Cellular internalization of insulin-like growth factor binding protein-3: distinct endocytic pathways facilitate re-uptake and nuclear localization.
Insulin-like growth factor binding protein-3 (IGFBP-3) is well established as a growth-inhibitory, apoptosis-inducing secreted molecule that acts via insulin-like growth factor (IGF)-independent as well as IGF-dependent pathways. Nuclear localization of IGFBP-3 has been observed and nuclear binding partners for IGFBP-3 demonstrated. However, little is known about the mechanism of IGFBP-3 internalization. We hypothesized that IGFBP-3 is first secreted then taken up again into cells and that its internalization could occur via binding to transferrin or caveolin. Incubation of cells with an IGFBP-3-neutralizing antibody demonstrated that nuclear translocation of endogenous IGFBP-3 requires IGFBP-3 secretion and re-uptake. Nuclear localization of exogenously added IGFBP-3 was rapid, occurring within 15 min, inhibited by co-incubation and extracellular sequestration with IGF-I, and dependent on the transferrin-binding C-terminal peptide region of IGFBP-3. Co-immunoprecipitation assays confirmed that IGFBP-3 binds transferrin but not directly to the transferrin receptor (TfR1); however, transferrin binds TfR1 and a ternary complex is formed. Specific binding to caveolin scaffolding docking sequence was confirmed utilizing radiolabeled IGFBP-3. Blocking TfR1-mediated endocytosis prevents both endogenous and exogenous IGFBP-3 re-uptake and inhibitors of caveolae formation also retard IGFBP-3 nuclear entry. Co-treatment with anti-transferrin receptor antibody and cholesterol depletion agents completely abolished endogenous and exogenous IGFBP-3 uptake. Suppression of IGFBP-3 internalization by TfR1 blockade inhibited IGFBP-3-induced apoptosis. Together, these data indicate that the actions of IGFBP-3 are mediated by internalization via distinct endocytic pathways.
Division of Pediatric Endocrinology, Mattel Children's Hospital, David Geffen School of Medicine, UCLA, Los Angeles, California 90095, USA.
Kuk-Wha
KW
Lee
Bingrong
B
Liu
Liqun
L
Ma
Heju
H
Li
Peter
P
Bang
H Phillip
HP
Koeffler
Pinchas
P
Cohen
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001665:Binding Sites; D051242:Caveolin 1; D022461:Caveolins / Q000737:chemistry / Q000378:metabolism; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism*; D004705:Endocytosis / Q000502:physiology*; D006801:Humans; D018972:Insulin-Like Growth Factor Binding Protein 3 / Q000737:chemistry / Q000378:metabolism*; D007334:Insulin-Like Growth Factor I / Q000378:metabolism; D008024:Ligands; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry; D021381:Protein Transport; D011990:Receptors, Transferrin / Q000378:metabolism; D011994:Recombinant Proteins; D014168:Transferrin / Q000737:chemistry / Q000378:metabolism
C495540:CAV1 protein, human; D051242:Caveolin 1; D022461:Caveolins; D018972:Insulin-Like Growth Factor Binding Protein 3; D008024:Ligands; D010446:Peptide Fragments; D011990:Receptors, Transferrin; D011994:Recombinant Proteins; D014168:Transferrin; D007334:Insulin-Like Growth Factor I