Abstract
Previous studies using in vitro procedures have not clearly established whether the estrogen receptor (ER) acts as a monomer or dimer in the cell. We have used the yeast two-hybrid system as an in vivo approach to investigate the dimerization of the estrogen receptor in the absence and presence of estrogen and anti-estrogens. This system is independent of ER binding to the estrogen response element. Two vectors, expressing GAL4 DNA binding domain-human ER and GAL4 transactivation domain-human ER, were constructed. Control experiments showed that each fusion protein had a high affinity binding site for estradiol-17 beta and could transactivate an ERE-LacZ reporter gene in yeast similar to the wild type ER. The two fusion proteins, GAL4 DB-hER and GAL 4 TA-hER, were expressed in the yeast strain, PCY2, which carries a GAL1 promoter-lacZ reporter. ER dimerization was measured via reconstitution of GAL4 through interaction of the fusion proteins, which transactivates LacZ through the GAL1 promoter. When both ER fusion proteins were expressed, beta-galactosidase activity was estradiol-17 beta-inducible. Furthermore, we showed that both tamoxifen and ICI 182,780 also induced beta-galactosidase activity, albeit lower than that induced by estradiol-17 beta. These results strongly argue that ER dimerization is ligand-dependent and the dimer can be induced by estradiol-17 beta, tamoxifen, or ICI 182,780. We also treated the yeast containing the two fusion proteins with estradiol-17 beta and tamoxifen or ICI 182,780 simultaneously to determine the effects on ER dimerization. beta-Galactosidase activity was lower when the yeast was treated with a higher ratio of tamoxifen or ICI 182,780 to estrogen than estradiol-17 beta alone. Taken together, we conclude that ER dimerization is ligand (estradiol-17 beta, tamoxifen, or ICI 182, 780)-dependent, and we suggest that estradiol-17 beta-induced dimers are destabilized when estradiol-17 beta is used with tamoxifen or ICI 182,780 simultaneously.
Highlights
Mediates the actions of estrogens in target cells
Neither GAL4 DNA binding (GAL4 DB)-Human ER cDNA (hER) Nor GAL4 TA-hER Alone Activate the Transcription of GAL1 Promoter Driving LacZ—Since estrogen receptor (ER) has two independent transcriptional activation domains (AF-1 and AF-2), as well as a DNA binding domain, it was unknown whether ER itself could activate transcription of the GAL1-lacZ reporter gene
Human ER cDNA was cloned into the GAL4 fusion vectors (GAL4 DB and GAL4 TA) and introduced into PCY2 yeast to express GAL4 DB-hER or GAL4 TA-hER, respectively. -Galactosidase activity in the yeast colonies selected with Trp or Leu dropout medium was determined using X-gal indicator (Table I)
Summary
Mediates the actions of estrogens in target cells. The ER is a member of a superfamily of related nuclear proteins which includes receptors for steroid hormones, thyroid hormones, vitamin D, the retinoids, and a number of proteins with high sequence homology but as yet unidentified ligands. Most of the data for and against dimerization of ER have been shown using gel mobility shift assays or complex assays in vitro, where the assay itself requires ER to bind DNA These assays have given conflicting evidence in demonstrating whether estrogen is required [4] or not required (10 –12) for high affinity binding of the ER to the ERE. The yeast two-hybrid system has been described by Fields and co-workers [21, 22] It involves the expression of a LacZ reporter gene under the control of a GAL4-activated promoter (GAL1 promoter) that depends on the reconstitution of GAL4 activity via protein-protein interactions. This system has been used successfully in screening a cDNA library [23,24,25] and has turned out to be a useful approach to study protein-protein interactions in vivo
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
