Endocrine signal transduction occurs through cascades that involve the action of both ligand-dependent and ligand-independent nuclear receptors. In insects, two such nuclear receptors are HR3 and E75 that interact to transduce signals initiated by ecdysteroids. We have cloned these nuclear receptors from the crustacean Daphnia pulex to assess their function as regulators of gene transcription in this ecologically and economically important group of organisms. Both nuclear receptors from D. pulex (DappuHR3 (group NR1F) and DappuE75 (group NR1D)) exhibit a high degree of sequence similarity to other NR1F and NR1D group members that is indicative of monomeric binding to the RORE (retinoid orphan receptor element). DappuE75 possesses key amino acid residues required for heme binding to the ligand-binding domain. Next, we developed a gene transcription reporter assay containing a luciferase reporter gene driven by the RORE. DappuHR3, but not DappuE75, activated transcription of the luciferase gene in this system. Co-transfection experiments revealed that DappuE75 suppressed DappuHR3-dependent luciferase transcription in a dose-dependent manner. Electrophoretic mobility shift assays confirmed that DappuHR3 bound to the RORE. However, we found no evidence that DappuE75 similarly bound to the response element. These experiments further demonstrated that DappuE75 prevented DappuHR3 from binding to the response element. In conclusion, DappuHR3 functions as a transcriptional activator of genes regulated by the RORE and DappuE75 is a negative regulator of this activity. DappuE75 does not suppress the action of DappuHR3 by occupying the response element but presumably interacts directly with the DappuHR3 protein. Taken together with the previous demonstration that daphnid HR3 is highly induced by 20-hydroxyecdysone, these results support the premise that HR3 is a major component of ecdysteroid signaling in some crustaceans and is under the negative regulatory control of E75.
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