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Volume 272, Number 49, Issue of December 5, 1997
pp. 30729-30734
(Received for publication, July 30, 1997, and in revised form, September 19, 1997)
From the Department of Pediatrics, School of Medicine, Oregon
Health Sciences University, Portland, Oregon 97201
ABSTRACT The insulin-like growth factors (IGFs) are
transported by a family of high-affinity binding proteins (IGFBPs) that
protect IGFs from degradation, limit their binding to IGF receptors,
and modulate IGF actions. The six classical IGFBPs have been believed to have no affinity for insulin. We now demonstrate that IGFBP-7/mac25, a newly identified member of the IGFBP superfamily that binds IGFs
specifically with low affinity is a high-affinity insulin binding
protein. IGFBP-7 blocks insulin binding to the insulin receptor and
thereby inhibiting the earliest steps in insulin action, such as
autophosphorylation of the insulin receptor INTRODUCTION Insulin-like growth factors
(IGFs)-I1 and -II are
structurally related to insulin, sharing approximately 50% amino acid
homology with insulin in the A- and B-chain regions but retaining a
connecting peptide, as well as a carboxyl-terminal extension (1). The anabolic and mitogenic actions of IGFs are mediated largely through the
type 1 IGF receptor, which, like the insulin receptor, is a
heterotetrameric, membrane-spanning tyrosine kinase (2).
It has been accepted dogma that, unlike insulin, the IGFs bind to a
family of binding proteins (IGFBPs) with high affinity and specificity
(3-5). The six well characterized IGFBPs have significant sequence
homology, including a GCGCCXXC motif as part of 10-12
conserved cysteines at the amino terminus and six conserved cysteines
at the carboxyl terminus. We have recently characterized two additional
secreted proteins with lower affinity for IGFs (IGFBP-7/mac25 and
IGFBP-8/CTGF) and two additional potential members of the IGFBP
superfamily (6-9). The structure of IGFBP-7 revealed the presence of
amino-terminal conserved sequences, including 11 cysteines, but lower
sequence homology in the normally well-conserved COOH terminus. IGFBPs
1-6 are believed to function as carrier proteins for IGFs, delaying
degradation of IGF peptides and increasing their half-lives and
inhibiting IGF access to receptors (10-14).
Proteolysis of IGFBPs, as observed during pregnancy, results in IGFBP
fragments with decreased affinity for IGFs and, thereby, promotes
enhanced access of IGFs to their receptors (15, 16). Recent studies
have demonstrated that several of these IGFBPs, especially IGFBP-3, may
also be capable of directly inhibiting cell growth in an
IGF-independent manner (17-23). Both intact IGFBP-3 and IGFBP-3
proteolytic fragments have been shown to be capable of blocking the
mitogenic effect of insulin (24, 25). Whether these effects reflect
inhibition of insulin signaling pathways or a direct effect of IGFBP-3
and its fragments on cell growth remains unclear.
We have, therefore, reevaluated the ability of IGFBPs 1-6, IGFBP-7,
and IGFBP-3 proteolytic fragments to bind insulin and have demonstrated
that IGFBP-7 and NH2-terminal fragment of IGFBP-3 (IGFBP-3(1-87)) not only bind insulin specifically but
also modulate insulin binding to its receptor and subsequently
inhibiting insulin-stimulated autophosphorylation of the insulin
receptor EXPERIMENTAL PROCEDURES High performance liquid chromatography-purified
hIGFBP-1 from human amniotic fluid was kindly provided by Dr. D. R. Powell (Baylor College of Medicine, Houston, TX) (26). Recombinant human IGFBP-3 (rhIGFBP-3), a nonglycosylated 29 kDa core protein expressed in Escherichia coli cells, was the generous gift
of Celtrix, Inc. (Santa Clara, CA) (27). Recombinant human IGFBP-2, -4, -5, and -6 were purchased from Austral Biologicals (San Ramon, CA). The
cDNA for IGFBP-7 (mac25) was cloned, and baculovirus recombinants
were made as described in (7). IGF-I was purchased from Bachem
California (Torrance, CA). IGF-II was kindly provided by Lilly. Bovine
insulin was purchased from Sigma. [(Gln6,
Ala7, Thr18, Leu19,
Leu27] IGF-II ([QAYLL]IGF-II), a synthetic IGF-II
analog, was synthesized as described previously (28). Human placentas
were stripped of their membranes, washed in 0.25 M sucrose,
and homogenized with a polytron (29). Crude microsomal membrane
preparations were obtained via differential centrifugation (30).
