Interactions of High Affinity Insulin-like Growth Factor-binding Proteins with the Type V Transforming Growth Factor-beta Receptor in Mink Lung Epithelial Cells*

Sandra M. Leal, Shuan Shian Huang, and Jung San HuangDagger

From the Department of Biochemistry and Molecular Biology, St. Louis University School of Medicine, St. Louis, Missouri 63104

    ABSTRACT
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Abstract
Introduction
References

High affinity insulin-like growth factor-binding proteins (IGFBP-1 to -6) are a family of structurally homologous proteins that induce cellular responses by insulin-like growth factor (IGF)-dependent and -independent mechanisms. The IGFBP-3 receptor, which mediates the IGF-independent growth inhibitory response, has recently been identified as the type V transforming growth factor-beta receptor (Tbeta R-V) (Leal, S. M., Liu, Q. L., Huang, S. S., and Huang, J. S. (1997) J. Biol. Chem. 272, 20572-20576). To characterize the interactions of high affinity IGFBPs with Tbeta R-V, mink lung epithelial cells (Mv1Lu cells) were incubated with 125I-labeled recombinant human IGFBPs (125I-IGFBP-1 to -6) in the presence of the cross-linking agent disuccinimidyl suberate and analyzed by 5% SDS-polyacrylamide gel electrophoresis and autoradiography. 125I-IGFBP-3, -4, and -5 but not 125I-IGFBP-1, -2, and -6 bound to Tbeta R-V as demonstrated by the detection of the ~400-kDa 125I-IGFBP·Tbeta R-V cross-linked complex in the cell lysates and immunoprecipitates. The analyses of 125I-labeled ligand binding competition and DNA synthesis inhibition revealed that IGFBP-3 was a more potent ligand for Tbeta R-V than IGFBP-4 or -5. Most of the high affinity 125I-IGFBPs formed dimers at the cell surface. The cell-surface dimer of 125I-IGFBP-3 preferentially bound to and was cross-linked to Tbeta R-V in the presence of disuccinimidyl suberate. IGFBP-3 did not stimulate the cellular phosphorylation of Smad2 and Smad3, key transducers of the transforming growth factor-beta type I/type II receptor (Tbeta R-I·Tbeta R-II) heterocomplex-mediated signaling. These results suggest that IGFBP-3, -4, and -5 are specific ligands for Tbeta R-V, which mediates the growth inhibitory response through a signaling pathway(s) distinct from that mediated by the Tbeta R-I and Tbeta R-II heterocomplex.

    INTRODUCTION
Top
Abstract
Introduction
References

High affinity insulin-like growth factor-binding proteins 1-6 (IGFBP-1 to -6)1 are a family of structurally homologous ~24-43-kDa proteins composed of three defined domains including a nonconserved central domain flanked by conserved cysteine-rich N- and C-terminal domains (1-3). Recently, several low affinity IGFBPs with sequence homology to the N-terminal domains of the high affinity IGFBPs have been identified and referred to as IGFBP-7 to -10 (4).

High affinity IGFBPs are produced by a variety of cell types and tissues (1-3). They coordinate and regulate the biological activities of IGF-I and IGF-II by serving as transporter proteins or carriers and by scavenging IGFs from IGF receptors (1-3). High affinity IGFBPs have also been shown to induce cellular responses in an IGF-independent manner (5-15). These IGF-independent actions of high affinity IGFBPs are believed to be mediated by specific cell-surface receptors or membrane proteins (6, 16-18). The IGFBP-3 receptor, which mediates the IGF-independent growth inhibitory response, has been recently identified as the type V TGF-beta receptor (Tbeta R-V) (19).

The Tbeta R-V is a 400-kDa non-proteoglycan membrane glycoprotein (20). It is a Ser-specific protein kinase and co-expresses with type I, type II, and type III TGF-beta receptors (Tbeta R-I, Tbeta R-II, and Tbeta R-III) in most cell types (21-23). The Tbeta R-V is a low affinity TGF-beta receptor with Kd of ~0.4 nM for TGF-beta 1 and TGF-beta 2 and ~5 nM for TGF-beta 3 (23, 24). Nevertheless, several lines of evidence suggest that Tbeta R-V is important in mediating TGF-beta -induced growth inhibitory responses. These include the following: 1) cells lacking Tbeta R-V but expressing Tbeta R-I, Tbeta R-II, and Tbeta R-III do not exhibit the growth inhibitory response to stimulation by exogenous TGF-beta , although exogenous TGF-beta is able to induce transcriptional activation of plasminogen activator inhibitor 1 and fibronectin in these cells (24); 2) Tbeta R-V mediates the growth inhibitory response in the absence of Tbeta R-I or Tbeta R-II, but both Tbeta R-I and Tbeta R-II are required for maximal growth inhibition (24); and 3) the cells lacking Tbeta R-V have been found to be carcinoma cells, whereas all normal cell types studied express Tbeta R-V (19, 21, 24). This implies that the loss of Tbeta R-V, which mediates negative growth regulation, may contribute to malignancy of certain carcinoma cells (19, 21, 24).

