Insulin-like growth factor-binding protein-5 enters vesicular structures but not the nucleus.
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 mum 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.
Jurgeit
Andreas
A
Division of Medical Biochemistry, Biocenter, Innsbruck Medical University, Fritz-Pregl-Strasse 3, 6020 Innsbruck, Austria.|Present address: Institute of Zoology, University of Zürich, Winterthurerstrasse 190, 8057 Zürich, Switzerland.
Berlato
Chiara
C
Division of Medical Biochemistry, Biocenter, Innsbruck Medical University, Fritz-Pregl-Strasse 3, 6020 Innsbruck, Austria.
Obrist
Peter
P
Department of Pathology, St. Vinzenz Hospital Zams, 6511 Zams, Austria.
Ploner
Christian
C
Division of Molecular Pathophysiology, Biocenter, Innsbruck Medical University, Fritz-Pregl-Strasse 3, 6020 Innsbruck, Austria.
Massoner
Petra
P
Department of Urology, Innsbruck Medical University, Anichstrasse 35, 6020 Innsbruck, Austria.
Schmölzer
Judith
J
Division of Medical Biochemistry, Biocenter, Innsbruck Medical University, Fritz-Pregl-Strasse 3, 6020 Innsbruck, Austria.
Haffner
Michael C
MC
Division of Medical Biochemistry, Biocenter, Innsbruck Medical University, Fritz-Pregl-Strasse 3, 6020 Innsbruck, Austria.
Klocker
Helmut
H
Department of Urology, Innsbruck Medical University, Anichstrasse 35, 6020 Innsbruck, Austria.
Huber
Lukas A
LA
Division of Cell Biology, Biocenter, Innsbruck Medical University, Fritz-Pregl-Strasse 3, 6020 Innsbruck, Austria.
Geley
Stephan
S
Division of Molecular Pathophysiology, Biocenter, Innsbruck Medical University, Fritz-Pregl-Strasse 3, 6020 Innsbruck, Austria.
Doppler
Wolfgang
W
Division of Medical Biochemistry, Biocenter, Innsbruck Medical University, Fritz-Pregl-Strasse 3, 6020 Innsbruck, Austria.
D021581:Active Transport, Cell Nucleus; D000818:Animals; D019556:COS Cells; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D002467:Cell Nucleus / Q000378:metabolism*; D002522:Chlorocebus aethiops; D003593:Cytoplasm / Q000378:metabolism; D004072:Digitonin / Q000378:metabolism*; D004847:Epithelial Cells / Q000378:metabolism; D006801:Humans; D018975:Insulin-Like Growth Factor Binding Protein 5 / Q000378:metabolism*; D008321:Mammary Glands, Animal / Q000378:metabolism; D051379:Mice; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D013997:Time Factors
D018975:Insulin-Like Growth Factor Binding Protein 5; D004072:Digitonin