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Thyroid Hormone Receptors
Steroid/Thyroid Hormone Receptors R D Ward and N L Weigel, Baylor College of Medicine, Houston, TX, USA ã 2013 Elsevier Inc. All rights reserved. This article is a revision of the previous edition article by Ramesh Narayanan and Nancy L. Weigel, volume 4, pp. 111–116, ã 2004, Elsevier Inc.
Glossary Agonists Natural or synthetic ligands that bind to the hormone-binding domain of the receptor and stimulate activity. Antagonists Ligands that compete with the agonists for binding to the hormone-binding domain, but do not cause activation of the receptor. Nuclear receptors Ligand-activated transcription factors characterized by conserved zinc-finger motifs in their DNA-binding domains and smaller conserved regions in the hormone-binding domain.
Overview of Nuclear Receptor Ligands and Mechanism of Action Figure 1 shows the structures of some of the ligands for members of the nuclear receptor family. Estradiol is the primary ligand for both estrogen receptors, estrogen receptor a (ERa) and ERb. Testosterone, which is closely related to estradiol, is one of the two major ligands for the androgen receptor (AR). The ligands for the thyroid hormone receptor (T3 and T4) and the vitamin D receptor (1,25(OH)2D3) are also shown. The five major classes of steroids are synthesized from pregnenolone, which is derived from cholesterol through the actions of the cholesterol side-chain cleavage enzyme (P450scc). The synthesis of the hormones is complex, with a number of alternate pathways leading to the same hormone. Testosterone and estradiol are derived from 17a-hydroxy pregnenolone. Testosterone, the major circulating androgen, is synthesized in the testes of males and in the ovaries of females. It is important for development of the male reproductive tract, fertility, and secondary male characteristics. Estradiol, the major circulating estrogen, is produced in the ovaries of females. Estradiol is important in the female reproductive tract, playing roles in breast and uterine development, fertility, and also in bone and other tissues. Progesterone is synthesized directly from pregnenolone, and the major site of synthesis in females is the ovary. Progesterone, acting through the progesterone receptor (PR), plays important roles in the breast and uterus and in the maintenance of pregnancy. Progesterone is a precursor of corticosterone and aldosterone, which are synthesized in the adrenal glands. The mineralocorticoid, aldosterone, is important for salt retention in the kidney. The glucocorticoid, cortisol, is produced in the adrenals from 17a-hydroxy pregnenolone and is important for regulation of carbohydrate metabolism; it also plays a role in suppressing immune responses. The secosteroid, 1,25(OH) 2D3, is derived from cholesterol through a ultraviolet-catalyzed reaction in the epidermis followed by sequential hydroxylations in the liver and kidney. The action of 1,25(OH)2D3 is
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Phosphorylation A posttranslational modification of an amino acid in which a phosphate group is added by a kinase. Sumoylation A posttranslational modification of an amino acid in which a SUMO protein is added via an enzymatic cascade to modify the function of the protein. Ubiquitination A posttranslational modification of an amino acid in which one or more ubiquitin monomers are added by the E3 enzyme. Ubiquitin frequently acts as a signal targeting the protein for degradation by proteosomes.
important for calcium homeostasis and also plays a role in the differentiation of a variety of tissues. Thyroid hormones are produced from tyrosines and iodide in the thyroid gland. Thyroid hormones have multiple actions in regulating metabolism, typically increasing oxidation rates. The hormones are transported through the blood to their sites of action. Figure 2 depicts the general mechanism of action of nuclear receptors. The ligands are all lipophilic compounds, which enter the cells by passive diffusion. They bind to their cognate intracellular receptors located either in the cytoplasm or the nucleus. The receptors can be divided into two classes – those that do not bind DNA in the absence of hormone (Figure 2(a)) and those that can bind to DNA in the absence of hormone (Figure 2(b)). The regulation of their activities differs somewhat. Classical steroid receptors such as AR, glucocorticoid receptor (GR), PR, mineralocorticoid receptor (MR), and ER belong to the first class; they are maintained in an inactive conformation capable of ligand binding by a complex of chaperone proteins including hsp90 and p23. Whether these complexes are nuclear or cytoplasmic depends upon the receptor. Upon ligand binding during the genomic pathway, the receptor changes its conformation, no longer binding to heat-shock proteins, homodimerizes and, if the unliganded receptor is localized in the cytoplasm, translocates to the nucleus. There, the receptor binds to DNA containing specific sequences called hormone response elements (HREs) that are typically found in the 50 flanking region of target genes. However, some HREs are more than 10 kb upstream and others are found in the introns of target genes. The receptor recruits components of the basal transcription machinery, as well as proteins termed coactivators that perform a variety of functions including histone acetylation that enhance transcription. In addition, these receptors can mediate a rapid, nongenomic activation of signaling pathways as discussed later. Unlike the receptors for classical steroids, thyroid receptors (TRs) are not bound to heat-shock proteins in the absence of hormone and, instead, are bound to the DNA as a heterodimer with the retinoid X receptor (RXR),
Signaling | Steroid/Thyroid Hormone Receptors
Testosterone OH
OH
Estradiol
309
OH
O T4
T3
NH2
NH2 OH
CH2CH
O
OH
O
CH2CH COOH
COOH 1,25-dihydroxyvitamin D3 OH
OH
OH
Figure 1 Structure of steroid and thyroid hormones. Estradiol is the ligand for estrogen receptor, testosterone is the ligand for androgen receptor, T3-3,5,3-L-triiodothyronine, T4-thyroxine are the ligands for thyroid receptor, and 1,25-dihydroxyvitamin D3 is the ligand for the vitamin D receptor.
another member of the steroid/thyroid hormone receptor superfamily (Figure 2(b)). In the absence of ligand, TR binds corepressors, which, in turn, bind histone deacetylases resulting in lower levels of histone acetylation and repression of target gene transcription. Hormone binding releases corepressors and promotes binding of coactivators, resulting in increased transcription. Agonist-bound receptors also are known to repress transcription of some target genes although mechanisms for repression are less well defined.
