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Department of Pediatrics, School of Medicine, Oregon Health Sciences University, Portland, Oregon 97201-3042
Address all correspondence and requests for reprints to: Gayathri R. Devi, Ph.D., AVI BioPharma, Inc., 4575 SW Research Way, Suite 200, Corvallis, Oregon 97333. E-mail: grdevi{at}avibio.com
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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IGFBP-3 may be posttranslationally modified by IGFBP-3 protease(s) present in biological fluids or culture media (plasmin, prostate-specific antigen (PSA), matrix metalloproteases) (9) and those whose activity has been demonstrated only in vitro like that of stromelysin 3, thrombin (10). Serum IGFBP proteases(s) have been detected in diabetes (11, 12), renal (13), pregnancy (14), malignancy (15, 16), and following traumatic conditions or invasive procedures, such as surgery. Cleavage sites in IGFBP-3 have been located at the beginning of the variable domain (residues 95–98), particularly residue 97, which is the cleavage site for PSA, plasmin, human serum, and thrombin and yields a fragment of approximately 16 kDa or 20 kDa (glycosylated IGFBP-3) (10). However, the COOH-terminal fragments, containing a highly basic heparin-binding domain, have only been detected in vitro by plasmin digestion of intact IGFBP-3 and these fragments seem to inhibit degradation of other binding proteins (17). It is recognized that IGFBP proteolysis also occurs in the normal state outside of the bloodstream (18, 19) and that, in the cell environment, it is an essential mechanism in regulating the bioavailability of IGF. Both intact IGFBP-3 and IGFBP-3 proteolytic fragments have been shown to be capable of blocking the mitogenic effect of IGFs (20). Whether these actions primarily represent IGF-dependent or IGF- independent remains to be determined.
Our laboratories have demonstrated that the NH2-terminal recombinant fragments of IGFBP-3 (1–87) and (1–97) retain the ability to bind IGF, albeit with substantially reduced affinity. Additionally, these fragments specifically bind insulin and modulate insulin binding to its receptor (21, 22). Based on these studies, it has been hypothesized that the conserved NH2- and COOH-terminal sequences, as well as the appropriate ternary structure formed by disulfide bonds in the six classical IGFBPs, are all required for high-affinity binding of IGFs. A recent study had indicated that a natural COOH-terminal fragment of human IGFBP-2 retained partial IGF-binding activity (23), and a COOH-terminal, 13-kDa IGFBP-5 fragment (isolated from hemofiltrate) showed similar results (24). However, there is limited information on the binding characteristics of the IGFBP-3 COOH-terminal domain and the resultant biological effects of proteolytic fragments containing either the NH2- or COOH-terminal residues.
In this study, we demonstrate the ability of COOHterminal fragments of IGFBP-3 to bind IGFs. The (98–264)IGFBP-3 fragment and the (1–97)NH2-terminal fragment are both characterized, furthermore, by the ability to bind insulin with low affinity, but with higher affinity than is the case for intact IGFBP-3. Additionally, we have examined the effect of intact IGFBP-3 and the IGFBP-3 fragments on IGF-I-stimulated autophosphorylation of the IGF-I receptor (IGFIR) ß-subunit and their ability to associate with the cell surface.
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Materials and Methods |
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Cell culture
NIH-3T3 cells overexpressing the human IGFIR were kindly gifted
from Dr. C. T. Roberts, Jr. (Department of Pediatrics, Oregon
Health Sciences University) and grown in DMEM with 10% FCS plus 500
µg/ml geneticin at 37 C with 5% CO2.
