From the Department of Biochemistry and Molecular Biology, St. Louis University School of Medicine, St. Louis, Missouri 63104
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ABSTRACT |
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High affinity insulin-like growth
factor-binding proteins (IGFBP-1 to -6) are a family of structurally
homologous proteins that induce cellular responses by insulin-like
growth factor (IGF)-dependent and -independent mechanisms.
The IGFBP-3 receptor, which mediates the IGF-independent growth
inhibitory response, has recently been identified as the type V
transforming growth factor- High affinity insulin-like growth factor-binding proteins 1-6
(IGFBP-1 to -6)1 are a family
of structurally homologous ~24-43-kDa proteins composed of three
defined domains including a nonconserved central domain flanked by
conserved cysteine-rich N- and C-terminal domains (1-3). Recently,
several low affinity IGFBPs with sequence homology to the N-terminal
domains of the high affinity IGFBPs have been identified and referred
to as IGFBP-7 to -10 (4).
High affinity IGFBPs are produced by a variety of cell types and
tissues (1-3). They coordinate and regulate the biological activities
of IGF-I and IGF-II by serving as transporter proteins or carriers and
by scavenging IGFs from IGF receptors (1-3). High affinity IGFBPs have
also been shown to induce cellular responses in an IGF-independent
manner (5-15). These IGF-independent actions of high affinity IGFBPs
are believed to be mediated by specific cell-surface receptors or
membrane proteins (6, 16-18). The IGFBP-3 receptor, which mediates the
IGF-independent growth inhibitory response, has been recently
identified as the type V TGF- The T To define the function of T Because IGFBP-3 is structurally homologous to other high affinity
IGFBPs and because four of six high affinity IGFBPs (IGFBP-3 to -6)
possess the putative TGF- Materials--
Na125I (17 Ci/mg),
[methyl-3H]thymidine (67 Ci/mmol), and
[32P]orthophosphate (500 mCi/ml) were purchased from ICN
Biochemicals, Inc. (Costa Mesa, CA). High and low molecular mass
protein standards, recombinant human IGFBP-1, avidin-agarose, and other
chemical reagents were purchased from Sigma. Disuccinimidyl suberate
(DSS) and sulfo-NHS-biotin were obtained from Pierce. Anti-Smad2,
anti-Smad3, and goat IgG were purchased from Santa Cruz Biotechnology,
Inc. (Santa Cruz, CA). Recombinant human TGF- 125I-IGFBP Affinity Labeling of Mv1Lu
Cells--
Mv1Lu cells grown to confluency in 35-mm Petri dishes
were incubated with 1 nM 125I-IGFBPs
(specific activity, 10-90 µCi/ng) in the presence of various
concentrations (1-100 nM) of unlabeled IGFBPs with and without 10 µM [methyl-3H]Thymidine Incorporation
Assay--
Mv1Lu cells were plated on 24-well cluster dishes at near
confluence in DMEM containing 10% FCS. Within 6-8 h after plating, cells were rinsed twice with serum-free DMEM and incubated with various concentrations of IGFBPs in DMEM containing 0.1%
FCS. After 18 h at 37 °C, the
[methyl-3H]thymidine incorporation into
cellular DNA was determined as described previously (19).
