Specific amino acid substitutions determine the differential contribution of the N- and C-terminal domains of insulin-like growth factor (IGF)-binding protein-5 in binding IGF-I

J.H. Shand, J. Beattie, D.J. Flint, H. Song, G.J. Allan, K. Phillips, S.M. Kelly

Research output: Contribution to journalArticle

45 Citations (Scopus)

Abstract

We have previously reported that two highly conserved amino acids in the C-terminal domain of rat insulin-like growth factor-binding protein (IGFBP)-5, Gly203 and Gln209, are involved in binding to insulin-like growth factor (IGF)-1. Here we report that mutagenesis of both amino acids simultaneously (C-Term mutant) results in a cumulative effect and an even greater reduction in IGF-I binding: 30-fold measured by solution phase IGF binding assay and 10-fold by biosensor analysis. We compared these reductions in ligand binding to the effects of specific mutations of five amino acids in the N-terminal domain (N-Term mutant), which had previously been shown by others to cause a very large reduction in IGF-I binding (1). Our results confirm this as the major IGF-binding site. To prove that the mutations in either N- or C-Term were specific for IGF-I binding, we carried out CD spectroscopy and showed that these alterations did not lead to gross conformational changes in protein structure for either mutant. Combining these mutations in both domains (N+C-Term mutant) has a cumulative effect and leads to a 126-fold reduction in IGF-I binding as measured by biosensor. Furthermore, the equivalent mutations in the C terminus of rat IGFBP-2 (C-Term 2) also results in a significant reduction in IGF-I binding, suggesting that the highly conserved Gly and Gln residues have a conserved IGF-I binding function in all six IGFBPs. Finally, although these residues lie within a major heparin-binding site in IGFBP-5 and -3, we also show that the mutations in C-Term have no effect on heparin binding.
Original languageEnglish
Pages (from-to)17859-17866
Number of pages7
JournalJournal of Biological Chemistry
Volume278
Issue number20
DOIs
Publication statusPublished - 16 May 2003

Keywords

  • amino acids
  • insulin-like growth factor
  • heparin binding

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