IGFBP-3 binds GRP78, stimulates autophagy and promotes the survival of breast cancer cells exposed to adverse microenvironments.

Despite the established role of insulin-like growth factor binding protein-3 (IGFBP-3) as a growth inhibitor in vitro, a high level of IGFBP-3 in breast tumor tissue is associated with the stimulation of xenograft growth in mice and poor prognosis in patients. To understand the contribution of IGFBP-3 to breast cancer progression, tandem affinity purification was used to identify novel interacting proteins. The endoplasmic reticulum protein, glucose-regulated protein 78 (GRP78), was shown to bind to IGFBP-3, confirmed by colocalization, coimmunoprecipitations, glutathione S-transferase (GST) pulldowns and a nanomolar binding affinity. GST pulldowns also indicated that the GRP78 ATPase domain mediated the interaction with IGFBP-3. The critical roles of GRP78 in the unfolded protein response and macroautophagy led to an investigation of possible links between IGFBP-3, GRP78 and cellular stress responses. IGFBP-3 was found to stimulate the survival of breast cancer cells subjected to glucose starvation and hypoxia. Pharmacological inhibitors and small interfering RNA knockdown established that the increased survival of IGFBP-3-expressing cells was dependent on an intact autophagy response, as well as GRP78. The contribution of autophagy was confirmed by the demonstration that IGFBP-3 expression increases both the formation of autophagic puncta and flux through the system. In conclusion, we have shown that IGFBP-3 stimulates autophagy and thereby promotes the survival of breast cancer cells exposed to conditions that represent the adverse microenvironments encountered by solid tumor cells in vivo.

The role of the M6P/IGF-II receptor in cancer: tumor suppression or garbage disposal?

The mannose 6-phosphate/insulin-like growth factor-II receptor (M6P/IGF-IIR) is an intriguing protein with multiple ligands and multiple functions. Approximately 90 - 95 % of the receptor is located intracellularly, with 5 - 10 % being on the cell surface. It has long been known to play an essential intracellular role in the transport of newly-synthesized lysosomal enzymes from the trans-Golgi network (TGN) to the lysosomes. More recently, however, the loss of this receptor has been described in some tumour types, suggesting that it may play a role in tumour suppression. The focus has therefore shifted to elucidating the role played by the cell surface receptor and its interaction with its diverse ligands in tumour growth and progression. The list of ligands is continuously increasing and includes growth factors such as IGF-II and transforming growth factor beta (TGFbeta). This review will address the question of whether the M6P/IGF-IIR plays a direct role in tumour suppression or merely plays an indirect role as a transporter for ligands designated for degradation in the lysosomes.

Mutagenesis of basic amino acids in the carboxyl-terminal region of insulin-like growth factor binding protein-5 affects acid-labile subunit binding.

Like insulin-like growth factor binding protein-3 (IGFBP-3), IGFBP-5 was recently shown to form ternary complexes with insulin-like growth factor (IGF) and the acid-labile subunit (ALS). Previous studies using IGFBP-5/IGFBP-6 chimeric proteins have identified major and minor ALS binding sites in the carboxyl-terminal and central regions, respectively of IGFBP-5. We now report that ALS binds to IGFBP-3 (K(a) = 1.1 +/- 0.1 liters/nmol) and IGFBP-5 (K(a) = 1.8 +/- 0.5 liters/nmol) with similar binding affinities. Using site-specific mutants, we have identified residues K(211)/R(214)/K(217)/R(218) within the carboxyl-terminal region of IGFBP-5 as being essential for ALS binding. Mutation of K(134)R(136) or K(138)K(139) in the central region of IGFBP-5 resulted in a small decrease in ALS binding.

Ligand-binding characteristics of recombinant amino- and carboxyl-terminal fragments of human insulin-like growth factor-binding protein-3.

Insulin-like growth factor-binding protein-3 (IGFBP-3) is a member of a family of structurally conserved proteins (IGFBP-1 to -6) which act as carriers and regulators of the mitogenic peptide hormones IGF-I and IGF-II. Members of the IGFBP family share conserved cysteine-rich amino- and carboxyl-terminal regions. The amino-terminal domain of these proteins is recognised to contain an IGF-binding determinant, but evidence to support a binding site in the carboxyl-terminal region of the protein is less rigorous. To further investigate this, we have synthesised both the amino-terminal (residues 1-88; N-88) and carboxyl-terminal (residues 165-264; C-165) domains of human IGFBP-3 in bacteria, as fusion proteins with a carboxyl-terminal FLAG peptide. Although only C-165 showed binding to IGF-I and -II by solution-binding assays, both N-88 and C-165 demonstrated binding to IGF-I and -II by biosensor analysis albeit with reduced affinities compared with full-length IGFBP-3. Only the carboxyl-terminal fragment (C-165) was able to form hetero-trimeric complexes with IGF-I and the acid-labile subunit (ALS). We conclude that the carboxyl-terminal domain of IGFBP-3 contains an IGF-binding determinant and can form ternary complexes with ALS.