The Steroid/Thyroid Hormone Receptor Superfamily The steroid/thyroid receptors are the largest family of ligandactivated transcription factors. In addition to the wellcharacterized steroid, thyroid, retinoid, and vitamin D receptors, the family contains receptors for numerous lipophilic metabolites and xenobiotics. A number of receptor family members are referred to as orphan nuclear receptors. These receptors were not initially identified on the basis that known hormones were regulating their physiological actions through a ligand-binding process, but rather through their sequence similarities to the known receptors. Investigators searched for additional steroid receptors using the conserved regions determined to be common to nuclear receptors in DNA-screening approaches and grouped them based on phylogeny. Although some orphan receptors were later found to be regulated by a ligand binding, for instance, peroxisome proliferator–activator receptor can respond to fatty acid derivatives and certain prostaglandins in addition to an array of synthetic ligands, some orphan receptors such as estrogen-related receptor and TR2 (testes receptor) still have no known ligand. Thus, these orphan receptors may either work independent of ligand or the ligand (s) responsible for their regulation have yet to be discovered.
Receptor Structure Despite some evolutionary and functional differences, the steroid receptor family members have many similarities especially in their structure. As shown in Figure 3(a), there are multiple domains in the receptors, with all receptors containing domains A–E and only a subset containing the additional F domain.
The N terminus The N terminus or the A/B domain of the receptor is the least conserved domain among the family members. This region is the most variable in length ranging from a few amino acids to more than 500. This region has an activation function, AF-1, which contributes to the transcriptional activity of the receptor through binding of coactivators. The position of this region within the N terminus differs among the receptors. AF-1 has been defined functionally by deletion analyses, but to date no common structural motifs have been identified in this region.
The DNA-binding domain The DNA-binding domain (DBD), region C, is important for the binding of receptor to the DNA and is the most highly conserved domain. This region has two type-2 zinc-finger motifs, which are responsible for DNA recognition and dimerization. Each finger is composed of four cysteines that coordinate with one zinc atom. Amino acids in this region also participate in receptor dimerization.
The hinge region Downstream of the DBD is the hinge region (D), which contains a nuclear localization signal. This is a short lysine-rich region, with a high homology to the simian virus 40 T-antigen nuclear localization signal. Additional functions of this region are receptor specific.
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Signaling | Steroid/Thyroid Hormone Receptors
GF
GFR
‘Non-genomic’
Shc Grb Sos Ras
Src SR SR
MAPK
SR hsp90
‘Nuclear’ Steroid hormone
SR hsp90 p23 TPR
CoA
mRNA
CoA CoA SR SR GTF HRE
(a)
Thyroid hormone CoR TR HRE
CoR
mRNA
CoA CoA RXR TR
GTF
HRE
(b)
Figure 2 Mechanism of steroid (a) and thyroid (b) hormone action. The two classes are distinguished, in part, by whether they are associated with heat-shock proteins (a) like classical steroid receptors or are bound to DNA in the absence of hormone (b) like thyroid hormone receptor. Classical receptors (a) can bind hormone, dimerize, and translocate to the nucleus in the ‘nuclear’ pathway. In some cases, they also mediate a rapid activation of signaling pathways in the ‘nongenomic’ pathway. In both the classical, genomic pathway (a) and the thyroid receptor pathway (b), binding of the agonist causes dissociation of proteins that repress activity and promotes a conformation that induces recruitment of coactivators stimulating transcription of the target gene. SR, steroid receptor; HRE, hormone response element; GTF, general transcription factor; RXR, retinoid X receptor; TR, thyroid receptor; CoA, coactivator; CoR, corepressor; Hsp90, heat shock protein 90; TPR, tetratricopeptide; Src, Src tyrosine kinase; GF, growth factor; GFR, growth factor receptor.
The ligand-binding domain The ligand-binding domain (E) is essential for the binding of ligand. The primary interaction site for the hsp complex is also in this domain. Also located in this region is the second activation function domain, AF-2, which is responsible for ligandmediated transcription of target genes. The relative importance of AF-2 and AF-1 in inducing transcription is receptor- and celltype specific. The structures of the hormone-binding domains
of several receptors have been determined using X-ray diffraction. The hormone-binding domain consists of a series of 12 a-helices. Binding of hormone causes a substantial conformational change in the receptor exposing AF-2 for interactions with coactivators. This domain also contains the strongest dimerization interface in most steroid receptors. The function of the F domain, located at the C terminus of some receptors such as the ER is not well defined.
