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Department of Pediatrics, Oregon Health Sciences University (C.K.B., E.M.W., Y.O., R.G.R.), Portland, Oregon 97201; and Department of Woman and Child Health, Karolinska Institute and Hospital (M.A., P.B.), Stockholm, Sweden
Address all correspondence and requests for reprints to: Caroline K. Buckway, M.D., Department of Pediatrics, NRC-5, Oregon Health Sciences University, 3181 SW Sam Jackson Park Road, Portland, Oregon 97201. E-mail: buckwayc{at}ohsu.edu
Abstract
The N-terminal domain is conserved in all members of the IGF-binding protein superfamily. Most recently, studies have demonstrated the importance of an IGF-binding protein N-terminal hydrophobic pocket for IGF binding. To examine more critically the amino acids important for IGF binding within the full-length IGF-binding protein-3 protein while minimizing changes in the tertiary structure, we targeted residues I56, L80, and L81 within the proposed hydrophobic pocket for mutation. With a single change at these sites to the nonconserved glycine there was a notable decrease in binding. A greater reduction was seen when both L80 and L81 were substituted with glycine, and complete loss of affinity for IGF-I and IGF-II occurred when all three targeted amino acids were changed to glycine. Furthermore, the ability of the IGF-binding protein-3 mutants to inhibit IGF-I-stimulated phosphorylation of its receptor was a reflection of their affinity for IGF, with the lowest affinity mutants having the least inhibitory effect.
These studies, thus, support the hypothesis that an N-terminal hydrophobic pocket is the primary site of high affinity binding of IGF to IGF-binding protein-3. The mutants provide a tool for future studies directed at IGF-dependent and IGF-independent actions of IGF-binding protein-3.
OVER THE LAST decade, the role of the IGF system in growth has been extensively studied. IGF-I and -II are potent mitogens that are tightly regulated in vivo by the six IGF-binding proteins (IGFBPs), for which they have high affinity (1, 2). IGFBP-3 is the most abundant binding protein in human serum and the major carrier of IGF-I in the circulation (3). Although IGFBP-3 can attenuate the effects of IGF-I by sequestering it from its receptor (4), IGFBP-3 may also act independently of IGF-I via a putative receptor (5), inhibiting cellular proliferation (6, 7, 8, 9, 10, 11). Using synthetic IGFBP-3 fragments, Yamanaka et al. (12) presented evidence that the nonconserved midregion of IGFBP-3 most likely contains the IGFBP-3 receptor-binding site and, presumably, the ability to inhibit cellular growth in an IGF-independent manner.
Structural analysis of IGFBP-1 to -6 reveals similar modular construction, with the greatest similarity (58%) in the N-terminus, including 12 cysteines in IGFBP-1 to -5 and 10 cysteines in IGFBP-6 (13). The mid region is highly variable, but the C-terminus has 34% similarity, containing another cysteine-rich region. Much of our knowledge of the function of these regions has been learned through the examination of proteolytic and synthetic IGFBP fragments and through directed mutational analysis. Several laboratories have shown from fragment analysis that the N-terminus contains an IGF-binding site (14, 15, 16, 17, 18, 19, 20), but a few others have also suggested another binding region in the C-terminus (21, 22, 23).
The three-dimensional structures of IGF-I (24) and IGF-II (25) have been known since the early 1990s, but other than the regional domains very little is known of the three-dimensional structures of the IGFBPs, with the exception of recent work by Kalus et al. (26). These investigators proposed an N-terminal hydrophobic patch (amino acids V49, Y50, P62, and K68 to L74) in IGFBP-5 critical for the binding of IGF-II based on analysis of an IGFBP-5 fragment (A40 to I92) by solution nuclear magnetic resonance (NMR) spectroscopy. Recent work after this study validated this hypothesis in full-length IGFBP-5 and also the conserved region in IGFBP-3 by substituting five amino acids in this pocket with neutral or nonhydrophobic residues, thereby reducing IGF-I affinity by more than 1000-fold (27).
