Nucleocytoplasmic shuttling of the ecdysteroid receptor (EcR) and of ultraspiracle (Usp) from Drosophila melanogaster in mammalian cells: Energy requirement and interaction with exportin

Abstract

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.

The importance of exportin and Ran for nucleocytoplasmic shuttling of the ecdysteroid receptor

“…Our previous observations that EcR export is leptomycin B sensitive (Betanska et al, 2007) are confirmed by our present experiments with siRNA targeting exportin1. Similar results have been described for the transport of adenovirus particles (Strunze (Macchi et al, 2004;Miki and Yoneda, 2004) or the NF-kB transcription factor Dorsal, a regulator of dorso-ventral axis formation in early Drosophila embryogenesis, is also known to be a substrate of exportin (Xylourgidis et al, 2006;DeLotto et al, 2007).…”

“…We demonstrated the presence of Ran in the cell lines of interest (Betanska et al, 2007) but the direct involvement of Ran in nucleocytoplasmic shuttling of the EcR was not demonstrated so far.…”

“…Since a complete loss of Ran is not tolerated, the reduced concentration of Ran in our experiments may be still sufficient for the transport of the smaller protein Usp but is not sufficient for the bigger EcR. Experiments with oligomycin indicate that this might be more likely since blocking energy supply with oligomycin, Usp is less abundant in the nucleus (Betanska et al, 2007). In addition, we have to take into consideration that there is also a Ran-independent nucleocytoplasmic transport of proteins (Sorokin et al, 2007).…”

Influence of cell cycle on ecdysteroid receptor in CHO‐K1 cells

“…In this context, it is interesting to note that no export signal could be detected in Usp (Gwóźdź et al, 2007), which is always localized quantitatively in the nucleus (Nieva et al, 2005;Gwóźdź et al, 2007). The export signal present in the ligand binding domain of EcR is unaccessible after heterodimerization with Usp (Betanska et al, 2007) resulting in increased nuclear localization of EcR in the presence of Usp (Nieva et al, 2005), thus allowing an indirect influence of Usp on cell cycle control. Posttranslational modification seems also to be involved.…”

“…(Lombardi et al, 2008). Intracellular distribution of nuclear receptor is controlled both by import and export (Betanska et al, 2007;Rinnab et al, 2008). In this context, it is interesting to note that no export signal could be detected in Usp (Gwóźdź et al, 2007), which is always localized quantitatively in the nucleus (Nieva et al, 2005;Gwóźdź et al, 2007).…”