Biotinylation of IGFBP-3 and Detection of the
125I-IGFBP-3-biotinylated IGFBP-3 Complex--
The
biotinylation of IGFBP-3 was carried out in a reaction mixture (50 µl) containing IGFBP-3 and sulfo-NHS-biotin (1:25, mol/mol) in 50 mM NaHCO3, pH 8.5. After 30 min at room
temperature, the reaction was terminated by the addition of glycine (10 mM). For dimer formation studies, Mv1Lu cells were
incubated with a premix of 8 nM 125I-IGFBP-3
and 2 nM biotinylated IGFBP-3 at 0 °C for 4 h
in the absence or presence of a 100-fold excess of unlabeled
IGFBP-3. Cells were rinsed twice with 1 ml of binding medium,
collected by scraping, and pelleted by centrifugation at 10,000 rpm for 5 min. The cell pellets were lysed in solubilization buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.4) containing 1%
Triton X-100 and mixed for 1 h at 4 °C followed by
centrifugation at 12,000 rpm for 10 min. The supernatant was
transferred to an Eppendorf tube containing 0.036 mg of avidin-agarose
(10 µl of suspension) and mixed at 4 °C for 1 h in the
absence or presence of D-biotin (20 µg or ~1.6
mM) (to estimate nonspecific binding). The
avidin-agarose beads bound by biotinylated IGFBP-3 and
125I-IGFBP-3-biotinylated IGFBP-3 complexes were washed
once with 1 ml of solubilization buffer containing Triton X-100 (0.2%)
and 0.5 M NaCl followed by two more washes with salt-free
solubilization buffer. Concentrated SDS sample buffer (2×) containing
[32P]Orthophosphate Metabolic Labeling and
Immunoprecipitation by Anti-Smad2 and Anti-Smad3 IgGs--
Mv1Lu cells
were plated on 100-mm Petri dishes in DMEM containing 10% FCS at near
confluency. After 16-18 h at 37 °C, cells (14 × 106 total) were rinsed twice with phosphate-free DMEM and
incubated with 5 ml of phosphate-free DMEM containing 0.2% dialyzed
FCS for 1 h at 37 °C. Cells were metabolically labeled with 1.0 mCi/ml [32P]orthophosphate in phosphate-free DMEM
containing 0.2% dialyzed FCS for 2 h at 37 °C. The
32P metabolically labeled cells were treated with
TGF- IGFBP-3, -4, and -5 Bind to T
The Kd of IGFBP-3 binding to T
IGFBP-3 was previously shown to inhibit growth of Mv1Lu cells as
measured by DNA synthesis (19). This inhibition appeared to be mediated
by T IGFBP-3 Forms Dimers at the Cell Surface That Preferentially Bind
to T
As described above, we demonstrated the covalently linked dimer
formation of 125I-IGFBP-3 or other 125I-IGFBPs
by taking advantage of the properties of covalent linking of
125I-IGFBPs prepared by the chloramine-T procedure. To
prove that the dimer formation is an inherent property of IGFBP-3, we
determined the formation of the IGFBP-3 dimer using an approach in
which IGFBP-3 was tagged with 125I or biotin. The formation
of the IGFBP-3 dimer was detected by identifying the
125I-IGFBP-3-biotinylated IGFBP-3 complex in the lysates of
Mv1Lu cells that were incubated with a premix of
125I-IGFBP-3 and biotinylated IGFBP-3 (4:1, mol/mol). After
2.5 h at 0 °C, the cell lysates were incubated with
avidin-agarose. After centrifugation, the avidin-agarose pellets were
analyzed by 7.5% SDS-PAGE under reducing conditions and
autoradiography. As shown in Fig. 6
(lane 1), 125I-IGFBP-3 was detected in the
avidin-agarose pellets of lysates of cells incubated with a premix of
125I-IGFBP-3 and biotinylated IGFBP-3. Very little
125I-IGFBP-3 was detected in the avidin-agarose pellets of
lysates of cells incubated with a premix of 125I-IGFBP-3
and biotinylated IGFBP-3 in the presence of a 100-fold excess of
unlabeled IGFBP-3 or ~1.6 mM biotin (Fig. 6, lanes
3 and 2). These results further verify the ability of
IGFBP-3 to form dimers at the cell surface.
TGF- IGFBP-3 Does Not Stimulate the Cellular Phosphorylation of
Smad2 and Smad3--
In Mv1Lu cells, Smad2 and Smad3 have been
identified as key signal transducers within the signal transduction
cascade initiated by the T High affinity IGFBPs are important modulators of IGF actions
(1-3). Accumulated evidence suggests that IGFBPs are also able to
induce cellular responses in an IGF-independent manner (5-15). The
IGF-independent actions for IGFBPs are believed to be mediated by
specific cell-surface receptors or membrane-binding proteins (1,
16-18). Several membrane-binding proteins for IGFBPs were identified,
but none of these proteins were well characterized (6, 16-18). We have
recently identified the IGFBP-3 receptor as T Among high affinity IGFBPs, four (IGFBP-3 to -6), which possess the
putative TGF- Several polypeptide growth factors are known to stimulate the
cytoplasmic kinase activities of their respective receptors by inducing
receptor dimerization through their dimeric structures (39-41). The
covalent dimeric structure is also known to be required for TGF- The major cell-surface binding sites for IGFBP-3 dimers appear to be
membrane proteins other than T The signaling mediated by T receptor (T
R-V) (Leal, S. M., Liu,
Q. L., Huang, S. S., and Huang, J. S. (1997) J. Biol.
Chem. 272, 20572-20576). To characterize the interactions of
high affinity IGFBPs with T
R-V, mink lung epithelial cells (Mv1Lu
cells) were incubated with 125I-labeled recombinant human
IGFBPs (125I-IGFBP-1 to -6) in the presence of the
cross-linking agent disuccinimidyl suberate and analyzed by 5%
SDS-polyacrylamide gel electrophoresis and autoradiography.