Anti-phosphotyrosine monoclonal antibody (4G10) was a generous gift
from Dr. B. J. Druker (Dept. of Hematology and Medical Oncology,
Oregon Health Sciences University).
125I-(A14)-monoiodinated insulin was purchased from
Amersham Corp. 125I-labeled growth hormone and
125I-prolactin were kindly provided by DSL (Webster, TX).
Na125I was obtained from Amersham Corp. Iodination was
performed by a modification of the chloramine-T technique to specific
activities of 350-500 µCi/µg for IGF-I and -II. Reagents used for
SDS-polyacrylamide gel electrophoresis (PAGE) were purchased from
Bio-Rad Laboratories (Richmond, CA).
NIH-3T3 cells overexpressing the human insulin
receptor were kindly gifted from Dr. C. T. Roberts, Jr. (Dept. of
Pediatrics, Oregon Health Sciences University) and grown in Dulbecco's
modified Eagle's medium + 10% fetal calf serum + 500 µg/ml
geneticin at 37 °C with 5% CO2. High Five cells were
maintained in ExCell 405 Media (JRH Biosciences, Lenexa, KS) at
27 °C. The wild-type baculovirus (AcNPv) was obtained from
Invitrogen Inc. (Carlsbad, CA). All tissue culture media and components
were purchased from Life Technologies, Inc.
Aliquots of reagents plus binding
buffer (50 mM Tris-HCl, pH 7.4) were incubated with
125I-insulin (20,000 cpm), 125I-IGF-I (20,000 cpm), or 125I-IGF-II (20,000 cpm) with or without indicated
reagents, at the concentrations indicated under "Results" and in
Figs. 1, 2, 3 for 16 h at 4 °C. At the end of incubation, the
cross-linking reagent disuccinimidyl suberate (Pierce) was added to a
final concentration of 0.5 mM. After cross-linking for 15 min, the samples were subjected to 12% SDS-PAGE and autoradiography.
Bands were quantified by densitometry, as calculated by a densitometer
(Bio-Rad).
[View Larger Version of this Image (69K GIF file)]
[View Larger Version of this Image (61K GIF file)]
[View Larger Version of this Image (78K GIF file)]
Polymerase chain reaction was
used to add a FLAG epitope sequence (DYKDDDDK) and a new stop codon
immediately following amino acid 87 of the mature IGFBP-3. After
sequencing, this fragment was subcloned into the baculovirus expression
vector pFASTBAC1 (Life Technologies, Inc.) and transfected into
Sf9 insect cells, and viral recombinants were identified by
immunoblotting with the anti-FLAG M2 antibody (Eastman Kodak, New
Haven, CT). Recombinant protein was purified from the media of High
Five cells after infecting at an m.o.i. = 3 for 3 days at 27 °C. The
media was collected, and the protein was purified over anti-FLAG M2
affinity beads according to the manufacturer protocol by elution with
the FLAG peptide. The purified protein was subjected to SDS-PAGE in a
15% gel and stained with Coomassie Blue or transferred to
nitrocellulose for immunodetection. Protein fractions were pooled,
concentrated, and quantitated by comparison with known amounts of BSA
by silver staining (Bio-Rad). Immunoblotting using the enhanced
chemiluminescence (ECL) detection technique was performed as described
in Wilson et al. (31) using a 1:3000 dilution of anti-FLAG
M2 antibody and incubating for 16 h at 4 °C and then in a
1:3000 dilution of anti-mouse IgG horseradish peroxidase (Amersham
Corp.) for 1 h at room temperature.
Ligand blotting was performed
as described in Hossenlopp et al. (32) with minor
modifications. Briefly, IGFBP-1, IGFBP-3, IGFBP-3(1-87),
and IGFBP-7 at the concentrations indicated in the text and figures in
SDS sample buffer (62.5 mM Tris-HCl, pH 6.8, 40% glycerol, and 2.3% SDS) with or without 100 mM dithiothreitol (DTT)
were analyzed by SDS-PAGE (12 or 15%). Separated proteins were
electroblotted onto nitrocellulose filters, which were incubated
overnight with 1.5 × 106 cpm of
125I-insulin or a mixture of 125I-IGF-I and
125I-IGF-II, washed, dried, and exposed to film (Biomax;
Eastman Kodak).
