INTRODUCTION

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The insulin-like growth factor-binding proteins (IGFBPs)¹ are a family of at least six related proteins involved in regulating the bioavailability of insulin-like growth factors, IGF-I and IGF-II. The IGFBP structure may be divided into cysteine-rich amino- and carboxyl-terminal domains, which show considerable structural conservation among IGFBP-1 to -6, and a central domain that is unique for each IGFBP. IGFBP-3, a glycoprotein of 40-45 kDa, is characterized by its ability to bind to another glycoprotein, the 85-kDa acid-labile subunit (ALS), in the presence of either IGF-I or IGF-II to form a ternary complex of 150 kDa (1). Because the majority of serum IGFs are bound in this form (2, 3), the ternary complex acts essentially as a circulating reservoir of IGFs and regulates the delivery of the IGFs to target tissues.

IGFBP-3 has been implicated at the cellular level as a modulator of IGF-I action (4, 5). Soluble IGFBP-3 can inhibit IGF-I activity by sequestering the peptide and consequently preventing interaction with its receptor. In contrast, potentiation of IGF-I action has been attributed to cell surface-associated IGFBP-3 (5). Several studies have also suggested that IGFBP-3 may modulate cell growth independently of IGFs (6-8). This IGF-independent growth-inhibitory effect was recently shown to be mediated by the direct induction of apoptosis by IGFBP-3 (9).

This multifaceted role of IGFBP-3 has led to extensive studies on its regulation, expression, distribution, and function in cultured cells and in animal models (3, 10-12). To date however, there have been few studies aimed at elucidating the structural determinants involved in the protein-protein and protein-cell interactions required for IGFBP-3 function. In this study we describe the generation and expression of cDNAs encoding the natural form, three deletion mutants, and two site-specific mutants of human IGFBP-3. The resulting recombinant proteins have allowed the delineation of domains involved in IGF-I and ALS binding and in cell surface association.

	MATERIALS AND METHODS

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Reagents-- All radiolabeled proteins used were prepared as described previously (13, 14). Restriction enzymes were from Promega Corp. (Madison, WI). T7 DNA polymerase was from Pharmacia Biotech Inc. (Uppsala, Sweden). Pfu DNA polymerase was from Stratagene (La Jolla, CA). Hexadimethrine bromide (Polybrene), dexamethasone, hypoxanthine, xanthine, thymidine, and mycophenolic acid were purchased from Sigma Chemical Co. (St. Louis, MO) and aminopterin from Life Technologies Inc. (Gaithersburg, MD). Nucleoside-free -modified Eagle's medium (-MEM) and fetal calf serum were from Cytosystems (North Ryde, NSW, Australia).

cDNA Constructs-- A 1,080-base pair EcoRI-PvuII fragment, excised from ibp.118 (15), containing the full coding sequence of hIGFBP-3 (provided by Dr. W. I. Wood, Genentech, South San Francisco, CA) was inserted into pSELECT (Promega). Using this recombinant plasmid as cDNA template and pairs of oligonucleotides (see below), fragments containing full or partial hIGFBP-3 coding sequences were amplified by Pfu DNA polymerase in polymerase chain reactions and cloned into the expression vector pMSG (Pharmacia). This generated expression plasmids rhIGFBP-3, rhIGFBP-3[89-264], rhIGFBP-3[185-264], and rhIGFBP-3[89-184] (Fig. 1A). Site-directed mutagenesis (16), employing oligonucleotides and the pSELECT-hIGFBP-3 plasmid as the mutagenesis vector, was carried out to introduce specific mutations in vitro. cDNA fragments amplified from these mutated plasmids were cloned into pMSG to generate expression plasmids rhIGFBP-3[²⁵³KED  RGD] and rhIGFBP-3[²²⁸KGRKR  MDGEA], each carrying the IGFBP-1 sequence analogous to the mutated region (Fig. 1A). The hIGFBP-3 coding sequences of each construct were verified by plasmid DNA sequencing (17).

View larger version (19K): [in this window] [in a new window]   Fig. 1.   Panel A, expression plasmids of rhIGFBP-3 and its derivatives. The structure of hIGFBP-3 is represented by the box at the top. The stippled box indicates the signal peptide sequence, the vertical lines represent cysteine residues, and the shaded boxes represent putative N-glycosylation sites. The regions of conserved sequences among the six IGFBPs are indicated above the cDNA; the numbers below are amino acid residue numbers. The black bars below show the extent of hIGFBP-3 cDNA sequences carried by each expression plasmid, and the numbered arrowed lines indicate the oligonucleotides used in amplifying these regions. The numbered broken lines represent oligonucleotides used to generate specific mutations in IGFBP-3. The sequences of the oligonucleotides and the construction of the plasmids are outlined under "Materials and Methods." Panel B, induction of rhIGFBP-3 expression in transfected cells by dexamethasone. Monolayer cultures of each transfected cell line were incubated in the medium in the absence (filled bars) or presence (open bars) of 10 µM dexamethasone, as described under "Materials and Methods." Media were collected after 72 h and assayed for IGFBP-3. Values shown are the mean ± S.E. for quadruplicate wells.