Preliminary evidence from our laboratory of analysis of small fragments of the IGFBP-3 N-terminus also led us to believe it to be the primary site of IGF binding. Based on the work by Kalus (26) as well as our preliminary IGFBP-3 deletional analysis, we targeted three amino acids within the hydrophobic pocket (I56, L80, and L81) likely to have significant effects on IGF affinity. We compared both conserved and nonconserved amino acid changes, mutating one, two, and three residues at a time. Our goal was to disrupt the binding site, but with the fewest number of residue alterations so that the least degree of disruption to the tertiary structure of the protein would occur. Our data support the existence of a high affinity hydrophobic IGF-binding site in the N-terminus of IGFBP-3, probably conserved throughout the IGFBPs, which, when disrupted, can affect the IGF-dependent biological functions of IGFBP-3.
Materials and Methods
Materials
[125I]IGF-I and -II were provided by Diagnostics Systems Laboratories, Inc. (Webster, TX). IGF-I and -II were purchased from Austral Biologicals (Santa Clara, CA). Reagents for SDS-PAGE were purchased from Bio-Rad Laboratories, Inc. (Hercules, CA). The BIAcore X instrument, sensor chip CM5 (research grade), HBS-EP buffer (0.01 M HEPES, 0.15 M NaCl, 3 mM EDTA, and 0.005% surfactant P20, pH 7.4), and the amine coupling kit containing N-hydroxysuccinimide, N-ethyl-N'-(3-diethylaminopropyl)carbodiimide, and ethanolamine hydrochloride were purchased from BIAcore AB (Uppsala, Sweden). For use in the BIAcore analysis, recombinant human IGF-I (rhIGF-I) was a gift from Genentech, Inc. (South San Francisco, CA), and rhIGF-II was a gift from Pharmacia Biotech (Uppsala, Sweden).
Cell culture
COS-7 cells were obtained from American Type Culture Collection (Manassas, VA) and grown in DMEM with 10% FCS at 37 C in 5% CO2. NIH-3T3 cells were a gift from Dr. C. T. Roberts, Jr. (Oregon Health Sciences University, Portland, OR), and were grown in DMEM with 10% FCS and 500 µg/ml G418 at 37 C in 5% CO2. All tissue culture media and components were purchased from Life Technologies, Inc. (Grand Island, NY), except FCS, which was obtained from HyClone Laboratories, Inc. (Logan, UT).
Generation, purification, and quantitation of recombinant IGFBP-3 deletion fragments, wild-type and mutants
The cDNAs of IGFBP-3-(1–46), -(1–75), -(1–80), and -(1–87) FLAG epitope-tagged fragments were generated by PCR amplification from the human IGFBP-3 cDNA and a C-terminal FLAG epitope sequence (DYKDDDDK). After sequencing sense and antisense strands, the fragments were subcloned into pGEX4T-1 (Amersham Pharmacia Biotech, Piscataway, NJ) and transformed into BL21DE3 Escherichia coli cells, cultured overnight in Luria-Bertoni broth/ampicillin, and induced with 2 mM isopropylthio-ß-D-galactoside. Cell lysates were then harvested, analyzed by SDS-PAGE staining with Coomassie blue and by Western immunoblot with M2 anti-FLAG antibody (Eastman Kodak Co., Rochester, NY).
The preparation of expression vector pBSSK:IGFBP-3, containing a full-length human IGFBP-3 cDNA with a C-terminal FLAG epitope sequence (DYKDDDDK), by PCR amplification has been described previously (28). Single stranded phagemid DNA was generated from pBSSK:IGFBP-3FLAG, and mutations were introduced using synthetic degenerate oligonucleotides as substrates for antisense DNA synthesis. The following complementary oligonucleotide was used to mutate I56 to V56 or G56 with an AclI site: agc gag ggc cag ccg tgc ggc rkc tac acc gaa cgt tgt ggc tcc ggc ctt cgc (r = a or c; k = t or g). The following complementary oligonucleotide was used to mutate L80 and L81 to V80 or G80 and/or V81 or G81 without a PstI site: gag gcg cga ccg ctg caa gcg skg skg gac ggc cgc ggg ctc tgc gt (s = c or g; k = t or g). Sense and antisense strands were sequenced, and preparations were subcloned into pCMV6 for transient transfections of COS-7 cells, into pGEX4T-1 for generation of E. coli glutathione-S-transferase (GST) fusion cell lysates as described above, and into pFASTBAC1 (Life Technologies, Inc., Grand Island, NY) for baculovirus-generated proteins as described below. The full-length IGFBP-3 triple G mutant was constructed by subcloning a G80G81 fragment into the pCMV6:G56 mutant with BamHI and BstAPI.