To define the function of Tbeta R-V, we developed specific peptide antagonists that showed higher affinity to Tbeta R-V than to other TGF-beta receptor types (25). The structural and functional analyses of these peptide antagonists revealed that a W/RXXD motif is essential for the antagonist activity. Multiple conjugation of the peptide antagonists to carrier proteins conferred TGF-beta agonist activity in growth inhibition but not in transcriptional activation (25). These results prompted us to identify structurally unrelated TGF-beta agonists that possess the W/RXXD motif. IGFBP-3 was the first TGF-beta agonist identified (19). IGFBP-3 possesses a putative TGF-beta active site motif (WCVD) near its C terminus (1-3).

Because IGFBP-3 is structurally homologous to other high affinity IGFBPs and because four of six high affinity IGFBPs (IGFBP-3 to -6) possess the putative TGF-beta active site motif (WCVD) near their C termini (1-3), we hypothesized that at least some of these IGFBPs might bind to Tbeta R-V, which may mediate the IGF-independent activities of these IGFBPs. To test this hypothesis, we characterized the interactions of IGFBP-1 to -6 with Tbeta R-V in mink lung epithelial cells (Mv1Lu cells). In this communication, we show that IGFBP-3, -4, and -5 but not IGFBP-1, -2, or -6 bind to Tbeta R-V as demonstrated by 125I-labeled ligand affinity labeling of Tbeta R-V in Mv1Lu cells. IGFBP-4 and -5 bind to Tbeta R-V with lower affinities than that of IGFBP-3. The cell surface-associated dimeric form of IGFBP-3 exhibits a preference for binding to Tbeta R-V. We also demonstrate that IGFBP-3-induced growth inhibition mediated by Tbeta R-V does not involve the stimulated phosphorylation of Smad2 and Smad3.

    EXPERIMENTAL PROCEDURES

Materials-- Na125I (17 Ci/mg), [methyl-3H]thymidine (67 Ci/mmol), and [32P]orthophosphate (500 mCi/ml) were purchased from ICN Biochemicals, Inc. (Costa Mesa, CA). High and low molecular mass protein standards, recombinant human IGFBP-1, avidin-agarose, and other chemical reagents were purchased from Sigma. Disuccinimidyl suberate (DSS) and sulfo-NHS-biotin were obtained from Pierce. Anti-Smad2, anti-Smad3, and goat IgG were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Recombinant human TGF-beta 1, IGFBP-2, IGFBP-4, IGFBP-5, and IGFBP-6 were obtained from Austral Biologicals (San Ramon, CA). Recombinant nonglycosylated human IGFBP-3 (expressed in Escherichia coli) was provided by Celtrix Pharmaceutical, Inc. (Santa Clara, CA). 125I-Labeled IGFBPs (125I-IGFBPs) and antiserum to Tbeta R-V were prepared according to our published procedures (19). The TGF-beta 1 peptide antagonist, beta 1-(41-65), was synthesized as described previously (25). Mv1Lu cells were grown and maintained in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal calf serum (FCS).

125I-IGFBP Affinity Labeling of Mv1Lu Cells-- Mv1Lu cells grown to confluency in 35-mm Petri dishes were incubated with 1 nM 125I-IGFBPs (specific activity, 10-90 µCi/ng) in the presence of various concentrations (1-100 nM) of unlabeled IGFBPs with and without 10 µM beta 1-(41-65) in binding medium (125 mM NaCl, 5 mM KCl, 5 mM MgSO4, 2 mM CaCl2, 50 mM Hepes, pH 7.5) at 0 °C for 4 h. After 125I-IGFBP affinity labeling in the presence of DSS (19), the 125I-IGFBP·Tbeta R-V complex was analyzed by 5% SDS-polyacrylamide gel electrophoresis (PAGE) under reducing conditions and autoradiography.

[methyl-3H]Thymidine Incorporation Assay-- Mv1Lu cells were plated on 24-well cluster dishes at near confluence in DMEM containing 10% FCS. Within 6-8 h after plating, cells were rinsed twice with serum-free DMEM and incubated with various concentrations of IGFBPs in DMEM containing 0.1% FCS. After 18 h at 37 °C, the [methyl-3H]thymidine incorporation into cellular DNA was determined as described previously (19).