125I-IGFBP-3, -4, and -5 but not 125I-IGFBP-1,
-2, and -6 bound to T
R-V as demonstrated by the detection of the
~400-kDa 125I-IGFBP·T
R-V cross-linked complex in the
cell lysates and immunoprecipitates. The analyses of
125I-labeled ligand binding competition and DNA synthesis
inhibition revealed that IGFBP-3 was a more potent ligand for T
R-V
than IGFBP-4 or -5. Most of the high affinity 125I-IGFBPs
formed dimers at the cell surface. The cell-surface dimer of
125I-IGFBP-3 preferentially bound to and was cross-linked
to T
R-V in the presence of disuccinimidyl suberate.
IGFBP-3 did not stimulate the cellular phosphorylation of Smad2
and Smad3, key transducers of the transforming growth factor-
type
I/type II receptor (T
R-I·T
R-II) heterocomplex-mediated
signaling. These results suggest that IGFBP-3, -4, and -5 are specific
ligands for T
R-V, which mediates the growth inhibitory response
through a signaling pathway(s) distinct from that mediated by the
T
R-I and T
R-II heterocomplex.
INTRODUCTION
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Abstract
Introduction
References
receptor (T
R-V) (19).
R-V is a 400-kDa non-proteoglycan membrane glycoprotein (20).
It is a Ser-specific protein kinase and co-expresses with type I, type
II, and type III TGF-
receptors (T
R-I, T
R-II, and T
R-III)
in most cell types (21-23). The T
R-V is a low affinity TGF-
receptor with Kd of ~0.4 nM for
TGF-
1 and TGF-
2 and ~5 nM
for TGF-
3 (23, 24). Nevertheless, several lines of
evidence suggest that T
R-V is important in mediating TGF-
-induced growth inhibitory responses. These include the following: 1) cells lacking T
R-V but expressing T
R-I, T
R-II, and T
R-III do not exhibit the growth inhibitory response to stimulation by exogenous TGF-
, although exogenous TGF-
is able to induce transcriptional activation of plasminogen activator inhibitor 1 and fibronectin in
these cells (24); 2) T
R-V mediates the growth inhibitory response in
the absence of T
R-I or T
R-II, but both T
R-I and T
R-II are
required for maximal growth inhibition (24); and 3) the cells lacking
T
R-V have been found to be carcinoma cells, whereas all normal cell
types studied express T
R-V (19, 21, 24). This implies that the loss
of T
R-V, which mediates negative growth regulation, may contribute
to malignancy of certain carcinoma cells (19, 21, 24).
R-V, we developed specific peptide
antagonists that showed higher affinity to T
R-V than to other TGF-
receptor types (25). The structural and functional analyses of
these peptide antagonists revealed that a
W/RXXD motif is
essential for the antagonist activity. Multiple conjugation of the
peptide antagonists to carrier proteins conferred TGF-
agonist
activity in growth inhibition but not in transcriptional activation
(25). These results prompted us to identify structurally unrelated
TGF-
agonists that possess the
W/RXXD motif. IGFBP-3
was the first TGF-
agonist identified (19). IGFBP-3 possesses a
putative TGF-
active site motif (WCVD) near
its C terminus (1-3).
active site motif
(WCVD) near their C termini (1-3), we
hypothesized that at least some of these IGFBPs might bind to T
R-V,
which may mediate the IGF-independent activities of these IGFBPs. To
test this hypothesis, we characterized the interactions of
IGFBP-1 to -6 with T
R-V in mink lung epithelial cells (Mv1Lu
cells). In this communication, we show that IGFBP-3, -4, and -5 but not
IGFBP-1, -2, or -6 bind to T
R-V as demonstrated by
125I-labeled ligand affinity labeling of T
R-V in Mv1Lu
cells. IGFBP-4 and -5 bind to T
R-V with lower affinities than that
of IGFBP-3. The cell surface-associated dimeric form of
IGFBP-3 exhibits a preference for binding to T
R-V. We also
demonstrate that IGFBP-3-induced growth inhibition mediated by T
R-V
does not involve the stimulated phosphorylation of Smad2 and Smad3.
EXPERIMENTAL PROCEDURES
1, IGFBP-2,
IGFBP-4, IGFBP-5, and IGFBP-6 were obtained from Austral Biologicals
(San Ramon, CA). Recombinant nonglycosylated human IGFBP-3 (expressed in Escherichia coli) was provided by Celtrix Pharmaceutical,
Inc. (Santa Clara, CA). 125I-Labeled IGFBPs
(125I-IGFBPs) and antiserum to T
R-V were prepared
according to our published procedures (19). The TGF-
1
peptide antagonist,
1-(41-65), was synthesized as
described previously (25). Mv1Lu cells were grown and maintained in
Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal calf
serum (FCS).
