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

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 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

Evolving Trends in Estrogen Receptor Biology 69

(A)The cells were fixed, permeabilised and exposed to rabbit anti-goat E-RAF IgG, followed by Cy3

(C)The merged figures created by confocal microscopy clearly showed that endoplasmic reticulum is

Fig. 1. **Intracellular association of E-RAF with endoplasmic reticulum in goat endometrial cells in culture.** A primary culture of goat endometrial cells was exposed to estradiol-free medium for 48 hours following which the cells were examined under a Leica confocal

Premkumar and Thampan (1995) examined the level of E-RAF in the uteri of pregnant rats during a full term of pregnancy. It was noticed that from day 1 of pregnancy the E-RAF titer in the uterus registered a steady increase. It reached an all time peak towards midpregnancy following which E-RAF level began to decline. The rate of decline was found to be very fast; two days before parturition the uterine E-RAF titer became virtually undetectable. It is known that progesterone is essential for maintaining the functional integrity of the pregnant uterus. The possibility, therefore, exists that the E-RAF titer is a reflection of the progesterone requirement of the pregnant uterus. The decline in E-RAF titer

(B)The cells were also stained with DiOC6 (3) to highlight the endoplasmic reticulum.

**7. E-RAF in pregnant rat uterus: significance of the findings** 

labeled anti rabbit IgG.

microscope.

the primary site of localization of E-RAF.

was the non activated estrogen receptor (naER),a glycoprotein and a tyrosine kinase sensitive to the presence of estradiol and primarily localized at the plasma membrane(Karthikeyan & Thampan,1994). The naER was the only estrogen receptor that could dimerise with E-RAF.The 3S peak on the other hand, represented the nuclear estrogen receptor II (nERII), a tyrosine kinase insensitive to the presence of estradiol. The nuclear estrogen receptor II failed to dimerise with E-RAF, the obvious reason being the changes induced in naER conformation during its transformation to nERII (Karthikeyan & Thampan,1995;Thampan et al.,1996). The naER to nERII transformation was accomplished by a 61kDa nuclear naER-transforming factor (naER-TF), originally reported by Jaya and Thampan (2000).
