**6. Factors regulating nuclear entry of E-RAF**

Endoplasmic reticulum is the primary site of localization of intracellular E-RAF.A 55kDa anchor protein, ap55, that binds estradiol with high affinity retains E-RAF at the endoplasmic reticulum (Govind et al.,2003 a,b). Figure 1 displays the immunolocalisation of E-RAF in the endoplasmic reticulum of a goat uterine cell.

E-RAF remains anchored to ap55, through the mediation of a 66kDa nuclear transport protein, tp66. The tp66 recognizes the NLS in E-RAF.Within the E-RAF-tp66 complex, tp66 is anchored by ap55 in an estrogen dependent manner. Presence of saturating levels of estradiol maintains a specific conformation of ap55 that keeps tp66-E-RAF complex anchored to it. Lowering of estradiol concentration results in altered ap55 conformation that facilitates the release of tp66-E-RAF complex from ap55. The complex moves to the nucleus during which tp66 gets docked to a 38kDa nuclear pore-complex protein, npcp38. E-RAF enters the nucleus.

E-RAF is a high affinity progesterone and cholesterol binding protein (Thampan et al., 2000). Under both conditions E-RAF dissociates from the ap55-tp66 complex and migrates to the nucleus (possibly also to the mitochondria as cholesterol bound form). Premkumar et al(1999)presented information on the functional domains of E-RAF.Nuclear run on transcription studies were carried out in order to identify the genes influenced by E-RAF.For this, subtractive hybridization approach was attempted (Jacob,2006). Free E-RAF which can be transported to the nuclei by tp66, and progesterone bound E-RAF that gets transported to the nuclei on its own displayed totally distinct response patterns. It was a 55kDa nuclear pore complex protein (npcp55) that docked progesterone bound E-RAF at the pore complex. On the contrary, the free E-RAF-tp66 complex was docked to npcp-38. While free E-RAF was found to enhance the expression of splicing factor(s) genes, a major gene that was shown to be influenced by progesterone bound E-RAF was the collagenase(s) gene(s). The gene(s) if any, that are under the regulatory influence of cholesterol bound E-RAF remain to be known. Also the nuclear pore complex protein that docks cholesterol bound E-RAF is to be identified.

It appears that cholesterol is a natural regulator of E-RAF mediated gene expression (Thampan et al,2000). The presence of an inhibitor that prevented the dimerisation between E-RAF and naER in goat uterus was recognized early in E-RAF studies. The inhibition in the formation of E-RAF –naER heterodimer and the subsequent decline in the nuclear binding of the receptor was the assay target employed for the identification of this inhibitor. GC-MS analysis of the purified molecule showed its identity as unmetabolised cholesterol (Thampan et al,2000).

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

Endoplasmic reticulum is the primary site of localization of intracellular E-RAF.A 55kDa anchor protein, ap55, that binds estradiol with high affinity retains E-RAF at the endoplasmic reticulum (Govind et al.,2003 a,b). Figure 1 displays the immunolocalisation of

E-RAF remains anchored to ap55, through the mediation of a 66kDa nuclear transport protein, tp66. The tp66 recognizes the NLS in E-RAF.Within the E-RAF-tp66 complex, tp66 is anchored by ap55 in an estrogen dependent manner. Presence of saturating levels of estradiol maintains a specific conformation of ap55 that keeps tp66-E-RAF complex anchored to it. Lowering of estradiol concentration results in altered ap55 conformation that facilitates the release of tp66-E-RAF complex from ap55. The complex moves to the nucleus during which tp66 gets docked to a 38kDa nuclear pore-complex protein, npcp38. E-RAF

E-RAF is a high affinity progesterone and cholesterol binding protein (Thampan et al., 2000). Under both conditions E-RAF dissociates from the ap55-tp66 complex and migrates to the nucleus (possibly also to the mitochondria as cholesterol bound form). Premkumar et al(1999)presented information on the functional domains of E-RAF.Nuclear run on transcription studies were carried out in order to identify the genes influenced by E-RAF.For this, subtractive hybridization approach was attempted (Jacob,2006). Free E-RAF which can be transported to the nuclei by tp66, and progesterone bound E-RAF that gets transported to the nuclei on its own displayed totally distinct response patterns. It was a 55kDa nuclear pore complex protein (npcp55) that docked progesterone bound E-RAF at the pore complex. On the contrary, the free E-RAF-tp66 complex was docked to npcp-38. While free E-RAF was found to enhance the expression of splicing factor(s) genes, a major gene that was shown to be influenced by progesterone bound E-RAF was the collagenase(s) gene(s). The gene(s) if any, that are under the regulatory influence of cholesterol bound E-RAF remain to be known. Also the nuclear pore complex protein that docks cholesterol bound E-RAF is to

It appears that cholesterol is a natural regulator of E-RAF mediated gene expression (Thampan et al,2000). The presence of an inhibitor that prevented the dimerisation between E-RAF and naER in goat uterus was recognized early in E-RAF studies. The inhibition in the formation of E-RAF –naER heterodimer and the subsequent decline in the nuclear binding of the receptor was the assay target employed for the identification of this inhibitor. GC-MS analysis of the purified molecule showed its identity as unmetabolised cholesterol

Thampan (2000).

enters the nucleus.

be identified.

(Thampan et al,2000).

**6. Factors regulating nuclear entry of E-RAF** 

E-RAF in the endoplasmic reticulum of a goat uterine cell.

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

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

(C)The merged figures created by confocal microscopy clearly showed that endoplasmic reticulum is the primary site of localization of E-RAF.

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 microscope.
