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Proteolysis of insulin-like growth factor (IGF)-binding protein-3 (IGFBP-3) in 150-kilodalton IGFBP complexes by a cation-dependent protease activity in adult rat serum promotes the release of bound IGF-I.

Insulin-like growth factor I (IGF-I) circulates in plasma predominantly in a 150-kDa complex together with IGF-binding protein-3 (IGFBP-3), an approximately 40-kDa glycoprotein that binds IGF-I with high affinity, and an 85-kDa acid-labile subunit that does not bind IGFs. The 150-kDa complex serves as a potential reservoir of IGF-1 by sequestering the growth factor in the vascular compartment. Before IGF-I can reach the tissues, however, it must be released from the complex in a form that can cross the capillary-endothelial barrier. The present study demonstrates that proteolysis of IGFBP-3 in the 150-kDa complex occurs in vitro and results in the release of IGF-I. Specific IGFBP-3 protease activity in adult rat serum was demonstrated by incubating the serum at 37 C in the presence and absence of various protease inhibitors, followed by ligand blotting. Intact and truncated (30-kDa) IGFBP-3 were almost completely proteolyzed after 12 h of incubation, whereas IGFBP-2 and IGFBP-4 in the serum were unchanged even after 48-h incubation. The IGFBP-3 protease activity was inhibited by EDTA, indicating cation dependence, and by 4-(2-aminoethyl)-benzenesulfonyl fluoride, a serine protease inhibitor. Fractionation of the serum after incubation indicated that IGFBP-3 was proteolyzed while it was part of the 150-kDa complex. Proteolysis of human IGFBP-3 (hIGFBP-3) also occurred within 150-kDa complexes when ternary complexes that had been reconstituted from recombinant hIGFBP-3, rat acid-labile subunit, and IGF-I were incubated with rat serum. Release of IGF-I from ternary complexes after proteolysis of hIGFBP-3 was indicated by the observation that some of the [125I]IGF-I initially associated with reconstituted 150-kDa complexes was transferred to endogenous rat IGFBPs during the incubation. Similar proteolysis of IGFBP-3 within 150-kDa complexes in vivo would provide a mechanism for mobilizing IGF-I from the circulating reservoir in plasma as well as for the turnover of IGFBP-3.