DEFINITION
Estrogen receptor refers to a group of receptors that are activated by the hormone 17β-estradiol (estrogen). The main function of the estrogen receptor is as a DNA-binding transcription factor that regulates gene expression. However, the estrogen receptor has additional functions independent of DNA binding.
ER GENE ORGANIZATION AND EXPRESSION PATTERN
There are two forms of estrogen receptors:
Estrogen receptor alpha or ESR1
Estrogen Receptor beta or ESR2
ERalpha and ERbeta ( NR3A1 and NR3A2, respectively) are the products of separate genes (ESR1 and ESR2, respectively) present on distinct chromosomes (locus 6q25.1 and locus 14q23-24.1, respectively) (Localization of the human oestrogen receptor gene to chromosome 6q24----q27 by in situ hybridization, 1986; The chicken oestrogen receptor sequence: homology with v-erbA and the human oestrogen and glucocorticoid receptors, 1986; Human estrogen receptor beta-gene structure, chromosomal localization, and expression pattern, 1997; Estrogen-related receptor alpha 1 actively antagonizes estrogen receptor-regulated transcription in MCF-7 mammary cells, 2002 ).
ESR1 and ESR2 comprise eight exons separated by seven intronic regions and spans more than 140 kilobases and approximately 40 kilobases, respectively. ERalpha is a protein of 595 aa, and its molecular weight is 68 kDa. ERbeta is a protein of 530 aa and its molecular weight is about 55 kDa.
ERalpha and ERbeta can be detected in a broad spectrum of tissues. In some organs, both receptor subtypes are expressed at similar levels, whereas in others, one or the other subtype predominates. In addition, both receptor subtypes may be present in the same tissue but in different cell types. ERalpha is mainly expressed in, for example, uterus, prostate (stroma), ovary (theca cells), testes(Leydig cells), epididymis, bone, breast, various regions of the brain, liver, and white adipose tissue. ERbeta is expressed in, for example, colon, prostate (epithelium), testis, ovary (granulosa cells), bone marrow, salivary gland, vascular endothelium, and certain regions of the brain.
A number of promoters have been described for ERbeta; some show tissue specific activation (Tissue-specific expression of human ERalpha and ERbeta in the male, 2001). For ERbeta, two promoters, ON and OK, have thus far been characterized (Cloning and characterization of human estrogen receptor beta promoter, 2000). Methylation of the ERbeta ON promoter has been shown to be inversely correlated with mRNA expression (Expression of estrogen receptor beta isoforms in normal breast epithelial cells and breast cancer: regulation by methylation, 2003). It is possible that additional, not yet identified promoters also regulate the expression of ERs. Moreover, various transcripts can be found in different tissues because of alternative splicing and different transcription start sites.
CHEMICAL STRUCTURES AND IMAGES
Human ERalpha and ERbeta, like all the members of the NR super-family, are modular proteins sharing common regions, named A/B, C, D, E, and F. These regions participate in the formation of independent but interacting functional domains: the N-terminal transactivation domain ( AF-1 ), the DNA binding domain ( DBD ), the dimerization domain(s), the nuclear localization sequence ( NLS ), and the Ligand binding domain ( LBD ) (ER beta: identification and characterization of a novel human estrogen receptor, 1996; The structure of the nuclear hormone receptors, 1999; Mechanisms of estrogen action, 2001; DNA recognition by nuclear receptors,2004; Potential Role of a Novel Transcriptional Coactivator PELP1 in Histone H1 Displacement in Cancer Cells, 2004).
The ER LBD is composed of 12 alpha helices that form the characteristic three-layer antiparallel alpha-helical sandwich with a small four-stranded beta sheet. Agonists bind to an internal cavity of the receptor that stabilizes the overall conformation of the LBD and induces a conformation of helix 12 that promotes coactivator binding (Figure below).
ERalpha and ERbeta share significant homology in their overall amino acid sequences and in DNA binding and ligand binding domains, but their AF-1 domains differ in both length and amino acid sequence, exhibiting a low amino acid identity(Mechanisms of estrogen action, 2001; Potential Role of a Novel Transcriptional Coactivator PELP1 in Histone H1 Displacement in Cancer Cells, 2004). This may indicate either that the A/B region has been added to the receptor genes after duplication of the ancestral gene or that the A/B region has diverged considerably during evolution (Evolution of vertebrate steroid receptors from an ancestral estrogen receptor by ligand exploitation and serial genome expansions, 2001). ERalpha and ERbeta have overlapping but nonidentical tissue distribution and expression levels, suggesting functionally distinct biological roles. In addition, the two receptor subtypes have opposite effects on the activation of estrogen responsive promoters containing an AP1 site, with ERalpha activating transcription and ERbeta attenuating transcription.
