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Endocrinology Vol. 139, No. 4 1943-1951
Copyright © 1998 by The Endocrine Society

ARTICLES

Multivalent Cations Depress Ligand Binding to Cell-Associated Insulin-Like Growth Factor Binding Protein-5 on Human Glioblastoma Cells*

Rebecca L. Sackett and Robert H. McCusker

The Department of Animal Sciences, Laboratory of Developmental Endocrinology, The University of Illinois, Urbana, Illinois 61801

Address all correspondence and requests for reprints to: Robert H. McCusker, Ph.D., 210 Meat Science Laboratory, 1503 South Maryland Drive, Urbana, Illinois 61801-4737. E-mail: rmccuske{at}staff.uiuc.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The current studies quantified the effect of the multivalent cations zinc, cadmium, lanthanum, chromium, and gold (Zn2+, Cd2+, La3+, Cr3+, and Au3+) on [125I]-insulin-like growth factor ([125I]-IGF) binding to T98G human glioblastoma cells. The major binding site for the IGFs on T98G cells is IGF binding protein-5 (IGFBP-5), as determined by affinity labeling. Competitive binding studies, using either [125I]-IGF-I or [125I]-IGF-II, indicated that La3+ and Cr3+ did not affect [125I]-IGF-I or [125I]-IGF-II binding to cell-associated IGFBP-5. Zn2+, Au3+, and Cd2+ depressed binding of both [125I]-IGF-I and [125I]-IGF-II. [125I]-IGF-I and [125I]-IGF-II binding resulted in nonlinear concave-down Scatchard plots, indicating the presence of high- and low-affinity equilibrium constant of association (Ka) sites. Assuming a preexisting asymmetric model with independent high (KaHi) and low (KaLo) sites; Zn2+, Cd2+, and Au3+ eliminated KaHi and Zn2+, and Au3+ lowered KaLo, compared with control values. The same results were found, independent of whether [125I]-IGF-I or [125I]-IGF-II was used. Similarly, assuming a ligand-induced model of negative cooperativity, all three cations eliminated the initial affinity for the high affinity sites (Ke), whereas Zn2+ and Au2+ reduced the final affinity for the low affinity sites (Kf). Dose-response studies indicated that Zn2+, Au3+, and Cd2+ depressed binding with half-maximal activities of approximately 20 µM, 14–60 µM, and 50–65 µM, respectively. Zn2+, Au3+, and Cd2+ bind to similar sites on proteins (a zinc-binding motif), indicating similar mechanisms of action. A zinc-binding motif is present within the IGFBPs but not the IGFs. We demonstrate, for the first time, that multivalent cations have the potential to modulate IGF activity by decreasing the amount of IGF bound to cell-associated IGFBP-5.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
INSULIN-LIKE growth factors I and II (IGF-I and IGF-II) mediate many of the anabolic effects of GH in vivo and are important growth factors for a variety of cells in vitro (1, 2). IGF binding proteins (IGFBPs) are considered as primary modulators of metabolism, because of their ability to bind both IGF-I and IGF-II. Both inhibitory and stimulatory actions have been described for the IGFBPs, each with distinct mechanisms. IGFBPs that are soluble in extracellular fluids decrease IGF activity in vitro. This is an effect whose mechanism is simple to explain. Soluble IGFBPs prevent IGFs from activating the type 1 IGF receptor on the cell surface. IGFBPs can also intensify IGF activity in vitro. How this occurs is still unclear, but it seems to involve IGFBP association with the cell surface (3). Possibly the least understood aspects of IGF action are: 1) the mechanisms by which the distribution of the IGFBPs between the extracellular fluids and the cell surface is controlled; and 2) how IGF distribution between cell-associated IGFBPs and the type 1 IGF receptor is regulated. Understanding factors that control this complex configuration is important in designing methods to enhance IGF-driven performance and the therapeutic value of the IGFs.

It is a well known that animals deficient in Zn2+ stop growing, compared with those with sufficient amounts of dietary Zn2+ (4). Zn2+ has a variety of functions, including its requirement for activity of numerous metalloenzymes (5). In addition, part of Zn2+’s growth regulatory activity includes controlling IGF levels in serum (4, 6, 7, 8, 9, 10, 11). It is also possible that one of the functions of Zn2+ is to control growth by modulating the activity of growth factors. Zn2+ enhances the metabolic and mitogenic activity of IGF-I but not insulin (12). Although the authors proposed an intracellular mechanism of action, if this were the case, Zn2+ should have enhanced the activity of insulin. Insulin and IGF-I share common intracellular signaling mechanisms (13).

