CLEAVAGE ANALYSIS OF INSULIN-LIKE GROWTH-FACTOR (IGF)-DEPENDENT IGF-BINDING PROTEIN-4 PROTEOLYSIS AND EXPRESSION OF PROTEASE-RESISTANT IGF-BINDING PROTEIN-4 MUTANTS

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 similar to 18 and 14 kDa. Edman degradation of the isolated proteins established the amino ter mini 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 IGPBP-4 molecules with single amino acid substitutions around this cleavage site were constructed and expressed, Wild-type and mutant IGFBPs-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 IGFEP-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 IGFBPs-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.