title: A triple mutant of the Drosophila ERR confers ligand-induced suppression of activity.
abstract: The steroid hormone (NR3) subfamily of nuclear receptors was until recently believed to be restricted to deuterostomes. However, a novel nuclear receptor belonging to the NR3 subfamily was recently identified in the Drosophila melanogaster genome, indicating the existence of an ancestor before the evolutionary split of deuterostomes and protostomes. This receptor, termed the Drosophila estrogen-related receptor (dERR), most closely resembles the human and mouse estrogen-related receptors (ERRs) in both the DNA binding domain (DBD) (approximately 85% identical) and the ligand binding domain (LBD) (approximately 35% identical). Here we describe the functional analysis and rational design of ligand responsive dERR mutants created by protein engineering of the LBD. On the basis of homology modeling, three amino acid residues in the LBD were identified and mutated to enable ligand-dependent suppression of transcriptional activity. Our results show that the Y295A/T333I/Y365L triple mutant is significantly suppressed by the known ERR inverse agonists 4-hydroxytamoxifen (OHT) and diethylstilbestrol (DES), in comparison to the wild-type dERR receptor, which was inefficiently suppressed by these substances. The coactivator mGRIP-1 (mouse glucocorticoid receptor interacting protein 1) was shown to significantly increase the activity of the triple mutant in transfection experiments, and the addition of OHT resulted in an efficient suppression of the activity. Accordingly, the ability to functionally interact with a coactivator is still maintained by the Y295A/T333I/Y365L mutant. These findings demonstrate the potential of using rational design and engineering of the LBD to study the function of a nuclear receptor lacking identified ligands.
authors.affiliation: Department of Cell and Molecular Biology, Medical Nobel Institute, Karolinska Institute, S-171 77 Stockholm, Sweden.
authors.forename: Tove
authors.initials: T
authors.lastname: Ostberg
authors.forename: Micael
authors.initials: M
authors.lastname: Jacobsson
authors.forename: Anneli
authors.initials: A
authors.lastname: Attersand
authors.forename: Alexander
authors.initials: A
authors.lastname: Mata de Urquiza
authors.forename: Lena
authors.initials: L
authors.lastname: Jendeberg
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D000818:Animals; D002467:Cell Nucleus / Q000378:metabolism; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism; D004252:DNA Mutational Analysis; D004054:Diethylstilbestrol / Q000494:pharmacology; D029721:Drosophila Proteins / Q000737:chemistry / Q000235:genetics* / Q000502:physiology*; D004331:Drosophila melanogaster / Q000235:genetics*; D017930:Genes, Reporter; D006801:Humans; D008024:Ligands; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D051958:Nuclear Receptor Coactivator 2; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D015202:Protein Engineering; D011506:Proteins / Q000235:genetics*; D011960:Receptors, Estrogen / Q000737:chemistry / Q000235:genetics* / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013629:Tamoxifen / Q000031:analogs & derivatives* / Q000494:pharmacology; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured
chemical_list: D000596:Amino Acids; D029721:Drosophila Proteins; C059917:ERD5 protein, human; C475538:ERR protein, Drosophila; D008024:Ligands; D051958:Nuclear Receptor Coactivator 2; D011506:Proteins; D011960:Receptors, Estrogen; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D013629:Tamoxifen; C016601:afimoxifene; D004054:Diethylstilbestrol; D004247:DNA