Influence of hormone on intracellular localization of the Drosophila melanogaster ecdysteroid receptor (EcR).
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.
Nieva
Claudia
C
Institute of General Zoology and Endocrinology, University of Ulm, Albert-Einstein-Allee 11, 89069 Ulm, Germany.
Spindler-Barth
Margarethe
M
Azoitei
Anca
A
Spindler
Klaus-Dieter
KD
D021581:Active Transport, Cell Nucleus; D000818:Animals; D016466:CHO Cells; D019556:COS Cells; D002467:Cell Nucleus / Q000378:metabolism*; D002522:Chlorocebus aethiops; D006224:Cricetinae; D003412:Cricetulus; D003593:Cytoplasm / Q000378:metabolism*; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D019281:Dimerization; D029721:Drosophila Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004441:Ecdysterone / Q000031:analogs & derivatives* / Q000378:metabolism; D008024:Ligands; D009154:Mutation; D017434:Protein Structure, Tertiary; D011987:Receptors, Steroid / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013997:Time Factors; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D015533:Transcriptional Activation; D014162:Transfection
D004268:DNA-Binding Proteins; D029721:Drosophila Proteins; D008024:Ligands; D011987:Receptors, Steroid; D014157:Transcription Factors; C066030:USP protein, Drosophila; C034808:ecdysteroid receptor; C055348:muristerone A; D004441:Ecdysterone; D004247:DNA