Requirement of co-factors for the ligand-mediated activity of the insect ecdysteroid receptor in yeast

Abstract

In insects, a steroid hormone 20-hydroxyecdysone has an important role in regulating critical events such as development and reproduction. The action of 20-hydroxyecdysone is mediated by its binding to the ecdysteroid receptor (EcR), which requires a heterodimeric partner, ultraspiracle protein (USP), a homologue of the retinoid X receptor (RXR). The EcR-USP heterodimer represents a functional receptor complex capable of initiating transcription of early genes. Our goal was to establish a ligand-dependent transactivation system in yeast utilizing an insect EcR-USP heterodimer. This has been achieved using mosquito Aedes aegypti AaEcR-USP. Expression of AaEcR alone, but not USP, resulted in constitutive transcription of the ecdysone reporter gene coupled with the Drosophila heat shock protein-27 ecdysone response elements. Removal of the N-terminal A/B domain of AaEcR abolished its constitutive transcription. Constitutive transcription was also eliminated in the presence of its heterodimeric partner, AaUSPa, AaUSPb or mammalian RXR. This suggests that the A/B domain is essential for the EcR ligand-independent transactivation and its interaction with the yeast transcription complex. A ligand-mediated transactivation of Aa( A/B)EcR-USP or Aa( A/B)EcR-RXR heterodimers in response to an ecdysteroid agonist RH-5992 was observed only in the presence of GRIP1, a mouse co-activator. In the presence of a co-repressor, SMRT, Aa( A/B)EcR-USP heterodimer exhibited a ligand-dependent repression activity. In addition, ligand-dependent transactivation systems for spruce budworm and fruit fly ecdysone receptors were also reported. This is the first report establishing the requirements of cofactors for a highly efficient ligand-dependent function of the insect EcR-USP in yeast. These findings open a way to study insect EcR-USP structure and function and to identify ligands that are specific for a certain group of insects, such as mosquitoes.

DNA‐binding properties of Drosophila ecdysone receptor isoforms and their modification by the heterodimerization partner ultraspiracle

“…The differences in DNA-binding capabilities of EcR isoforms confirm that the N-terminal and the DNA-binding domains influence each other and that the interaction of these domains modifies receptor activity, e.g., the transcriptional capability as previously described for vertebrate receptors (Tung et al, 2006;Zhou and Cidlowski, 2005). Transcriptional activity is further modulated by isoform-specific interaction of the AB-domain of EcR with the C-terminus as shown by FRET (Tremmel, personal communication), which in addition to co-modulator interaction (Tsai et al, 1999;Tran et al, 2001;Maki et al, 2004) may explain the discrepancy between DNA-binding and transcriptional activity.…”

Fine-tuning of secondary arbor development: the effects of the ecdysone receptor on the adult neuronal lineages of theDrosophilathoracic CNS

“…When 20E is absent, co-repressors may bind to EcR/USP to shut down transcription (Dressel et al, 1999;Tsai et al, 1999). The binding of 20E releases co-repressors, after which co-activators are recruited to promote transcriptional activation (Bai et al, 2000;Dressel et al, 1999;Tran et al, 2001;Tsai et al, 1999). Ecdysone responses are also modulated by the specific blending of the three different EcR isoforms expressed (Talbot et al, 1993;Truman et al, 1994).…”

Transcriptional activity of ecdysone receptor isoforms is regulated by modulation of receptor stability and interaction with Ab‐ and C‐domains of the heterodimerization partner ultraspiracle

“…Although some co-repressors seem to be rather conserved between vertebrates and insects (Tsai et al, 1999;Tran et al, 2001b), the mixture of co-modulators certainly differs between vertebrates and insects. Differences also exist between different insect tissues and comodulator concentrations and activities vary depending on the physiological state of the target cells.…”

“…Therefore, these cells were routinely used for the characterization of the molecular properties of EcR and Usp (Henrich et al, 2003(Henrich et al, , 2009Beatty et al, 2006;Betanska et al, 2007Betanska et al, , 2009Dutko-Gwóźdź et al, 2008;Nieva et al, 2007Nieva et al, , 2008. At least some accessorial proteins like comodulators, which are required for receptor action, seem to be sufficiently conserved to allow structurefunction studies (Tsai et al, 1999;Tran et al, 2001b;Henrich et al, 2009). Because the presence and the combinations of different comodulators varies in different target tissues, and their activities, which are regulated by posttranslational modification (Hermanson et al, 2002;Lonard and O'Malley, 2007), depend on the physiological state, selection of a typical insect-specific environment seems questionable.…”