20,000 cpm of
125I-insulin were preincubated for 2 h at 4 °C with
unlabeled IGFBP-3, IGFBP-7, and insulin at concentrations indicated in
the figure and then incubated with 50 µg of microsomal placental crude membranes in 500 µl of binding buffer (50 mM
Tris-HCl, 0.5% BSA, pH 7.4) for 16 h at 4 °C. Samples were
centrifuged at 10,000 × g for 10 min at 4 °C. After
samples were centrifuged, pellets were counted in a Confluent NIH-3T3 cells
stably transfected with the human insulin receptor cDNA were
exposed for 3 min to 100 ng/ml insulin, which had been preincubated
with IGFBP-3, IGFBP-3(1-87), or IGFBP-7 for 2 h at
4 °C. The reaction was quenched by solubilization buffer (1%
Nonidet P-40, 20 mM Tris-HCl, pH 8.0, 1 mM
EDTA, 150 mM NaCl, 10% glycerol, 12 units/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride, and 1 mM
Na3VO4). Solubilized proteins were separated by
SDS-PAGE (8%) and visualized by immunoblot analysis. For immunoblot analysis, the filters were blocked in TBS-T containing 5% non-fat dry
milk for 1 h at room temperature and then incubated with
anti-phosphotyrosine monoclonal antibody (1.5 µg/ml) diluted by
Tris-buffered saline + 0.1% Triton X-100 overnight at 4 °C. The
filters were then rinsed and incubated in a 1:3000 dilution of goat
anti-mouse IgG-conjugated horseradish peroxidase (Amersham Corp.) for
1 h at room temperature. Immunoreactive proteins were visualized
using the ECL detection system. Alternatively, solubilized proteins
were immunoprecipitated with anti-IRS-1 antibody (Upstate
Biotechnology, Lake Placid, NY) and were subjected to immunoblot using
anti-phosphotyrosine antibody as above. In parallel, solubilized
proteins were subjected to immunoblot using anti-IRS-1 antibody.
Data were analyzed with a two-tailed
Student's t-test, using the software program Instat 2.01 (Graphpad, San Diego, CA). Values are expressed as means ± S.D.
RESULTS To evaluate the
affinity of IGFBPs 1-7 for IGFs and insulin, the affinity
cross-linking was performed with 125I-IGF-I + 125I-IGF-II or 125I-insulin. Affinity
cross-linking of IGFBP-7 with 125I-insulin shows that
IGFBP-7 binds insulin in a dose-dependent manner (Fig.
1A). As shown in Fig.
1B, top panel, the expected sizes of individual
IGFBPs coupled to 7 kDa 125I-IGF-I or
125I-IGF-II were detected on the SDS-PAGE gel. In a similar
way, Fig. 1B, bottom panel, showed
125I-insulin bound to IGFBPs 1-7 with proper size of
bands. This data demonstrates that IGFBPs 1-6 also bind insulin but
with significantly reduced affinity compared with their affinities for
IGFs. In contrast, the ratio of insulin:IGF binding for IGFBP-7 was
approximately 500-fold higher than for IGFBPs 1-6 by densitometric
analysis.
To establish the specificity of IGFBP binding to insulin, we attempted
to cross-link IGFBP-1, -3, and -7 with 125I-prolactin (Fig.
2B) and
125I-growth hormone (Fig. 2C), as well as with
125I-insulin (Fig. 2A). Although these IGFBPs
failed to bind the other proteins, binding of 125I-insulin
was confirmed. Furthermore, analysis of competitive affinity
cross-linking revealed that the relative affinities of IGFBP-7 are:
insulin Insulin binding to IGFBPs can be further demonstrated by
use of WLB. It appears that insulin binding to IGFBPs is much less effective in WLBs, especially in the absence of DTT compared with that
seen in solution binding, as assessed by affinity cross-linking (Fig.
1). This presumably reflects changes in the tertiary structure of
IGFBPs during SDS-PAGE. However, WLB demonstrated that reduction of
IGFBPs -1, -3, and -7 with DTT resulted in a marked decrease in
affinity for IGF but increased affinity for insulin (Fig.
4). These data indicate that disulfide
bonds and the tertiary structure of IGFBP molecules are, at least in
part, responsible for the relatively low affinity for insulin and
high affinity for IGF. To further test this hypothesis, we expressed
human IGFBP-3(1-87) in a baculovirus system, which
was epitope-tagged with the FLAG sequence at the COOH end (Fig.
