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 protein-3 (IGFBP-3) cell binding and action in cultured bovine fibroblasts. When cells were preincubated for 48 h with 50 nM recombinant human (rh) IGFBP-3, IGF-I-stimulated [3H]aminoisobutyric acid ([125H]AIB) uptake was enhanced 2- to 3-fold. The addition of cytoskeletal disrupting agents during the preincubation with rhIGFBP-3 did not affect IGFBP-3 potentiation of IGF-I action, nor did a variety of serine, aspartate, and metalloproteinase inhibitors. On the other hand, ammonium chloride and chloroquine, weak bases that neutralize the pH of acidic cell compartments, blocked IGFBP-3 potentiation of IGF-I-stimulated [3H]AIB uptake. Chloroquine and ammonium chloride had no effect alone and did not inhibit IGF-I receptor binding or action in the absence of rhIGFBP-3. Bafilomycin A, a specific inhibitor of ATP-dependent hydrogen ion pumps, also inhibited IGFBP-3 potentiation of IGF-I-stimulated [3H]AIB uptake. Competitive [125I]IGF-I binding and affinity cross-linking experiments suggested structure/function changes in cell-bound IGFBP-3 that were altered in the presence of chloroquine and bafilomycin. Heparin markedly decreased initial IGFBP-3 cell adherence, but could not promote dissociation of IGFBP-3 from cells after the 48-h preincubation. Moreover, heparin did not inhibit IGFBP-3 potentiation of IGF-I action. In summary, these data indicate that IGFBP-3 undergoes specific pH-dependent structural and/or environmental modifications that mediate the enhancing effect of IGFBP-3 on IGF-I action in bovine fibroblasts. They also suggest that IGFBP-3 binding to heparin-like molecules on the cell surface is not directly involved in this process.
Conover
C A
CA
Division of Endocrinology and Metabolism, Mayo Clinic, Rochester, Minnesota 55905, USA.
Clarkson
J T
JT
Bale
L K
LK
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000621:Aminoisobutyric Acids / Q000378:metabolism; D000643:Ammonium Chloride / Q000494:pharmacology; D000818:Animals; D001667:Binding, Competitive; D002417:Cattle; D002478:Cells, Cultured; D002738:Chloroquine / Q000494:pharmacology; D003432:Cross-Linking Reagents; D004357:Drug Synergism; D005347:Fibroblasts / Q000378:metabolism; D006031:Glycosylation; D006493:Heparin / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D018972:Insulin-Like Growth Factor Binding Protein 3 / Q000378:metabolism* / Q000494:pharmacology*; D007334:Insulin-Like Growth Factor I / Q000378:metabolism / Q000494:pharmacology*; D007457:Iodine Radioisotopes; D011994:Recombinant Proteins / Q000494:pharmacology
D000621:Aminoisobutyric Acids; D003432:Cross-Linking Reagents; D018972:Insulin-Like Growth Factor Binding Protein 3; D007457:Iodine Radioisotopes; D011994:Recombinant Proteins; D000643:Ammonium Chloride; D007334:Insulin-Like Growth Factor I; D002738:Chloroquine; D000255:Adenosine Triphosphate; D006493:Heparin