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Differential Effects of Insulin-Like Growth Factor (IGF)-Binding Protein-3 and Its Proteolytic Fragments on Ligand Binding, Cell Surface Association, and IGF-I Receptor Signaling¹

Department of Pediatrics, School of Medicine, Oregon Health Sciences University, Portland, Oregon 97201-3042

Address all correspondence and requests for reprints to: Gayathri R. Devi, Ph.D., AVI BioPharma, Inc., 4575 SW Research Way, Suite 200, Corvallis, Oregon 97333. E-mail: grdevi{at}avibio.com

Insulin-like growth factor (IGF)-binding protein-3 (IGFBP-3), the predominant IGF carrier protein in circulation, is posttranslationally modified in vivo by IGFBP-3 protease(s) into a number of fragments. Based on the ascertained and predicted recognition sites for known IGFBP-3 proteases, FLAG-epitope tagged intact IGFBP-3, NH₂terminal ^(1–97), intermediate fragment ^(88–148), and COOH-terminal fragments ^(98–264 and ^(184–264) were generated in a baculovirus and/or Escherichia coli expression system and examined, by Western ligand blot and affinity cross-linking assays, for their ability to bind IGF and insulin. The NH₂- and COOH-terminal fragments bound both IGF and insulin specifically (albeit with significantly reduced affinity) for IGF but higher affinity for insulin, when compared with intact IGFBP-3. The effect of IGFBP-3 and the fragments on IGF-I receptor (IGFIR) signaling pathways was studied by testing IGF-I-induced receptor autophosphorylation in IGFIR-overexpressing NIH-3T3 cells. IGFBP-3 showed a dose-dependent inhibition of autophosphorylation of the ß-subunit of IGFIR. The ^(1–97) NH₂-terminal fragment inhibited IGFIR autophosphorylation at high concentrations, and this effect seems largely attributable to sequestration of IGF-I. In contrast, no inhibition of IGF-I-induced IGFIR autophosphorylation was detectable with the ^(98–264) and ^(184–264) COOH-terminal fragments, despite their ability to bind IGF. However, unlike the ^(1–97)NH₂-terminal fragment, the COOH-terminal fragments of IGFBP-3 retained their ability to associate with the cell surface, and this binding was competed by heparin, similar to intact IGFBP-3.

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