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

Abstract

Salivary glands of 3rd-instar larvae of D. melanogaster as well as Drosophila Kc tissue culture cells were 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 MW 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 .apprx. 15%. Ponasterone A competed with ecdysterone for the bonding. Ponasterone A itself reacted upon photoactivation with the .beta.-ecdysterone receptor protein in Drosophila tissue culture cells. Ecdysterone is bonded upon irradiation to specific hormone-controlled puffs of polytene chromosomes of D. melanogaster 3rd-instar larvae [Gronemeyer and Pongs (1980)]. Because the molecular target of the ecdysterone photoreaction, has been identified 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. The translocated hormone-receptor complex resides in the nucleus as long as the hormone is present in the tissue culture medium.