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• W2088085585 abstract "To identify the molecular mechanism by which insulin-like growth factor binding protein-4 (IGFBP-4) exerts its inhibitory effects on insulin-like growth factor (IGF) actions, we localized and determined the role of the IGF binding domain in modulating IGF actions in human osteoblasts. Deletion analysis using IGFBP-4 expressed in bacteria revealed that the N-terminal sequence Leu72–Ser91 was essential for IGF binding. The C-terminal fragments (His121–Glu237 or Arg142–Glu237) did not bind to IGF but loss of these regions decreased IGF binding activity. Detailed deletion analysis identified the residues Cys205–Val214as the motif to facilitate IGF binding. Mitogenic studies revealed that an IGFBP-4 mutant (His74 replaced by Pro74) and an N-terminal peptide (N terminus to Thr71) with little IGF binding activity failed to inhibit IGF-II-induced human osteoblast proliferation. An N-terminal peptide (N terminus to Asn182) with reduced IGF binding activity inhibited IGF action but with lower potency. In contrast, an IGFBP-4 mutant (His74 replaced with Ala74) exhibited similar IGF binding activity and potency in inhibiting the activity of IGF-II compared with the wild type. Therefore, the N-terminal sequence (Leu72–Ser91) and the C-terminal sequence (Cys205–Val214) are necessary to form the high affinity IGF binding domain, which is the major structural determinant of the IGFBP-4 function. To identify the molecular mechanism by which insulin-like growth factor binding protein-4 (IGFBP-4) exerts its inhibitory effects on insulin-like growth factor (IGF) actions, we localized and determined the role of the IGF binding domain in modulating IGF actions in human osteoblasts. Deletion analysis using IGFBP-4 expressed in bacteria revealed that the N-terminal sequence Leu72–Ser91 was essential for IGF binding. The C-terminal fragments (His121–Glu237 or Arg142–Glu237) did not bind to IGF but loss of these regions decreased IGF binding activity. Detailed deletion analysis identified the residues Cys205–Val214as the motif to facilitate IGF binding. Mitogenic studies revealed that an IGFBP-4 mutant (His74 replaced by Pro74) and an N-terminal peptide (N terminus to Thr71) with little IGF binding activity failed to inhibit IGF-II-induced human osteoblast proliferation. An N-terminal peptide (N terminus to Asn182) with reduced IGF binding activity inhibited IGF action but with lower potency. In contrast, an IGFBP-4 mutant (His74 replaced with Ala74) exhibited similar IGF binding activity and potency in inhibiting the activity of IGF-II compared with the wild type. Therefore, the N-terminal sequence (Leu72–Ser91) and the C-terminal sequence (Cys205–Val214) are necessary to form the high affinity IGF binding domain, which is the major structural determinant of the IGFBP-4 function. insulin-like growth factor IGF binding protein human IGF human osteoblasts glutathione S-transferase isopropyl-1-thio-β-d-galactopyranoside high performance liquid chromatography polyacrylamide gel electrophoresis polymerase chain reaction. Insulin-like growth factors (IGFs)1 play a critical role in promoting the differentiation and proliferation of a variety of cell types including human osteoblasts (hOBs) (1Mohan S. Bautista C.M Wergedal J.E. Baylink D.J. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 8338-8349Crossref PubMed Scopus (352) Google Scholar, 2Mohan S. Growth Regul. 