Insulin-like growth factor-independent effects mediated by a C-terminal metal-binding domain of insulin-like growth factor binding protein-3.
Insulin-like growth factors (IGFs) play a central role in the integration of proliferative and survival responses of most mammalian cell types. IGF-binding protein-3 (IGFBP-3) influences IGF action directly as a carrier of IGFs but also modulates these actions indirectly via independent mechanisms involving interactions with plasma, extracellular matrix and cell surface molecules, conditional proteolysis, cellular uptake, and nuclear transport. Here we demonstrate that a short C-terminal metal-binding domain (MBD) of IGFBP-3 mediates binding to metals. MBD epitopes, sequestered in the intact molecule, are unmasked by incubation in the presence of ferrous (but not ferric or zinc) ions. An isolated 14-mer MBD peptide triggered apoptotic effects in stressed HEK293 cells as effectively as IGFBP-3. The MBD, which encompasses a nuclear localization sequence and an adjacent putative caveolin-binding sequence, mobilizes rapid cellular uptake and nuclear localization of unrelated proteins such as green fluorescent protein and streptavidin-horseradish peroxidase conjugate. Metal ions stimulate MBD-mediated cellular/nuclear uptake in vivo. Cross-linking studies showed a direct physical interaction of MBD with integrins alphav and beta1, caveolin-1, and transferrin receptor. MBD-mediated protein mobilization and pro-apoptotic effects are inhibited by nystatin but not chlorpromazine, suggesting an involvement of caveolar-mediated endocytosis. However, MBD effects are inhibited by antibodies to transferrin receptor or integrins. These results are discussed with particular reference to the cell target specificity of IGFBP-3 in disease processes such as cancer and atherosclerosis.
Singh
Baljit
B
Protigen, Inc, Sunnyvale, California 94085, USA.
Charkowicz
Dona
D
Mascarenhas
Desmond
D
D000595:Amino Acid Sequence; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology; D001665:Binding Sites; D051242:Caveolin 1; D022461:Caveolins / Q000378:metabolism; D002460:Cell Line; D003001:Cloning, Molecular; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins; D006801:Humans; D018972:Insulin-Like Growth Factor Binding Protein 3 / Q000737:chemistry* / Q000378:metabolism*; D007334:Insulin-Like Growth Factor I / Q000378:metabolism; D007335:Insulin-Like Growth Factor II / Q000378:metabolism; D007501:Iron / Q000378:metabolism; D007700:Kinetics; D008164:Luminescent Proteins / Q000378:metabolism; D008670:Metals / Q000378:metabolism; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D011990:Receptors, Transferrin / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism
C495540:CAV1 protein, human; D051242:Caveolin 1; D022461:Caveolins; D018972:Insulin-Like Growth Factor Binding Protein 3; D008164:Luminescent Proteins; D008670:Metals; D010446:Peptide Fragments; D011990:Receptors, Transferrin; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D007334:Insulin-Like Growth Factor I; D007335:Insulin-Like Growth Factor II; D007501:Iron