pFASTBAC1 preps were transformed into DH10Bac E. coli cells. The amplified DNA was transfected into Sf9 insect cells (American Type Culture Collection). HIGH-5 insect cells (Invitrogen, Carlsbad, CA) were infected with P2 virus. The media were harvested on the third day and incubated with an anti-M2 antibody affinity column overnight at 4 C. The FLAG-tagged protein was then eluted by using FLAG peptide as described previously (29). Eluted fractions were analyzed on 12% SDS-PAGE under nonreducing conditions, followed by staining with Coomassie blue. Fractions were pooled and quantitated by two methods: 1) comparison with known quantities of baculovirus IGFBP-3 by silver staining (Bio-Rad Laboratories, Inc.), and 2) IGFBP-3 immunoradiometric assay (IRMA; Diagnostics Systems Laboratories, Inc.).
Transient transfections of COS-7 cells
Cells were plated in six-well plates, grown to 50–70% confluence, and transfected with a 1:2 ratio of cDNA and Mirrus Transit LT-1 (PanVera, Madison, WI). Medium was changed to serum-free medium after 16 h, then collected 48 h later, and cellular debris was removed by centrifugation. COS-7 cells have some endogenous IGFBP-3 secretion, so the conditioned medium was immunoprecipitated with M2 anti-FLAG antibody to pull out only the IGFBP-3 that had resulted from transient transfections.
Western ligand blot analysis
Samples of E. coli-generated GST fusion cell lysates, purified baculovirus-expressed proteins, or conditioned medium at the concentrations indicated in the figure legends were mixed with Laemmli sample buffer without a reducing agent and heated at 95 C for 5 min, then electrophoresed on a 12% SDS gel and electroblotted onto nitrocellulose. For dot blots, 2.5 µl sample were dotted directly onto the nitrocellulose membrane. Membranes were then blocked for 1 h at 21 C in 1% BSA/TBS-T (Tris-buffered saline-0.1% Tween 20), then incubated at 4 C overnight with 1 x 106 cpm [125I]IGF-I, [125I]IGF-II, or a mixture of the two. The membranes were then washed, dried, and exposed to Biomax MS film (Eastman Kodak Co.). The same membranes were then probed with antibodies as described below. Bands were quantified using an image analyzer (GS-700) equipped with MultiAnalyst version 1.0.2 software (Bio-Rad Laboratories, Inc.).
Western immunoblot analysis
Samples of E. coli-generated GST fusion cell lysates, purified baculovirus-expressed proteins, whole cell lysates, or conditioned medium at the concentrations indicated in the figure legends were mixed with Laemmli sample buffer with or without a reducing agent and heated at 95 C for 5 min, then subjected to SDS-PAGE (8% or 12% gels) and electroblotted onto nitrocellulose membranes. For dot blots, 2.5 µl sample were dotted directly onto the nitrocellulose membrane. The membranes were then blocked for 1 h at 21 C in 4% milk/TBS-T, followed by overnight incubation at 4 C with anti-IGFBP-3 monoclonal antibody (Diagnostics Systems Laboratories, Inc.), anti-GST polyclonal antibody (Amersham Pharmacia Biotech, Piscataway, NJ), anti-PY20 monoclonal antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), or antiinsulin receptor substrate-1 (anti-IRS-1) polyclonal antibody (Upstate Biotechnology, Inc., Lake Placid, NY), all at 1:3000 dilutions. Membranes were washed with TBS-T and incubated for 1 h at 21 C with a 1:3000 dilution of horseradish peroxidase-linked antirabbit or antimouse IgG secondary antibodies (Amersham Pharmacia Biotech, Piscataway, NJ). Proteins were detected by enhanced chemiluminescence reagents, according to the manufacturer’s protocol (NEN Life Science Products, Boston, MA).
