Fibronectin Binds Insulin-like Growth Factor-binding Protein 5 and Abolishes Its Ligand-dependent Action on Cell Migration
2004; Elsevier BV; Volume: 279; Issue: 6 Linguagem: Inglês
10.1074/jbc.m311586200
ISSN1083-351X
AutoresQijin Xu, Ben Yan, Shenghua Li, Cunming Duan,
Tópico(s)Fibroblast Growth Factor Research
ResumoInsulin-like growth factor-binding protein 5 (IGFBP-5) is a secreted protein that binds to insulin-like growth factors (IGFs) and modulates IGF actions on cell proliferation, differentiation, survival, and motility. IGFBP-5 also regulates these cellular events through IGF-independent mechanisms. To elucidate the molecular mechanisms governing these diverse actions of IGFBP-5, we screened a human cDNA library by a yeast two-hybrid system using IGFBP-5 as bait and identified fibronectin (FN) as a potential IGFBP-5-interacting partner. The complex formation of IGFBP-5 and FN was established by glutathione S-transferase pull-down, solution, and solid phase binding assays using glutathione S-transferase-IGFBP-5 and native IGFBP-5 in vitro and by co-immunoprecipitation in vivo. Binding assay using deletion mutants indicated that the IGFBP-5 C domain binds to the 10th and 11th type I repeats of FN. IGFBP-5 potentiated IGF-I-induced cell migration in FN-null, but not in wild-type, mouse embryonic cells. When FN was reintroduced either as an adhesive substrate or in solution to the FN-null cells, the potentiating effect of IGFBP-5 on IGF-I-induced cell migration was abolished. Binding of IGFBP-5 to FN had no effect on the ability of IGFBP-5 to bind IGF-I, but it increased the proteolytic degradation of IGFBP-5. Inhibition of IGFBP-5 proteolysis restored the potentiating effect of IGFBP-5. These results suggest that FN and IGFBP-5 bind to each other, and this binding negatively regulates the ligand-dependent action of IGFBP-5 by triggering IGFBP-5 proteolysis. Insulin-like growth factor-binding protein 5 (IGFBP-5) is a secreted protein that binds to insulin-like growth factors (IGFs) and modulates IGF actions on cell proliferation, differentiation, survival, and motility. IGFBP-5 also regulates these cellular events through IGF-independent mechanisms. To elucidate the molecular mechanisms governing these diverse actions of IGFBP-5, we screened a human cDNA library by a yeast two-hybrid system using IGFBP-5 as bait and identified fibronectin (FN) as a potential IGFBP-5-interacting partner. The complex formation of IGFBP-5 and FN was established by glutathione S-transferase pull-down, solution, and solid phase binding assays using glutathione S-transferase-IGFBP-5 and native IGFBP-5 in vitro and by co-immunoprecipitation in vivo. Binding assay using deletion mutants indicated that the IGFBP-5 C domain binds to the 10th and 11th type I repeats of FN. IGFBP-5 potentiated IGF-I-induced cell migration in FN-null, but not in wild-type, mouse embryonic cells. When FN was reintroduced either as an adhesive substrate or in solution to the FN-null cells, the potentiating effect of IGFBP-5 on IGF-I-induced cell migration was abolished. Binding of IGFBP-5 to FN had no effect on the ability of IGFBP-5 to bind IGF-I, but it increased the proteolytic degradation of IGFBP-5. Inhibition of IGFBP-5 proteolysis restored the potentiating effect of IGFBP-5. These results suggest that FN and IGFBP-5 bind to each other, and this binding negatively regulates the ligand-dependent action of IGFBP-5 by triggering IGFBP-5 proteolysis. Insulin-like growth factor-binding proteins (IGFBPs) 1The abbreviations used are: IGFBPinsulin-like growth factor-binding proteinIGFinsulin-like growth factorVSMCvascular