1993; 3: 67-70PubMed Google Scholar, 3Rosen C.J. Donahue L.A. Hunter S.J. Proc. Soc. Exp. Bio. Med. 1994; 206: 83-102Crossref PubMed Scopus (199) Google Scholar, 4Canalis E. Bone. 1993; 14: 273-276Crossref PubMed Scopus (94) Google Scholar). The functions of IGFs depend not only on the amount of IGF produced but also on the level of IGF binding proteins (IGFBP) which modulate their actions (5Rechler M.M. Vitam. Horm. 1993; 47: 1-14Crossref PubMed Scopus (444) Google Scholar, 6Mohan 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 (260) Google Scholar, 7LaTour D. Mohan S. Linkhart T.A. Baylink D.J. Strong D. Mol. Endocrinol. 1990; 4: 1806-1814Crossref PubMed Scopus (218) Google Scholar, 8Campbell P.G. Novak J.F. J. Cell. Physiol. 1991; 149: 93-300Crossref Scopus (65) Google Scholar, 9Jones J.I. Clemmons D.R. Endocr. Rev. 1995; 16: 3-34Crossref PubMed Google Scholar) as well as the specific IGFBP proteases that regulate the availability of the IGFBPs (10Durham S.K. Kiefer M.C. Rggs B.L. Conover C.A. J. Bone Miner. Res. 1994; 9: 111-117Crossref PubMed Scopus (85) Google Scholar, 11Kanzaki S. Hilliker S. Baylink D.J. Mohan S. Endocrinology. 1994; 134: 383-392Crossref PubMed Scopus (109) Google Scholar, 12Nam T.J. Busby Jr., W.H. Clemmons D.R. Endocrinology. 1996; 137: 5530-5536Crossref PubMed Scopus (33) Google Scholar, 13Fowlkes J.L. Thraikill K.M. Serra D.M. Suzuki K. Nagase H. Prog. Growth Factor Res. 1995; 6: 255-263Abstract Full Text PDF PubMed Scopus (107) Google Scholar, 14Fowlkes J. Freemark M. Endocrinology. 1992; 131: 2071-2076Crossref PubMed Scopus (71) Google Scholar). Although hOBs produce multiple IGFBPs, IGFBP-4 is particularly important with regard to local reduction of IGF function based on its abundance (10Durham S.K. Kiefer M.C. Rggs B.L. Conover C.A. J. Bone Miner. Res. 1994; 9: 111-117Crossref PubMed Scopus (85) Google Scholar, 15Malpe R. Baylink D.J. Linkhart T.A. Wergedal J.E. Mohan S. J. Bone Miner. Res. 1997; 12: 423-430Crossref PubMed Scopus (70) Google Scholar) and biological potency (1Mohan S. Bautista C.M Wergedal J.E. Baylink D.J. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 8338-8349Crossref PubMed Scopus (352) Google Scholar, 6Mohan 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 (260) Google Scholar). In vitro studies demonstrate that IGFBP-4 inhibits IGF-stimulated osteoblast cell proliferation (1Mohan S. Bautista C.M Wergedal J.E. Baylink D.J. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 8338-8349Crossref PubMed Scopus (352) Google Scholar, 6Mohan 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 (260) Google Scholar). The importance of IGFBP-4 as a negative regulator of hOB cell proliferation is also attested to by studies on the regulation of IGFBP-4 production. In cultured osteoblasts, agents that inhibit osteoblast cell proliferation (cAMP, 1,25-dihydroxyvitamin D3, and parathyroid hormone) increase IGFBP-4 levels (7LaTour D. Mohan S. Linkhart T.A. Baylink D.J. Strong D. Mol. Endocrinol. 1990; 4: 1806-1814Crossref PubMed Scopus (218) Google Scholar, 16Scharla S.H. Strong D.D. Mohan S. Baylink D.J. Linkhart T.A. Endocrinology. 1991; 129: 3139-3146Crossref PubMed Scopus (126) Google Scholar, 17Scharla S.H. Strong D.D. Rosen C. Mohan S. Hilick M. Baylink D.J. Linkhart T.A. J. Clin. Endocrinol. Metab. 1993; 77: 1190-1197Crossref PubMed Google Scholar, 18Scharla S.H. Mohan S. Strong D.D. Baylink D.J. Linkhart T.A. Cohn D.V. Gennari C. Tashjian Jr., A.H. Calcium Regulating Hormones and Bone Metabolism. Excerpta Medica, New York1992: 223-227Google Scholar), while agents that increase cell proliferation (progesterone, IGFs, transforming growth factor-β, and bone morphogenetic protein-7) decrease IGFBP-4 concentrations in the conditioned medium (10Durham S.K. Kiefer M.C. Rggs B.L. Conover C.A. J. Bone Miner. Res. 1994; 9: 111-117Crossref PubMed Scopus (85) Google Scholar, 19Durham S.K. Riggs B.L. Conover C.A. J. Clin. Endocrionol. Metab. 