The goal of the current work was to test a new mechanism whereby trace nutrients (Zn2+ and Cr3+) and other multivalent cations interact with the endocrine system, primarily by controlling the binding of IGF to IGFBPs. The objective was met by quantifying [125I]-IGF binding to cell surface-associated IGFBP-5. Cells were originally grown in zinc-deficient conditions before cation supplementation during the binding assay. We report here that several cations are capable of reducing the binding affinity of both IGF-I and IGF-II with cell-associated IGFBP-5. We have previously demonstrated that soluble-released IGFBP-3 has a 10-fold higher affinity for IGF-I than that of cell-associated IGFBP-3 (14). Also, La3+ and Zn2+ prevent the loss of cell-associated IGFBP-3 and IGFBP-5 and, like IGFBP-3, cell-associated IGFBP-5 has a lower affinity than soluble IGFBP-5 (15). Hence, there is a step-wise change in ligand affinity (Ka ranking; soluble IGFBPs >> cell-associated IGFBPs > cell-associated IGFBPs + zinc). The combined ability to prevent the loss of cell-associated IGFBP-5 and to reduce the affinity of the IGF-IGFBP-5 interaction at physiological concentration endows Zn2+ with the ability to maintain a reserve of IGFBP-5 at cell surface and, at the same time, make bound IGF more available to the receptor, because receptor affinity is not depressed by Zn2+ (16). Overall, these abilities of Zn2+ probably serve to deliver IGF to the type 1 IGF receptors.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture
T98G (human glioblastoma) cells were acquired from the American Type Culture Collection (no. 1690; Rockville, MD). Cells were grown in Eagles Minimum Essential Medium (EMEM) plus 10% calf serum, 100 µg/ml pyruvate, 30 µg/ml asparagine, 21 µg/ml serine, 10 U/ml penicillin, and 10 µg/ml streptomycin (14). EMEM does not contain zinc or any of the multivalent cations tested in this study. Thus, cells were grown in zinc-deficient conditions before binding assays (10% circulating levels supplied by the calf serum). Reagents were purchased from either Gibco BRL (Gaithersburg, MD) or Sigma Chemical Co. (St. Louis, MO), unless indicated.

Binding assay
Assays were performed using confluent 7–8 day cultures, as previously described (14), with some modifications. Cells were rinsed 3 times with PBS at 4 C, then incubated with Earle’s balanced salt solution for 3 h. This incubation releases loosely attached IGFBPs from the cell surface and minimizes release during the subsequent incubation (14). Cultures were then rinsed twice with PBS and once with Earle’s balanced salt solution. This is a more extensive rinsing protocol than previously used, and it removes loosely attached IGFBPs from the cell surface. Cultures also were incubated at 4 C, instead of the previous 7 C, to prevent IGFBP loss from the cell surface. This rigorous washing procedure and change in temperature were introduced to prevent loss of IGFBPs during the binding assays and to eliminate the ability of La3+ and Zn2+ to alter the number of cell-associated binding sites by preventing IGFBP release (15). Cultures were then incubated with assay buffer (AB) (EMEM without sodium bicarbonate, with 20 mM HEPES, 1% BSA, pH 7.4) containing either [125I]-IGF-I, [125I]-R3-IGF-I, or [125I]-IGF-II (70,000–80,000 cpm, ~0.6 ng/ml) and varying doses of unlabeled IGF-I or IGF-II (1,000 ng/ml for nonspecific binding) with or without 200 µM of the indicated cation. The dose of added cations varied only in one experiment, as indicated. All cations were added as chloride salts in water with the same amount of water added to controls (5 µl/well) and did not affect pH of the AB. Because all assays are performed at 4 C, the cations did not affect cell number/well, cell viability, or protein content and none of the treatments detached cells from the wells. In all but part of the affinity labeling study, the wells contained 10 µg/ml sodium insulin (Gibco BRL), to prevent binding to the type 1 IGF receptor (14). After 3 h (confirmed to be at equilibrium by preliminary studies), the AB was collected into 12 x 75-mm tubes, and the cells were rinsed three times with PBS. NaOH (0.3 M) was added for 1 h to solubilize the cells. The cells were transferred to 12 x 75-mm tubes and counted, to quantify cell surface bound [125I]-IGF. To quantify IGF bound to IGFBPs released into the AB, bound and free [125I]-IGF were separated by PEG precipitation (14). Results of the PEG precipitation assay are not presented, but the procedure was conducted for all assays. There was no specific binding of the [125I]-IGFs to released IGFBPs (data not shown).

Affinity labeling
Affinity labeling was performed with disuccinimidyl suberate (Pierce, Rockford, IL), as previously described (14). Samples (30 µl) of cells solubilized directly in Laemmli buffer (50 µl/cm2) with 6% ß-mercaptoethanol were electrophoresed through 5–18% gradient SDS-polyacrylamide gels and were then exposed to PhosphoImager Screens (Molecular Dynamics, Sunnyvale, CA).

