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 insuli n-like growth factor (IGF)-dependent protease that cleaves IGF-binding protein-4 (IGFBP-4) into two fragments of similar to 18 and 14 kDa. E dman degradation of the isolated proteins established the amino ter mi ni of the reaction products, Sequence analysis of the 14-kDa carboxyl terminal half of IGFBP-4 suggested cleavage after methionine at positi on 135 of the mature protein, Four variant IGPBP-4 molecules with sing le amino acid substitutions around this cleavage site were constructed and expressed, Wild-type and mutant IGFBPs-4 bound IGF-I and IGF-II w ith equivalent affinities and, in the intact state, were equally effec tive inhibitors of IGF-I action, However, the IGFEP-4 mutants were rel atively resistant to IGF-dependent proteolysis, A 5-6-h incubation in human fibroblast conditioned medium in the presence of IGF-II was suff icient for near total hydrolysis of wild-type IGFBP-4, whereas the mut ant IGFBPs-4 were only minimally affected at this time, After a 24-h i ncubation with IGF-II, all mutant IGFBPs-4 showed extensive proteolysi s, generating 18- and 14-kDa fragments, Pre-exposure of human fibrobla sts in serum-free conditioned medium to IGF-II for 5 h potentiated sub sequent IGF-I stimulation of DNA synthesis, When added with IGF-II, th e protease-resistant mutant IGFBPs-4, but not wild-type IGFBP-4, suppr essed IGF-II enhancement of IGF-I-stimulated DNA synthesis, These biol ogical studies suggest that the IGFBP-4/IGFBP-4 protease system may pl ay a role modulating local cellular response to IGF-I.