1-(41-65) in binding medium
(125 mM NaCl, 5 mM KCl, 5 mM
MgSO4, 2 mM CaCl2, 50 mM Hepes, pH 7.5) at 0 °C for 4 h. After
125I-IGFBP affinity labeling in the presence of DSS (19),
the 125I-IGFBP·T
R-V complex was analyzed by 5%
SDS-polyacrylamide gel electrophoresis (PAGE) under reducing conditions
and autoradiography.
-mercaptoethanol was added to the agarose beads. The bead suspension
was boiled for 5 min and vortexed vigorously to release biotinylated
IGFBP-3 and 125I-IGFBP-3 from the beads. The agarose beads
were pelleted, and the supernatant was analyzed by 12% SDS-PAGE and autoradiography.
1 (10 ng/ml or 0.4 nM) or IGFBP-3 (1 µg/ml or ~33 nM) for an additional 3 h at
37 °C, rinsed twice with 5 ml of cold phosphate-buffered saline, and
lysed with lysis buffer (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.5% Triton X-100, 50 mM NaF, 10 mM sodium pyrophosphate, 1 mM sodium orthovanadate, 1 mM
phenylmethylsulfonyl fluoride, and 20 µg/ml aprotinin) for 10 min at
0 °C followed by repeated aspiration through a 21-gauge needle. The
cell lysates were centrifuged at 10,000 rpm for 15 min, and the
supernatant was precleared with 1 µg of goat IgG and protein
G-Sepharose at 4 °C. The precleared cell lysates were incubated with
anti-Smad2 and anti-Smad3 IgGs (2 µg) for 2.5 h at 4 °C and
incubated with protein G-Sepharose for an additional 1 h. The
protein G-Sepharose beads were rinsed 4 times with 1 ml of lysis
buffer, suspended in 40 µl of SDS sample buffer containing
-mercaptoethanol, and boiled for 5 min. The immunoprecipitates were
analyzed by 7.5% SDS-PAGE and autoradiography.
RESULTS
R-V with Different
Affinities--
Mv1Lu cells have been used as a model cell system to
investigate T
R-V and other TGF-
receptor types and
TGF-
-induced cellular responses (19, 20, 24, 26). To determine the
interactions of T
R-V with IGFBPs, we first performed ligand affinity
labeling of T
R-V in Mv1Lu cells using 125I-labeled
recombinant human IGFBP-1 to -6 (125I-IGFBP-1 to -6). After
incubation of Mv1Lu cells with 5 nM
125I-IGFBP-1, -2, -3, -4, -5, or -6 at 0 °C for 3 h, the ~400-kDa 125I-IGFBP·T
R-V complex was
cross-linked with DSS and identified by 5% SDS-PAGE under reducing
conditions and autoradiography. As shown in Fig.
1, the 125I-IGFBP·T
R-V
complex was detected in the lysates of cells that were affinity-labeled
with 125I-IGFBP-3, 125I-IGFBP-4, or
125I-IGFBP-5 (lanes 1, 3, and
5). The specificity of the affinity labeling of T
R-V was
supported by blocking with
1-(41-65), a specific
TGF-
antagonist (25) (lanes 2, 4, and
6). The 125I-IGFBP·T
R-V complex was not
detected in the lysates of cells affinity-labeled with
125I-IGFBP-1, -2, or -6 (lanes 7, 9,
and 11). The 125I-IGFBP·T
R-V complex was
verified by its immunoprecipitation with specific antiserum to T
R-V
(Fig. 2, lanes 2,
5, and 8). The immunoprecipitation of the
125I-IGFBP-3·T
R-V complex by antiserum to T
R-V was
previously reported (19). It is of importance to note that all
125I-IGFBPs except 125I-IGFBP-2 formed
covalently linked dimers that were stable after treatment at 100 °C
for 5 min in 0.1% SDS containing
-mercaptoethanol and subsequent
SDS-PAGE (Fig. 1).
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Fig. 1.
125I-IGFBP affinity labeling in
Mv1Lu cells. Cells were incubated with 5 nM
125I-IGFBP-1 to -6 in the presence (+) and
absence ( ) of 10 µM
1-(41-65). After 3 h at 0 °C, the
125I-IGFBP affinity labeling was carried out and analyzed
by 5% SDS-PAGE under reducing conditions and autoradiography. The
arrows indicate the location of the ~400-kDa
125I-IGFBP·T
R-V complex. The asterisks
denote the locations of the covalently linked 125I-IGFBP
dimers.
