Steroid hormones exert a wide variety of effects on growth, development, and differentiation, including important regulatory and behavioral functions within the reproductive system, central nervous system, and adrenal axis. These hormones act by binding to specific intracellular receptor proteins that function as signal transducers and transcription factors to modulate expression of target genes.^1,2 Molecular cloning of the steroid hormone receptor genes in humans has been accomplished over the past 15 years (Table 1). Sequence comparison has revealed that steroid hormone receptors belong to a diverse family of ligand-activated gene regulators that share a highly conserved structure and common mechanisms affecting gene transcription.³ The evolutionary relationship among the steroid and nuclear receptors has been deduced by the high conservation in their DNA-binding domains (DBDs) and in their less-conserved ligand-binding domains (LBDs) and indicates that this large group of proteins arose from a common ancestral molecule.³

TABLE 1. Members of the Steroid Hormone and Nuclear Receptor Gene Superfamily in Mammalian Tissues

  Receptors with known ligands^{1,2,4,26,156,236,237}

  ERα
  ERβ
  AR
  PR
  GR
  MR
  VDR
  TRs (α1,α2,β)
  c-erb A1
  Rev-erbAα (Rev-Erb)
  RARs (α,β,γ)²³⁷
  RXRs (α,β,γ)²³⁷
  PPARs (α,β,γ)

The steroid/nuclear hormone receptor superfamily (Table 1), includes more than 150 receptors for the gonadal and adrenal steroids; nonsteroidal ligands such as thyroid hormones, vitamin D, retinoic acid, and fatty acids; and numerous "orphan" receptors whose endogenous ligands, if necessary, are unknown or are just being identified.^1,4,5 Until recently, only one estrogen receptor (ER) was thought to mediate the physiologic effects of estrogens. However, a second gene encoding a closely related but distinct ER, called ERβ, was identified in rat prostate⁶ and in humans.⁷ The original ER is called ERα. ERα and ERβ can form heterodimers and homodimers in vitro and in vivo.⁸ The superfamily also includes the ERBA1 and ERBA2 oncogene proteins that bind to DNA but lack a functional LBD⁹ and other orphan receptors, such as short heterodimer partner, that lack a DBD but have a LBD.¹⁰ The steroid receptors are considered class I members of the nuclear receptor superfamily, and the other receptors are class II receptors.¹ A variety of mechanisms for achieving tissue-specific gene expression in response to steroid hormones have evolved to ensure diversity through the interaction of these receptors with other cellular proteins and gene elements.

In this chapter, we summarize current information on the steroid/nuclear hormone receptors, focussing primarily on receptors for the sex steroid hormones.

To understand how steroid hormone receptors regulate gene function, it is important to know the structure of the receptor proteins and the identity and cellular function of the genes that they regulate. Members of the steroid receptor superfamily share amino acid homology and a common structure (Fig. 1). Receptors in this superfamily contain several key structural elements that enable them to bind to their respective ligands with high affinity and specificity, recognize and bind to discrete response elements within the DNA sequence of target genes with high affinity and specificity, and regulate gene transcription.²

Molecular cloning of the complementary DNA (cDNA) for each of the major steroid receptors has greatly enhanced our understanding of the structure-function associations for these molecules. The receptor proteins have five or six domains designated A through F, from the N terminus to the C terminus, encoded by 8 to 9 exons. The receptors contain three major functional domains that have been shown experimentally to operate as independent "cassettes,"¹¹ unrestricted according to position within the molecule. The three major functional domains (Fig. 2) of the receptor are a variable N terminus (domains A and B) that confers immunogenicity and modulates transcription in a gene and cell-specific manner through its N-terminal activation function-1 (AF-1); a central DBD (consisting of the C domain), composed of two functionally distinct zinc fingers through which the receptor physically interacts directly with the DNA helix; and the LBD (domains E and, in some receptors, F) that contains activation function-2 (AF-2). The F domain is thought to play a role in distinguishing estrogen agonists from antagonists, perhaps through interaction with cell-specific factors.¹² Domain-swapping experiments in which the DBD of ERα was switched with that of the glucocorticoid receptor (GR) yielded a chimeric receptor that bound to specific DNA sequences bound by GR but upregulated of transcription of glucocorticoid-responsive target genes when treated with estrogen,¹³ demonstrating the specificity of the DBD in target gene regulation.

The amino-terminal domain is hypervariable (<15% homology among steroid receptors) in size and amino acid sequence, is 25 to 603 amino acids long, and constitutes the major source of size differences between receptors.¹⁴ The AF-1 domain in this region is involved in activation of gene transcription but does not depend on ligand binding. In rat GR, the AF-1 region is called tau 1 or enh2 and constitutes amino acids 108 through 317. Deletion of the C-terminal LBD of GR yields constitutive (hormone-independent) transcriptional activation, implying that the N-terminal regions harbor autonomous transcriptional activation functions.¹⁵

Some steroid receptors exist as isoforms, encoded by the same gene but differing in their N terminus. The progesterone receptor (PR) and androgen receptor (AR) exist in two distinct forms, A and B, synthesized from the same mRNA by alternate splicing. The two PR receptor isoforms differ by 128 amino acids in the N-terminal region, yielding PR-A (90 kd) and PR-B (120 kd), which have strikingly different capacities to regulate transcription.¹⁶ In contrast, AR-A and AR-B isoforms show minimal differences in activation of a reporter gene in response to androgen agonists or antagonists in transiently transfected cells.¹⁷

The central core or DBD is highly conserved and shows 60% to 95% homology among steroid receptors.¹ The DBD varies in size from 66 to 70 amino acids and is hydrophilic because of its high content of basic amino acids.¹¹ The major function of this region is to bind to specific hormone response elements (HREs) of the target gene. DNA binding is achieved through the tetrahedral coordination of zinc by four cysteine residues in each of two extensions, which form two structurally distinct zinc fingers (Fig. 3).¹⁸ Zinc fingers are common among gene regulatory proteins.¹⁸ Specificity of HRE binding is determined by the more highly conserved hydrophilic first zinc finger (C1),¹⁴ and the second zinc finger (C2) is involved in dimerization and stabilizing DNA binding by ionic interactions with the phosphate backbone of the DNA.¹⁴ The D box is involved in HRE half-site spacing recognition. The highly conserved DBD shared by AR, GR, mineralocorticoid receptor (MR), and PR enables them to bind to the same HRE, called the glucocorticoid response element (GRE). The more C-terminal part of the C2 zinc finger and amino acids in the hinge region are involved in receptor dimerization in coordination with amino acids in the LBD.