125Iodine labeling
IGF-I, IGF-II (Bachem, Torrance, CA), and R3-IGF-I (Gro-Pep, Adelaide, Australia) were iodinated by incubating 5 µg peptide with 2 mCi [125iodine] (Amersham, Arlington Heights, IL) and 12 µg/ml chloramine-T in 0.5 M sodium phosphate buffer (pH 7.4). Free [125I] was separated from the [125I]-labeled IGF with Millipore (UFC3 LGC 25) 10,000-kDa nominal molecular mass cut-off filter units, which retain more than 95% of labeled peptide with more than 85% recovery of [125I]-labeled IGF. Iodinations were performed on the peptides at the same time, with specific activities ranging between 186 and 337 µCi/µg, as determined by tri-chloroacetic acid precipitability. Mean specific activities were 258, 259, and 263 µCi/µg for [125I]-IGF-I, [125I]-IGF-II, and [125I]-R3-IGF-I, respectively. R3-IGF-I binds poorly to IGFBPs but with normal affinity to type 1 IGF receptors (unpublished data in Gro-Pep brochure).

Data analysis
Scatchard analysis (17) was performed as previously described (15). Average affinity and fractional occupancy for each point along the competition curve was calculated as described by DeMeyts and Roth (18). These data were imported into the SigmaPlot 2D curve fit program (Jandel Scientific, Jan Rafael, CA) and fit to Gaussian Cumulative Formula 8012, to calculate Ke and Kf. Ke and Kf are the affinities (Ka) at fractional binding site occupancies () of 0 and 1 (i.e. the affinity of unoccupied and saturated IGFBPs, respectively). Thus, KaHi and KaLo are affinity binding constants, to describe nonlinear Scatchard plots caused by the presence of two distinct binding sites with preexistent differences in affinity (Preexistent asymmetric model); whereas, Ke and Kf represent the high- and low-affinity binding constants, assuming that the nonlinear plots are a result of a ligand-induced decrease in affinity (Ligand-induced Sequential Model) (18). (B, bound, F, Free, Ro, receptor number). Graphically, is presented as log (B/Ro) (18). Hill coefficients are used as a measure of the linearity of the Scatchard plots, with values less than 1 indicating possible negative cooperativity, and were calculated as described by De Lean and Rodbard (19). Data were further analyzed using SAS and the General Linear Models procedure. Means were compared by Duncans’ multiple range test.

Similar effects on binding affinities resulted, independent of the analysis (Preexistent vs. ligand-induced); i.e. if KaHi was decreased, so was Ke. Thus, why both analyses? Binding constants for the low-affinity sites (KaLo and Kf) were similar, independent of the model, as expected (18). However, KaHi and Ke differed by 3- to 5-fold (KaHi always being greater than Ke, with negative cooperativity). Thus, affinity differences among IGFBPs, and between IGFBPs and receptors reported from different studies, will depend on the type of analysis performed and whether all assays were sensitive enough to detect nonlinear Scatchard plots. Until the cause of the nonlinear Scatchard plots is defined, constants from both analyses are necessary for comparison of findings.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Affinity labeling
Affinity labeling of T98G cell surfaces was performed with [125I]-IGF-I, [125I]-IGF-II, and [125I]-R3-IGF-I (Fig. 1Go). In the presence of insulin, both [125I]-IGF-I (A) and [125I]-IGF-II (B) bind to a single protein, forming a band at 37,000 Mr. This protein was previously identified as IGFBP-5 (20). Band intensity was decreased, with either unlabeled IGF-I or IGF-II, indicating specificity. Type 1 IGF receptors are present on these cells, as indicated by the affinity labeling of 306,000 Mr and 116,000 Mr bands with [125I]-R3-IGF-I in the absence of insulin (C). [125I]-R3-IGF-I did not bind to cell-associated IGFBP-5. In the absence of insulin, more [125I]-IGF-I than [125I]-IGF-II bound the type 1 IGF receptor, and when directly compared, the IGFBP-5 band is more intense with [125I]-IGF-II than with [125I]-IGF-I (D). Na-insulin (10 µg/ml) was added to all wells in all subsequent assays, to prevent binding to the type 1 IGF receptor. Thus, IGFBP-5 is the only binding site available for [125I]-IGF-I and [125]-IGF-II binding to T98G cells in subsequent assays. There was no evidence of a band, either the size of the type 2 IGF receptor or high-molecular-weight band, which bound [125I]-IGF-II > [125I]-IGF-I, that could represent the type 2 IGF receptor.