Cell Culture and Transfection-- CHO cells were grown in -MEM supplemented with 10% (v/v) fetal calf serum at 37 °C. For transfection, the cells were plated out at 6 × 10⁵ cells/75-cm² flask, incubated for 24 h, and then transfected with 20 µg of DNA of each rhIGFBP-3 expression plasmid or pMSG in the presence of 100 µg of Polybrene (18). The plasmids contain the guanine phosphoribosyltransferase (gpt) gene, which confers resistance to mycophenolic acid. The transfected cells were cultured in GPT selection medium for 21 days to select for stable transfectants. The medium for GPT selection consists of -MEM supplemented with 10% (v/v) fetal calf serum, 250 µg/ml xanthine, 25 µg/ml mycophenolic acid, 2 µg/ml aminopterin, 10 µg/ml thymidine, and 15 µg/ml hypoxanthine. Expression of the recombinant proteins is driven by the mouse mammary tumor virus long terminal repeat promoter on pMSG which has a glucocorticoid-responsive element, hence expression is inducible by dexamethasone. Following the selection period, the mixed population of each transfected cell line was grown to confluence and the media changed to serum-free -MEM supplemented with 0.1% (w/v) bovine serum albumin (BSA) and 10 µM dexamethasone. After 72 h, the conditioned media were collected and stored at 15 °C before assaying for rhIGFBP-3 by a radioimmunoassay (RIA) specific for hIGFBP-3 (13).

Purification of IGFBP-3 Variants-- Serum-free media conditioned for 48-72 h by each of the transfected CHO populations were collected and clarified by centrifugation at 15,300 × g for 20 min. A mixture of protease inhibitors (500 units/ml aprotinin, 5 µg/ml ₂-macroglobulin, 0.5 µg/ml leupeptin, 0.5 mg/ml Na₂EDTA) was added to the medium.

Immuno- and Ligand Blotting-- Conditioned media were concentrated by centrifugation through either Centricon-3 or Centricon-10 microconcentrators (Amicon Inc., Beverley, MA) and the IGFBP-3 concentration in each sample determined by RIA. Each protein (approximately 50 ng) was reconstituted in 50 µl of Laemmli sample buffer, heated at 95 °C for 5 min, and fractionated under nonreducing conditions on a 12% SDS-polyacrylamide gel overnight at 100 V (20). The proteins were then transferred to Hybond-C Extra supported nitrocellulose (Amersham, Bucks, UK) by electroblotting using a Multiphor II Novablot unit (Pharmacia). After transfer, the blot was incubated at 37 °C for 3 h in Tris-buffered saline (TBS, 10 mM Tris, 150 mM NaCl, pH 7.4) containing 1% (w/v) BSA and then probed with anti-hIGFBP-3 antiserum at a final concentration of 1:10,000 (prepared in TBS containing 1% (w/v) BSA and 0.05% (v/v) Nonidet P-40). The blot was washed three times in TBS, once in TBS containing 0.05% Nonidet P-40, and a further three times in TBS and then incubated for 2 h at 22 °C in ¹²⁵I-protein A (1 × 10⁶ cpm/50 ml of TBS containing 1% (w/v) BSA and 0.05% (v/v) Nonidet P-40). Following the wash regime used above, the dried blot was placed against Hyperfilm MP autoradiographic film (Amersham) for 1-3 days at 70 °C.

Affinity Labeling-- 10 ng of each rhIGFBP-3 variant was incubated with ¹²⁵I-IGF-I or ¹²⁵I-IGF-II (0.4 × 10⁶ cpm) in the presence of unlabeled IGF-I or IGF-II, respectively, at concentrations over the range of 10¹¹ to 10⁶ M. The reactions were made up to a final volume of 45 µl with 50 mM sodium phosphate (pH 6.5) containing 0.05% (w/v) BSA. The complexes were cross-linked with 0.25 mM disuccinimidyl suberate (Pierce, Rockford, IL), and after 30 min incubation at 4 °C, the reactions were terminated by the addition of 2 µl of 1.0 M Tris base. Reactions were heated to 95 °C before electrophoresis on either 10% or 12% SDS-PAGE. The gels were processed for autoradiography, and radiolabeled protein bands were quantified by densitometry (Video Densitometer model 620, Bio-Rad).

Binding Assays-- Binary and ternary complex formation in the presence of rhIGFBP-3 or variants was measured essentially as described previously (21). Briefly, reactions containing ¹²⁵I-IGF-I or ¹²⁵I-IGF-II (10,000 cpm) and rhIGFBP-3 analogs over the concentration range of 0-5 ng in a total volume of 0.3 ml of 50 mM sodium phosphate containing 1% (w/v) BSA, were incubated at 22 °C for 2 h. The binary complexes were immunoprecipitated with 0.5 µl of IGFBP-3 antibody and 2.5 µl of goat anti-rabbit immunoglobulin in the presence of a 4% final concentration of polyethylene glycol. Ternary complex formation was measured by incubating ¹²⁵I-ALS (10,000 cpm) with mixtures of IGF-I (50 ng) and varying concentrations of rhIGFBP-3 or variants (over the range of 0-20 ng) in a total volume of 0.3 ml of 50 mM sodium phosphate containing 1% (w/v) BSA at 22 °C for 2 h. Ternary complexes were then separated from unbound tracer as described above.

