Nucleocytoplasmic shuttling of ecdysteroid receptor

Abstract

Ecdysteroids are a class of steroid hormones that coordinate development, reproduction, and other essential processes in insects through a heterodimer consisting of ecdysteroid receptor (EcR) and ultraspiracle (Usp). Both proteins are transcription factors and members of the nuclear receptor superfamily. Karyopherins represent a family of proteins, which regulates nucleocytoplasmic shuttling. Importins are responsible for nuclear import, and recognize cargoes which possess a nuclear localization signal (NLS). Exportins bind to and ferry proteins that contain a nuclear export signal (NES). The molecular interactions of transport receptors with their cargos are regulated by the small GTPase Ran. The intracellular distribution of EcR and Usp in mammalian cell lines and the role of transport proteins in the nucleocytoplasmic shuttling of these nuclear receptors were analysed. Usp was found exclusively in the nucleus, whereas the localization of EcR was dependent on time and presence of ultraspiracle. Nevertheless, both proteins could enter the nucleus separately and interacted with importin alpha1. Moreover, experiments with Ran knock down revealed an essential and direct role of Ran in EcR, but not Usp import. Export of EcR was mediated by exportin-1 (CRM1) as shown by co-immunoprecipitation experiments, and the inhibiting effect of leptomycin B. No export signal and no binding to exportin-1 could be identified for Usp. Additionally, first results suggest that cell cycle may influence the intracellular transport of EcR. The data proved that intracellular localization is individually regulated for Usp and EcR.