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  		Figure 1. Affinity labeling of T98G cell surfaces with [125I]-IGF. T98G cells were affinity labeled with either [125I]-IGF-I (A, D), [125I]-IGF-II (B, D), or [125I]-R3-IGF-I (C). Sodium insulin was added at 10 µg/ml during the binding of [125I]-IGF-I (A) and [125I]-IGF-II (B) but not [125I]-R3-IGF-I (C). Unlabeled IGFs were added at 200 ng/ml to indicate specificity of binding (+IGF-I or +IGF-II). For direct comparison, samples from a separate cross-linking experiment, in which cells were labeled with [125I]-IGF-I or [125I]-IGF-II, were run in parallel lanes (D; insulin was not present). Samples were electrophoresed through gradient SDS-polyacrylamide resolving gels. Molecular weight markers were run in parallel lanes to determine the size of receptors and IGFBPs bands. Sizes of IGF-specific affinity labeled bands are indicated by large bold letters, and the locations of molecular weight markers by small letters.

 
Specificity of IGF binding to cell surface IGFBP-5
[125I]-IGF-I binding was specific, as indicated by competition with unlabeled IGF-I (Fig. 2Go, top left). La3+ did not effect specific binding, whereas Zn2+ markedly decreased specific binding. [125I]-IGF-II binding (Fig. 2Go, bottom left) was specific, as indicated by competition with unlabeled IGF-II. Again, only Zn2+ decreased binding. From repeated assays (Table 1Go), La3+ did not affect (but Zn2+ significantly decreased) [125I]-IGF-I and [125I]-IGF-II binding to cell-associated IGFBP-5. In addition, cell-associated IGFBP-5 binds approximately 5-fold more [125I]-IGF-II than [125I]-IGF-I (P < 0.05).



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  		Figure 2. Effect of La3+ and Zn2+ on specific binding of [125I]-IGF to T98G cell-associated IGFBP-5. La3+ (200 µM) or Zn2+ (200 µM) were added to cultures, and then binding was accomplished by a 3-h incubation with tracer. Binding of [125I]-IGF-I (top) and [125I]-IGF-II (bottom) are shown. Results were taken from representative assays with homologous ligand competition. Specific cpm bound are shown (left). Averages of duplicates from single assays are presented. Scatchard plots for the data in the left panels are presented (middle). Average affinity plots for the same data are also shown (right).

 

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  		Table 1. Effect of La3+ and Zn2+ on IGF binding to cell-associated IGFBP-5

 
Dose dependency
Three cations were eventually identified that depressed IGF binding to cell-associated IGFBP-5. Increasing amounts of Zn2+, Cd2+, and Au3+ were tested to determine the potency of their effects. Zn2+, Au3+, and Cd2+ depressed the specific binding of [125I]-IGF-I, with half-maximal effects at approximately 20 µM, 15 µM, and 50 µM, respectively (Fig. 3Go, top); and specific binding of [125I]-IGF-II, with half-maximal effects at approximately 20 µM, 60 µM, and 65 µM, respectively (Fig. 3Go, bottom). Thus, all three cations depressed binding in the low micromolar range, but the important finding is that Zn2+ was active at similar doses for both IGF-I and IGF-II and that this represents physiological (serum) levels of zinc. To determine the mechanism of action, Scatchard analysis was performed at a maximally active dose of 200 µM for the active cations and with two other inactive cations (La3+ and Cr3+) serving as controls.



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  		Figure 3. Dose-dependent effect of cations on the binding of [125I]- IGF-I and IGF-II to T98G cell-associated IGFBP-5. Increasing amounts of the indicated cation were added to wells containing either [125I]-IGF-I (top) or [125I]-IGF-II (bottom). Specific binding was determined for each dose of each cation. Data represent the mean for duplicate determinations. SD of the 200 µM points are presented to indicate the type of variation.

 
Preexistent asymmetric model
Scatchard plots. Nonlinear Scatchard plots for [125I]-IGF-I binding to cell-associated IGFBP-5 in control and La3+ treated cultures indicated the presence of both high and low-affinity sites with IGF-I competition (Fig. 2Go, top middle). The Scatchard plots were linear and shallow in the presence of Zn2+. Scatchard analysis for [125I]-IGF-II binding data (Fig. 2Go, bottom middle) also indicated the presence of high and low-affinity sites for control and La3+ cultures, but not Zn2+.