Detection of Cell-associated IGFBP-3-- Cell surface association of rhIGFBP-3 produced endogenously by the transfected cell lines was measured by an immunological assay described previously (22). Briefly, cells were plated at 2 × 10⁴ cells/well in 24-well plates for 48 h. Cultures were changed to serum-free media supplemented with 0.1% (w/v) BSA and incubated for a further 48 h. The cell monolayers were then washed and incubated with either hIGFBP-3 antibody (R-100) or normal rabbit serum (as control for nonspecific effects) diluted 1:5,000 in 0.5 ml of medium. After a 16-h incubation at 22 °C, the cell monolayers were washed again before incubation with ¹²⁵I-labeled protein A (20,000 cpm in 0.5 ml of medium) for 2 h. Unbound tracer was removed by washing, and the cells were solubilized with 0.5% (w/v) SDS. The cell lysates were collected, and radioactivity was determined in a -counter.

Statistical Analysis-- Statistical analysis was carried out using StatView 4.02 (Abacus Concepts Inc., Berkeley, CA). Differences between groups were evaluated by Fisher's Protected Least Significant Difference test after analysis of variance, and a significant difference was defined as p < 0.05.

	RESULTS

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rhIGFBP-3 proteins, detectable by RIA, were secreted by each of the transfected cell lines, and the expression of these proteins was inducible up to 7-fold by dexamethasone (Fig. 1B). The differences in levels of stimulation by dexamethasone are probably due to the heterogeneous population of transfectants in each cell line. There was no detectable hIGFBP-3 in media conditioned by CHO cells transfected with pMSG as a control. The polyclonal IGFBP-3 antibody recognized all variant proteins including the deletion variants, indicating that an antigenic epitope is present in the amino-terminal portion of the protein, the only region common to all variants. In the RIA, the full-length analogs and rhIGFBP-3 yielded displacement curves that were parallel to pure serum-derived IGFBP-3. In contrast, the deletion variants displayed parallelism up to 1-2 ng, but the displacement curves became nonparallel at higher concentrations (data not shown). All samples were assayed under conditions where parallelism was observed. Under these conditions, the RIA was considered the best method available for quantifying low amounts of these proteins.

Immunoblots of the five variants are shown in Fig. 2A. rhIGFBP-3, rhIGFBP-3[²⁵³KED  RGD], and rhIGFBP-3[²²⁸KGRKR  MDGEA] produced a 40-45-kDa doublet as well as a 30-kDa form, essentially identical to serum-derived hIGFBP-3. rhIGFBP-3[185-264] migrated as a doublet of 30-35 kDa. Adding an estimated mass of 10-15 kDa of carbohydrate to the calculated 19-kDa core protein, the observed bands of rhIGFBP-3[185-264] corresponded well with the predicted size of 29-34 kDa. The doublet evident in rhIGFBP-3[185-264] probably represents different glycoforms of the protein consistent with the current view that the 40-45-kDa doublet of IGFBP-3 consists of glycoforms (23); IGFBP-3 contains three potential N-glycosylation sites at ⁸⁹NAS, ¹⁰⁹NAS, and ¹⁷²NFS (15). rhIGFBP-3[89-264] corresponded to an apparent size of approximately 15 kDa, whereas rhIGFBP-3[89-184] migrated as a major band at 21 kDa and a minor band at 17 kDa. Because the deduced molecular masses for the 89-264 and 89-184 variants are 9 and 18 kDa, respectively, it would appear that the proteins are migrating aberrantly on SDS-PAGE. Presumably, the smaller 17-kDa form of rhIGFBP-3[89-184] represents a proteolyzed form of the protein, comparable to the 30-kDa form of rhIGFBP-3, rhIGFBP-3[²⁵³KED  RGD], and rhIGFBP-3[²²⁸KGRKR  MDGEA].

View larger version (46K): [in this window] [in a new window]   Fig. 2.   Immuno- and ligand blotting of various hIGFBP-3. Samples were prepared and processed for immunoblotting (panel A) or ligand blotting (panels B and C) as described under "Materials and Methods." Relative migration distances of molecular mass standards are indicated on the left of each panel. Panel A, samples are natural, serum-derived hIGFBP-3 (lane 1), media conditioned by CHO cells transfected with pMSG (lane 2), rhIGFBP-3 (lane 3), rhIGFBP-3[89-264] (lane 4), rhIGFBP-3[185-264] (lane 5), rhIGFBP-3[89-184] (lane 6), rhIGFBP-3[253KED  RGD] (lane 7), and rhIGFBP-3[228KGRKR  MDGEA] (lane 8). The probe used was specific hIGFBP-3 antibody. Panel B, samples are serum-derived hIGFBP-3 (lanes 1 and 4) and media conditioned by CHO cells transfected with pMSG (lanes 2 and 5) or rhIGFBP-3 (lanes 3 and 6). Samples in lanes 4-6 were immunoprecipitated with hIGFBP-3 antiserum before electrophoresis. Panel C, samples are identical to those in panel A except that they were immunoprecipitated with hIGFBP-3 antiserum as described under "Materials and Methods." The ligand used in panels B and C was 125I-IGF-I.