Signaling | Steroid/Thyroid Hormone Receptors
AF-2
AF-1
1
N
A/B
C
D
E/F
777
C GR
(a)
(b)
5⬘ AGAACAnnnTGTTCT 3⬘ 3⬘ TCTTGTnnnACAAGA 5⬘
GRE
5⬘ AGGTCAnnnTGACCT 3⬘ 3⬘ TCCAGTnnnACTGGA 5⬘
ERE
5⬘ AGGTCAnxAGGTCA 3⬘ 3⬘ TCCAGTnxTCCAGT 5⬘
VDRE, TRE
Figure 3 Receptor structure and DNA binding elements. (a) Shows the common structural features of nuclear receptors using GR as an example. The A/B region contains the AF-1, a region important for transcriptional activation. C is the DNA-binding domain, the most conserved region in the nuclear receptors. D contains a nuclear localization sequence. E contains the hormone-binding domain and second activation function AF-2. Some receptors also contain a C-terminal extension, termed the F domain, whose physiological function is not well described. (b) Shows sequences of consensus hormone response elements. The consensus sequence for a GRE (binds GR, AR, PR) and an ERE (binds ER) are shown. Vitamin D receptor and the thyroid hormone receptors bind to direct repeats separated by three and four nucleotides, respectively. Other receptors bind to direct or inverted repeats with a spacing of 0–6 (nx indicates that the half-site may be separated by 0–6 nucleotides). GRE, glucocorticoid response element; ERE, estrogen response element; VDRE, vitamin D response element; TRE, thyroid response element; GR, glucocorticoid receptor; AF, activation function.
Receptor Binding to DNA All of the classical receptors bind to their cognate HREs as dimers. The consensus binding sequence for AR, PR, GR, and MR, shown in Figure 3(b), contains two half-sites separated by three nucleotides with the sites oriented to form a palindrome. ER recognizes a related pair of half-sites with the same spacing and orientation. Each monomer binds to a half-site. The class-II receptors, including vitamin D receptor (VDR) and TR, bind to pairs of half-sites whose sequences are identical to the ER halfsite, but whose orientation (direct or inverted repeats) and spacing (0–6 nucleotides) determine the specificity of binding. TR and VDR each heterodimerize with RXR and bind to the 30 end (half of the HRE), whereas RXR binds to the 50 end (half of the response element). Although these sequences represent the consensus binding sites, natural sequences may differ significantly and promoters may contain combinations of HREs as well as individual half-sites all of which contribute to the final activity.
Steroid Receptor Coregulators When the receptor binds to the DNA, it recruits a series of protein complexes that facilitate recruitment of proteins required for basal transcription of the receptors. Receptors bind proteins or protein complexes that modulate receptor activity through alterations in chromatin structure; these are termed coactivators and corepressors. Coactivators are defined as proteins that interact with the receptors and increase their ability to transactivate the target gene. The mechanism by which
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individual coactivators achieve this can vary. More than 100 candidate coactivators have been identified. Although some coactivators function only with steroid receptors and a small subset of other transcription factors, others are used by many transcription factors. The best characterized of the steroid receptor coactivators (SRCs) is the p160 family of coactivators: SRC1, SRC-2 (GRIP1, TIF2), and SRC-3 (Rac3, AIB1). These bind to the receptor recruiting additional coactivators, including cAMP response element binding (CREB)-binding protein (CBP), CBP-associated factor (p/CAF), and coactivator-associated arginine methyltransferase (CARM-1). CBP/p300, p/CAF and some of the p160 proteins are histone acetyl transferases and their binding increases local histone acetylation. Other coactivators include the DRIP/TRAP (D receptor interacting protein/thyroid hormone receptor-associated protein) complex. Many coactivators interact with AF-2 located in the ligand-binding domain (LBD). In other cases, coactivators interact with the AF-1 region and some interact with both domains. Interactions with AF2 are typically mediated by LXXLL (L ¼ leucine and X ¼ any amino acid) motifs in the coactivator. Another class of proteins, termed corepressor, reduces the activation of target gene transcription through interaction with the receptors. The best-characterized nuclear receptor corepressors are nuclear receptor corepressor (NcoR) and silencing mediator of retinoid and thyroid receptors (SMRTs). These proteins bind histone deacetylase complexes and also interact with class II receptors in the absence of hormone resulting in local reductions in histone acetylation. Corepressors do not bind to unliganded steroid receptors. However, steroid receptor antagonists cause changes in the conformation of the hormone-binding domain that induce binding of the corepressors.
Steroid Receptor Agonists and Antagonists Although steroid receptor family members are important for normal physiological processes, there are a number of instances in which it is desirable to block the actions of selected steroid receptors. These include breast cancer (ER) and prostate cancer (AR). Thus, although natural antagonists of steroid receptor action have not been identified, much effort has been devoted to identifying compounds that will antagonize hormone action. These compounds compete with the natural ligand for binding to the hormone-binding domain of the receptors. Although some antagonists block dissociation from heat-shock protein complexes or destabilize the receptor, most of the antagonists promote dissociation from heat-shock proteins and cause the receptors to bind to DNA. However, the conformation induced by the antagonist differs from that induced by agonist. This prevents recruitment of coactivators to AF-2 and, instead, promotes recruitment of corepressors. In some cases, it is desirable to maintain the activity of a receptor in some tissues while inhibiting activity in other tissues. The most common example of this is the need for tissue-specific regulation of ER activity. Estradiol is important for maintaining bone mass and postmenopausal women frequently develop osteoporosis. However, estradiol can promote uterine cancer and may also be detrimental in breast. Thus, a great deal of effort has been devoted to developing selective estrogen receptor modulators (SERMs) which have tissue-specific agonistic and antagonistic activities.
