(Received for publication, August 26, 1994; and in revised form, November 1, 1994)
From the
Cell-surface retention is a newly identified mechanism
associated with the secretion of certain polypeptide growth factors and
cytokines. This novel form of secretion appears to be mediated by
cell-surface retention sequences (CRS) in the polypeptide molecules. To
test the hypothesis that high-affinity CRS-binding proteins (CRS-BPs)
are responsible for the cell-surface retention, we identified and
characterized the high-affinity binding sites on various cell types for I-labeled CRS peptide (sis) and CRS peptide
(VEGF), each of which contained the putative CRS motifs of
platelet-derived growth factor B (c-sis) and vascular
endothelial cell growth factor, respectively. Scatchard plot analysis
revealed a single class of high-affinity binding sites with K
= 0.5-0.7 nM and
22,000-55,000 sites/cell. High-affinity binding activity
could be demonstrated between pH 4.5 and 8.0, but was much greater
below 6.0 (maximum pH 5.0-5.5). The ligand binding activity was
inhibited by heparin, polylysine, and protamine, but not by cytochrome c. CRS-BPs responsible for the high-affinity binding were
identified as 60-72-kDa proteins by ligand affinity labeling.
CRS-BPs were purified from human SK-Hep cells and bovine liver plasma membranes by Triton X-100 extraction followed by affinity column chromatography on wheat germ lectin-Sepharose 4B and CRS peptide (sis)-Affi-Gel 10. Purified CRS-BPs exhibited ligand binding properties (pH profile and inhibitor sensitivity) similar to those of the high-affinity binding sites for CRS peptides on cultured cells. The major CRS-BPs (p60, p66, and p72) purified from bovine liver plasma membranes were found to have identical N-terminal amino acid sequence and were assumed to represent different forms of the same gene product, which we have designated CRS-BP1.
Cell-surface retention during secretion is a newly identified
and novel aspect of the secretion of certain polypeptide growth factors
and
cytokines(1, 2, 3, 4, 5, 6, 7) .
These growth factors and cytokines are secreted, but remain associated
with the cell surface rather than released into the
medium(1, 2, 3, 4, 5, 6, 7) .
Such cell surface-bound proteins can be released by incubation with
suramin, heparin, or high salt(1, 3, 5) . The
functional significance of this novel aspect of secretion is not known.
However, in v-sis-transformed cells, cell-surface retention of
the v-sis gene product facilitates its binding to the
platelet-derived growth factor (PDGF) ()receptor(1) . This efficient interaction between
the cell surface-bound v-sis gene product and the PDGF
receptor on the cell surface may play a role in the autocrine growth of
v-sis-transformed cells(1) .
The v-sis gene product (PDGF-B) was first reported to undergo cell-surface
retention during secretion(1) , and this result was confirmed
by others. PDGF-B (c-sis)(2, 3) ,
PDGF-A (alternatively spliced longer version of PDGF-A) (2, 3) , vascular endothelial cell growth factor
(VEGF)-189 (or VEGF-206), Int-2(5) , Wnt-1(6) , and Cyr
61 (7) proteins have all been shown to exhibit this novel
secretion. Among these secretory growth factors and cytokines, the
v-sis gene product, PDGF-B (c-sis),
PDGF-A
, and VEGF-189 (or VEGF-206) are structurally
homologous growth factors and members of the PDGF
superfamily(8, 9, 10, 11, 12, 13) .
Int-2 protein is a member of the fibroblast growth factor
family(14) . Wnt-1 protein is a proto-oncogene
product(15) , and Cyr 61 protein is a growth factor-inducible
early gene product(16) . No amino acid sequence homology is
found among PDGF family, Int-2, Wnt-1, and Cyr 61 proteins, implying
that these secretory proteins may carry analogous sequence motifs
mediating their cell-surface retention. Studies of the PDGF-B
(c-sis) deletion mutation have revealed that PDGF-B
(c-sis) possesses a cell-surface retention sequence (CRS) at
residues 219-229 or 212-226(2, 3) . This
11- or 15-amino acid residue CRS contains a 7- or 8-basic amino acid
residue sequence that is homologous to sequences present in
PDGF-A
and VEGF-189 (or VEGF-206). Both PDGF-A
and VEGF-189 (or VEGF-206) have been shown to remain associated
with the cell surface following
secretion(2, 3, 4) . Their alternatively
spliced shorter versions (PDGF-A
, VEGF-121, and VEGF-165),
which lack putative CRS, have been shown to be secreted directly into
the medium(2, 4, 17) . Deletion of the
putative CRS in PDGF-A
also results in the promotion of
secretion into the medium(17) . These observations argue that
the putative CRS of PDGF-A
and VEGF-189 (or VEGF-206) are
required for their cell-surface retention.
