## 3 Estrogen Receptors  
### Identification  
Discovery and subsequent cloning of the estrogen receptor were paramount steps not only for the estrogen signaling field but also for the discovery of the nuclear receptor superfamily. In 1960, a revolutionary theory of a plausible receptor for estrogen started to take shape. By using tritium-labeled estrogen, which is highly radioactive, [PERSON] and colleagues paved the way for the discovery and identification of such molecule [21]. When physiological amounts of tritium-labeled estrogen (90 ng) were administered to immature female rats, the uterus and vagina (tissues that respond to estrogen treatment with dramatic growth) showed higher uptake (100-200 times higher concentration than blood) and longer  
Figure 1: Traditional Mechanisms of Estrogen Signaling. Genomic signaling: Estrogen diffuses through the cell membrane into the cytosol of the cell where it binds to its receptors. Estrogen-Estrogen Receptor (E-ER) complex moves to the nucleus where it forms homo- and/or heterodimer complexes. This complex either (1) binds to the specific Estrogen Responsive Elements (ERE) in the DNA or (2) recruits transcription factors which then alter transcription. Nongenotropic signaling: (3) Estrogen binds to G protein-coupled estrogen receptor (in the plasma membrane) and activates downstream signaling mechanisms. These three signaling mechanisms ultimately lead to altered gene expression. retention of the radioactively labeled hormone (peak levels lasted for almost 2 h). The liver and kidneys (tissues in which substantial steroid metabolism and degradation occurs) also show high levels of radioactivity; however, the peak concentration was observed at 15 min in these organs. These results revealed two major breakthroughs: (1) at low levels in the blood, estrogen targets specific tissues such as the uterus; and (2) estradiol is not metabolized in the uterus. With the results from these experiments, [PERSON] and colleagues proposed that estrogen was held in the cytoplasm by a specific protein, which we now refer to as the estrogen receptor, and it is this complex that moves to the nucleus where it exerts its action. In 1966, six years later, [PERSON] and [PERSON] reported data on the isolation of a macro-molecule bound to estrogen, with the characteristic of a receptor, which was only found in the uterus, but not in the small intestine nor blood serum. The binding was antagonized by diethylstilbestrol, but not by non-estrogenic steroids, testosterone, or corticosterone [22]. Twenty years later, in 1986, in collaboration with [PERSON]'s group, the gene for estrogen receptor was cloned. The clone, now referred to as ER\(\alpha\), encoded 595 amino acid residues with a calculated molecular weight of 66 kDa [23]. For several decades after its discovery, ER\(\alpha\) was the only estrogen receptor known to the scientific community; therefore, the discovery of a second estrogen receptor in 1996 was a surprise to the field. [PERSON] et al. identified and cloned the ER\(\beta\) from a rat prostate cDNA library while screening for additional androgen receptors [24]. The ER\(\beta\) clone encoded 485 amino acid residues with a calculated molecular weight of 54.2 kDa. Authors reported that this novel estrogen receptor had a high homology at the DNA binding site (95%) to the already known estrogen receptor (ER\(\alpha\)) and a high affinity for estradiol (Kd = 0.6 nM). Using in situ hybridization, they reported high expression in the prostate, in epithelial cells of the secretory alveoli, and the granulosa cells in the primary, secondary, and mature follicles of the ovary [24]. Like other nuclear receptors, the two ERs seem to have opposite effects. In a classic paper, [PERSON] et al. (1999) eloquently demonstrated that ER\(\beta\) functions as a trans-dominant inhibitor of ER\(\alpha\) at sub-saturating hormone levels. The authors transfected HepG2 and HeLa cells with ER plasmids.