Generation and purification of recombinant IGFBP-3 proteolytic
fragments
(1–97)IGFBP-3 and (88–148)IGFBP-3
FLAG-epitope tagged fragments were generated and purified in
baculovirus and tested to be 99% pure, as described earlier (25, 26). The cDNAs for the COOH-terminal fragments
(98–264) and (184–264) were generated by PCR
amplification from the human IGFBP-3 cDNA and a FLAG epitope sequence
(DYKDDDDK), and a stop codon was added immediately following amino
acid 264. The signal peptide sequences of IGFBP-3 cDNA were ligated to
NH2-termini of IGFBP-3 fragments. After
sequencing, the 98–264 amplicon was then subcloned into the
baculovirus expression vector pFASTBAC1 (Life Technologies, Inc., Grand Island, NY) and transformed into DH10Bac
Escherichia coli cells. The amplified DNA was transfected
into Sf9 insect cells (ATCC, Manassas, VA), and
large-scale protein purification was begun by infecting the P2 virus
into 108 HI-5 insect cells
(Invitrogen, Carlsbad, CA), at a multiplicity of
infection of 3, at 27 C for 3 days. The media from the infected cells
were collected and concentrated, and the resultants were bound to an
anti-M2 antibody column overnight at 4 C, and the FLAG tagged
(98–264) protein was then eluted by using FLAG peptide
(0.5 µg/ml), as described earlier (27). The purified
protein was subjected to SDS-PAGE in a 15% gel and stained with
Coomasie blue. Further, the fragment was also identified by
immunoblotting with the M2 anti-FLAG antibody (Eastman Kodak Co., New Haven, CT) and anti-IGFBP-3 monoclonal antibody
(Diagnostic Systems Laboratories, Inc.). Eluted
fractions from an anti-M2 antibody column were pooled, concentrated,
and quantitated by comparison with known amounts of BSA and
IGFBP-3Escherichia coli after silver
staining.
The (184–264)IGFBP-3 amplicon, after sequencing, was subcloned in the C-terminal end of glutathione S-transferase (GST) in the plasmid pGEX4T and transformed into Escherichia coli cells. The culture was grown overnight in LB-ampicillin and induced with 2 mM IPTG, and the cell lysates of the (184–264) GST fusion protein were prepared. The lysates were incubated with GST Sepharose beads for 1 h at RT and then washed. Purity and concentration of the fragments were determined by comparison with known amounts of BSA standards after silver staining. Further, the purified protein was subjected to SDS-PAGE in a 15% gel and stained with Coomasie Blue and also transferred to nitrocellulose and identified by immunoblotting with M2 anti-FLAG antibody (1:3000 dilution).
Affinity cross-linking
Intact IGFBP-3 or the NH2- and
COOH-terminal fragments were incubated with
125I-IGF-I or 125I-insulin
(50,000 cpm), in the presence or absence of unlabeled ligand, in a
100-µl vol for 16 h at 4 C and then cross-linked with 0.5
mM disuccinimidyl suberate (DSS) for 15 min at 4 C. The
samples were then subjected to SDS-PAGE (12% or 15% gels) under
reducing conditions, and autoradiography on Biomax MS film
(Eastman Kodak Co.). Bands were quantified by densitometry
(Bio-Rad Laboratories, Inc.).
Western ligand blot analysis
Ligand blotting was performed as described by Hossenlopp at al
(28), with minor modifications. Briefly, samples of intact
IGFBP-3 (1–97), IGFBP-3, and
(98–264)IGFBP-3 fragments, at the concentrations indicated
in the figure legends, were subjected to SDS-PAGE (12% or 15% gel)
under reducing or nonreducing conditions, electroblotted onto
nitrocellulose filters, incubated with 1.5 x
106 cpm of 125I-insulin or
a mixture of 125I-IGF-I and
125I-IGF-II, washed, dried, and exposed to film
(Biomax).
Monolayer 125I-IGF-I affinity cross-linking
125I-IGF-I (100,000 cpm) was preincubated
in a microfuge tube for 2 h at 4 C, in the presence or absence of
cold IGF-I, intact IGFBP-3 (30 nM) (98–264),
IGFBP-3 (250 nM) (184–264), IGFBP-3 (250
nM), or (1–97)IGFBP-3 (250
nM), in binding buffer (50 mM HEPES, 150
mM NaCl, 0.5% BSA). Confluent NIH-3T3 cells stably
transfected with the human IGFIR cDNA (NIH-3T3-IGFIR cells) were
incubated in serum free medium overnight. The cells were washed once
with PBS. The cells were incubated with the
125I-IGF-I/IGFBP-3 combinations in triplicate
wells for 3 h at 15 C. The cells were then washed with PBS and
cross-linked with DSS for 15 min at 4 C, and the reaction was quenched
with 100 mM Tris/HCl. The cells were solubilized with
sample buffer. The covalent ligand-receptor
(125I-IGF-I-IGFIR) complex in the lysates was
resolved on a nonreducing 6% SDS-PAGE, followed by autoradiography.