Biotinylation of IGFBP-3 and Detection of the 125I-IGFBP-3-biotinylated IGFBP-3 Complex-- The biotinylation of IGFBP-3 was carried out in a reaction mixture (50 µl) containing IGFBP-3 and sulfo-NHS-biotin (1:25, mol/mol) in 50 mM NaHCO3, pH 8.5. After 30 min at room temperature, the reaction was terminated by the addition of glycine (10 mM). For dimer formation studies, Mv1Lu cells were incubated with a premix of 8 nM 125I-IGFBP-3 and 2 nM biotinylated IGFBP-3 at 0 °C for 4 h in the absence or presence of a 100-fold excess of unlabeled IGFBP-3. Cells were rinsed twice with 1 ml of binding medium, collected by scraping, and pelleted by centrifugation at 10,000 rpm for 5 min. The cell pellets were lysed in solubilization buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.4) containing 1% Triton X-100 and mixed for 1 h at 4 °C followed by centrifugation at 12,000 rpm for 10 min. The supernatant was transferred to an Eppendorf tube containing 0.036 mg of avidin-agarose (10 µl of suspension) and mixed at 4 °C for 1 h in the absence or presence of D-biotin (20 µg or ~1.6 mM) (to estimate nonspecific binding). The avidin-agarose beads bound by biotinylated IGFBP-3 and 125I-IGFBP-3-biotinylated IGFBP-3 complexes were washed once with 1 ml of solubilization buffer containing Triton X-100 (0.2%) and 0.5 M NaCl followed by two more washes with salt-free solubilization buffer. Concentrated SDS sample buffer (2×) containing beta -mercaptoethanol was added to the agarose beads. The bead suspension was boiled for 5 min and vortexed vigorously to release biotinylated IGFBP-3 and 125I-IGFBP-3 from the beads. The agarose beads were pelleted, and the supernatant was analyzed by 12% SDS-PAGE and autoradiography.

[32P]Orthophosphate Metabolic Labeling and Immunoprecipitation by Anti-Smad2 and Anti-Smad3 IgGs-- Mv1Lu cells were plated on 100-mm Petri dishes in DMEM containing 10% FCS at near confluency. After 16-18 h at 37 °C, cells (14 × 106 total) were rinsed twice with phosphate-free DMEM and incubated with 5 ml of phosphate-free DMEM containing 0.2% dialyzed FCS for 1 h at 37 °C. Cells were metabolically labeled with 1.0 mCi/ml [32P]orthophosphate in phosphate-free DMEM containing 0.2% dialyzed FCS for 2 h at 37 °C. The 32P metabolically labeled cells were treated with TGF-beta 1 (10 ng/ml or 0.4 nM) or IGFBP-3 (1 µg/ml or ~33 nM) for an additional 3 h at 37 °C, rinsed twice with 5 ml of cold phosphate-buffered saline, and lysed with lysis buffer (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.5% Triton X-100, 50 mM NaF, 10 mM sodium pyrophosphate, 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, and 20 µg/ml aprotinin) for 10 min at 0 °C followed by repeated aspiration through a 21-gauge needle. The cell lysates were centrifuged at 10,000 rpm for 15 min, and the supernatant was precleared with 1 µg of goat IgG and protein G-Sepharose at 4 °C. The precleared cell lysates were incubated with anti-Smad2 and anti-Smad3 IgGs (2 µg) for 2.5 h at 4 °C and incubated with protein G-Sepharose for an additional 1 h. The protein G-Sepharose beads were rinsed 4 times with 1 ml of lysis buffer, suspended in 40 µl of SDS sample buffer containing beta -mercaptoethanol, and boiled for 5 min. The immunoprecipitates were analyzed by 7.5% SDS-PAGE and autoradiography.

