Lipid Microdomains Are Required Sites for the Selective Endocytosis and Nuclear Translocation of IFN-γ, Its Receptor Chain IFN-γ Receptor-1, and the Phosphorylation and Nuclear Translocation of STAT1α

Abstract

IFN-γ contains a nuclear localization sequence that may play a role in the nuclear transport of activated STAT1α via a complex of IFN-γ/IFN-γ receptor (IFNGR)-1/STAT1α 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 STAT1α. 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-γ 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-γ and IFNGR-1 as well as the tyrosine phosphorylation and nuclear translocation of STAT1α, but did not affect the binding of IFN-γ to these cells. In the Jurkat T lymphocyte cell line, which does not express caveolin-1, nuclear translocation of IFNGR-1 and STAT1α 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-γ, suggesting that the IFNGR subunits are recruited to lipid microdomains by IFN-γ 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 STAT1α or the nuclear translocation of IFN-γ or IFNGR-1. Thus, membrane lipid microdomains play an important role in IFN-γ-initiated endocytic events involving IFNGR-1, and the nuclear translocation of IFN-γ, IFNGR-1, and STAT1α.