Affinity cross-linking
E. coli-generated GST fusion cell lysates of full-length IGFBP-3 or mutants were incubated with 50,000 cpm [125I]IGF-I in the presence or absence of unlabeled IGF-I (100 nM) overnight at 4 C and then cross-linked with 0.5 mM disuccinimidyl suberate for 15 min at 4 C. The samples were quenched with 100 mM Tris, pH 7.4, and were then subjected to 12% SDS-PAGE and autoradiography on Biomax MS film (Eastman Kodak Co.).
Solution binding assay
Increasing amounts (0–100 ng/ml) of purified baculovirus
IGFBP-3 or mutant proteins in duplicate were incubated in 500 µl
buffer (50 mM Tris, pH 7.4, and 0.5% BSA) with 10,000 cpm
[125I]IGF-I at 4 C overnight. One milliliter of
activated charcoal solution (0.5% activated charcoal, 0.2 mg/ml
protamine sulfate, and 1% BSA in PBS) was added for 10 min and then
centrifuged for 10 min at 4000 rpm at 4 C to separate bound and free
IGF-I. A 
-counter was used to measure the radioactivity of the
supernatants.
BIAcore analysis
Equal volumes of N-hydroxysuccinimide and N-ethyl-N'-(3-diethylaminopropyl)carbodiimide were mixed, and 35 µl of the mixture were injected over the surface of the sensor chip to activate the carboxymethylated dextran. Eighty-three microliters of purified baculovirus-generated wild-type or mutant IGFBP-3 solution (15 µg/ml in 10 mM sodium acetate, pH 4.5) were injected over the activated surface, followed by 35 µl ethanolamine to deactivate remaining active carboxyl groups. The immobilization procedure was carried out at 25 C at a constant flow rate of 5 µl/min. The first of the two flow cells of each chip was used as an in-line blank reference cell. The carboxymethylated dextran in the reference cell was activated and deactivated as described above, but without any ligand bound. All experiments were carried out at 25 C at a constant flow rate of 10 µl/min HBS-EP buffer. Thirty-five microliters of the analyte (IGF-I or IGF-II) diluted in HBS-EP buffer were injected over the immobilized wild-type or mutant IGFBP-3, followed by a 5-min period when buffer was passed over the surface. Six concentrations of IGF-I and IGF-II were passed over each chip (3.13, 6.25, 12.5, 25, 50, and 100 nM). All kinetic assays were followed by an injection of 15 µl 0.1 M HCl to dissociate the remaining ligand from the binding protein. The experiment was performed a total of three times. BIAevaluation 3.0 software and SigmaStat were used for data analysis, and a 1:1 mass transfer curve-fitting model was used in the evaluation.
IGF-I-induced IGF-I receptor autophosphorylation assay
Confluent monolayers of NIH-3T3 IGF-I receptor cells were incubated in serum-free medium overnight. Purified baculovirus IGFBP-3 and mutant proteins (250 ng) were incubated with IGF-I (100 ng) in 1 ml DMEM and 0.05% BSA for 30 min at 21 C and then added to the cells for 10 min. The reaction was quenched with solubilization buffer [50 mM Tris (pH 7.5), 2.5 mg/ml sodium deoxycholate, 150 mM sodium chloride, 1 mM sodium orthovanadate, 20 mM sodium fluoride, and 1% Nonidet P-40]. Samples were normalized for protein concentration using a DC protein assay (Bio-Rad Laboratories, Inc.) and were separated on 8% SDS-PAGE under reducing conditions. Anti-PY20 or anti-IRS-1 antibodies were used for Western immunoblot.
Results
Expression and analysis of E. coli-generated GST fusion IGFBP-3 deletion fragments
Fragments of the N-terminus of IGFBP-3 were constructed by PCR
amplification and expressed as GST fusion cell lysates in E.
coli. Amplification of the peptides was confirmed by SDS-PAGE and
Coomassie staining. Binding to [125I]IGF-I was
screened by dot-blot analysis (Fig. 1
, upper panel) as explained in Materials and
Methods. Binding was strongly detectable for both full-length
IGFBP-3 and for IGFBP-3-(1–87) GST fusion fragment. No binding was
detectable for the smaller fragments (1–80, 1–75, and 1–46).
