Studies on the role of human insulin-like growth factor-II (IGF-II)-dependent IGF binding protein (hIGFBP)-4 protease in human osteoblasts using protease-resistant IGFBP-4 analogs.
To characterize the insulin-like growth factor binding protein-4 (IGFBP-4) protease produced by human osteoblasts (hOBs), we localized and determined the role of the proteolytic domains in human IGFBP-4 (hIGFBP-4) in modulating IGF-II actions. N-terminal amino acid sequence and mass spectrometric analyses of the 6xHis-tagged IGFBP-4 proteolytic fragments revealed that Met135-Lys136 was the only cleavage site recognized by the IGF-II-dependent IGFBP-4 protease produced by hOBs. This cleavage site was confirmed by the finding that deletion of His121 to Pro141 blocked proteolysis. However, unexpectedly, deletion of Pro94 to Gln119 containing no cleavage site had no effect on IGF-II binding activity but blocked proteolysis. Addition of the synthetic peptide corresponding to this region at concentrations of 250 or 1000 molar excess failed to block IGFBP-4 proteolysis. These data suggest that residues 94-119 may be involved in maintaining the IGFBP-4 conformation required to expose the cleavage site rather than being involved in direct protease-substrate binding. To determine the physiological significance of the IGF-II-dependent IGFBP-4 protease, we compared the effect of the wild-type IGFBP-4 and the protease-resistant IGFBP-4 analogs in blocking IGF-II-induced cell proliferation in normal hOBs, which produce IGFBP-4 protease, and MG63 cells, which do not produce IGFBP-4 protease. It was found that protease-resistant IGFBP-4 analogs were more potent than the wild-type protein in inhibiting IGF-II-induced cell proliferation in hOBs but not in MG63 cells. These data suggest that IGFBP-4 proteolytic fragments are not biologically active and that IGFBP-4 protease plays an important role in regulating IGFBP-4 bioavailability and consequently the mitogenic activity of IGFs in hOBs.
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Musculoskeletal Diseases Center, J.L. Pettis Memorial Veterans' Medical Center, Loma Linda University, Loma Linda, California, USA.
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Strong
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Baylink
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Mohan
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D000595:Amino Acid Sequence; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D006801:Humans; D018974:Insulin-Like Growth Factor Binding Protein 4 / Q000737:chemistry* / Q000494:pharmacology; D007335:Insulin-Like Growth Factor II / Q000494:pharmacology*; D013058:Mass Spectrometry; D008666:Metalloendopeptidases / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D010006:Osteoblasts / Q000187:drug effects* / Q000378:metabolism; D010446:Peptide Fragments / Q000494:pharmacology; D011266:Pregnancy-Associated Plasma Protein-A; D011485:Protein Binding; D013379:Substrate Specificity
D018974:Insulin-Like Growth Factor Binding Protein 4; D010446:Peptide Fragments; D007335:Insulin-Like Growth Factor II; D008666:Metalloendopeptidases; D011266:Pregnancy-Associated Plasma Protein-A