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 pa rticipate in IGF binding, but only recently have some of the critical resid ues in the IGFBP sequence involved in ligand binding been identified. Here we describe two highly conserved amino acids in the C-terminal domain of ra t 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 wil d-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 respectivel y. 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 seque nces, we suggest that they are likely to be involved in binding IGF-I in al l six binding proteins. In addition, these two non-basic residues lie withi n the ECM binding region (201-218) of IGFBP-5, demonstrating that the C-ter minus contains partially overlapping IGF-I and ECM binding sites. We theref ore propose that heparin binding to basic amino acids in IGFBP-5 between 20 1-218 may physically occlude subsequent interaction between IGF-I and Gly20 3/Gln209, and that this may explain previous work of others showing reduced affinity of ECM-bound IGFBP-5 for IGF-I.