Role of N- and C-terminal residues of insulin-like growth factor (IGF)-binding protein-3 in regulating IGF complex formation and receptor activation

Abstract

Insulin-like growth factor-binding protein-3 (IGFBP-3), the major IGFBP in the circulation, sequesters IGF in a stable ternary complex with the acid-labile subunit. The high affinity IGF-binding site is proposed to reside within an N-terminal hydrophobic domain in IGFBP-3, but C-terminal residues have also been implicated in the homologous protein IGFBP-5. We have mutated in various combinations Leu77, Leu80, and Leu81 in the N terminus and Gly217 and Gln223 in the C terminus of IGF-BP-3. All mutants retained immunoreactivity toward a polyclonal IGFBP-3 antibody, whereas IGF ligand blotting showed that all of the mutants had reduced binding to IGFs. Both solution IGF binding assays and BIAcore analysis indicated that mutations to the N-terminal region caused greater reduction in IGF binding activity than C-terminal mutations. The combined N- and C-terminal mutants showed undetectable binding to IGF-I but retained <10% IGF-II binding activity. Reduced ternary complex formation was seen only in mutants that had considerably reduced IGF-I binding, consistent with previous studies indicating that the binary IGF·IGFBP-3 complex is required for acid-labile subunit binding. Decreased IGF binding was also reflected in the inability of the mutants to inhibit IGF-I signaling in IGF receptor overexpressing cells. However, when present in excess, IGFBP-3 analogs defined as non-IGF-binding by biochemical assays could still inhibit IGF signaling. This suggests that residual binding activity of IGFBP-3 mutants may still be sufficient to inhibit IGF biological activity and questions the use of such analogs to study IGF-independent effects of IGFBP-3.