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The studies with deletion fragments indicated that N-terminal
residues were crucial to the binding of IGF, consistent with our prior
studies and with the NMR solution binding, which predicted a binding
pocket in the N-terminus of IGFBP-5. In IGFBP-3, therefore, we
mutated three of the amino acids (I56, L80, and L81) in this region in
various combinations either to a conserved residue, valine, or to a
nonconserved residue, glycine (Fig. 2).
Initial binding studies were performed by dot-blot analysis (Fig. 3A, upper panel) of E.
coli GST fusion proteins as described above and confirmed by
Western ligand blots on 12% SDS-PAGE (Fig. 3B, upper
panel). Both methods showed minimal change in binding when the
large, nonpolar, hydrophobic residue, valine, was substituted for I56
or for L80L81. However, a clear reduction in binding was observed with
substitution of I56 or L80L81 by glycine, a small polar amino acid.
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). The binding of
[125I]IGF-I to the G56 mutant was specific, as
it could be competed with unlabeled IGF-I.
To examine any differences between E. coli and mammalian
expressed proteins, we tested conditioned medium collected from COS-7
cells that had been transiently transfected with the cDNAs of our
glycine mutants. The medium was immunoprecipitated with the M2
anti-FLAG antibody before analysis to remove any endogenous IGFBP-3
that may have been produced by the COS-7 cells. On Western ligand blot,
reductions in binding were evident for all of the glycine-substituted
mutants, with G81 the least affected and with IGF binding by G80G81
(double G) and G56G80G81 (triple G) abolished (Fig. 4).
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Based upon the results of the above-described screening binding studies, we chose to produce the following four FLAG-tagged, mutant human IGFBP-3 proteins in a baculovirus system: G56, G80, G80G81 (double G), and G56G80G81 (triple G). The proteins were purified over an M2 FLAG antibody affinity column. The purity of the pooled fractions was verified by silver staining of protein subjected to SDS-PAGE and was quantitated relative to known quantities of baculovirus-generated human IGFBP-3. Quantitation was confirmed subsequently by IRMAs. The mutations introduced did not interfere with the ability of the peptide to be recognized by the anti-IGFBP-3 antibodies used in the assay, and parallel curves of the mutants were generated with each assay run, suggesting minimal disruption of the tertiary structure and the epitopes recognized by the antibodies.
When quantified by densitometry (Fig. 5, graph), bands detected by Western ligand analysis (Fig. 5, upper
panel) showed a 60% reduction in binding of IGF-I for G56, a 70%
reduction for G80, and a reduction to background level for the double G
and triple G mutants. No binding was seen for the double G or triple G
mutants, even with exposures of up to 1 wk. Similar levels of binding
were seen when the ligand used was IGF-II (data not shown).
Verification of the quantity of protein loaded (50 ng/lane) was made by
immunoblot with anti-IGFBP-3 monoclonal antibody (Fig. 5, lower
panel). Similar quantitation results for mutant proteins were
observed on immunoblots using an anti-IGFBP-3 polyclonal antibody
produced in our laboratory (data not shown).
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).
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. By this
methodology, wild-type IGFBP-3 had a Kd of
0.79 x 10-9 for IGF-I and 0.69 x
10-9 for IGF-II. The G56 mutant had a 1.7-fold
lower affinity for IGF-I, whereas the G80 mutant had a 4.3-fold lower
affinity for IGF-I, with statistically significant increases in the
dissociation rates; both single mutants preserved normal affinity for
IGF-II. The double G mutant had only minimal binding to IGF-I and -II,
at least 3–4 orders of magnitude lower affinity. The triple G mutant
had no detectable binding of either IGF-I or IGF-II.
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IGFBP-3 is known to inhibit IGF-I-stimulated phosphorylation of
the type I IGF receptor (IGF-IR) when added to culture medium
(4). As the mutants have varying affinities for IGF, we
hypothesized they may well have different abilities to modulate this
IGF-dependent action. Tyrosine phosphorylation of the ß-subunit of
the IGF-IR was seen when COS-7 cells were treated with 100 ng/ml IGF-I,
but was blocked when IGF-I and 250 ng/ml wild-type IGFBP-3 were
preincubated and then used to treat the cells. Likewise, the G56 mutant
still bound IGF-I with sufficient affinity to inhibit phosphorylation.