1994; 79: 1752-1758Crossref PubMed Google Scholar, 20Knutsen R. Honda Y. Strong D.D. Sampath T.K. Baylink D.J. Mohan S. Endocrinology. 1995; 136: 857-865Crossref PubMed Google Scholar). In vivo studies on serum regulation of IGFBP-4 have shown that the serum IGFBP-4 levels are elevated with age and in type II osteoporosis patients (21Honda, Y., Landale, E., Strong, D. D., Baylink, D. J. & Mohan, S. (1996) J. Clin. Endocrinol. Metab.1389–1396Google Scholar, 22Rosen C. Donahue L.R. Hunter S. Holick M. Kavookjian H. Kirschenbaum A. Mohan S. Baylink D.J. J. Clin. Endocrinol. Metab. 1992; 74: 24-27Crossref PubMed Scopus (57) Google Scholar, 23Rajaram S. Baylink D.J. Mohan S. Endocr. Rev. 1997; 18: 801-831Crossref PubMed Scopus (963) Google Scholar). In addition to the role of IGFBP-4 in modulating IGF actions in bone, the inhibitory effect of IGFBP-4 on the mitogenic effect of IGFs has also been demonstrated in other cell types, such as human fibroblasts (24Conover C.A. Durham S.K. Zapf J. Masiarz F.R. Kiefer M.C. J. Biol. Chem. 1995; 270: 4395-4400Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar), neuronal cells (25Cheung P.T. Smith E.P. Shimasaki S. Ling N. Chernausek S.D. Endocrinology. 1991; 129: 1006-1015Crossref PubMed Scopus (107) Google Scholar), and colon carcinoma cells (26Culouscou J.M. Shyoab M. Cancer Res. 1991; 51: 2813-2819PubMed Google Scholar). These findings suggest that IGFBP-4 is a key component of the IGF system in a variety of tissues including bone.The mechanisms by which IGFBPs stimulate or inhibit IGF actions have not been clearly defined and may vary among the IGFBPs. In this regard, recent evidence suggests that some of the IGFBPs may exert IGF-independent actions (6Mohan 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 (260) Google Scholar, 27Lalou C. Lassarre C. Binoux M. Endocrinology. 1996; 137: 3206-3212Crossref PubMed Scopus (148) Google Scholar, 28Valentinis B. Bhala A. Deangelis T. Baserga R. Cohen P. Mol. Endocrinol. 1995; 9: 361-367Crossref PubMed Google Scholar, 29Mohseni-Zadeh S. Binoux M. Endocrinology. 1997; 138: 3069-3072Crossref PubMed Google Scholar), in addition to modulating IGF actions (6Mohan 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 (260) Google Scholar, 8Campbell P.G. Novak J.F. J. Cell. Physiol. 1991; 149: 93-300Crossref Scopus (65) Google Scholar, 30Chernausek S.D. Smith C.E. Duffin K.L. Busby W.H. Wright G. Clemmons D.R. J. Biol. Chem. 1995; 270: 11377-11382Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). Studies in our laboratory demonstrate that IGFBP-4 primarily acts to inhibit cell proliferation by an IGF-dependent mechanism (6Mohan 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 (260) Google Scholar). The findings which support this concept include: 1) IGFBP-4 has been shown to compete with IGF receptors for IGF binding in both cells in monolayer culture and in purified type I IGF receptor preparations (6Mohan 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 (260) Google Scholar), 2) IGFBP-4 has no effect on cell proliferation induced by IGF-I or -II analogs that exhibit reduced affinity for IGFBP-4 (6Mohan 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 (260) Google Scholar), and 3) IGFBP-4 proteolytic fragments with reduced IGF binding activity are unable to inhibit IGF-induced cell proliferation (10Durham S.K. Kiefer M.C. Rggs B.L. Conover C.A. J. Bone Miner. Res. 1994; 9: 111-117Crossref PubMed Scopus (85) Google Scholar, 30Chernausek S.D. Smith C.E. Duffin K.L. Busby W.H. Wright G. Clemmons