5A). While the affinity for
IGF was greatly diminished in IGFBP-3(1-87), the affinity
for insulin was significantly increased in IGFBP-3(1-87),
compared with that of intact IGFBP-3 (Fig. 5, B and
C). Reduction of IGFBP-3(1-87) with DTT
resulted in a modest increase in affinity for insulin, suggesting that
disulfide bonds within the IGFBP-3(1-87) fragment still
limit, at least in part, the binding affinity for insulin. These
results suggest that disulfide bonds between the NH2 and
COOH termini result in a ternary structure for the IGFBPs that confers
high affinity for IGFs. To enhance the affinity for insulin, the
IGFBP-3 molecule must be modified by either disrupting the ternary
structure (under reducing conditions) or by eliminating or modifying
the COOH-terminal domain (IGFBP-3(1-87) or IGFBP-7). A
schematic model is depicted in Fig.
6.
[View Larger Version of this Image (52K GIF file)]
[View Larger Version of this Image (33K GIF file)]
[View Larger Version of this Image (33K GIF file)]
We next investigated the
biological relevance of IGFBP-7 binding to insulin. Insulin receptor
binding assays were performed using 125I-insulin and crude
human placental microsomal membranes in the presence or absence of
unlabeled insulin, IGFBP-3, or IGFBP-7. IGFBP-3, at concentrations as
high as 300 pmol, did not block insulin binding. The specific binding
of 125I-insulin to human placental insulin receptors was
inhibited, however, by IGFBP-7 (60% by 100 pmol, and 90% by 300 pmol)
(Fig. 7), indicating that IGFBP-7 can
compete with insulin receptors for binding of insulin. Inhibition of
insulin binding to placental membranes by IGFBP-7 was further confirmed
by affinity cross-linking studies (data not shown). Thus, 100-300 pmol
of IGFBP-7, but not IGFBP-3, inhibit insulin binding to placental
membranes. This discrepancy between IGFBP-3 and IGFBP-7 action
presumably reflects the higher affinity of the latter protein for
insulin, as suggested in Fig. 1. Although care must be exercised in
estimating affinities from cross-linking experiments, it appears likely
that IGFBP-7 binds insulin more effectively than does IGFBP-3 although
with lower affinity than that exhibited by the insulin receptor.
[View Larger Version of this Image (38K GIF file)]
The effect of IGFBPs on insulin receptor signaling pathways, such as
receptor autophosphorylation and IRS-1 phosphorylation, was tested.
IGFBP-3(1-87) and IGFBP-7 blunted insulin-stimulated
autophosphorylation of the insulin receptor
[View Larger Version of this Image (37K GIF file)]
DISCUSSION Current dogma proposes that a major difference between insulin and
IGFs is that only IGFs have the ability to bind IGFBPs 1-6 with high
affinity. In addition, we recently have shown that IGFBP-7 also binds
both IGF-I and IGF-II but with at least 5-25-fold lower affinity than
do the other IGFBPs. However, it has been shown that several IGFBPs may
play roles in regulating insulin action. IGFBP-1 production is
suppressed by insulin and carbohydrate and stimulated by hypoglycemia,
suggesting that IGFBP-1 might play a counterregulatory role in glucose
homeostasis. Proteolyzed IGFBP-3 fragments, but not intact IGFBP-3,
inhibited the mitogenic effect of insulin, suggesting that proteolyzed
IGFBP-3 fragments may have the ability to regulate insulin signaling
pathways, despite having significantly reduced affinity for IGFs. In
the present studies, we have tested the ability of IGFBPs to bind
insulin and regulate insulin action. We demonstrate that IGFBPs have
the ability to bind insulin, and that fragmented IGFBP-3 has increased affinity for insulin, resulting in the inhibition of insulin action. Our further studies have revealed that IGFBP-3 fragments, either generated by plasmin digestion or present in normal human urine, bind
insulin with relatively high affinity, supporting the physiological relevance of IGFBP-3 fragments on insulin
action.2
Thus, recent studies have demonstrated that the current view of IGFBPs
must be modified by the following observations: 1) IGFBP proteolysis
results in peptide fragments with decreased affinity for IGFs (15, 16);
2) additional related proteins with low affinity for IGFs indicate the
existence of an IGFBP superfamily (7, 9); 3) IGFBPs can bind insulin,
as well as IGFs, and the affinity for insulin can be increased by
modification of the protein by reduction of disulfide bonds or
proteolysis of the protein; and 4) IGFBPs can, under certain
conditions, inhibit insulin binding to its receptor and reduce the
resulting stimulation of receptor and IRS-1 phosphorylation.