Binding constants. The effect of La3+ and Zn2+ on ligand affinity (Ka) of cell-associated IGFBP-5 was quantified (Fig. 4Go). Ka was determined from Scatchard plots for both high (KaHi) and low (KaLo) affinity sites from 5–6 assays for each ligand combination. KaHi was greater than 7-fold that of KaLo for [125I]-IGF-I binding with IGF-I competition (Fig. 4Go, top left). In the presence of Zn2+, the high-affinity site was absent and KaLo was depressed compared with controls. La3+ did not affect KaHi or KaLo. For [125I]-IGF-I binding with IGF-II competition1 (Fig. 4Go, top middle), KaHi was approximately 14-fold that of KaLo. Zn2+ eliminated KaHi and depressed KaLo. La3+ did not affect KaHi or KaLo. For [125I]-IGF-II binding with IGF-II competition (Fig. 4Go, top right), KaHi was approximatley 10-fold that of KaLo. In the presence of Zn2+, KaHi was present in only one of 5 determinations and was reduced to {approx}50% of control. Zn2+ decreased KaLo. La3+ did not affect KaHi or KaLo. Thus Zn2+ had a similar effect on Ka independent of the ligand combination. KaHi was 2-fold higher for IGF-II than for IGF-I (homologous ligand combinations). KaLo was similar for both ligands.



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  		Figure 4. Effect of La 3+ and Zn2+ on binding constants for T98G cell-associated IGFBP-5; Preexistent asymmetric model. Binding was accomplished through a 3-h incubation with either [125I]-IGF-I or [125I]-IGF-II, as indicated. The strength (Ka) values of both high- and low-affinity sites are presented (top row), as are the number (Ro) of each site (middle row) and their respective binding capacities (Ka x Ro; bottom row). Results of each graph are the average of five experiments for competition with IGF-I and the average of six experiments, for competition with IGF-II. La3+ and Zn2+ were added at 200 µM. Values are means ± SD. *, Bars within high or low affinity and within ligand combination differ from control, P < 0.05. ¶, Values were present in only one of six determinations.

 
Number of binding sites. Scatchard analysis was used to estimate the number of binding sites (Ro). For [125I]-IGF-I binding with IGF-I competition, high-affinity sites (RoHi) were absent in the presence of Zn2+ (Fig. 4Go, row 2 left) whereas Zn2+ increased the number of low-affinity sites compared with control. La did not affect RoHi or RoLo. For [125I]-IGF-I binding with unlabeled IGF-II competition, again RoHi was absent in the presence of Zn2+ and Zn2+ increased RoLo compared with control. La3+ did not affect RoHi or RoLo (Fig. 4Go, row 2 middle). Analysis of data for [125I]-IGF-II binding allowed for one estimate of RoHi in the presence of Zn2+. RoHi was unaffected by Zn2+. In all other experiments RoHi was absent when Zn2+ was added (Fig. 4Go, row 2 right). La3+ did not affect RoHi or RoLo. Zn2+ increased RoLo compared with control. For all three ligand combinations, the increase in RoLo, caused by Zn2+ was equivalent to the decrease in RoHi indicating a conversion of high-affinity sites to low-affinity sites. The lack of a change in total number of binding sites, indicates that the cations do not release IGFBPs from the cell surface or detach cells from the wells.

For all three ligand combinations, the number of low-affinity binding sites (RoLo) was greater than or equal to 3-fold that of the number of high-affinity binding sites (RoHi). Also, the number of sites (RoHi or RoLo) for [125I]-IGF-II binding was approximately 2-fold higher than that found using IGF-I competition for [125I]-IGF-I binding. The 2-fold greater number of binding sites and 2-fold higher affinity for [125I]-IGF-II compared with [125I]-IGF-I coincides with the approximately 5-fold greater specific binding of the former and greater band intensity of cell-associated IGFBP-5 (Fig. 1Go and Table 1Go, respectively).

Binding capacity. Capacity (Ka x Ro) for both high- and low-affinity sites was calculated (Fig. 4Go, bottom row). La3+ did not affect binding capacity of either site, independent of ligand. In only one experiment, [125I]-IGF-II vs. IGF-II, was capacity of the high-affinity site available in the presence of Zn2+; it was reduced to approximately 50% of control. In all other experiments, including those with [125I]-IGF-II, the high-affinity site was absent in the presence of Zn2+. In two of the three ligand combinations, Zn2+ depressed the binding capacity of the low-affinity site. For all three ligand combinations, the capacity of the high-affinity sites of controls was more than 2-fold that of low-affinity sites.

Ligand-induced cooperative model
Concave-down Scatchard plots could be caused by ligand-induced negative cooperativity (18). Thus, average affinity was calculated for each point and plotted (Fig. 2Go, right). Average affinity of control and La3+ cultures decreased with increasing unlabeled ligand for both [125I]-IGF-I (top right) and [125]-IGF-II (bottom right). Average affinity plots were flat in the presence of Zn2+. When these and other data were curve-fitted, Ke and Kf were estimated as the affinity of unoccupied and filled binding sites, respectively (Fig. 5Go). Ke was absent in the presence of Zn2+ with [125I]-IGF-I as the ligand, but one value was obtained for [125I]-IGF-II binding, which was approximately 65% of control. For all combinations, Zn2+ depressed Kf to approximately 60% of control. La3+ did not significantly affect either Ke or Kf. Kf was approximately 2-fold higher for IGF-II than for IGF-I (homologous ligand combinations). Ke was similar for both ligands.