D.R. J. Biol. Chem. 1995; 270: 11377-11382Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). Although these studies provide indirect evidence that binding of IGFBP-4 to an IGF is essential for inhibiting IGF action, this has not been verified by using IGFBP-4 analogs that do not bind the IGFs.The amino acids that constitute the IGF binding domain have not been identified for any of the six known high affinity IGFBPs. A few reports have indicated that both the N-terminal and C-terminal regions are critical for IGF binding (31Brinkman A. Kortleve D.J. Zwarthoff E.C. Drop S.L.S. Mol. Endocrinol. 1991; 5: 987-994Crossref PubMed Scopus (34) Google Scholar, 32Hobba G.D. Forbes B.E. Parkinson E.J. Francis G.L. Wallace J.C. J. Biol. Chem. 1996; 271: 30529-30536Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 33Spencer E.M. Chan K. Prog. Growth Factor Res. 1995; 6: 209-214Abstract Full Text PDF PubMed Scopus (41) Google Scholar, 34Wang J.F Hampton B. Mehlman I. Burgess W.H. Rechler M.M. Biochem. Biophys. Res. Commun. 1988; 157: 718-726Crossref PubMed Scopus (53) Google Scholar, 35Huhtala M.L. Koistinen R. Palomaki P. Partanen P. Bohn H. Seppala M. Biochem. Biophys. Res. Commun. 1986; 141: 263-270Crossref PubMed Scopus (42) Google Scholar, 36Brinkman A. Kortleve D.J. Schuller A.G.P. Zwarthoff E.C. Drop S.L.S. FEBS Lett. 1991; 29: 264-268Crossref Scopus (25) Google Scholar). Interestingly, the sequences and residues important for IGF binding appear to differ among the IGFBPs studied to date (31Brinkman A. Kortleve D.J. Zwarthoff E.C. Drop S.L.S. Mol. Endocrinol. 1991; 5: 987-994Crossref PubMed Scopus (34) Google Scholar, 32Hobba G.D. Forbes B.E. Parkinson E.J. Francis G.L. Wallace J.C. J. Biol. Chem. 1996; 271: 30529-30536Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 33Spencer E.M. Chan K. Prog. Growth Factor Res. 1995; 6: 209-214Abstract Full Text PDF PubMed Scopus (41) Google Scholar, 34Wang J.F Hampton B. Mehlman I. Burgess W.H. Rechler M.M. Biochem. Biophys. Res. Commun. 1988; 157: 718-726Crossref PubMed Scopus (53) Google Scholar, 35Huhtala M.L. Koistinen R. Palomaki P. Partanen P. Bohn H. Seppala M. Biochem. Biophys. Res. Commun. 1986; 141: 263-270Crossref PubMed Scopus (42) Google Scholar, 36Brinkman A. Kortleve D.J. Schuller A.G.P. Zwarthoff E.C. Drop S.L.S. FEBS Lett. 1991; 29: 264-268Crossref Scopus (25) Google Scholar). Recombinant IGFBP-1 with a 15-residue deletion in the C-terminal region did not exhibit IGF binding activity (31Brinkman A. Kortleve D.J. Zwarthoff E.C. Drop S.L.S. Mol. Endocrinol. 1991; 5: 987-994Crossref PubMed Scopus (34) Google Scholar). Chemical modification experiments of the bovine IGFBP-2 suggest that Tyr60 in the N-terminal region is important for IGF binding (32Hobba G.D. Forbes B.E. Parkinson E.J. Francis G.L. Wallace J.C. J. Biol. Chem. 1996; 271: 30529-30536Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). Studies of the IGF binding site in IGFBP-3 indicate that both N-terminal and C-terminal fragments of IGFBP-3 bind to IGF (33Spencer E.M. Chan K. Prog. Growth Factor Res. 1995; 6: 209-214Abstract Full Text PDF PubMed Scopus (41) Google Scholar). Analysis of the IGF binding activity of IGFBP-4 proteolytic fragments revealed that a N-terminal fragment binds to IGFs with a 15-fold reduced affinity compared with the intact form (37Cheung P.T. Wu J. Banach W. Chernausek S.D. Endocrinology. 