Another set of the same samples of cell lysates was run on a 15%
SDS-PAGE under reducing conditions and immunoblotted with M2
anti-FLAG antibody or anti-IGFBP-3 monoclonal antibody, and the
cell-associated bands were detected with enhanced chemiluminescence
(Amersham Pharmacia Biotech).
Determination of cell-surface association of intact IGFBP-3 and the
fragments
Confluent monolayers of NIH-3T3-IGFIR cells were incubated in
serum free medium overnight. Intact IGFBP-3 (30 nM);
fragments 98–264 (250 nM), 184–264 (250 nM),
or 1–97 (250 nM) in the presence or absence of 100 µg/ml
heparin (Sigma), in binding buffer, was added to
the cells. In a similar experiment, cells were treated with heparin
(100 µg/ml) for 1 h, before addition of the peptides as listed
above. The treatments were carried out at 15 C for 3 h. The cells
were washed with PBS and cross-linked with DSS, as described above. The
solubilized cell lysates were then run on a 15% SDS-PAGE and
immunoblotted with anti-IGFBP-3 monoclonal antibody and detected with
enhanced chemiluminescence.
IGF-I-induced IGFIR autophosphorylation assay
Confluent monolayers of serum-starved NIH-3T3-IGFIR cells were
exposed for 5 min to 7 nM IGF-I, which had been
preincubated with/without intact IGFBP-3 (1–97), IGFBP-3, or (98–264)IGFBP-3 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 U/ml aprotinin, phenylmethylsulfonyl fluoride, and 1
mM Na3VO4].
Solubilized proteins (25 µl of the cell lysates) were separated by
SDS-PAGE (7.5%) under reducing conditions and visualized by immunoblot
analysis. For immunoblot analysis, the filters were blocked in
Tris-buffered saline (TBS) with 2% gelatin for 1 h at room
temperature and then incubated with antiphosphotyrosine monoclonal
antibody (1.5 µg/ml) diluted by TBS + 0.1% Triton X-100 (TBST) for
1 h at room temperature. The filters were then rinsed in 1 x
TBST and incubated in a 1:5000 dilution of goat antimouse
IgG-conjugated horseradish peroxidase (Amersham Pharmacia Biotech) for 1 h at room temperature. Immunoreactive
proteins were visualized using an enhanced chemiluminescence detection
system.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. The purified proteins were
immunoblotted with anti-FLAG M2 or anti-IGFBP-3 monoclonal antibody for
estimation of their molecular weights. Intact IGFBP-3 and all the
fragments were detectable by anti-FLAG M2 antibody under reducing (Fig. 1B) and nonreducing conditions. Dimerized forms of the proteins were
identified in anti-FLAG M2 immunoblots run under nonreducing conditions
(data not shown). Small discrepancies between the Mr
for intact IGFBP-3 (1–97), IGFBP-3
(98–264), IGFBP-3, and (88–148)IGFBP-3
proteins seen on the immunoblots relative to the predicted Mr, which is
purely based on amino acid composition of the proteins, may have arisen
because of N-linked glycosylation. There are three potential
N-glycosylation sites (Fig. 1A): Asn89,
Asn109, and Asn172, in
IGFBP-3 (29). The anti-IGFBP-3 monoclonal antibody
detected intact IGFBP-3 and (98–264)IGFBP-3 under both
nonreducing (Fig. 1B) and reducing conditions (data not shown). The
fragment 1–97 was detectable only under nonreducing conditions. The
184–264 and 88–148 fragments were not detected effectively with this
antibody.
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demonstrate
that 125I-IGF-I can be cross-linked to the
(1–97)IGFBP-3 and (98–264)IGFBP-3
fragments in a dose-dependent manner. Significant IGF cross-linking was
observed at 50 nM concentrations of
(1–97)IGFBP-3, which was completely saturated by 100
nM concentrations. In the case of
(98–264)IGFBP-3, a dosedependent increase in IGF
binding with increasing protein concentrations with saturation of
binding occurred by 500-nM concentration range. The
expected sizes of the individual proteolytic fragment coupled to 7-kDa
125I-IGF-I were detected, shown as 25- and 41-kDa
bands, respectively. A faint band at 39 kDa is potentially a dimerized
form of 1–97 fragment cross-linked to IGF-I.