Immunoblot studies with antibodies specific for IGFBP-7 have
demonstrated the presence of the mature protein in the conditioned media of a number of cell lines, as well as in cerebrospinal fluid, urine, amniotic fluid, and serum (31). Quantitative studies await the
development of radioimmunoassays. Proteolytic fragments of IGFBP-3, as
well as other IGFBPs, were originally identified in pregnancy serum but
subsequently have been found in a wide variety of clinical conditions,
including critically ill patients, patients with catabolic disease,
diabetes mellitus, or noninsulin-dependent diabetes
mellitus (15, 16, 33-38). Many of these diseases are characterized by
insulin resistance of varying degrees, as measured by decreased insulin
receptor tyrosine kinase activity and defective insulin receptor
signaling (39-42). It is tempting to speculate that this insulin
resistance might reflect increased concentrations of IGFBP-3 fragments
or IGFBPs with enhanced affinity for insulin, such as IGFBP-7. These
proteins may compete with insulin receptors for peptide binding, much
as IGFBPs sequester IGF peptides from IGF receptors. These observations
thus support a novel model for the interactions of the IGF and insulin
pathways and suggest a potential new approach to our understanding of
the pathophysiology of insulin resistance.
FOOTNOTES ACKNOWLEDGEMENTS We thank P. Rotwein, C. T. Roberts, Jr.,
and B. J. Druker for critical comments, and T. Oda for technical
advice.
REFERENCES
Inhibition of Insulin Receptor Activation by Insulin-like Growth
Factor Binding Proteins*
,
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
subunit and
phosphorylation of IRS-1, indicating that IGFBP-7 is a functional
insulin-binding protein. The affinity of other IGFBPs for insulin can
be enhanced by modifications that disrupt disulfide bonds or remove the
conserved COOH terminus. Like IGFBP-7, an
NH2-terminal fragment of IGFBP-3
(IGFBP-3(1-87)), also binds insulin with high affinity and
blocks insulin action. IGFBPs with enhanced affinity for insulin might
contribute to the insulin resistance of pregnancy, type II diabetes
mellitus, and other pathological conditions.
subunit and phosphorylation of IRS-1.
Fig. 1.
Affinity cross-linking of IGFBP with
125I-insulin or 125I-IGFs. A, 2-200
pmol of IGFBP-7 were incubated with 125I-insulin (20,000 cpm) for 16 h at 4 °C. At the end of incubation, disuccinimidyl
suberate was added to a final concentration of 0.5 mM.
After cross-linking for 15 min at 4 °C, the samples were subjected
to 12% SDS-PAGE and autoradiography. B, IGFBPs 1-7, at the
concentrations indicated in the figure, were incubated with a mixture
125I-IGF-I (10,000 cpm) and 125I-IGF-II (10,000 cpm) (top), or 125I-insulin (20,000 cpm)
(bottom) and cross-linked as in Fig. 1A.
Fig. 2.
Specificity of IGFBP binding to
125I-insulin. Autoradiogram of
125I-insulin (A), 125I-prolactin
(B), and 125I-labeled growth hormone
(C) cross-linked to 20 pmol of IGFBP-1, -3, or -7, as in
Fig. 1.
Fig. 3.
Competitive affinity cross-linking of IGFBP-7
and -3. A, 125I-insulin was cross-linked to 20 pmol of IGFBP-7 (top) or IGFBP-3 (bottom) alone
or in the presence of unlabeled IGF-I or insulin. B,
125I-IGF-II was cross-linked to 20 pmol of IGFBP-7
(top) or 1 pmol of IGFBP-3 (bottom) alone or in
the presence of unlabeled IGF-II, [QAYLL] IGF-II, or insulin.
Cross-linking was performed as in Fig. 1.
-counter.