smooth muscle cellFNfibronectinGSTglutathione S-transferaseSFMserum-free defined mediumECMextracellular matrix.1The abbreviations used are: IGFBPinsulin-like growth factor-binding proteinIGFinsulin-like growth factorVSMCvascular smooth muscle cellFNfibronectinGSTglutathione S-transferaseSFMserum-free defined mediumECMextracellular matrix. are a family of secreted proteins that bind to IGFs and modulate their distribution, stability, and cellular actions (1Clemmons D.R. Endocr. Rev. 2001; 22: 800-817Crossref PubMed Scopus (122) Google Scholar, 2Firth S.M. Baxter R.C. Endocr. Rev. 2002; 23: 824-854Crossref PubMed Scopus (1432) Google Scholar). IGFBP-5 is the most evolutionarily conserved member in this gene family (3Duan C. Ding J. Schlueter P. Li Y. Zhang J. Royer T. Acta Zool. Sinica. 2003; 49: 421-431Google Scholar). Like other IGFBPs, IGFBP-5 binds to IGFs in the extracellular environment with high affinity (1Clemmons D.R. Endocr. Rev. 2001; 22: 800-817Crossref PubMed Scopus (122) Google Scholar). In the blood, IGFBP-5 forms a ternary complex with IGF and acid labile subunit that controls the efflux of IGFs from the vascular space and prolongs IGF half-lives (2Firth S.M. Baxter R.C. Endocr. Rev. 2002; 23: 824-854Crossref PubMed Scopus (1432) Google Scholar). Locally produced IGFBP-5 provides a means of localizing IGFs on target cells and can alter the biological activities of IGFs by modulating their interaction with the cell surface IGF receptors. IGFBP-5 has been shown to inhibit the proliferative responses of skeletal muscle cells and breast cancer cells to IGF-I (4Rozen F. Yang X.F. Huynh H. Pollak M J. Natl. Cancer Inst. 1997; 89: 652-656Crossref PubMed Scopus (90) Google Scholar, 5Ewton D.Z. Coolican S.A. Mohan S. Chernausek S.D. Florini J.R. J. 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Chem. 1998; 273: 16836-16842Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 12Duan C. Hawes S.B. Prevette T. Clemmons D.R. J. Biol. Chem. 1996; 271: 4280-4288Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). More recent studies have shown that IGFBP-5 also stimulates bone cell growth and mesangial cell and VSMC migration through an IGF-independent mechanism(s) (13Abrass C.K. Berfield A.K. Andress D.L. Am. J. Physiol. 1997; 273: F899-F906PubMed Google Scholar, 14Berfield A.K. Andress D.L. Abrass C.K. Kidney Int. 2000; 57: 1991-2003Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 15Miyakoshi N. Richman C. Kasukawa Y. Linkhart T.A. Baylink D.J. Mohan S. J. Clin. Invest. 2001; 107: 73-81Crossref PubMed Scopus (184) Google Scholar, 16Hsieh T. Gordon R.E. Clemmons D.R. Busby Jr., W.H. Duan C. J. Biol. Chem. 2003; 278: 42886-42892Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). insulin-like growth factor-binding protein insulin-like growth factor vascular smooth muscle cell fibronectin glutathione S-transferase serum-free defined medium extracellular matrix. insulin-like growth factor-binding protein insulin-like growth factor vascular smooth muscle cell fibronectin glutathione S-transferase serum-free defined medium extracellular matrix. Although studies describing various cellular actions of IGFBP-5 have been reported for over a decade, the molecular mechanisms governing these actions are still not well understood. To identify proteins that bind and modulate IGFBP-5 actions, we screened a human aorta cDNA library by a yeast two-hybrid system using human IGFBP-5 as bait and identified fibronectin (FN) as a potential IGFBP-5-interacting partner. Further biochemical and functional studies show that FN and IGFBP-5 directly bind to each other, and this