The hinge region or D domain is a 40- to 50-amino acid sequence separating the DNA-binding and LBDs that contains sequences for receptor dimerization and ligand-dependent and -independent nuclear localization sequences (NLSs).¹⁹ The hinge region interacts with nuclear corepressor proteins,²⁰ and with L7/SPA, a 27-kd protein that increases the partial agonist activity of certain antagonist-liganded steroid hormone receptors, such as tamoxifen-liganded ERα, RU486-occupied PR, or RU486-occupied GR.²¹

The carboxyl-terminal domain or LBD is poorly conserved, is 218 to 264 amino acids long, and is hydrophobic. This region contains the ligand-binding site and dictates hormone binding specificity.¹ Greater structural similarity between steroid hormone ligands generally indicates greater amino acid sequence homology in the LBD. Information from the x-ray crystal structures of the LBDs of the retinoic acid receptor (RAR), thyroid hormone receptor (TR), and ERα in the presence or absence of their cognate ligands has shown that the LBD has a compact structure consisting of 12 α helices with a "pocket" into which the ligand fits.^22,23,24,25 Binding of the ligand within the pocket alters the conformation of the LBD with helix 12, forming a "lid" over the pocket, trapping the ligand in a hydrophobic environment, and forming a surface on the LBD with which coactivator proteins interact. Helix 12 in indispensable for AF-2 function. For ERα, 17β-estradiol (E₂) and the antiestrogen or select estrogen receptor modulator (SERM), raloxifene, form different amino acid contacts within the pocket.²⁵ This results in different positioning of helix 12 in the LBD that is thought to permit interaction with coactivators, such as SRC-1, in the presence of E₂ but not raloxifene or, by inference , antiestrogens such as tamoxifen.²⁵

Two human GR isoforms, GRα and GRβ, derived from the same gene by differential splicing at the C terminus, have been reported.²⁶ GRβ was reported to localize in the cell nucleus in the absence of ligand and to block hGRα activity.²⁷ Likewise, a novel isoform of ERβ, called ERβ2, containing an in-frame insertion of an exon of 54 nucleotides, resulting in an insertion of 18 amino acids in the LBD, was identified first by screening rat prostate cDNA library and is also expressed in human cell lines.²⁸ ERβ2 binds E₂ with lower affinity (K_d = 8 nM) than ERβ1 (K_d = 1 nM).

Sequences within the LBD form the binding site for heat shock protein 90 (HSP90) that blocks the DBD in the cytosolic, nonliganded GR.²⁹ The CII and CIII regions (Fig. 2) show homology among members of the steroid/nuclear receptor superfamily and are important in forming the ligand binding pocket.³⁰

The C-terminal AF-2 transactivation domain is highly conserved within the nuclear receptor superfamily³¹ and is recognized by various transcriptional coactivators.^32,33 AF-2 is localized to the most C-terminal end of the E domain. A third transactivation domain called AF-2a or tau2 has been localized to the N-terminal region of the LBD of ERα³¹ and GR.³⁴ Deletion experiments revealed a role for AF-2a and the DBD in targeting rat GR to the nuclear matrix,³⁵ an interconnected ribonuclear-protein network within the nucleus that is thought to play an important roles in transcription of active genes by stabilizing the assembly of the transcriptional machinery.³⁶

Although individual domains of steroid/nuclear receptors can be exchanged and function when spliced with nonrelated transcription factors, forming chimeric proteins, experiments on ERα³⁷ and GR³⁸ show that these receptors function optimally when intact. The N and C terminals of the receptor interact with each other to increase transcriptional activation.³⁷

The transcription of DNA to messenger RNA (mRNA) is the most important process regulated by steroid hormones. All genes share a common basic design (Fig. 4), composed of a structural region in which the DNA encodes the specific amino acids of the protein and regulatory region that interacts with various proteins to control the rate of transcription. Several key elements in the regulatory region of the target gene must be activated before mRNA synthesis can occur. These elements, called cis-acting elements because they are located on the same DNA as the gene itself, are usually located near the 5' end (beginning) of the gene and consist of four main groups: promoters, hormone-responsive enhancers, silencers, and hormone-independent enhancers.³⁹

The promoter, essential for gene activation, sets the basal rate of transcription and controls the accuracy of transcription initiation.⁴⁰ The promoter is located closest to the transcription start site and consists of two sub-elements: the TATA box and the upstream promoter. Located further upstream are one or more HREs, the specific DNA-binding sites to which steroid receptors bind, conferring hormone sensitivity to the gene.² Silencers are elements that inhibit transcription of adjacent genes in the absence of hormone activation. Hormone-independent enhancers are DNA sequences bound by other transcription factors that can further increase the rate of gene expression.⁴¹ The synergistic interaction between regulatory cis-acting elements permits fine tuning of the rates of transcription of target genes in response to the local cellular and hormonal milieu.

HREs are consensus 13- to 15-bp DNA sequences derived from alignment of genes responsive to a particular steroid hormone. HREs have two "half-sites" that each bind the C1 zinc finger of one receptor monomer. Steroid hormone receptors bind DNA as homodimers with each monomer binding to adjacent major grooves on the same side of the DNA helix.¹⁸ Class II nuclear receptors may interact with a different class II nuclear receptor, forming a heterodimer and thereby creating a more stable complex with much higher affinity that is thought to enhance transcriptional activity significantly.^1,4 On the basis of sequence homology and functional similarity, there are three classes of hormone response elements within the steroid hormone receptor superfamily (Table 2).

