Identification of the extracellular matrix binding sites for insulin-like growth factor-binding protein 5

Fibroblast extracellular matrix (ECM) contains two forms of insulin-like growth factor-binding proteins (IGFBPs), IGFBP-3 and IGFBP-5, These studies were undertaken to identify the regions within IGFBP-5 that mediate its binding to fibroblast ECM, Synthetic peptides were prepared that were homologous with two regions of basic amino acids within IGFBP-5 (Arg(201)-Arg(218) and Ala(131)-Thr(141)), Increasing concentrations of both peptides competed with IGFBP-5 for binding to ECM but the Arg(201)-Arg(218) peptide was more potent, Mutagenesis was used to define the effect of substituting for these basic residues on ECM binding, Substitution for two peptide B residues K134A and R136A reduced binding by 40%, Substitution of a single basic residue within the peptide A region (K211N) reduced binding to ECM by 49%, Substitution for R211N, K134A, and R136A reduced binding by 52%. More extensive substitutions in the peptide A region, e.g. K211N,R214A,K217A,R218N, resulted in a greater (e.g. 88%) decrease. The positional location of basic residues appeared to be more important than the total number of substitutions since the mutant K202N,R206A,R207A had a 79% reduction in ECM binding, Two basic regions of IGFBP-5 contribute to its binding to ECM, but the region containing amino acids 201-218 has a greater contribution. ECM binding is mediated by charged residues and acts to stabilize IGFBP-5 by protecting it from proteolysis.