Role of N- and C-terminal residues of insulin-like growth factor (IGF)-binding protein-3 in regulating IGF complex formation and receptor activation.
Insulin-like growth factor-binding protein-3 (IGFBP-3), the major IGFBP in the circulation, sequesters IGF in a stable ternary complex with the acid-labile subunit. The high affinity IGF-binding site is proposed to reside within an N-terminal hydrophobic domain in IGFBP-3, but C-terminal residues have also been implicated in the homologous protein IGFBP-5. We have mutated in various combinations Leu(77), Leu(80), and Leu(81) in the N terminus and Gly(217) and Gln(223) in the C terminus of IGF-BP-3. All mutants retained immunoreactivity toward a polyclonal IGFBP-3 antibody, whereas IGF ligand blotting showed that all of the mutants had reduced binding to IGFs. Both solution IGF binding assays and BIAcore analysis indicated that mutations to the N-terminal region caused greater reduction in IGF binding activity than C-terminal mutations. The combined N- and C-terminal mutants showed undetectable binding to IGF-I but retained <10% IGF-II binding activity. Reduced ternary complex formation was seen only in mutants that had considerably reduced IGF-I binding, consistent with previous studies indicating that the binary IGF.IGFBP-3 complex is required for acid-labile subunit binding. Decreased IGF binding was also reflected in the inability of the mutants to inhibit IGF-I signaling in IGF receptor overexpressing cells. However, when present in excess, IGFBP-3 analogs defined as non-IGF-binding by biochemical assays could still inhibit IGF signaling. This suggests that residual binding activity of IGFBP-3 mutants may still be sufficient to inhibit IGF biological activity and questions the use of such analogs to study IGF-independent effects of IGFBP-3.
Kolling Institute of Medical Research, University of Sydney, Royal North Shore Hospital, St. Leonards, New South Wales 2065, Australia.
Xiaolang
X
Yan
Briony E
BE
Forbes
Kerrie A
KA
McNeil
Robert C
RC
Baxter
Sue M
SM
Firth
D000595:Amino Acid Sequence; D000818:Animals; D001681:Biological Assay; D002478:Cells, Cultured; D017931:DNA Primers / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D005973:Glutamine / Q000737:chemistry; D005998:Glycine / Q000737:chemistry; D007328:Insulin / Q000378:metabolism; D018972:Insulin-Like Growth Factor Binding Protein 3 / Q000737:chemistry* / Q000378:metabolism; D018975:Insulin-Like Growth Factor Binding Protein 5 / Q000737:chemistry; D007700:Kinetics; D007930:Leucine / Q000737:chemistry; D008024:Ligands; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D041681:NIH 3T3 Cells; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D013997:Time Factors
D017931:DNA Primers; D007328:Insulin; D018972:Insulin-Like Growth Factor Binding Protein 3; D018975:Insulin-Like Growth Factor Binding Protein 5; D008024:Ligands; D011994:Recombinant Proteins; D005973:Glutamine; D007930:Leucine; D005998:Glycine