IGF Fragments

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, NH2-
terminal (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 b-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. (