The molecular
mechanism(s) by which the secretory proteins with CRS are transported
to the cell surface and retained there is not understood. Based on the
studies of v-sis product synthesis and secretion by
pulse-chase experiments, we hypothesized that the v-sis gene
product (a secretory protein with CRS) became associated with the
cell-surface components (CRS-binding proteins) in the trans-Golgi complex and exocytosis vesicles, after which the
associated complexes appeared at and remained at the cell surface (1) . Recently, Raines and Ross (18) identified binding
sites on human smooth muscle cells and umbilical vein endothelial cells
for an I-labeled tyrosinated pentadecapeptide and
eicosapeptide that contained the CRS motifs of PDGF-B (c-sis)
and PDGF-A
, respectively. The large number (10
to 10
/cell) and low affinity (K
= 2-4 µM) of the binding sites
identified were consistent with the suggestion that heparan sulfate
proteoglycan (HS-PG) was responsible for the binding in the assays.
Khachigian et al.(19) also recently identified
binding sites on different cell types for an
Ilabeled
tyrosinated nonadecapeptide containing the CRS motif of
PDGF-A
. 3T3 fibroblasts were found to express a large
number of low-affinity binding sites (1.6
10
/cell)
with an apparent K
of 0.6
µM(19) . The large population of sites bound with
low affinity was consistent with HS-PG-binding sites in these assays.
To test the hypothesis that specific high-affinity binding proteins
mediate the cell-surface retention of secretory proteins with
CRS(1) , we attempted to identify, purify, and characterize the
putative CRS-binding proteins (CRS-BPs) in cultured cells and tissues.
In this study, we demonstrate that cultured cells show
22,000-55,000 high-affinity binding sites/cell for
CRS-containing peptide ligands with an apparent K
of 0.5-0.7 nM. Heparin and basic
polypeptides, polylysine and protamine (but not cytochrome c),
are potent inhibitors of the ligand binding to these high-affinity
binding sites. The major CRS-BPs, which are responsible for
high-affinity CRS peptide binding, were identified as protein species
with molecular masses of 60-72 kDa by
I-labeled
ligand affinity labeling. We also report that the major CRS-BPs (p60,
p66, and p72) purified from bovine liver plasma membranes possess
identical N-terminal amino acid sequence. CRS-BPs p60, p66, and p72 are
assumed to represent different forms of the same gene product, which we
have designated CRS-BP1. The antiserum raised against the N-terminal
peptide of CRS-BP1 immunoprecipitates a ligand affinity-labeled CRS-BP
on cultured HepG2 cells.
In some experiments, after binding and cross-linking, the reaction mixture was incubated with 3 µl of 1 M Tris-HCl, pH 7.4, 1 M glycine, which was used to inactivate excess EDAC. After 5 h at 4 °C, 100 µl of wheat germ lectin-Sepharose 4B (50% suspension in 20 mM Tris-HCl, pH 7.4, 0.15 M NaCl, and 0.2% Triton X-100) was added to the reaction mixture. The gel suspension was incubated at 4 °C for 5 h. Following centrifugation, the gels were washed five times with 20 mM Tris-HCl, pH 7.4, 0.15 M NaCl, and 0.2% Triton X-100 and incubated with 100 µl of 0.4 MN-acetylglucosamine in 20 mM Tris-HCl, pH 7.4, 0.15 M NaCl, and 0.2% Triton X-100 (2 h at 4 °C). The N-acetylglucosamine eluents were analyzed by 7% SDS-polyacrylamide gel electrophoresis.
The CRS-BP-containing fractions from wheat germ lectin-Sepharose 4B column chromatography were pooled and applied onto a column (15-ml volume) of CRS peptide (sis)-Affi-Gel 10. After washing with buffer B, the column was eluted with 0.5 M NaCl in buffer B. CRS-BPs were recovered in the 0.5 M NaCl eluents.