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  		Figure 5. Effect of La 2+ and Zn2+ on binding constants for T98G cell-associated IGFBP-5: Ligand-induced sequential negative cooperativity model. Average-affinity plots were used to calculate Ke and Kf; the affinity of empty ( = 0) and filled (y = 1) binding sites, respectively (18). Results represent the means ± SD of five or six assays. La3+ and Zn2+ were added at 200 µM. ¶, Ke was present in only one of six determinations.

 
Effect of other multivalent cations
Table 2Go shows the effect of other multivalent cations on IGF binding to cell-associated IGFBP-5. Gold (Au3+) and cadmium (Cd2+) were tested because they bind to proteins via Zn2+-binding motifs, and chromium (Cr3+) was chosen as an additional control cation. Au3+ decreased [125I]-IGF-I and [125I]-IGF-II binding (in some cases, to the point of nondetectable specific binding). KaHi was absent in the presence of Au3+ for all three ligand combinations. Where quantifiable (using [125I]-IGF-II vs. IGF-II), Au3+ decreased KaLo, compared with control. In this case, the number of binding sites for [125I]-IGF-II was also markedly decreased by the addition of Au3+ because of the loss of high-affinity sites (RoHi) without a corresponding increase in RoLo. IGFBP-5 binding of both [125I]-IGF-I and [125I]-IGF-II was depressed by Cd2+, compared with control. Again, KaHi was absent in the presence of Cd2+. Cd2+ did not consistently alter KaLo. RoHi was absent and RoLo was not affected by Cd2+. Thus, there was no binding capacity for the high-affinity sites in the presence of Au3+ or Cd2+, but the capacity of the low-affinity site was less or invariably affected. Cr3+ was without effect on either [125I]-IGF-I or [125I]-IGF-II binding. Similar to Cr3+, we found that Hg2+, Mn2+, Se3+, and Ag1+ were without effect on IGF binding to T98G cells (not shown).


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  		Table 2. Effect of multivalent cations on IGF binding to T98G cell-associated IGFBP-5; Scatchard analysis for preexistent asymmetry model

 
When tested, using the ligand-induced cooperative model, Au3+ and Cd2+ eliminated Ke (Table 3Go). Au3+ (but not Cd2+) depressed Kf. Cr3+ did not affect either Ke or Kf.


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  		Table 3. Effect of multivalent cations on IGF binding to T98G cell-associated IGFBP-5; ligand-induced sequential negative cooperativity model

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The results from this work are the first to show a specific interaction of multivalent cations with cell-associated IGFBP-5. Several unique findings resulted from this work. Cell-associated IGFBP-5 binds more [125I]-IGF-II than [125I]-IGF-I, because of a greater affinity of IGF-II for the IGFBP and because there are approximately 2-fold more binding sites for [125I]-IGF-II. Scatchard plots for IGF-I and IGF-II binding were nonlinear concave-down. Thus, binding constants for both high- and low-affinity sites were calculated. The high-affinity sites for cell-associated IGFBP-5 (KaHi or Ke) are absent in the presence of either Zn2+, Cd2+, or Au3+. These sites are normal when either La3+ or Cr3+ are present. Binding to the low-affinity site is depressed by Zn2+ and Au3+, because of a reduction in affinity (KaLo or Kf). The effect of Zn2+ seems to be a conversion of high-affinity sites into low-affinity sites, as evidenced by the equivalent changes in number of the two binding sites (the increase in the number of low-affinity sites is equal to the decrease in the number of high-affinity sites). However, in one experiment, the conversion was not complete, and a high-affinity site for [125I]-IGF-II binding could be quantified although Ka was depressed, compared with controls. The finding of lowered binding of [125I]-IGF to the cell-associated IGFBP-5, in response to multivalent cations, is entirely new. Thus, we have identified a novel mechanism by which IGF binding activity can be controlled via modulation of IGFBP affinity.

The effects of Zn2+, Au3+, and Cd2+ are probably by direct interaction with the IGFBPs. This conclusion is based on the fact that IGF-I and IGF-II lack histidines, including the two of insulin (B5 and B10), which impart Zn2+ binding activity (21) and, hence, lack the physical properties to either dimerize or bind Zn2+ (22) and, presumably, to bind either Au3+ or Cd2+. Also, Zn2+ does not depress IGF-I, IGF-II, or R3-IGF-I binding to the type 1 IGF receptor (16). Most zinc-binding proteins contain cysteine or histidine residues that coordinate Zn2+ binding (23). The cysteines or histidines within the metal-binding motif are usually separated by 1–3 amino acids, with 2 being the most common (ex. CxxC). Because of a loop in the tertiary structure of the protein, 2 of these motifs come into close proximity, forming a tetrahedrally-coordinated binding site for Zn2+. The loop is usually formed by 20–30 amino acids (i.e. CxxC... 20–30 amino acids... CxxC). This type of tetrahedrally coordinated site binds Zn2+, Cd2+, and Au3+ (24). Zn2+ binding sites involving only cysteine residues are found only within proteins in which the Zn2+ is present to stabilize tertiary structure (5, 25).