TABLE 2. Hormone Response Element Binding Sites for Steroid/Nuclear Receptors

The response elements for the PRs, ARs, GRs, and MRs are closely related and are collectively referred to as the GRE, consisting of a palindromic (symmetric) sequence 5'-GGTACAnnnTGTTCT-3', in which n stands for any nucleotide.⁴² AR, PR, GR, and MR show subtle differences in DNA base contact points to GREs.⁴² Examples of genes containing one or more GREs whose transcription is upregulated by glucocorticoids include the much-studied mouse mammary tumor virus (MMTV) promoter,⁴³ tyrosine aminotransferase,⁴⁴ and enzymes involved in gluconeogenesis.⁴⁵ Examples of genes that are specifically inhibited by glucocorticoids through "negative GREs" include proopiomelanocortin,⁴⁶ gonadotropin-releasing hormone (GnRH),⁴⁷ and prolactin.⁴⁸

The minimal consensus estrogen response element (ERE) sequence is also palindromic, 5'-GGTCAnnnTGACC-3',⁴⁹ and differs in only 2 bp from the GRE. Extension of the length of the ERE palindrome, such as 5'-CAGGTCAnnnTGACCTG-3', and the sequences immediately flanking the ERE are important in determining the affinity with which ERα binds the ERE.⁵⁰ Examples of genes whose promoters contain functional EREs include those encoding the much studied Xenopus vitellogenin A1⁵¹ and human genes encoding pS2, a marker for human breast cancer diagnostics;⁵² FOS;⁵³ MYC;⁵³ and PR.⁵⁴

The response elements for the various class II nuclear receptors, such as TR, RAR, retinoid X receptor (RXR), and vitamin D receptor (VDR), are composed of direct repeats of the half-site 5'-AGGTCA-3' with no space in between the half-sites (DR0) or separated by a gap of 1 to 5 nucleotides (DR1 to DR5).⁵⁵ The number of nucleotides separating the half-sites determines the specificity of class II nuclear receptor binding.^1,4 Many class II nuclear receptors require RXR for hormonal activation of transcription.⁴

Arrival and Entry of Steroid Hormones Into Target Tissue Cells

Steroid hormones are small, hydrophobic, and lipid-soluble molecules derived from cholesterol. They circulate in blood free or bound (95%) to plasma carrier proteins.^56,57,58 Sex hormone-binding globulin (SHBG), also known as testosterone-estradiol-binding globulin (TeBG), and androgen-binding protein (ABP) are encoded by the same gene.⁵⁹ ABP is produced by the Sertoli cells of the testis, and SHBG is produced by the liver and is present in the circulatory system.⁵⁸ SHBG binds most gonadal steroids, and corticosteroid-binding globulin (CBG or transcortin) binds glucocorticoids and progesterone with different affinities. When circulating levels of steroid hormones exceed the binding capacity of their respective binding proteins, they can then bind nonspecifically and with low affinity to albumin, from which they can readily dissociate and enter target cells.⁶⁰ The unbound and loosely albumin-bound steroids are believed to be the most biologically important fractions because the steroid is free to diffuse or be actively transported through the capillary wall and lipid plasma membrane bilayer. Extracellular binding proteins may modulate hormone response by regulating the amount of steroid available to the cell.⁶⁰ The binding capacity of binding globulins is influenced by endocrine status and other factors.⁵⁷

SHBG binds to a specific cell membrane receptor called sex hormone-binding globulin receptor (SHBG-R) and activates adenylyl cyclase, increasing intracellular cAMP.⁵⁸ Binding of SHBG to SHBG-R also transfers SHBG into the cell as a consequence of receptor-mediated endocytosis.⁶¹ The interaction of SHBG with SHBG-R is inhibited when steroids are bound to SHBG, suggesting that SHBG is an allosteric protein.⁶¹ However, if unliganded SHBG is allowed to bind to its receptor on intact cells and an appropriate steroid hormone then is introduced, adenylate cyclase is activated and intracellular cAMP increases.⁶² After the steroid hormone is in the cytoplasm, it is not clear whether a transport protein is required for movement of the hydrophobic steroid molecule through the aqueous cytoplasm to the receptor, regardless of whether the receptor is cytoplasmic or nuclear in location. The currently accepted model is that the steroid hormone diffuses freely in the cytoplasm.

Intracellular Localization of the Steroid/Nuclear Receptors

Early attempts to isolate steroid hormone receptors led to a major controversy regarding their intracellular localization in the unliganded state. Reports published before 1984 suggested that, before hormone treatment, steroid receptors in target tissues were located predominantly in the cytosolic fraction as large protein complexes of 300 to 400 kd. After hormone exposure, receptors were detected primarily in the nuclear fraction.⁶³ It was initially proposed that unoccupied and untransformed receptors were located in the cytoplasm until ligand binding, which caused their translocation into the nucleus. The reported presence of ERs and PRs in cytosol is now acknowledged to be largely artifactual, the result of homogenization and centrifugation processes used to isolate the receptor protein.⁶⁴

The newly synthesized unliganded receptor is highly unstable and moves into the nucleus, targeted by its NLS, or associates with the HSP90 complex of cytoplasmic proteins.⁶⁵ Although newly synthesized receptor proteins would be expected to contribute a small amount to cytoplasmic levels of receptor, most steroid/nuclear receptors in the unliganded state reside in the nucleus. The exceptions are the GRs and MRs, which in the unliganded state reside in the cytoplasm in a complex containing HSP90, HSP70, and a variety of receptor-associated proteins.⁶⁶ HSP90 is a general molecular chaperone involved in the folding of various proteins. The HSP90 complex of proteins has chaperonin activity that facilitates hormone binding and subsequent proper folding of the GR.⁶⁷ The HSP90 dimer is thought to stabilize the receptor, protecting it from protease degradation; block the DBD and the NLS; and maintain the receptor in an inactive state until ligand binding occurs (Fig. 5).⁶⁸ HSP90 is also required for ligand binding by the steroid receptors.⁶⁶ In addition to HSP90, unliganded steroid receptors extracted from animal tissues or mammalian cells showed that GRs and PRs are complexed with a number of other proteins, including HSP70, FKB59, p60, p48 (Hip), and p23.^66,67 These proteins are thought to be required for the assembly and maintenance of ligand-sensitive aporeceptor complexes.

