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.
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.
Spencer
E M
EM
Davies Medical Center, San Francisco, CA 94114, USA.
Chan
K
K
D001665:Binding Sites; D006801:Humans; D018972:Insulin-Like Growth Factor Binding Protein 3 / Q000737:chemistry*; D018969:Insulin-Like Growth Factor Binding Proteins / Q000737:chemistry*; D007334:Insulin-Like Growth Factor I / Q000378:metabolism; D008958:Models, Molecular; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry; D013329:Structure-Activity Relationship
D018972:Insulin-Like Growth Factor Binding Protein 3; D018969:Insulin-Like Growth Factor Binding Proteins; D011994:Recombinant Proteins; D007334:Insulin-Like Growth Factor I