Identification and characterization of the ecdysterone receptor in Drosophila melanogaster by photoaffinity labeling

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Salivary glands of third-instar larvae of Drosophila melanogaster as well as Drosophila Kc tissue culture cells have been irradiated in the presence of ecdysterone. Irradiation covalently links ecdysterone to a single cellular protein, which is similar, if not identical, in salivary glands and in Kc cells. This protein has a molecular weight of 130,000 and it has the characteristics of a typical hormone-receptor molecule in terms of hormone-binding properties, translocation into the nucleus, and sedimentation characteristics. The yield of the photoinduced bonding of ecdysterone to receptor protein is around 15%. Ponasterone A competed with ecdysterone for the bonding. Also, ponasterone A itself reacted upon photoactivation with the β-ecdysterone receptor protein in Drosophila tissue culture cells. We have previously shown that ecdysterone can be bonded upon irradiation to specific hormone-controlled puffs of polytene chromosomes of D. melanogaster third-instar larvae [Gronemeyer, H. & Pongs, O. (1980) Proc. Natl. Acad. Sci. USA 77, 2108-2112]. Because we have now identified the molecular target of the ecdysterone photoreaction, these data show that a hormone-receptor complex translocates to the nucleus and directly binds to the genes, which are under hormonal control. A quantitative assay of hormone-receptor complex in Kc cells before and after hormone stimulation showed that ecdysterone does not regulate the synthesis and the available amount of its receptor. It was also observed that the translocated hormone-receptor complex resides in the nucleus as long as the hormone is present in the tissue culture medium.

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