On activation by hormone binding and the release of HSP90 and the other GR-associated proteins, the hormone-receptor monomer is released from the complex, dimerizes, and translocates to the nucleus. In contrast to GR, immunohistochemical localization experiments showed that ERα,⁶⁹ ERβ,⁶ PR,⁷⁰ and AR⁷¹ are primarily nuclear in the absence of hormone treatment. All class II nuclear receptors are nuclear in the absence of ligand.^1,4

Although each of the steroid receptors (GR, MR, PR, ERα, AR) has been found in association with HSP90, with the exception of GR and MR, hormone binding does not appear to be required for translocation of these receptors to the nucleus.⁶⁶ The role of the HSP90 complex in ER function is not clear. Purification of ERα after chemical cross-linking in intact MCF-7 human breast cancer cells revealed one ERα monomer complexed with two molecules of HSP90 and one molecule of p59 (FKBP52) but no HSP70 or 40-kd cyclophilin.⁷² HSP90 is not required for ligand-dependent transcriptional activation by ERα.⁷³ Although HSP70 was shown to be required for purified, recombinant ERα to bind EREs in vitro,⁷⁴ other experiments imply that HSP70 targets inappropriately folded, artificially overexpressed nuclear proteins⁷⁵ and is not required for ERα-ERE binding.⁷⁶

Entry of Steroid/Nuclear Receptors Into the Nucleus

Steroid hormone receptors within living cells are dynamic. They shuttle between the cytoplasm and the nucleus.⁶⁶ The hormone receptor enters the nucleus by two processes: passive diffusion through the an "ever opened" central channel of the nuclear pore or active transport that is mediated by interaction of the NLSs on the receptor proteins with the NLS receptor-HSP90 complex.⁷⁷ The NLS-steroid hormone receptor-NLS receptor-HSP90 complex binds to the nuclear pore complex by means of nucleoporins in an ATP-dependent process.⁷⁸ The receptor is then trapped by binding to intranuclear components.⁷⁷ Steroid hormone receptor complexes have been demonstrated in association with nuclear membranes and with chromatin components including histones,⁷⁹ nonhistone basic proteins, DNA, and ribonucleoproteins and with nuclear matrix.⁷⁸

The TR, VDR, RAR, RXR, and other class II nuclear receptors do not form high-molecular-weight complexes but are believed to enter the nucleus directly and become tightly associated with chromatin.^1,4 However, later experiments may cause rethinking of this model. The subcellular localization of human TRβ1 fused at its N terminus to green fluorescent protein (GFP) was followed in living cells.⁸⁰ In the absence of the thyroid hormone T₃, more GFP-TRβ1 was present in the cytoplasm, and when the cells were treated with T₃, the GFP-TRβ1 was predominately localized in the nucleus.⁸⁰ Because GFP-TRβ1 bound T₃ and DNA in a manner identical to wild-type TRβ1 and because T₃ treatment moved all of the GFP-TRβ1 into the cell nucleus, the investigators concluded that their findings reflect the behavior of endogenous TR.⁸⁰ With the exception of the ERBA1 oncogene product and some of the orphan receptors that function constitutively or are activated by growth factor-mediated phosphorylation, unliganded class II nuclear receptors are not transcriptionally active until liganded, because the class II nuclear receptors, including RAR, RXR, and TR, are constitutively bound by corepressor proteins that silence transcription until the appropriate ligand is bound.^32,81,82

Ligand-Dependent Activation of Steroid/Nuclear Receptors

The LBD of the receptor may act as a repressor of receptor function because deletion of the LBD from GRs and PRs causes constitutive gene activation.^83,84 Ligand binding to the receptor stimulates dissociation of the receptor-HSP90 complex,² which facilitates conformational changes in the receptor (activation) that exposes the DBD and promotes dimerization of the receptor. Binding of the hormone to the receptor may be only one of several factors that activates or transforms the receptor, enabling it to bind as a dimer to specific hormone response elements located adjacent to or sometimes at a far distance from the transcription start site of the regulated gene.

Type II nuclear receptors (i.e. TR, VDR, RAR, RXR, and the orphan receptors) do not interact with HSP90. These receptors are bound to DNA in the absence of ligand and are associated with corepressor proteins, such as NCoR^81,82 and SMRT.⁸⁵ Corepressor proteins NCoR and SMRT are associated with a complex of proteins that have histone deacetylase activity that are believed to repress gene expression by maintaining chromatin in a more condensed conformation.^86,87,88

Hormone-dependent phosphorylation of steroid hormone receptors may play an important role in binding of the receptor to its specific response element on the gene and subsequent activation of transcription. PR, GR, ERα, and VDR are all phosphorylated after binding to their respective ligands.⁸⁹

Ligand-Independent Activation of Steroid Hormone Receptors

Although steroid and nuclear receptors are classically activated by ligand binding and are subsequently phosphorylated, a second mode of activation in the absence of ligand has been detected for certain receptors. Phosphorylation of certain steroid hormone/nuclear receptors in response to cell membrane-activated signaling cascades activates the receptor in the absence of the cognate ligand.⁸⁹ Examples of peptide hormones and growth factors that activate steroid receptors by triggering intracellular phosphorylation cascades include dopamine, epidermal growth factor (EGF), insulin, and insulin-like growth factor.⁸⁹ Activation of β₂-adrenergic receptors by the antiinflammatory, antiasthmatic drugs salbutamol and salmeterol was demonstrated to activate GR, resulting in nuclear translocation and transactivation of a GRE-driven reporter gene.⁹⁰ Signal transduction took place through activation of a cAMP cascade mediated by the cell membrane β₂-adrenergic receptor. Likewise activation of the protein kinase A (PKA) pathway stimulated transcription by the MR in a ligand-independent manner.⁹¹ PR appears to be refractory to activation induced by phosphorylation cascades.⁸⁹

Binding of Steroid/Nuclear Receptors to Hormone Response Elements and DNA Bending