Phylogenetic analysis show that several steroid receptor sub-families are present in early metazoans. Thus, it appears that the super-family underwent an ‘explosive expansion’ during early metazoan evolution (Figure below). ERalpha and ERbeta arose by a series of gene duplications from an ancestral NR, containing both DBD and ligand-binding domain (LBD) . Interestingly, many steroidogenic and steroid-inactivating enzymes arose at the same time. Paradoxically, the gross difference in sequence divergence rates suggests that the ancestral steroid receptor was a functional ER and not a progesterone receptor; the sequence of the ancestral steroid receptor was conserved among descendant ERs. Indeed, the progesterone receptor should be the most ancient receptor because progesterone is the first active Delta4 steroid, and progesterone is a precursor for other steroids, including 17-beta-estradiol ( Recent insights into the origins of adrenal and sex steroid receptors, 2002 ). Thus, upstream steroids, such as Delta4 progesterone, cortisol , and testosterone were inactive towards ER because a receptor with a ligand-binding domain for these steroids had not yet evolved. The presence of these upstream ‘orphan’ ligands was a selective force for the evolution in duplicated receptors of a new steroid binding activity. After one to three duplications of the ER gene, followed by considerable sequence divergence, receptors emerged that gave these intermediate compounds novel signaling functions. Redundant receptors created by gene duplication could then diverge in sequence from their ancestors and evolve affinity for these steroids, creating signaling functions for what were once intermediates. This process is called ligand exploitation , because it involves the cooption of existing metabolites to serve as novel hormones by duplicated receptors. Ligand exploitation can occur as ancestral receptors, regulating cellular processes through direct transcriptional activation via signal transduction pathways in the cytosol or the membrane, as extant steroid receptors, or as both ancestral and extant ( Evolution of vertebrate steroid receptors from an ancestral estrogen receptor by ligand exploitation and serial genome expansions, 2001 , Evolution of hormone-receptor complexity by molecular exploitation, 2006 , Protein Evolution by Molecular Tinkering: Diversification of the Nuclear Receptor Superfamily from a Ligand-Dependent Ancestor, 2010 , Resurrecting the Ancestral Steroid Receptor: Ancient Origin of Estrogen Signaling, 2003 )
POST-TRANSLATIONAL MODIFICATIONS INVOLVED IN NON-GENOMIC SIGNALING AND IN REGULATION OF ESTROGEN RECEPTOR FUNCTIONS
A significant number of post-translational modifications of ERs have also been described, including phosphorylation, acetylation, SUMOylation, and ubiquitination, which affect receptor activity and/or stability. In particular, here we will talk about the role of Cysteine S-palmitoylation in non-genomic signaling.
Palmitoylation exerts diverse effects on the protein structure and function . Indeed, the attachment of this long chain fatty acid (palmitic acid) increases protein hydrophobicity and thereby facilitates membrane association and protein targeting to membrane microdomains (e.g., lipid rafts and caveolae) that are enriched in cholesterol and saturated fatty acid chains, allowing the lipid molecules to pack tightly together and form a “liquid ordered” phase. This enhances protein/protein and protein/lipid interactions which appear relevant for efficient signal transduction. Palmitoylation also influences intracellular protein trafficking and protein activity. Protein palmitoylation is thought to be an enzymatic reaction, mediated by palmitoyltransferase (PAT).
Generally, there is no consensus sequence for palmitoylation, but the common denominator for most palmitoylated proteins is a membrane targeting sequence encompassing the target Cys, which consists of positively charged residues and lipid anchors or transmembrane domains (Membrane association of estrogen receptor alpha and beta influences 17beta-estradiol-mediated cancer cell proliferation, 2008 ).
On the basis of multiple alignment analysis by ClustalW, the amino acid sequence encompassing the solvent-exposed Cys447 and Cys399 residues present in the LBD of ER alpha and ER beta, respectively, are the unique which follow the requirement, because of the presence of an hydrophobic patch (Leu zipper-like) and an hydrophilic positively charged Lys residue (K) are present close to the S-palmitoyable Cys ( C ) (Figure above) (Does palmitoylation target estrogen receptors to plasma membrane caveolae?, 2003).
Intriguingly, GRs and PR display an amino acid sequence highly homologous to that encompassing the S-palmitoylated ERalpha Cys447 and ERbeta Cys399 residues, suggesting that GRs and PR may undergo S-palmitoylation (Steroid hormone rapid signaling: the pivotal role of S-palmitoylation, 2006; A conserved mechanism for steroid receptor translocation to the plasma membrane, 2007). This chemical modification could account for membrane-starting rapid signals activated upon corticosteroids or progesterone binding (Figure below).
By contrast, ERRs and MR display a high amino acid sequence homology, but the S-palmitoylable Cys residue is replaced by Leu or Thr or Ile. This is in agreement with the exclusive nuclear localization of orphan ERRs devoid of rapid action(s).
