**9. Multiple intracellular sites for E-RAF action**

Recent observation regarding the positive presence of E-RAF in goat uterine mitochondria is indicative of a possible functional role for E-RAF in the mitochondria. Confocal microscopic studies conducted on goat endometrial cells in culture with exposure to varying concentrations of estradiol or progesterone showed that while 3-5nM concentrations of estradiol helped in the nuclear entry of E-RAF, progesterone mediated nuclear entry was

biopsies of mammary tissue. Figure 4 presents the results of a recent study carried out in this direction where the tissue sections were exposed first to anti E-RAF IgG and subsequently to FITC labeled secondary antibody. There is a dominating presence of E-RAF

A cryostat section of human breast cancer biopsy was fixed, permeabilised and exposed overnight to rabbit anti goat E-RAF IgG at 40C.Further exposure of the section to goat anti rabbit IgG labeled with FITC was conducted in the dark for 1 hr following which the nuclei were stained with propidium iodide. The sections were examined using a Leica fluorescence microscope. The green fluorescence indicates the cytoplasmic presence of E-RAF.The nuclei are stained red with propidium iodide. However the presence of E-RAF in the nuclei is marked by the transition of the red colour to light

If what I discussed in the previous paragraph regarding the brain derived regulator of E-RAF gene expression is proven correct, it is possible to suggest that defects in this regulatory protein mechanism and its action could lead to uncontrolled expression of the E-RAF gene. How does this enhanced expression of E-RAF gene influence mammary cancer progression remains to be seen. One of the major molecular targets of progesterone bound E-RAF is the collagenase gene. Whether the progesterone bound E-RAF mediated enhancement in collagenase gene expression has any role in mammary cancer metastasis is yet to be examined.

Recent observation regarding the positive presence of E-RAF in goat uterine mitochondria is indicative of a possible functional role for E-RAF in the mitochondria. Confocal microscopic studies conducted on goat endometrial cells in culture with exposure to varying concentrations of estradiol or progesterone showed that while 3-5nM concentrations of estradiol helped in the nuclear entry of E-RAF, progesterone mediated nuclear entry was

Fig. 4. **Detection of E-RAF in human breast cancer biopsy.** 

**9. Multiple intracellular sites for E-RAF action** 

orange and even yellow.

in the cytoplasm and also in the nuclei (primarily stained with propidium iodide).

found to be effective only in the presence of 15-16nM progesterone. The corresponding effects produced in the presence of different concentrations of cholesterol remains to be seen. The postulate that the enhancement in E-RAF titer in the pregnant uterus is an indirect reflection of the progesterone production in the uterus during the first half of pregnancy takes into account the possibility that mitochondrial steroidogenesis in the uterine cell is under E-RAF control. As mentioned earlier, E-RAF may function as a cholesterol transporter to the mitochondrial steroidogenic site, eventually facilitating the conversion of cholesterol to pregnenolone and progesterone. The nuclear genes influenced by cholesterol-bound E-RAF could well be those the products of expression of which are constituents of the mitochondrial steroidogenic complex like cytochrome P450(Fig.5)

(1) Nucleus (2) Genes (3) Endoplasmic reticulum (4) Mitochondria (5) (5A) and (5B) Nuclear pore complex proteins that bind free E-RAF, progesterone-bound E-RAF and cholesterol bound E-RAF respectively. (6) ap55 (6A) Estradiol-bound ap55 (7) tp66 (7A) tp66-E-RAF complex (8) Free E-RAF (9) Progesterone bound E-RAF (10) Cholesterol bound E-RAF.

## Fig. 5. **Mode of action of E-RAF in the target cell.**

The primary site of intracellular location of E-RAF is the endoplasmic reticulum where it remains anchored to the anchor protein 55(ap55) through the mediation of tp66 (transport protein 66) in an estrogen dependent manner.tp66 transports E-RAF to the nucleus, after dissociation from ap55, when the intracellular level of estrogen declines. When bound by cholesterol or progesterone, E-RAF dissociates from tp66 and moves independently to the nucleus. The nuclear entry of E-RAF again is regulated by nuclear pore complex proteins (npcp). Apparently it is the conformation of E-RAF that determines the identity of npcp with which it should interact. There is a distinct possibility that within the nucleus free E-RAF, progesterone bound E-RAF and cholesterol bound E-RAF recognize

Evolving Trends in Estrogen Receptor Biology 75

The studies reported from our laboratory (Sreeja & Thampan, 2004 b) have indicated that the internalization of naER from the plasma, following estradiol binding to the receptor was a clathrin-coated vesicle (CCV)-mediated mechanism. A 55 kDa protein of the CCV, apparently carrying the internalization signal (Trowbridge at al, 1993) is the target protein for naER in CCV. The internalized naER interacts with a 58kDa nuclear transport protein, the actin binding p58, that recognizes the nuclear localization signal (NLS) on the receptor. Prior to recognizing p58, the site involved on the naER is bound by Hsp-90.Estradiol binding to naER promotes dissociation of Hsp-90 from the receptor (Anilkumar at al., 2010). Confocal microscopic studies presented in this study showed that in goat endometrial cells in culture exposure of the cells to estradiol resulted in the intracellular movement of both naER and Hsp-90. It was observed that both naER and Hsp-90 entered the nuclei within a matter of 3 hours following the exposure of the cells to estradiol. The functional significance of Hsp-90 in the nuclei remains to be known.naER is transformed into nuclear estrogen receptor II (nERII) within the nucleus. It is evident that this change in identity is associated with a distinct structural change in the protein. Possibly, this transformation that takes place

Long before naER discovery became a reality, a nuclear receptor that was distinctly different from the classical estrogen receptors had come to my notice. It was observed that when uterine nuclei from ovarectomized rats were exposed to 10nM 3H-estradiol,at 30-370C, the hormone-binding component moved out of the nuclei and reached the outer medium within a span of 5 minutes after hormonal exposure (Thampan,1985;1988). What became apparent in the subsequent studies was that the hormone was bound to a class of ribonucleoproteins (RNP) that moved out of the nuclei following exposure to estradiol. Invivo studies involving ovariectomized rats demonstrated that the RNP that moved out of the nuclei was found associated with cytoplasmic polysomes. The results gave a clear indication to the possibility that a new class of estrogen receptors existed whose primary functional role was in posttranscriptional control mechanisms like splicing, nucleocytoplasmic transport of RNP and

The subsequent studies reported by our group(Jacob et al.,2006) presented systematic observations on both naER and nERII and concluded that the latter was a transformed form of the former. The observed differences between the two proteins are being listed below(Table 1). The methods employed for purifying the two proteins were identical. Going back to the observations related to E-RAF function (Thampan,1989), it may be recalled that the estrogen receptor function detected in close proximity to rat uterine nuclear RNA polymerases displayed both naER and nERII characteristics with nERII representing the major share of this activity. The naER existed in dimerisation with E-RAF.Later reports by Karthikeyan and Thampan (1996) showed that nERII tyrosine phosphorylated three subunits of nuclear RNA polymerase II.A re-examination of the 1989 report(Thampan,1989) will reveal that the naER/nERII interaction was not restricted to RNA polymerase II alone. There was very clear evidence to support the hypothesis that the receptor interacted with all four classes of nuclear RNA polymerases. Therefore, it may be speculated that nERII –mediated tyrosine

phosphorylation involved specified subunits of all 4 categories of the enzyme.

**11. Protein protein interactions during naER internalization following** 

**estradiol binding** 

the translation.

within the nucleus is chaperoned by Hsp-90.

**12. Nuclear estrogen receptor II (nERII)** 

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

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.