IGFBP-1 through -5 and IGFBP-7 have cysteine pairs that could form such a tetrahedrally-coordinated Zn2+ binding site. Two motifs (CxxC and CxCCxxC) are found in the N-terminal region of IGFBP-1, 2, 3, 4, 5, and 7 and are separated by 23–38 amino acids (26, 27). IGFBP-6 has a CxxC and CxxxxxC motif. Additionally, IGFBPs may bind Zn2+ if one or both of these N-terminal motifs coordinate with the CxC motif found near the C-terminus of IGFBP-1 through 6 (but not IGFBP-7). Thus, we propose the presence of a Zn2+ binding site within IGFBPs with structure-altering properties. No such motifs are found in the IGFs.

Scatchard analysis resulted in nonlinear plots for IGF binding to cell-associated IGFBP-5. There are two possible models that could explain this finding: ligand-induced negative cooperativity or the presence of two preexistent independent forms of cell-associated IGFBP-5. The two models cannot be distinguished by equilibrium binding experiments; thus the data were analyzed for both possibilities. Binding of IGF-I and insulin to their appropriate receptors also gives nonlinear concave-down Scatchard plots because of negative cooperativity of ligand binding (28). It is tempting to speculate that the nonlinear Scatchard plots for binding to cell surface IGFBPs could be caused by a similar phenomenon. In support of a cooperative model for the nonlinear Scatchard plots, consider that IGF-II enhances [125I]-IGF-II ligand dissociation from cell-associated IGFBPs (29), providing evidence for negative cooperativity. In addition, low levels of unlabeled IGF-I do not decrease but increase [125I]-IGF-II binding to human fibroblasts, and IGF-I only competes for binding at very high levels (14). Because of this phenomenon, the current work does not contain data using the combination of unlabeled IGF-I and [125I]-IGF-II. This unusual binding phenomenon indicates that binding cooperativity must be involved in IGF binding to cell-associated IGFBPs, and this further warrants analysis of data using both types of binding models. Independent of the model, Zn2+, Cd2+, and Au3+ caused similar effects on ligand binding.

Binding data for IGFBP-3 purified from bovine placental membrane, cell-associated IGFBP-3, and IGFBP-I purified from amniotic fluid fit a two-site model (14, 30, 31) with KaHi and KaLo differing by approximately 10-fold for [125I]-IGF-I binding. This is similar to the KaHi/KaLo ratio for cell-associated IGFBP-5 in the current study. Therefore, the finding of two binding affinities for cell-associated IGFBP-5 is not unusual. The two binding states of IGFBP-1 have been attributed to phosphorylation of the IGFBP (KaHi phosphorylated, KaLo non-(de)phosphorylated) (31), fitting a preexistent independent model for binding. However, nonphosphorylated, nonglycosylated IGFBP-3 and native IGFBP-3 have similar affinities for IGF-I (32). Thus, the cause of the nonlinear Scatchard data for membrane purified IGFBP-3, cell-associated IGFBP-3, and cell-associated IGFBP-5 is still open to speculation.

T98G cells synthesize and secrete IGFBP-2, -3, -4, and -5 (20). Only one IGFBP (IGFBP-5) was affinity labeled on the cell surface. Whether a combination of IGFBPs on the cell surface, not detected by affinity labeling, explains the curvilinear Scatchard plots could not be definitely addressed by the current work. However, the difference in binding of [125I]-IGF-I and [125I]-IGF-II is clearly caused by differential binding of the ligands to IGFBP-5 on the cell surface (Fig. 1Go). The mechanism of the nonlinear Scatchard plots for cell-associated IGFBP-5 can only be defined by additional kinetic experiments. The greater binding of [125I]-IGF-II vs. [125I]-IGF-I is consistent with a higher affinity of soluble recombinant IGFBP-5 for [125I]-IGF-II (33). The reason for the difference in number of binding sites between the two ligands is unknown. A comparative number of binding sites for the two labeled ligands has not been previously reported.

Of the three active cations, Zn2+ is most likely to play a physiological role and to modulate IGF activity. In support of this, Zn2+ altered IGFBP binding at physiological levels (half-maximal effect at serum concentrations). However, most (>90%) of the Zn2+ in serum is bound by albumin. Free Zn2+ in serum is approximately 1 µM. However, there is no information available on total or, in particular, free Zn2+ in the pericellular fluids surrounding nonendothelial cells. Neither is there information on the affinity of Zn2+ binding to IGFBPs, to compare with albumin. Such values await further analysis, which is underway. Relative affinities would determine the percentage of each protein with bound zinc when in direct competition for available Zn2+; although, because of its high molar concentration, the vast majority of zinc is bound by albumin in serum.