    RESULTS

IGFBP-3, -4, and -5 Bind to Tbeta R-V with Different Affinities-- Mv1Lu cells have been used as a model cell system to investigate Tbeta R-V and other TGF-beta receptor types and TGF-beta -induced cellular responses (19, 20, 24, 26). To determine the interactions of Tbeta R-V with IGFBPs, we first performed ligand affinity labeling of Tbeta R-V in Mv1Lu cells using 125I-labeled recombinant human IGFBP-1 to -6 (125I-IGFBP-1 to -6). After incubation of Mv1Lu cells with 5 nM 125I-IGFBP-1, -2, -3, -4, -5, or -6 at 0 °C for 3 h, the ~400-kDa 125I-IGFBP·Tbeta R-V complex was cross-linked with DSS and identified by 5% SDS-PAGE under reducing conditions and autoradiography. As shown in Fig. 1, the 125I-IGFBP·Tbeta R-V complex was detected in the lysates of cells that were affinity-labeled with 125I-IGFBP-3, 125I-IGFBP-4, or 125I-IGFBP-5 (lanes 1, 3, and 5). The specificity of the affinity labeling of Tbeta R-V was supported by blocking with beta 1-(41-65), a specific TGF-beta antagonist (25) (lanes 2, 4, and 6). The 125I-IGFBP·Tbeta R-V complex was not detected in the lysates of cells affinity-labeled with 125I-IGFBP-1, -2, or -6 (lanes 7, 9, and 11). The 125I-IGFBP·Tbeta R-V complex was verified by its immunoprecipitation with specific antiserum to Tbeta R-V (Fig. 2, lanes 2, 5, and 8). The immunoprecipitation of the 125I-IGFBP-3·Tbeta R-V complex by antiserum to Tbeta R-V was previously reported (19). It is of importance to note that all 125I-IGFBPs except 125I-IGFBP-2 formed covalently linked dimers that were stable after treatment at 100 °C for 5 min in 0.1% SDS containing beta -mercaptoethanol and subsequent SDS-PAGE (Fig. 1).


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Fig. 1.   125I-IGFBP affinity labeling in Mv1Lu cells. Cells were incubated with 5 nM 125I-IGFBP-1 to -6 in the presence (+) and absence (-) of 10 µM beta 1-(41-65). After 3 h at 0 °C, the 125I-IGFBP affinity labeling was carried out and analyzed by 5% SDS-PAGE under reducing conditions and autoradiography. The arrows indicate the location of the ~400-kDa 125I-IGFBP·Tbeta R-V complex. The asterisks denote the locations of the covalently linked 125I-IGFBP dimers.


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Fig. 2.   Immunoprecipitation of the 125I-IGFBP-3, -4, and -5 affinity-labeled Tbeta R-V in Mv1Lu cells. Cells were incubated with 10 nM 125I-IGFBP-3 (A), 25 nM 125I-IGFBP-4 (B), or 25 nM 125I-IGFBP-5 (C) both with and without 10 µM beta 1-(41-65), a TGF-beta /IGFBP-3 antagonist. After 3 h at 0 °C and affinity labeling, cell lysates were immunoprecipitated with antiserum to Tbeta R-V or non-immune (Control serum) serum (19). The immunoprecipitates were analyzed by 5% SDS-PAGE under reducing conditions and autoradiography. The brackets indicate the locations of the 125I-IGFBP·Tbeta R-V complexes. The asterisk denotes the location of the 125I-IGFBP-3 dimer. The arrows indicate the location of dye front.

The Kd of IGFBP-3 binding to Tbeta R-V was previously estimated to be ~6 nM (19). To determine the relative affinities of IGFBP-4 and -5 to Tbeta R-V in Mv1Lu cells, we performed competition experiments using 125I-IGFBP-3 (1 nM) as the ligand and unlabeled IGFBP-3, -4, and -5 as competitors. As shown in Fig. 3A, increasing concentrations of unlabeled IGFBP-3 quantitatively inhibited 125I-IGFBP-3 binding to Tbeta R-V as determined by 125I-IGFBP-3 affinity labeling of Tbeta R-V. The quantitative analysis of this inhibition revealed that unlabeled IGFBP-3 blocked the 125I-IGFBP-3 binding with an IC50 of ~6 nM, which is identical with the estimated Kd of IGFBP-3 binding to Tbeta R-V (19) (Fig. 3B). Unlabeled IGFBP-4 and -5 weakly inhibited 125I-IGFBP-3 binding to Tbeta R-V with an IC50 of >= 100 nM (Fig. 3B). We also determined the effects of various concentrations of unlabeled IGFBP-1, -2, and -6 on 125I-IGFBP-3 binding to Tbeta R-V. Unlabeled IGFBP-1, -2, and -6 did not show any significant effect on the binding of 125I-IGFBP-3 to Tbeta R-V at concentrations up to 100 nM (data not shown). These results suggest that IGFBP-3 binds to Tbeta R-V with higher affinity than IGFBP-4 and -5 and that IGFBP-1, -2, and -6 are not ligands for Tbeta R-V.


