Ultraspiracle promotes the nuclear localization of ecdysteroid receptor in mammalian cells

Abstract

The heterodimer consisting of ecdysteroid receptor (EcR) and ultraspiracle (USP), both of which are members of the nuclear receptor superfamily, is considered to be the functional ecdysteroid receptor. Here we analyzed the subcellular distribution of EcR and USP fused to fluorescent proteins. The experiments were carried out in mammalian COS-7, CHO-K1 and HeLa cells to facilitate investigation of the subcellular trafficking of EcR and USP in the absence of endogenous expression of these two receptors. The distribution of USP tagged with a yellow fluorescent protein (YFP-USP) was almost exclusively nuclear in all cell types analyzed. The nuclear localization remained constant for at least 1 day after the first visible signs of expression. In contrast, the intracellular distribution of EcR tagged with a yellow fluorescent protein (YFP-EcR) varied and was dependent on time and cell type, although YFP-EcR alone was also able to partially translocate into the nuclear compartment. Coexpression of YFP-EcR with USP tagged with a cyan fluorescent protein (CFP-USP) resulted in exclusively nuclear localization of both proteins in all cell types analyzed. The USP-induced nuclear localization of YFP-EcR was stable for at least 20 hours. These experiments suggest that USP has a profound effect on the subcellular distribution of EcR.

The variety of complexes formed by EcR and Usp nuclear receptors in the nuclei of living cells

“…Therefore, earlier we had analyzed the subcellular trafficking of the EcRB1 isoform and Usp in mammalian cells (CHO-K1, COS-7 and HeLa) which are routinely used for EcR/Usp heterologous studies (Gwozdz et al, 2007;Nieva et al, 2005). The experiments showed that the intracellular distribution of EcR was dependent on time and cell type.…”

“…Taking into account current data about nuclear receptors (Laudet and Gronemeyer, 2002), including our recently published data (Gwozdz et al, 2007;Nieva et al, 2005), it is reasonable to assume that the EcR/Usp complexes are formed due to the direct interaction between the ligand binding Page 15 of 27 A c c e p t e d M a n u s c r i p t 15 domains (LBDs) of both receptors. However, the interaction that brings together EcR and Usp and thus localizes them in the nucleus, may not be a direct interaction.…”

“…Since EcR in the absence of Usp can be distributed within the whole cell (Nieva et al, 2005), at least in the case of the CHO-K1 cell line, it is reasonable to assume that the nuclear localization of the EcR/Usp complex is determined by the exclusively nuclear Usp. As shown M a n u s c r i p t 16 previously, EcR contains several putative nuclear export signals (NESs) within its LBD (Gwozdz et al, 2007).…”

“…Constructs containing the full-length EcRB1 isoform and USP from Drosophila melanogaster tagged with fluorescent proteins YFP and CFP have been previously described (Nieva et al, 2005).…”

“…Determining the molecular mechanisms responsible for the intracellular distribution of the EcR/Usp complex and/or its constituents is of great importance. However, there are few data concerning this phenomenon (Gwozdz et al, 2007;Nieva et al, 2005), possibly due to the fact that an experimental approach enabling direct investigation of EcR-Usp interactions in living cells has not been described.…”