Binding Sites and Binding Properties of Binary and Ternary Complexes of Insulin-like Growth Factor-II (IGF-II), IGF-binding Protein-3, and Acid-labile Subunit

Abstract We have examined regions of rat IGF-binding protein-3 (IGFBP-3) important for complex formations using two kinds of deletion mutants, three kinds of chimera molecules between rat IGFBP-3 and rat IGFBP-2, and a synthetic peptide (41 residues, Glu52-Ala92) derived from rat IGFBP-3. Solid-phase binding assays using 96-well microtiter plates were designed to quantitate the relative binding affinities. It was found that not only the IGFBP-3 derivatives with the amino-terminal, cysteine-rich domain (N domain) but also the synthetic peptide maintained affinity for IGF-II. Ternary complex formation was observed with full-length IGFBP-3 and chimera IGFBP, the carboxyl-terminal cysteine-rich domain (C domain) of which was derived from IGFBP-3, unlike the mutants lacking the C domain and the chimera IGFBPs, the C domain of which was derived from IGFBP-2. These results were confirmed by affinity cross-linking experiments. Furthermore, the IGFBP-3 derivatives that possessed the C domain of IGFBP-3 bound to the acid-labile subunit, even in the absence of IGFs. Finally, we observed sites in IGF-II important for the ternary complex formation using various IGF-II mutants. These IGF-II mutants, which contained a substitution of Tyr27 for Leu, had extremely reduced activity. These results strongly suggest that: 1) the N domain, containing at least Glu52-Ala92, of rat IGFBP-3 is important for binding to IGF-II; 2) the C domain of IGFBP-3 is essential for binding to the acid-labile subunit both in the presence and absence of IGF-II; and 3) Tyr27 of IGF-II is important for the ternary complex formation.