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Fig. 3.   Effects of unlabeled IGFBP-3, -4, and -5 on the formation of the 125I-IGFBP-3·Tbeta R-V complex in Mv1Lu cells. Cells were incubated with 1 nM 125I-IGFBP-3 in the presence of various concentrations of unlabeled IGFBP-3, -4, or -5 at 0 °C for 3 h. The 125I-IGFBP·3-Tbeta R-V complex was then cross-linked with DSS and analyzed by 5% SDS-PAGE under reducing conditions and autoradiography (A) or quantitation using a PhosphorImager (B). The relative level of the 125I-IGFBP-3·Tbeta R-V complex in cells incubated with 125I-IGFBP-3 in the absence of unlabeled IGFBPs was taken as 100% formation of the 125I-IGFBP-3·Tbeta R-V complex. The figure is representative of three experiments that gave comparable results. The relative level of the 125I-IGFBP-3·Tbeta R-V complex in the presence of 3 nM unlabeled IGFBP-5 was ~134%, which was unusually high and not reproducible.

IGFBP-3 was previously shown to inhibit growth of Mv1Lu cells as measured by DNA synthesis (19). This inhibition appeared to be mediated by Tbeta R-V because the IGFBP-3-induced growth inhibition was blocked in the presence of beta 1-(41-65), a specific TGF-beta peptide antagonist that blocked IGFBP-3 binding to Tbeta R-V (19). Because IGFBP-3, -4, and -5 bind to Tbeta R-V with different affinities, we determined the relative potencies of IGFBP-3, -4, and -5 for DNA synthesis inhibition in Mv1Lu cells. As shown in Fig. 4, at 1 µg/ml (~33 nM) IGFBP-3 inhibited ~50% of DNA synthesis of Mv1Lu cells, whereas IGFBP-4 and -5 produced ~15-20% inhibition of DNA synthesis at the same concentration. The potent DNA synthesis inhibitory activity of IGFBP-3 is consistent with its high affinity to Tbeta R-V. We also determined the effects of IGFBP-1, -2, and -6 on DNA synthesis of Mv1Lu cells. IGFBP-1, -2, and -6 exhibited ~5-10% inhibition of DNA synthesis of Mv1Lu cells at 1 µg/ml (data not shown). This inhibition may be because of scavenging endogenous IGFs from the IGF-1 receptor. IGF-1 is a weak growth factor or mitogen for Mv1Lu cells under these experimental conditions.


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Fig. 4.   Effects of IGFBP-3, -4, and -5 on DNA synthesis of Mv1Lu cells. Cells were incubated with various concentrations of IGFBP-3, -4, or -5. After 16 h at 37 °C, the [methyl-3H]thymidine incorporated into cellular DNA was determined. The experiment was performed in triplicate cell cultures. The error bars represent means ± S.E. The data were obtained from three independent experiments.

IGFBP-3 Forms Dimers at the Cell Surface That Preferentially Bind to Tbeta R-V-- As demonstrated in Fig. 1, most high affinity 125I-IGFBPs formed covalently linked dimers at the cell surface. This experiment was performed in the presence of the cross-linking agent DSS. Because 125I-labeled proteins prepared by the chloramine-T procedure are known to acquire the properties of covalent linking during 125I-labeling (27-29), the covalently linked dimers of 125I-IGFBPs may be spontaneously produced in a DSS-independent manner during the incubation (3 h at 0 °C) of cells with 125I-IGFBPs that were also prepared using chloramine T. To test this possibility, we investigated the formation of the covalently linked dimer of 125I-IGFBP-3 in aqueous solution and at the cell surface of Mv1Lu cells in the absence of added cross-linking agents. As shown in Fig. 5, less than 2% of 125I-IGFBP-3 (5 nM) spontaneously formed covalently linked dimers in binding medium (lane 1). However, approximately 20% of the 125I-IGFBP-3 associated with the cell surface was found to be covalently linked dimers (lane 2). These results indicate that the formation of the covalently linked 125I-IGFBP-3 dimer does not require the presence of cross-linking agents. These results also suggest that the 125I-IGFBP-3 dimer formation may be enhanced at the cell surface. Alternatively, the 125I-IGFBP-3 dimer may associate with the cell surface of Mv1Lu cells with an affinity higher than that of the 125I-IGFBP-3 monomer.


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Fig. 5.   Formation of the covalently linked 125I-IGFBP-3 dimer in binding medium and at the cell surface of Mv1Lu cells in the absence of added cross-linking agents. Five nM 125I-IGFBP-3 was incubated with or without Mv1Lu cells in binding medium containing 0.2% bovine serum albumin. After 3 h at 0 °C, the cell lysates and an aliquot of the binding medium were analyzed by 10% SDS-PAGE under reducing conditions and autoradiography. The arrows indicate the locations of the covalently linked 125I-IGFBP dimer and 125I-IGFBP-3 monomers. The relative intensities of covalently linked IGFBP-3 dimers and monomers were quantitated using a PhosphorImager.

