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.
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, Gly(203) and Gln(209), 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 (). 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.
Shand
John H
JH
Hannah Research Institute, Ayr KA6 5HL, Scotland, United Kingdom.
Beattie
James
J
Song
Hyuk
H
Phillips
Kirsten
K
Kelly
Sharon M
SM
Flint
David J
DJ
Allan
Gordon J
GJ
D000818:Animals; D001665:Binding Sites; D015374:Biosensing Techniques; D002417:Cattle; D002942:Circular Dichroism; D004305:Dose-Response Relationship, Drug; D005973:Glutamine / Q000737:chemistry; D005998:Glycine / Q000737:chemistry; D006493:Heparin / Q000378:metabolism; D006801:Humans; D018975:Insulin-Like Growth Factor Binding Protein 5 / Q000378:metabolism*; D007334:Insulin-Like Growth Factor I / Q000378:metabolism*; D007700:Kinetics; D008024:Ligands; D009682:Magnetic Resonance Spectroscopy; D051379:Mice; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D013552:Swine; D013997:Time Factors; D014466:Ultraviolet Rays
D018975:Insulin-Like Growth Factor Binding Protein 5; D008024:Ligands; D011994:Recombinant Proteins; D005973:Glutamine; D007334:Insulin-Like Growth Factor I; D006493:Heparin; D005998:Glycine