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). Quantitative analysis of the radioactive
bands in these gels was done by densitometric analysis, and the data
were plotted, as a function of IGF-I or insulin concentration, to
construct 125I-IGF-I displacement curves (Fig. 2C). Calculation of the unlabeled IGF-I concentrations required to
achieve 50% displacement of binding (IC50)
indicated that (1–97)IGFBP-3 fragment had an
IC50 value of 0.2–0.3 µM. The
(98–264)IGFBP-3 fragment binding to
125I-IGF-I was completely displaced by 0.15
µM unlabeled IGF-I (Fig. 2B) and, using lower
concentrations of unlabeled IGF-I in similar experiments, revealed the
IC50 to be in the range of 0.1–0.15
µM (Fig. 2C). In addition, insulin was also able to
compete for 125I-IGF-I binding to the fragments,
especially to (1–97)IGFBP-3. The
IC50 values calculated for unlabeled insulin to
compete for 125I-IGF-I binding revealed that the
1–97 fragment had an IC50 value of about 0.3
µM insulin, which was at least 3-fold lower than that of
the 98–264 fragment (IC50 = 0.8–1
µM), as shown in Fig. 2C. In summary, the
(1–97)IGFBP-3 showed slightly lower binding affinity for
IGF-I, relative to (98–264)IGFBP-3, and
125I-IGF-I binding to the fragments was
equipotently displaced by insulin.
Further, the ability of the fragments to bind IGF-I and IGF-II was
assessed by Western ligand blotting. The data in Fig. 3 demonstrate that both the 1–97 and
98–264 fragments were able to bind to a mixture of IGF-I and IGF-II at
concentrations as low as 5–10 pmol. It is to be noted, however, that
the intensity of the major radiolabeled bands was much lower than that
observed with intact IGFBP-3 at similar concentrations. The 20-kDa
faint band observed in the case of (1–97)IGFBP-3 is
probably a different glycosylation product of the 16-kDa fragment.
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). Unlabeled insulin was able to
dose-dependently inhibit 125I-insulin binding to
both the NH2 and COOH-terminal fragments (Fig. 4B), although even higher concentrations of unlabeled insulin could not
completely displace 125I-insulin binding to the
(98–264)IGFBP-3, suggesting a slow dissociation rate.
Calculation of the unlabeled insulin concentrations required to achieve
IC50 indicated that the
(1–97)IGFBP-3 had an IC50 value
ranging between 0.3–0.4 µM, whereas nearly 1
µM unlabeled insulin was required to cause 50%
displacement of 125I-insulin binding to the
(98–264) fragment (Fig. 4c). IGF-I was also able to
compete for the 125I-insulin binding to the
fragments, although higher concentrations of IGF-I were required
in the case of (98–264)IGFBP-3. In summary, the
(1–97)IGFBP-3 showed significantly high affinity for
insulin, relative to the (98–264)IGFBP-3 fragment.
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IGFBP-3 and 1–97 fragment inhibit IGF-I interaction with the
IGFIR
To determine, whether the ability of intact IGFBP-3 and the amino-
and carboxyterminal fragments to bind IGFs in vitro lead to
sequestration of IGFs in vivo, a
125I-IGF-I monolayer affinity cross-linking assay
was done in the NIH-3T cells overexpressing the IGFIR (NIH-3T3-IGFIR).
The data in Fig. 5A shows that
125I-IGF-I specifically cross-links with the
IGFIR shown as a 230-kDa band under nonreducing conditions.
125I-IGF-I binding to IGFIR was completely
displaced by 100 nM unlabeled IGF-I. Further, the
IGF-I-IGFIR complex formation was completely inhibited by preincubation
of the iodinated IGF-I with unlabeled IGFBP-3 (30
nM) and about 90% inhibited by preincubating
with 250 nM concentration of (1–97)
NH2-terminal fragment. The cross-linked band was
not inhibited, however, by preincubation of the 125I-IGF-I
with 250 nM of (98–264)IGFBP-3 or the
(184–264)IGFBP-3 fragment.
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show that the carboxyterminal
fragments (98–264), (184–264), and intact
IGFBP-3 molecules associated with the cell surface in the presence of
IGF-I. (1–97)IGFBP-3, however, showed no cell-associated
band.