binding abolishes the ligand-dependent action of IGFBP-5 on cell migration. Materials—All of the chemicals and reagents were obtained from Fisher unless noted otherwise. Recombinant human IGF-I, IGFBP-4, and IGFBP-5 were purchased from GroPep Ltd (Adelaide, Australia). Human plasma FN was obtained from Chemicon International Inc. (Temecula, CA). Fetal bovine serum was purchased from Invitrogen. The Matchmaker™ two-hybrid system 3 and the human aorta Gal4 activation domain-cDNA library were purchased from Clontech Laboratories (Palo Alto, CA). Yeast Two-hybrid Assay—The Matchmaker™ two-hybrid system 3 (Clontech) was used to identify clones that interact with human IGFBP-5. The bait, pGBKT7-IGFBP-5, generated by inserting full-length human IGFBP-5 cDNA into the NcoI and BamHI sites of the pGBKT7, was used to screen a human aorta cDNA library constructed in the pACT2 vector (Clontech). Positive clones were selected by growth on a drop-out minimal medium lacking tryptophan, leucine, histidine, and adenine and by activation of X-α-galactosidase gene. All of the positive clones identified in the screen were retested twice under high stringency. The resulted positive cDNA clones were recovered from the host cells and sequenced at the University of Michigan DNA Sequencing Core. The IGFBP-5 and FN interaction was further studied by co-transforming yeasts with pGBKT7-IGFBP-5 and pGADT7-FN plasmids. Transformed cells were plated on synthetic drop-out minimal medium or the tryptophan- and leucine-deficient medium. The liquid cultures were diluted to an A600 of 1.0, and 10-fold serial dilutions of each yeast culture were spotted onto drop-out minimal medium and grown at 30 °C for 2-3 days. Plasmid Construction—DNA fragments corresponding to the N (containing residues 1-80 of the mature protein), L (residues 81-169), C (residues 170-252), NL (residues 1-169), and LC (residues 81-252) domains of human IGFBP-5 were generated by PCR amplification using human IGFBP-5 cDNA as template and the following primer sets: P1 and P2 (N domain), P3 and P4 (L domain), P5 and P6 (C domain), P1 and P4 (NL domains), and P3 and P6 (LC domains). The sequences of these primers are shown in Table I. The resulted PCR products were subcloned into the NcoI and BamHI restriction sites of the pGBKT7 vector to produce fusion proteins with the Gal4 DNA-binding domain. The cDNAs encoding various FN C-terminal regions, namely Fn-A, Fn-B, Fn-C, Fn-C/I10+11+12, Fn-C/I10, Fn-C/I11, Fn-C/I12, Fn-C/I10+11, and Fn-C/I11+12 (see Fig. 3 for schematic diagrams) were generated by PCR amplification using the cloned FN plasmid as template and the following primer sets: primers P7 and P8 (Fn-A), P7 and P9 (Fn-B), P10 and P8 (Fn-C), P10 and P15 (Fn-C/I10+11+12), P10 and P11 (Fn-C/I10), P12 and P13 (Fn-C/I11), P14 and P15 (Fn-C/I12), P11 and P13 (Fn-C/I10+11), and P12 and P8 (Fn-C/I11+12) (see Table I). The FN cDNAs were subcloned into the BamHI and XhoI sites of the pGADT7 vector to produce fusion proteins with the Gal4 activation domain. Plasmid DNA was isolated using Qiagen Miniprep kits (Qiagen) and confirmed by DNA sequencing.Table IPrimer sequences The underlined sequences were used to introduce a NcoI cloning site; the bold sequences were used to introduce a BamHI cloning site; and the italic sequences were used to introduce a XhoI cloning site.Primer namesPrimer