1994; 135: 1328-1335Crossref PubMed Scopus (47) Google Scholar). Taken together, these findings suggest that the structure, location, and perhaps, the number of IGF binding sites in the IGFBPs may differ.The purpose of this study was to characterize the hIGFBP-4 IGF binding domain and evaluate if the IGF binding domain is essential for the inhibitory effect of IGFBP-4 on hOB proliferation. To evaluate the critical regions in IGFBP-4 which are essential for IGF binding, we generated various IGFBP-4 analogs by protein engineering and used these analogs for evaluation of IGF binding and biological activity.DISCUSSIONThis is a first study that shows the results of the systematic analysis of the IGF binding activity of N-terminal-, mid-molecule-, and C-terminal-deleted recombinant IGFBP-4 analogs. Our findings demonstrate that although the N-terminal but not the C-terminal recombinant IGFBP-4 fragments bind radiolabeled IGF-I and IGF-II, structural elements in both the N-terminal and the C-terminal regions of IGFBP-4 are essential for high affinity IGF binding. The findings of this study also demonstrate that disruption of the IGF binding domain abolishes the inhibitory effect of IGFBP-4 on IGF-induced cell proliferation in serum-free cultures of human osteoblasts.It is generally accepted that the IGF binding site for the various high affinity IGFBPs is in the N-terminal region, based on the findings that N-terminal IGFBP proteolytic fragments or recombinant N-terminal IGFBP peptides retain various IGF binding activities (6Mohan 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 (260) Google Scholar, 27Lalou C. Lassarre C. Binoux M. Endocrinology. 1996; 137: 3206-3212Crossref PubMed Scopus (148) Google Scholar, 33Spencer E.M. Chan K. Prog. Growth Factor Res. 1995; 6: 209-214Abstract Full Text PDF PubMed Scopus (41) Google Scholar, 37Cheung P.T. Wu J. Banach W. Chernausek S.D. Endocrinology. 1994; 135: 1328-1335Crossref PubMed Scopus (47) Google Scholar). As a first step toward identifying the critical sequences in IGFBP-4 for IGF-I and IGF-II binding, we generated a series of N-terminal and C-terminal fragments of IGFBP-4 by recombinant DNA technology and evaluated their IGF binding activity by Western ligand blot analysis using radiolabeled IGF-I and IGF-II as tracers. Our findings demonstrate that a number of N-terminal fragments encoded by exon 1 and 2 of IGFBP-4 retained IGF binding activity but none of the C-terminal IGFBP-4 fragments tested exhibited measurable IGF binding activity (Figs. Figure 1, Figure 2, Figure 3). These findings are consistent with the hypothesis that the IGF binding site in IGFBP-4 is located in the N-terminal region. In contrast to our findings, Spencer and Chan (33Spencer E.M. Chan K. Prog. Growth Factor Res. 1995; 6: 209-214Abstract Full Text PDF PubMed Scopus (41) Google Scholar) have recently reported that a C-terminal fragment of recombinant IGFBP-3 encoding residues 151–263, expressed in bacteria, exhibits IGF binding activity. Moreover, an IGFBP-2 C-terminal proteolytic fragment (148–270) purified from Life BRL-3A conditioned medium also retained partial IGF-I and -II binding activity (34Wang J.F Hampton B. Mehlman I. Burgess W.H. Rechler M.M. Biochem. Biophys. Res. Commun. 1988; 157: 718-726Crossref PubMed Scopus (53) Google Scholar). Therefore, while hIGFBP-4 does not seem to contain an independent IGF binding site in the C-terminal region, other IGFBPs may contain additional IGF binding sites in this region.It is not known whether the structural elements that are involved in IGF binding are similar or different for the six high affinity IGFBPs. In this study, we found that an N-terminal fragment Gly−5to Ser91 but not Gly−5 to Thr71binds to the IGFs, suggesting that the region Leu72–Ser91 is critical for IGFBP-4 binding to the IGFs. To further confirm the importance of this region in IGF binding, we replaced His74, a basic residue