Requirement of co-factors for the ligand-mediated activity of the insect ecdysteroid receptor in yeast.
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(Delta A/B)EcR-USP or Aa(Delta 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(Delta 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 co-factors 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.
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LifeSensors Inc., 271 Great Valley Parkway, Malvern, Pennsylvania 19355, USA.
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D000330:Aedes / Q000235:genetics / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics; D029721:Drosophila Proteins; D004331:Drosophila melanogaster / Q000235:genetics; D004353:Drug Evaluation, Preclinical; D026461:Ecdysteroids / Q000494:pharmacology; D017344:Genes, Insect; D017930:Genes, Reporter; D066298:In Vitro Techniques; D007306:Insecticides / Q000494:pharmacology; D008024:Ligands; D009363:Neoplasm Proteins / Q000235:genetics; D018168:Receptors, Retinoic Acid / Q000235:genetics; D011987:Receptors, Steroid / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D047488:Retinoid X Receptors; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics; D015533:Transcriptional Activation / Q000187:drug effects
D017931:DNA Primers; D004268:DNA-Binding Proteins; D029721:Drosophila Proteins; D026461:Ecdysteroids; D007306:Insecticides; D008024:Ligands; D009363:Neoplasm Proteins; D018168:Receptors, Retinoic Acid; D011987:Receptors, Steroid; D011994:Recombinant Proteins; D047488:Retinoid X Receptors; D014157:Transcription Factors; C066030:USP protein, Drosophila; C034808:ecdysteroid receptor