated the ~140-kDa
PDGF
-type receptor but not the 180-kDa (mature form) and p160-kDa
(precursor) PDGF
-type receptors (Fig. 7, lane 2). Interestingly,
more 32P-labeled ~140-kDa PDGF
-type receptor was
co-immunoprecipitated by antiserum to CRSBP-1 than that
immunoprecipitated by antiserum to the PDGF
-type receptor. These
results suggest that CRSBP-1 forms complexes with a proteolytically
processed form (identified as a ~140-kDa phosphorylated protein) of
the PDGF
-type receptor in SSV-NIH 3T3 cells and that the
proteolytically cleaved PDGF
-type receptor·CRSBP-1 complex reacts
more strongly with antiserum to CRSBP-1 than with antiserum to the PDGF
-type receptor. The ~140-kDa proteolytically processed form of the
PDGF
-type receptor was detected in the cell lysates of SSV-NIH 3T3
cells but not normal NIH 3T3 cells (22). Only the 180-kDa mature form
and 160-kDa precursor form of the PDGF
-type receptor were detected in the immunoprecipitates (using antiserum to the PDGF
-type receptor) of normal NIH 3T3 cell lysates as described previously (22).
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Fig. 7.
Co-immunoprecipitation of the ~140-kDa
PDGF -type receptor with CRSBP-1 in SSV-NIH
3T3 cells. Cells grown on Petri dishes were lysed and the cell
lysates were immunoprecipitated with antiserum to the PDGF
-type
receptor (lane 1) and antiserum to CRSBP-1 (lane
2). The immunoprecipitates were reacted with
[
-32P]ATP, then washed and dissociated in the presence
of 1% SDS. Following dilution to a final SDS concentration of 0.1%,
all samples were subjected to a second immunoprecipitation with
antiserum to the PDGF
-type receptor. The immunoprecipitates were
analyzed by 7.5% SDS-PAGE and autoradiography. The arrows
indicate the locations of the precursor (160 kDa) and mature form (180 kDa) and a proteolytically processed form (140 kDa) of the PDGF
-type receptor. The bracket indicates the location of the
32P-labeled heavy chain of rabbit IgG.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-type receptor (1, 22).
-type receptor by the v-sis
gene product during routing of both ligand and the receptor from the ER
to plasma membranes or extracellular compartment (1, 22). We hypothesize that CRSBP-1, which is mainly localized in TGN and endosomes/prelysosomal compartments, may form a transient ternary complex with the v-sis gene product and PDGF
-type
receptor in these compartments (Fig. 8).
This hypothesis is supported by the following evidence: 1) CRSBP-1 was
co-immunoprecipitated with the v-sis gene products
proteolytically generated at and/or after TGN and the ~140-kDa PDGF
-type receptor in SSV-NIH 3T3 cells but not in normal and other
transformed NIH 3T3 cells which do not express the v- or
c-sis gene product. The ~140-kDa PDGF
-type receptor is
an intracellular product of SSV-NIH 3T3 cells and resistant to trypsin
digestion (22, 33). 2) Suramin treatment of SSV-NIH 3T3 cells blocked
the co-immunoprecipitation of CRSBP-1 and the v-sis gene
product or the ~140-kDa PDGF
-type receptor (33). Suramin has been
recently shown to enter TGN and endosomes/prolysosome compartments of
SSV-NIH 3T3 cells and other cell types (37, 38), and 3) all three
proteins (CRSBP-1, the v-sis gene product and the PDGF
-type receptor) exhibited rapid turnover in cells expressing these
proteins. No turnover of the PDGF
-type receptor and CRSBP-1 was
observed in cells lacking expression of the v-sis gene
product (e.g. normal and other transformed NIH 3T3 cells). CRSBP-1 exhibits broad pH activity for ligand binding (8). pH 6.0-6.5
(the luminal pH of TGN and exocytic vessicles) is not optimal for
binding of the v-sis gene product to the PDGF
-type receptor. However, it is possible that these slightly acidic pH environments would not have significant effects on the transfer of the
v-sis gene product from the occupied CRSBP-1 to the PDGF
-type receptor. CRSBP-1 may play an important role in the
interaction of the v-sis gene product and the PDGF
-type
receptor in the lumen of TGN and exocytic vessicles especially under
non-overexpression conditions.
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Fig. 8.
Model for the interactions of CRSBP-1 with
the v-sis gene product and PDGF
-type receptor in SSV-NIH 3T3 cells. In
SSV-NIH 3T3 cells, CRSBP-1 forms complexes with the v-sis
gene product and presents it to the unoccupied PDGF
-type receptor
at the TGN and cell surface. At the TGN, CRSBP-1 may stabilize the
activated and proteolytically cleaved PDGF
-type receptor in the
ternary complex. The persistent signal transduction mediated by this
proteolytically cleaved PDGF
-type receptor (identified as a
~140-kDa phosphorylated protein) may play an important role in
autocrine transformation of the cells. The activation of cell surface
PDGF
-type receptors by the v-sis gene product, which is
retained by CRSBP-1 at the cell surface, transduces signals to enhance
the growth of the transformed cells. Suramin appears to be able to
block the interactions of the v-sis gene product with
CRSBP-1 and the PDGF
-type receptor at the cell surface and TGN (1,
8, 22, 33).