Less inhibition of IGF-I receptor phosphorylation was seen with the
other three mutants relative to the degree of loss of IGF affinity;
using the triple G mutant, no significant inhibition was observed (Fig. 7).
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Several studies of fragments of IGFBP-3 have provided evidence that IGF binds to both the N-terminus and the C-terminus, but there are no definitive three-dimensional structural analyses of the IGFBPs, including their interactions with the IGFs. The IGF affinity for the N-terminus is clearly less than that of intact IGFBP-3, but is present, as shown in our laboratory, both for 1–87 and 1–97 fragments (20, 30) and for the C-terminal 98–264 IGFBP-3 fragment (23). In general, most investigators agree that both the N-terminus and the C-terminus of the IGFBPs are required to be in the appropriate conformation, stabilized by disulfide bonds, for high affinity IGF binding to occur.
Previous work by Kalus et al. (26) predicted a hydrophobic patch on the N-terminus of IGFBP-5 (residues V49, Y50, P62, and K68 to L74) to be the primary IGF-binding site, based upon studies with solution NMR spectroscopy, using a fragment of IGFBP-5 and IGF-II as the ligand. This work was validated by the finding of a full-length IGFBP-5 mutant with five amino acids changed within this pocket and with reduced affinity for IGF (27); similar results were shown for a full-length IGFBP-3 mutant with the same five residues mutated. Our studies have more definitively defined the crucial amino acids and substantiate the theory of a primary N-terminal IGF-binding site within the IGFBPs, but do not address the necessity of the C-terminus. Based upon N-terminal conservation among the six high affinity binding proteins, we chose to examine three of the amino acids of IGFBP-3 (I56, L80, and L81) corresponding to V49, L73, and L74 within the hydrophobic patch of IGFBP-5 proposed by Kalus et al. (26).
The choice of these mutations was based on several factors. First, the dot-blot screen of the smaller N-terminal fragments showed binding to the IGFBP-3-(1–87) fragment, but not to the IGFBP-3-(1–80) fragment. Deletion of residues 81–87 thus appeared to disrupt a critical region for IGF binding, and amino acid 81 is one of the proposed residues within the hydrophobic pocket. Second, several other reports suggest that this region of the N-terminus has significance. For example, Hashimoto et al. (19) found amino acids E52 to A92 of IGFBP-3 important for binding to IGF-II. In IGFBP4, L72 to S91 were essential for binding in studies by Qin et al. (31), whereas residues C205 to V214 were facilitators of binding rather than primary sites. Third, on the IGFBP-5 fragment described by Kalus et al. (26), side-chains extend into solution from residues V49, L70, and L74, creating a hydrophobic surface corresponding to I56, L77, and L81 in IGFBP-3. In addition, IGFBP-5 residues V49, L70, L73, and L74 had several specific intermolecular interactions with IGF-II by NMR spectra. These studies provided a rationale for speculation concerning which amino acids within the N-terminus have singularly or in combination the greatest effect on IGF affinity without altering the disulfide bonds of these cysteine-rich binding proteins.
Our initial screening studies support the importance of hydrophobic residues for IGF affinity. Valine is a large nonpolar amino acid, as are isoleucine and leucine. When valine was substituted for either isoleucine or leucine, it did not dramatically affect binding. However, when glycine, a small polar residue, was used instead of valine, it produced very different results, with more marked reductions in binding, suggesting that a change from hydrophobic to nonhydrophobic residues was more relevant than the amino acid itself. Binding studies using purified baculovirus-generated protein showed that the substitution of glycine for leucine at position 80 led to the single greatest reduction in affinity and, furthermore, only two mutations (double G mutant) were necessary to reduce binding markedly to both IGF-I and -II by Western ligand blot. On more sensitive solution binding assays, binding was still not detected for the double G or the triple G mutant. BIAcore analysis further validated these findings and, more specifically, provided kinetics data supporting a loss of affinity by the mutants. The lower affinities of the G56 and G80 mutants for IGF-I were statistically significant for increased rates of dissociation, indicating that although IGF-I is able to bind these mutants, the complexes are possibly not as stable as wild-type IGFBP-3. Interestingly, IGF-II binding was not affected for the single mutations. However, for both IGF-I and -II there was absolutely no binding detectable for the triple G mutant and only very minimal binding found for the double G mutant.
