Cleavage analysis of Insulin-like Growth Factor (IGF)-dependent IGF-binding protein-4 proteolysis and expression of protease-resistant IGF-binding protein-4 mutants

Cheryl A. Conover, Susan K. Durham, Jürgen Zapf, Frank R. Masiarz, Michael C. Kiefer

Research output: Contribution to journalArticle

87 Scopus citations

Abstract

Cultured human fibroblasts and osteoblast-like cells secrete an insulin-like growth factor (IGF)-dependent protease that cleaves IGF-binding protein-4 (IGFBP-4) into two fragments of ∼18 and 14 kDa. Edman degradation of the isolated proteins established the amino termini of the reaction products. Sequence analysis of the 14-kDa carboxyl-terminal half of IGFBP-4 suggested cleavage after methionine at position 135 of the mature protein. Four variant IGFBP-4 molecules with single amino acid substitutions around this cleavage site were constructed and expressed. Wild-type and mutant IG-FBPs-4 bound IGF-I and IGF-II with equivalent affinities and, in the intact state, were equally effective inhibitors of IGF-I action. However, the IGFBP-4 mutants were relatively resistant to IGF-dependent proteolysis. A 5-6-h incubation in human fibroblast conditioned medium in the presence of IGF-II was sufficient for near total hydrolysis of wild-type IGFBP-4, whereas the mutant IGFBPs-4 were only minimally affected at this time. After a 24-h incubation with IGF-II, all mutant IGFBPs-4 showed extensive proteolysis, generating 18- and 14-kDa fragments. Pre-exposure of human fibroblasts in serum-free conditioned medium to IGF-II for 5 h potentiated subsequent IGF-I stimulation of DNA synthesis. When added with IGF-II, the protease-resistant mutant IG-FBPs-4, but not wild-type IGFBP-4, suppressed IGF-II enhancement of IGF-I-stimulated DNA synthesis. These biological studies suggest that the IGFBP-4/IGFBP-4 protease system may play a role modulating local cellular response to IGF-I.

Original languageEnglish (US)
Pages (from-to)4395-4400
Number of pages6
JournalJournal of Biological Chemistry
Volume270
Issue number9
DOIs
StatePublished - Mar 3 1995

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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