The mitogenic and anabolic actions of the insulin-like growth factors (IGFs) are modulated by a minimum of six serum binding proteins (BPs) . The major carrier of IGF peptides in plasma is IGFBP-3, which normally complexes with IGF-I (or -II) and an acid-labile subunit, to form a 150K complex. We have recently shown, by western ligand blotting techniques, that there is a dramatic reduction of intact IGFBP-3 in the sera of pregnant women, mice and rats, and that this decrease can be attributed to the presence of a pregnancy-associated protease (P-A-P) which cleaves IGFBP-3 into smaller fragments. We have demonstrated a similar mechanism in seminal plasma, and in several malignancy-related proteases. We propose that proteolysis of IGFBP-3 is a regulatory process, whereby alterations in the affinity of IGFBP-3 for IGF peptides modulates access of these IGFs to cell membrane receptors. We plan to identify and characterize IGFBP-3 proteases, with special attention to the P-A-P and the protease activity of seminal plasma, which we have tentatively ldentified as prostate-specific antigen (PSA). P-A-P will be purified and its cDNA cloned. Employing an IGFBP protease assay developed in our, laboratories, together with PSA and purified P-A-P, we will perform enzyme kinetic studies, identify specific protease inhibitors, determine cleavage site specificity, and quantitate protease activity. The source of P-A-P will be determined by decidual, trophoblast and hepatic explant and cell cultures, and hormonal regulation of protease activity will be studied. Immunohistochemical, in situ hybridization, and Northern blot studies will be performed to identify cells which produce IGFBP-3 and/or IGFBP-3 proteases. Finally, the IGFBP-3 fragments will be characterized in terms of: 1) their size; 2) their affinity for IGF-I and -II; 3) their ability to complex with the acid-labile subunit to form the normal ternary complex; and 4) their effect upon the ability of IGF-I and -II to bind to membrane receptors and exert biological action.
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