Growth inhibition by insulin-like growth factor-binding protein-3 in T47D breast cancer cells requires transforming growth factor-beta (TGF-beta ) and the type II TGF-beta receptor.

This study explores the relationship between anti-proliferative signaling by transforming growth factor-beta (TGF-beta) and insulin-like growth factor-binding protein-3 (IGFBP-3) in human breast cancer cells. In MCF-7 cells, the expression of recombinant IGFBP-3 inhibited proliferation and sensitized the cells to further inhibition by TGF-beta1. To investigate the mechanism, we used T47D cells that lack type II TGF-beta receptor (TGF-betaRII) and are insensitive to TGF-beta1. After introducing the TGF-betaRII by transfection, the basal proliferation rate was significantly decreased. Exogenous TGF-beta1 caused no further growth inhibition, but immunoneutralization of endogenous TGF-beta1 restored the proliferation rate almost to the control level. The addition of IGFBP-3 did not inhibit the proliferation of control cells but caused dose-dependent inhibition in TGF-betaRII-expressing cells when exogenous TGF-beta1 was also present. Similarly, receptor-expressing cells showed dose-dependent sensitivity to exogenous TGF-beta1 only in the presence of exogenous IGFBP-3. This indicates that in these cells, anti-proliferative signaling by exogenous IGFBP-3 requires both the TGF-betaRII and exogenous TGF-beta1. To investigate this synergism, the phosphorylation of TGF-beta signaling intermediates, Smad2 and Smad3, was measured. Phosphorylation of each Smad was stimulated by TGF-beta1 and, independently, by IGFBP-3 with the two agents together showing a cumulative effect. These data suggest that IGFBP-3 inhibitory signaling requires an active TGF-beta signaling pathway and implicate Smad2 and Smad3 in IGFBP-3 signal transduction.

Insulin-like growth factor-binding protein-3 modulates expression of Bax and Bcl-2 and potentiates p53-independent radiation-induced apoptosis in human breast cancer cells.

We report that transfection of insulin-like growth factor-binding protein-3 (IGFBP-3) cDNA in human breast cancer cell lines expressing either mutant p53 (T47D) or wild-type p53 (MCF-7) induces apoptosis. IGFBP-3 also increases the ratio of pro-apoptotic to anti-apoptotic members of the Bcl-2 family. In MCF-7, an increase in Bad and Bax protein expression and a decrease in Bcl-x(L) protein and Bcl-2 protein and mRNA were observed. In T47D, Bax and Bad proteins were up-regulated; Bcl-2 protein is undetectable in these cells. As T47D expresses mutant p53 protein, these modulations of pro-apoptotic proteins and induction of apoptosis are independent of p53. The effect of IGFBP-3 on the response of T47D to ionizing radiation (IR) was examined. These cells do not G(1) arrest in response to IR and are relatively radioresistant. Transfection of IGFBP-3 increased the radiosensitivity of T47D and increased IR-induced apoptosis but did not effect a rapid G(1) arrest. IR also caused a much greater increase in Bax protein in IGFBP-3 transfectants compared with vector controls. Thus, IGFBP-3 increases the expression of pro-apoptotic proteins and apoptosis both basally and in response to IR, suggesting it may be a p53-independent effector of apoptosis in breast cancer cells via its modulation of the Bax:Bcl-2 protein ratio.

Nuclear import of insulin-like growth factor-binding protein-3 and -5 is mediated by the importin beta subunit.