ER, GR, and PR sequences were further confirmed to be palmitoylable by analysis with CSS-Palm . Although SHRs may contain putative sites displaying a higher chance for palmitoylation (higher score) when analyzed by CSS-Palm, they were discarded accounting for protein conformation, disulphide bond formation, Cys accessibility, as well as Cys-metal coordination (Steroid hormone rapid signaling: the pivotal role of S-palmitoylation, 2006).
Cycles of palmitoylation and de-palmitoylation affect protein activation allowing their movement(s) within membrane subdomains. Ligand (e.g., E2) binding modulates ERalpha and ERbeta palmitoylation. E2 binding to ERalpha and ERbeta decreases the ER palmitoylation rate (t1/2 ∼30 min) and induces structural modification(s) impairing PAT recognition (i.e., ER palmitoylation).
CELLULAR FUNCTIONS OF ESTROGEN RECEPTOR ALPHA AND BETA PALMITOYLATION
Current evidence indicates that the small population of ERalpha and ERbeta localized at the plasma membrane exists within caveolar rafts, interacting with specific membrane proteins (Membrane association of estrogen receptor alpha and beta influences 17beta-estradiol-mediated cancer cell proliferation, 2008 ). ERalpha can further associate with specialized proteins including the modulator of the non-genomic activity of estrogen receptor (MNAR ), Shc , EGFR and IGF-1R , and striatin . Shc and striatin have been reported to interact with the A/B domain of ERalpha , while MNAR acts as an adapter coupling ER to "Src": http://en.wikipedia.org/wiki/Src_%28gene%29 .
Palmitoylation enables ERalpha to reside at the plasma membrane and to interact with caveolin-1. Upon E2 binding, ERalpha undergoes slow de-palmitoylation and dissociates from caveolin-1, facilitating ERalpha movement to other membrane micro-domains. Thus, ERalpha could be re-located by docking to other partner proteins (i.e., Shc/IGF-1 receptor and Src/p85).
ERK/MAPK and PI3K/AKT pathways, rapidly activated by ERalpha–E2 complex, also have a critical role in E2 action as a survival agent. In fact, these pathways enhance the expression of the anti-apoptotic protein Bcl-2 , block the activation of the p38/MAPK , reduce the pro-apoptotic caspase-3 activation, and promote G1-to-S phase transition via the enhancement of the cyclin D1 expression (Distinct nongenomic signal transduction pathways controlled by 17beta-estradiol regulate DNA synthesis and cyclin D(1) gene transcription in HepG2 cells, 2002 ; Plasma membrane estrogen receptors signal to antiapoptosis in breast cancer, 2000 ; Nongenotropic, sex-nonspecific signaling through the estrogen or androgen receptors: dissociation from transcriptional activity, 2001 ; Survival versus apoptotic 17beta-estradiol effect: role of ER alpha and ER beta activated non-genomic signaling, 2005 ).
On the other hand, E2 induces ERbeta de-palmitoylation and increases ERbeta level and its association with caveolin-1 . As a whole, these data raise the intriguing possibility that the short A/B domain of ERbeta could facilitate the E2-induced association between ERbeta and caveolin-1, impairing its association with MNAR and Src. However, E2 increases the association of ERbeta to a cytosolic kinase, p38 kinase, a member of the mitogen-activated protein kinase (MAPK) family, inducing p38 activation .
Estrogen receptor alpha and beta non-genomic effects in physiology
The physiological significance of rapid membrane-starting pathways has been clarified, at least for some E2 targets:
- nervous system, E2 affects neural functions (e.g., cognition, behavior, stress responses, and reproduction) in part by inducing such rapid responses (Steroid hormone interactions with target cells: cross talk between membrane and nuclear pathways, 2003 ).
- skeleton, ERalpha, present in caveolae of bone-forming osteoblasts, transmits survival signals through activation of the Src/Shc/ERK pathway and prolongs the life span of osteoblasts (Kinase-mediated regulation of common transcription factors accounts for the bone-protective effects of sex steroids, 2003 ). At the same time, E2 delivers a pro-apoptotic signal to bone-resorbing osteoclasts, shortening their life span (Reversal of bone loss in mice by nongenotropic signaling of sex steroids, 2002 ; Sex steroids and bone, 2002 ).
- Liver, rapid E2-induced signals (i.e., PLC/PKC ) are deeply linked to the expression of the LDL receptor and to a decreased level of LDL-cholesterol (Activation of IP-protein kinase C-alpha signal transduction pathway precedes the changes of plasma cholesterol, hepatic lipid metabolism and induction of low-density lipoprotein receptor expression in 17-beta-oestradiol-treated rats, 2001 ; Role of tyrosine kinase signaling in estrogen-induced LDL receptor gene expression in HepG2 cells, 2002 ).