IGFs [QAYLL] IGF-II (an IGF-II analog whose
affinity for IGFBPs 1-6 is normally 100-fold less than that of native
IGF-II). In contrast, affinities for IGFBP-3 are: IGFs 
insulin > [QAYLL] IGF-II (Fig. 3,
A and B). The binding of IGFBP-3 or -7 to
125I-IGFs or 125I-insulin was competitively
displaced by both IGFs and insulin, suggesting that the insulin binding
site may be at, or near, the IGF binding site. IGFBP-7 was found to
have similar affinity for IGFs and insulin (IC50 = 20-34
nM). Previous studies have suggested that IGF binding sites
reside on both the NH2- and COOH-terminal regions of the
IGFBP-3 molecule; each binding site is individually capable of
low-affinity binding, whereas high-affinity binding of IGFs requires
the presence of both binding domains. Taken together, our data suggest
that the low affinity of IGFBP-7 for IGFs and the relatively high
affinity for insulin, compared with those of IGFBPs 1-6, can be
attributed to the lack of conserved amino acid sequence and cysteines
at the COOH terminus of IGFBP-7 (only 1 of the 6 cysteines is
conserved) although IGFBP-7 retains 11 of the 12 cysteines found in the
IGFBPs at the NH2 terminus. High-affinity binding of IGFBPs
to IGFs thus appears to require proper structural configuration
involving both NH2- and COOH-terminal ligand-binding domains.
Fig. 4.
Western ligand blot of IGFBP-1, -3, and
-7. IGFBP-1, -3, and -7 at the concentrations indicated in the
figures with or without 100 mM DTT were processed by
SDS-PAGE. A, 12%; B and C,
15%.
Fig. 5.
Characterization of
IGFBP-3(1-87). A, the purified FLAG-tagged
IGFBP-3(1-87) was subjected to SDS-PAGE (15%) and then to
immunoblot with anti-M2 antibody. IGFBP-3 and
IGFBP-3(1-87) were subjected to SDS-PAGE with or without
DTT, and electroblotted onto filters, which were incubated with
125I-insulin (B) or 125I-IGFs
(C).
Fig. 6.
Schematic representation of IGFBP binding to
insulin and IGFs. [1], the conserved
NH2-terminal and COOH-terminal sequences and the
appropriate ternary structures formed by disulfide bonds result in high
affinity for IGFs but low affinity for insulin in IGFBPs 1-6.
[2], IGFBP-7 lacks the ternary structure necessary for
high affinity IGF binding but can still bind insulin; reduction of
disulfide bridges produces a structure that is further conducive to
insulin binding. [3, 4], when IGFBPs 1-6 are either
reduced or fragmented, the insulin binding site at the NH2
terminus of the IGFBP molecule is exposed, resulting in increased
affinity for insulin and diminished affinity for IGFs.
Fig. 7.
Insulin receptor binding. 20,000 cpm of
125I-insulin were preincubated for 2 h at 4 °C with
unlabeled IGFBP-3, IGFBP-7, and insulin at concentrations indicated in
the figure and then incubated with 50 µg of microsomal placental
crude membranes for 16 h at 4 °C. After samples were
centrifuged, pellets were counted in a -counter. Data corresponding
to means ± S.D. for three independent experiments are
shown.
subunit in a
dose-dependent manner, with 55 and 80% inhibition at
concentrations of 100 pmol of IGFBP-3(1-87) or IGFBP-7,
respectively (Fig. 8A).
IGFBP-3 was ineffective at these concentrations, as predicted by the
inability of IGFBP-3 to inhibit insulin binding to placental membranes.
Similarly, the insulin-stimulated phosphorylation of IRS-1 was
inhibited 65 and 85% by 100 pmol of IGFBP-3(1-87) or
IGFBP-7, respectively (Fig. 8B). These differences did not reflect reduced concentrations of IRS-1 among samples tested, as
demonstrated by IRS-1 immunoblots. Taken together, our data demonstrate
that IGFBP-7 and IGFBP-3(1-87) have the ability to bind
insulin and subsequently inhibit insulin binding to the insulin
receptor, resulting in the inhibition of insulin-stimulated
autophosphorylation of the insulin receptor subunit and
phosphorylation of IRS-1.
Fig. 8.