The remaining 400 µl of the cell lysates was mixed with 100 µl of wheat germ lectin-Sepharose 4B (50% gel suspension). After mixing at 4 °C for 16 h, the gels were washed five times with 20 mM Tris-HCl, pH 7.4, 0.15 M NaCl containing 0.2% Triton X-100 and incubated with 100 µl of 0.4 MN-acetylglucosamine in 20 mM Tris-HCl, pH 7.4, 0.15 M NaCl, and 0.2% Triton X-100 at 4 °C for 2 h. The N-acetylglucosamine eluents were then analyzed by 7% SDS-polyacrylamide gel electrophoresis.
Figure 1:
Binding of I-CRS peptide (sis) to SK-Hep cells. A shows the concentration
dependence of the binding of
I-CRS peptide (sis)
to SK-Hep cells at pH 7.4. The nonspecific binding was determined in
the presence of 1 mM suramin. Each data point represents the
mean ± S.D. from three assays. B shows the Scatchard
plot analysis of the specific binding data from A.
Since
CRS peptides are basic in nature and heparin has been shown to displace
the cell surface-bound growth factors(4, 5) , the
effects of heparin and basic polypeptides on the binding of I-CRS peptide (VEGF) to SK-Hep cells were examined. As
shown in Fig. 2(A and B), heparin,
polylysine, and protamine (but not cytochrome c) inhibited the
I-CRS peptide (VEGF) binding to SK-Hep cells. The
half-maximal inhibition concentrations (IC
) of heparin,
polylysine, and protamine were estimated to be
0.4,
8, and
1 µg/ml, respectively. Similar IC
values for
heparin and basic polypeptides were also obtained when
I-CRS peptide (sis) was used as the ligand (data
not shown).
Figure 2:
Effects of heparin (A) and basic
polypeptides (B) on the binding of I-CRS peptide
(VEGF) to SK-Hep cells. The binding of
I-CRS peptide
(VEGF) to SK-Hep cells was carried out at pH 7.4 using 1 nM
I-CRS peptide (VEGF) and various concentrations of
heparin or basic polypeptides. The specific binding of
I-CRS peptide (VEGF) in the absence of heparin or basic
polypeptides is taken as 100% bound.
To investigate whether HS-PG was responsible for the
high-affinity binding sites, the effect of pretreatment with
heparitinase on the ligand binding activity of the high-affinity
binding sites was examined. Without pretreatment of SK-Hep cells with
heparitinase, the specific binding at 1 nMI-CRS
peptide (sis) was estimated to be 1250 ± 100 cpm/well.
After pretreatment with heparitinase, the specific binding of
I-CRS peptide (sis) (1 nM) was
determined to be 1219 ± 152 cpm/well. This result suggests that
the heparitinase pretreatment does not affect the high-affinity binding
of
I-CRS peptide (sis) to SK-Hep cells and that
the high-affinity binding of
I-CRS peptide (sis)
is not mediated through HS-PG.
Figure 3:
Effect of pH on the binding of I-CRS peptide (sis) to SK-Hep cells. The binding
assay was performed in Tris acetate buffer, pH 4.5-8.0, with 1
nM
I-CRS peptide (sis) ± 1
mM suramin. The specific binding of
I-CRS
peptide (sis) at the pH indicated was determined after
incubation at 0 °C for 2.5 h.
Figure 4:
Ligand affinity labeling of CRS-BPs on
SK-Hep cells. CRS-BPs on SK-Hep cells were affinity-labeled by I-CRS peptide (VEGF) in the presence (+) and absence
(-) of 1 mM suramin using 10 mM EDAC.
Affinity-labeled CRS-BPs were analyzed by 8% SDS-polyacrylamide gel
electrophoresis followed by autoradiography. The bracket indicates the location of the major
I-CRS peptide
(VEGF)
CRS-BP affinity-labeled complexes (63-75
kDa).
In preliminary
experiments, we found that CRS-BPs in plasma membranes could be
extracted with Triton X-100, but not by high salt (2 M NaCl)
or high pH (0.5 M NaCO
, pH 11.5). This
result suggests that CRS-BPs are membrane proteins. For isolation of
CRS-BPs, Triton X-100 was therefore used to solubilize CRS-BPs in
plasma membranes and was included in the solvent buffers for the
subsequent chromatographies.