Estrogen receptor alpha and beta non-genomic effects in cancer
It is now accepted that the ER-dependent mechanisms underlying cancer effects are mainly the membrane-starting rapid effects.
- Breast cancer ERalpha are known promoters of breast cancer development, perhaps in conjunction with progesterone and its receptor.
E2 treatment of mammary derived ERalpha-positive-MCF-7 cells triggers association of ERalpha with Src and p85, the regulatory subunit of PI3K leading to DNA synthesis (PI3-kinase in concert with Src promotes the S-phase entry of oestradiol-stimulated MCF-7 cells, 2001 ). ERbeta appears to act as a dominant regulator of E2 signalling, and when coexpressed with ERalpha, it causes a concentration-dependent reduction in ERalpha-mediated transcriptional activation (Estrogen signaling: a subtle balance between ER alpha and ER beta, 2003 ). When ERbeta is introduced into these cells, E2-induced proliferation is inhibited (Estrogen receptor beta inhibits 17beta-estradiol-stimulated proliferation of the breast cancer cell line T47D, 2004 ). Ductal cells in the mammary gland appear to be one example of cells where ERalpha and ERbeta oppose each other on proliferation and the proliferative response to E2 is determined by the ERalpha/ERbeta ratio ( Loss of ERbeta expression as a common step in estrogen-dependent tumor progression, 2004 ). Another important evidence is that, in some breast cancers, ERbeta transcript is regulate by promoter methylation (Quantitative analysis of estrogen receptor-alpha and -beta messenger RNA expression in breast carcinoma by real-time polymerase chain reaction, 2000 ; Stable transfection of an estrogen receptor beta cDNA isoform into MDA-MB-231 breast cancer cells, 2003 ).Moreover, ER alpha is involved in the response to DNA damage (Figure above) (Plasma membrane estrogen receptors, 2009 ). In fact, E2 and membrane ERalpha were recently shown to block ATR and ATM signaling and the activation of downstream kinases and phosphatases. This occurs when E2 signals through membrane ERalpha and PI3K/AKT to phosphorylate the TopBP1 protein, a protein necessary for ATR activity. Phosphorylation at the single AKT site of TopBP1 prevents its enhanced interaction with ATR. E2/Era also causes additional chromosomal breaks in the setting of DNA damage induced by gamma radiation, probably resulting from the steroid receptor blocking the ATR signaling cascade prompted by DNA damage.
- Epatocellular carcinoma In HepG2 cells multiple and parallel membrane starting pathways are rapidly activated by the ERalpha–E2 complex (Biphasic estradiol-induced AKT phosphorylation is modulated by PTEN via MAP kinase in HepG2 cells, 2003 ). The blockade of PLC/PKC, MAPK/ERK, and PI3K/AKT pathways completely prevents the E2-induced DNA synthesis. Cyclin D1 expression, a well-defined target for E2 action, which has a pivotal role in the progression of E2-induced proliferation, has been reported to occur in these cells independently of the transcriptional activity of ERalpha . The disruption of rapid-membrane starting ERK/MAPK and PI3K/AKT kinase pathway completely prevents both E2 induction of cyclin D1 expression at AP-1 and Sp1 sites sustaining that a synergy does exist also between E2 indirect genomic and rapid mechanisms.
- Colon cancer ERbeta is the predominant ER form in both normal and malignant human colon tissue, as well as several human colon cancer-derived cell lines (Estrogen receptor beta mRNA in colon cancer cells: growth effects of estrogen and genistein, 2000 ; Nitric oxide impairs the 17beta-estradiol-induced apoptosis in human colon adenocarcinoma cells, 2006 ). ERbeta levels are reportedly lower in colon tumors compared with normal colon tissue, and loss of ERbeta is associated with advanced stages of colon cancer and tumor cell de-differentiation, thus suggesting a protective role for ERbeta in colon tumorigenesis (Oestrogen receptor beta (ERbeta) is abundantly expressed in normal colonic mucosa, but declines in colon adenocarcinoma paralleling the tumour's dedifferentiation, 2003 ;
Bardin et al., 2004; Loss of expression of oestrogen receptor beta in colon cancer and its association with Dukes' staging, 2005 ). Clinical studies indicate that the incidence of colon cancer is lower in women than in men (Cancer statistics, 2004, 2004 ), and data from the Women’s Health Initiative indicate a significantly reduced incidence of colon cancer in postmenopausal women receiving combined ‘Hormone Replacement Therapy’ (HRT; estrogen plus progestin) (Risks and benefits of estrogen plus progestin in healthy postmenopausal women: principal results From the Women's Health Initiative randomized controlled trial, 2002 ), this being consistent with a protective role of ERbeta.
