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 modulati ng IGF-II actions. N-terminal amino acid sequence and mass spectrometric an alyses of the 6xHis-tagged IGFBP-4 proteolytic fragments revealed that Met1 35-Lys136 was the only cleavage site recognized by the IGF-II-dependent IGF BP-4 protease produced by hOBs, This cleavage site was confirmed by the fin ding that deletion of His121 to Pro141 blocked proteolysis. However, unexpe ctedly, deletion of Pro94 to Gln119 containing no cleavage site had no effe ct on IGF-II binding activity but blocked proteolysis. Addition of the synt hetic 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 req uired to expose the cleavage site rather than being involved in direct prot ease-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-induc ed cell proliferation in normal hOBs, which produce IGFBP-4 protease, and M G63 cells, which do not produce IGFBP-4 protease, It was found that proteas e-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 IGF BP-4 bioavailability and consequently the mitogenic activity of IGFs in hOB s.