Since many cell-surface receptor
proteins are glycoproteins, which can be absorbed by wheat germ lectin
affinity gel(20, 26) , the Triton X-100 extracts of
suramin-prewashed SK-Hep cells or bovine liver plasma membranes were
mixed with a 30-ml gel volume of wheat germ lectin-Sepharose 4B gel.
After mixing overnight, the gel suspension was packed onto a column.
After washing extensively with buffer, the column was eluted with 0.2 MN-acetylglucosamine. As shown in Fig. 5A
(SK-Hep cells) and Fig. 6A (bovine liver plasma membranes), a
single protein peak was detected by the absorbance at 595 nm (Bio-Rad
reagent assay) in the eluents of 0.2 M N-acetylglucosamine. This protein peak contained CRS-BPs,
which were identified by the I-CRS peptide (VEGF)
cross-linking assay. As shown in Fig. 5B (SK-Hep cells)
and Fig. 6B (bovine liver plasma membranes), the major
CRS-BPs were identified as the
I-CRS peptide (VEGF)
cross-linked complexes with molecular masses of 58-63 and
69-75 kDa. These CRS-BP-containing fractions were then pooled and
subjected to ligand affinity column chromatography on CRS peptide (sis)-Affi-Gel 10 (Fig. 5C and Fig. 6C). After washing with buffer, the affinity
column was eluted with 0.5 M NaCl (Fig. 5C for
SK-Hep cells and Fig. 6C for bovine liver plasma
membranes). The major CRS-BPs, identified as the
I-CRS
peptide (VEGF) cross-linked complexes with molecular masses of
58-63 and 69-75 kDa, were found in the 0.5 M NaCl
eluents (Fig. 5D (lanes 1-10) and 6D (lanes3-12)), but not in the flow-through
fractions (Fig. 6D, lanes1 and 2). The
I-CRS peptide (VEGF)
CRS-BP
complexes with molecular masses of 69-75 kDa appeared to
comigrate with
I-CRS peptide (VEGF)
BSA complexes (Fig. 5D (lanes11 and 12)
and 6D (lanes13 and 14)). The
I-CRS peptides (VEGF)
BSA complexes were generated
during storage of
I-CRS peptide (VEGF) and cross-linking
with EDAC (BSA was used as the carrier protein). The preparations of
CRS-BPs purified by ligand affinity column chromatography were used for
the following activity and biochemical characterization of CRS-BPs.
Figure 5:
Wheat
germ lectin-Sepharose 4B column chromatography (A and B) and CRS peptide (sis)-Affi-Gel 10 column
chromatography (C and D) of CRS-BPs from SK-Hep
cells. A shows the wheat germ lectin-Sepharose 4B column
chromatography of Triton X-100 extracts of SK-Hep cells. After
exhaustively washing with the equilibration buffer, CRS-BPs were eluted
with 0.2 MN-acetylglucosamine. The protein
concentrations of chromatographic fractions were spectrophotometrically
monitored at 595 nm using Bio-Rad protein assay reagent. The arrow indicates the start of elution with 0.2 MN-acetylglucosamine. The flow rate was 20 ml/h, and the
fractional volume was 1 ml. The fractions indicated by the hatchedbox were pooled and subjected to CRS peptide (sis)-Affi-Gel 10 column chromatography. B shows the
detection of CRS-BPs in the column fractions shown in A by
cross-linking with I-CRS peptide (VEGF) and EDAC.
Cross-linking with
I-CRS peptide (VEGF) was performed in
the presence (+) and absence(-) of 1 mM suramin.
The brackets indicate the locations of
I-CRS
peptide
CRS-BP complexes (58-63 and 69-75 kDa). C shows the CRS peptide (sis)-Affi-Gel 10 column
chromatography of CRS-BPs obtained by wheat germ lectin-Sepharose 4B
column chromatography. The fractional volume prior to elution with 0.5 M NaCl was 0.8 ml, and the fractional volume in the 0.5 M NaCl elution was 1 ml. The flow rate was 20 ml/h. D shows
the detection of CRS-BPs by cross-linking with
I-CRS
peptide (VEGF) and EDAC. Cross-linking in the absence of fractions
(buffer alone) was used as control (lanes11 and 12). The brackets indicate the locations of
I-CRS peptide (VEGF)
CRS-BPs complexes (58-63
and 69-75 kDa).
