The small G protein Ran, which is important for nucleocytoplasmic shuttling of proteins is present, but does not interact with EcR, Usp, and EcR/Usp. As shown by oligomycin treatment, EcR, Usp, and EcR/Usp import is energy dependent. Export of EcR and EcR/Usp is mediated by exportin-1 (CRM-1) as shown by the inhibiting effect of leptomycin B (LMB). Usp remains in the nucleus for more than 24 h. Nuclear retainment of EcR and Usp is energy dependent as shown by treatment with oligomycin. No export signal could be identified for Usp. The data confirm that EcR and Usp can enter the nucleus independently and that intracellular localization is regulated individually for each receptor. It is also demonstrated that the export signal of EcR is inaccessible after heterodimerization with Usp.
Nuclear localization of the ecdysteroid receptor (EcR) is increased in HeLa cells if exportin-1 (CRM1), a predominant carrier for export of proteins and RNA from the nucleus into the cytoplasm, is knocked down by siRNA against exportin. However, knockdown of the small G protein Ran, which is essential for nuclear transport, leads to an arrest of EcR in the cytoplasm, but does not prevent efficient nuclear import of the most important heterodimerization partner of EcR, ultraspiracle (Usp). Like in vertebrate cells, EcR is also distributed heterogeneously in Drosophila melanogaster S2 cells but shifted exclusively to the nucleus, if Usp is present.
CHO-K1 cells are routinely used for characterization of ecdysone receptor (EcR) function, because these vertebrate cells are devoid of endogenous ecdysone receptor protein. Moreover, the endogenous expression of RXR, the vertebrate orthologue of Ultraspiracle (Usp), the most important heterodimerization partner, is neglectable. In contrast to insect cells, there is also no influence of moulting hormone on CHO-K1 cells on cell proliferation either in the absence or presence of transiently expressed EcR. In contrast to Usp, which is exclusively found in nuclei, EcR is heterogeneously distributed between cytoplasm and nuclei in non-synchronized cells. Synchronization of CHO-K1 cells by nocodazole revealed that the cell cycle influences receptor concentration with lowest amounts in late S-phase and G2/M phase and intracellular distribution of the receptor protein showing a minimum of receptors present in nuclei during S-phase. EcR, but not Usp reduces cyclin D1 expression and cyclin D1 concentration is impaired by cyclin D1. Coimmunoprecipitation studies reveal physical interaction of EcR and cyclin D1.
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