that is conserved in IGFBP-4 from different species, with Pro74 in order to disrupt the IGFBP-4 structure in this region. This structural disruption led to a greater than 50-fold reduction in IGF-I and IGF-II binding activity (Fig. 6). Sequence comparison of IGFBPs derived from different species indicates that this region is highly conserved among IGFBP-4s derived from human, bovine, and rat, whereas some of other IGFBPs from different species demonstrate more variability in this region (39Landale E.C. Strong D.D. Mohan S. Baylink D.J. Growth Factors. 1995; 12: 245-250Crossref PubMed Scopus (13) Google Scholar, 40Roghani M. Hossenlopp P. Balland A. Binoux M. FEBS Lett. 1989; 255: 253-258Crossref PubMed Scopus (99) Google Scholar) (Fig. 8). Since the sequence, Leu72–Ser91 resided in an area that contained both conserved and variable amino acid sequences among the six IGFBPs, it is speculated that the IGF binding domain in IGFBP-4 is different from that of other IGFBPs. Although deletion and initial mutagenesis studies suggest that amino acids, particularly Glu90 and Ser91, in the region Leu72–Ser91 are functionally significant in contributing to the formation of an IGF binding domain and that this region may represent the IGF binding domain, additional site-directed mutagenesis studies involving single and multiple amino acid substitutions will be required in order to determine the contribution of each residue to IGF binding.Our findings also demonstrate that, although C-terminal fragments of IGFBP-4 do not exhibit measurable IGF binding, the C-terminal region is required for high affinity binding of IGFBP-4 to the IGFs. Based on the findings that the deletion of Val214–Cys205caused at least a 6-fold reduction in IGF binding activity (Fig. 3), we predict that this region in IGFBP-4 is critical for high affinity IGF binding. Consistent with these data, other studies have shown that the C-terminal domain in IGFBP-3 is essential for IGF binding (27Lalou C. Lassarre C. Binoux M. Endocrinology. 1996; 137: 3206-3212Crossref PubMed Scopus (148) Google Scholar, 29Mohseni-Zadeh S. Binoux M. Endocrinology. 1997; 138: 3069-3072Crossref PubMed Google Scholar, 33Spencer E.M. Chan K. Prog. Growth Factor Res. 1995; 6: 209-214Abstract Full Text PDF PubMed Scopus (41) Google Scholar). Furthermore, substitution of Cys226 to Tyr226in IGFBP-1 led to dimerization and a loss of IGF binding activity (31Brinkman A. Kortleve D.J. Zwarthoff E.C. Drop S.L.S. Mol. Endocrinol. 1991; 5: 987-994Crossref PubMed Scopus (34) Google Scholar). Comparison of the amino acid sequence of the six high affinity IGFBPs from different species revealed that a Cys-containing sequence, Cys205–Tyr206–Cys207–Val208–Asp209, is highly conserved among the high affinity IGFBPs (Fig. 8). Further studies are needed, however, to evaluate whether this conserved motif represents a critical domain in facilitating the binding of IGFs to the N-terminal motif in IGFBP-4.IGFBP-4 is unique among the six known high affinity IGFBPs by having two extra cysteine residues (Cys110 and Cys117) in the variable region encoded by exon 2 (7LaTour D. Mohan S. Linkhart T.A. Baylink D.J. Strong D. Mol. Endocrinol. 1990; 4: 1806-1814Crossref PubMed Scopus (218) Google Scholar, 39Landale E.C. Strong D.D. Mohan S. Baylink D.J. Growth Factors. 