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Tamoxifen (a SERM) has been used in the treatment of breast cancer, but increases the risk of uterine cancer. A newer SERM, raloxifene, is an antagonist in both breast and uterus, but acts as an agonist in bone.
mRNA ER
ER
ERE
Cross-Talk between Nuclear Receptors and Cell Signaling Pathways
(a)
Phosphorylation and other Posttranslational Modifications of Receptors and Coactivators The nuclear receptors and their coactivators are phosphoproteins; in some cases, enhanced cell signaling is sufficient to induce the transcriptional activity of the receptor in the absence of normal levels of ligand. The ability to be activated by cellsignaling pathways alone is receptor specific, although changes in cell signaling modulate the activity of all of the receptors. Estrogen receptors are activated both by growth factor pathways and by activation of protein kinase A. Other receptors such as GR, require hormone for activity. Recent studies show that there are many mechanisms to regulate receptor activity through posttranslational modification. Examples of posttranslational modification include phosphorylation, sumoylation, and ubiquitination. Phosphorylation of steroid receptors on Ser, Thr, and Tyr residues can occur in the absence of ligand or can be induced by ligand binding and/or kinase signaling to regulate various functions, including protein stability, hormone sensitivity, DNA binding, subcellular localization, and protein interactions. Lysine residues can be modified by ubiquitination, sumoylation, or acetylation and can regulate protein turnover, subcellular localization, DNA-binding, transcriptional potential, or affect chromatin structure that controls the availability of promoter regions to regulatory proteins. Posttranslational modifications of coactivators are also important for regulation of receptor-specific functions.
Functional Interactions between Nuclear Receptors and Signal-Regulated Transcription Factors In addition to altering transcription through direct binding to DNA, nuclear receptors alter transcription through interactions with other transcription factors. In some cases, these protein– protein interactions enhance transcription, while in others they prevent binding of the transcription factor to its DNA target site in a process referred to as transrepression. For instance, GR can interact with additional transcription factors in order to prevent these proteins from binding to and activating their respective targets. This type of process has been shown to be important for the antiinflammatory actions of GR. In other cases, the receptor binds to the factors on their DNA target sites influencing (either þ or ) the transcription of a target gene. GR also can inhibit the transcription of certain genes by direct binding to negative GREs in the promoter regions of these genes. Figure 4 shows the comparison between two of these pathways for ER. In the upper panel is the classical DNA-binding-dependent induction of transcription. We next depict the ability of ER to induce transcription through interactions with AP-1 complexes. In this instance, both estradiol as well as SERMs will stimulate the activity of AP-1.
mRNA
ER
Fos
Jun
AP-1 RE (b)
Figure 4 Mechanisms of transcription activation by nuclear receptors. (a) Shows the classical pathway for transcriptional activation with a steroid receptor dimer binding directly to a hormone response element. (b) Shows an alternative mode of activation. In this case, a receptor such as the estrogen receptor binds to another transcription factor in a process referred to as tethering and influences transcription through its interactions with the transcription factor and the recruitment of coregulator proteins to the complex. ER, estrogen receptor; AP-1 RE, AP-1 response element; ERE, estrogen response element.
Nuclear Receptor Stimulation of Cell-Signaling Pathways Both of the pathways above can be considered genomic pathways in that the nuclear receptor acts by directly altering transcription. Nuclear receptors can also act through stimulating kinase activity, although the final downstream target may be a change in transcription. These actions are rapid (minutes) and are termed nongenomic (Figure 2). This process is thought to occur close to the cell membrane and involves the crosstalk of steroid receptors with various adaptor proteins, G proteins, and receptor tyrosine kinases. There is evidence that activation of nuclear receptors can lead to downstream activation of mitogen-activated protein kinase. In some cases, this is through activation of Src kinase and in others through generation of a ligand for a growth factor receptor. There are numerous other examples of these rapid actions. Induction by estradiol of nitric oxide synthase activity in endothelial cells is a rapid response that does not require transcription. Thus, steroid/thyroid hormones alter cellular activities through multiple mechanisms.
See also: Signaling: Mitogen-Activated Protein Kinase Family; Thyroid-Stimulating Hormone/Luteinizing Hormone/FollicleStimulating Hormone Receptors; Vitamin D Receptor.
Further Reading Faus H and Haendler B (2006) Post-translational modifications of steroid receptors. Biomedicine and Pharmacotherapy 60: 520–528.
Signaling | Steroid/Thyroid Hormone Receptors
Hammes SR and Levin ER (2007) Extranuclear steroid receptors: Nature and actions. Endocrine Reviews 28: 726–741. McDonnell DP, Connor CE, Wijayaratne A, Chang CY, and Norris JD (2002) Definition of the molecular and cellular mechanisms underlying the tissue-selective agonist/antagonist activities of selective estrogen receptor modulators. Recent Progress in Hormone Research 57: 295–316. Tsai MJ and O’Malley BW (1994) Molecular mechanisms of action of steroid/thyroid receptor superfamily members. Annual Review of Biochemistry 63: 451–486. Weigel NL and Moore NL (2007) Kinases and protein phosphorylation as regulators of steroid hormone action. Nuclear Receptor Signalling 5: e005.
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Whitfield GK, Jurutka PW, Haussler CA, and Haussler MR (1999) Steroid hormone receptors: Evolution, ligands, and molecular basis of biologic function. Journal of Cellular Biochemistry – Supplement 32–33: 110–122. Wolf IM, Heitzer MD, Grubisha M, and DeFranco DB (2008) Coactivators and nuclear receptor transactivation. Journal of Cellular Biochemistry 104: 1580–1586.
