©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Cleavage Analysis of Insulin-like Growth Factor (IGF)-dependent IGF-binding Protein-4 Proteolysis and Expression of Protease-resistant IGF-binding Protein-4 Mutants (*)

(Received for publication, July 15, 1994; and in revised form, December 12, 1994)

Cheryl A. Conover (1)(§) Susan K. Durham (1) Jürgen Zapf (2) Frank R. Masiarz (3) Michael C. Kiefer (3) (4)

From the  (1)Endocrine Research Unit, Division of Endocrinology and Metabolism, Mayo Clinic and Mayo Foundation, Rochester, Minnesota 55905, the (2)Metabolic Unit, Department of Medicine, University Hospital, CH-8091 Zurich, Switzerland, (3)Chiron Corporation, Emeryville, California 94608, and (4)LXR Biotechnology Inc., Richmond, California 94804

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Cultured human fibroblasts and osteoblast-like cells secrete an insulin-like growth factor (IGF)-dependent protease that cleaves IGF-binding protein-4 (IGFBP-4) into two fragments of 18 and 14 kDa. Edman degradation of the isolated proteins established the amino termini of the reaction products. Sequence analysis of the 14-kDa carboxyl-terminal half of IGFBP-4 suggested cleavage after methionine at position 135 of the mature protein. Four variant IGFBP-4 molecules with single amino acid substitutions around this cleavage site were constructed and expressed. Wild-type and mutant IGFBPs-4 bound IGF-I and IGF-II with equivalent affinities and, in the intact state, were equally effective inhibitors of IGF-I action. However, the IGFBP-4 mutants were relatively resistant to IGF-dependent proteolysis. A 5-6-h incubation in human fibroblast conditioned medium in the presence of IGF-II was sufficient for near total hydrolysis of wild-type IGFBP-4, whereas the mutant IGFBPs-4 were only minimally affected at this time. After a 24-h incubation with IGF-II, all mutant IGFBPs-4 showed extensive proteolysis, generating 18- and 14-kDa fragments. Pre-exposure of human fibroblasts in serum-free conditioned medium to IGF-II for 5 h potentiated subsequent IGF-I stimulation of DNA synthesis. When added with IGF-II, the protease-resistant mutant IGFBPs-4, but not wild-type IGFBP-4, suppressed IGF-II enhancement of IGF-I-stimulated DNA synthesis. These biological studies suggest that the IGFBP-4/IGFBP-4 protease system may play a role modulating local cellular response to IGF-I.


INTRODUCTION

Insulin-like growth factor-binding protein-4 (IGFBP-4) (^1)is expressed and secreted by a variety of cell types and is an effective inhibitor of IGF action in vivo and in vitro(1, 2, 3, 4, 5, 6, 7) . Regulation of IGFBP-4 bioavailability occurs at the level of IGFBP-4 gene expression and also through post-translational modification of the secreted protein. We (7, 9, 10) and others (8, 11) have identified an IGF-dependent IGFBP-4 protease secreted by human fibroblasts and human osteoblast-like cells that cleaves the IGFBP-4 molecule (24 kDa unreduced, 32 kDa reduced) into two fragments of 18 and 14 kDa. This specific proteolytic cleavage decreases the affinity of IGFBP-4 for IGF peptide, resulting in increased IGF bioactivity(7) . Thus, the IGFBP-4/IGFBP-4 protease system, by virtue of its tight and focused control of IGF action, could be important in regulating localized cell growth.

In this study, we identify an IGFBP-4 cleavage site by sequencing purified 18- and 14-kDa reaction products of IGF-dependent IGFBP-4 proteolysis. Using this information, we constructed and expressed variant IGFBP-4 molecules with amino acid substitutions around the cleavage site. These ``proteolysis'' mutants were employed to study the role of the IGFBP-4/IGFBP-4 protease system in modulating cellular response to IGFs.


EXPERIMENTAL PROCEDURES

Materials

Recombinant human IGF-I and IGF-II were purchased from Amgen Biologicals (Thousand Oaks, CA) and Bachem California (Torrance, CA), respectively. Crystalline human and bovine insulins were provided by Lilly. IGFBP-4 antiserum was generated against rhIGFBP-4 in rabbits and is highly specific for IGFBP-4(5, 7, 9) . Human and bovine fibroblasts (GM03652 and GM06034) were obtained from the Human Genetic Mutant Cell Repository (Camden, NJ) and cultured as described previously(7, 8, 9, 10, 11, 12) . Human osteoblasts from normal adult donors were derived from trabecular bone obtained during orthopedic surgery(9) .