The ability of the rhIGFBP-3 proteins to bind IGF-I was examined by ligand blot using ¹²⁵I-IGF-I. A preliminary experiment revealed that there were two forms of IGF-I-binding protein present in the media conditioned by cells transfected with the vector, pMSG (Fig. 2B). Based on the apparent sizes of these proteins (approximately 23 and 28 kDa), we assume that they are glycoforms of CHO-derived IGFBP-4. Immunoprecipitation of the sample with hIGFBP-3 antibody before electrophoresis removed these proteins (Fig. 2B). Ligand blotting of immunoprecipitated IGFBP-3 variant proteins (Fig. 2C) indicated that rhIGFBP-3[²⁵³KED  RGD] and rhIGFBP-3[²²⁸KGRKR  MDGEA] have an IGF-I binding function. In contrast, none of the deletion variants showed detectable IGF binding by this method.

To examine their binding properties in more detail, the full-length rhIGFBP-3 analogs were purified from conditioned media by heparin-Sepharose chromatography followed by reverse phase HPLC. The wild-type protein was eluted from the heparin column as a single peak by 0.75 M NaCl (Fig. 3A). rhIGFBP-3[²⁵³KED  RGD] showed a similar elution profile (Fig. 3B). In contrast, rhIGFBP-3[²²⁸KGRKR  MDGEA] eluted at a concentration of only 0.5 M (Fig. 3C), and rhIGFBP-3[89-184] eluted as two peaks at 0.5 and 0.75 M NaCl (Fig. 3D). Among the deletion analogs, the 89-184 variant showed a marked reduction in binding activity, but the other two analogs did not bind to heparin-Sepharose at all (data not shown). Endogenous IGFBP-4 from CHO cells also bound to heparin-Sepharose and eluted at 0.5 M NaCl (data not shown), consistent with previous studies (24). The CHO-derived IGFBP-4 was separated from rhIGFBP-3[²²⁸KGRKR  MDGEA] on reverse phase HPLC.

View larger version (38K): [in this window] [in a new window]   Fig. 3.   Elution profiles of rhIGFBP-3 from heparin-Sepharose chromatography. Media containing rhIGFBP-3 (panel A, ), rhIGFBP-3[253KED  RGD] (panel B, ), rhIGFBP-3[228KGRKR  MDGEA] (panel C, ), and rhIGFBP-3[89-184] (panel D, ) were applied to 1-ml columns equilibrated in 50 mM sodium phosphate (pH 6.5) buffer. The column was washed with 50 mM sodium phosphate (pH 6.5) buffer, and adherent proteins were eluted in a stepwise fashion with the same buffer (10 ml) containing increasing concentrations of sodium chloride (). The 2-ml fractions were assayed for rhIGFBP-3 in the RIA.

Because of the poor binding of the rhIGFBP-3 deletion analogs to heparin-Sepharose, these analogs were purified from an antibody affinity column followed by reverse phase HPLC, as described under "Materials and Methods." The proteins purified by either heparin or antibody affinity were analyzed by SDS-PAGE to check for protein integrity and purity. Dose-response curves for the binding of various IGFBP-3 analogs to either ¹²⁵I-IGF-I (Fig. 4A) or ¹²⁵I-IGF-II (Fig. 4B) were obtained from solution binding assays. In both instances, there was little or no detectable binding of the deletion analogs to either IGF ligand, consistent with the results obtained from ligand blotting (Fig. 2C). Competitive binding curves were generated for IGFBP-3 forms that showed IGF binding, to compare their relative affinities for IGF-I (Fig. 4C) and IGF-II (Fig. 4D). Data from these assays were analyzed by Scatchard plot, and the derived binding affinities are summarized in Table I. Consistent with the previously determined affinities for IGF-I and IGF-II binding by serum-derived IGFBP-3 (19, 25), rhIGFBP-3 displayed a higher affinity for IGF-II than for IGF-I. However, the difference in K_a values was not statistically significant (p = 0.06). This may result from different post-translational modifications on the IGFBP-3 from different sources. The RGD mutant had significant decreases in its affinities for both IGF-I and IGF-II (4- and 6-fold, respectively; Table I). The MDGEA mutant showed significant reduction in IGF-II affinity (3-fold), but its affinity to IGF-I was near normal (Table I).