Relevant Websites http://www.nursa.org – The Nuclear Receptor Signaling Atlas.
Glossary Agonists Natural or synthetic ligands that bind to the hormone-binding domain of the receptor and stimulate activity. Antagonists Ligands that compete with the agonists for binding to the hormone-binding domain, but do not cause activation of the receptor. Nuclear receptors Ligand-activated transcription factors characterized by conserved zinc-finger motifs in their DNA-binding domains and smaller conserved regions in the hormone-binding domain.
Overview of Nuclear Receptor Ligands and Mechanism of Action Figure 1 shows the structures of some of the ligands for members of the nuclear receptor family. Estradiol is the primary ligand for both estrogen receptors, estrogen receptor a (ERa) and ERb. Testosterone, which is closely related to estradiol, is one of the two major ligands for the androgen receptor (AR). The ligands for the thyroid hormone receptor (T3 and T4) and the vitamin D receptor (1,25(OH)2D3) are also shown. The five major classes of steroids are synthesized from pregnenolone, which is derived from cholesterol through the actions of the cholesterol side-chain cleavage enzyme (P450scc). The synthesis of the hormones is complex, with a number of alternate pathways leading to the same hormone. Testosterone and estradiol are derived from 17a-hydroxy pregnenolone. Testosterone, the major circulating androgen, is synthesized in the testes of males and in the ovaries of females. It is important for development of the male reproductive tract, fertility, and secondary male characteristics. Estradiol, the major circulating estrogen, is produced in the ovaries of females. Estradiol is important in the female reproductive tract, playing roles in breast and uterine development, fertility, and also in bone and other tissues. Progesterone is synthesized directly from pregnenolone, and the major site of synthesis in females is the ovary. Progesterone, acting through the progesterone receptor (PR), plays important roles in the breast and uterus and in the maintenance of pregnancy. Progesterone is a precursor of corticosterone and aldosterone, which are synthesized in the adrenal glands. The mineralocorticoid, aldosterone, is important for salt retention in the kidney. The glucocorticoid, cortisol, is produced in the adrenals from 17a-hydroxy pregnenolone and is important for regulation of carbohydrate metabolism; it also plays a role in suppressing immune responses. The secosteroid, 1,25(OH) 2D3, is derived from cholesterol through a ultraviolet-catalyzed reaction in the epidermis followed by sequential hydroxylations in the liver and kidney. The action of 1,25(OH)2D3 is
308
Phosphorylation A posttranslational modification of an amino acid in which a phosphate group is added by a kinase. Sumoylation A posttranslational modification of an amino acid in which a SUMO protein is added via an enzymatic cascade to modify the function of the protein. Ubiquitination A posttranslational modification of an amino acid in which one or more ubiquitin monomers are added by the E3 enzyme. Ubiquitin frequently acts as a signal targeting the protein for degradation by proteosomes.
important for calcium homeostasis and also plays a role in the differentiation of a variety of tissues. Thyroid hormones are produced from tyrosines and iodide in the thyroid gland. Thyroid hormones have multiple actions in regulating metabolism, typically increasing oxidation rates. The hormones are transported through the blood to their sites of action. Figure 2 depicts the general mechanism of action of nuclear receptors. The ligands are all lipophilic compounds, which enter the cells by passive diffusion. They bind to their cognate intracellular receptors located either in the cytoplasm or the nucleus. The receptors can be divided into two classes – those that do not bind DNA in the absence of hormone (Figure 2(a)) and those that can bind to DNA in the absence of hormone (Figure 2(b)). The regulation of their activities differs somewhat. Classical steroid receptors such as AR, glucocorticoid receptor (GR), PR, mineralocorticoid receptor (MR), and ER belong to the first class; they are maintained in an inactive conformation capable of ligand binding by a complex of chaperone proteins including hsp90 and p23. Whether these complexes are nuclear or cytoplasmic depends upon the receptor. Upon ligand binding during the genomic pathway, the receptor changes its conformation, no longer binding to heat-shock proteins, homodimerizes and, if the unliganded receptor is localized in the cytoplasm, translocates to the nucleus. There, the receptor binds to DNA containing specific sequences called hormone response elements (HREs) that are typically found in the 50 flanking region of target genes. However, some HREs are more than 10 kb upstream and others are found in the introns of target genes. The receptor recruits components of the basal transcription machinery, as well as proteins termed coactivators that perform a variety of functions including histone acetylation that enhance transcription. In addition, these receptors can mediate a rapid, nongenomic activation of signaling pathways as discussed later. Unlike the receptors for classical steroids, thyroid receptors (TRs) are not bound to heat-shock proteins in the absence of hormone and, instead, are bound to the DNA as a heterodimer with the retinoid X receptor (RXR),
Signaling | Steroid/Thyroid Hormone Receptors
Testosterone OH
OH
Estradiol
309
OH
O T4
T3
NH2
NH2 OH
CH2CH
O
OH
O
CH2CH COOH
COOH 1,25-dihydroxyvitamin D3 OH
OH
OH
Figure 1 Structure of steroid and thyroid hormones. Estradiol is the ligand for estrogen receptor, testosterone is the ligand for androgen receptor, T3-3,5,3-L-triiodothyronine, T4-thyroxine are the ligands for thyroid receptor, and 1,25-dihydroxyvitamin D3 is the ligand for the vitamin D receptor.
another member of the steroid/thyroid hormone receptor superfamily (Figure 2(b)). In the absence of ligand, TR binds corepressors, which, in turn, bind histone deacetylases resulting in lower levels of histone acetylation and repression of target gene transcription. Hormone binding releases corepressors and promotes binding of coactivators, resulting in increased transcription. Agonist-bound receptors also are known to repress transcription of some target genes although mechanisms for repression are less well defined.
