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Heparin-Binding, Highly Basic Regions within the Thyroglobulin Type-1 Repeat of Insulin-Like Growth Factor (IGF)-Binding Proteins (IGFBPs) -3, -5, and -6 Inhibit IGFBP-4 Degradation¹

Department of Pediatrics, University of Kentucky College of Medicine (J.L.F., K.M.T.), Lexington, Kentucky 40536; Bios-Chile (C.G-N), Santiago, Chile; and the Department of Pediatrics, Duke University Medical Center (C.K.R., D.M.S.), Durham, North Carolina 27710

Address all correspondence and requests for reprints to: John L. Fowlkes, M.D., Department of Pediatrics, Division of Endocrinology, University of Kentucky College of Medicine, J462 Kentucky Clinic, 740 South Limestone Avenue, Lexington, KY 40536-0284.

MC3T3-E1 murine osteoblasts produce insulin-like growth factor (IGF)-binding protein-4 (IGFBP-4)-degrading proteinase activity, which is inhibited by IGFBP-3 and a highly basic, C-terminal domain of IGFBP-3. Of all the other five IGFBPs, IGFBP-5 and -6 share the highest degree of homology with this domain of IGFBP-3; therefore, we investigated whether these two IGFBPs inhibit IGFBP-4 degradation. Both IGFBP-5 and IGFBP-6 inhibit the degradation of ¹²⁵I-IGFBP-4 by MC3T3-E1-conditioned media, and their inhibitory effects are variably reversed by IGFs. Synthetic peptides containing highly basic, C-terminal regions of IGFBP-5 and IGFBP-6 inhibit ¹²⁵I-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 the least potent. In contrast, a homologous, yet neutral, IGFBP-4 peptide does not inhibit ¹²⁵I-IGFBP-4 proteolysis, confirming the role of basic residues in the inhibitory process. The IGFBP-3, -5, and -6 peptides, each of which contains the heparin-binding consensus sequence XBBBXXBX, bind heparin, yet the IGFBP-3 and -5 peptides bind heparin with the highest affinities, whereas the IGFBP-6 peptide binds heparin with 10-fold less affinity. Consistent with these regions being involved in proteinase inhibition, heparin completely reverses their inhibitory effects on ¹²⁵I-IGFBP-4 proteolysis. Together, these data demonstrate that IGFBP-3, -5, and -6 can function as IGF-reversible inhibitors of IGFBP-4 proteolysis, likely through homologous, highly basic, heparin-binding domains contained within the conserved thyroglobulin type-1 motif present in the C-termini of these IGFBPs.

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