sequencesP15′-GTAACCATGGCACTGGGCTCCTTCGTGCAC-3′P25′-CTATGGATCCTCAGCAAACCCCGCGGCCGTG-3′P35′-TATCCCATGGCTCTCAACGAAAAGAGCTAC-3′P45′-ATCAGGATCCTCACTGCTCAGACTCCTGTCT-3′P55′-TATGCCATGGCAGGCCCCTGCCGCAGACAC-3′P65′-ATGCGGATCCTCACTCAACGTTGCTGCTGTC-3′P75′-GCTCGGATCCAAGGAACCGAATATACAATTTATGTC-3′P85′-TGGCGCTCGAGCTTACTCTCGGGAATCTTCTCTGTC-3′P95′-GGGGTCTCGAGCCGAGTCATCCGTAGGTTGGTTCAA-3′P105′-GCTCGGATCCAATGCTTTGACCCCTACACAGTT-3′P115′-TCTAGCTCGAGCACATCTGAAATGACCACTTCC-3′P125′-ATCATGGATCCAATGCCATGACAATGGTGTGAAC-3′P135′-CTCATCTCGAGCACACTTGAATTCTCCTTTTCC-3′P145′-AGAATGGATCCAAGACCCTCATGAGGCAACGTGT-3′P155′-GTATCTCGAGCGTCACAGCGCCAGCCCCGCTG-3′ Open table in a new tab Cell Culture—Wild-type (MEC+/+) and FN-null mouse embryonic cells (MEC-/-) were a gift from Dr. Jane Sottile (University of Rochester). These cells were grown in a 1:1 mixture of Cellgro (Mediatech, Herndon, VA) and Aim V (Invitrogen). This defined medium is serum-free and contains no adhesion molecules or growth factors. Both wild-type and FN-null cells were grown in tissue culture dishes precoated with 50 μg/ml type I collagen (Sigma). Porcine VSMC cells were grown in Dulbecco's minimum essential medium supplemented with 4 mml-glutamine, penicillin (100 units/ml), streptomycin (100 μg/ml), and 10% fetal bovine serum. For GST pull-down or co-immunoprecipitation assays, the cells were lysed in 1× binding buffer (30 mm Tris acetate, 10 mm sodium phosphate, pH 7.4, 0.1% Tween 20, 1 mm EDTA, 2 μg/ml leupeptin, 4 μg/ml aprotinin, 1 μg/ml pepstatin A, and 1 mm phenylmethylsulfonyl fluoride) and used immediately after lysis. GST Pull-down Assay—The cDNA fragment encoding full-length human IGFBP-5 was cloned into the NcoI and SalI sites of the pGEX-KG vector to produce a fusion protein with GST at the C terminus of IGFBP-5. JM109 Escherichia coli cells were transformed with pGEX-KG-IGFBP5 and grown in LB broth at 37 °C to an A600 of 0.4-0.6. Following the addition of isopropyl-1-thio-β-d-galactopyranoside to a final concentration of 0.1 mm, the cells were incubated at 30 °C for 2 h. After harvesting, the cells were resuspended in French press buffer (50 mm Tris, pH 8.0, 0.1 m NaCl, 1 mm EDTA, 0.05% Tween 20, 1 mm EDTA, 2 μg/ml leupeptin, 4 μg/ml aprotinin, 1 μg/ml pepstatin A, and 1 mm phenylmethylsulfonyl fluoride). The cells were broken by French press and centrifuged at 20,000 × g for 20 min at 4 °C, and the supernatant (S1) was collected. S1 was further centrifuged at 100,000 × g for 45 min, and that supernatant (S2) was collected. 10 ml of S2 was loaded onto 0.6 ml glutathione-Sepharose beads (Amersham Biosciences) at 4 °C overnight. After centrifugation at 500 × g for 5 min, the beads were extensively washed, resuspended in 0.6 ml of French press buffer, and used for the pull-down assay. 500 μl of cell lysates or serum or 10 μg of pure plasma FN in 250 μl of binding buffer were incubated with GST or GST-IGFBP-5 immobilized on glutathione-Sepharose beads (∼15 μg/20 μl) at 4 °C for 3 h. After the incubation, the beads were separated by centrifugation, washed with binding buffer, and boiled in Laemmli loading buffer. The eluted proteins were resolved by SDS-PAGE and analyzed by immunoblot analysis as previously reported (12Duan C. Hawes S.B. Prevette T. Clemmons D.R. J. Biol. Chem. 1996; 271: 4280-4288Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). Co-immunoprecipitation—500 μl of cell lysates were incubated and precipitated with 2 μg of a monoclonal human FN antibody (Pierce) or 2 μg of mouse IgG following previously published method (17Duan C. Bauchat J.R. Hsieh T. Cir. Res. 2000; 86: 15-23Crossref PubMed Scopus (148) Google Scholar). The co-precipitated proteins were separated by SDS-PAGE followed by immunoblot analysis using an IGFBP-5 polyclonal antibody (Diagnostic Systems Laboratories, Inc., Webster, TX). Similarly, equal amounts of the cell lysates were immunoprecipitated with the IGFBP-5 antibody and analyzed by immunoblot. Migration Assay—The cell migration assays were performed using 24-well cell culture inserts (Falcon) coated with 0.1% gelatin. In some experiments, the gelatin-coated inserts were further incubated with human FN (15 μg/ml) at room temperature for 2 h. The free FN was removed by washing. Wild-type or FN-null cells were washed with 1× phosphate-buffered saline and trypsinized. After trypsinization, the cells were washed once in 1× phosphate-buffered saline. 