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

We have examined regions of rat IGF-binding protein-3 (IGFBP-3) import ant 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, Glu(52)-Ala(92)) derived from rat IGFB P-3. Solid-phase binding assays using 96-well microtiter plates were d esigned to quantitate the relative binding affinities. It was found th at not only the IGFBP-3 derivatives with the amino-terminal, cysteine- rich domain (N domain) but also the synthetic peptide maintained affin ity for IGF-II. Ternary complex formation was observed with full-lengt h IGFBP-3 and chimera IGFBP, the carboxyl-terminal cysteine-rich domai n (C domain) of which was derived from IGFBP-3, unlike the mutants lac king the C domain and the chimera IGFBPs, the C domain of which was de rived from IGFBP-2. These results were confirmed by affinity crosslink ing experiments. Furthermore, the IGFBP-3 derivatives that possessed t he C domain of IGFBP-3 bound to the acid-labile subunit, even in the a bsence 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 Tyr(27) for Leu, had extrem ely reduced activity. These results strongly suggest that: 1) the N do main, containing at least Glu(52)-Ala(92), of rat IGFBP-3 is important for binding to IGF-II; 2) the C domain of IGFBP-3 is essential for bi nding to the acid-labile subunit both in the presence and absence of I GF-II; and 3) Tyr(27) of TGF-II is important for the ternary complex f ormation.