Sequence Analysis of IGFBP-4 Proteolytic Products

Large-scale proteolysis of IGFBP-4 was based on the cell-free IGFBP-4 protease assay described previously(7, 9) . 25 µg of rhIGFBP-4 were incubated with 200 µl of human fibroblast or human osteoblast-like cell conditioned medium and 0.8 µg of IGF-II for 72 h at 37 °C. Cell conditioned media incubated without rhIGFBP-4 or IGF-II were employed as controls. IGFBP-4 fragments were identified by Western immunoblotting of 2 µl of the reaction mixture with specific IGFBP-4 antiserum(5, 7) . The majority of the proteolysis sample was subjected to SDS-PAGE using a 1.5-mm-thick 15% slab gel. The sample was diluted with 4 times sample buffer containing dithiothreitol and heated for 10 min at 60 °C. The entire volume was placed in a single well using a 5-slot comb. After electrophoresis for 1 h at 25 mA, the current was increased to 50 mA, and electrophoresis was terminated after another 2.5 h. The proteins were transferred to an Applied Biosystems ProBlott polyvinylidene difluoride membrane and localized with Coomassie Blue. The 14- and 18-kDa bands were excised with a razor blade and subjected to Edman degradation using an Applied Biosystems Model 473A sequencer equipped with a BLOTT cartridge.

Oligonucleotide Synthesis

PCR and DNA sequencing primers were synthesized by the phosphoramidite method using an Applied Biosystems Model 380B synthesizer, purified by PAGE, and desalted on Sep-Pak C(18) cartridges (Waters Associates). The PCR mutagenesis primers were 45-mer oligonucleotides with the following sequences: M1, 5`CTCCCGGGGCGCCCCATTGACCTTCATCTGGCCCCCACTGGT-3`; M2, 5`-CTCCCGGGGCGCCCCATTGACCTGCATCTTGCCCCCACTGGT-3`; M3, 5`-CTCCCGGGGCGCCCCATTGACCTTCAGCTTGCCCCCACTGGT-3`; and M4, 5`-CTCCCGGGGCGCCCCATTGACCTTCTCCTTGCCCCCACTGGT-3`. The NarI-containing synthetic DNA fragment for pBluescript SK consisted of two oligonucleotides: RL1-27 (5`-AATTCGATATCAAGCTTGGCGCCACCG-3`) and RL2-27 (5`-TCGACGGTGGCGCCAAGCTTGATATCG-3`).

IGFBP-4 Mutant Constructions

The IGFBP-4 mutants were generated in several steps. cDNA encoding the COOH-terminal portion of IGFBP-4 (amino acids 140-237) was excised from pBS24Ub-IGFBP-4 (5) with NarI and SalI and ligated into pBlsc-Nar to generate pBlscBP4(140-237). pBlsc-Nar was made by digesting pBluescript SK (Stratagene, La Jolla, CA) with EcoRI and SalI and replacing the small multiple cloning site segment (containing the ClaI site) with a synthetic double-stranded DNA fragment containing a NarI site flanked by EcoRI and SalI sites. Mutations were generated in IGFBP-4 cDNA by PCR using mismatched primers (M1-M4). PCR was performed according to the suppliers of the PCR kit (Perkin-Elmer). Thirty cycles of PCR were performed in a Perkin-Elmer DNA thermal cycler with each cycle consisting of a 1-min denaturation step at 94 °C, a 2-min annealing step at 55 °C, and a 3-min extension step at 72 °C. An additional 7-min extension step was included after the last cycle. The 5`-PCR primer and the IGFBP-4 cDNA used as template were from the pBS24Ub-IGFBP-4 construction(5) . The 3`-reverse PCR primers (M1-M4) were complementary to the nucleotide sequence encoding amino acids 129-143 of mature IGFBP-4, but contained mismatches at appropriate codons to generate the mutants shown in Table 2. The M3 and M4 PCRs also contained 2.5% formamide(5) . The PCR products were treated as described (5) and then digested with SstII and NarI (located within the nucleotide sequences encoding amino acids 139-141), gel-purified, and ligated to SstII/NarI-digested pBlsc-BP4(140-237) to generate full-length IGFBP-4 mutants. These constructs were sequenced to confirm the mutations. The full-length IGFBP-4 mutant cDNAs were then excised with SstII and SalI, gel-purified, ligated into the yeast expression vector pBS24Ub, and introduced into Escherichia coli strain HB101 as described(5) .