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Fig. 2.
Immunoprecipitation of the
125I-IGFBP-3, -4, and -5 affinity-labeled
T R-V in Mv1Lu cells. Cells were incubated
with 10 nM 125I-IGFBP-3 (A), 25 nM 125I-IGFBP-4 (B), or 25 nM 125I-IGFBP-5 (C) both with and
without 10 µM
1-(41-65), a
TGF-
/IGFBP-3 antagonist. After 3 h at 0 °C and affinity
labeling, cell lysates were immunoprecipitated with antiserum to
T
R-V or non-immune (Control serum) serum (19). The
immunoprecipitates were analyzed by 5% SDS-PAGE under reducing
conditions and autoradiography. The brackets indicate the
locations of the 125I-IGFBP·T
R-V complexes. The
asterisk denotes the location of the
125I-IGFBP-3 dimer. The arrows indicate the
location of dye front.
R-V was previously
estimated to be ~6 nM (19). To determine the relative
affinities of IGFBP-4 and -5 to T
R-V in Mv1Lu cells, we performed
competition experiments using 125I-IGFBP-3 (1 nM) as the ligand and unlabeled IGFBP-3, -4, and -5 as
competitors. As shown in Fig.
3A, increasing concentrations of unlabeled IGFBP-3 quantitatively inhibited 125I-IGFBP-3
binding to T
R-V as determined by 125I-IGFBP-3 affinity
labeling of T
R-V. The quantitative analysis of this inhibition
revealed that unlabeled IGFBP-3 blocked the 125I-IGFBP-3
binding with an IC50 of ~6 nM, which is
identical with the estimated Kd of IGFBP-3 binding
to T
R-V (19) (Fig. 3B). Unlabeled IGFBP-4 and -5 weakly
inhibited 125I-IGFBP-3 binding to T
R-V with an
IC50 of
100 nM (Fig. 3B). We also
determined the effects of various concentrations of unlabeled IGFBP-1,
-2, and -6 on 125I-IGFBP-3 binding to T
R-V. Unlabeled
IGFBP-1, -2, and -6 did not show any significant effect on the binding
of 125I-IGFBP-3 to T
R-V at concentrations up to 100 nM (data not shown). These results suggest that IGFBP-3
binds to T
R-V with higher affinity than IGFBP-4 and -5 and that
IGFBP-1, -2, and -6 are not ligands for T
R-V.
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Fig. 3.
Effects of unlabeled IGFBP-3, -4, and -5 on
the formation of the
125I-IGFBP-3·T R-V complex in
Mv1Lu cells. Cells were incubated with 1 nM
125I-IGFBP-3 in the presence of various concentrations of
unlabeled IGFBP-3, -4, or -5 at 0 °C for 3 h. The
125I-IGFBP·3-T
R-V complex was then cross-linked with
DSS and analyzed by 5% SDS-PAGE under reducing conditions and
autoradiography (A) or quantitation using a PhosphorImager
(B). The relative level of the
125I-IGFBP-3·T
R-V complex in cells incubated with
125I-IGFBP-3 in the absence of unlabeled IGFBPs was taken
as 100% formation of the 125I-IGFBP-3·T
R-V complex.
The figure is representative of three experiments that gave comparable
results. The relative level of the 125I-IGFBP-3·T
R-V
complex in the presence of 3 nM unlabeled IGFBP-5 was
~134%, which was unusually high and not reproducible.
R-V because the IGFBP-3-induced growth inhibition was blocked in
the presence of
1-(41-65), a specific TGF-
peptide antagonist that blocked IGFBP-3 binding to T
R-V (19). Because IGFBP-3, -4, and -5 bind to T
R-V with different affinities, we determined the relative potencies of IGFBP-3, -4, and -5 for DNA synthesis inhibition in Mv1Lu cells. As shown in Fig.
4, at 1 µg/ml (~33 nM)
IGFBP-3 inhibited ~50% of DNA synthesis of Mv1Lu cells, whereas
IGFBP-4 and -5 produced ~15-20% inhibition of DNA synthesis at the
same concentration. The potent DNA synthesis inhibitory activity of
IGFBP-3 is consistent with its high affinity to T
R-V. We also
determined the effects of IGFBP-1, -2, and -6 on DNA synthesis of Mv1Lu
cells. IGFBP-1, -2, and -6 exhibited ~5-10% inhibition of DNA
synthesis of Mv1Lu cells at 1 µg/ml (data not shown). This inhibition
may be because of scavenging endogenous IGFs from the IGF-1 receptor.
