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 (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.
Parker
A
A
Department of Medicine, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599, USA.
Clarke
J B
JB
Busby
W H
WH
Clemmons
D R
DR
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D001667:Binding, Competitive; D002460:Cell Line; D018076:DNA, Complementary / Q000235:genetics; D005109:Extracellular Matrix / Q000378:metabolism*; D005347:Fibroblasts / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D018972:Insulin-Like Growth Factor Binding Protein 3 / Q000235:genetics / Q000378:metabolism; D018975:Insulin-Like Growth Factor Binding Protein 5 / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation
D018076:DNA, Complementary; D018972:Insulin-Like Growth Factor Binding Protein 3; D018975:Insulin-Like Growth Factor Binding Protein 5