Protein Expression and Purification

These methods have been previously described(5) . Briefly, yeast lysates were purified by IGF-I affinity chromatography and subsequent HPLC on a Nucleosil 10 C(18) column (Macherey Nagel, Düren, Germany). Fractions were analyzed by SDS-PAGE with Western ligand blotting and silver staining. The IGFBP-4 fractions that appeared pure by these analyses were pooled, and the protein content was determined by the method of Lowry et al.(28) and by weighing the lyophilized material.

Western Ligand Blot Analysis

Unreduced conditioned medium samples (50 µl) were processed by SDS-PAGE using a 7.5-15% linear gradient gel, and separated proteins were electroblotted onto nitrocellulose filters. Filters were blocked, labeled with I-IGF overnight at 4 °C, and visualized by autoradiography according to the method of Hossenlopp et al.(13) and as described previously(7, 9, 10) . Unstained molecular weight standards (Bio-Rad) were processed in parallel, and proteins were stained using India ink(14) . Films were scanned with an UltroScan XL laser densitometer; absorbance curves were integrated, and molecular size was determined using GelScan XL software (Pharmacia Biotech Inc.).

Western Immunoblot Analysis

Reduced (+100 mM dithiothreitol) samples were electrophoresed and transferred as described above for Western ligand blots. Filters were blocked with 3% bovine serum albumin overnight at 4 °C, incubated for 2 h with IGFBP-4 antiserum (1:500 final dilution), and then incubated for 2 h with goat anti-rabbit IgG-alkaline phosphatase conjugate (1:300 final dilution) as described previously(7, 9) . Antigen-antibody reactions were visualized using Vectastain ABC immunoblotting reagents following the manufacturer's instructions (Vector Laboratories, Inc., Burlingame, CA).

Soluble IGF Binding Assay

In each assay, rhIGFBP-4 and the four IGFBP-4 mutants (0.08 nM) were incubated with I-IGF (30,000 cpm, 0.02 nM) and various concentrations of unlabeled IGF overnight at 4 °C. (The concentration of IGFBP-4 was based on a preliminary titration experiment to determine a concentration near but not at the plateau of maximal radioligand binding.) 1% activated charcoal containing 0.2 mg/ml protamine sulfate was added, and the samples were centrifuged at 4 °C to separate bound from free IGF-I(7, 15) . A control value for binding in buffer alone (nonspecific binding) was subtracted from the total bound radioactivity to determine a specific binding value. Nonspecific binding ranged from 3 to 10% of total counts added.

Aminoisobutyric Acid Uptake

[^3H]Aminoisobutyric acid (AIB) uptake was determined as described previously(7) . Confluent bovine fibroblasts were washed three times with Hanks' balanced saline solution containing 1.75 g/liter NaHCO(3), 20 mM Hepes (pH 7.4), and 0.1% bovine serum albumin. The medium was changed to that containing IGF-I or insulin with and without rhIGFBP-4 or mutant IGFBP-4, and the monolayers were incubated at 37 °C for 6 h. [^3H]AIB (0.5 µCi/ml, 8 µM) was added, and incubation was continued for 12 min. Cultures were placed on ice, and cells were washed quickly four times with cold phosphate-buffered saline. Monolayers were solubilized in 0.25 N NaOH, and aliquots were taken for liquid scintillation counting. Results are expressed as the percentage of total counts in the incubation medium that were taken up by the cells.

Thymidine Incorporation

[^3H]Thymidine incorporation was determined as described previously(16) . Confluent human fibroblast cultures were washed twice with a 1:1 mixture (v/v) of Waymouth medium/Dulbecco's modified Eagle's medium plus 0.1% bovine serum albumin, preincubated in this serum-free medium (SFM) for 6 h, washed again, and exposed to SFM for 40 h. IGF-II or an equivalent volume of SFM, with and without rhIGFBP-4 or mutant IGFBP-4, was added to this medium for 5 h. IGF-I or insulin was then added to the conditioned medium, and [^3H]thymidine incorporation was measured at 22-26 h. Results are expressed as the percentage of total counts in the incubation medium that were incorporated into acid-precipitable material.

