**4. Search for the "receptor-activator" protein and the discovery of E-RAF**

There was a line of thinking that originated from Notides'(Notides & Nielson,1974)and Yamamoto's (Yamamoto,1974)laboratories that in estrogen action there was a possibility for the involvement of a DNA binding X-protein in converting the non-DNA binding estrogen receptor to a DNA binding form. Based on these observations and consideration of a potential possibility that a non-hormone binding transcription factor could be involved in the "activation" process, Thampan and Clark (1981,1983) presented the first ever experimental evidence for the existence of an estrogen receptor activation factor (E-RAF) in the rat uterus. A parallel thinking that contributed to the design of experiments was the already available information that many transcription factors were moderately basic proteins and also that such proteins failed to bind to DEAE cellulose. It was this information

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

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

Sai Padma et al (2000)and Sai Padma & Thampan(2000) observed that there were three nuclear proteins that contributed to the regulated entry of ERα into the nuclei. (a) the p55 that recognized the NLS on ERα(b)a 28kDa protein,p28 that bound to the NLS signal on ERα and thereby prevented the p55-ERα interaction;(c)a 73 kDa protein,p73 that bound to the hormone binding domain(HBD)on ERα.Under hormone free conditions,p28 remained bound to the ERα NLS,blocking the NLS recognition by p55.Estradiol binding to the HBD and the consequent conformational change in the HBD brought the HBD-bound p73 in close interaction with p28.This resulted in the dissociation of p28 from the NLS which was subsequently occupied by p55.The interaction culminated in the nuclear entry of ERα,also

**4. Search for the "receptor-activator" protein and the discovery of E-RAF** 

There was a line of thinking that originated from Notides'(Notides & Nielson,1974)and Yamamoto's (Yamamoto,1974)laboratories that in estrogen action there was a possibility for the involvement of a DNA binding X-protein in converting the non-DNA binding estrogen receptor to a DNA binding form. Based on these observations and consideration of a potential possibility that a non-hormone binding transcription factor could be involved in the "activation" process, Thampan and Clark (1981,1983) presented the first ever experimental evidence for the existence of an estrogen receptor activation factor (E-RAF) in the rat uterus. A parallel thinking that contributed to the design of experiments was the already available information that many transcription factors were moderately basic proteins and also that such proteins failed to bind to DEAE cellulose. It was this information

sucrose gradients containing 0.3M KCl (Shyamala & Gorski, 1969).

group gave additional validity to the role of p55 in the nuclear entry of ERα.

**3. The role of estradiol in the nuclear entry of ERα**

mediated by the cytoskeletal elements, actin and tubulin(14).

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

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 manner.
