Transcription activation by the ecdysone receptor (EcR/USP): identification of activation functions

Mol Endocrinol. 2003 Apr;17(4):716-31. doi: 10.1210/me.2002-0287. Epub 2003 Jan 16.

Abstract

The ecdysone receptor is a heterodimer of the two nuclear receptors EcR and ultraspiracle (USP). We have identified the regions of Drosophila EcR and USP responsible for transcriptional activation of a semisynthetic Eip71CD promoter in Kc cells. The isoform-specific A/B domains of EcR-B1 and B2, but not those of EcR-A or USP, exhibit strong activation activity [activation function 1 (AF1)], both in isolation and in the context of the intact receptor. AF1 activity in isoform B1 derives from dispersed elements; the B2-specific AF1 consists of a 17-residue amphipathic helix. AF2 function was studied using a two-hybrid assay in Kc cells, based on the observation that potent hormone-dependent activation by the EcR/USP ligand-binding domain heterodimer requires the participation of both partners. Mutagenesis reveals that AF2 function depends on EcR helix 12, but not on the cognate USP region. EcR helix 12 mutants (F645A and W650A) exhibit a dominant negative phenotype. Thus, in the setting tested, the ecdysone receptor can activate transcription using the AF1 regions of EcR-B1 or -B2 and the AF2 region of EcR. USP acts as an allosteric effector for EcR, but does not contribute any intrinsic function.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Binding Sites
  • Cell Line
  • Conserved Sequence
  • DNA-Binding Proteins / genetics
  • DNA-Binding Proteins / metabolism*
  • Drosophila Proteins / genetics
  • Drosophila Proteins / metabolism*
  • Ecdysone / pharmacology
  • Gene Expression Regulation / drug effects
  • Genes, Dominant
  • Molecular Sequence Data
  • Mutation
  • Promoter Regions, Genetic
  • Protein Conformation
  • Protein Isoforms
  • Receptors, Steroid / genetics
  • Receptors, Steroid / metabolism*
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Transcription Factors / genetics
  • Transcription Factors / metabolism*
  • Transcriptional Activation*
  • Two-Hybrid System Techniques

Substances

  • DNA-Binding Proteins
  • Drosophila Proteins
  • Protein Isoforms
  • Receptors, Steroid
  • Recombinant Proteins
  • Transcription Factors
  • USP protein, Drosophila
  • ecdysone receptor
  • Ecdysone