5.0 × 104 cells in 200 μl of the serum-free defined medium (SFM) containing IGFBP-5 (200 ng/ml) or SFM were loaded into the upper wells. In some experiments, FN and/or protease inhibitors were also added into the upper wells. 800 μl of the defined medium, with or without IGF-I (50 ng/ml), was loaded into each bottom well. After 6 h of incubation at 37 °C, the cells were removed from the upper sides of the inserts. The migrated cells on the underside were stained in toluidine blue, and the total number of migratory cells was counted. The results were expressed as percentages of the values from the SFM control group. Binding Assays—For solid phase binding assays, 96-well plates (Falcon) were coated with FN (5 μg/well) or the vehicle overnight at 4 C. After coating, the plates were washed three times with the binding buffer and blocked with 0.1% bovine serum albumin in 1× phosphate-buffered saline for 2 h at room temperature. After washing, pure IGFBP-5 (10 ng/well in 100 μl of 1× phosphate-buffered saline) was added and incubated for 3 h at 4 C. After removing the unbound IGFBP-5 by washing, increasing amounts of 125I-IGF-I (0-100,000 cpm) were added and incubated for 1 h at 37 °C. The bound 125I-IGF-I were recovered by adding 200 μl of 0.1 n NaOH followed with 200 μl of 1% SDS and measured in an automatic Gamma Counter (ICN Biomedicals, Inc., Huntsville, AL). Immobilized GST or GST-IGFBP-5 beads (15 μg/20 μl) was incubated with 125I-IGF-I (50,000 cpm) in the presence and absence of human FN (10 μg) in 1× binding buffer at a final volume of 120 μl. After incubation for 14 h at 4 °C, the beads were pelleted by centrifugation at 500 × g for 1 min and washed three times in binding buffer. The 125I-IGF-I precipitated was measured in an automatic Gamma Counter. Alternatively, immobilized GST or GST-IGFBP-5 (15 μg) was incubated with pure FN (10 μg) in the presence or absence of 2 μg of IGF-I for 14 h at 4 °C. The level of bound FN in the pulled down complex was determined by immunoblot analysis. Solution binding experiments were also carried out using 300 ng of pure human IGFBP-5, 125I-IGF-I (50,000 cpm), and human FN (8 μg) in 1× binding buffer at a final volume of 120 μl. The bound 125I-IGF-I in the complex was separated from free 125I-IGF-I by immunoprecipitation using the FN antibody. Proteolysis Assay—To determine the possible effect of FN on the stability of IGFBP-5, MEC-/- cells were grown to subconfluence in 6-cm cell culture dishes. After being washed three times with SFM, the cells were incubated in 400 μl of SFM without or with IGFBP-5 (1.0 μg/ml), FN (8.0 μg/ml), or IGFBP-5 plus FN at 37 °C overnight. The media were collected and concentrated through a Centricon-10 microconcentrator (Millipore, Bedford, MA). The samples were analyzed by immunoblot analysis. Statistical Analysis—The differences among groups were analyzed by one-way analysis of variance followed by Fisher's protected least significance difference test using Statview (Abacus Concept, Inc.). Identification of FN as an IGFBP-5-interacting Protein—A total of 4.3 million colonies from a human aorta library were screened with a chimeric bait containing the Gal4 DNA-BD and full-length human IGFBP-5. 