As described above, we demonstrated the covalently linked dimer formation of 125I-IGFBP-3 or other 125I-IGFBPs by taking advantage of the properties of covalent linking of 125I-IGFBPs prepared by the chloramine-T procedure. To prove that the dimer formation is an inherent property of IGFBP-3, we determined the formation of the IGFBP-3 dimer using an approach in which IGFBP-3 was tagged with 125I or biotin. The formation of the IGFBP-3 dimer was detected by identifying the 125I-IGFBP-3-biotinylated IGFBP-3 complex in the lysates of Mv1Lu cells that were incubated with a premix of 125I-IGFBP-3 and biotinylated IGFBP-3 (4:1, mol/mol). After 2.5 h at 0 °C, the cell lysates were incubated with avidin-agarose. After centrifugation, the avidin-agarose pellets were analyzed by 7.5% SDS-PAGE under reducing conditions and autoradiography. As shown in Fig. 6 (lane 1), 125I-IGFBP-3 was detected in the avidin-agarose pellets of lysates of cells incubated with a premix of 125I-IGFBP-3 and biotinylated IGFBP-3. Very little 125I-IGFBP-3 was detected in the avidin-agarose pellets of lysates of cells incubated with a premix of 125I-IGFBP-3 and biotinylated IGFBP-3 in the presence of a 100-fold excess of unlabeled IGFBP-3 or ~1.6 mM biotin (Fig. 6, lanes 3 and 2). These results further verify the ability of IGFBP-3 to form dimers at the cell surface.


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Fig. 6.   Complex formation of 125I-IGFBP-3 with biotinylated IGFBP-3 in Mv1Lu cells. Cells were incubated with a premix of 125I-IGFBP-3 and biotinylated IGFBP-3 (4:1, mol/mol) in the presence and absence of a 100-fold excess of unlabeled IGFBP-3. After 3 h at 0 °C, the cells were washed; the cell lysates were incubated with avidin-agarose with or without ~1.6 mM biotin at 4 °C for 1 h. After centrifugation, the pellets were analyzed by 12% SDS-PAGE under reducing conditions and autoradiography. The arrow indicates the location of 125I-IGFBP-3.

TGF-beta is known to stimulate cellular responses by inducing hetero-oligomerization of TGF-beta receptors through its covalent dimeric structure (30, 31). We hypothesize that IGFBP-3 inhibits cellular growth by a similar mechanism in which the dimeric form of IGFBP-3 is required for activation of Tbeta R-V. To test this hypothesis, we determined the binding of the dimeric form of IGFBP-3 to Tbeta R-V in Mv1Lu cells in the presence and absence of DSS. As shown in Fig. 7, the covalently linked dimer of 125I-IGFBP-3 was detected in the medium (lane 1) and lysates (lane 3) of cells incubated with 125I-IGFBP-3 without added cross-linking agents. It is of importance to note that the exposure times for the autoradiograms of the medium and cell lysates were 16 and 2 h, respectively, to have comparable intensities of covalently linked 125I-IGFBP-3 dimers. In the presence of the cross-linking agent DSS, most of the covalently linked dimer associated with the cell surface was cross-linked to Tbeta R-V (lane 4 versus 3). These results suggest that the cell surface-associated dimeric form of IGFBP-3 preferentially binds to Tbeta R-V. These results are also consistent with our previous observation that both Tbeta R-V and 125I-IGFBP-3 dimers were immunoprecipitated by specific antiserum to Tbeta R-V after the 125I-IGFBP-3-affinity labeling of Tbeta R-V in Mv1Lu cells (19).


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Fig. 7.   Binding and cross-linking of the cell surface covalently linked 125I-IGFBP-3 dimer to Tbeta R-V in Mv1Lu cells. Cells were incubated with 5 nM 125I-IGFBP-3 at 0 °C for 3 h and then treated with (+) or without (-) DSS (0.3 mM) for an additional 15 min. The medium and cell lysates were analyzed by 5% SDS-PAGE under reducing conditions and autoradiography. The exposure times for the autoradiograms of the medium and cell lysates were 16 and 2 h, respectively. Arrows indicate the locations of the 125I-IGFBP-3·Tbeta R-V cross-linked complex and covalently linked 125I-IGFBP-3 dimers. The crescent shape of the 125I-IGFBP-3 dimer in the medium (lanes 1 and 2) is because of the influence from a large quantity of bovine serum albumin in the binding medium, which migrates closely with the 125I-IGFBP-3 dimer on the SDS-polyacrylamide gel.