Although insulin-like growth factor-binding protein (IGFBP)-3 and IGFBP-5 are known to modulate cell growth by reversibly sequestering extracellular insulin-like growth factors, several reports have suggested that IGFBP-3, and possibly also IGFBP-5, have important insulin-like growth factor-independent effects on cell growth. These effects may be related to the putative nuclear actions of IGFBP-3 and IGFBP-5, which we have recently shown are transported to the nuclei of T47D breast cancer cells. We now describe the mechanism for nuclear import of IGFBP-3 and IGFBP-5. In digitonin-permeabilized cells, where the nuclear envelope remained intact, nuclear translocation of wild-type IGFBP-3 appears to occur by a nuclear localization sequence (NLS)-dependent pathway mediated principally by the importin beta nuclear transport factor and requiring both ATP and GTP hydrolysis. Under identical conditions, an NLS mutant form of IGFBP-3, IGFBP-3[(228)KGRKR --> MDGEA], was unable to translocate to the nucleus. In cells where both the plasma membrane and nuclear envelope were permeabilized, wild-type IGFBP-3, but not the mutant form, accumulated in the nucleus, implying that the NLS was also involved in mediating binding to nuclear components. By fusing wild-type and mutant forms of NLS sequences (IGFBP-3 [215-232] and IGFBP-5 [201-218]) to the green fluorescent protein, we identified the critical residues of the NLS necessary and sufficient for nuclear accumulation. Using a Western ligand binding assay, wild-type IGFBP-3 and IGFBP-5, but not an NLS mutant form of IGFBP-3, were shown to be recognized by importin beta and the alpha/beta heterodimer but only poorly by importin alpha. Together these results suggest that the NLSs within the C-terminal domain of IGFBP-3 and IGFBP-5 are required for importin-beta-dependent nuclear uptake and probably also accumulation through mediating binding to nuclear components.

Characterization of an amino-terminal fragment of insulin-like growth factor binding protein-3 and its effects in MCF-7 breast cancer cells.

This study describes the purification, characterization and actions of a peptide derived from proteolysis of IGFBP-3 by an enzyme secreted by MCF-7 breast cancer cells. One millilitre of cell-conditioned medium at pH 5.5 fully proteolysed 10 microg plasma-derived IGFBP-3, yielding an immunoreactive fragment of apparent molecular mass 21 kDa by SDS-PAGE. After purification to homogeneity by IGF-I affinity chromatography and reverse-phase HPLC, sequence analysis revealed the amino-terminus of IGFBP-3, and mass spectrometry indicated a molecular mass of 12 295 Da. Analysis of the corresponding fragment generated by proteolysis of a non-glycosylated IGFBP-3 mutant indicated a molecular mass of 9855 Da, consistent with cleavage after Arg97. This suggests that the fragment derived from glycosylated IGFBP-3 contains approximately 2.5 kDa carbohydrate on Asn89. IGFBP-3[1-97] formed binary complexes with IGFs, but with reduced efficiency compared with intact IGFBP-3. IGFBP-3[1-97] at 11 nM inhibited IGF-I-stimulated DNA synthesis by 50-60% in MCF-7 breast cancer cells, similar to the inhibition observed with the intact protein. In the absence of IGF-I, DNA synthesis was inhibited by IGFBP-3[1-97], but not intact IGFBP-3. This suggests that the IGFBP-3 protease in MCF-7 cell medium can generate an inhibitor of IGF-dependent and independent breast cancer cell growth.

Decreased insulin-like growth factor-II/mannose 6-phosphate receptor expression enhances tumorigenicity in JEG-3 cells.

The insulin-like growth factor-II/mannose-6 phosphate receptor (IGF-II/M6PR) is believed to bind and degrade the potent mitogen IGF-II, a growth factor for many tumors. This receptor has been shown to be mutated and/or lost in a significant percentage of a variety of tumors, implying that it may act as a negative regulator of cell growth. In this study, we demonstrate that down-regulation of this receptor, mediated by antisense IGF-II/M6PR cDNA transfection into JEG-3 choriocarcinoma cells, results in increased growth rate in vitro and increased tumor growth rate in vivo. These findings demonstrate that a decrease in IGF-II/M6PR expression results in a growth advantage in JEG-3 cells and are consistent with the hypothesis that the IGF-II/M6PR is an inhibitor of tumor growth.

The acid-labile subunit of the serum insulin-like growth factor-binding protein complexes. Structural determination by molecular modeling and electron microscopy.