Tyrosine phosphorylation assay. A,
confluent NIH-3T3 cells stably transfected with the human insulin
receptor cDNA were exposed for 3 min to 100 ng/ml insulin, which
had been preincubated with IGFBP-3, IGFBP-3(1-87), or
IGFBP-7 for 2 h at 4 °C. After quenching, solubilized proteins were separated by SDS-PAGE (8%) and visualized by immunoblot analysis using an anti-phosphotyrosine monoclonal antibody. B,
alternatively, solubilized proteins were immunoprecipitated with
anti-IRS-1 antibody and were subjected to immunoblot using
anti-phosphotyrosine antibody. In parallel, solubilized proteins were
subjected to immunoblot using anti-IRS-1 antibody. Data corresponding
to means ± S.D. for three independent experiments are shown and
are depicted relative to maximum stimulation (100 ng/ml insulin), which
was assigned a value of 100%.
Present address: Dept. of Pediatrics, Okayama University Medical
School, 2-5-1 shikata-cho, Okayama 700, Japan.
§
To whom correspondence should be addressed: Dept. of Pediatrics,
School of Medicine, Oregon Health Sciences University, 3181 S. W. Sam Jackson Park Rd., NRC5, Portland, OR 97201. Tel.: 503-494-1930; Fax: 503-494-1933; E-mail: ohy{at}ohsu.edu.
1
The abbreviations used are: IGF, insulin-like
growth factor; IGFBP, IGF binding protein; IRS-1, insulin receptor
substrate-1; PAGE, polyacrylamide gel electrophoresis; m.o.i.,
multiplicity of infection; WLB, Western ligand blot; DTT,
dithiothreitol.
2
P. Vorwerk, Y. Yamanaka, G. Spagnoli, Y. Oh, and
R. G. Rosenfeld, submitted for publication.
Volume 272, Number 49,
Issue of December 5, 1997
pp. 30729-30734
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.
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 A. Lopez-Bermejo, J. Khosravi, C. L. Corless, R. G. Krishna, A. Diamandi, U. Bodani, E. M. Kofoed, D. L. Graham, V. Hwa, and R. G. Rosenfeld Generation of Anti-Insulin-Like Growth Factor-Binding Protein-Related Protein 1 (IGFBP-rP1/MAC25) Monoclonal Antibodies and Immunoassay: Quantification of IGFBP-rP1 in Human Serum and Distribution in Human Fluids and Tissues J. Clin. Endocrinol. Metab., July 1, 2003; 88(7): 3401 - 3408. [Abstract] [Full Text] [PDF]
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 P. Vorwerk, B. Hohmann, Y. Oh, R. G. Rosenfeld, and R. M. Shymko Binding Properties of Insulin-Like Growth Factor Binding Protein-3 (IGFBP-3), IGFBP-3 N- and C-Terminal Fragments, and Structurally Related Proteins mac25 and Connective Tissue Growth Factor Measured Using a Biosensor Endocrinology, May 1, 2002; 143(5): 1677 - 1685. [Abstract] [Full Text] [PDF]
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 P Vorwerk, H Wex, B Hohmann, K Mohnike, U Schmidt, and U Mittler Expression of components of the IGF signalling system in childhood acute lymphoblastic leukaemia Mol. Pathol., February 1, 2002; 55(1): 40 - 45. [Abstract] [Full Text] [PDF]
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 C. H. Lang, G. J. Nystrom, and R. A. Frost Burn-induced changes in IGF-I and IGF-binding proteins are partially glucocorticoid dependent Am J Physiol Regulatory Integrative Comp Physiol, January 1, 2002; 282(1): R207 - R215. [Abstract] [Full Text] [PDF]
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C. K. Buckway, E. M. Wilson, M. Ahlsen, P. Bang, Y. Oh, and R. G. Rosenfeld Mutation of Three Critical Amino Acids of the N-Terminal Domain of IGF-Binding Protein-3 Essential for High Affinity IGF Binding J. Clin. Endocrinol. Metab., October 1, 2001; 86(10): 4943 - 4950. [Abstract] [Full Text] [PDF]
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G. R. Devi, D.-H. Yang, R. G. Rosenfeld, and Y. Oh Differential Effects of Insulin-Like Growth Factor (IGF)-Binding Protein-3 and Its Proteolytic Fragments on Ligand Binding, Cell Surface Association, and IGF-I Receptor Signaling Endocrinology, November 1, 2000; 141(11): 4171 - 4179. [Abstract] [Full Text] [PDF]
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 R. C. Pereira, D. Durant, and E. Canalis Transcriptional regulation of connective tissue growth factor by cortisol in osteoblasts Am J Physiol Endocrinol Metab, September 1, 2000; 279(3): E570 - E576. [Abstract] [Full Text] [PDF]