The Steroid/Thyroid Hormone Receptor Superfamily The steroid/thyroid receptors are the largest family of ligandactivated transcription factors. In addition to the wellcharacterized steroid, thyroid, retinoid, and vitamin D receptors, the family contains receptors for numerous lipophilic metabolites and xenobiotics. A number of receptor family members are referred to as orphan nuclear receptors. These receptors were not initially identified on the basis that known hormones were regulating their physiological actions through a ligand-binding process, but rather through their sequence similarities to the known receptors. Investigators searched for additional steroid receptors using the conserved regions determined to be common to nuclear receptors in DNA-screening approaches and grouped them based on phylogeny. Although some orphan receptors were later found to be regulated by a ligand binding, for instance, peroxisome proliferator–activator receptor can respond to fatty acid derivatives and certain prostaglandins in addition to an array of synthetic ligands, some orphan receptors such as estrogen-related receptor and TR2 (testes receptor) still have no known ligand. Thus, these orphan receptors may either work independent of ligand or the ligand (s) responsible for their regulation have yet to be discovered.
Receptor Structure Despite some evolutionary and functional differences, the steroid receptor family members have many similarities especially in their structure. As shown in Figure 3(a), there are multiple domains in the receptors, with all receptors containing domains A–E and only a subset containing the additional F domain.
The N terminus The N terminus or the A/B domain of the receptor is the least conserved domain among the family members. This region is the most variable in length ranging from a few amino acids to more than 500. This region has an activation function, AF-1, which contributes to the transcriptional activity of the receptor through binding of coactivators. The position of this region within the N terminus differs among the receptors. AF-1 has been defined functionally by deletion analyses, but to date no common structural motifs have been identified in this region.
The DNA-binding domain The DNA-binding domain (DBD), region C, is important for the binding of receptor to the DNA and is the most highly conserved domain. This region has two type-2 zinc-finger motifs, which are responsible for DNA recognition and dimerization. Each finger is composed of four cysteines that coordinate with one zinc atom. Amino acids in this region also participate in receptor dimerization.
The hinge region Downstream of the DBD is the hinge region (D), which contains a nuclear localization signal. This is a short lysine-rich region, with a high homology to the simian virus 40 T-antigen nuclear localization signal. Additional functions of this region are receptor specific.
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Signaling | Steroid/Thyroid Hormone Receptors
GF
GFR
‘Non-genomic’
Shc Grb Sos Ras
Src SR SR
MAPK
SR hsp90
‘Nuclear’ Steroid hormone
SR hsp90 p23 TPR
CoA
mRNA
CoA CoA SR SR GTF HRE
(a)
Thyroid hormone CoR TR HRE
CoR
mRNA
CoA CoA RXR TR
GTF
HRE
(b)
Figure 2 Mechanism of steroid (a) and thyroid (b) hormone action. The two classes are distinguished, in part, by whether they are associated with heat-shock proteins (a) like classical steroid receptors or are bound to DNA in the absence of hormone (b) like thyroid hormone receptor. Classical receptors (a) can bind hormone, dimerize, and translocate to the nucleus in the ‘nuclear’ pathway. In some cases, they also mediate a rapid activation of signaling pathways in the ‘nongenomic’ pathway. In both the classical, genomic pathway (a) and the thyroid receptor pathway (b), binding of the agonist causes dissociation of proteins that repress activity and promotes a conformation that induces recruitment of coactivators stimulating transcription of the target gene. SR, steroid receptor; HRE, hormone response element; GTF, general transcription factor; RXR, retinoid X receptor; TR, thyroid receptor; CoA, coactivator; CoR, corepressor; Hsp90, heat shock protein 90; TPR, tetratricopeptide; Src, Src tyrosine kinase; GF, growth factor; GFR, growth factor receptor.
The ligand-binding domain The ligand-binding domain (E) is essential for the binding of ligand. The primary interaction site for the hsp complex is also in this domain. Also located in this region is the second activation function domain, AF-2, which is responsible for ligandmediated transcription of target genes. The relative importance of AF-2 and AF-1 in inducing transcription is receptor- and celltype specific. The structures of the hormone-binding domains
of several receptors have been determined using X-ray diffraction. The hormone-binding domain consists of a series of 12 a-helices. Binding of hormone causes a substantial conformational change in the receptor exposing AF-2 for interactions with coactivators. This domain also contains the strongest dimerization interface in most steroid receptors. The function of the F domain, located at the C terminus of some receptors such as the ER is not well defined.