IGFBP-3 Does Not Stimulate the Cellular Phosphorylation of Smad2 and Smad3-- In Mv1Lu cells, Smad2 and Smad3 have been identified as key signal transducers within the signal transduction cascade initiated by the Tbeta R-I·Tbeta R-II heterocomplex following stimulation by TGF-beta (32). The phosphorylation of Smad2 and Smad3 by Tbeta R-I is essential for their complex formation with Smad4 and subsequent translocation to the nucleus where they regulate transcriptional activities required for cell cycle arrest and other cellular responses (32, 33). Because Tbeta R-V forms complexes with Tbeta R-I (24), the phosphorylation of Smad2 and Smad3 may also be involved in the signaling mediated by the Tbeta R-I·Tbeta R-V heterocomplex (24). To test this possibility, we investigated the effect of IGFBP-3 on the phosphorylation of Smad2 and Smad3 in Mv1Lu cells. As shown in Fig. 8, the phosphorylation of Smad2 and Smad3 was not affected by IGFBP-3 treatment (lane 4 versus 2), but TGF-beta treatment enhanced the phosphorylation of Smad2 and Smad3 by ~7- and ~2-fold, respectively (lane 3 versus 2). This result suggests that the IGFBP-3-induced growth inhibition, which is mediated by Tbeta R-V, may involve a signaling pathway that is distinct from that mediated by the Tbeta R-I·Tbeta R-II heterocomplex.


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Fig. 8.   Effect of IGFBP-3 on the phosphorylation of Smad2 and Smad3 in Mv1Lu cells. Cells labeled metabolically with [32P]orthophosphate were treated with or without IGFBP-3 (33 nM) or TGF-beta (0.1 nM) at 37 °C for 2 h. The 32P-labeled cell lysates were immunoprecipitated with a mixture of anti-Smad2 and anti-Smad3 IgGs or control IgG. The immunoprecipitates were analyzed by 7.5% SDS-PAGE under reducing conditions. The arrows indicate the locations of 32P-labeled Smad2 and Smad3. The bars indicate the locations of the immunologically cross-reacted proteins that were not immunoprecipitated by control IgG. The relative levels of phosphorylated Smad2 and Smad3 in cells treated with and without IGFBP-3 or TGF-beta were quantitated by a PhosphorImager.


    DISCUSSION

High affinity IGFBPs are important modulators of IGF actions (1-3). Accumulated evidence suggests that IGFBPs are also able to induce cellular responses in an IGF-independent manner (5-15). The IGF-independent actions for IGFBPs are believed to be mediated by specific cell-surface receptors or membrane-binding proteins (1, 16-18). Several membrane-binding proteins for IGFBPs were identified, but none of these proteins were well characterized (6, 16-18). We have recently identified the IGFBP-3 receptor as Tbeta R-V, which mediates the IGF-independent growth inhibitory response induced by IGFBP-3 (19). In this communication, we show that IGFBP-4 and -5 are also specific ligands for Tbeta R-V, although their affinities for Tbeta R-V are weaker than that of IGFBP-3. The Tbeta R-V is likely the same receptor for IGFBP-5, which has been recently identified in mouse osteoblasts (34). The Tbeta R-V and putative IGFBP-5 receptor in osteoblasts share similar properties including the following: 1) they have almost identical molecular weights (~400,000) (19-25, 34), 2) both show ligand (TGF-beta /IGFBP-3 and IGFBP-5)-stimulated serine-specific autophosphorylation and kinase activity toward caseins2 (23, 24, 34), and 3) the Tbeta R-V is expressed in most cell types including osteoblasts (21).3

Among high affinity IGFBPs, four (IGFBP-3 to -6), which possess the putative TGF-beta active site motif WCVD near their C termini, were initially predicted to bind to Tbeta R-V. However, although IGFBP-3, -4, and -5 were found to interact with Tbeta R-V in Mv1Lu cells, IGFBP-6 did not. The inability of IGFBP-6 to interact with Tbeta R-V may be because of its unique structure. IGFBP-6 contains 10 of 12 N-terminal cysteine residues conserved in other high affinity IGFBPs and possesses additional O-linked carbohydrate moieties in the central domain and possibly near the C-terminal end (1-3, 35, 36). These distinct structural features may yield a conformation that does not allow the WCVD motif in IGFBP-6 to interact with Tbeta R-V. It is also possible that the WCVD motif is not the only determinant required for the interactions of IGFBPs with Tbeta R-V. The WCVD motif is contained within the thyroglobulin type-1 repeat of IGFBP-3 (37). Thyroglobulin, which contains multiple WCVD motifs per monomer, has recently been shown to exhibit an authentic TGF-beta antagonist/agonist activity after activation by acidic pH/denaturing agent treatments and chemical modifications (38). This implies that certain structural configurations of the WCVD motif are required for optimal interaction with Tbeta R-V.

