DIFFERENTIAL FUNCTIONAL ACTIVITIES OF RAINBOW-TROUT AND HUMAN ESTROGEN-RECEPTORS EXPRESSED IN THE YEAST SACCHAROMYCES-CEREVISIAE

The cDNA of rainbow trout estrogen receptor (rtER), highly and stably expressed in yeast, Saccharomyces cerevisiae, was used to analyse the biological activity of the receptor. The rtER mRNA encoded a 65-kDa pr otein which was immunorevealed by a specific antibody and migrated wit h the authentic rtER major protein form detected in trout liver. Yeast rtER bound estradiol with high affinity and the dissociation constant (K-d = 1.35 nM) was very similar to the value measured from trout liv er extracts but 3-5-fold higher than the K-d found for human estrogen receptor (hER). This indicates therefore that the rtER has a lower est radiol affinity compared to the human receptor. While the hER K-d rema ined unchanged at both 4 degrees C or 22 degrees C, it was slightly mo dified at 30 degrees C. The K-d measured for rtER at 22 degrees C and 30 degrees C were about 2-fold, and 12-fold higher, respectively, than the K-d obtained at 4 degrees C suggesting an alteration of the rtER affinity for its ligand at elevated temperature. To examine the estrog en-receptor-mediated activation of transcription in yeast, reporter pl asmids integrated or not in the yeast genome were used. The reporter g enes consist of one, two, or three copies of estrogen-responsive eleme nts (ERE) upstream of the yeast proximal CYC1 or URA3 promoters fused to the lacZ gene of Escherichia coli coding for beta-galactosidase. Th e induction of beta-galactosidase activity for all reporter genes was strictly dependent on the presence of rtER and estrogens. The activati on of transcription mediated by rtER responded in an estradiol-dose-de pendent manner as in animal cells. However, compared to hER, the estra diol concentration necessary to achieve maximal activation was 10-fold higher. This is probably a consequence of the lower estradiol-affinit y for rtER compared to hER. The levels of induction of the reporter ge nes containing two or three ERE were strongly enhanced compared to the one ERE construct. This is in agreement with the synergistic effect p reviously described for multiple ERE. The magnitudes of transcriptiona l induction mediated by rtER and hER were similar when the reporter ge ne containing three ERE was used but changed when the one ERE construc t was used. In this case transcriptional activation mediated by rtER w as 10-20-fold lower. This suggests that rtER requires protein/protein interaction for its stabilization on DNA. Antiestrogens were able to b ind rtER and promote gene transcription. However, to produce effects c omparable to those obtained with estrogens, much higher concentrations were required. This may imply none-theless that antihormones were cap able of provoking efficient interactions of rtER with the transcriptio nal machinery.