Signaling | Steroid/Thyroid Hormone Receptors
AF-2
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N
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C
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E/F
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(a)
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5⬘ AGAACAnnnTGTTCT 3⬘ 3⬘ TCTTGTnnnACAAGA 5⬘
GRE
5⬘ AGGTCAnnnTGACCT 3⬘ 3⬘ TCCAGTnnnACTGGA 5⬘
ERE
5⬘ AGGTCAnxAGGTCA 3⬘ 3⬘ TCCAGTnxTCCAGT 5⬘
VDRE, TRE
Figure 3 Receptor structure and DNA binding elements. (a) Shows the common structural features of nuclear receptors using GR as an example. The A/B region contains the AF-1, a region important for transcriptional activation. C is the DNA-binding domain, the most conserved region in the nuclear receptors. D contains a nuclear localization sequence. E contains the hormone-binding domain and second activation function AF-2. Some receptors also contain a C-terminal extension, termed the F domain, whose physiological function is not well described. (b) Shows sequences of consensus hormone response elements. The consensus sequence for a GRE (binds GR, AR, PR) and an ERE (binds ER) are shown. Vitamin D receptor and the thyroid hormone receptors bind to direct repeats separated by three and four nucleotides, respectively. Other receptors bind to direct or inverted repeats with a spacing of 0–6 (nx indicates that the half-site may be separated by 0–6 nucleotides). GRE, glucocorticoid response element; ERE, estrogen response element; VDRE, vitamin D response element; TRE, thyroid response element; GR, glucocorticoid receptor; AF, activation function.
Receptor Binding to DNA All of the classical receptors bind to their cognate HREs as dimers. The consensus binding sequence for AR, PR, GR, and MR, shown in Figure 3(b), contains two half-sites separated by three nucleotides with the sites oriented to form a palindrome. ER recognizes a related pair of half-sites with the same spacing and orientation. Each monomer binds to a half-site. The class-II receptors, including vitamin D receptor (VDR) and TR, bind to pairs of half-sites whose sequences are identical to the ER halfsite, but whose orientation (direct or inverted repeats) and spacing (0–6 nucleotides) determine the specificity of binding. TR and VDR each heterodimerize with RXR and bind to the 30 end (half of the HRE), whereas RXR binds to the 50 end (half of the response element). Although these sequences represent the consensus binding sites, natural sequences may differ significantly and promoters may contain combinations of HREs as well as individual half-sites all of which contribute to the final activity.
Steroid Receptor Coregulators When the receptor binds to the DNA, it recruits a series of protein complexes that facilitate recruitment of proteins required for basal transcription of the receptors. Receptors bind proteins or protein complexes that modulate receptor activity through alterations in chromatin structure; these are termed coactivators and corepressors. Coactivators are defined as proteins that interact with the receptors and increase their ability to transactivate the target gene. The mechanism by which
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individual coactivators achieve this can vary. More than 100 candidate coactivators have been identified. Although some coactivators function only with steroid receptors and a small subset of other transcription factors, others are used by many transcription factors. The best characterized of the steroid receptor coactivators (SRCs) is the p160 family of coactivators: SRC1, SRC-2 (GRIP1, TIF2), and SRC-3 (Rac3, AIB1). These bind to the receptor recruiting additional coactivators, including cAMP response element binding (CREB)-binding protein (CBP), CBP-associated factor (p/CAF), and coactivator-associated arginine methyltransferase (CARM-1). CBP/p300, p/CAF and some of the p160 proteins are histone acetyl transferases and their binding increases local histone acetylation. Other coactivators include the DRIP/TRAP (D receptor interacting protein/thyroid hormone receptor-associated protein) complex. Many coactivators interact with AF-2 located in the ligand-binding domain (LBD). In other cases, coactivators interact with the AF-1 region and some interact with both domains. Interactions with AF2 are typically mediated by LXXLL (L ¼ leucine and X ¼ any amino acid) motifs in the coactivator. Another class of proteins, termed corepressor, reduces the activation of target gene transcription through interaction with the receptors. The best-characterized nuclear receptor corepressors are nuclear receptor corepressor (NcoR) and silencing mediator of retinoid and thyroid receptors (SMRTs). These proteins bind histone deacetylase complexes and also interact with class II receptors in the absence of hormone resulting in local reductions in histone acetylation. Corepressors do not bind to unliganded steroid receptors. However, steroid receptor antagonists cause changes in the conformation of the hormone-binding domain that induce binding of the corepressors.
Steroid Receptor Agonists and Antagonists Although steroid receptor family members are important for normal physiological processes, there are a number of instances in which it is desirable to block the actions of selected steroid receptors. These include breast cancer (ER) and prostate cancer (AR). Thus, although natural antagonists of steroid receptor action have not been identified, much effort has been devoted to identifying compounds that will antagonize hormone action. These compounds compete with the natural ligand for binding to the hormone-binding domain of the receptors. Although some antagonists block dissociation from heat-shock protein complexes or destabilize the receptor, most of the antagonists promote dissociation from heat-shock proteins and cause the receptors to bind to DNA. However, the conformation induced by the antagonist differs from that induced by agonist. This prevents recruitment of coactivators to AF-2 and, instead, promotes recruitment of corepressors. In some cases, it is desirable to maintain the activity of a receptor in some tissues while inhibiting activity in other tissues. The most common example of this is the need for tissue-specific regulation of ER activity. Estradiol is important for maintaining bone mass and postmenopausal women frequently develop osteoporosis. However, estradiol can promote uterine cancer and may also be detrimental in breast. Thus, a great deal of effort has been devoted to developing selective estrogen receptor modulators (SERMs) which have tissue-specific agonistic and antagonistic activities.