IGF-1 is a weak growth factor or mitogen for Mv1Lu cells under these
experimental conditions.
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Fig. 4.
Effects of IGFBP-3, -4, and -5 on DNA
synthesis of Mv1Lu cells. Cells were incubated with various
concentrations of IGFBP-3, -4, or -5. After 16 h at 37 °C, the
[methyl-3H]thymidine incorporated into
cellular DNA was determined. The experiment was performed in triplicate
cell cultures. The error bars represent means ± S.E.
The data were obtained from three independent experiments.
R-V--
As demonstrated in Fig. 1, most high affinity
125I-IGFBPs formed covalently linked dimers at the cell
surface. This experiment was performed in the presence of the
cross-linking agent DSS. Because 125I-labeled proteins
prepared by the chloramine-T procedure are known to acquire the
properties of covalent linking during 125I-labeling
(27-29), the covalently linked dimers of 125I-IGFBPs may
be spontaneously produced in a DSS-independent manner during the
incubation (3 h at 0 °C) of cells with 125I-IGFBPs that
were also prepared using chloramine T. To test this possibility, we
investigated the formation of the covalently linked dimer of
125I-IGFBP-3 in aqueous solution and at the cell surface of
Mv1Lu cells in the absence of added cross-linking agents. As shown in Fig. 5, less than 2% of
125I-IGFBP-3 (5 nM) spontaneously formed
covalently linked dimers in binding medium (lane 1).
However, approximately 20% of the 125I-IGFBP-3 associated
with the cell surface was found to be covalently linked dimers
(lane 2). These results indicate that the formation of the
covalently linked 125I-IGFBP-3 dimer does not require the
presence of cross-linking agents. These results also suggest that
the 125I-IGFBP-3 dimer formation may be enhanced
at the cell surface. Alternatively, the 125I-IGFBP-3 dimer
may associate with the cell surface of Mv1Lu cells with an affinity
higher than that of the 125I-IGFBP-3 monomer.
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Fig. 5.
Formation of the covalently linked
125I-IGFBP-3 dimer in binding medium and at the cell
surface of Mv1Lu cells in the absence of added cross-linking
agents. Five nM 125I-IGFBP-3 was incubated
with or without Mv1Lu cells in binding medium containing 0.2% bovine
serum albumin. After 3 h at 0 °C, the cell lysates and an
aliquot of the binding medium were analyzed by 10% SDS-PAGE under
reducing conditions and autoradiography. The arrows indicate
the locations of the covalently linked 125I-IGFBP dimer and
125I-IGFBP-3 monomers. The relative intensities of
covalently linked IGFBP-3 dimers and monomers were quantitated using a
PhosphorImager.
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Fig. 6.
Complex formation of 125I-IGFBP-3
with biotinylated IGFBP-3 in Mv1Lu cells. Cells were incubated
with a premix of 125I-IGFBP-3 and biotinylated IGFBP-3
(4:1, mol/mol) in the presence and absence of a 100-fold excess of
unlabeled IGFBP-3. After 3 h at 0 °C, the cells were washed;
the cell lysates were incubated with avidin-agarose with or without
~1.6 mM biotin at 4 °C for 1 h. After
centrifugation, the pellets were analyzed by 12% SDS-PAGE under
reducing conditions and autoradiography. The arrow indicates
the location of 125I-IGFBP-3.
is known to stimulate cellular responses by inducing
hetero-oligomerization of TGF-
receptors through its covalent dimeric structure (30, 31). We hypothesize that IGFBP-3 inhibits cellular growth by a similar mechanism in which the dimeric form of
IGFBP-3 is required for activation of T
R-V. To test this hypothesis, we determined the binding of the dimeric form of IGFBP-3 to T
R-V in
Mv1Lu cells in the presence and absence of DSS. As shown in Fig.
7, the covalently linked dimer of
125I-IGFBP-3 was detected in the medium (lane 1)
and lysates (lane 3) of cells incubated with
125I-IGFBP-3 without added cross-linking agents. It is of
importance to note that the exposure times for the autoradiograms of
the medium and cell lysates were 16 and 2 h, respectively, to have comparable intensities of covalently linked 125I-IGFBP-3
dimers. In the presence of the cross-linking agent DSS, most of the
covalently linked dimer associated with the cell surface was
cross-linked to T
R-V (lane 4 versus 3). These
results suggest that the cell surface-associated dimeric form of
IGFBP-3 preferentially binds to T
R-V. These results are also
consistent with our previous observation that both T
R-V and
125I-IGFBP-3 dimers were immunoprecipitated by specific
antiserum to T
R-V after the 125I-IGFBP-3-affinity
labeling of T
R-V in Mv1Lu cells (19).
