Cell Biology and Metabolism
Identification of the Extracellular Matrix Binding Sites for Insulin-like Growth Factor-binding Protein 5*

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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 (Arg201-Arg218 and Ala131-Thr141). Increasing concentrations of both peptides competed with IGFBP-5 for binding to ECM but the Arg201-Arg218 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 K211N, 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,K206A,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.

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This work was supported by National Institutes of Health Grant AG-02331. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.