**2. The concept of steroid hormone receptor activation**

The favorite theme of the 60's and early 70's in descriptions of steroid hormone action, particularly with reference to estrogen action, used to be that the receptor primarily existed in the cytosol. Upon hormone binding and the consequent "receptor activation" the receptor entered the nucleus and interacted with the genetic elements. This "two-step mechanism", independently proposed by the research groups led by Jensen and Gorski (Jensen & DeSombre, 1973; Shyamala & Gorski, 1969; Mohla et al, 1972) formed the basis for all subsequent discussions on intracellular movements of the receptor –steroid hormone

Evolving Trends in Estrogen Receptor Biology 67

that primarily led to the separation of E-RAF from the estrogen receptor that it dimerises with during DEAE-cellulose chromatography. Thampan and Clark (1981) reported that a 3S protein of the rat uterine cytosol, that appeared in the DEAE cellulose flow through fraction, promoted the DNA binding of a specific class of non-DNA binding estrogen receptor. Thampan (1987,1989) in his reports on the purification of E-RAF observed that E-RAF

While both forms displayed identical molecular weight of 66kDa, their molecular shapes appeared to be different as displayed by the results of gel filtration chromatography and also in their dissimilar sedimentation behavior in linear sucrose density gradients. Functional assays were carried out in which the proteins were incubated with labeled DNA, which was subsequently exposed to S1 nuclease in order to digest the single stranded regions. The results showed that while E-RAF II destabilized DNA double helix and enhanced strand separation, the reverse property (stabilization of double helical structure) was found associated with E-RAF I. In vitro transcription assays involving isolated nuclear RNA polymerases also highlighted this differential behavior of the two molecular forms. While E-RAF II enhanced transcription, in a system containing nuclear RNA polymerase purified from goat uterine nuclei, E-RAF I inhibited transcription in a dose-dependent

**5. A vision into the molecular identity of the type I and type II nuclear** 

type I and type II estrogen binding sites in rat uterine nuclei (Eriksson et al.,1978).

Following DEAE Sephadex-A25 chromatography of nuclear sonicates, the fractions collected were subjected to the estradiol exchange assay as well as RNA polymerase assay with calf thymus DNA as the template .RNA polymerase peaks representing I, II, IIIa and IIIb were clearly demonstrated in the DEAE-Sephadex A-25column fractions. Also demonstrated was the estrogen binding function associated with all four peaks of RNA polymerase activity. The 'receptor' activity associated with the RNA polymerase II was subjected to further analysis. Sucrose density gradient analysis displayed two peaks of activity, a small peak at 5S and a large peak at 3S.While the 5S peak was distinctly DNA binding, the 3S peak which represented the major share of receptor activity, remained non DNA binding. Subsequent studies (Thampan,1989) have demonstrated that the DNA binding function of the 5S peak was due to the presence of E-RAF and an estrogen receptor that dimerised with E-RAF while the non DNA binding 3S fraction was represented by a receptor that did not dimerise with E-RAF.The same studies have concluded, subsequently that the receptor of the 5S peak

The report in which functional characterization of E-RAF was described (Thampan,1989), also presented a method for the assay for E-RAF in association with the nuclear RNA polymerases. Nuclear RNA polymerases were extracted from isolated rat uterine nuclei and subjected to partial purification through chromatography on DEAE Sephadex A-25 and elution with linear (NH4)2SO4 gradient. Ovariectomized rats were used in this study. While control rats received injection of the vehicle alone, experimental animals were subjected to subcutaneous injections of 3μg estradiol-17β for a duration of one hour. The RNA polymerase fractions derived from both control and experimental nuclei and eluted from DEAE-Sephadex A-25 column were subjected to the nuclear exchange assay that was developed earlier by Clark and coworkers (Clark & Peck,1979;Clark et al.,1979). It was through this nuclear exchange assay that Clark's group had demonstrated the existence of

existed in two molecular forms, E-RAF II and I.

manner.

**estrogen binding sites** 

complex. It was proposed that the cytosolic receptor existed as a high molecular weight form that sedimented at 8-9S in low salt linear sucrose gradients. Later studies have revealed that in this cytosolic form, the receptor with an average sedimentation value of 4S, remained in association with heat shock protein 90(hsp 90) when there was no hormone bound to it (Pratt, 1990; Pratt &Toft, 1997). Hormone binding to the receptor initiated dissociation of the receptor form Hsp-90, which formed a key event in steroid receptor activation (Pratt, 1990). One of the major structural changes noticed in the receptor during its activation was the transformation of the 4S receptor to a form that sedimented at 5S in sucrose gradients containing 0.3M KCl (Shyamala & Gorski, 1969).

The 4S-5S conversion was the target of several hypotheses that attempted to explain the molecular event. In the Hsp-90 model, it was clear that association of the receptor with Hsp-90 prevented the nuclear migration of the receptor the reason for which was not clear at that time. It was the first ever report on the sequencing of amino acids of the human estrogen receptor α (ER α) by Chambon's group at Strasburgh that paved the way for a number of active studies in this direction (Green & Chambon, 1987 a, b). The identification of the nuclear localization signal (NLS) in ERα (Kumar et al., 1986; Kumar et al., 1987) was one such landmark observation. Thampan's group subsequently extended the studies using ERα isolated from goat uterus and purified and characterized a 55kDa protein (p55) that apparently recognized the nuclear localization signal (NLS) on ERα and initiated the nuclear entry of the receptor (Nirmala & Thampan,1995 a,b). The studies reported by Thampan's group gave additional validity to the role of p55 in the nuclear entry of ERα.
