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) 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.
Hashimoto
R
R
Basic Technology Research Laboratory, Daiichi Pharmaceutical Co., Ltd., 16-13, Kitakasai 1-chome, Edogawa-ku, Tokyo 134, Japan.
Ono
M
M
Fujiwara
H
H
Higashihashi
N
N
Yoshida
M
M
Enjoh-Kimura
T
T
Sakano
K
K
D000818:Animals; D001665:Binding Sites; D005260:Female; D018972:Insulin-Like Growth Factor Binding Protein 3 / Q000235:genetics / Q000378:metabolism*; D007335:Insulin-Like Growth Factor II / Q000378:metabolism*; D016296:Mutagenesis; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship
D018972:Insulin-Like Growth Factor Binding Protein 3; D011994:Recombinant Proteins; D007335:Insulin-Like Growth Factor II