The sequences specifically recognized by the various steroid/nuclear receptors are described in Table 2. When steroid/nuclear receptors bind their cognate HRE, the DNA is deformed, causing a bend in the DNA. DNA bending appears to be important in cellular processes mediated by multiprotein complexes, including transcription in prokaryotes and eukaryotes⁹²; DNA bending is thought to facilitate interactions between components of the transcription complex bound to different sites and to promote DNA looping to allow single proteins to contact multiple DNA elements. ERα binding to an ERE results in a bend of the DNA toward the major groove.⁹³ Other steroid receptors, including GR⁹⁴ and PR⁹⁵, also induce DNA bending. Similarly, class II nuclear receptors, including TR and RXR, induce DNA bending.⁹⁶

That the topology of DNA is important for steroid hormone receptor recognition of HREs is reinforced by the observation that the nonhistone chromosomal protein HMG-1, which recognizes irregular DNA structure, enhances the binding of PR, ERα, AR, and GR to their respective response elements in vitro but has no effect on the binding of TR, RAR, RXR, or VDR to their target sequences.^95,97 Moreover, coexpression of HMG-1 or HMG-2 increased PR-mediated transcription in transiently transfected mammalian cells by 7- to 10-fold without altering the basal promoter activity of target reporter genes.⁹⁷

Direct Regulation of Gene Transcription by Steroid Hormone Receptors

Initiation of transcription is a complex event occurring through the cooperative interaction of multiple factors at the target gene promoter (Fig. 4). When bound to the specific HRE on the DNA, the hormone-receptor complex interacts with basal transcription factors and with other proteins to stabilize basal transcription factor binding and promote the assembly of the transcription initiation complex. Once the transcription initiation complex is in place, the enzyme RNA polymerase II is recruited to the transcription start site, where it begins transcribing the DNA sequence into mRNA.

INTERACTION OF STEROID/NUCLEAR RECEPTORS WITH BASAL TRANSCRIPTION FACTORS.

For RNA polymerase II to initiate transcription, basal transcription factors TFIIA, TFIIB, TFIID, TFIIE, TRIIF, and TFIIH must assemble on the core promoter.³⁹ TFIID consists of the TATA box binding protein (TBP) and at least eight tightly associated factors (TAFs) of 18 to 250 kd.⁹⁸ Because the amount of RNA polymerase II is limited in the nucleus, genes must compete for it by assembling an appropriate set of cis-acting elements, HREs, and binding sites for sequence-specific transcription factors, such as AP-1⁹⁹ and Sp1.¹⁰⁰ The net result is the synthesis of new mRNAs that move into the cytoplasm and are translated into new proteins that may alter cell function (acting in an intracrine manner) or may be modified further and secreted by the cell to act as endocrine, autocrine, or paracrine factors.

Steroid receptors interact with basal transcription factors TFIIB, TBP, and various TAFs of TFIID.¹⁰¹ Studies indicate that steroid and nuclear receptors use different domains for interaction with basal transcription factors.¹⁰¹ These interactions help stabilize the assembly of the RNA polymerase II preinitiation complex.

INTERACTION OF STEROID/NUCLEAR RECEPTORS WITH COACTIVATORS.

Steroid hormone receptors interact with multiple proteins when bound to DNA or in solution in vitro. Early experiments showed that overexpression of one type of steroid hormone receptor could inhibit or "squelch" the activation of transcription mediated by a different steroid hormone receptor, hinting that steroid receptors compete for limited amounts of a factor required for transcription.¹⁰² During the past 4 years, at least 15 different coactivators have been identified. Examples of coactivators are listed in Table 3. These proteins have also been called receptor-interacting proteins (RIPs) and receptor-associated proteins (RAPs); however, not all RIPs are coactivators. By definition, coactivators are considered to interact directly with the steroid/nuclear receptor and enhance transcription.³² The first coactivators for steroid receptors to be discovered, the yeast SWI/SNF proteins,¹⁰³ may not be strictly coactivators but instead may serve as "bridging factors" that interact between coactivators and basal transcription factors. Several coactivators have general transcriptional activator function (e.g. CBP); they enhance transcription by different types of transcription factors, including steroid receptors.

TABLE 3. Coactivator Proteins That Interact With Steroid Hormone Receptors

Steroid receptors can interact with a number of different coactivators. Some of these coactivators, such as SRC-1, CBP, and TIF2, play a critical role in ligand-activated transcription.^32,101 Coactivator proteins contain one or more copies of a nuclear receptor binding motif, also called the nuclear receptor box, consisting of the amino acids LXXLL, which physically interacts with the steroid/nuclear receptors. Steroid receptors show different affinities for the various coactivators¹⁰⁴ and use different amino acids to contact the coactivators.¹⁰⁵ The discovery that coactivators SRC-1, ACTF, and CREB/p300/CBP¹⁰⁶ have histone acetyltransferase (HAT) activity¹⁰⁷ indicates a possible mechanism for enhanced transcription. Many transcriptional regulatory proteins have intrinsic HAT activity.¹⁰⁸ HATs acetylate lysine residues on the N-terminal tails of histones H3 and H4 in chromatin, resulting in a weaker association of histones with DNA, altering nucleosomal conformation and stability in a manner that facilitates transcriptional activation by RNA polymerase II.¹⁰⁹ SRC-1, CBP/p300, CREB, and other coactivators are believed to form a ternary complex with liganded steroid receptors to increase the rate of hormone-responsive gene transcription.¹¹⁰ Several HAT activities may be tethered to hormone-activated receptors on the promoter, yielding synergistic transactivation. Not all genes are affected by histone acetylation,¹⁰⁸ and steroid and nuclear receptors show different affinities of interaction with coactivators.¹¹¹

The currently accepted model suggests that different target cells express different levels of coactivators and corepressors that, along with the amount of receptor protein and ligand, could allow fine tuning of target gene transcription in response to steroid hormones. Northern blot analysis confirmed the idea that different rat tissues¹¹² and cell lines¹¹³ express different amounts of the mRNA for p300, CBP, SRC-1, RIP140, SMRT, and NCoR.