Figure 6:
Wheat germ lectin-Sepharose 4B column
chromatography (A and B) and CRS peptide (sis)-Affi-Gel 10 column chromatography (C and D) of CRS-BPs from bovine liver plasma membranes. A shows wheat germ lectin-Sepharose 4B column chromatography. The
chromatographic conditions are the same as described in the legend of Fig. 5A. B shows the identification of CRS-BPs
by cross-linking with I-CRS peptide (VEGF) and EDAC. C shows the CRS peptide (sis)-Affi-Gel 10 column
chromatography of CRS-BPs obtained by wheat germ lectin-Sepharose 4B
column chromatography. The conditions for elution of CRS-BPs are
essentially identical to those described in the legend of Fig. 5C. D shows the identification of CRS-BPs
in the fractions from CRS peptide (sis)-Affi-Gel 10
chromatography by cross-linking with
I-CRS peptide (VEGF)
and EDAC.
Figure 7:
Inhibition of the I-CRS
peptide (VEGF) binding to CRS-BPs by unlabeled CRS peptides. The
binding of
I-CRS peptide (VEGF) to CRS-BPs purified from
SK-Hep cells (A) and `bovine liver plasma membranes (B) was determined by the
I-CRS peptide (VEGF)
cross-linking assay. In the assay, the concentrations of
I-CRS peptide (VEGF) and unlabeled CRS peptides were 10
nM and -fold molar excess as indicated, respectively. The
I-CRS peptide (VEGF) cross-linked complexes were
identified by 8% SDS-polyacrylamide gel electrophoresis followed by
autoradiography. The brackets indicate the locations of
I-CRS peptide (VEGF)
CRS-BP complexes (58-63
and 69-75 kDa).
As shown in Fig. 3, intact SK-Hep cells showed a high-affinity binding
activity for I-CRS peptides within a broad pH range. If
purified CRS-BPs are responsible for the binding activity of SK-Hep
cells for
I-CRS peptides, purified CRS-BPs should show a
similar pH activity. To test this prediction, purified CRS-BPs were
incubated with
I-CRS peptide (VEGF) at various pH values
(5.0, 5.5, 6.0, 6.5, 7.0, 7.5, and 8.0). After 2.5 h at 0 °C, the
I-CRS peptide (VEGF)
CRS-BP complexes were
cross-linked by 10 mM EDAC and analyzed by 8%
SDS-polyacrylamide gel electrophoresis. As shown in Fig. 8, the
complex formation of
I-CRS peptide (VEGF) and CRS-BPs
occurred at various pH values from 5.0 to 8.0, with a maximum at pH
5.0-5.5. This pH profile is similar to that of the
I-CRS peptide (VEGF) binding activity observed in intact
SK-Hep cells (Fig. 3). CRS-BPs that were identified as the
I-CRS peptide (VEGF) cross-linked complexes with
molecular masses of 69-75 kDa showed a maximal ligand binding
activity at pH 5.0-5.5 and appeared to be the major CRS-BPs at
this pH. It is of importance to note that the cross-linking efficiency
of EDAC does not vary greatly within the pH range from 5.0 to 8.0 under
the cross-linking conditions(27) .
Figure 8:
Effect of pH on the I-CRS
peptide (VEGF) binding to CRS-BPs purified from bovine liver plasma
membranes. The binding of
I-CRS peptide (VEGF) (10
nM) to CRS-BPs was carried out at various pH values (in buffer
B and Mes buffer for pH 6.5-8.0 and pH 5.0-6.0,
respectively) in the presence (+) and absence (-) of 1
mM suramin. The
I-CRS peptide (VEGF)
CRS-BP
complexes were cross-linked using EDAC and analyzed by 8%
SDS-polyacrylamide gel electrophoresis followed by autoradiography. The brackets indicate the locations of
I-CRS peptide
(VEGF)
CRS-BP cross-linked complexes (58-63 and 69-75
kDa).
