Lipid microdomains are required sites for the selective endocytosis and nuclear translocation of IFN-gamma, its receptor chain IFN-gamma receptor-1, and the phosphorylation and nuclear translocation of STAT1alpha.
IFN-gamma contains a nuclear localization sequence that may play a role in the nuclear transport of activated STAT1alpha via a complex of IFN-gamma/IFN-gamma receptor (IFNGR)-1/STAT1alpha with the nuclear importer nucleoprotein interactor 1. In this study, we examine the mechanism of endocytosis of IFNGR-1 and the relationship of its nuclear translocation to that of STAT1alpha. In untreated WISH cells, both IFNGR-1 and IFNGR-2 were constitutively localized within caveolae-like microdomains isolated from plasma membrane. However, treatment of cells with IFN-gamma resulted in rapid migration of IFNGR-1, but not IFNGR-2, from these microdomains. Filipin pretreatment, which specifically inhibits endocytosis from caveolae-like microdomains, inhibited the nuclear translocation of IFN-gamma and IFNGR-1 as well as the tyrosine phosphorylation and nuclear translocation of STAT1alpha, but did not affect the binding of IFN-gamma to these cells. In the Jurkat T lymphocyte cell line, which does not express caveolin-1, nuclear translocation of IFNGR-1 and STAT1alpha were similarly inhibited by filipin pretreatment. Isolation of lipid microdomains from Jurkat cells showed that both IFNGR-1 and IFNGR-2 were associated with lipid microdomains only after stimulation with IFN-gamma, suggesting that the IFNGR subunits are recruited to lipid microdomains by IFN-gamma binding in lymphocytes (Jurkat) in contrast to their constitutive presence in epithelial (WISH) cells. In contrast, treatments that block clathrin-dependent endocytosis did not inhibit either activation or nuclear translocation of STAT1alpha or the nuclear translocation of IFN-gamma or IFNGR-1. Thus, membrane lipid microdomains play an important role in IFN-gamma-initiated endocytic events involving IFNGR-1, and the nuclear translocation of IFN-gamma, IFNGR-1, and STAT1alpha.
Subramaniam
Prem S
PS
Department of Microbiology and Cell Science, University of Florida, Gainesville, FL 32611, USA. prem@ufl.edu
Johnson
Howard M
HM
D021581:Active Transport, Cell Nucleus / Q000187:drug effects; D001665:Binding Sites; D002460:Cell Line; D004705:Endocytosis; D005372:Filipin / Q000494:pharmacology; D006801:Humans; D050832:Interferon-Stimulated Gene Factor 3; D007371:Interferon-gamma / Q000378:metabolism* / Q000494:pharmacology; D021962:Membrane Microdomains / Q000276:immunology* / Q000378:metabolism*; D010766:Phosphorylation; D017471:Receptors, Interferon / Q000378:metabolism*; D011994:Recombinant Proteins; D015398:Signal Transduction; D014157:Transcription Factors / Q000378:metabolism*; D000096743:Interferon gamma Receptor
D005372:Filipin; D050832:Interferon-Stimulated Gene Factor 3; D007371:Interferon-gamma; D017471:Receptors, Interferon; D011994:Recombinant Proteins; D014157:Transcription Factors; D000096743:Interferon gamma Receptor; C079868:gamma interferon activation factor