Statistics

Statistical comparisons were performed using analysis of variance and the Newman-Keuls test for multiple comparisons. Results are considered statistically significant at p < 0.05.


RESULTS

Sequence of rhIGFBP-4 Cleavage Products

Large-scale proteolysis of 25 µg of rhIGFBP-4 was performed in human fibroblast and human osteoblast-like cell conditioned media (sources of IGF-dependent IGFBP-4 protease) (7, 9) in the presence of IGF-II. Two fragments were detected by immunoblotting with IGFBP-4 antiserum. Edman degradation of the two proteins yielded the NH(2)-terminal amino acid sequences shown in Table 1. The 18-kDa IGFBP-4 fragment corresponded to the NH(2)-terminal half of the protein. The 15 NH(2)-terminal amino acids are identical to those of native IGFBP-4 (where X = Cys), except for an additional NH(2)-terminal Arg that occurs in 90% of rhIGFBP-4, as previously noted(5) . The 14-kDa IGFBP-4 fragment corresponded to the COOH-terminal half of the mature IGFBP-4 molecule. The NH(2)-terminal amino acid of the 14-kDa fragment suggests cleavage after methionine at position 135 of the mature protein. Incubation of rhIGFBP-4 either in human fibroblast or human osteoblast-like cell conditioned medium generated fragments with identical sequences.



Construction, Expression, and Characterization of IGFBP-4 Mutants

Four mutant human IGFBP-4 cDNAs were generated by PCR using mismatched primers and were expressed in yeast as ubiquitin fusion proteins. We have previously shown that this expression system produces high levels of biologically active rhIGFBP-4, -5, and -6(5) . The mutations were designed to introduce single amino acid changes around the IGFBP-4 cleavage site and are shown in Table 2(M1-M4). All four mutants were expressed at the same high levels and had the same apparent molecular mass as nonmutated rhIGFBP-4 (24 kDa) when analyzed by nonreducing SDS-PAGE (Fig. 1). In addition, the mutants displayed an HPLC elution profile similar to that of rhIGFBP-4(5) .


Figure 1: SDS-PAGE analysis of wild-type and mutant rhIGFBPs-4. 300 ng of HPLC-purified rhIGFBP-4 (lanea), M1 (laneb), M2 (lanec), M3 (laned), and M4 (lane e) were fractionated on a 7.5-15% acrylamide gel under nonreducing conditions, and the gel was silver-stained. Migration positions of molecular size markers (in kilodaltons) are shown on the left.



All four IGFBP-4 mutants were able to bind radiolabeled IGF-I and IGF-II on Western ligand blotting. Furthermore, the affinity for IGF-I and IGF-II was not appreciably altered by the mutations. IGFs were equipotent in competing for radiolabeled IGF-I and IGF-II binding to ``wild-type'' and mutant IGFBPs-4 (Fig. 2). 50% displacement of I-IGF-I and I-IGF-II from each IGFBP-4 mutant was seen with unlabeled IGF at 0.05 and 0.06 nM, respectively. Scatchard analysis of the data from three experiments estimated an equilibrium constant of 2 times 10M for rhIGFBP-4, which agrees with our earlier study(5) . Equilibrium constants for the IGFBP-4 mutants did not differ significantly from that for rhIGFBP-4 (Table 3).


Figure 2: Competitive inhibition of I-IGF-I and I-IGF-II binding to IGFBP-4 mutants. Various concentrations of unlabeled IGF-I or IGF-II were added to compete for I-IGF-I binding (leftpanel) and IIGF-II binding (rightpanel) to M1 (), M2 (&cjs3570;), M3 (box), M4 (circle), and wild-type rhIGFBP-4 () as described under ``Experimental Procedures.'' Results are means of three determinations expressed as percent of maximum specific I-IGF-I binding (26, 29, 22, 32, and 18%) or I-IGF-II binding (33, 31, 30, 38, and 35%) for wild-type rhIGFBP-4 and M1-M4, respectively.





