The estrogen receptor (ER) can be activated as a transcription factor either by binding of cognate estrogenic ligand or, indirectly, by a variety of other extracellular signals. As a first step towards elucidating the mechanism of ‘steroid‐independent activation’ of the ER by the epidermal growth factor (EGF), we have mapped the ER target domain and determined the signaling pathway. We show that the N‐terminal transcriptional activation function AF‐1, but not the C‐terminal AF‐2, is necessary for the EGF response. Both the EGF‐induced hyperphosphorylation and the transcriptional activation of the unliganded ER depend on a phosphorylatable serine residue at position 118. However, its phosphorylation is not sufficient and, hence, there must be other target domains or proteins which fulfill an additional requirement for EGF signaling through the ER. Using dominant‐negative Ras and MAP kinase kinase (MAPK kinase) and constitutively active MAPK kinase mutants, we show that EGF activates the ER by signaling through the MAPK pathway suggesting that MAPK directly phosphorylates the critical serine 118. Our results also imply that the steroid‐independent activation of a variety of ER mutants, which arise during the malignant progression of breast tumors, may contribute to tamoxifen resistance.

Heat-shock protein 90 (Hsp90) is an abundant and highly conserved molecular chaperone that is essential for viability in eukaryotes. Hsp90 fulfills a housekeeping function in contributing to the folding, maintenance of structural integrity and proper regulation of a subset of cytosolic proteins. A remarkable proportion of its substrates are proteins involved in cell cycle control and signal transduction. Hsp90 acts with a cohort of Hsp90 co-chaperones that modulate its substrate recognition, ATPase cycle and chaperone function. The large conformational flexibility of Hsp90 and a multitude of dynamic co-chaperone complexes contribute to generating functional diversity, and allow Hsp90 to assist a wide range of substrates.

We have detected nuclear localization signals within the 795 amino acid rat glucocorticoid receptor. Using a transient expression assay, we monitored by immunofluorescence the subcellular distribution of receptor derivatives and beta‐galactosidase‐receptor fusion proteins. Two distinct nuclear localization signals, NL1 and NL2, were defined. NL1 maps to a 28 amino acid segment closely associated, but not coincident with the DNA binding domain; NL2 resides within a 256 amino acid region that also includes the hormone binding domain. Most importantly, nuclear localization of fusion proteins containing either the full‐length receptor or the NL2 region alone is fully hormone‐dependent; similar results were obtained with the wild‐type receptor, provided the analysis was performed in medium lacking serum and phenol red. The rate of hormone‐induced nuclear localization of an NL2‐containing fusion protein is consistent with the rapid kinetics of hormone‐regulated transcription mediated by the receptor. Thus, hormonal control of nuclear localization contributes to the modulation of glucocorticoid receptor transcriptional regulatory activity.

Signalling by steroid hormones is mediated by receptor proteins that bind hormonal ligands and regulate the transcription of specific genes. The heat-shock protein hsp90 seems to associate selectively with unliganded receptors (aporeceptors), but it has not been determined whether this interaction affects receptor function in vivo. To address the role of hsp90, we have taken advantage of the capacity of mammalian steroid receptors to function in yeast. We constructed a strain of Saccharomyces cerevisiae in which hsp90 expression was regulatable and could be reduced more than 20-fold relative to wild type. At low levels of hsp90, aporeceptors seem to be mostly hsp90-free, yet fail to enhance transcription; on hormone addition, the receptors are activated but with markedly reduced efficiency. Thus hsp90 does not inhibit receptor function solely by steric interference; rather, hsp90 seems to facilitate the subsequent response of the aporeceptor to the hormonal signal. This is the first biological evidence that hsp90 acts in the signal transduction pathway for steroid receptors.