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Signaling | Steroid/Thyroid Hormone Receptors
Tamoxifen (a SERM) has been used in the treatment of breast cancer, but increases the risk of uterine cancer. A newer SERM, raloxifene, is an antagonist in both breast and uterus, but acts as an agonist in bone.
mRNA ER
ER
ERE
Cross-Talk between Nuclear Receptors and Cell Signaling Pathways
(a)
Phosphorylation and other Posttranslational Modifications of Receptors and Coactivators The nuclear receptors and their coactivators are phosphoproteins; in some cases, enhanced cell signaling is sufficient to induce the transcriptional activity of the receptor in the absence of normal levels of ligand. The ability to be activated by cellsignaling pathways alone is receptor specific, although changes in cell signaling modulate the activity of all of the receptors. Estrogen receptors are activated both by growth factor pathways and by activation of protein kinase A. Other receptors such as GR, require hormone for activity. Recent studies show that there are many mechanisms to regulate receptor activity through posttranslational modification. Examples of posttranslational modification include phosphorylation, sumoylation, and ubiquitination. Phosphorylation of steroid receptors on Ser, Thr, and Tyr residues can occur in the absence of ligand or can be induced by ligand binding and/or kinase signaling to regulate various functions, including protein stability, hormone sensitivity, DNA binding, subcellular localization, and protein interactions. Lysine residues can be modified by ubiquitination, sumoylation, or acetylation and can regulate protein turnover, subcellular localization, DNA-binding, transcriptional potential, or affect chromatin structure that controls the availability of promoter regions to regulatory proteins. Posttranslational modifications of coactivators are also important for regulation of receptor-specific functions.
Functional Interactions between Nuclear Receptors and Signal-Regulated Transcription Factors In addition to altering transcription through direct binding to DNA, nuclear receptors alter transcription through interactions with other transcription factors. In some cases, these protein– protein interactions enhance transcription, while in others they prevent binding of the transcription factor to its DNA target site in a process referred to as transrepression. For instance, GR can interact with additional transcription factors in order to prevent these proteins from binding to and activating their respective targets. This type of process has been shown to be important for the antiinflammatory actions of GR. In other cases, the receptor binds to the factors on their DNA target sites influencing (either þ or ) the transcription of a target gene. GR also can inhibit the transcription of certain genes by direct binding to negative GREs in the promoter regions of these genes. Figure 4 shows the comparison between two of these pathways for ER. In the upper panel is the classical DNA-binding-dependent induction of transcription. We next depict the ability of ER to induce transcription through interactions with AP-1 complexes. In this instance, both estradiol as well as SERMs will stimulate the activity of AP-1.
mRNA
ER
Fos
Jun
AP-1 RE (b)
Figure 4 Mechanisms of transcription activation by nuclear receptors. (a) Shows the classical pathway for transcriptional activation with a steroid receptor dimer binding directly to a hormone response element. (b) Shows an alternative mode of activation. In this case, a receptor such as the estrogen receptor binds to another transcription factor in a process referred to as tethering and influences transcription through its interactions with the transcription factor and the recruitment of coregulator proteins to the complex. ER, estrogen receptor; AP-1 RE, AP-1 response element; ERE, estrogen response element.
Nuclear Receptor Stimulation of Cell-Signaling Pathways Both of the pathways above can be considered genomic pathways in that the nuclear receptor acts by directly altering transcription. Nuclear receptors can also act through stimulating kinase activity, although the final downstream target may be a change in transcription. These actions are rapid (minutes) and are termed nongenomic (Figure 2). This process is thought to occur close to the cell membrane and involves the crosstalk of steroid receptors with various adaptor proteins, G proteins, and receptor tyrosine kinases. There is evidence that activation of nuclear receptors can lead to downstream activation of mitogen-activated protein kinase. In some cases, this is through activation of Src kinase and in others through generation of a ligand for a growth factor receptor. There are numerous other examples of these rapid actions. Induction by estradiol of nitric oxide synthase activity in endothelial cells is a rapid response that does not require transcription. Thus, steroid/thyroid hormones alter cellular activities through multiple mechanisms.
See also: Signaling: Mitogen-Activated Protein Kinase Family; Thyroid-Stimulating Hormone/Luteinizing Hormone/FollicleStimulating Hormone Receptors; Vitamin D Receptor.
Further Reading Faus H and Haendler B (2006) Post-translational modifications of steroid receptors. Biomedicine and Pharmacotherapy 60: 520–528.
Signaling | Steroid/Thyroid Hormone Receptors
Hammes SR and Levin ER (2007) Extranuclear steroid receptors: Nature and actions. Endocrine Reviews 28: 726–741. McDonnell DP, Connor CE, Wijayaratne A, Chang CY, and Norris JD (2002) Definition of the molecular and cellular mechanisms underlying the tissue-selective agonist/antagonist activities of selective estrogen receptor modulators. Recent Progress in Hormone Research 57: 295–316. Tsai MJ and O’Malley BW (1994) Molecular mechanisms of action of steroid/thyroid receptor superfamily members. Annual Review of Biochemistry 63: 451–486. Weigel NL and Moore NL (2007) Kinases and protein phosphorylation as regulators of steroid hormone action. Nuclear Receptor Signalling 5: e005.
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Whitfield GK, Jurutka PW, Haussler CA, and Haussler MR (1999) Steroid hormone receptors: Evolution, ligands, and molecular basis of biologic function. Journal of Cellular Biochemistry – Supplement 32–33: 110–122. Wolf IM, Heitzer MD, Grubisha M, and DeFranco DB (2008) Coactivators and nuclear receptor transactivation. Journal of Cellular Biochemistry 104: 1580–1586.
Relevant Websites http://www.nursa.org – The Nuclear Receptor Signaling Atlas.