The IGFBP-4 mutants were potent inhibitors of IGF-I-stimulated [^3H]AIB uptake in cultured bovine fibroblasts. Bovine fibroblasts are exquisitely responsive to IGF-I and do not degrade IGFBP-4 during the bioassay; therefore, this system can be used to evaluate function of the intact IGFBP-4 molecule(7) . As indicated in Fig. 3, the presence of 10 nM wild-type IGFBP-4, M1, M2, M3, or M4 completely inhibited the 7-fold increase in [^3H]AIB uptake stimulated by 2 nM IGF-I. Half-maximal effectiveness was seen with 4 nM mutant and wild-type IGFBPs-4 (data not shown)(7) . Exogenous wild-type and mutant IGFBPs-4 had no effect alone and did not influence insulin-stimulated [^3H]AIB uptake in these cells. When added with IGF-I, a 5-fold molar excess of wild-type and mutant IGFBPs-4 inhibited IGF-I stimulation of [^3H]thymidine incorporation in human fibroblasts by 70% (Table 4).


Figure 3: Effect of mutant IGFBP-4 on IGF-I- and insulin-stimulated [^3H]AIB uptake in bovine fibroblasts. Bovine fibroblasts were washed and incubated for 6 h with 2 nM IGF-I or 100 nM insulin with or without the indicated IGFBP-4 at 10 nM. [^3H]AIB uptake was measured as described under ``Experimental Procedures.'' Results are means ± S.E. of three determinations. The asterisks indicate a significant effect of IGFBP-4 (p < 0.05)





IGFBP-4 Proteolysis

The IGFBP-4 mutants were tested for their susceptibility to IGF-dependent IGFBP-4 proteolysis in a cell-free assay. We have previously demonstrated that, in this assay, the IGF-II-induced loss of detectable IGFBP-4 by Western ligand blotting reflects proteolysis(7, 9, 10) . Levels of wild-type IGFBP-4 were decreased 89% during a 6-h incubation in human fibroblast conditioned medium (source of protease) with IGF-II, whereas levels of M1 and M2 were relatively unaffected by this incubation (Fig. 4). Greater than 80% of M1 and M2 remained detectable after a 6-h incubation in human fibroblast conditioned medium with IGF-II. After a 24-h incubation with IGF-II, all IGFBPs-4 were apparently proteolyzed. Wild-type and mutant IGFBP-4 levels did not change appreciably over the 24-h incubation period in human fibroblast conditioned medium in the absence of IGF-II. Similar results were obtained with M3 and M4. Densitometric analyses of three time course experiments of IGF-dependent proteolysis of these IGFBPs-4 indicated 50% proteolysis of rhIGFBP-4 by 2.5 h, whereas 50% proteolysis of the IGFBP-4 mutants ranged from 10 to 12 h. After 24 h, 70% or more of the IGFBPs-4 were hydrolyzed (Fig. 5). Immunoblotting with specific antiserum to IGFBP-4 detected 18- and 14-kDa fragments in wild-type and mutant IGFBP-4 incubation experiments of 24 h in the presence of IGF-II (Fig. 6).


Figure 4: Cell-free IGFBP-4 protease assay. 50 ng of wild-type rhIGFBP-4 and mutant IGFBP-4 (M1 and M2) were incubated at 37 °C in human fibroblast conditioned medium under cell-free conditions without(-) or with (+) 5 nM IGF-II for the indicated times. Samples were analyzed by Western ligand blotting. The arrow indicates the migration position of 24-kDa IGFBP-4.




Figure 5: Time course for cell-free IGF-dependent IGFBP-4 proteolysis. Proteolysis of wild-type rhIGFBP-4 (), M1 (), M2 (&cjs3570;), M3 (box), and M4 (circle) with time was determined as described in the legend to Fig. 4. Results are expressed as percent of intact IGFBP-4 at t = 0. Each point represents the mean value of three separate experiments.




Figure 6: Immunoblot analysis using IGFBP-4 antiserum. Wild-type and mutant IGFBPs-4 (100 ng) were incubated for 24 h in human fibroblast conditioned medium under cell-free conditions without(-) or with (+) 5 nM IGF-II. Reduced samples were electrophoresed and transferred to nitrocellulose, and the filter was incubated with antiserum to IGFBP-4 (1:500 dilution) as described under ``Experimental Procedures.'' Migration positions of unstained molecular size marker (in kilodaltons) are shown on the left. Arrows indicate 18- and 14-kDa IGFBP-4 fragments.



