The internalized interleukin-1 activation complex in fibroblasts localizes to the Golgi apparatus.
In order to investigate binding and internalization of interleukin-1 (IL-1) by confocal laser scanning microscopy, we established a model system comprising an IL-1 receptor type I (IL-1R1) overexpressing transfectant of the murine fibrosarcoma cell line L929 (L929R1) and an N-terminal FLAG-tagged human recombinant IL-1 alpha (FLAG-IL-1 alpha). The function of the transfected receptors was shown by their IL-1-induced association with a kinase activity. The biological activity of the purified FLAG-IL-1 alpha was comparable to the unmodified molecule. L929RI cells were exposed to saturating concentrations of FLAG-IL-1 alpha. Two-color fluorescence analysis revealed increasing cell surface binding of FLAG-IL-1 alpha to the receptor over 30 min. This was followed by internalization and accumulation of the ligand/receptor complex at the Golgi apparatus. After 3 hr the receptor signal significantly decreased and patches of FLAG-epitopes reappeared on the cell surface, no longer colocalized with IL-1R1. Thus, in this model, the previously assumed nuclear accumulation of IL-1 was not detected but rather localization of the internalized IL-1/IL-1R1-complex to the Golgi apparatus was found. Direct effects of IL-1 on the nucleus or the nuclear membrane therefore are unlikely.
Praast
G
G
Tumorimmunology Program, German Cancer Research Center, Heidelberg, Germany.
Hofmeister
R
R
Grube
K
K
Hans
W
W
Kühlwein
T
T
Krammer
P H
PH
Falk
W
W
D000818:Animals; D016367:Baculoviridae / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D018076:DNA, Complementary / Q000235:genetics; D004789:Enzyme Activation; D005455:Fluorescent Antibody Technique; D006056:Golgi Apparatus / Q000378:metabolism*; D006801:Humans; D007375:Interleukin-1 / Q000031:analogs & derivatives / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D010770:Phosphotransferases / Q000378:metabolism; D017472:Receptors, Interleukin-1 / Q000235:genetics / Q000378:metabolism*; D053573:Receptors, Interleukin-1 Type I; D011994:Recombinant Proteins / Q000378:metabolism; D014162:Transfection; D014407:Tumor Cells, Cultured
D018076:DNA, Complementary; D007375:Interleukin-1; D017472:Receptors, Interleukin-1; D053573:Receptors, Interleukin-1 Type I; D011994:Recombinant Proteins; D010770:Phosphotransferases