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

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. Recen tly, the pregnancy-associated plasma protein (PAPP)-A has been determined t o be the IGF-II-dependent IGFBP-4 protease produced by human fibroblasts. T his study sought to define the mechanism by which IGF-II enhances IGFBP-4 p roteolysis. Addition of PAPP-A antibody blocked the IGFBP-4 proteolytic act ivity in hOB conditioned medium (CM), suggesting that PAPP-A is the major I GFBP-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 re quirement of the presence of IGF-II in the reaction. In contrast, prior inc ubation of the partially purified IGFBP-4 protease from either hOB CM or hu man pregnancy serum with IGF-II did not lead to IGFBP-4 proteolysis unless IGF-II was readded to the assays. To further confirm that the interaction b etween IGF-II and IGFBP-4 is required for IGFBP-4 protease activity, we pre pared IGFBP-4 mutants, which contained the intact cleavage site (Met135-Lys 136) but lacked the IGF binding activity, by deleting the residues Leu72-Hi s74 in the IGF binding domain or Cys183-Glu237 that contained an IGF bindin g 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. Conv ersely, an IGFBP-4 mutant with His74 replaced by an Ala, which exhibited no rmal IGF binding activity, was effectively cleaved in the presence of IGF-I I. Taken together, these findings provided strong evidence that the interac tion 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. (C) 2000 Academic Press.