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

The heterodimer of the ecdysone receptor (EcR) and ultraspiracle (Usp), members of the nuclear receptor superfamily, is considered to be functional receptor for the ecdysteroids that coordinate essential biological processes in insects. In this work we have applied a bimolecular fluorescence complementation (BiFC) method to directly analyze the formation of the EcR/Usp complex. The BiFC experiments were carried out in mammalian cells which are routinely used for heterologous studies of the EcR/Usp complex, including experiments on EcR-based artificial molecular gene switches. BiFC analysis, supported by flow cytometry, revealed that EcR–Usp interaction is nuclei-restricted. If expressed separately, Usp and EcR are able to form nuclear complexes in the absence of the cognate dimerization partner. We have observed that Muristerone A that is widely used for the induction of ecdysteroid-dependent transcription in mammalian cells, does not significantly change the number of EcR/Usp and EcR/EcR complexes, and it does not influence their subcellular localization.