The presence of IGFBPs on the cell surface provides not only specific binding sites, which can alter the kinetics of IGF-I and IGF-II binding, but also may be important in enhancing IGF activity (3). IGFBPs in extracellular fluids negatively modulate IGF activity by controlling the amount of IGF that is available to bind to cell surface binding sites (14, 30). Despite some adherence to cell surfaces, exogenously added IGFBP-5 inhibited DNA synthesis of SV 40-transformed fibroblasts, presumably by inhibiting the effect of endogenously produced IGF-I (34). In contrast, IGFBP-5 within extracellular matrix potentiates the hyperplastic effect of IGF-I on human fibroblasts (35). IGFBP-5 also increases the rate of DNA synthesis of normal mouse osteoblasts (36, 37). Both intact and carboxytruncated IGFBP-5 adhered to these cells, both increased the rate of DNA synthesis when added alone, and both potentiated the affect of added IGF-I or IGF-II. IGFBP-5 increased the rate of DNA synthesis, even in the presence of an equimolar concentration of IGFBP-3 (IGFBP-3 alone inhibited DNA synthesis and the effect of added IGF-I). The stimulatory effect of IGFBP-5 in the absence of added IGF may indicate an IGF-independent mode of action for IGFBP-5 via an IGFBP cell surface receptor, although the possibility that IGFBP-5 potentiates the effect of endogenously secreted IGFs cannot be dismissed. Similarly, IGFBP-5 increased the rate of DNA synthesis of MC3T3-E1 mouse osteoblastic cells in the absence or presence of added IGF-I or IGF-II (38). IGFBP-5 adhered to MC3T3-E1 bone cells and increased [125I]-IGF-I and [125I]-IGF-II binding to cell surfaces. The stimulatory effect of IGFBP-5 in the absence of added IGF again may indicate an IGF-independent action or, likely, the ability to potentiate the activity of the IGF-I secreted by these cells (39). Interestingly, the MC3T3-E1 cells secrete IGFBP-5 (40), and Zn2+ potentiates the effect of IGF on these cells in the absence of added IGFBP-5 (12). Thus, cell-associated IGFBP-5 may have IGF-independent actions, but it most likely provides a mechanism for optimal presentation of IGF to the cell surface. In addition, Zn2+ helps to retain IGFBP-5 on the surface of T98G cells (15). This finding, together with a Zn2+-induced decrease in affinity of cell-associated IGFBP-5 for the IGFs, may optimize IGF delivery to the cell surface and then act as a means to release IGFs to enhance receptor activation.

In conclusion, the results presented here should help to develop a more comprehensive theory on how IGFBPs interact to enhance IGF activity, by a mechanism that involves trace nutrients, specifically zinc. IGFBPs probably bind to the cell surface to prevent diffusion of IGFs from the pericellular space. When Zn2+ lowers the affinity of IGFBPs for IGF at the cell surface, it should release IGFs. Zn2+ is active, in this respect, at physiological (serum) concentrations. In vivo, low extracellular Zn2+ could result in IGFs remaining tightly bound to IGFBPs, with a greater percentage of these released from the cell surface, making IGFs less available for receptor activation. Such a scenario during Zn2+ deficiency would result in depressed growth. Similar to an in vivo model for Zn2+ deficiency, in which extracellular (serum) levels are low, our cells are grown and maintained in one-tenth serum Zn2+ concentrations before the assay (see Materials and Methods). The present work, therefore, represents a model for Zn2+ deficiency and indicates that Zn2+ supplementation will decrease IGF association with IGFBPs at the cell surface. Hence, Zn2+ may ultimately be necessary for growth by increasing IGF activity, not only by a possible intracellular pathway (12), but by an extracellular mechanism of action.


   Footnotes
 
1 The authors realize that Scatchard analysis was designed for data with homologous combinations of labeled and unlabeled ligands. However, because there is no method to analyze nonhomologous combinations, Scatchard analysis was used to quantify what is a normal in vivo competition between IGF-I and IGF-II. The data generated in this manner indicate that Ka reflects that of the unlabeled ligand, IGF-II, whereas Ro and binding capacity reflect that of the labeled ligand, [125I]-IGF-I. Additionally, ED50 (dose at 50% competition) values cannot be used because they assume a similar number of binding sites. Homologous ligand comparisons indicate that this is not the case with binding sites being [125I]-IGF-II > [125I]-IGF-I. Back

Received August 28, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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