Evidence that the interaction between insulin-like growth factor (IGF)-II and IGF binding protein (IGFBP)-4 is essential for the action of the IGF-II-dependent IGFBP-4 protease

Arch Biochem Biophys. 2000 Jul 15;379(2):209-16. doi: 10.1006/abbi.2000.1872.

Abstract

A variety of human cell types, including human osteoblasts (hOBs), produce an IGFBP-4 protease, which cleaves IGFBP-4 in the presence of IGF-II. Recently, the pregnancy-associated plasma protein (PAPP)-A has been determined to be the IGF-II-dependent IGFBP-4 protease produced by human fibroblasts. This study sought to define the mechanism by which IGF-II enhances IGFBP-4 proteolysis. Addition of PAPP-A antibody blocked the IGFBP-4 proteolytic activity in hOB conditioned medium (CM), suggesting that PAPP-A is the major IGFBP-4 protease in hOB CM. Pre-incubation of IGFBP-4 with IGF-II, followed by removal of unbound IGF-II, led to IGFBP-4 proteolysis without further requirement of the presence of IGF-II in the reaction. In contrast, prior incubation of the partially purified IGFBP-4 protease from either hOB CM or human pregnancy serum with IGF-II did not lead to IGFBP-4 proteolysis unless IGF-II was re-added to the assays. To further confirm that the interaction between IGF-II and IGFBP-4 is required for IGFBP-4 protease activity, we prepared IGFBP-4 mutants, which contained the intact cleavage site (Met135-Lys136) but lacked the IGF binding activity, by deleting the residues Leu72-His74 in the IGF binding domain or Cys183-Glu237 that contained an IGF binding enhancing motif. The IGFBP-4 protease was unable to cleave these IGFBP-4 mutants, regardless of whether or not IGF-II was present in the assay. Conversely, an IGFBP-4 mutant with His74 replaced by an Ala, which exhibited normal IGF binding activity, was effectively cleaved in the presence of IGF-II. Taken together, these findings provided strong evidence that the interaction between IGF-II and IGFBP-4, rather than the direct interaction between IGF-II and IGFBP-4 protease, is required for optimal IGFBP-4 proteolysis.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Antibodies / immunology
  • Antibodies / pharmacology
  • Binding Sites
  • Catalysis / drug effects
  • Culture Media, Conditioned / metabolism
  • Female
  • Humans
  • Immunoblotting
  • Insulin-Like Growth Factor Binding Protein 4 / antagonists & inhibitors
  • Insulin-Like Growth Factor Binding Protein 4 / chemistry
  • Insulin-Like Growth Factor Binding Protein 4 / genetics
  • Insulin-Like Growth Factor Binding Protein 4 / metabolism*
  • Insulin-Like Growth Factor II / metabolism*
  • Insulin-Like Growth Factor II / pharmacology
  • Metalloendopeptidases / immunology
  • Metalloendopeptidases / isolation & purification
  • Metalloendopeptidases / metabolism*
  • Molecular Weight
  • Mutation
  • Osteoblasts / enzymology
  • Osteoblasts / metabolism
  • Peptide Fragments / chemistry
  • Peptide Fragments / metabolism
  • Pregnancy
  • Pregnancy-Associated Plasma Protein-A / immunology
  • Protein Binding
  • Protein Structure, Tertiary
  • Recombinant Proteins

Substances

  • Antibodies
  • Culture Media, Conditioned
  • Insulin-Like Growth Factor Binding Protein 4
  • Peptide Fragments
  • Recombinant Proteins
  • Insulin-Like Growth Factor II
  • Metalloendopeptidases
  • Pregnancy-Associated Plasma Protein-A