Fibronectin Binds Insulin-like Growth Factor-binding Protein 5 and Abolishes Its Ligand-dependent Action on Cell Migration

Abstract

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 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. Cell. Physiol. 1998; 177: 47-57Crossref PubMed Scopus (96) Google Scholar, 6Butt A.J. Dickson K.A. McDougall F. Baxter R.C. J. Biol. Chem. 2002; 278: 29676-29685Abstract Full Text Full Text PDF Scopus (118) Google Scholar). In fibroblasts, osteoblasts, and vascular smooth muscle cells (VSMCs), however, IGFBP-5 potentiates the effect of IGFs on cell growth (7Jones J.I. Gockerman A. Busby Jr., W.H. Camacho-Hubner C. Clemmons D.R. J. Cell Biol. 1993; 121: 679-687Crossref PubMed Scopus (481) Google Scholar, 8Andress D.L. Loop S.M. Zapf J. Kiefer M.C. Biochem. Biophys. Res. Commun. 1993; 195: 25-30Crossref PubMed Scopus (117) Google Scholar, 9Mohan S. Nakao Y. Honda Y. Landale E. Leser U. Dony C. Lang K. Baylink D.J. J. Biol. Chem. 1995; 270: 20424-20431Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar, 10Richman S. Baylink D.J. Lang K. Dony C. Mohan S. Endocrinology. 1999; 140: 4699-4705Crossref PubMed Google Scholar, 11Duan C. Clemmons D.R. J. Biol. 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 were drop-out minimal medium and at for to the of the C and of human IGFBP-5 were generated by using human IGFBP-5 cDNA as and the and and and and and and The of these are shown in The resulted were into the NcoI and BamHI sites of the vector to proteins with the Gal4 The various FN and 3 for were generated by using the FN as and the and and and and and and and and and and The FN were into the BamHI and sites of the vector to proteins with the Gal4 activation DNA was using and by DNA The were used to a NcoI the were used to a BamHI and the were used to a in a Cell and FN-null mouse embryonic cells were a from of These cells were in a of and defined medium is serum-free and no or growth and FN-null cells were in with type I VSMC cells were in medium with and bovine or co-immunoprecipitation the cells were in binding and and used Assay—The cDNA full-length human IGFBP-5 was into the NcoI and sites of the vector to a protein with at the C of IGFBP-5. cells were with and in at to an A600 of the of to a of the cells were at for the cells were in and The cells were by and at for at and the was was further at for and that was of was at at for 5 the were in of and used for the of cell or serum or of plasma FN in of binding were with or on at for 3 the the were by with binding and in The proteins were by and by as reported 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). of cell were and with of a human FN or of mouse C. J.R. T. Res. 2000; PubMed Scopus Google Scholar). The proteins were by by using an IGFBP-5 of the cell were with the IGFBP-5 and by Assay—The cell migration assays were using cell with In the were further with human FN at for The FN was by or FN-null cells were with and the cells were in cells in of the serum-free defined medium IGFBP-5 or were into the In FN were also into the of the defined with or IGF-I was into each of at the cells were from the of the The cells on the were in and the of cells was The results were as of the from the Binding solid phase binding were with FN or the at C. the were with the binding and with bovine serum in for at IGFBP-5 in of was and for 3 at C. the IGFBP-5 by of were and for at The were recovered by of with of and in an or was with in the and of human FN in binding at a of for at the were by at for and in binding The was in an or was with FN in the or of of IGF-I for at The of FN in the complex was by binding were also using of human IGFBP-5, and human FN in binding at a of The in the complex was from by using the FN the effect of FN on the of IGFBP-5, cells were to in cell with the cells were in of or with IGFBP-5 FN or IGFBP-5 FN at The were and through a The were by were by of by using of FN as an IGFBP-5-interacting of from a human aorta library were screened with a bait the Gal4 and full-length human IGFBP-5. of the positive clones that to human FN and DNA these clones into To this a from each was selected and into yeasts with the pGBKT7-IGFBP-5 or the All of the yeast on and When plated on the the cells with IGFBP-5 and FN were to 5 and The IGFBP-5 and FN interaction was further by and solution binding shown in with plasma but not FN. but not was to FN from bovine serum that binds to plasma FN. FN not in a in the plasma but it is also in a in the of To IGFBP-5 is also of with cellular assays were using are to and FN Duan C. Clemmons D.R. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). The that was to bind cellular FN secreted by these cells The IGFBP-5 and FN complex formation was also using native IGFBP-5 and FN To IGFBP-5 and FN interact with each other under co-immunoprecipitation were using These cells IGFBP-5 and FN Duan C. Clemmons D.R. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). with a FN resulted in the of IGFBP-5 The of IGFBP-5 and FN was also in a co-immunoprecipitation not the ability of IGFBP-5 to bind the of FN has no effect on IGFBP-5 and IGF-I or was with in the or of FN. The to IGFBP-5 was from by and The are the means of each in the of FN in the complex was by IGFBP-5 binds to IGF-I and FN. FN was with in the or of IGFBP-5. The from by using FN and was of IGF-I not the IGFBP-5 and FN or was with FN in the or of The FN to was by and by using a FN FN was used as positive for the The IGFBP-5 and FN Binding by the 10th and 11th I in FN and the IGFBP-5 C of the FN clones that in a from to the type type I and the cells with IGFBP-5 and a FN that the well on the medium To the that binding to IGFBP-5, and were the type and the type I repeats and the The results indicated that but not was to interact with IGFBP-5 type I repeats and and the we further forms of with IGFBP-5 as as that the type I but not the are To the of and that to each of the type I were but was of IGFBP-5 and were generated and The results that but not was of IGFBP-5 binding These results suggest that and are the domain in FN. IGFBP-5 of a a and a domain with no To of IGFBP-5 is in binding interaction with IGFBP-5 DNA to the and of IGFBP-5 were generated and into the vector to When the various pGBKT7-IGFBP-5 were into cells with cells with IGFBP-5 C domain and domain were to on the medium growth was with of the full-length IGFBP-5. The cells with FN and IGFBP-5 and not under the These suggest that C domain of IGFBP-5 is and for binding interaction with FN. Binding with FN the of IGFBP-5 in IGF-I-induced Cell not cells FN and it into the we used FN-null mouse embryonic cells to the functional of the IGFBP-5 and FN These cells not FN but are of a FN in the of FN J. J. Cell 1998; PubMed Google Scholar). can in defined medium growth factor or When to an IGF-I the and cells migration IGF-I a and a over the in and cells, The of IGFBP-5 with IGF-I at an resulted in a in the FN-null cells was that of the IGF-I In IGFBP-5 had no effect on IGF-I-induced cell migration in cells The of IGFBP-5 had no effect on cell migration in either IGFBP-5 potentiated IGF-I action in the FN-null cells but not in the cells. To these biological of IGFBP-5 were to the or of cells were to migration assay using with or FN. The of FN not the migration it alter the responses to IGF-I, but it the potentiating effect of IGFBP-5 on IGF-I-induced migration the of FN the potentiating effect of IGFBP-5 The effect of FN to for IGFBP-5, FN not the ability of to inhibit IGF-I-induced cell migration in cells In the effect of was in the of FN. FN Binding the of IGFBP-5 to the FN and IGFBP-5 binding the ability of IGFBP-5 to cell we binding of IGFBP-5 to FN inhibit binding to IGF-I and the potentiating effect of IGFBP-5. To this the effect of FN on the IGFBP-5 and IGF-I binding was in a solid phase binding native IGFBP-5 was in the or of FN with bovine serum of were and The was by and was was in the of IGFBP-5, that IGF-I not bind to FN. to the IGFBP-5 in a and a was at indicated that the of IGFBP-5 for IGF-I binding was in the of FN. In the of the was These results suggest that the of FN not the IGF-I and IGFBP-5 To IGFBP-5 can bind to FN and IGF-I was with in the or of FN. was used