The carboxyterminal fragments have the ability to associate to the
cell surface
Since, compared with the NH2-terminal
fragment, the (98–264) COOH-terminal IGFBP-3 fragment
failed to inhibit binding of IGF-I to the IGFIR, we wanted to study the
ability of the fragments to associate with the cell surface in the
absence of IGF-I. Monolayer cross-linking was carried out with the
FLAG-epitope tagged intact IGFBP-3 (1–97) or
(98–264) fragments in NIH-3T3-IGFIR cells, and the
cell-associated proteins were detected by immunoblotting the cell
lysates with anti-IGFBP-3 monoclonal antibody. The
(98–264) carboxyterminal fragment and intact IGFBP-3
molecules associated with the cell surface.
(1–97)IGFBP-3, however, showed no cell-associated band
(Fig. 6, lanes 2 and 5). Further, there
was no detectable shift in molecular weights of the cross-linked
proteins when compared with control (Fig. 1B) noncross-linked protein
preparations.
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). Similar results were
observed in both types of experiments, i.e. heparin blocked
the cell surface association of intact IGFBP-3 (Fig. 6, lanes 3 and 4)
and the (98–264)IGFBP-3 fragment (Fig. 6, lanes 6 and
7).
Inhibition of IGFIR signaling
Since intact IGFBP-3 and its fragments have the ability to bind
IGFs and thereby impede its interaction with the IGFIR, we analyzed the
potential biological manifestation of this interaction on IGFIR
signaling. This was carried out by testing the effect of IGFBP-3 and
its fragments on IGF-I-induced IGFIR autophosphorylation in
NIH-3T3-IGFIR cells. Control experiments with IGF-I revealed that 5-min
treatments with 7–14 nM of the peptide showed maximal
intensity autophosphorylation of the 95-kDa band of the ß-subunit of
IGFIR in antiphosphotyrosine immunoblots (Fig. 7A).
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). Quantification of the
inhibition of the phosphorylated subunit of IGFIR was carried out by
densitometrically analyzing the specific 95-kDa band and the 116-kDa
nonspecific band in each gel. The ratio of the two band intensities was
used to normalize and calculate the percentage of maximal
IGF-I-stimulated IGFIR autophosphorylation detected in the
presence of IGFBP-3 and the IGFBP-3 fragments (Fig. 7C).
IGFBP-3 caused 50% inhibition of the IGF-I-induced
autophosphorylation at 5–7 nM concentration range, and by
15–20 nM IGFBP-3 concentrations, complete inhibition of
IGFIR autophosphorylation was observed. In contrast, the
(1–97)IGFBP-3 fragment inhibited receptor
autophosphorylation only at higher concentrations (50–70% inhibition
at 100–250 nM concentrations). The
(98–264)IGFBP-3 and (184–264)IGFBP-3
fragments, however, did not show any significant inhibition of
IGF-I-induced IGFIR autophosphorylation, even at 250 nM
concentrations (Fig. 7, B and C), although these fragments were able to
bind IGF-I in binding assays. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The concept that interaction between NH2- and COOH-terminal domains is essential for high-affinity IGF binding was initially conceived based on observations that proteolysis of IGFBPs in biological fluids results in fragments that have diminished or no binding affinities for IGFs (33). The in vitro generation of recombinant fragments or fragments isolated by limited proteolysis supports the in vivo data. A 16-kDa fragment corresponding to the NH2-terminus and a small portion of intermediate region, generated by proteolytically modifying IGFBP-4, specifically cross-linked to both IGF-I and II, although with a 20-fold lower affinity than intact IGFBP-4 (34, 35). Similarly, a carboxytruncated 23-kDa IGFBP-5 fragment from osteoblast-like cells demonstrated decreased IGF binding affinity (36, 37). Deletion mutagenesis of the carboxyterminal domains of IGFBP-1 and IGFBP-4 has resulted in a decrease in IGF affinity, thereby demonstrating the importance of the highly conserved Cys-Try-Cys-Val motif in the carboxyterminal region (38, 39). The present study is the first to clearly demonstrate the ability of the 28-kDa (98–264), IGFBP-3 intermediate+COOH-terminal proteolytic fragment to bind both IGFs and insulin in two different procedures, affinity cross-linking and Western ligand blot. Interestingly, the binding of the (98–264)IGFBP-3 fragment to 125I-IGF-I or 125I-insulin was competitively displaced by both IGF-I and insulin, though with different affinities, suggesting that the insulin and IGF binding sites are probably not identical but overlap or reside closely on the IGFBP-3 molecule. This is in contrast with the (1–97)IGFBP-3 fragment, where insulin and IGF-I were approximately equipotent in displacing 125I-IGF-I.