The ligand binding
activities of the high-affinity binding sites (or CRS-BPs) on SK-Hep
cells were found to be inhibited by heparin and basic polypeptides. The
effects of heparin and basic polypeptides on the complex formation at
pH 5.0 of I-CRS peptide (VEGF) and purified CRS-BPs were
therefore investigated. At pH 5.0, the
I-CRS peptide
(VEGF)
CRS-BP complexes with molecular masses of 69-75 kDa
were identified as the major complexes. As shown in Fig. 9, like
in intact cells, the ligand binding activities of purified CRS-BPs were
inhibited by heparin, polylysine, protamine, and myelin basic protein,
but not by cytochrome c. The inhibition of
I-CRS
peptide (VEGF)
CRS-BP complex formation by heparin and basic
polypeptides suggests that electrostatic forces are involved in the
interactions of
I-CRS peptides and CRS-BPs. To further
define the electrostatic interactions between CRS peptides and CRS-BPs,
we investigated the complex formation at pH 5.0 of
I-CRS
peptide (VEGF) and CRS-BPs in the presence of different concentrations
of NaCl. As shown in Fig. 10, at 300 mM NaCl, very
little complex formation of
I-CRS peptide (VEGF) and
CRS-BPs was detected on the autoradiograph. Interestingly, the presence
of 50 mM NaCl appeared to enhance complex formation. About
1.5-2.0-fold enhancement was consistently observed when compared
with that observed in the absence of NaCl. These results support that
electrostatic interactions are involved in the complex formation of
I-CRS peptides and CRS-BPs. Because cytochrome c, which is a basic protein with a pI of 11.7 (28) ,
failed to inhibit the complex formation of
I-CRS peptides
and CRS-BPs, these results also suggest that in addition to being basic
in nature, certain structural features (e.g. clustering of
basic amino acid residues in a certain configuration) are required for
the basic polypeptides to inhibit complex formation.
Figure 9:
Effects of heparin and basic polypeptides
on the binding at pH 5.0 of I-CRS peptide (VEGF) to
CRS-BPs purified from bovine liver plasma membranes. The binding of
I-CRS peptide (VEGF) (10 nM) to CRS-BPs purified
from bovine liver plasma membranes was performed at pH 5.0 in the
absence and presence of CRS peptides, heparin, basic polypeptides, or
suramin as indicated. The
I-CRS peptide
(VEGF)
CRS-BP complexes were cross-linked and then analyzed by 8%
SDS-polyacrylamide gel electrophoresis followed by autoradiography. The bracket indicates the location of the
I-CRS
peptide (VEGF)
CRS-BP complexes (69-75 kDa). The relative
intensity of CRS-BPs (69-75 kDa) was quantitated by a
PhosphorImager.
Figure 10:
Effect of NaCl concentration on the
binding at pH 5.0 of I-CRS peptide (VEGF) to CRS-BPs
purified from bovine liver plasma membranes. The binding of
I-CRS peptide (VEGF) to CRS-BPs purified from bovine
liver plasma membranes, which had been ultrafiltrated to remove
endogenous NaCl, was carried out at pH 5.0 in the presence of various
concentrations of NaCl with (+) or without(-) 1 mM suramin. The
I-CRS peptide (VEGF)
CRS-BP
complexes were cross-linked by EDAC and then analyzed by 8%
SDS-polyacrylamide gel electrophoresis followed by autoradiography. The bracket indicates the location of
I-CRS peptide
(VEGF)
CRS-BP complexes (69-75 kDa). The relative intensity
of CRS-BP complexes (69-75 kDa) was quantitated by a
PhosphorImager.
Figure 11:
SDS-polyacrylamide gel patterns of
CRS-BPs purified from bovine liver plasma membranes (A) and
their I-CRS peptide (VEGF) cross-linked complexes (B). A shows the SDS gel pattern of purified CRS-BPs
(total protein,
0.3 µg) after electrophoresis and silver
staining. The arrows indicate the locations of the major
CRS-BPs (60, 66, and 72 kDa). B shows the autoradiograph of
the
I-CRS peptide (VEGF) cross-linked complexes of
CRS-BPs purified from bovine liver plasma membranes. After binding and
cross-linking at pH 7.4, the
I-CRS peptide (VEGF)
cross-linked complexes were subjected to wheat germ lectin-Sepharose 4B
absorption and elution with 0.2 MN-acetylglucosamine
prior to 7% SDS-polyacrylamide gel electrophoresis. The arrows indicate the locations of the major
I-CRS peptide
(VEGF)
CRS-BP complexes (63, 69, and 75
kDa).