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Fig. 7.
Binding and cross-linking of the cell surface
covalently linked 125I-IGFBP-3 dimer to
T R-V in Mv1Lu cells. Cells were incubated
with 5 nM 125I-IGFBP-3 at 0 °C for 3 h
and then treated with (+) or without (
) DSS
(0.3 mM) for an additional 15 min. The medium and cell
lysates were analyzed by 5% SDS-PAGE under reducing conditions and
autoradiography. The exposure times for the autoradiograms of the
medium and cell lysates were 16 and 2 h, respectively.
Arrows indicate the locations of the
125I-IGFBP-3·T
R-V cross-linked complex and covalently
linked 125I-IGFBP-3 dimers. The crescent shape of the
125I-IGFBP-3 dimer in the medium (lanes 1 and
2) is because of the influence from a large quantity of
bovine serum albumin in the binding medium, which migrates closely with
the 125I-IGFBP-3 dimer on the SDS-polyacrylamide gel.
R-I·T
R-II heterocomplex following
stimulation by TGF-
(32). The phosphorylation of Smad2 and Smad3 by
T
R-I is essential for their complex formation with Smad4 and
subsequent translocation to the nucleus where they regulate
transcriptional activities required for cell cycle arrest and other
cellular responses (32, 33). Because T
R-V forms complexes with
T
R-I (24), the phosphorylation of Smad2 and Smad3 may also be
involved in the signaling mediated by the T
R-I·T
R-V
heterocomplex (24). To test this possibility, we investigated the
effect of IGFBP-3 on the phosphorylation of Smad2 and Smad3 in Mv1Lu
cells. As shown in Fig. 8, the
phosphorylation of Smad2 and Smad3 was not affected by IGFBP-3
treatment (lane 4 versus 2), but TGF-
treatment enhanced the phosphorylation of Smad2 and Smad3 by ~7- and
~2-fold, respectively (lane 3 versus 2). This
result suggests that the IGFBP-3-induced growth inhibition, which is
mediated by T
R-V, may involve a signaling pathway that is distinct
from that mediated by the T
R-I·T
R-II heterocomplex.
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Fig. 8.
Effect of IGFBP-3 on the phosphorylation of
Smad2 and Smad3 in Mv1Lu cells. Cells labeled metabolically with
[32P]orthophosphate were treated with or without IGFBP-3
(33 nM) or TGF- (0.1 nM) at 37 °C for
2 h. The 32P-labeled cell lysates were
immunoprecipitated with a mixture of anti-Smad2 and anti-Smad3 IgGs or
control IgG. The immunoprecipitates were analyzed by 7.5% SDS-PAGE
under reducing conditions. The arrows indicate the locations
of 32P-labeled Smad2 and Smad3. The bars
indicate the locations of the immunologically cross-reacted proteins
that were not immunoprecipitated by control IgG. The relative levels of
phosphorylated Smad2 and Smad3 in cells treated with and without
IGFBP-3 or TGF-
were quantitated by a PhosphorImager.
DISCUSSION
R-V, which
mediates the IGF-independent growth inhibitory response induced by
IGFBP-3 (19). In this communication, we show that IGFBP-4 and -5 are also specific ligands for T
R-V, although their affinities for
T
R-V are weaker than that of IGFBP-3. The T
R-V is likely the same
receptor for IGFBP-5, which has been recently identified in mouse
osteoblasts (34). The T
R-V and putative IGFBP-5 receptor in
osteoblasts share similar properties including the following: 1) they
have almost identical molecular weights (~400,000) (19-25, 34), 2)
both show ligand (TGF-
/IGFBP-3 and IGFBP-5)-stimulated
serine-specific autophosphorylation and kinase activity toward
caseins2 (23, 24, 34), and 3)
the T
R-V is expressed in most cell types including osteoblasts
(21).3
active site motif WCVD near their C termini, were
initially predicted to bind to T
R-V. However, although IGFBP-3, -4, and -5 were found to interact with T
R-V in Mv1Lu cells,
IGFBP-6 did not. The inability of IGFBP-6 to interact with
T
R-V may be because of its unique structure. IGFBP-6 contains 10 of
12 N-terminal cysteine residues conserved in other high affinity IGFBPs
and possesses additional O-linked carbohydrate moieties in
the central domain and possibly near the C-terminal end (1-3, 35, 36). These distinct structural features may yield a conformation that does
not allow the WCVD motif in IGFBP-6 to interact with T
R-V. It is
also possible that the WCVD motif is not the only determinant required
for the interactions of IGFBPs with T
R-V. The WCVD motif is
contained within the thyroglobulin type-1 repeat of IGFBP-3 (37).