View larger version (33K): [in this window] [in a new window]   Fig. 4.   Formation of the binary complex in the presence of various rhIGFBP-3 analogs. The analogs shown are rhIGFBP-3 (), rhIGFBP-3[89-264] (), rhIGFBP-3[185-264] (), rhIGFBP-3[89-184] (), rhIGFBP-3[253KED  RGD] (), and rhIGFBP-3[228KGRKR  MDGEA] (). Binding of 125I-IGF-I (panel A) or 125I-IGF-II (panel B) to increasing amounts of rhIGFBP-3 is shown. The binding curves are representatives of at least two independent measurements for each analog. Competition for the binding of 125I-IGF-I (panel C) or 125I-IGF-II (panel D) to 0.5 ng of each rhIGFBP-3 by increasing concentrations of unlabeled IGF-I or IGF-II, respectively, is shown. B/B0 represents the ratio of 125I-IGF bound to rhIGFBP-3 in the presence of unlabeled IGF to that bound in the absence of unlabeled IGF. Data points shown are mean ± S.E. of at least three independent measurements.

To investigate the possibility that the deletion analogs may have low affinities for IGF which were undetectable by solution binding, ¹²⁵I-IGF-I or ¹²⁵I-IGF-II was covalently cross-linked to the analogs in the presence of competing unlabeled IGF-I or IGF-II, respectively. The samples were analyzed on SDS-PAGE, and the resulting band intensities were quantified by scanning densitometry, to generate displacement curves. Typical electrophoretograms for rhIGFBP-3 are shown (Fig. 5, panels A and F). By this technique, half-maximal displacement of bound IGF-I or IGF-II tracer from rhIGFBP-3 (Fig. 5, B and G, respectively) was seen at unlabeled ligand concentrations similar to those in the solution binding assays (Fig. 4, C and D), confirming the validity of the affinity labeling technique.

View larger version (35K): [in this window] [in a new window]   Fig. 5.   Affinity cross-linking of [125I]IGF-I (top panels) or [125I]IGF-II (bottom panels) to rhIGFBP-3 analogs in the presence of increasing concentrations of unlabeled IGF-I or IGF-II, respectively. After affinity labeling as described under "Materials and Methods," the samples were processed by SDS-PAGE. Representative autoradiographs of rhIGFBP-3 cross-linked to 125I-IGF-I (panel A) and 125I-IGF-II (panel F) are shown. Relative migration distances of molecular mass standards are indicated on the left. The intensities of the bands were quantified by scanning densitometry, and the resulting displacement curves are shown for rhIGFBP-3 (panels B and G, ), rhIGFBP-3[185-264] (panels C and H, ), rhIGFBP-3[89-184] (panels D and I, ), and rhIGFBP-3[89-264] (panels E and J, ). B/B0 represents the ratio of 125I-IGF bound to rhIGFBP-3 in the presence of unlabeled IGF to that bound in the absence of unlabeled IGF. Data shown are mean ± S.E. of three independent measurements.

As shown in Fig. 5, all three deletion analogs were cross-linked to either ¹²⁵I-IGF-I or ¹²⁵I-IGF-II, and the tracers were displaceable by unlabeled IGF-I or IGF-II. However, the affinities were 20-60-fold lower than that of rhIGFBP-3. The concentrations of IGF-I required to displace 50% of tracer binding to the various analogs were 0.12 ± 0.02 nM (rhIGFBP-3), 2.02 ± 0.96 nM (185-264), 4.42 ± 1.19 nM (89-184), and 7.00 ± 1.22 nM (89-264). The relative concentrations of IGF-II required to displace 50% of tracer binding to the various analogs were 0.06 ± 0.03 nM (rhIGFBP-3), 2.43 ± 0.61 nM (185-264), 2.14 ± 0.59 nM (89-184), and 2.50 ± 0.94 nM (89-264).

Fig. 6A shows the dose-response curves of the various IGFBP-3 analogs for binding to ALS in the presence of excess (50 ng) IGF-I, i.e. for ternary complex formation. As predicted by the lack of binding in IGF-I solution binding assays above (Fig. 4A), the deletion variants also displayed barely detectable levels of ALS binding. Similar results were obtained when the binding assays were performed in the presence of IGF-II (data not shown). On the other hand, rhIGFBP-3[²⁵³KED  RGD] exhibited a dose-response curve parallel to that of rhIGFBP-3, but the curve had shifted to the right, indicating that the ALS binding activity of this variant was decreased compared with rhIGFBP-3. This may reflect its decreased affinity for IGF-I (Table I). Although rhIGFBP-3[²²⁸KGRKR  MDGEA] demonstrated a smaller, nonsignificant decrease in affinity for IGF-I compared with rhIGFBP-3[²⁵³KED  RGD] (Table I), its binding to ALS was decreased markedly (Fig. 6A), suggesting that the mutation in this analog predominantly affects its ALS binding function.

View larger version (18K): [in this window] [in a new window]   Fig. 6.   Formation of the ternary complex in the presence of various rhIGFBP-3 analogs. Panel A, binding of 125I-ALS to 50 ng of IGF-I in the presence of increasing rhIGFBP-3 concentrations. The IGFBP-3 analogs shown are rhIGFBP-3 (), rhIGFBP-3[89-264] (), rhIGFBP-3[185-264] (), rhIGFBP-3[89-184] (), rhIGFBP-3[253KED  RGD] (), and rhIGFBP-3[228KGRKR  MDGEA] (). The binding curves shown are representatives of at least two independent measurements for each analog. Panel B, Scatchard plots of ALS binding to 50 ng of IGF-I in the presence of 0.5 ng of rhIGFBP-3 (), rhIGFBP-3[253KED  RGD] (), or rhIGFBP-3[228KGRKR  MDGEA] (). The plots shown are representatives of three independent measurements for each analog.

