**10. Identification of the estrogen receptor that dimerizes with E-RAF**

The experimental observations on the goat uterine E-RAF signals a clear indication that a special class of estrogen receptor dimerised with E-RAF within the nucleus. A DNA cellulose binding assay was developed in which the non-DNA binding estrogen receptor was labeled with 3H-estradiol and the binding of the hormone-receptor complex to DNA cellulose in the presence of E-RAF was quantitated. Anuradha et al (1994) reported on the isolation and characterization of a 66kDa, high affinity estrogen binding protein from the goat uterus. The receptor displayed the same affinity to bind estradiol as that of the estrogen receptor α (ER α). In view of its inability to bind to DNA on its own, this new estrogen receptor was designated as non-activated estrogen receptor (naER).

A method was developed for the isolation of the non-DNA binding estrogen receptor that dimerized with E-RAF.The method involved preparation of goat uterine cytosol, collection of the DNA-Sepharose unadsorbed fraction, successive ion exchange chromatography over DEAE cellulose and phosphocellulose and finally Hsp 90 Sepharose chromatography in the presence of sodium molybdate, achieving final elution with zero molybdate buffer (Anuradha et al.,1994). While showing its distinctiveness over ERα, as a non DNA binding protein, the naER further demonstrated its function as a glycoprotein and a tyrosine kinase (Karthikeyan & Thampan,1996). The tyrosine kinase property was sensitive to the presence of estradiol: the enzyme activity was totally inhibited in the presence of the hormone at concentrations which saturated its binding sites. The observation was a clear indication to the possibility that the naER tyrosine kinase activity can become functional only after naER undergoes a critical structural change within the cell.

Direct biochemical analysis showed that plasma membrane is the primary site of localization of naER (Karthikeyan & Thampan,1996). The possibility of plasma membrane being a site of intracellular localization of estrogen receptor was first proposed by Pietras and Szego (1975,1977) several years ago. Sreeja and Thampan (2004 a,b) demonstrated that naER dissociated from the plasma membrane following exposure to estradiol. This was shown to be an estrogen-specific phenomenon since non-estrogenic steroids failed to bring about the dissociation while the non steroidal estrogen, diethylstilbestrol was as effective as estradiol-17β in inducing naER dissociation from the plasma membrane. What was unique in this observation was that the dissociation of naER appeared to be an energy dependent process. The involvement of a Ca++/Mg++dependent ATPase in the process was evident. Enhancement of the ATPase activity was dependent on exposure of the membrane to estradiol and the activity was inhibited by the flavanoid, quercetin(Sreeja & Thampan,2004 a).

and regulate the expression of specific sets of genes, possibly influenced by specific

Mitochondria appears to be the other target of cholesterol bound E-RAF.The possibility exists that E-RAF functions as a cholesterol transporter to mitochondria, favoring the conversion of cholesterol to pregnenolone and progesterone.This higher titer of E-RAF should be reflected in higher production of uterine progesterone that could eventually contribute to the maintenance of the pregnant uterus during the first half of pregnancy.

The experimental observations on the goat uterine E-RAF signals a clear indication that a special class of estrogen receptor dimerised with E-RAF within the nucleus. A DNA cellulose binding assay was developed in which the non-DNA binding estrogen receptor was labeled with 3H-estradiol and the binding of the hormone-receptor complex to DNA cellulose in the presence of E-RAF was quantitated. Anuradha et al (1994) reported on the isolation and characterization of a 66kDa, high affinity estrogen binding protein from the goat uterus. The receptor displayed the same affinity to bind estradiol as that of the estrogen receptor α (ER α). In view of its inability to bind to DNA on its own, this new estrogen

A method was developed for the isolation of the non-DNA binding estrogen receptor that dimerized with E-RAF.The method involved preparation of goat uterine cytosol, collection of the DNA-Sepharose unadsorbed fraction, successive ion exchange chromatography over DEAE cellulose and phosphocellulose and finally Hsp 90 Sepharose chromatography in the presence of sodium molybdate, achieving final elution with zero molybdate buffer (Anuradha et al.,1994). While showing its distinctiveness over ERα, as a non DNA binding protein, the naER further demonstrated its function as a glycoprotein and a tyrosine kinase (Karthikeyan & Thampan,1996). The tyrosine kinase property was sensitive to the presence of estradiol: the enzyme activity was totally inhibited in the presence of the hormone at concentrations which saturated its binding sites. The observation was a clear indication to the possibility that the naER tyrosine kinase activity can become functional only after naER undergoes a critical structural change within the

Direct biochemical analysis showed that plasma membrane is the primary site of localization of naER (Karthikeyan & Thampan,1996). The possibility of plasma membrane being a site of intracellular localization of estrogen receptor was first proposed by Pietras and Szego (1975,1977) several years ago. Sreeja and Thampan (2004 a,b) demonstrated that naER dissociated from the plasma membrane following exposure to estradiol. This was shown to be an estrogen-specific phenomenon since non-estrogenic steroids failed to bring about the dissociation while the non steroidal estrogen, diethylstilbestrol was as effective as estradiol-17β in inducing naER dissociation from the plasma membrane. What was unique in this observation was that the dissociation of naER appeared to be an energy dependent process. The involvement of a Ca++/Mg++dependent ATPase in the process was evident. Enhancement of the ATPase activity was dependent on exposure of the membrane to estradiol and the activity was inhibited by the flavanoid, quercetin(Sreeja &

**10. Identification of the estrogen receptor that dimerizes with E-RAF** 

receptor was designated as non-activated estrogen receptor (naER).

acceptor proteins.

cell.

Thampan,2004 a).
