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Amino- and Carboxyl-Terminal Fragments of Insulin-Like Growth Factor (IGF) Binding Protein-3 Cooperate to Bind IGFs with High Affinity and Inhibit IGF Receptor Interactions

Kolling Institute of Medical Research, University of Sydney, Royal North Shore Hospital, St. Leonards, New South Wales 2065, Australia

Address all correspondence and requests for reprints to: Sue M. Firth, Ph.D., Kolling Institute of Medical Research, Royal North Shore Hospital, St. Leonards, New South Wales 2065, Australia. E-mail: sfirth{at}med.usyd.edu.au

Both the amino-terminal and carboxyl-terminal domains of IGF binding protein (IGFBP)-3 are believed to contribute to high-affinity IGF binding. To investigate cooperativity in IGF binding by these domains, we expressed IGFBP-3 fragments 1–88 (NBP-3) and 185–264 (CBP-3) as FLAG and hexahistidine-tagged fusion proteins, respectively. IGF-I and IGF-II bound to NBP-3 poorly and to CBP-3 with moderate affinities, approximately 1 liter/nmol. Coincubating the fragments in equimolar concentrations caused a significant cooperative increase in IGF binding, demonstrated by immunoprecipitation with IGFBP-3, FLAG, or hexahistidine antibodies. Equimolar NBP-3 + CBP-3 bound IGF-II with an affinity (12.2 liter/nmol) only 4-fold lower than that of the IGFBP-3-IGF-II complex and IGF-I with an affinity (3.2 liter/nmol) 13-fold lower than IGFBP-3-IGF-I. Heterotrimeric complexes of NBP-3, CBP-3, and IGF, also demonstrated by affinity labeling, bound acid-labile subunit poorly. Coprecipitation assays with iodinated NBP-3 or CBP-3 indicated that the fragments cannot interact unless IGF is also present. Complexing with NBP-3 + CBP-3 inhibited IGF stimulation of type 1 IGF receptor activity and IGF-II binding to the type II receptor. This study demonstrates that isolated amino-terminal and carboxyl-terminal domains of IGFBP-3 cooperate in the presence of IGFs to form high-affinity complexes that retain the ability to block IGF activity.

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