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 signaling.
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 beta-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.
Devi
G R
GR
Department of Pediatrics, School of Medicine, Oregon Health Sciences University, Portland 97201-3042, USA. grdevi@avibio.com
Yang
D H
DH
Rosenfeld
R G
RG
Oh
Y
Y
D016475:3T3 Cells; D000818:Animals; D001667:Binding, Competitive; D002462:Cell Membrane / Q000378:metabolism*; D019281:Dimerization; D010450:Endopeptidases / Q000378:metabolism*; D006801:Humans; D007328:Insulin / Q000378:metabolism; D018972:Insulin-Like Growth Factor Binding Protein 3 / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D007334:Insulin-Like Growth Factor I / Q000378:metabolism / Q000494:pharmacology; D007335:Insulin-Like Growth Factor II / Q000378:metabolism; D051379:Mice; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D010766:Phosphorylation; D017526:Receptor, IGF Type 1 / Q000378:metabolism*; D011994:Recombinant Proteins; D015398:Signal Transduction / Q000187:drug effects*
D007328:Insulin; D018972:Insulin-Like Growth Factor Binding Protein 3; D010446:Peptide Fragments; D011994:Recombinant Proteins; D007334:Insulin-Like Growth Factor I; D007335:Insulin-Like Growth Factor II; D017526:Receptor, IGF Type 1; D010450:Endopeptidases; C080473:insulin-like growth factor binding protein-3 protease