BINDING OF MUTANTS OF HUMAN INSULIN-LIKE GROWTH FACTOR-II TO INSULIN-LIKE GROWTH-FACTOR BINDING PROTEINS-1-6

A family of six specific insulin-like growth factor binding proteins ( IGFBPs) modulates the biological actions of the insulin-like growth fa ctors, IGF-I and IGF-II. In the present study, we determined the bindi ng affinity of purified human IGFBPs 1-6 for recombinant human IGF-II mutants whose binding to IGF-I, IGF-II/mannose 6-phosphate, and insuli n receptors was previously reported (Sakano, K., Enjoh, T., Numata, F. , Fujiwara, H., Marumoto, Y., Higashihashi, N., Sato, Y., Perdue, J. F ., and Fujita-Yamaguchi, Y. (1991) J. Biol. Chem. 266, 20626-20635). O f the regions studied, the most important determinants of IGF-II bindi ng to the IGFBPs were A-domain residues 4850 and B-domain residue 26. Substitution of residues 48-50 with the analogous residues from human insulin (Thr-Ser-Ile) reduced binding to IGFBP-1, -5, and -6 more than 50-fold and to IGFBP-4 by 15-50-fold; binding to IGFBP-2 and -3 was r educed 6-12-fold. The same substitution markedly reduced binding to th e IGF-II/mannose 6-phosphate receptor but not to IGF-I or insulin rece ptors. Although substitution of residues 54 and 55 with the analogous residues from IGF-I (Arg-Arg) abolished binding to the IGF-II/mannose 6-phosphate receptor, binding to IGFBPs was not substantially affected . Substitution of Phe26 with Ser or Leu, which decreased binding to th e IGF-I and insulin receptors, reduced binding to IGFBP-1 and -6 up to 80-fold, but had lesser effects on the other IGFBPs. [Leu27]IGF-II an d [Leu43]IGF-II, which had a more markedly reduced affinity for the IG F-I and insulin receptors than did [Ser26]IGF-II, were bound by the IG FBPs with relatively unchanged affinity compared with IGF-II. Thus, th e determinants of IGF-II binding to IGFBPs partially overlap those for the IGF-II/mannose 6-phosphate receptor and overlap those for the IGF -I receptor to a lesser extent. IGFBP-I and IGFBP-6 are most sensitive to changes in IGF-II structure, although IGFBP-I binds IGF-I and IGF- II with equal affinity, whereas IGFBP-6 has a marked preferential bind ing affinity for IGF-II. IGF-II mutants with selective impairment in r ecognition by specific IGFBPs or receptors will provide a useful tool for dissecting the role of the different IGF binding macromolecules in the mediation of IGF-II actions.