Isoform-specific regulation of a steroid hormone nuclear receptor by an E3 ubiquitin ligase in Drosophila melanogaster

The steroid hormone 20-hydroxyecdysone (20E) regulates gene transcription through the heterodimeric nuclear receptor composed of ecdysone receptor (EcR) and Ultraspiracle (USP). The EcR gene encodes three protein isoforms-A, B1, and B2-with variant N-terminal domains that mediate tissue and developmental stage-specific responses to 20E. Ariadne-1a is a conserved member of the RING finger family of ubiquitin ligases first identified in Drosophila melanogaster. Loss-of-function mutations at key cysteines in either of the two RING finger motifs, as well as general overexpression of this enzyme, cause lethality in pupae, which suggests a requirement in metamorphosis. Here, we show that Ariadne-1a binds specifically the isoform A of EcR and ubiquitylates it. Coimmunoprecipitation experiments indicate that the full sequence of EcRA is required for this binding. Protein levels of EcRA and USP change in opposite directions when those of ARI-1a are genetically altered. This is an isoform-specific, E3-dependent regulatory mechanism for a steroid nuclear receptor. Further, qRT-PCR experiments show that the ARI-1a levels lead to the transcriptional regulation of Eip78C, Eip74EF, Eip75B, and Br-C, as well as that of EcR and usp genes. Thus, the activity of this enzyme results in the regulation of dimerizing receptors at the protein and gene transcription levels. This fine-tuned orchestration by a conserved ubiquitin ligase is required during insect metamorphosis and, likely, in other steroid hormone-controlled processes across species. 2011 by the Genetics Society of America