**1. Introduction**

140 Biochemistry

Yamamoto, S., T. Tsukamoto, A. Terai, H. Kurazono, Y. Takeda and O. Yoshida (1997).

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"Genetic evidence supporting the fecal-perinealurethral hypothesis in cystitis

ERs are members of the nuclear receptor superfamily and have a broad range of biological roles, such as growth, differentiation and physiology of the reproductive system (Pearce & Jordan, 2004). These enzymes also have roles in non-reproductive tissues such as bone, cardiovascular system, brain and liver (Heldring *et al.*, 2007). Until 1996, only one human estrogen receptor (ER) was known. That year Kuiper et al. discovered a novel nuclear estrogen receptor cloned from rat prostate. The known ER was renamed and called ERα to differentiate it from the novel ER, ERβ (Kuiper *et al.*, 1996). The complete human ERβ cDNA sequence was published in 1998 by Ogawa et al (Ogawa *et al.*, 1998a).

#### **1.1 Estrogen receptors and signalling function**

Estrogen receptors are products of distinct genes localized on different chromosomes; human ERα is encoded on chromosome 6q24-q27 (Gosden *et al.*, 1986), while the gene encoding human ERβ is localized on chromosome 14q22-q24 (Enmark *et al.*, 1997). Despite their distinct localization, the gene organization of the two receptors is well conserved. ESR1 (ERα) and ESR2 (ERβ) genes contain eight exons, separated by seven long intronic sequences. As members of the nuclear receptor superfamily, ERs contain 6 regions in their protein structure common for all nuclear receptors, namely: A, B, C, D, E and F which form functionally different but interacting domains (figure 1). Exon 1 encodes the A/B region in ERα and ERβ, exons 2 and 3 encode part of the C region. Exon 4 encodes the remaining part of region C, the whole of region D and part of region E. Exons 5 to 8 contain the rest of region E and region F is encoded by part of exon 8 [reviewed in (Ascenzi *et al.*, 2006)].

Although ERα and ERβ are encoded separately they share a high degree of homology. The most conserved domain among ERs is the DNA binding domain (DBD) corresponding to the C region, with 96% homology between α and β ER subtypes. The DBD is responsible for binding to specific DNA sequences (Estrogen Responsive Elements or EREs) in target gene promoter regions. High structure similarity in this region suggests similar target promoter sites for both receptors. The A/B region located in the N-terminus of the protein encompasses the AF-1 domain responsible for ligand independent transactivation. The AF-1 domain is the least conserved part among the two ERs with only 30% homology and it is functional only in the ERα subtype (Hall & McDonnell, 1999). The C-terminus of the protein contains the ligand dependent transactivation domain AF-2, the ligand binding domain (LBD) and the homo-/heterodimerization site. Homology between the E/F regions of both proteins is only 53%, explaining differences in ligand binding affinities between the two receptors. The hinge region localized in the D domain contains the nuclear localization signal of the ERs as well as post translational modification sites (Sentis *et al.*, 2005). Information on structure/function relationship of this region is very limited and it appears to be a variable and not well conserved part of the ERs (only 30% homology).

Fig. 1. Proteomic format, domain structure of human ERα (A) and ERβ (B). Based on Matthews and Gustafsson (Matthews & Gustafsson, 2003).

Estrogen (E2) binding to the receptor induces the LBD to undergo a conformational change, upon which the receptor dimerizes, binds to DNA, and stimulates gene expression (Cowley *et al.*, 1997; Katzenellenbogen & Katzenellenbogen, 2000).

#### **1.2 Estrogen receptor distribution**

The distribution of ERs varies both between and within human tissues (see Table 1). The cardiovascular system, brain, and bones express both receptors. ERβ is predominant in the male reproductive system. Expression of both ERα and ERβ has been found in all major human uterine cell types at every menstrual stage. However, expression varies from cell-type to cell-type with expression of ERα mRNA generally being higher than that of ERβ (Matsuzaki *et al.*, 1999). Changes in expression of estrogen receptors has been found in certain tumour types. Normal mammary tissue in man predominantly expresses ERβ mRNA, whereas most ER-positive breast tumours appear to exhibit increased ratios of ERα/ERβ (Leygue *et al.*, 1998).

Although ERα and ERβ are encoded separately they share a high degree of homology. The most conserved domain among ERs is the DNA binding domain (DBD) corresponding to the C region, with 96% homology between α and β ER subtypes. The DBD is responsible for binding to specific DNA sequences (Estrogen Responsive Elements or EREs) in target gene promoter regions. High structure similarity in this region suggests similar target promoter sites for both receptors. The A/B region located in the N-terminus of the protein encompasses the AF-1 domain responsible for ligand independent transactivation. The AF-1 domain is the least conserved part among the two ERs with only 30% homology and it is functional only in the ERα subtype (Hall & McDonnell, 1999). The C-terminus of the protein contains the ligand dependent transactivation domain AF-2, the ligand binding domain (LBD) and the homo-/heterodimerization site. Homology between the E/F regions of both proteins is only 53%, explaining differences in ligand binding affinities between the two receptors. The hinge region localized in the D domain contains the nuclear localization signal of the ERs as well as post translational modification sites (Sentis *et al.*, 2005). Information on structure/function relationship of this region is very limited and it appears

to be a variable and not well conserved part of the ERs (only 30% homology).

Fig. 1. Proteomic format, domain structure of human ERα (A) and ERβ (B). Based on

Estrogen (E2) binding to the receptor induces the LBD to undergo a conformational change, upon which the receptor dimerizes, binds to DNA, and stimulates gene expression (Cowley

The distribution of ERs varies both between and within human tissues (see Table 1). The cardiovascular system, brain, and bones express both receptors. ERβ is predominant in the male reproductive system. Expression of both ERα and ERβ has been found in all major human uterine cell types at every menstrual stage. However, expression varies from cell-type to cell-type with expression of ERα mRNA generally being higher than that of ERβ (Matsuzaki *et al.*, 1999). Changes in expression of estrogen receptors has been found in certain tumour types. Normal mammary tissue in man predominantly expresses ERβ mRNA, whereas most ER-positive breast tumours appear to exhibit increased ratios of ERα/ERβ (Leygue *et al.*, 1998).

Matthews and Gustafsson (Matthews & Gustafsson, 2003).

*et al.*, 1997; Katzenellenbogen & Katzenellenbogen, 2000).

**1.2 Estrogen receptor distribution** 

Likewise, an increased ratio of ERα/ERβ mRNA has been demonstrated in ovarian carcinoma compared with normal tissue or cysts (Bardin *et al.*, 2004). High concentrations of ERβ have also been found within the human gut (Enmark *et al.*, 1997).

Therefore, the ultimate estrogenic effect of a certain compound on cells or tissues will be dependent on the receptor phenotype of these cells or tissues.


Table 1. Tissue distribution of ER subtypes in humans.