Thyroglobulin, which contains multiple WCVD motifs per monomer, has
recently been shown to exhibit an authentic TGF-
antagonist/agonist
activity after activation by acidic pH/denaturing agent treatments and
chemical modifications (38). This implies that certain structural
configurations of the WCVD motif are required for optimal interaction
with T
R-V.
activities (42). Most 125I-labeled IGFBPs form covalently
linked dimers at the cell surface. Approximately 20% of cell
surface-associated 125I-IGFBP-3 is estimated to be in the
form of covalently linked dimers, whereas less than 2% exists as the
covalently linked dimer in binding medium. This suggests that the cell
surface association enhances the dimer formation of IGFBP-3. Assuming
that the efficiency of the spontaneous covalent linking of
125I-IGFBP-3 is ~20%, it is estimated that almost 100%
of the cell surface-associated 125I-IGFBP-3 are dimers. The
cell-surface dimeric form of IGFBP-3 appears to be the active form of
IGFBP-3 for binding to T
R-V.
R-V because cells lacking T
R-V
(human colorectal carcinoma cells) express these binding sites
(19). Interestingly, the binding of 125I-IGFBP-3 and
125I-IGFBP-5 dimers to their major cell-surface binding
sites is blocked by
1-(41-65), a specific TGF-
peptide antagonist, whereas the binding of 125I-IGFBP-1 and
-4 dimers to their major binding sites is resistant to the blocking by
the TGF-
peptide antagonist (Fig. 1). This suggests that the major
cell-surface binding sites for IGFBP-3 and IGFBP-5 dimers are distinct
from those for other IGFBPs dimers. This suggestion has been supported
by the observation that heparin inhibits the binding of
125I-IGFBP-3 and -5 dimers but not
125I-IGFBP-1 and -4 dimers to cell-surface
binding sites (18, 19).4 The
functions of these major cell-surface binding sites are unknown. However, one function may involve presentation of IGFBPs to their respective cell-surface receptors. In the case of IGFBP-3, these binding sites may present IGFBP-3 to T
R-V as demonstrated in Fig. 7.
This would explain the observation that heparin inhibits the binding of
125I-IGFBP-3 to both the major cell-surface binding sites
and to the T
R-V (18, 19).4
R-V has been difficult to define because
of the co-expression of T
R-I, T
R-II, T
R-III, and T
R-V in
the same cells. The identification of IGFBP-3 as well as IGFBP-4 and -5 as specific ligands for T
R-V has enabled us to investigate the
signaling mediated by T
R-V in cells containing other TGF-
receptors. In this communication, we show that IGFBP-3 does not stimulate the cellular phosphorylation of Smad2 and Smad3, both of
which play key roles in the signaling mediated by the T
R-I and
T
R-II heterocomplex (31, 32). This result is consistent with the
observation that IGFBP-3 induces growth inhibition but not
transcriptional activation of plasminogen activator inhibitor-1 in
Mv1Lu cells (19). The TGF-
-induced expression of plasminogen activator inhibitor-1 is mainly mediated by the T
R-I·T
R-II
complex (24). Furthermore, IGFBP-3 has been shown to inhibit the growth of mutant mink lung epithelial cells (DR26 and R-1B cells), which express T
R-V but lack the expression of the functional T
R-II or
T
R-I (24).
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ACKNOWLEDGEMENTS |
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We thank Celtrix Pharmaceutical, Inc. for providing recombinant nonglycosylated human IGFBP-3, Drs. William S. Sly and Frank E. Johnson for critical review of the manuscript, and 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 CA 38808 and a predoctoral fellowship (to S. M. L.) from the American Heart Association, Missouri Affiliate.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.
4 S. M. Leal, S. S. Huang, and J. S. Huang, unpublished results.
3 S. S. Huang and J. S. Huang, unpublished results.
2 T. Zhao, Q. Liu, S. S. Huang, and J. S. Huang, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are:
IGFBP, insulin-like
growth factor-binding protein;
IGF, insulin-like growth factor;
TGF-, transforming growth factor-
;
T
R-V, type V TGF-
receptor;
DSS, disuccinimidyl suberate;
DMEM, Dulbecco's modified
Eagle's medium;
FCS, fetal calf serum;
PAGE, polyacrylamide gel
electrophoresis.
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REFERENCES |
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