FACTORS REGULATING INSULIN-LIKE GROWTH FACTOR-BINDING PROTEIN-3 BINDING, PROCESSING, AND POTENTIATION OF INSULIN-LIKE GROWTH-FACTOR ACTION

In this study, we investigated the effects of various biochemical and pharmacological agents on insulin-like growth factor (IGF)-binding pro tein-3 (IGFBP-3) cell binding and action in cultured bovine fibroblast s. When cells were preincubated for 48 h with 50 nm recombinant human (rh) IGFBP-2, IGF-I-stimulated [H-3]aminoisobutyric acid ([H-125]AIB) uptake was enhanced 2- to 3-fold. The addition of cytoskeletal disrupt ing agents during the preincubation with rhIGFBP-3 did not affect IGFB P-3 potentiation of IGF-I action, nor did a variety of serine, asparta te, and metalloproteinase inhibitors. On the other hand, ammonium chlo ride and chloroquine, weak bases that neutralize the pH of acidic cell compartments, blocked IGFBP-3 potentiation of IGF-I-stimulated [H-3]A IB uptake. Chloroquine and ammonium chloride had no effect alone and d id not inhibit IGF-I receptor binding or action in the absence of rhIG FBP-3. Bafilomycin A, a specific inhibitor of ATP-dependent hydrogen i on pumps, also inhibited IGFBP-3 potentiation of IGF-I-stimulated [H-3 ]AIB uptake. Competitive [I-125]IGF-I binding and affinity cross-linki ng experiments suggested structure/function changes in cell-bound IGFB P-3 that were altered in the presence of chloroquine and bafilomycin. Heparin markedly decreased initial IGFBP-3 from cells after the 48-h p reincubation. Moreover, heparin did not inhibit IGFBP-3 potentiation o f IGF-I action. In summary, these data indicate that IGFBP-3 undergoes specific pH-dependent structural and/or environmental modifications t hat mediate that enhancing effect of IGFBP-3 on IGF-I action in bovine fibroblasts. They also suggest that IGFBP-3 binding to heparin-like m olecules on the cell surface is not directly involved in this process.