A 3-dimensional model for the insulin-like growth factor binding proteins (IGFBPs); Supporting evidence using the structural determinants of the IGF binding site on IGFBP-3

Abstract

It is generally accepted but not established that the insulin-like growth factor (IGF) binding site on the IGF binding proteins (IGFBPs) is in the N-terminal region. However, other workers have reported C-terminal fragments with IGF binding determinants. Therefore, we tested the hypothesis that both the N- and C-terminal regions of IGFBPs are involved in binding. Using a protein A gene fusion system, a cDNA encoding residues 1–147 (N147) was cloned into the plasmid pRIT2T and expressed in E. coli as a fusion protein. Since an Asn N-terminal to the gly of IGFBP-3 had been engineered into the cDNA construct, protein A was cleaved from N147 by hydroxylamine. Purified N147 was refolded in a DTT/cystamine redox system at pH 8.4 under nitrogen atmosphere. Both ligand binding Westerns and solution binding assays demonstrated that the recombinant derived N147 bound IGFs. The 147 and 176 residue N-terminal fragments, including a C-terminal fragment (residues 151–263) of IGFBP-3 were also expressed in pichia (yeast) as glycosylated proteins. Solution binding assays showed that they all bound labelled IGF-1. In conclusion, IGFBP-3 contains at least two binding determinants, one on the N- and one on the C-terminal domain. There may also be a possible contribution from the intermediate (I) domain. Our molecular genetic approach to mapping the binding region for IGFs on IGFBP-3 can now be tested on the other mutants we have prepared. Subsequently, site directed mutagenesis can be used to pinpoint key functional residues.