Several polypeptide growth factors are known to stimulate the cytoplasmic kinase activities of their respective receptors by inducing receptor dimerization through their dimeric structures (39-41). The covalent dimeric structure is also known to be required for TGF-beta activities (42). Most 125I-labeled IGFBPs form covalently linked dimers at the cell surface. Approximately 20% of cell surface-associated 125I-IGFBP-3 is estimated to be in the form of covalently linked dimers, whereas less than 2% exists as the covalently linked dimer in binding medium. This suggests that the cell surface association enhances the dimer formation of IGFBP-3. Assuming that the efficiency of the spontaneous covalent linking of 125I-IGFBP-3 is ~20%, it is estimated that almost 100% of the cell surface-associated 125I-IGFBP-3 are dimers. The cell-surface dimeric form of IGFBP-3 appears to be the active form of IGFBP-3 for binding to Tbeta R-V.

The major cell-surface binding sites for IGFBP-3 dimers appear to be membrane proteins other than Tbeta R-V because cells lacking Tbeta R-V (human colorectal carcinoma cells) express these binding sites (19). Interestingly, the binding of 125I-IGFBP-3 and 125I-IGFBP-5 dimers to their major cell-surface binding sites is blocked by beta 1-(41-65), a specific TGF-beta peptide antagonist, whereas the binding of 125I-IGFBP-1 and -4 dimers to their major binding sites is resistant to the blocking by the TGF-beta peptide antagonist (Fig. 1). This suggests that the major cell-surface binding sites for IGFBP-3 and IGFBP-5 dimers are distinct from those for other IGFBPs dimers. This suggestion has been supported by the observation that heparin inhibits the binding of 125I-IGFBP-3 and -5 dimers but not 125I-IGFBP-1 and -4 dimers to cell-surface binding sites (18, 19).4 The functions of these major cell-surface binding sites are unknown. However, one function may involve presentation of IGFBPs to their respective cell-surface receptors. In the case of IGFBP-3, these binding sites may present IGFBP-3 to Tbeta R-V as demonstrated in Fig. 7. This would explain the observation that heparin inhibits the binding of 125I-IGFBP-3 to both the major cell-surface binding sites and to the Tbeta R-V (18, 19).4

The signaling mediated by Tbeta R-V has been difficult to define because of the co-expression of Tbeta R-I, Tbeta R-II, Tbeta R-III, and Tbeta R-V in the same cells. The identification of IGFBP-3 as well as IGFBP-4 and -5 as specific ligands for Tbeta R-V has enabled us to investigate the signaling mediated by Tbeta R-V in cells containing other TGF-beta receptors. In this communication, we show that IGFBP-3 does not stimulate the cellular phosphorylation of Smad2 and Smad3, both of which play key roles in the signaling mediated by the Tbeta R-I and Tbeta R-II heterocomplex (31, 32). This result is consistent with the observation that IGFBP-3 induces growth inhibition but not transcriptional activation of plasminogen activator inhibitor-1 in Mv1Lu cells (19). The TGF-beta -induced expression of plasminogen activator inhibitor-1 is mainly mediated by the Tbeta R-I·Tbeta R-II complex (24). Furthermore, IGFBP-3 has been shown to inhibit the growth of mutant mink lung epithelial cells (DR26 and R-1B cells), which express Tbeta R-V but lack the expression of the functional Tbeta R-II or Tbeta R-I (24).

    ACKNOWLEDGEMENTS

We thank Celtrix Pharmaceutical, Inc. for providing recombinant nonglycosylated human IGFBP-3, Drs. William S. Sly and Frank E. Johnson for critical review of the manuscript, and John H. McAlpin for typing the manuscript.

    FOOTNOTES

* This work was supported by National Institutes of Health Grant CA 38808 and a predoctoral fellowship (to S. M. L.) from the American Heart Association, Missouri Affiliate.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, St. Louis University School of Medicine, 1402 South Grand Blvd., St. Louis, MO 63104. Tel.: 314-577-8135; Fax: 314-577-8156; E-mail: huangjs{at}wpogate.slu.edu.

4 S. M. Leal, S. S. Huang, and J. S. Huang, unpublished results.

3 S. S. Huang and J. S. Huang, unpublished results.

2 T. Zhao, Q. Liu, S. S. Huang, and J. S. Huang, unpublished results.

    ABBREVIATIONS

The abbreviations used are: IGFBP, insulin-like growth factor-binding protein; IGF, insulin-like growth factor; TGF-beta , transforming growth factor-beta ; Tbeta R-V, type V TGF-beta receptor; DSS, disuccinimidyl suberate; DMEM, Dulbecco's modified Eagle's medium; FCS, fetal calf serum; PAGE, polyacrylamide gel electrophoresis.

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