Functional characterization of two Ultraspiracle forms (CtUSP-1 and CtUSP-2) from Chironomus tentans

Two forms, CtUSP-1 and CtUSP-2, of the Chironomus tentans homolog of Ultraspiracle (new nomenclature: Chironomus NR2B4) were described and verified as components of the functional ecdysteroid receptor. The two forms differed from each other in the most N-terminal regions of the A/B domain and were tested for several properties. Both forms showed the ability to heterodimerize with CtEcR and interact with a variety of direct repeat and palindromic EcREs, and both conferred specific ligand binding when heterodimerized with EcR. CtUSP-2 showed a twofold higher ponasterone-binding potential than CtUSP-1. Both USP forms demonstrated the ability to activate ecdysteroid-inducible transcription in HeLa cells and the variations in the A/B domain of these forms were not associated with detectable differences in transcriptional activation. Thus, the two forms function similarly. Among species for which USP forms have been reported, Chironomus is the most closely related one evolutionarily to Drosophila. Despite this proximity, a variety of structural differences were noted in both the A/B and E domains of USP between the two species. The Chironomus USP forms lack many of the amino acid residues associated with the ligand-dependent AF2 transactivation function found in all other RXRs and USPs reported so far.