To define the structural relationship of p60,
p66, and p72, the N-terminal amino acid sequence of each of these
CRS-BPs was analyzed using automated Edman degradation after further
purification by 7% SDS-polyacrylamide gel electrophoresis followed by
polyvinylidene difluoride membrane transblotting. CRS-BPs p60, p66, and
p72 were found to share the same N-terminal sequence of
HN-Ser-Leu-Arg-Ser-Glu-Glu. These results suggest that p60
and p66 may be the proteolytic products of p72. Alternatively, these
CRS-BPs may be the products due to differential glycosylation (or other
post-translational modifications) of the same gene product.
Figure 12:
Immunoprecipitation of ligand
affinity-labeled CRS-BPs on HepG2 cells by antiserum to CRS-BP1.
Confluent HepG2 cells were incubated with 10 nMI-CRS peptide (VEGF) in the presence (+) and
absence (-) of 1 mM suramin. The
I-CRS
peptide (VEGF)
CRS-BP complexes were then cross-linked by EDAC,
concentrated by wheat germ lectin-Sepharose 4B absorption, and analyzed
by 7% SDS-polyacrylamide gel electrophoresis followed by
autoradiography (lanes3 and 4). One-fifth
of the cells were immunoprecipitated by anti-N-terminal peptide
antiserum to CRS-BP1 and then analyzed by 7% SDS-polyacrylamide gel
electrophoresis followed by autoradiography (lanes1 and 2). The arrow indicates the location of a
I-CRS peptide (VEGF)
CRS-BP complex with a molecular
mass of 75 kDa.
Cell-surface retention is an intriguing aspect of secretion
of some proteins that has been defined by pulse-chase
experiments(1, 2, 3, 4, 5, 6, 7) .
During chase, this class of newly synthesized S-labeled
secretory proteins undergo processing of carbohydrate moieties in the
endoplasmic reticulum and Golgi complex and appear and remain at the
cell surface for many
hours(1, 2, 3, 4, 5, 6, 7) .
The cell surface-bound secretory proteins can be released upon
treatment with suramin or heparin or by protease
digestion(1, 3, 5) . Upon longer incubations
(days), the cell surface-bound secretory proteins either are released
(mainly as degraded forms) or diffuse slowly and become associated with
extracellular matrix(1, 29) . Several secretory growth
factors and cytokines, which have been shown to undergo cell-surface
retention by pulse-chase experiments, possess putative CRS motifs (Table 2). The CRS motifs appear to share some of the following
structural features. 1) Four (or 5) or more residues out of a stretch
of 7 (or 8) contiguous residues including the N- and C-terminal
residues are basic amino acids. 2) No acidic amino acid is included in
these 7 or 8 contiguous amino acid residues. 3) No more than 2
contiguous residues are nonbasic amino acid residues. 4) Most of the
CRS motifs are located in the C-terminal parts of the molecules. The
CRS motifs appeared to be homologous to nucleus localization sequence
motifs (31) and may function as nucleus localization sequences
when appropriately expressed(32, 33) .
The
persistent cell-surface retention of secretory proteins with CRS
implies high-affinity binding sites for cell-surface retention of these
proteins. Attempts to identify these high-affinity binding sites
resulted in little success due to the presence of an enormous number
(10 to 10
sites/cell) of low-affinity (K
0.6-4 µM) binding
sites(18, 19) . In the presence of a relatively high
number of low-affinity binding sites(18, 19) , the
higher affinity binding sites cannot be detected by binding analysis
using a 100-fold molar excess of unlabeled ligand to estimate the
nonspecific binding. The 100-fold molar excess of unlabeled ligand only
increases the ligand binding to the low-affinity sites at the low
concentrations of radioactive ligand so that the apparent nonspecific
bindings are higher than the total bindings of the ligand.
To
identify the high-affinity binding sites for CRS peptides, we
determined the suramin-displaceable specific binding of I-CRS peptides on cells. Suramin inhibits the
high-affinity binding of
I-CRS peptides. Scatchard plot
analysis of the suramin-displaceable specific binding revealed a single
class of high-affinity binding sites for
I-CRS peptides
on various cell types. The ligand binding activity of these
high-affinity binding sites is not influenced by pretreatment of cells
with heparitinase, suggesting that HS-PG is not responsible for the
high-affinity binding of
I-CRS peptides. This suggestion
has been supported by the observation of cross-linked complex formation
of
I-CRS peptide (VEGF) and high-affinity binding sites
(CRS-BPs). HS-PG has not been reported to be able to form cross-linked
complexes with proteins under cross-linking conditions(34) .