1995; 12: 245-250Crossref PubMed Scopus (13) Google Scholar). In order to evaluate whether this region containing the two extra cysteine residues contributes to IGF binding activity, we prepared an IGFBP-4 analog in which the region Pro94–Gln119 was deleted. Based on the finding that the deletion of this variable region had no effect on the IGF binding activity (Fig. 5), we concluded that the two unique Cys residues contained in the mid-region of hIGFBP-4 were not important in the formation of the IGFBP-4 + IGF complex.The findings of our study are also consistent with the concept that IGFBP-4 contains a single binding site for both IGF-I and IGF-II. Our findings that the deletion of Leu72–Ser91results in the loss of both IGF-I and IGF-II binding (Figs. 1 and 2) and that substitution of His74 to Pro74essentially abolished both IGF-I and IGF-II binding (Fig. 6) support the above idea. In addition, previous studies have shown that both unlabeled IGF-I and IGF-II are equally effective in displacing either of the IGF tracers bound to IGFBP-4 (1Mohan S. Bautista C.M Wergedal J.E. Baylink D.J. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 8338-8349Crossref PubMed Scopus (352) Google Scholar). In contrast to IGFBP-4, IGFBP-6 binds to IGF-II but not IGF-I with high affinity (40Roghani M. Hossenlopp P. Balland A. Binoux M. FEBS Lett. 1989; 255: 253-258Crossref PubMed Scopus (99) Google Scholar). In addition, IGFBP-2 and IGFBP-5 bind to IGF-II with higher affinity than does IGF-I (23Rajaram S. Baylink D.J. Mohan S. Endocr. Rev. 1997; 18: 801-831Crossref PubMed Scopus (963) Google Scholar). Thus, it is possible that IGFBP-2, IGFBP-5, and IGFBP-6 may contain separate IGF binding sites, one for IGF-I and the other for IGF-II. Future studies on identification of the IGF-I and IGF-II binding domain for those IGFBPs that bind IGF-I and IGF-II with different affinities are needed to evaluate whether separate binding sites are present for IGF-I and IGF-II in some of the IGFBPs.The findings of this study that the C-terminal region in IGFBP-4 is essential for high affinity IGF binding are consistent with the earlier reports that hIGFBP-4 proteolytic fragments bind IGFs with little or no affinity compared with the intact protein (10Durham S.K. Kiefer M.C. Rggs B.L. Conover C.A. J. Bone Miner. Res. 1994; 9: 111-117Crossref PubMed Scopus (85) Google Scholar, 14Fowlkes J. Freemark M. Endocrinology. 1992; 131: 2071-2076Crossref PubMed Scopus (71) Google Scholar, 24Conover C.A. Durham S.K. Zapf J. Masiarz F.R. Kiefer M.C. J. Biol. Chem. 1995; 270: 4395-4400Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 37Cheung P.T. Wu J. Banach W. Chernausek S.D. Endocrinology. 1994; 135: 1328-1335Crossref PubMed Scopus (47) Google Scholar). These findings together with the observation that there is significant sequence conservation in the cysteine rich N-terminal and C-terminal regions of IGFBPs suggest that both the N-terminal and the C-terminal domains act in a cooperative manner to bind to the IGFs. However, the molecular mechanism by which the C-terminal motif in IGFBP-4 enhances IGF binding can only be speculated. In this regard, it is possible that the N-terminal sequence Leu72–Ser91 and the C-terminal sequence Cys205–Val214 may contribute to the overall IGFBP-4 tertiary structure that is important for the high affinity association of this molecule with an IGF. Alternatively, the intact IGF binding domain, located in the N-terminal region, may become more accessible to the ligand in the presence of the C-terminal region. X-ray crystallographic studies and site-directed mutagenesis of IGFBP-4 are required to elucidate the molecular mechanism by which C-terminal region of IGFBP-4 interacts with the N-terminal region to form a high affinity IGF binding site.In previous studies, we found that IGFBP-4 inhibited the binding of IGF tracer to purified IGF receptors (6Mohan 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 (260) Google Scholar). Based on these data