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C. H. Lang, X. Liu, G. J. Nystrom, and R. A. Frost Acute response of IGF-I and IGF binding proteins induced by thermal injury Am J Physiol Endocrinol Metab, June 1, 2000; 278(6): E1087 - E1096. [Abstract] [Full Text] [PDF]
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J. C. Irwin, L.-F. Suen, B.-H. Cheng, R. Martin, P. Cannon, C. L. Deal, and L. C. Giudice Human Placental Trophoblasts Secrete a Disintegrin Metalloproteinase Very Similar to the Insulin-Like Growth Factor Binding Protein-3 Protease in Human Pregnancy Serum Endocrinology, February 1, 2000; 141(2): 666 - 674. [Abstract] [Full Text] [PDF]
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K. L. Haugk, H.-M. P. Wilson, K. Swisshelm, and L. S. Quinn Insulin-Like Growth Factor (IGF)-Binding Protein-Related Protein-1: An Autocrine/Paracrine Factor That Inhibits Skeletal Myoblast Differentiation but Permits Proliferation in Response to IGF Endocrinology, January 1, 2000; 141(1): 100 - 110. [Abstract] [Full Text] [PDF]
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V. Hwa, Y. Oh, and R. G. Rosenfeld The Insulin-Like Growth Factor-Binding Protein (IGFBP) Superfamily Endocr. Rev., December 1, 1999; 20(6): 761 - 787. [Abstract] [Full Text]
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 D. M. Sutkowski, R. L. Goode, J. Baniel, C. Teater, P. Cohen, A. M. McNulty, H. M. Hsiung, G. W. Becker, and B. L. Neubauer Growth Regulation of Prostatic Stromal Cells by Prostate-Specific Antigen J Natl Cancer Inst, October 6, 1999; 91(19): 1663 - 1669. [Abstract] [Full Text] [PDF]
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 R. G. Rosenfeld, V. Hwa, L. Wilson, A. Lopez-Bermejo, C. Buckway, C. Burren, W. K. Choi, G. Devi, A. Ingermann, D. Graham, et al. The Insulin-like Growth Factor Binding Protein Superfamily: New Perspectives Pediatrics, October 1, 1999; 104(4): 1018 - 1021. [Abstract] [Full Text]
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 S. Cianfarani, D. Germani, and F. Branca Low birthweight and adult insulin resistance: the "catch-up growth" hypothesis Arch. Dis. Child. Fetal Neonatal Ed., July 1, 1999; 81(1): 71F - 73. [Full Text]
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 A. Degeorges, F. Wang, H. F. Frierson Jr., A. Seth, L. W. K. Chung, and R. A. Sikes Human Prostate Cancer Expresses the Low Affinity Insulin-like Growth Factor Binding Protein IGFBP-rP1 Cancer Res., June 1, 1999; 59(12): 2787 - 2790. [Abstract] [Full Text] [PDF]
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R. C. Pereira and E. Canalis Parathyroid Hormone Increases mac25/Insulin-Like Growth Factor-Binding Protein-Related Protein-1 Expression in Cultured Osteoblasts Endocrinology, May 1, 1999; 140(5): 1998 - 2003. [Abstract] [Full Text]
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H. K. How, A. Yeoh, T. C. Quah, Y. Oh, R. G. Rosenfeld, and K.-O. Lee Insulin-Like Growth Factor Binding Proteins (IGFBPs) and IGFBP-Related Protein 1-Levels in Cerebrospinal Fluid of Children with Acute Lymphoblastic Leukemia J. Clin. Endocrinol. Metab., April 1, 1999; 84(4): 1283 - 1287. [Abstract] [Full Text]
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Y. Yamanaka, J. L. Fowlkes, E. M. Wilson, R. G. Rosenfeld, and Y. Oh Characterization of Insulin-Like Growth Factor Binding Protein-3 (IGFBP-3) Binding to Human Breast Cancer Cells: Kinetics of IGFBP-3 Binding and Identification of Receptor Binding Domain on the IGFBP-3 Molecule Endocrinology, March 1, 1999; 140(3): 1319 - 1328. [Abstract] [Full Text]
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R. C. Pereira, F. Blanquaert, and E. Canalis Cortisol Enhances the Expression of mac25/Insulin-Like Growth Factor-Binding Protein-Related Protein-1 in Cultured Osteoblasts Endocrinology, January 1, 1999; 140(1): 228 - 232. [Abstract] [Full Text]
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