We hypothesized that the cell-surface retention of secretory proteins with CRS results from the consistent interaction with high-affinity binding sites (or binding proteins) during intracellular routing of both secretory proteins and high-affinity binding sites (or binding proteins) from the trans-Golgi complex to the plasma membrane(1) . If the cell-surface high-affinity binding sites are the same components involved in the interaction with secretory proteins in the intracellular organelles (the trans-Golgi complex and exocytosis vesicles), the high-affinity binding sites should show a broad pH activity. As expected, the cell-surface high-affinity binding sites were found to exhibit a broad pH activity, with a maximum at pH 5.0-5.5. This result suggests that the high-affinity binding sites (or binding proteins) have the potential to interact with secretory proteins with CRS in the intracellular organelles, that have lower luminal pH.
For biochemical
characterization of CRS-BPs, we have developed a purification procedure
involving Triton X-100 extraction of liver plasma membranes or cultured
cells and sequential affinity column chromatography on wheat germ
lectin-Sepharose 4B and CRS peptide (sis)-Affi-Gel 10. This
procedure enabled us to obtain 20 µg of CRS-BPs from
5 g
protein of liver plasma membranes. Since the cross-linking assay for
CRS-BPs is semiquantitative, we are unable to estimate the exact yield
and -fold purification of CRS-BPs purified by this procedure. However,
the recovery of CRS-BPs from each of the column chromatographies on
wheat germ lectin-Sepharose 4B and CRS peptide (sis)-Affi-Gel
10 was estimated to be >80% by applying a known quantity of purified
CRS-BPs onto these affinity columns.
Major CRS-BPs purified from
bovine liver plasma membranes and SK-Hep cells are glycoproteins
(retained by wheat germ lectin-Sepharose 4B gel) with molecular masses
of 60, 66, and 72 kDa, which are designated p60, p66, and p72,
respectively. These CRS-BPs form I-CRS peptide (VEGF)
cross-linked complexes with molecular masses of 63, 69, and 75 kDa,
respectively. Based on the molecular mass (
3 kDa) of
I-CRS peptide (VEGF), it is estimated that CRS-BPs form
1:1 stoichiometric complexes with
I-CRS peptide (VEGF).
N-terminal amino acid sequence analysis revealed that CRS-BPs p60, p66,
and p70 purified from bovine liver plasma membranes possess the same
N-terminal amino acid sequence, suggesting that p60 and p66 may be the
degradation products of p72. Alternatively, these CRS-BPs may be
differential post-translational modification products of the same gene
product.
Several lines of evidence support the suggestion that the
major CRS-BPs purified are involved in the high-affinity binding of I-CRS peptide (VEGF) to the cell surface of cultured
cells. These include the following. 1) The molecular masses
(63-75 kDa) of
I-CRS peptide (VEGF) cross-linked
complexes of purified major CRS-BPs are very similar to those of
I-CRS peptide (VEGF) affinity-labeled binding sites on
intact SK-Hep cells; 2) purified major CRS-BPs show a pH activity (with
a maximum at pH 5.0-5.5) similar to that of cell-surface
high-affinity binding sites; 3) the ligand binding activities of
purified CRS-BPs and high-affinity binding sites are inhibited by
polylysine, protamine, and heparin, but not cytochrome c; 4)
antiserum to the N-terminal peptide of CRS-BP1 immunoprecipitates a
I-CRS peptide (VEGF)
CRS-BP cross-linked complex on
intact HepG2 cells; and 5) CRS-BP1 is identified as an integral
membrane protein based on the observations that CRS-BPs in plasma
membranes cannot be extracted by high salt or high pH (11.5) and that
CRS-BP1 is recovered in the detergent phase during phase partition in
Triton X-114. (
)
This study is the first to identify and characterize high-affinity binding sites for CRS peptides on cultured cells and to isolate and characterize CRS-BPs from SK-Hep cells and bovine liver plasma membranes. The finding of the acidic pH activity of the high-affinity binding sites or CRS-BPs supports the hypothesis that the high-affinity binding sites (or CRS-BPs) interact with secretory proteins with CRS in the intracellular organelles during intracellular routing of both proteins from the trans-Golgi complex to the plasma membrane or extracellular compartment. Experiments are currently in progress to investigate the interaction of CRS-BP1 with the sis gene product in the intracellular organelles.