The ~140-kDa PDGF -type receptor which was co-immunoprecipitated
with CRSBP-1 was previously identified by 32P-metabolic
labeling followed by immunoprecipitation using antiserum to the PDGF
-type receptor in SSV-NIH 3T3 cells (22). The ~140-kDa PDGF
-type receptor is a proteolytically cleaved product of the 180-kDa
form (mature form) of the PDGF
-type receptor which was resistant to
endoglycosidase H digestion but sensitive to endoglycosidase F
digestion (22).2 The endoglycosidase F digestion of the
~140-kDa PDGF
-type receptor yielded a product of molecular weight
(~120,000) which is smaller than that (molecular mass ~140,000) of
the endoglycosidase F-digested 180-kDa PDGF
-type receptor
(22).2 Compared with the 180- and 160-kDa PDGF
-type
receptors, this ~140-kDa PDGF
-type receptor appeared to have a
high phosphorylation activity (>10 fold) based on the estimation of
the ratio of
[32P]orthophosphate/[35S]methionine
metabolic labeling (22). Since the ~140-kDa PDGF
-type receptor
was only found in v-sis-transformed cells (22, 36), it may
play an important role in mediating the signal transduction involved in
the autocrine transformation of SSV-NIH 3T3 cells.
The mechanism for the production of the ~140-kDa PDGF -type
receptor is unknown. We hypothesize that some of CRSBP-1 molecules at
TGN form a transient ternary complex with the newly synthesized v-sis gene product and PDGF
-type receptor and that some
of CRSBP-1 molecules form a binary complex with the v-sis
gene product (Fig. 8). The PDGF
-type receptor in the ternary
complex undergoes proteolysis at its ligand-binding domain to produce
the ~140-kDa form which is identified on SDS-polyacrylamide gel
following immunoprecipitation with antiserum to a C-terminal region of
the PDGF
-type receptor. However, the whole molecule of the
proteolytically cleaved PDGF
-type receptor is held together by
noncovalent forces and associates tightly with the v-sis
gene product and CRSBP-1. The proteolytically cleaved PDGF
-type
receptor which possesses potent kinase activity may be stabilized at
the TGN in complexes with CRSBP-1 and v-sis gene product. In
contrast, the CRSBP-1·v-sis gene product binary complex is
transported to the cell surface, where it presents the v-sis
gene product to unoccupied PDGF
-type receptors during its cell
surface internalization and recycling (1) (Fig. 8). During transport
through exocytotic vesicles which have acidic luminal pH, the
CRSBP-1·v-sis gene product binary complex is stable (8).
Recently, Valgeirsdóttir et al. (30) characterized the
PDGF receptor-mediated signal transduction in SSV-NIH 3T3 cells. Their
analysis of a panel of known PDGF -receptor signaling molecules revealed the presence of suramin-sensitive and -insensitive pathways of
PDGF receptor-mediated signaling (30). Since suramin enters and
accumulates in intracellular acidic compartments (37, 38), suramin-sensitive signaling should be mediated by the PDGF
-type receptor activated by the v-sis gene product at TGN as well
as the cell surface. The suramin-insensitive signaling should be mediated by the PDGF
-type receptor activated by the
v-sis gene product in cis and medial Golgi complex and the
ER. No evidence has been found to suggest that suramin is able to enter
these compartments. We propose that the CRSBP-1 v-sis gene
product and proteolytically cleaved PDGF
-type receptor (identified
as a 140-kDa protein) ternary complex reported here is involved in mediating suramin-sensitive signaling in the TGN and
endosomes/prelysosomal compartments (Fig. 8).
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ACKNOWLEDGEMENTS |
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We thank Drs. William S. Sly and Frank E. Johnson for critical review of the manuscript. We also thank John H. McAlpin for typing the manuscript.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant CA38808.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: Dept. of Biochemistry and Molecular Biology, St. Louis University School of Medicine, 1402 South Grand Blvd., St. Louis, MO 63104. Tel.: 314-577-8135; Fax: 314-577-8156; E-mail: huangjs{at}wpogate.slu.edu.
2 C. Boensch, S. S. Huang, D. T. Connolly, and J. S. Huang, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: CRS, cell surface retention sequence; CRSBP, CRS-binding protein; SSV, simian sarcoma virus; ER, endoplasmic reticulum; TGN, trans-Golgi network; STI, soybean trypsin inhibitor; DMEM, Dulbecco's modified Eagle's medium; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; NRK, normal rat kidney.
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REFERENCES |
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