28 of the 60 positive clones encoded polypeptides that corresponded to human FN (Fig. 1A). Restriction enzyme digestion and DNA sequencing analyses grouped these clones into two types (Fig. 1A). To verify this interaction, a plasmid from each group was selected and co-transformed into yeasts with the plasmid pGBKT7-IGFBP-5 or the empty pGBKT7 vector. All of the yeast transformants grew on leucine- and tryptophan-deficient plates (Fig. 1B, left panel), indicating successful transformation. When plated on the selective medium, only the cells co-transformed with both IGFBP-5 and FN were able to grow (Fig. 1B, right panel, sections 5 and 6). The IGFBP-5 and FN interaction was further analyzed by GST pull-down and solution binding assays. As shown in Fig. 2A (top panel), when incubated with pure plasma FN, GSTIGFBP-5, but not GST, pulled down FN. Likewise, GSTIGFBP-5, but not GST, was able to pull down FN from fetal bovine serum (Fig. 2A, middle panel), suggesting that GSTIGFBP-5 binds to plasma FN. FN not only exists in a soluble protomeric form in the blood (i.e. plasma FN), but it is also present in a multimeric form in the ECM of local tissues (cellular FN). To determine whether IGFBP-5 is also capable of interacting with cellular FN, GST pull-down assays were performed using VSMCs, which are known to synthesize and secrete FN (18Zheng B. Duan C. Clemmons D.R. J. Biol. Chem. 1998; 273: 8994-9000Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). The result showed that GST-IGFBP-5 was able to bind cellular FN secreted by these primarily cultured cells (Fig. 2A, bottom panel). The IGFBP-5 and FN complex formation was also confirmed using purified native IGFBP-5 and FN (see Fig. 6). To test whether IGFBP-5 and FN interact with each other under physiological conditions, co-immunoprecipitation experiments were performed using primarily cultured VSMCs. These cells synthesize both IGFBP-5 and FN (18Zheng B. Duan C. Clemmons D.R. J. Biol. Chem. 1998; 273: 8994-9000Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). Immunoprecipitation with a FN antibody resulted in the co-precipitation of IGFBP-5 (Fig. 2B, upper panel). The association of IGFBP-5 and FN was also confirmed in a reciprocal co-immunoprecipitation experiment (Fig. 2B, lower panel).Fig. 6FN does not affect the ability of IGFBP-5 to bind IGF-I.A, the presence of FN has no effect on IGFBP-5 and IGF-I interaction. Immobilized GST or GST-IGFBP-5 beads was incubated with 125I-IGF-I in the presence or absence of FN. The 125I-IGF-I bound to GST IGFBP-5 was separated from free 125I-IGF-I by GST pull-down and measured. The values are the means ± S.E. of two separate experiments, each performed in duplicate. Lower panel, the presence of FN in the pull-down complex was confirmed by Western blot. IB, immunoblot. B, IGFBP-5 simultaneously binds to IGF-I and FN. FN was incubated with 125I-IGF-I in the presence or absence of pure IGFBP-5. The bound 125I-IGF-I separated from free 125I-IGF-I by immunoprecipitation using FN antibody and was measured. C, addition of IGF-I does not affect the IGFBP-5 and FN binding. Immobilized GST or GST-IGFBP-5 was incubated with FN in the presence or absence of IGF-I. The FN bound to GSTIGFBP-5 was separated by GST pull-down and analyzed by immunoblot using a FN antibody. Pure FN (far right lane) was used as positive control for the immunoblot.