as a high of the was recovered from the complex In was in the the of FN not the of to indicated that the FN was in the complex that the proteins are in the further studied the complex formation using native IGFBP-5, and FN. was with FN in the or of IGFBP-5. FN was using a FN shown in was in the complex in the of IGFBP-5. When IGFBP-5 was a high of the was in the that IGFBP-5 binds to FN and IGF-I FN and were in the or of of The FN was and by was no in the of FN to in the and of IGF-I These suggest that IGFBP-5 can bind to IGF-I and and these are from each FN Binding the of IGFBP-5 by IGFBP-5 binding with FN alter IGFBP-5 stability, IGFBP-5 was to cells and in the or of FN. shown in and no IGFBP-5 was in these cells under serum-free or in the FN In the of the IGFBP-5 the In most of the IGFBP-5 was into in the of FN To the that FN or other in the FN IGFBP-5, IGFBP-5 was with FN in the serum-free medium. degradation of IGFBP-5 was To the of the effect of IGFBP-5 is to proteolysis with migration assays were in the of a shown in the of these not the to IGF-I, but it restored the potentiating effect of IGFBP-5 on IGF-I-induced migration. In this we have a the ligand-dependent action of IGFBP-5 in cell migration. show that IGFBP-5 and FN a complex by The binding of IGFBP-5 to FN is from the IGFBP-5 and IGF binding and is by the 10th and 11th type I repeats of FN and the IGFBP-5 C IGFBP-5 potentiated IGF-I-induced cell migration in the FN-null but not mouse embryonic cells. When FN was reintroduced to the cells, either in adhesive or in solution, the potentiating effect of IGFBP-5 on IGF-I-induced cell migration was abolished. effect of FN was to IGFBP-5 the of FN not the ability of to inhibit IGF-I-induced cell migration under the These results that the binding of IGFBP-5 with FN abolishes the ability of IGFBP-5 to IGF-I-induced cell migration. have that IGFBP-5 and FN directly bind to each other in vitro and are in the complex in vivo. Although this is with a that IGFBP-5 binds to FN on in vitro (7Jones J.I. Gockerman A. Busby Jr., W.H. Camacho-Hubner C. Clemmons D.R. J. Cell Biol. 1993; 121: 679-687Crossref PubMed Scopus (481) Google it with a recent that but not IGFBP-5 or binds to FN in the of human 2002; Full Text PDF PubMed Scopus Google Scholar). In the by 2002; Full Text PDF PubMed Scopus Google the not biochemical assays to FN and IGFBP-5 The that IGFBP-5 was not with FN was on the of of IGFBP-5 and FN in these was in the and is most the of is by of the and FN over the the is also that the interaction IGFBP-5 and FN IGFBP-5 has been reported to have high affinity for extracellular in fibroblasts, bone cells, and (7Jones J.I. Gockerman A. Busby Jr., W.H. Camacho-Hubner C. Clemmons D.R. J. Cell Biol. 1993; 121: 679-687Crossref PubMed Scopus (481) Google Scholar, A. C. J. Busby W.H. Clemmons D.R. Biol. Cell. 1998; PubMed Scopus Google Scholar, 9Mohan S. Nakao Y. Honda Y. Landale E. Leser U. Dony C. Lang K. Baylink D.J. J. Biol. Chem. 1995; 270: 20424-20431Abstract Full Text Full Text PDF PubMed Scopus (261) 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). To this it is of to that FN was with the in yeast two-hybrid screen of a human aorta cDNA the positive clones were FN. IGFBP-5 and FN are by vascular smooth and are in Duan C. Clemmons D.R. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google the of FN is of a interaction these proteins a high of FN in The of IGF-I actions by IGFBP-5 has been in a of cell (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). ability of IGFBP-5 has been to (7Jones J.I. Gockerman A. Busby Jr., W.H. Camacho-Hubner C. Clemmons D.R. J. Cell Biol. 1993; 121: 679-687Crossref PubMed Scopus (481) Google Scholar, 9Mohan S. Nakao Y. Honda Y. Landale E. Leser U. Dony C. Lang K. Baylink D.J. J. Biol. Chem. 1995; 270: 20424-20431Abstract Full Text Full Text PDF PubMed Scopus (261) 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, A. C. J. Busby W.H. Clemmons D.R. Biol. Cell. 1998; PubMed Scopus Google Scholar). by Clemmons and A. C. J. Busby W.H. Clemmons D.R. Biol. Cell. 1998; PubMed Scopus Google Scholar, T. J. A. Busby T. Clemmons D.R. