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 protein which was immunorevealed by a specific antibody and migrated with the authentic rtER major protein form detected in trout liver. Yeast rtER bound estradiol with high affinity and the dissociation constant (Kd = 1.35 nM) was very similar to the value measured from trout liver extracts but 3-5-fold higher than the Kd found for human estrogen receptor (hER). This indicates therefore that the rtER has a lower estradiol affinity compared to the human receptor. While the hER Kd remained unchanged at both 4 degrees C or 22 degrees C, it was slightly modified at 30 degrees C. The Kd measured for rtER at 22 degrees C and 30 degrees C were about 2-fold, and 12-fold higher, respectively, than the Kd obtained at 4 degrees C suggesting an alteration of the rtER affinity for its ligand at elevated temperature. To examine the estrogen-receptor-mediated activation of transcription in yeast, reporter plasmids integrated or not in the yeast genome were used. The reporter genes consist of one, two, or three copies of estrogen-responsive elements (ERE) upstream of the yeast proximal CYC1 or URA3 promoters fused to the lacZ gene of Escherichia coli coding for beta-galactosidase. The induction of beta-galactosidase activity for all reporter genes was strictly dependent on the presence of rtER and estrogens. The activation of transcription mediated by rtER responded in an estradiol-dose-dependent manner as in animal cells. However, compared to hER, the estradiol concentration necessary to achieve maximal activation was 10-fold higher. This is probably a consequence of the lower estradiol-affinity for rtER compared to hER. The levels of induction of the reporter genes containing two or three ERE were strongly enhanced compared to the one ERE construct. This is in agreement with the synergistic effect previously described for multiple ERE. The magnitudes of transcriptional induction mediated by rtER and hER were similar when the reporter gene containing three ERE was used but changed when the one ERE construct was used. In this case transcriptional activation indicated by rtER was 10-20 fold lower. This suggests that rtER requires protein/protein interaction for its stabilization on DNA. Antiestrogens were able to bind rtER and promote gene transcription. However, to produce effects comparable to those obtained with estrogens, much higher concentrations were required. This may imply nonetheless that antihormones were capable of provoking efficient interactions of rtER with the transcriptional machinery.
Petit
F
F
Laboratoire de Biologie Moléculaire, URA CNRS 256, Université de Rennes I, France.
Valotaire
Y
Y
Pakdel
F
F
D000818:Animals; D004958:Estradiol / Q000378:metabolism; D004965:Estrogen Antagonists / Q000494:pharmacology; D006801:Humans; D017686:Oncorhynchus mykiss; D011960:Receptors, Estrogen / Q000235:genetics / Q000502:physiology*; D011994:Recombinant Proteins / Q000378:metabolism / Q000494:pharmacology; D012441:Saccharomyces cerevisiae / Q000235:genetics; D014157:Transcription Factors / Q000502:physiology
D004965:Estrogen Antagonists; D011960:Receptors, Estrogen; D011994:Recombinant Proteins; D014157:Transcription Factors; D004958:Estradiol