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.

Origin and diversification of steroids: Co-evolution of enzymes and nuclear receptors

“…This leaves unresolved how ligand activation of the vertebrate ER evolved from a constitutively active ancestor, which appears to be a complex process because a mutant human ERR that binds E2 [46] and a mutant Drosophila ERR [61] that binds diethylstilbestrol have been constructed and neither activates transcription with the bound ligand. The mutant human ERR complexed with E2 is constitutively active [46].…”

“…The mutant human ERR complexed with E2 is constitutively active [46]. Interestingly, binding of diethylstilbestrol to the mutant Drosphila ERR represses constitutive activity [61]. Further complicating the relationship between binding of E2 to the ER and transcriptional activity is the evidence that, point mutations in ER lead to a constitutively active receptor [62].…”

The C-terminal tail of the Hedgehog receptor Patched regulates both localization and turnover

“…S-CTD produced by in vitro translation either before (CTD) or after incubation with glutathione beads (CTD + GST proteins) that were bound by bacteria-generated GST or GST fusions proteins, as indicated. ERR is the cytoplasmic domain of the estrogen receptor-related protein (CG7404) (Ostberg et al 2003), and was used as an unrelated control. Pull-downs were with 10-µg beads except for lane 5 (5 µg).…”

Molecular Cloning and Expression of an Estrogen Receptor-Related Receptor Gene in the Ant Polyrhachis vicina (Hymenoptera: Formicidae)

“…There are only two introns in the DERR gene (Ostberg et al 2003), seven introns in both of the PvERR and the predicted AmERR gene, the latter being similar to the mammalian ERRs. This may imply that the number of introns tends to increase during evolution.…”