Abstract
In the absence of hormone the ecdysteroid receptor (EcR) is distributed between the cytoplasm and the nucleus. Addition of the hormone muristerone A increases nuclear localization of wild type EcR within 5-10 min. Mutation of M504 to alanine, an amino acid, which is essential for ligand binding and which is situated in helix 5 of the ligand binding domain, abolishes hormone binding but still allows nuclear localization at only slightly reduced levels in the absence of hormone, whereas nuclear localization of EcR(M504R) is nearly abolished. Cotransfection with ultraspiracle (USP), the invertebrate ortholog of RXR, leads to exclusively nuclear localization of wild type EcR and EcR(M504A) indicating that basal heterodimerization in the absence of hormone is still possible. In the presence of Usp, EcR(M504R) is only partially localized in the nucleus. EMSA experiments show that the ligand muristerone A enhances binding of wild type EcR, but only slightly of mutated EcRs, to the canonical hsp 27 ecdysone response element. This is confirmed by transactivation studies. The results indicate that the architecture of the E-domain of EcR is important for nuclear localization even in the absence of a ligand.
