Ecdysteroid receptor and ultraspiracle from Chironomus tentans (Insecta) are phosphoproteins and are regulated differently by molting hormone.

Three different isotypes of the ecdysteroid receptor (cEcR) (66, 68 and 70 kDa) and several molecular variants of the dimerization partner "ultraspiracle" (cUSP) (58-77 kDa) can be separated electrophoretically in homogenates of the epithelial cell line from Chironomus tentans. After phosphatase treatment the bands with the lowest electrophoretic mobility disappear in both cases. Phosphorylation occurs exclusively at ser/thr in EcR and USP. Binding studies with 3H-ponasterone A using 0.4 M NaCl extracts revealed two classes of high-affinity binding (KD1 = 0.47 and KD2 = 7.2 nM) competable either with 20-OH-ecdysone or muristerone A. At least KD2 and Bmax2 are unchanged after dephosphorylation. In hormonally naive cells a considerable part of EcR and USP is already present in nuclei. The phosphorylation pattern of both transcription factors is the same in cytosol and nuclear fractions. Incubation with 20-OH-ecdysone (1 microM, up to 4 days) does not alter the extent and mode of phosphorylation of EcR, although EcR concentration increases. In contrast USP concentration remains constant but phosphorylation is enhanced.