We have shown that IGFBP-3 causes a dose-dependent inhibition of IGF-I-induced IGFIR autophosphorylation in NIH-3T3 cells overexpressing the IGFIR. This inhibition occurs at an 1:1 molar ratio of IGFBP-3 to IGF-I, suggesting an IGF-dependent mechanism of modulation of receptor signaling. The (1–97) NH2-terminal fragment retained the ability to modulate IGF-I binding and signaling via the IGFIR by inhibiting IGF-I-stimulated IGFIR autophosphorylation, albeit at 50-fold higher concentrations than intact IGFBP-3. That this inhibition of IGFIR signaling is largely attributable to sequestration of IGF-I is strongly supported by the observations that both intact IGFBP-3 and (1–97)IGFBP-3 compete with 125I-IGF-I binding/cross-linking to the receptor in monolayer affinity cross-linking experiments.
Interestingly, the (98–264) fragment unlike the (1–97)IGFBP-3, failed to show any inhibition of IGFIR signaling, despite its ability to bind IGFs, as revealed by in vitro binding analysis. The COOH-terminal fragments (98–264) and (184–264) also failed to compete for 125I-IGF-I binding and cross-linking to the IGFIR, compared with intact IGFBP-3 and the (1–97) NH2-terminal IGFBP-3 fragment. We speculate that the inability of the fragments containing the COOH-terminal domain of IGFBP-3 to inhibit IGF-I binding to the IGFIR could be attributable to the following mechanisms: 1) the COOH-terminal fragment binds IGF-I, and the entire complex is still capable of binding to and autophosphorylating the IGFIR, implying that the binding site on IGF-I for the receptor and for the carboxyterminal region of IGFBP-3 are different; and 2) the COOH-terminal domain of IGFBP-3 possesses an extracellular matrix (ECM) binding region, and it is possible that in the cellular environment, the fragments containing the COOH-terminal domains are more prone to associate with the cell surface and are not available to sequester IGF-I, especially given their low affinity for IGF. To test these hypotheses, the ability of the FLAG-epitope tagged fragments to associate with the cell surface was studied in the presence of IGF-I, with subsequent cross-linking and by analysis of cell lysates on immunoblots probed with anti-IGFBP-3 or M2 anti-FLAG antibody. Our data indicate that the COOH-terminal fragments (98–264) and (184–264) have the ability to associate to the cell surface in the presence of IGF-I, unlike the NH2 fragment (1–97)IGFBP-3, which showed no cell-surface association. Further, there was no shift in molecular weight of the cell-surface associated bands, and heparin blocked the binding of both intact and the (98–264)IGFBP-3 fragment to the cell surface, ruling out the possibility of interaction of the fragments with any receptor molecule and thereby supporting the second hypothesis. This is in agreement with an earlier study (40), which reported that an IGFBP-3 deletion fragment, lacking the 184–264 region, failed to show any cell-surface association. There are two putative heparin-binding motifs in IGFBP-3, located at amino acids 148–153 and 219–226 in the central and carboxylterminal regions, respectively, and the carboxylterminal motif has been shown to have 4-fold higher affinity for heparin (41). Recently, Bramani et al., 1999 (42), have identified two nonbasic residues (Gly203 and Gln209) within the ECM binding region (201–218) in the carboxyterminal region of IGFBP-5, mutations of which cause 8- to10-fold reduction in affinity for human IGF-I. This region is a highly conserved domain in IGFBP-5, -3, and -6 and is known to contain the heparin-binding domain. The authors have proposed that the IGF-I and ECM binding sites partially overlap, and heparin binding to the basic amino acids might interfere with IGF-I interaction in vivo.
