Insulin-like growth factor-binding protein-5 enters vesicular structures but not the nucleus

Andreas Jurgeit, Chiara Berlato, Peter Obrist, Christian Ploner, Petra Massoner, Judith Schmölzer, Michael C. Haffner, Helmut Klocker, Lukas A. Huber, Stephan Geley, Wolfgang Doppler

Research output: Contribution to journalArticle

Abstract

In addition to its extracellular function as a secreted protein, IGF-binding protein (IGFBP)-5 has been postulated to act as a signaling molecule in the nucleus. This study aims to assess the significance of this postulated nuclear localization. By confocal immunofluorescence microscopy, we detected IGFBP-5 in the vesicular compartment of mammary epithelial cells in culture, while no nuclear staining was observed. Immunohistochemistry performed on paraffin sections of the involuting mammary gland revealed IGFBP-5 positive staining of epithelial cells only outside the nucleus. To evaluate the contribution of reuptake of extracellular IGFBP-5, T47D cells were incubated with Alexa Fluor 647-labeled IGFBP-5. The protein was taken up into intracellular vesicles and again was neither detectable in the cytoplasm outside of vesicular structures nor in the nucleus. Quantification of the time and concentration dependence of uptake by immunoblotting revealed that the process was saturable at IGFBP-5 concentrations between 1 and 2 μm and partially reversible with 30% remaining in the cell after a 1-h chase. The observation of nuclear uptake of IGFBP-5 was restricted to artificial conditions such as expression of non-secreted forms of IGFBP-5 or selective permeabilization of the plasma membrane by digitonin.

Original languageEnglish (US)
Pages (from-to)1815-1828
Number of pages14
JournalTraffic
Volume8
Issue number12
DOIs
StatePublished - Dec 2007
Externally publishedYes

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Insulin-Like Growth Factor Binding Protein 5
Epithelial Cells
Staining and Labeling
Digitonin
Confocal microscopy
Cell membranes
Human Mammary Glands
Fluorescence Microscopy
Immunoblotting
Confocal Microscopy
Paraffin
Cytoplasm
Proteins
Breast
Cell Culture Techniques
Immunohistochemistry
Cell Membrane
Observation
Molecules

Keywords

  • Digitonin
  • Insulin-like growth factor-binding protein
  • Mammary epithelium
  • Nuclear uptake
  • Secretion
  • Selective permeabilization
  • Vesicular transport

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology
  • Structural Biology
  • Molecular Biology
  • Genetics

Cite this

Jurgeit, A., Berlato, C., Obrist, P., Ploner, C., Massoner, P., Schmölzer, J., ... Doppler, W. (2007). Insulin-like growth factor-binding protein-5 enters vesicular structures but not the nucleus. Traffic, 8(12), 1815-1828. https://doi.org/10.1111/j.1600-0854.2007.00655.x

Insulin-like growth factor-binding protein-5 enters vesicular structures but not the nucleus. / Jurgeit, Andreas; Berlato, Chiara; Obrist, Peter; Ploner, Christian; Massoner, Petra; Schmölzer, Judith; Haffner, Michael C.; Klocker, Helmut; Huber, Lukas A.; Geley, Stephan; Doppler, Wolfgang.

In: Traffic, Vol. 8, No. 12, 12.2007, p. 1815-1828.

Research output: Contribution to journalArticle

Jurgeit, A, Berlato, C, Obrist, P, Ploner, C, Massoner, P, Schmölzer, J, Haffner, MC, Klocker, H, Huber, LA, Geley, S & Doppler, W 2007, 'Insulin-like growth factor-binding protein-5 enters vesicular structures but not the nucleus', Traffic, vol. 8, no. 12, pp. 1815-1828. https://doi.org/10.1111/j.1600-0854.2007.00655.x
Jurgeit A, Berlato C, Obrist P, Ploner C, Massoner P, Schmölzer J et al. Insulin-like growth factor-binding protein-5 enters vesicular structures but not the nucleus. Traffic. 2007 Dec;8(12):1815-1828. https://doi.org/10.1111/j.1600-0854.2007.00655.x
Jurgeit, Andreas ; Berlato, Chiara ; Obrist, Peter ; Ploner, Christian ; Massoner, Petra ; Schmölzer, Judith ; Haffner, Michael C. ; Klocker, Helmut ; Huber, Lukas A. ; Geley, Stephan ; Doppler, Wolfgang. / Insulin-like growth factor-binding protein-5 enters vesicular structures but not the nucleus. In: Traffic. 2007 ; Vol. 8, No. 12. pp. 1815-1828.
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abstract = "In addition to its extracellular function as a secreted protein, IGF-binding protein (IGFBP)-5 has been postulated to act as a signaling molecule in the nucleus. This study aims to assess the significance of this postulated nuclear localization. By confocal immunofluorescence microscopy, we detected IGFBP-5 in the vesicular compartment of mammary epithelial cells in culture, while no nuclear staining was observed. Immunohistochemistry performed on paraffin sections of the involuting mammary gland revealed IGFBP-5 positive staining of epithelial cells only outside the nucleus. To evaluate the contribution of reuptake of extracellular IGFBP-5, T47D cells were incubated with Alexa Fluor 647-labeled IGFBP-5. The protein was taken up into intracellular vesicles and again was neither detectable in the cytoplasm outside of vesicular structures nor in the nucleus. Quantification of the time and concentration dependence of uptake by immunoblotting revealed that the process was saturable at IGFBP-5 concentrations between 1 and 2 μm and partially reversible with 30{\%} remaining in the cell after a 1-h chase. The observation of nuclear uptake of IGFBP-5 was restricted to artificial conditions such as expression of non-secreted forms of IGFBP-5 or selective permeabilization of the plasma membrane by digitonin.",
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