Indirect Regulation of Gene Transcription by Interaction of Steroid/Nuclear Receptors With Other Transcription Factors

In addition to their direct action on target gene expression mediated by direct DNA binding, steroid receptors interact physically with different transcription factors to alter target gene transcription without the steroid receptor interacting directly with DNA. The best studied example of this "transcriptional crosstalk" is the interaction of the AP-1 transcription factor with GR.¹¹⁴ Depending on the cell type¹¹⁵ and the composition of the AP-1 complex, GR synergizes with AP-1 (JUN-JUN homodimer) or suppresses the FOS-JUN heterodimer.¹¹⁶

Similarly, ERα liganded by the antiestrogen tamoxifen activates promoters through AP-1 sites in genes such as that encoding human collagenase.¹¹⁷ This contrasts with the inability of tamoxifen to activate transcription from promoters bearing classic EREs. Tamoxifen agonism at AP-1 sites is cell type specific; it occurs in cell lines of uterine but not of breast origin. The DBD of ERα is required for tamoxifen activation at AP-1 sites. Conversely, the AP-1 components JUN- and FOS-inhibited, E₂-dependent, ERα-stimulated reporter gene activity in transiently transfected MCF-7 or CV-1 cells transfected with ERα.¹¹⁸ DNA binding experiments revealed that ERα-ERE binding was inhibited by the JUN protein and that ERαinhibited JUN-DNA binding.¹¹⁸

E₂ signals in opposite ways through ERα and ERβ from an AP-1 site: with ERα, E₂ activated transcription, whereas with ERβ, E₂ inhibited transcription.¹¹⁹ Moreover, in contrast to ERα, the antiestrogens tamoxifen, raloxifene, and ICI 164,384 were potent transcriptional activators with ERβ at an AP-1 site. The two ERs differ in how they respond to ligand and response element, suggesting that ERα and ERβ may play different roles in gene regulation.¹¹⁹

Another transcription factor with which ERα interacts directly to activate gene transcription is Sp1. A number of estrogen-responsive genes are activated by ERα-Sp1 interaction, including cathepsin D,¹²⁰ RARα,¹²¹ and FOS.¹²² ERα and Sp1 physically interact and this interaction increases Sp1-DNA binding in the presence or absence of E₂. In contrast, transactivation of Sp1-driven promoter-reporter constructs is E₂ dependent.¹²³ These results indicate that transcriptional activation requires more than ERα-Sp1 interaction and increased DNA binding. A likely interpretation is that coactivators are required.

Repression of Target Gene Transcription by Steroid/Nuclear Receptors

Binding of the hormone-receptor complex can also repress transcription of target genes.³² One mechanism by which a steroid receptor represses gene transcription is by binding to the same, or an overlapping, DNA binding site as that used by a different activator protein, competitively blocking activator binding to DNA. One example of this mechanism is the mutual inhibition by GR and AP-1 on the proliferin gene promoter that contains a composite GRE-AP-1 binding site.¹¹⁶

Competition for limiting amounts of coactivators is another mechanism by which steroid hormones inhibit gene expression. The mutual inhibition of AP-1 and steroid receptor transactivation, including AR, ERα, GR, and PR,¹¹⁴ is achieved through competition for limiting amounts of the coactivators CBP/p300 in the nucleus.¹²⁴ Likewise, GR and ERα block NF-κB-mediated transactivation.¹¹⁴ GR and other steroid receptors interact directly with NF-κB, and NF-κB inhibits GR-, ERα-, and PR-mediated transcription. Later experiments indicate that GR and NF-κB compete for limited amounts of the coactivators CBP and SRC-1.¹²⁵

Another mechanism for repression by steroid receptors is by binding to the HRE and recruiting corepressor proteins that quench the activity of activators bound to the promoter. The corepressors N-CoR^81,82 and SMRT⁸⁵ were first identified by their binding to unliganded TR, RAR, and RXR (Table 4). Tethering of these corepressors to the DNA by their interaction with the DNA-bound TR, RAR, or RXR recruits other components of the corepressor complex that mediate the actual events of transcriptional silencing.¹²⁶ Binding of TR or RAR ligands cause dissociation of NCoR, but only when the receptors are bound to DNA.⁸² Unlike coactivator complexes that have endogenous HAT activity, corepressors to not appear to have enzymatic activity. Corepressors NCoR and SMRT, appear to function by recruiting histone deacetylase (HDAC)-con-taining multiprotein repression complexes to the promoter.¹²⁷ Deacetylation of chromatin helps maintain a condensed state of nucleosomal structure that blocks the binding of components of the RNA polymerase II transcription initiation complex.

TABLE 4. Corepressor Proteins That Interact With Steroid/Nuclear Receptors

A variety of mechanisms are likely to be involved in hormone antagonist action. First, the antagonist activity of an antihormone may depend on the cell or tissue type. For instance, antiestrogen-liganded ERα binds EREs with high affinity,^50,128 but in certain cell types, such as MCF-7 human breast cancer cells, transcription is not activated,¹²⁹ whereas in endometrial cells, tamoxifen stimulates transcription.¹³⁰ The proposed mechanism for these observations involves the inability of antiestrogen-liganded ERα to interact with coactivator proteins, such as SRC-1¹³¹ and TIF1a.¹³² Another possibility is that antiestrogen-liganded ERα may recruit corepressor proteins to the promoter, inhibiting chromatin changes mediated by HDACs that promote a "loosening" of nucleosomal structure.¹³³ The corepressor NCoR interacts with the tamoxifen metabolite 4-hydroxytamoxifen (4-OHT)-liganded ER.¹³⁴ Breast cancer cells that are resistant to tamoxifen inhibition of cell proliferation show reduced NCoR levels compared with tamoxifen-sensitive cells, suggesting a mechanism whereby cells become resistant to tamoxifen.¹³⁴ Similarly, NCoR and SMRT repress the agonist activity of antiprogestin RU486-liganded PR or tamoxifen-liganded ERα.²¹

Nonnuclear Steroid Hormone Receptors and Nongenomic Effects of Steroid Hormones

Although most of the effects of steroid hormones are mediated through their interaction with their cognate receptors and subsequent effects on target gene transcription, certain rapid effects of steroid hormones are incompatible with a transcriptional mechanism.¹³⁵ Although highly controversial, specific binding sites for estrogen, glucocorticoids, and vitamin D receptors have been reported in the plasma membrane of various target cell types.^136,137

