HEPARIN-BINDING, HIGHLY BASIC REGIONS WITHIN THE THYROGLOBULIN TYPE-1REPEAT OF INSULIN-LIKE-GROWTH-FACTOR (IGF)-BINDING PROTEIN-3, PROTEIN-5, AND PROTEIN-6 (IGFBPS) INHIBIT IGFBP-4 DEGRADATION

MC3T3-E1 murine osteoblasts produce insulin-like,growth factor (IGF)-b inding protein-4, (IGFBP-4)-degrading proteinase activity, which is in hibited by IGFBP-3 and a highly basic, C-terminal domain of IGFBP-3. O f all the other five IGFBPs, IGFBF-5 and -6 share the highest degree o f homology with this domain of IGFBP-3; therefore, we investigated whe ther these two IGFBPs inhibit IGFBP-4 degradation. Both IGFBP-5 and IG FBP-6 inhibit the degradation of I-125-IGFBP-4 by MC3T3-E1-conditioned media, and their inhibitory effects are variably reversed by IGFs. Sy nthetic peptides containing highly basic. C-terminal regions of IGFBP- 5 and IGFBP-6 inhibit I-125-IGFBP-4 degradation, as does an homologous IGFBP-3 peptide, yet each peptide displays a different IC50, with the IGFBP-5 peptide being the most potent and the IGFBP-6 peptide being t he least potent. In contrast, a homologous, yet neutral, IGFBP-4 pepti de does not inhibit I-125-IGFBP-4 proteolysis, confirming the role of basic residues in the inhibitory process. The IGFBP-3, -5, and -6 pept ides, each of which contains the heparin-binding consensus sequence XB BBXXBX. bind heparin, yet the IGFBP-3 and -5 peptides bind heparin wit h the highest affinities, whereas the IGFBP-6 peptide binds heparin wi th similar to 10-fold less affinity. Consistent with these regions bei ng involved in proteinase inhibition, heparin completely reverses thei r inhibitory effects on I-125-IGFBP-4 proteolysis. Together, these dat a demonstrate that IGFBP-3, -5, and -6 can function as IGF-reversible inhibitors of IGFBP-4 proteolysis, likely through homologous, highly b asic, heparin-binding domains contained within the conserved thyroglob ulin type-1 motif present in the C-termini of these IGFBPs.