In the absence of x-ray crystallography or two-dimensional NMR spectroscopy, it is impossible to exclude the possibility that the observed loss of binding may be secondary to an alteration of the tertiary structure of the protein. However, there are several reasons to believe the disruption of structure to be minimal. Throughout our studies the mutant proteins were easily detectable on immunoblot by both a rabbit anti-IGFBP-3 polyclonal antibody and a monoclonal anti-IGFBP-3 antibody, suggesting that no disruption of the epitopes recognized by these antibodies occurred. Similarly, the quantity of the mutants was accurately measured by an IRMA that used a goat anti-IGFBP-3 polyclonal antibody, and with each assay run, curves of dilutions of mutants and native IGFBP-3 remained parallel. Furthermore, the IGFBP-3 and -5 mutants constructed by Imai et al. (27) that substituted five amino acids with neutral or nonhydrophobic ones both had similar decreases in affinity. They were able to show that IGF-I could not inhibit proteolysis in the IGFBP-5 mutant, whereas it did so in the native form, suggesting that the five-amino acid change did not affect the proteolytic cleavage sites, for which accurate conformation of the protein would be crucial.
Functionally, our results also support the conclusion that IGF-dependent actions of IGFBP-3 are via the sequestration of IGF, thereby inhibiting stimulation of the receptor. This appears to be primarily a function of the N-terminus, rather than the mid region or C-terminus, as previously shown using proteolytic fragments (23). Moreover, our data relate the degree of affinity for IGF with the degree of inhibition of phosphorylation. The lower the affinity a mutant IGFBP-3 has for IGF, the more IGF is able to access the receptor and activate phosphorylation. Very recent studies (32) have suggested that an IGF-independent pathway may also exist in MCF-7 breast cancer cells in which IGFBP-3 inhibits type I IGF-R phosphorylation. Increasing amounts of IGFBP-3, -1, and -5 were used to treat the cells, followed by stimulation with wild-type and mutant forms of IGF-I that have less affinity for the IGFBPs. Only IGFBP-3 inhibited phosphorylation of the receptor by all types of IGF-I tested. In the future similar studies using mutant IGFBP-3 should prove insightful regarding mechanisms and function for both IGF-dependent actions and IGF-independent actions of IGFBP-3.
In conclusion, these data serve to clarify the importance of three amino acids within the N-terminus of IGFBP-3 for IGF affinity and delineate the individual and additive effects of these residues. These results further support the existence of an N-terminal hydrophobic patch as the primary binding site of IGF to IGFBP-3 and suggest that this is similar in the other high affinity binding proteins, given the significant conservation of amino acids within this region.
Acknowledgments
We acknowledge the assistance of Donna Graham and Allison Watts in producing the purified baculovirus proteins, Katherine Pratt for performing the IGFBP-3 IRMAs, Eric Kofoed for technical assistance with the phosphorylation studies, Christine Carlsson-Skwirut for assistance with the BIAcore studies, and Dr. Vivian Hwa for helpful discussion.
Footnotes
This work was supported by NIH grants CA-58110 and DK-51513 (to R.G.R.) and 5T32-HD-07497 (to C.K.B.), Department of Defense Grants 17-96-1-6304 and 17-97-1-7204 (to Y.O.), and grants from the Swedish Medical Research Council (no. 11364), the Karolinska Institute Research Foundation, the Swedish Society of Medicine, the Wera Ekström’s Foundation, the Swedish Freemason’s Foundation, the Märta and Gunnar V. Philipson’s Foundation, the Milk Drop Foundation in Stockholm, and HRH Crown Princess Lovisa’s Society of Pediatric Health Care (to P.B.).