Estrogen receptors have been reported in the plasma membrane of GH3 pituitary cells.¹³⁸ Exposure of GH3 pituitary cells to estradiol was reported to elicit a rapid (within 5 minute) release of prolactin in a manner that cannot be accounted for by genomic effects of estradiol mediated through nuclear ERs. Binding sites for estrogen with different biochemical properties from the classic nuclear receptor have been reported in the endoplasmic reticulum of uterine tissues.¹³⁹ Estradiol appears to have receptor-mediated (genomic) and nongenomic effects in the brain.¹⁴⁰ The cardiovascular protective effects of estradiol are thought to be mediated at least in part by nongenomic ER and involve increased intracellular cAMP,¹⁴¹ inhibition of Ca+ 2 influx,¹⁴² and stimulation of nitric oxide release.¹⁴³ Estradiol has exerts direct beneficial effects on human oocytes during in vitro maturation, and these effects at least partly result from steroid action at the oocyte surface.¹⁴⁴

A study compared nuclear and membrane expression of ERα and ERβ by expressing the cDNAs for each receptor type in Chinese hamster ovary (CHO) cells.¹⁴⁵ ERα and ERβ were expressed in the nucleus and in the cell membrane. However, the membrane receptor number was only 2% of the nuclear receptors. E₂ bound to the nuclear and membrane forms of each isoform with identical affinity, but E₂ bound ERβ with lower affinity than ERα.¹⁴⁵ E₂ binding to CHO-ERα- or CHO-ERβ-activated G-coupled membrane receptors Gαq and Gαs and rapidly (within 5 to 10 minutes) stimulated corresponding increases in inositol phosphate and cAMP.¹⁴⁵ Membrane ERα and ERβ showed distinct differences in their activities: JUN N-terminal kinase activity was stimulated by E₂ in ERβ-expressing CHO but was inhibited in CHO-ERα cells.¹⁴⁵ Further studies are needed to extend these results on the role of a membrane ER to human tissues.

GRs and MRs appear to have nongenomic effects. GRs bind to cytoskeletal structures,¹⁴⁶ and glucocorticoids stimulate the rapid onset of polymerization of actin in a nongenomic manner that involves decreased intracellular cAMP.¹⁴⁷ GRs may activate mitochondrial gene expression in hepatic cells.¹⁴⁸ Rapid stress-induced changes in male amphibian reproductive behavior appear to be mediated by MRs on neuronal membranes.¹⁴⁹

One of the membrane steroid receptors that has been well characterized is the membrane PR in amphibian oocytes and in spermatids.¹⁵⁰ This receptor differs from the nuclear PR. The novel membrane PR mediates rapid changes in Ca+ 2 conductance in human sperm plasma membrane.¹⁵¹ A high-affinity progesterone-binding membrane protein of 200 kd has been described in pig liver.¹⁵² In addition to membrane PR action, metabolites of progesterone and deoxycorticosterone act as positive allosteric modulators of the γ-aminobutyric acid (GABA)A receptor complex in the cortex of rat brain.¹⁵³ This means that these hormones bind to an effector site (not the GABA binding site) and increase the affinity of GABA binding to the GABA_A receptor. Progesterone also binds to the G-coupled oxytocin receptor and inhibits peptide-mediated signaling.¹⁵⁴ The role for these steroid membrane receptor effects in human health is just beginning to be explored.

Diversity of tissue responses to steroid hormone action, despite conservation of structure and function, is achieved through a variety of mechanisms. Some receptors are expressed in only a limited number of cell types (e.g. gonadal steroid receptors), and others are found in a large number of cell types (e.g. glucocorticoid and thyroid hormone receptors). Other tissue-specific gene regulatory proteins (e.g. transcription factors, coactivators, corepressors) are involved in the modulation of gene transcription by steroid/nuclear receptors. Cell-specific posttranslational modification of receptors is another mechanism to ensure tissue diversity of hormone responses.⁸⁹ Multiple receptor isoforms for hormones may also account for tissue-specific gene expression.²⁸ Receptor isoforms have been shown to arise through alternative splicing of mRNA from a single gene (PR and AR),^17,155 from multiple genes (ER), or a combination of both (TR).¹⁵⁶

Interactions with other transcription factors, coactivators, and corepressors appear to play a significant role in determining specificity. Even though receptors for several different hormones (i.e. AR, GR, MR, and PR) can bind a common HRE sequence, they may exhibit different interactions with other DNA-bound bound factors and coactivators, thereby achieving different modulation of target gene expression.³²

The half-life of steroid hormone receptors ranges from 2 to 4 hours for ERα,¹⁵⁵ 4 hours for AR,¹⁵⁷ 7 to 10 hours for PR,¹⁵⁸ and 19 hours for GR.¹⁵⁹ The relatively long half-life of the steroid hormone receptors strongly suggests that the receptor proteins are recycled before eventual degradation.¹⁶⁰

Steroid hormones generally autoregulate their receptor levels.¹⁶¹ Desensitization or downregulation of receptor numbers, measured by decreased ligand binding capacity, occurs in response to exposure to high levels of ligand and involves the reduction in receptor mRNA levels, decreasing the number of available receptors. The receptor gene may be negatively regulated by the hormonal ligand itself through its receptor protein interacting with specific HREs in the gene.¹⁶² Upregulation or self-priming may occur in an analogous fashion.¹⁶² Steroid hormones can regulate receptor levels for other hormones (e.g. E₂ increases PR levels in estrogen-responsive tissues).⁵⁴ Progesterone can downregulate its own receptors, as well as ERα¹⁶³ and ERβ.¹⁶⁴ This increase or decrease in receptor levels in homologous or heterologous regulation can be caused by alterations in receptor gene transcription or decay rates for receptor mRNA or protein. Binding of the cytosolic GR complex to very long 3'-untranslated regions of its receptor mRNA has been reported to cause premature degradation.¹⁶⁵

