Carola Dony

Structure of the IGF-binding domain of the insulin-like growth factor-binding protein-5 (IGFBP-5): implications for IGF and IGF-I receptor interactions.

Binding proteins for insulin‐like growth factors (IGFs) IGF‐I and IGF‐II, known as IGFBPs, control the distribution, function and activity of IGFs in various cell tissues and body fluids. Insulin‐like growth factor‐binding protein‐5 (IGFBP‐5) is known to modulate the stimulatory effects of IGFs and is the major IGF‐binding protein in bone tissue. We have expressed two N‐terminal fragments of IGFBP‐5 in Escherichia coli; the first encodes the N‐terminal domain of the protein (residues 1–104) and the second, mini‐IGFBP‐5, comprises residues Ala40 to Ile92. We show that the entire IGFBP‐5 protein contains only one high‐affinity binding site for IGFs, located in mini‐IGFBP‐5. The solution structure of mini‐IGFBP‐5, determined by nuclear magnetic resonance spectroscopy, discloses a rigid, globular structure that consists of a centrally located three‐stranded anti‐parallel β‐sheet. Its scaffold is stabilized further by two inside packed disulfide bridges. The binding to IGFs, which is in the nanomolar range, involves conserved Leu and Val residues localized in a hydrophobic patch on the surface of the IGFBP‐5 protein. Remarkably, the IGF‐I receptor binding assays of IGFBP‐5 showed that IGFBP‐5 inhibits the binding of IGFs to the IGF‐I receptor, resulting in reduction of receptor stimulation and autophosphorylation. Compared with the full‐length IGFBP‐5, the smaller N‐terminal fragments were less efficient inhibitors of the IGF‐I receptor binding of IGFs.

Recombinant Human Insulin-Like Growth Factor-Binding Protein-5 Stimulates Bone Formation Parameters in Vitro and in Vivo

Insulin-like growth factor-binding protein-5 (rhIGFBP-5) is stored in bone and stimulates osteoblast cell proliferation in vitro. Bone formation is dependent on the number and activity of osteoblasts. We therefore evaluated the ability of recombinant human (rh) IGFBP-5 to increase osteoblast activity in vitro; both alkaline phosphatase (ALP) activity and osteocalcin levels showed a dose-dependent increase. In in vivo time-course studies, daily sc administration of 50 μg rhIGFBP-5/day/mouse significantly increased serum osteocalcin levels by day 7, and these levels were sustained through day 21. We further evaluated whether rhIGFBP-5 was as effective as IGF-I. Daily sc administration of rhIGFBP-5 (50 μg/day), IGF-I (13 μg/day), or IGF-I plus rhIGFBP-5 complex for 9 days increased serum osteocalcin levels by 58%, 65%, and 81% (P < 0.001 in all) and femoral bone extract ALP activity by 85% (P < 0.001), 29% (P < 0.05), and 13% (P = NS), respectively, and decreased carboxyl-terminal cross-linked telopeptide of...