The acid-labile subunit (ALS) is a glycosylated 85-kDa member of the leucine-rich repeat (LRR) protein superfamily and circulates in ternary complexes with the insulin-like growth factors (IGFs) and their binding proteins (IGFBPs). These complexes are thought to regulate the serum IGFs by restricting IGF movement out of the circulation. However, little is known about how ALS binds to IGFBP-3 or -5, which link the IGFs to ALS. To investigate potential sites of interaction, the ALS structure has been modeled with the crystal structure of the LRR protein porcine ribonuclease inhibitor as a template. ALS is predicted to be a donut-shaped molecule with an internal diameter of 1.7 nm, an external diameter of 7.2 nm, and a thickness of 3.6 nm. These dimensions are supported by rotary shadowing electron microscopy of ALS. The internal face is lined with a substantial region of electronegative surface potential that could interact with the positively charged region on IGFBP-3 known to be involved in ALS binding. The model also predicts that three potential N-linked oligosaccharide sites within the LRR domain are clustered together, which may be important in light of recent studies showing ALS glycan involvement in complex formation with IGFBP-3.

The IGF axis and programmed cell death.

Insulin-like growth factors (IGF) are mitogenic peptides that have been implicated as positive regulators of cellular proliferation. In recent years, several studies have suggested an additional role for the IGF axis in the regulation of apoptosis. Signalling through the IGF receptor has been shown to have a potent survival function and protect cells from a variety of apoptotic stimuli. The actions of IGF are regulated by a family of high-affinity IGF binding proteins (IGFBP), which sequester the IGF from the IGF receptor. However, there is some evidence that one of these binding proteins, IGFBP-3, may have its own pro-apoptotic effects that are independent of its ability to modulate IGF bioavailability. In addition, it has been suggested that the tumour suppressor p53, a crucial mediator of apoptosis in response to cellular stress, may elicit several of its apoptotic effects through manipulation of components of the IGF axis. This review summarizes what is currently known about the role of the IGF system in the regulation of apoptosis, highlighting its implications in the context of tumorigenesis.

Adenoviral-mediated expression of human insulin-like growth factor-binding protein-3.

Insulin-like growth factors (IGFs) in the circulation are predominantly sequestered into ternary complexes comprising IGF, IGF-binding protein-3 (IGFBP-3), and the acid-labile subunit (ALS). Besides its role in regulating IGF bioavailability in the circulation, IGFBP-3 has both IGF-dependent and IGF-independent actions on cell proliferation. As part of our studies into the structure-function relationships of the multifunctional IGFBP-3, we have evaluated the efficiency of an adenovirus-mediated expression system for rapid, medium-scale production of functional, glycosylated IGFBP-3. Replication-deficient adenovirus containing human IGFBP-3 cDNA was generated using standard techniques. Secreted, recombinant IGFBP-3 (IGFBP-3(Ad)) was purified from the culture medium of virus-infected cells by IGF-I affinity chromatography followed by reverse-phase HPLC. When analyzed by SDS-PAGE, IGFBP-3(Ad) was similar in size (43- to 45-kDa glycoform doublet) to IGFBP-3(Pl) derived from plasma. In addition, IGFBP-3(Ad) was detected by immunoblot using an antibody specific for human IGFBP-3 and by ligand blot using radiolabeled IGF-I. IGFBP-3(Ad) had similar affinities for IGF-I and ALS and an approximately 25% decreased affinity for IGF-II compared to IGFBP-3(Pl). IGFBP-3(Ad) showed no significant difference in its susceptibility to an IGFBP-3 protease present in medium conditioned by MCF-7 breast cancer cells compared to IGFBP-3(Pl), but appeared more resistant to the IGFBP-3 protease present in pregnancy serum. IGFBP-3(Ad) also exhibited increased binding to T47D cells which may be related to the glycosylation state of the protein.

Characterisation of recombinant glycosylation variants of insulin-like growth factor binding protein-3.

There are three potential N-glycosylation sites in the non-conserved central region of the insulin-like growth factor binding protein-3 (IGFBP-3) sequence (N89AS, N109AS, N172FS). IGFBP-3 exists as two glycoforms which reduce to a single form on enzymatic deglycosylation. To determine the functional significance of the carbohydrate chains, the N-glycosylation sites were mutated singly and in combinations by substituting Asn residues with Ala. Each recombinant glycoform was detected by radioimmunoassay, indicating that glycosylation is not essential for secretion in Chinese hamster ovary cells. Ligand blotting of the conditioned media using [125I]IGF-I indicated that all seven mutants are active. On the basis of the number and molecular masses of the bands detected for each glycoform, there is approximately 4, 4.5 and 5 kDa of carbohydrate on Asn89, Asn109 and Asn172 respectively, with variable occupancy of Asn172. Ternary complex formation by the glycovariants in the presence of ALS and excess IGF-I was not significantly different from that of fully glycosylated recombinant human (rh)IGFBP-3 [Ka (fully glycosylated)=12.5+/-4.1 l/nmol; mean Ka (all mutants)=22.1+/-3.0 l/nmol]. In contrast, Asn to Asp substitutions decreased acid-labile subunit (ALS) binding activity. Cell-surface association experiments indicate that glycosylation may influence the partitioning of IGFBP-3 between the extracellular milieu and the cell surface. Therefore, while the carbohydrate units appear to be non-essential to ALS or IGF binding, they may modulate other biological activities of IGFBP-3.

