Amino acids within the extracellular matrix (ECM) binding region (201-218) of rat insulin-like growth factor binding protein (IGFBP)-5 are important determinants in binding IGF-I.
The highly conserved N-and C-terminal domains of IGFBPs are believed to participate in IGF binding, but only recently have some of the critical residues in the IGFBP sequence involved in ligand binding been identified. Here we describe two highly conserved amino acids in the C-terminal domain of rat IGFBP-5 that are involved in binding IGF-I. Site-directed mutagenesis was used to produce two mutants, G203K and Q209A, of rIGFBP-5. Relative to wild-type rIGFBP-5, an 8-fold reduction in affinity for human IGF-I was found for recombinant G203K protein in both IGF-I ligand blots and solution phase ligand binding assays, and a 7-and 6-fold reduction for Q209A respectively. This shows that Gly203 and Gln209 in IGFBP-5 are important determinants in binding IGF-I, and due to their complete conservation in all IGFBP sequences, we suggest that they are likely to be involved in binding IGF-I in all six binding proteins. In addition, these two non-basic residues lie within the ECM binding region (201-218) of IGFBP-5, demonstrating that the C-terminus contains partially overlapping IGF-I and ECM binding sites. We therefore propose that heparin binding to basic amino acids in IGFBP-5 between 201-218 may physically occlude subsequent interaction between IGF-I and Gly203/Gln209, and that this may explain previous work of others showing reduced affinity of ECM bound IGFBP-5 for IGF-I.
Bramani
S
S
Hannah Research Institute, Ayr, KA6 5HL, UK.
Song
H
H
Beattie
J
J
Tonner
E
E
Flint
D J
DJ
Allan
G J
GJ
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D005109:Extracellular Matrix / Q000378:metabolism*; D006801:Humans; D018975:Insulin-Like Growth Factor Binding Protein 5 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007334:Insulin-Like Growth Factor I / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011485:Protein Binding; D011869:Radioligand Assay; D051381:Rats; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D018975:Insulin-Like Growth Factor Binding Protein 5; D007334:Insulin-Like Growth Factor I