and the data that IGFBP-4 had no effect on cell proliferation induced by those IGF analogs that exhibited >100-fold reduced affinity for binding to IGFBP-4, we proposed that IGFBP-4 inhibits IGF actions by preventing the binding of IGF to its receptor. Consistent with this hypothesis, we have now found that the IGFBP-4 analog in which Pro74 has been substituted to His74 resulted in greater than 50-fold reduction in IGF-I and -II binding activity (Fig. 6 and Table I) and that this analog had a negligible effect on cell proliferation induced by IGF-II (Fig. 7). In contrast, another IGFBP-4 analog in which His74 is replaced by Ala74 resulted in no loss of either IGF binding activity (Fig. 6) or the ability to inhibit IGF-II-induced osteoblast cell proliferation (Fig. 7). The truncated peptide, His6-BP-4(−5/182), which exhibited at least 5-fold reduction in IGF-II binding, inhibited 43% of the IGF-II-stimulated cell proliferation at 300 ng/ml. However, this potency was much lower compared with that of the wild type IGFBP-4, which inhibited 94% of the IGF-II-stimulated cell proliferation at 150 ng/ml. The truncated peptide, His6-BP-4(−5/71), which had no IGF binding activity, did not inhibit IGF-II-stimulated cell proliferation even at 1,000 ng/ml. These data demonstrate that the binding of IGF to IGFBP-4 is essential for IGFBP-4 to modulate its inhibitory effect on IGF-induced cell proliferation and that the IGF binding domain may represent the major structural determinant of IGFBP-4 biological activity.Table ISummary of the IGF binding activity of IGFBP-4 analogs prepared in this studyPeptides sequenceFusion proteinExpected molecular massIGF-I and IGF-II bindingIGF-II binding activitykDa% of the wild type hIGFBP-4Recombinant wild type IGFBP-4Gly-5 to Glu 237His6 and GST31.0 (51.0)+100Recombinant IGFBP-4 N-terminal fragmentsGly−5/Val214His6and GST28.6 (48.6)+≈100Gly−5/Lys204His627.5+<15Gly−5/Asn182His625.0+<15Gly−5/Ser167His623.3+<15Gly−5/Ser153His621.7+<15Gly−5/Pro141GST20.5+NQGly−5/Lys120His6 and GST18.2 (38.2)+<15Gly−5/Ser91His6and GST15.1 (35.1)+<15Gly−5/Gln89GST(34.8)+NQGly−5/Glu84GST(34.3)+NQGly−5/Gly75GST(33.3)+NQGly−5/Thr71His6 and GST12.9 (32.9)−0Gly−5/Tyr49GST(30.5)−0Recombinant IGFBP-4 C-terminal fragmentsArg142/Glu237GST(35.6)−0His121/Glu237GST(37.8)−0Recombinant IGFBP-4 fragments with mid-region deletionsΔPro94-Gln119His6 and GST29.2 (49.2)+≈100ΔHis121/Pro141His6 and GST29.8 (49.8)+≈100Recombinant full-length IGFBP-4 peptides with point mutationsHis74/Ala74His631.0+≈100His74/Pro74His631.0+≈2Addition of the His6 tag usually slows down the migration of the fused proteins by several kilodaltons. In this study, the apparent molecular mass of the His6-BP-4(−5/237) was approximately 31 kDa, 5 kDa larger than the calculated molecular mass of 26 kDa. Consequently, 5 kDa was added to the calculated molecular mass of each His6-tagged IGFBP-4 peptide. The predicted molecular mass of GST-IGFBP-4 fusion proteins shown in parentheses were the sum of the molecular mass of the GST peptide (25 kDa) and the calculated molecular mass of the IGFBP-4 peptide. The predicted sizes of most recombinant IGFBP-4 peptides roughly match their apparent masses on SDS-PAGE gel although minor inconsistency exists in certain analogs, possibly due to the structural modifications.IGF binding activity was" @default.
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• W2088085585 date "1998-09-01" @default.
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• W2088085585 title "Structure-Function Analysis of the Human Insulin-like Growth Factor Binding Protein-4" @default.
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