Effect of IGFBP-4 Mutants on IGF-II Enhancement of IGF-I-stimulated Mitogenesis

Preincubation with low concentrations of IGF-II enhances IGF-I-stimulated DNA synthesis and cell replication in human fibroblasts and osteoblasts(9, 16) . This potentiating effect of IGF-II is independent of direct interaction of IGF-II with type I and II IGF receptors and is associated with structural/functional changes in pericellular IGFBPs, most notably a specific loss in medium IGFBP-4(16) . We postulated that IGF-II-induced IGFBP-4 proteolysis contributes to the observed enhancement of IGF-I action. To test this hypothesis, we determined the effect of exogenous wild-type IGFBP-4 and the mutant IGFBPs-4 resistant to proteolysis on IGF-II potentiation of IGF-I action. Human fibroblasts were washed and changed to serum-free medium for 40 h to allow secretion and accumulation of IGFBP-4 and ``functionally dormant'' IGFBP-4 protease. IGF-II was added to the medium for 5 h, with or without the different IGFBP-4 preparations, before the subsequent addition of IGF-I or insulin. As shown in Fig. 7, the addition of IGF-I in the absence of IGF-II provoked a weak mitogenic response under these conditions. However, pretreatment with IGF-II enhanced IGF-I-stimulated [^3H]thymidine incorporation 3-fold. The addition of wild-type IGFBP-4 with IGF-II did not alter this augmentative effect. However, the addition of M1, M2, or M4 significantly inhibited IGF-II-enhanced IGF-I stimulation. These IGFBP-4 mutants had no effect alone and did not influence insulin-stimulated [^3H]thymidine incorporation. Similar results were obtained with M3 in a separate experiment (data not shown). As noted in Table 4, in the absence of proteolysis, wild-type and mutant IGFBPs-4 equivalently inhibited IGF-I-stimulated [^3H]thymidine incorporation.


Figure 7: IGF-II-enhanced, IGF-I-stimulated [^3H]thymidine incorporation in human fibroblasts: effect of IGFBP-4 mutants. Human fibroblasts were washed and changed to SFM for 40 h. 4 nM IGF-II (shaded and dotted bars) or an equivalent amount of SFM (solidbars), with or without the indicated IGFBP-4 (25 nM), was added to the medium, and incubation was continued for 5 h. IGF-I (5 nM) or SFM (control (C)) was then added, and [^3H]thymidine incorporation was measured at 22-26 h as described under ``Experimental Procedures.'' Results are means ± S.E. of three determinations. The asterisks indicate a significant effect of IGFBP-4 (p < 0.05).




DISCUSSION

These data demonstrate that the IGF-dependent IGFBP-4 protease secreted by human fibroblasts and human osteoblast-like cells cleaves the IGFBP-4 molecule at a single site on the carboxyl-terminal side of methionine 135, producing fragments of 18 and 14 kDa. Furthermore, studies with IGFBP-4 mutated at this cleavage site indicate that inhibition of this proteolytic processing of IGFBP-4 can have biological consequences.

Sequence analysis of the 14-kDa proteolysis product predicted a precise cleavage event between methionine and lysine of IGFBP-4 (Met-Lys). Tissue kallikreins are known to cleave peptide bonds between methionine and lysine and between arginine and serine in kininogen to release lysylbradykinin(17) . Thus, these results suggest that IGF-dependent IGFBP-4 cleavage may be the product of the activity of a hydrolytic enzyme with a specificity similar to that of the kallikreins. Preliminary data of Chernausek et al.(18) also suggest that the IGFBP-4 protease secreted by B104 rat neuroblastoma cells may be a kallikrein-like enzyme. However, IGF-dependent proteolysis of IGFBP-4 in human fibroblast or osteoblast-like cell conditioned medium was not inhibited by conventional trypsin- or chymotrypsin-like serine protease inhibitors. EDTA and 1,10-phenanthroline were by far the most effective inhibitors (7, 9) , and proteolytic activity could be restored with calcium in EDTA-treated samples and with zinc in 1,10-phenanthroline-treated samples. (^2)These protease inhibitor results may indicate a novel calcium-dependent metalloprotease with specificity similar to that of kallikreins. Alternatively, it is possible that the specific cleavage occurs several residues upstream of methionine 135 and that the 14-kDa fragment is further processed by the action of an aminopeptidase. COOH-terminal analysis of the 18-kDa IGFBP-4 fragment may help establish the involvement of additional enzymes. Similar immunoreactive fragments (Fig. 6) and the fact that sequencing of the 18- and 14-kDa products generated from IGF-dependent proteolysis of the four IGFBP-4 mutants yielded the same amino termini as wild-type rhIGFBP-4 (^3)argue against a secondary cleavage site being utilized as a result of a block in the mutated site. Regardless, our results clearly show that the cleavage site is located in domain 2 of IGFBP-4, which is the central nonconserved region of the IGFBPs(19, 20, 21, 22) . The conserved domains (domains 1 and 3) or NH(2)- and COOH-terminal portions of the IGFBPs are known to be important for IGF binding(23, 24) , leaving the possibility that the nonconserved domain 2 could be involved in regulating the activity and/or tissue specificity of each IGFBP. Our observations that IGFBPs-4 mutated in domain 2 have binding affinities similar to those of wild-type IGFBP-4 yet are partially resistant to proteolysis support this concept.