To compare the relative affinities of ALS for the IGFBP-3 variants, we determined the displacement of ¹²⁵I-ALS from the ternary complexes, formed in the presence of 50 ng of IGF-I and the IGF-binding IGFBP-3 variants, by increasing concentrations of unlabeled ALS (data not shown). Representative Scatchard plots derived from these competition curves are shown in Fig. 6B and the binding parameters summarized in Table I. Whereas rhIGFBP-3[²⁵³KED  RGD] had an affinity for ALS (21.7 ± 4.5 × 10⁹ liters/mol) similar to rhIGFBP-3 (24.3 ± 5.2 × 10⁹ liters/mol), rhIGFBP-3[²²⁸KGRKR  MDGEA] showed a 90% reduction in ALS affinity (2.5 ± 0.9 × 10⁹ l/mol, p < 0.05), indicating the importance of these basic residues in ALS binding.

It has been shown previously that IGFBP-3 synthesized by fibroblasts can associate with the cell surface and can be displaced by the addition of IGF-I (22). The interaction between the various endogenously produced rhIGFBP-3 forms and the cell surface was examined (Fig. 7). Cell surface-associated rhIGFBP-3 proteins were only detected in cell lines transfected with rhIGFBP-3, rhIGFBP-3[89-184], and rhIGFBP-3[²⁵³KED  RGD]. The absence of cell-associated forms of recombinant proteins in cells expressing the 89-264 and 185-264 variants indicates that the carboxyl-terminal region of the protein is necessary for cell surface association. Furthermore, the basic residues (²²⁸KGRKR) in the carboxyl-terminal region, shown above to be important determinants of affinity for ALS, are also integral to the cell association domain, as mutation of these residues abolished the ability of rhIGFBP-3[²²⁸KGRKR  MDGEA] to interact with the cell surface. Consistent with previous evidence indicating that IGF-I displaces cell surface-associated IGFBP-3 into the extracellular medium (22), rhIGFBP-3- and rhIGFBP-3[²⁵³KED  RGD]-transfected cells showed a decrease (approximately 40 and 60%, respectively) in cell-associated binding proteins when incubated with IGF-I (Fig. 7). On the other hand, there was no difference in the levels of cell-associated protein when cells transfected with rhIGFBP-3[89-184] were incubated in the presence or absence of IGF-I. This is in accord with previous observations that displacement of cell surface-associated IGFBP-3 by IGF-I requires a direct interaction between the two proteins because rhIGFBP-3[89-184] has a greatly reduced affinity for IGF-I.

View larger version (23K): [in this window] [in a new window]   Fig. 7.   Association of rhIGFBP-3 to the cell surfaces of transfected cells. Cultures of transfected cells were untreated (open bars) or treated (filled bars) with 50 ng/ml of IGF-I for 48 h. Cell surface-associated IGFBP-3 was detected by the binding of hIGFBP-3 antibody followed by 125I-protein A. The cells were solubilized, and the radioactivity in the cell lysates was determined. Data are expressed as a percentage of control (untreated) values. Results shown are the mean ± S.E. of three separate wells in a single experiment; similar results were obtained from two repeat experiments.

	DISCUSSION

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Comparison of the primary sequences of the six well characterized IGFBPs indicates that the strongest homology is in the amino- and carboxyl-terminal regions of the proteins, whereas the central regions are unique to each protein. The amino- and carboxyl-terminal regions contain 18 cysteine residues, at identical positions in the IGFBPs (IGFBP-6 lacks two of the residues), which probably confer similar constraints on the structure-function of these proteins (26). The IGF binding domain could therefore involve either or both of these regions. In this study we examined the role of the carboxyl-terminal conserved region in the binding of IGF-I, IGF-II, and ALS by generating three deletion variants of IGFBP-3.

The deletion of either the conserved carboxyl-terminal region (rhIGFBP-3[185-264]) or the nonconserved central region (rhIGFBP-3[89-184]) or the combined deletion of both regions (rhIGFBP-3[89-264]) abolished IGF-I and IGF-II binding when analyzed by either ligand blotting or solution binding. However, all three deletion analogs showed some affinity for both IGF ligands in affinity labeling experiments, in agreement with a previous preliminary report, unsupported by data, that rhIGFBP-3[89-264] and rhIGFBP-3[162-264] are capable of binding to IGF-I when analyzed by a similar method (27). It was also reported that deletion of amino acid residues 92-184 or 92-223 did not abolish IGF-I binding. When competitive binding curves for IGF-I or IGF-II binding to rhIGFBP-3 were compared for the solution binding and affinity labeling methods, similar affinity estimates (as determined by half-maximal displacement of radioligand) were obtained, providing some validation of the affinity labeling technique. However, the failure of binding assays to detect IGF binding to the deletion mutants unless stabilized by affinity cross-linking suggests that these relatively low affinity interactions must have quite rapid off-rates.