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The IGFBP-5 and FN Binding Interaction Is Mediated by the 10th and 11th Type I Repeats in FN and the IGFBP-5 C domain—Sequence alignments of the 28 FN clones revealed that they overlapped in a region ranging from residue 1931 to the C-terminal end (Fig. 1A). This region contains two type III repeats, three type I repeats, and the C-terminal tail. Indeed, cells co-transformed with IGFBP-5 and Fn-A, a truncated FN construct that includes the overlapping region, grew well on the selective medium (Fig. 3). To determine the specific structural motif(s) that mediates its binding to IGFBP-5, two truncated constructs, Fn-B and Fn-C, were tested. Fn-B covers the two type III repeats, and Fn-C spans the type I repeats and the tail region. The results indicated that Fn-C, but not Fn-B, was able to interact with IGFBP-5 (Fig. 3). Because Fn-C includes three type I repeats (I10, I11, and I12) and the C-terminal tail, we examined further truncated forms of FN-C, i.e. Fn-C/I10+11+12. Fn-C/I10+11+12 interacted with IGFBP-5 as strong as FN-A, suggesting that the type I repeats, but not the C-terminal tail, are important. To determine the role of individual repeat, Fn-C/I10, Fn-C/I11, and Fn-C/I12, three constructs that correspond to each of the three type I repeats, were tested, but none was capable of IGFBP-5 binding. Next, Fn-C/I10+11 and Fn-C/I11+12 were generated and tested. The results showed that Fn-C/I10+11, but not Fn-C/I11+12, was capable of IGFBP-5 binding (Fig. 3). These results suggest that I10 and I11 are the IGFBP-5-binding domain in FN. IGFBP-5 consists of three domains: a highly cysteine rich N-terminal domain, a cysteine-rich C-terminal domain, and a central (L) domain with no cysteine residues. To determine which domain(s) of IGFBP-5 is involved in its binding interaction with FN, five truncated IGFBP-5 DNA fragments corresponding to the N, L, C, NL, and LC domains of IGFBP-5 were generated and subcloned into the pGBKT7 vector to generate GAL4 DNA-BD fusion proteins. When the various pGBKT7-IGFBP-5 domain-specific plasmids were introduced into cells together with pGADT7-FN, cells co-transformed with IGFBP-5 C domain and LC domain were able to grow on the selective medium (Fig. 4). Their growth was comparable with those of the full-length IGFBP-5. The cells co-transformed with FN and IGFBP-5 N, L, and NL domains did not grow under the same conditions (Fig. 4). These data suggest that C domain of IGFBP-5 is both required and sufficient for its binding interaction with FN. Binding with FN Abolishes the Action of IGFBP-5 in Potentiating IGF-I-induced Cell Migration—Because most, if not all, adherent cells constitutively produce FN and deposit it into the ECM, we used FN-null mouse embryonic cells (MEC-/-) to determine the functional importance of the IGFBP-5 and FN interaction. These cells do not produce any FN but are capable of assembling a FN matrix when cultured in the presence of exogenous FN (19Sottile J. Hocking D.C. Swiatek P.J. J. Cell Sci. 1998; 111: 2933-2943Crossref PubMed Google Scholar). They can be cultured in defined medium without any growth factor or adhesion molecule. When subjected to an IGF-I gradient, the MEC-/- and MEC+/+ cells both showed elevated migration (Fig. 5A). At 50 ng/ml, IGF-I caused a 86 ± 13% (p < 0.05) and a 92 ± 15% (p < 0.05) increase over the control in MEC-/- and MEC+/+ cells, respectively. The addition of IGFBP-5 together with IGF-I at an equal molar concentration resulted in a 202 ± 12% increase in the FN-null cells (Fig. 5A, left panel). This value was significantly higher than that of the IGF-I alone group (p = 0.01). In contrast, exogenous IGFBP-5 had no effect on IGF-I-induced cell migration in MEC+/+ cells (Fig. 5A, right panel). The addition of IGFBP-5 