Previous studies with mini-receptor constructs and with isolated domains or proteolytic fragments of the IGF2R (43) urokinase receptor (44), GH receptor (45), talin (46), to name a few, have confirmed the involvement of two or more ligand contact regions. Similarly, in the case of IGFBP-3, it seems that the IGF- and insulin-binding domains are bipartite and possibly overlapping. In our biological system, the stoichiometry of IGFBP-3 binding to IGF-I seems to be 1:1. We postulate that both NH2- and COOH-terminal domains have residues that are capable of binding IGF-I and insulin with low affinity. However, there is simultaneous interaction of the two so-called half sites, in intact IGFBP-3, which creates a high-affinity IGF binding site on the molecule. Simultaneously, this interaction leads to a markedly reduced ability of intact IGFBP-3 to bind insulin, possibly because of masking of the residues that interact with insulin, as a result of tertiary conformational change (21). With respect to IGF binding to IGFBP-3, it is unclear whether the NH2- and COOH-terminal domains contribute equally. We predict the presence of functional residues in the NH2-terminus (1–97) and COOH-terminus (149–264) that confer high affinity by cooperative or conformational changes; the structural residues that mediate the necessary noncovalent interactions may reside in the NH2-, intermediate or COOH-terminus of the IGFBP-3 molecule. Alternatively, given the striking similarity of the NH2-terminal domains and the fact that this region is encoded by a single exon in all of the classical IGFBPs and the IGFBP-rPs (low-affinity IGF binders), it is possible that the NH2-terminus is the critical functional component involved in binding IGFs, and that conformational effects imposed on the NH2-terminus by the COOH-terminal domain are required for high-affinity binding.
In summary, the present study, along with previous work from our and other laboratories, clearly demonstrates the ability of the IGFBP-3 aminoterminal fragment to bind IGF and insulin and to inhibit IGFIR and insulin receptor autophosphorylation (21, 22), revealing that this 16-kDa fragment may be capable of both IGF-I-dependent and IGF-independent roles in modulating cell growth. However, the carboxylterminal fragments, which also have the ability to bind both IGF-I and insulin in vitro, fail to prevent binding of either IGF-I or insulin (data not shown) to their respective receptors, because of the tendency of these fragments for cell surface association via the heparin-binding domain. Also intriguing is the identification of a thyroglobulin-like motif in the COOH-terminal regions of IGFBP superfamily, which has also been found in the superfamily of protein inhibitors of cysteine proteinases (47, 48). Whether this highly conserved thyroglobulin type-I element indeed acts as an inhibitor of cysteine proteinases in these proteins, remains to be established. The intermediate region of IGFBP-3 does not seem to bind IGFs or insulin, and its role in high-affinity binding to IGFs is probably related to its ability to promote proper tertiary structure and optimal interactions between the amino- and carboxyterminal residues. Further, it has been demonstrated that the intermediate region of IGFBP-3 is involved in the specific interaction between IGFBP-3 and its putative cell-surface receptor (26). Taken together, it is tempting to speculate that various forms of IGFBP-3 fragments resulting from proteolysis by IGFBP-3 specific proteases will have different effects on the IGF-IGFIR axis, as well as potential IGF-independent actions.
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Acknowledgments |
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Footnotes |
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2 Present address: AVI BioPharma Inc., 4575 SW Research Way, Suite
200, Corvallis Oregon 97333.
3 Present address: Department of Surgery, Chonbuk National University
Medical School, Chonju, Korea.
Received March 28, 2000.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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S. S. Huang, F.-M. Tang, Y.-H. Huang, I-H. Liu, S.-C. Hsu, S.-T. Chen, and J. S. Huang Cloning, Expression, Characterization, and Role in Autocrine Cell Growth of Cell Surface Retention Sequence Binding Protein-1 J. Biol. Chem., October 31, 2003; 278(44): 43855 - 43869. [Abstract] [Full Text] [PDF]
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 L. D. Payet, X.-H. Wang, R. C. Baxter, and S. M. Firth Amino- and Carboxyl-Terminal Fragments of Insulin-Like Growth Factor (IGF) Binding Protein-3 Cooperate to Bind IGFs with High Affinity and Inhibit IGF Receptor Interactions Endocrinology, July 1, 2003; 144(7): 2797 - 2806. [Abstract] [Full Text] [PDF]
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J. A. Kricker, C. L. Towne, S. M. Firth, A. C. Herington, and Z. Upton Structural and Functional Evidence for the Interaction of Insulin-Like Growth Factors (IGFs) and IGF Binding Proteins with Vitronectin Endocrinology, July 1, 2003; 144(7): 2807 - 2815. [Abstract] [Full Text] [PDF]
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S. M. Firth and R. C. Baxter Cellular Actions of the Insulin-Like Growth Factor Binding Proteins Endocr. Rev., December 1, 2002; 23(6): 824 - 854. [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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 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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