Deletion of the central region from IGFBP-3 (89-184) appears to have decreased both IGF-I and IGF-II binding by approximately 40-fold. Whether this nonconserved region is directly involved in the binding interactions or simply helps to maintain the spatial configuration of the amino- and carboxyl-terminal domains is unknown. Deletion of the carboxyl-terminal region (185-264) alone affected IGF-II binding (~40-fold reduction) more than IGF-I binding (~20-fold reduction). This region is likely to make a specific contribution to the IGF binding site (particularly IGF-II) because carboxyl-terminal fragments of IGFBP-2 have been shown to retain considerable affinity for IGF-II but little for IGF-I (28). The amino-terminal domain must contribute similarly to the IGF binding site because the 89-264 variant retained some binding activity.

The specific mutations in rhIGFBP-3[²⁵³KED  RGD] and rhIGFBP-3[²²⁸KGRKR  MDGEA] did not abolish either IGF-I or IGF-II binding as determined by solution binding assays. The relative binding affinities of rhIGFBP-3[²⁵³KED  RGD] for IGF-I and IGF-II were decreased by approximately 4- and 6-fold, respectively. In both of the site-specific IGFBP-3 variants, the altered amino acids were replaced by analogous sequences from IGFBP-1. Interestingly, it has been reported that when the RGD sequence in IGFBP-1 was replaced by KED (the corresponding IGFBP-3 sequence), the IGFBP-1 variant lost its IGF-I binding activity which was attributed to the formation of dimers by the IGFBP-1 variant. When the RGD sequence in IGFBP-1 was replaced by KGD, however, the mutation did not abolish IGF-I binding (29). The mutation in rhIGFBP-3[²²⁸KGRKR  MDGEA] decreased its affinity for IGF-I and IGF-II by 2- and 3-fold, respectively (Table I). Taken together, these results would suggest that these mutated sequences, compared with the deletion analogs, make a relatively minor contribution to the IGF-I and IGF-II binding sites. The carboxyl-terminal region, where these specific mutations reside, appears to be more important for IGF-II binding than IGF-I binding, which is consistent with the relative IGF-I and IGF-II binding abilities of the deletion analogs.

In support of the participation of carboxyl-terminal residues in IGFBP binding activity, it has been reported that deletions of the carboxyl-terminal region of IGFBP-1 abolished IGF-I binding (29). Others have reported that natural proteolytic truncation of either the amino or carboxyl terminus of IGFBPs has adverse effects on IGF binding (30-33). Therefore it seems clear that both amino- and carboxyl-terminal regions of the IGFBPs participate in the formation of the ligand binding domain. Although the loss of IGF binding by the deletion variants may in part be the result of conformational changes caused by the large deletions, the structural disruption is apparently not excessive, as the epitopes on these proteins are still recognized by an hIGFBP-3 antibody. This antibody not only shows high specificity for hIGFBP-3 among the six hIGFBPs but also for IGFBP-3 from higher primates compared with other species (12).

The only deletion variant to associate with the cell surface was the central domain deletion rhIGFBP-3[89-184], indicating that the carboxyl-terminal region, but not the central region, is essential for this function of IGFBP-3. Furthermore, the carbohydrate moieties on IGFBP-3 are not required for cell surface association because all of the potential N-linked glycosylation sites have been removed from rhIGFBP-3[89-184]. This is in agreement with a previous study showing that nonglycosylated rhIGFBP-3 expressed in Escherichia coli can associate with the cell surface of bovine fibroblasts (5). The association of the central region deletion variant with the cell surface suggests that the structural integrity of the carboxyl-terminal domain was retained despite the deletion of one-third of the molecule.

In various studies, IGFBPs-1 to -5 have all been shown to bind to cells (22, 24, 34-36). The RGD sequence within the carboxyl-terminal region of IGFBP-1 and IGFBP-2 serves as a recognition motif on proteins that adhere to the cell surface via integrin receptors. It has been shown that IGFBP-1 associates with the ₅₁ integrin and can stimulate the migration of transfected CHO cells (37). The RGD motif is not present in the sequence of native IGFBP-3. The observation that cell-associated IGFBP-3 could be displaced from human fibroblasts by heparin suggested that IGFBP-3 may interact with proteoglycans or other negatively charged molecules on the cell surface (22), possibly via the carboxyl-terminal portion of the protein that is relatively abundant in basic amino acid residues. However, recent evidence suggests that the heparin-inhibitable cell binding of IGFBP-3 may not be to heparan sulfate or chondroitin sulfate glycosaminoglycans (38). This highly basic carboxyl-terminal region is also present in IGFBP-5 and has been shown to be involved in the binding of glycosaminoglycans (39).