alone had no effect on cell migration in either group. Therefore, IGFBP-5 potentiated IGF-I action in the FN-null cells but not in the wild-type cells. To determine whether these different biological effects of IGFBP-5 were due to the presence or absence of FN, MEC-/- cells were subjected to migration assay using inserts precoated with or without FN. The introduction of FN did not change the basal migration rate, nor did it alter the chemotactic responses to IGF-I, but it abolished the potentiating effect of IGFBP-5 on IGF-I-induced migration (Fig. 5B). Likewise, the addition of soluble FN abolished the potentiating effect of IGFBP-5 (Fig. 5C). The effect of FN appeared to be specific for IGFBP-5, because FN did not abolish the ability of IGFBP-4 to inhibit IGF-I-induced cell migration in MEC-/- cells (Fig. 5D). In fact, the inhibitory effect of IGFBP-4 was even more pronounced in the presence of FN. FN Binding Does Not Alter the Ability of IGFBP-5 to Bind IGF-I—Because the FN and IGFBP-5 binding attenuates the ability of IGFBP-5 to potentiate IGF-induced cell migration, we wondered whether binding of IGFBP-5 to FN would inhibit its binding to IGF-I and consequently abolish the potentiating effect of IGFBP-5. To test this idea, the effect of FN on the IGFBP-5 and IGF-I binding was examined in a solid phase binding assay. Pure native IGFBP-5 (10 ng/well) was immobilized onto 96-well plates in the presence or absence of FN (5 μg/well). After blocking with bovine serum albumin, increasing amounts of 125I-IGF-I (0-100,000 cpm) were added and incubated. The free 125I-IGF-I was removed by washing, and bound 125I-IGF-I was measured. There was little 125I-IGF-I detected in the absence of IGFBP-5, suggesting that IGF-I does not bind to FN. 125I-IGF-I bound to the immobilized IGFBP-5 in a dose-dependent manner, and a plateau was reached at concentration higher than 40,000 cpm. Scatchard analyses indicated that the Kd value of IGFBP-5 for IGF-I binding was 3.8 × 10-9m in the absence of FN. In the presence of FN, the Kd value was 3.0 × 10-9m. These results suggest that the presence of FN does not significantly affect the IGF-I and IGFBP-5 binding. To determine whether IGFBP-5 can bind to FN and IGF-I simultaneously, 125I-IGF-I was incubated with GST-IGFBP-5 in the presence or absence of FN. GST was used as a control. A high level of the added 125I-IGF-I (21,690 ± 8147 cpm) was recovered from the GST-IGFBP-5 pull-down complex (Fig. 6A). In comparison, there was little 125I-IGF-I in the GST group (1,298 ± 684 cpm). Again, the presence of FN did not change the amount of 125I-IGF-I bound to GST-IGFBP-5 (20,719 ± 6756 cpm). Western immunoblotting indicated that the added FN was present in the GST-IGFBP-5 complex (Fig. 6A), indicating that the three proteins are present in the same complex. We further studied the complex formation using purified, native IGFBP-5, and FN. 125I-IGF-I was incubated with FN in the presence or absence of pure IGFBP-5. After incubation, FN was precipitated using a FN antibody. As shown in Fig. 6B, there was little 125I-IGF-I in the immunoprecipitation complex in the absence of IGFBP-5. When IGFBP-5 was added, a high level of the added 125I-IGF-I (10,056 ± 3611 cpm) was found in the immunoprecipitation complex, suggesting that IGFBP-5 binds to FN and IGF-I simultaneously. Finally, FN and GSTIGFBP-5 were incubated in the presence or absence of excess amount of IGF-I. The GST-IGFBP-5-bound FN was pulled down and determined by immunoblotting. There was no significant difference in the amount of FN bound to GST-IG
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