Biological studies using IGFBPs-4 with single amino acid mutations around the predicted cleavage site indicated that we were at or near the correct site. The expressed wild-type and mutant IGFBPs-4 bound IGF-I and IGF-II with equivalent affinities and were effective inhibitors of IGF-I action when assessed for function of the intact IGFBP-4 molecule. One way in which the mutant IGFBPs-4 differed from wild-type IGFBP-4 was in their relative resistance to IGF-dependent proteolysis. A 5-6-h incubation in human fibroblast conditioned medium in the presence of IGF-II was sufficient for near total hydrolysis of wild-type IGFBP-4, whereas the mutant IGFBPs-4 were only minimally affected.

As demonstrated in the assays for function of intact IGFBP-4, the IGFBP-4 mutants were not super-inhibitors of IGF action. However, resistance to proteolysis corresponded with increased effectiveness of mutant IGFBP-4 as a physiological inhibitor of cellular IGF-I action. Pre-exposure of human fibroblasts to IGF-II results in marked enhancement of subsequent IGF-I-stimulated DNA synthesis and cell replication via effects on pericellular IGFBPs(16) . The addition of wild-type rhIGFBP-4, which is rapidly degraded in the human fibroblast system(7) , did not affect IGF-II potentiation of IGF-I action. The finding in the present study that protease-resistant IGFBP-4 mutants suppressed this effect indicates that IGFBP-4 proteolysis contributes to IGF-II enhancement of IGF-I action. In addition, functional interplay between other IGFBPs and local agents or distinct biological effects of 18- and/or 14-kDa IGFBP-4 fragments are likely to be important and were not addressed in this study. The latter possibility is particularly intriguing since IGFBP-3 and IGFBP-5 fragments generated through proteolytic processing appear to be stimulatory, whereas the intact forms of IGFBP are clearly inhibitory(25, 26, 27) . Future studies are aimed at acquiring a better understanding of the IGFBP-4/IGFBP-4 proteolytic system and its role in the control of cell growth.


FOOTNOTES

*
This work was supported in part by National Institutes of Health Grants DK-43258 (to C. A. C.) and DK-07352 (to S. K. D.), the Mayo Foundation, and Swiss National Science Foundation Grant 32-31281.91 (to J. Z.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence and reprint requests should be addressed: Endocrine Research Unit, 5-164 West Joseph, Mayo Clinic, Rochester, MN 55905. Tel.: 507-255-6415; Fax: 507-255-4828.

(^1)
The abbreviations used are: IGFBP-4, insulin-like growth factor-binding protein-4; rhIGFBP-4, recombinant human IGFBP-4; IGF, insulin-like growth factor; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; HPLC, high pressure liquid chromatography; AIB, aminoisobutyric acid; SFM, serum-free medium.

(^2)
C. A. Conover, S. K. Durham, J. T. Clarkson, and L. K. Bale, unpublished data.

(^3)
C. A. Conover, P. Pemberton, J. Zapf, and M. C. Kiefer, unpublished data.


ACKNOWLEDGEMENTS

We acknowledge the excellent technical assistance of Scott H. Chamberlain, Katherine E. Landsberg, Laurie Bale, and Jay Clarkson. We also thank Phil Pemberton (LXR Biotechnology Inc.) for sequencing mutant IGFBP-4 proteolysis products and for helpful suggestions and comments regarding proteases.


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