N-Linked glycosylation and sialylation of the acid-labile subunit. Role in complex formation with insulin-like growth factor (IGF)-binding protein-3 and the IGFs.

Over 75% of the circulating insulin-like growth factors (IGF-I and -II) are bound in 140-kDa ternary complexes with IGF-binding protein-3 (IGFBP-3) and the 84-86-kDa acid-labile subunit (ALS), a glycoprotein containing 20 kDa of carbohydrate. The ternary complexes regulate IGF availability to the tissues. Since interactions of glycoproteins can be influenced by their glycan moieties, this study aimed to determine the role of ALS glycosylation in ternary complex formation. Complete deglycosylation abolished the ability of ALS to associate with IGFBP-3. To examine this further, seven recombinant ALS mutants each lacking one of the seven glycan attachment sites were expressed in CHO cells. All the mutants bound IGFBP-3, demonstrating that this interaction is not dependent on any single glycan chain. Enzymatic desialylation of ALS caused a shift in isoelectric point from 4.5 toward 7, demonstrating a substantial contribution of anionic charge by sialic acid. Ionic interactions are known to be involved in the association between ALS and IGFBP-3. Desialylation reduced the affinity of ALS for IGFBP-3. IGF complexes by 50-80%. Since serum protein glycosylation is often modified in disease states, the dependence of IGF ternary complex formation on the glycosylation state of ALS suggests a novel mechanism for regulation of IGF bioavailability.

Insulin-like growth factor-binding protein (IGFBP)-3 and IGFBP-5 share a common nuclear transport pathway in T47D human breast carcinoma cells.

Insulin-like growth factor-binding proteins (IGFBPs) play an integral role in modifying insulin-like growth factor actions in a wide variety of cell types. Recent evidence suggests that IGFBP-3 and IGFBP-5 also have effects on cell growth that are insulin-like growth factor-independent. In investigating possible mechanisms for this effect, the intracellular trafficking of IGFBP-3 and IGFBP-5, both of which contain sequences with the potential for nuclear localization, was studied in T47D cells. Nuclear uptake of fluorescently labeled IGFBP-3 and IGFBP-5 was observed in a proportion of T47D cells that appeared to be rapidly dividing. IGFBP-1 and IGFBP-2, which do not possess the putative domain for nuclear translocation, were not transported to the nuclei of T47D cells. When T47D cells were preincubated with excess unlabeled IGFBP-3, nuclear localization of labeled IGFBP-3 or IGFBP-5 was not detected, indicating that their nuclear translocation involves a common pathway. Inhibition of receptor-mediated endocytosis did not affect nuclear uptake of IGFBP-3, suggesting that it uses an alternative non-classical import pathway for transport across the plasma membrane. In addition, a variant form of IGFBP-3 with a mutation in the putative nuclear localization sequence was unable to translocate to the nuclei of T47D cells, suggesting that nuclear translocation of IGFBP-3 was dependent on these carboxyl-terminal basic residues.

Development of resistance to insulin-like growth factor binding protein-3 in transfected T47D breast cancer cells.

Insulin-like growth factor binding protein-3 (IGFBP-3) has antiproliferative effects in many cell types but paradoxical growth stimulation has also been reported. In early passages following transfection of T47D breast cancer cells with IGFBP-3 cDNA, the proliferation rate and serum-stimulated DNA synthesis were significantly reduced compared to control cells. Cell cycle analysis indicated that growth-inhibited IGFBP-3-producing cells accumulated in G1 phase. After several passages, the transfected cells became resistant to the inhibitory effects of IGFBP-3 and showed transiently enhanced proliferation rates despite an increased IGFBP-3 concentration in the medium. IGFBP-3 proteolysis did not account for its decreased antiproliferative activity in resistant cells. We hypothesize that development of resistance to the antiproliferative action of IGFBP-3 might be an important step in the malignant progression of breast cancer cells.

Structural determinants of ligand and cell surface binding of insulin-like growth factor-binding protein-3.