There are two putative heparin binding motifs in IGFBP-3, located at amino acids 148-153 and 219-226 in the central and carboxyl-terminal regions, respectively. A recent study (40) showed that synthetic peptides containing either one of these heparin binding consensus sequences bound heparin and that the peptide containing the carboxyl-terminal motif had ~4-fold higher affinity for heparin. The inability of rhIGFBP-3[185-264] to bind heparin-Sepharose would suggest that the carboxyl-terminal region contributes structurally to the major heparin binding site. This is supported by the finding that rhIGFBP-3[89-184] bound to heparin-Sepharose, although with less avidity than rhIGFBP-3. Furthermore, heparin binding was affected when amino acid residues adjacent to the carboxyl-terminal putative heparin binding motif were mutated in rhIGFBP-3[²²⁸KGRKR  MDGEA].

Partial replacement of the carboxyl-terminal basic region in hIGFBP-3 with the homologous acidic residues of hIGFBP-1 (rhIGFBP-3[²²⁸KGRKR  MDGEA]) abolished its ability to associate with the cell surface. On the other hand, the presence of the analogous RGD sequence in IGFBP-3 did not appear to affect the ability of the protein to bind to the cell surface. This suggests that the amino acid residues ²²⁸KGRKR form a key part of the cell surface association domain of IGFBP-3, lending further support to the study in which synthetic peptides corresponding to the basic region were shown to decrease IGFBP-3 and IGFBP-5 binding to the cell surface (24).

Previous studies from this laboratory have shown by several independent methods, gel permeation chromatography, affinity labeling, and solution binding assays with immunoprecipitation of complexes, that little or no specific binding of human ALS to hIGFBP-3 occurs in the absence of IGF-I or IGF-II. This concept has been challenged recently on the basis of studies with rat ALS and a partially proteolyzed form of rat IGFBP-3 (41) and a study using human nonglycosylated IGFBP-3 (42), both of which were interpreted to show that ALS may bind to IGFBP-3 in the absence of IGFs. Whatever the explanation for these conflicting results, our studies with natural human ALS and IGFBP-3 consistently support the notion that the binary IGF·IGFBP-3 complex, rather than IGFBP-3 itself, forms a high affinity binding site for ALS. Indeed, in the presence of IGF-I variants (for example, with substitutions in the B domain) with low affinity for IGFBP-3, the total binding of ALS is low, because little binary (IGF·IGFBP-3) complex forms, but the affinity of ALS for this complex is not reduced (14). In the presence of IGF-I, the deletion variants that had low affinities for IGF-I (decreased by 20-60-fold) showed very low or no binding to ALS, which is consistent with the requirement of an IGF·IGFBP-3 complex for ALS binding. rhIGFBP-3[²⁵³KED  RGD], which had a 4-fold decrease in IGF-I affinity, displayed normal ALS binding affinity compared with rhIGFBP-3. In contrast, rhIGFBP-3[²²⁸KGRKR  MDGEA] showed markedly impaired ALS binding function, attributable to a loss of affinity for ALS, even though its IGF-I binding is relatively normal. These results specifically implicate IGFBP-3 residues 228-232, but not 253-255, in the interaction with ALS.

The two mutations, therefore, have quite distinct effects on the protein-protein interactions within the ternary complex. The KED  RGD mutation has altered the capacity of IGFBP-3 to bind to IGF-I without affecting the affinity of ALS. This implies that when the binary complex between rhIGFBP-3[²⁵³KED  RGD] and IGF-I has formed, the mutation has no bearing on the structural integrity of the ALS binding site, because the affinity of ALS remains unchanged. In contrast, the KGRKR  MDGEA mutation appears to disrupt the ALS binding site, as ALS affinity for this variant was decreased. ALS binding is known to be sensitive to increasing ionic strength (25, 43), and the KGRKR  MDGEA mutation introduces a significant charge reversal in this region of IGFBP-3, suggesting that interaction between key charged residues may be important. Although binding determinants in the ALS structure have not been elucidated, there is a region of acidic residues in the amino-terminal region of human ALS (²³DDDADE) (44) which might interact with the highly basic region in IGFBP-3, and preliminary molecular modeling studies suggest that within the leucine-rich repeating region of ALS, there may be a surface with an accumulation of negative charge.²

In summary, this study has shown that the protein-protein and protein-cell interactions of IGFBP-3 are complex and involve distinct domains of the protein. The structural integrity of the IGF-I binding site is disrupted significantly by deletion of either the central or carboxyl-terminal region of IGFBP-3, but more specific mutations of the carboxyl-terminal region can reduce IGF-I binding. The IGF-I and ALS binding sites are functionally distinct as shown by contrasting the binding characteristics of the ²⁵³RGD variant, with decreased IGF-I binding but normal ALS affinity, and the ²²⁸MDGEA variant, with near normal IGF binding and greatly reduced ALS affinity. Finally, although gross deletions affected the ability of IGFBP-3 to bind to IGF-I and consequently ALS, the deletion of amino acid residues 89-184 did not alter its interaction with the cell surface. We therefore conclude that the carboxyl-terminal region and in particular, ²²⁸KGRKR, is essential for this function. The availability of IGFBP-3 mutants with selective reduction in the affinity for IGFs, on the one hand, and reduced binding to ALS and the cell surface on the other, will provide powerful tools to help elucidate further the dual roles of IGFBP-3 as a transporter of IGFs and a regulator of cell function.