Abbreviations: GST, Glutathione-S-transferase; HBS-EP buffer, 0.01 M HEPES, 0.15 M NaCl, 3 mM EDTA, and 0.005% surfactant P20, pH 7.4; IGFBP, IGF-binding protein; IRMA, immunoradiometric assay; IRS-1, insulin receptor substrate-1; NMR, nuclear magnetic resonance; rhIGF, recombinant human IGF; TBS-T, Tris-buffered saline-0.1% Tween 20.
Received February 5, 2001.
Accepted July 2, 2001.
References
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 B. Liu, K.-W. Lee, H. Li, L. Ma, G. L. Lin, R. A.S. Chandraratna, and P. Cohen Combination Therapy of Insulin-Like Growth Factor Binding Protein-3 and Retinoid X Receptor Ligands Synergize on Prostate Cancer Cell Apoptosis In vitro and In vivo Clin. Cancer Res., July 1, 2005; 11(13): 4851 - 4856. [Abstract] [Full Text] [PDF]
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 L O'Rear, L Longobardi, M Torello, B K Law, H L Moses, F Chiarelli, and A Spagnoli Signaling cross-talk between IGF-binding protein-3 and transforming growth factor-{beta} in mesenchymal chondroprogenitor cell growth J. Mol. Endocrinol., June 1, 2005; 34(3): 723 - 737. [Abstract] [Full Text] [PDF]
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 L. M. Morimoto, P. A. Newcomb, E. White, J. Bigler, and J. D. Potter Variation in Plasma Insulin-Like Growth Factor-1 and Insulin-Like Growth Factor Binding Protein-3: Genetic Factors Cancer Epidemiol. Biomarkers Prev., June 1, 2005; 14(6): 1394 - 1401. [Abstract] [Full Text] [PDF]
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L. M. Morimoto, P. A. Newcomb, E. White, J. Bigler, and J. D. Potter Insulin-like Growth Factor Polymorphisms and Colorectal Cancer Risk Cancer Epidemiol. Biomarkers Prev., May 1, 2005; 14(5): 1204 - 1211. [Abstract] [Full Text] [PDF]
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J. V. Silha, Y. Gui, S. Mishra, A. Leckstrom, P. Cohen, and L. J. Murphy Overexpression of Gly56/Gly80/Gly81-Mutant Insulin-Like Growth Factor-Binding Protein-3 in Transgenic Mice Endocrinology, March 1, 2005; 146(3): 1523 - 1531. [Abstract] [Full Text] [PDF]
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X. Yan, B. E. Forbes, K. A. McNeil, R. C. Baxter, and S. M. Firth Role of N- and C-terminal Residues of Insulin-like Growth Factor (IGF)-binding Protein-3 in Regulating IGF Complex Formation and Receptor Activation J. Biol. Chem., December 17, 2004; 279(51): 53232 - 53240. [Abstract] [Full Text] [PDF]
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 H.-S. Kim, A. R. Ingermann, J. Tsubaki, S. M. Twigg, G. E. Walker, and Y. Oh Insulin-Like Growth Factor-Binding Protein 3 Induces Caspase-Dependent Apoptosis through a Death Receptor-Mediated Pathway in MCF-7 Human Breast Cancer Cells Cancer Res., March 15, 2004; 64(6): 2229 - 2237. [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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L. Longobardi, M. Torello, C. Buckway, L. O'Rear, W. A. Horton, V. Hwa, C. T. Roberts Jr., F. Chiarelli, R. G. Rosenfeld, and A. Spagnoli A Novel Insulin-Like Growth Factor (IGF)-Independent Role for IGF Binding Protein-3 in Mesenchymal Chondroprogenitor Cell Apoptosis Endocrinology, May 1, 2003; 144(5): 1695 - 1702. [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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A. Spagnoli, M. Torello, S. R. Nagalla, W. A. Horton, P. Pattee, V. Hwa, F. Chiarelli, C. T. Roberts Jr., and R. G. Rosenfeld Identification of STAT-1 as a Molecular Target of IGFBP-3 in the Process of Chondrogenesis J. Biol. Chem., May 17, 2002; 277(21): 18860 - 18867. [Abstract] [Full Text] [PDF]
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