Among the well defined insulin-like growth factor (IGF)-binding proteins (IGFBPs), IGFBP-3 is characterized by its interaction with an acid-labile glycoprotein (ALS) in the presence of IGFs. To identify the structural determinants on IGFBP-3 required for ligand binding and cell association, five recombinant human IGFBP-3 variants were expressed in Chinese hamster ovary cells: deletions of amino acids 89-264, 89-184, and 185-264, and site-specific mutations 228KGRKR --> MDGEA and 253KED --> RGD. The basic carboxyl-terminal region of IGFBP-3 was required for binding to heparin. The deletion variants had greatly decreased IGF binding ability as assessed by ligand blotting and solution binding assays; affinity cross-linking indicated at least a 20-fold decrease in IGF affinity. The RGD mutant had a 4-6-fold reduced affinity for both IGFs, but the MDGEA mutant bound IGF-I with near normal affinity and IGF-II with a 3-fold reduction in affinity. The three deletion variants were incapable of binding ALS; but of the site-specific variants, the MDGEA mutant bound ALS with 90% lower affinity (Ka = 2.5 +/- 0.9 liters/nmol) than seen for rhIGFBP-3 (Ka = 24.3 +/- 5.2 liters/nmol), whereas the RGD mutation had no effect on ALS affinity (Ka = 21.7 +/- 4.5 liters/nmol). The ability of IGFBP-3 to associate with the cell surface was lost in variants lacking residues 185-264 and in the 228KGRKR --> MDGEA mutant. We conclude that residues 228-232 of IGFBP-3 are essential for cell association and are required for normal ALS binding affinity.

Modulation of human IGF binding protein-3 activity by structural modification.

To delinate regions of IGFBP-3 involved in ligand and cell-surface binding. DNAs encoding human IGFBP-3 [1-264] and several variants were transfected into CHO cells. Of three deletion (delta) mutants. IGFBP-3 [1-88], [1-184], and [delta 89-184], none bound IGF-I tracer by ligand blotting, although all were detectable by immunoblotting. No ALS binding was detectable, as predicted by the lack of IGF binding. Normal-sequence IGFBP-3 associated with the CHO cells and was partly displaceable by IGF-I. Whereas IGFBP-3 [1-88] and [1-184] failed to cell-associate, the non-IGF-binding central deletion variant [delta 89-184] did associate with CHO cells but was not displaced by IGF-I. To further examine the role of the carboxy-terminal domain in cell-association, the basic sequence IGFBP-3 [228-232] (KGRKR) was altered to the corresponding IGFBP-1 residues MDGEA, a major charge reversal. This variant showed reduced IGF-I binding, and bound ALS with decreased affinity as determined by Scatchard analysis. It showed no cell binding, implicating the basic domain in cell-association. We conclude that, whereas the central and carboxy-terminal domain deletions fail to bind IGF-I, the ability to cell associate requires the carboxy-terminal but not the central domain. Specifically, the basic region [228-232] is essential for cell binding, and also affects IGF-I binding, and independently, ALS affinity.

The role of glycosylation in the action of IGFBP-3.

There are three potential N-glycosylation sites (Asn-X-Ser/Thr) located in the nonconserved central region of the human IGFBP-3 sequence (Asn89, Asn109, Asn172-sites 1, 2 and 3, respectively). Upon ligand blotting with IGFs, IGFBP-3 appears as two bands (40-45 kDa) representing different glycosylated forms. We have mutated the N-glycosylation sites in permutations of three single, three double and one triple mutations and expressed these variant cDNAs. Each mutant protein was detected by radioimmunoassay, indicating that glycosylation is not required for the secretion of the protein from CHO cells. Ligand blotting using [125I]IGF-I indicated that all seven mutants retained IGF-I binding. Based on the molecular weights of the variant proteins, there are approximately 4, 5 and 6 kDa of carbohydrate on sites 1, 2 and 3, respectively. Furthermore, the two forms of IGFBP-3 represent the protein glycosylated either at all three sites or at Asn89 and Asn109 only. There appears to be no difference between the mutants and the fully-glycosylated rhIGFBP-3 in their acid-labile subunit (ALS) binding. Analysis of variance confirmed that the association constant for ALS was not significantly changed by any mutation [Ka (fully-glycosylated) = 12.5 +/- 4.1 nM-1; mean Ka (all mutants) = 22.1 +/- 3.0 nM-1]. While glycosylation does not appear to play a role in IGFBP-3 ligand binding, it may affect the turnover rate of the protein or be involved in rendering the protein resistant to proteolysis.