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar, T. A. Busby W.H. Clemmons D.R. J. Biol. Chem. Full Text PDF PubMed Google have shown that binding of IGFBP-5 to or binding affinity for and other have to the that binding of the complex to cell to a in the affinity of IGFBP-5 for IGF-I, in the of IGF-I to and potentiating the IGF IGFBP-5 potentiates IGF-I action is also to IGFBP-5 binds to a of and and these proteins the cellular to IGF-I Busby Jr., W.H. C. Clemmons D.R. Endocrinology. 2000; PubMed Scopus Google Scholar, A. Clemmons D.R. Endocrinology. 2002; PubMed Scopus Google Scholar). binding of IGFBP-5 to these proteins is from ability to bind to it was that the of the IGF-I effect by IGFBP-5 is to the increased of the IGF-I to the IGF-I and of the to the cell through the binding of IGFBP-5 to these that the binding of IGFBP-5 to FN abolishes the ability of IGFBP-5 to IGF-I-induced cell migration is from the reported mechanisms. The of IGFBP-5 actions by FN to in the binding affinity the binding of IGFBP-5 to FN is from interaction with IGFs and In indicated that IGFBP-5 can bind to IGF-I and FN to a ternary is in with a recent on J. Clin. Endocr. 2001; Google and is with studies that FN binds to the IGFBP-5 C Although conserved in the C domain are also in IGF binding S. H. J. E. D.J. J. 1999; 23: PubMed Scopus Google Scholar, J. H. K. S.M. D.J. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google the high affinity binding for IGF is in the IGFBP-5 domain C. Y. J. Dony C. Lang K. T.A. J. 1998; PubMed Scopus Google Scholar, Y. A. U. Busby Jr., W.H. Clemmons D.R. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). The of the action of IGFBP-5 by FN is to the IGFBP-5 proteolysis with FN. is by the that of IGFBP-5 and FN resulted in a in the of the IGFBP-5 and the of IGFBP-5 the of restored the effect of IGFBP-5 on IGF-I-induced cell migration. In cell IGFBP-5 is or into with affinity for IGF-I 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, C. Busby W.H. Clemmons D.R. J. Biol. Chem. Full Text PDF PubMed Google Scholar). The IGFBP-5 proteolytic in medium has been to W.H. A. C. Clemmons D.R. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). In and were reported to have IGFBP-5 proteolytic Zapf J. Kiefer M.C. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar, Andress D.L. Am. J. Physiol. 1997; 273: Google Scholar, C. J.R. Natl. U. S. A. 1999; PubMed Scopus Google Scholar, C. 2001; PubMed Scopus Google Scholar, C. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). The for the IGFBP-5 in cells is not at but it is that the IGFBP-5 proteolysis in these cells FN is was degradation of IGFBP-5 in the of we that binding of IGFBP-5 with FN a of the IGFBP-5 and it to a IGFBP-5 mechanisms have been for the IGF of by Zapf J. Kiefer M.C. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar, C. J.R. Natl. U. S. A. 1999; PubMed Scopus Google and the degradation by the protein binding S.A. P. P. Cell. Biol. 2000; PubMed Scopus Google Scholar). is also that the of FN a that IGFBP-5. Further studies are to identify the IGFBP-5 and to elucidate the molecular mechanisms the IGFBP-5 proteolysis. In this has that FN and IGFBP-5 directly bind to each other, and this interaction negatively regulates the ligand-dependent action of IGFBP-5 by triggering IGFBP-5 proteolysis. The of FN as an IGFBP-5-interacting protein has not a the actions of IGFBP-5, but it has also to FN an in the of cell cell cell and cell by with and K. J. E. Natl. U. S. A. 1995; PubMed Scopus Google Scholar, A. J. Invest. 1997; Full Text PDF PubMed Scopus Google Scholar, S.B. E. J. Cell 1998; PubMed Google Scholar, J. J. Cell 2000; PubMed Google Scholar). recent that the of human breast cancer cells to FN was increased by but by IGFBP-5 C. J. Cell 2002; PubMed Scopus Google Scholar). More studies are to elucidate the potential of IGFBP-5 binding on these FN actions and to the of FN actions by IGFBP-5 to of the well actions of IGFBP-5 on cell differentiation, and of for the FN-null and mouse embryonic cells. are to and of for in the of protein and and Gordon of for of this