Steroid and nuclear hormone receptors have been detected in virtually every major organ and tissue in the mammalian body, including the brain. Much new information on the location and function of the steroid hormone receptors has been derived from techniques that allow manipulation of a specific mouse gene in vitro to generate targeted gene disruption of the gene for a given receptor, creating homozygous "gene knockout" mice.¹⁶⁶ The technique disrupts the linear gene by inserting an antibiotic resistance gene (e.g. neomycin) into one coding region (one exon) of the gene. The mutant DNA is inserted into genomic DNA by homologous recombination in mouse embryonic stem cells to generate transgenic mice. The mRNA for the targeted gene is truncated or nonsensical. This technique has resulted in ERα, ERβ, MR, PR, and GR knockout mice.¹⁶⁶

Estrogen Receptor-α Knockout Mice

To the surprise of many investigators, loss of ERα expression was not lethal and had no effects on the ratio of male to female mice born.¹⁶⁷ ERα knockout (ERKO) mice survived to adulthood and developed grossly normal external genitalia, but both sexes were infertile.¹⁶⁸ Females have hypoplastic uteri and hyperemic ovaries with no corpora lutea.¹⁶⁸ Serum levels of estradiol in the ERKO females are more than 10-fold higher than those in the wild type, consistent with a syndrome of hormone insensitivity.¹⁶⁷ ERKO females have 10-fold higher circulating E₂ and elevated luteinizing hormone (LH) but not follicle-stimulating hormone (FSH).¹⁶⁹ Ovarian histology is abnormal. Mammary glands of adult ERKO female mice lacks branching and terminal end bud formation.¹⁶⁹ Maternal behavior, as measured by retrieving of pups removed from their mothers, is reduced in ERKO females.¹⁷⁰ In some cases, pups were killed by the ERKO females, which was not seen in wild-type animals.¹⁷⁰ Aggression toward other females was increased, and female-typical lordosis behavior was reduced.¹⁷⁰

Adult ERKO males exhibit a number of alterations in reproductive tract histology, including atrophied, degenerated seminiferous tubules and diluted, infertile sperm.¹⁷¹ The mice exhibit decreased sperm counts and significantly lower testicular weight than wild-type males.¹⁶⁹ The reproductive capacity of sperm from ERKO males is significantly compromised in in vitro fertilization experiments. ERKO males appear to have normal mounting behavior toward wild-type females but exhibit an almost complete lack of intromission and ejaculation. ERKO males are consistently less aggressive than wild-type mice.^170,172 These findings indicate that ERα gene expression during development plays a major role in the organization of male-typical aggressive and emotional behaviors in addition to simple sexual behaviors.¹⁷² E₂-protected wild-type and ERKO female mice in response to carotid arterial injury, indicating that ERα may not be required for the protective actions of E₂ in the vascular system.¹⁷³

There has been one reported human case with an ERα mutation.¹⁷⁴ The male patient exhibited severe osteoporosis and insufficient closure of the epiphyseal growth plates.

Estrogen Receptor-β Knockout Mice

Mice lacking ERβ (BERKO) develop normally and are indistinguishable grossly and histologically from their littermates.¹⁷⁵ Breeding experiments with young, sexually mature females show that they are fertile and exhibit normal sexual behavior, but they have fewer and smaller litters than wild-type mice.¹⁷⁵ Superovulation experiments indicate that this reduction in fertility is the result of reduced ovarian efficiency.¹⁷⁵ The mutant females have normal mammary gland development and normal lactation.¹⁷⁵ Adult male mice show no overt abnormalities and reproduced normally. Older mutant males display signs of prostate and bladder hyperplasia. The investigators concluded that ERβ is essential for normal ovulation efficiency but is not essential for female or male sexual differentiation, fertility, or lactation.¹⁷⁵

Progesterone Receptor Knockout Mice

Mice carrying a null mutation of the PR gene (PRKO) exhibit several reproductive abnormalities, including anovulation, attenuated lordotic behavior, uterine hyperplasia, and lack of mammary gland development.¹⁷⁶ There were no effects on the viability or sexual differentiation of homozygous PR gene-disrupted mice.¹⁷⁶ The female homozygous for PR disruption are completely infertile, whereas males exhibit no apparent effects on fertility. Serum LH levels in PRKO mice were found to be elevated by approximately twofold over basal (metestrus) values in wild-type mice.¹⁷⁷ In contrast, basal FSH levels were not different in PRKO and wild-type mice. Basal levels of E₂ and progesterone in serum were likewise similar in the two groups, as were hypothalamic LHRH concentrations. Basal PRL levels were slightly higher in PRKO versus wild-type mice. These results confirm the essential role of PRs in the regulation of hypothalamic or pituitary processes that govern gonadotropin secretion.¹⁷⁷

Glucocorticoid Receptor Knockout Mice

Most of the mice homozygous for disruption of GR die shortly after birth because of severe lung atelectasis.¹⁷⁸ Additional defects were found in the adrenals, liver, brain, bone marrow, and thymus and in the feedback-regulation of the hypothalamic-pituitary axis.¹⁷⁹ However, disruption of the ability of GR to dimerize is not lethal.¹⁸⁰

Mineralocorticoid Receptor Knockout Mice

MR–/– mice, obtained by targeted gene disruption, died between days 8 and 13 after birth after exhibiting signs of pseudohypoaldosteronism, and the pups died of dehydration because of renal sodium and water loss.¹⁸¹ The MR–/– mice showed severe dehydration, hyperkalemia, hyponatremia, and high plasma levels of renin, angiotensin II, and aldosterone.¹⁸² The MR knockout mice showed significant increases in the expression level of several renal angiotensin system components—renin, angiotensinogen, and angiotensin II receptor (AT1)—but no alteration in angiotensin I-converting enzyme was detected in the kidney.¹⁸²

Androgen Receptor Insensitivity

No one has created an AR knockout mouse. A natural deficiency of AR occurs in the Tfm mouse,¹⁸³ which has an AR mutation that results in androgen insensitivity syndrome (AIS) that is an X-linked inherited disease.¹⁶⁶ Various mutations in the AR in humans have been shown to cause AIS.¹⁸⁴ Male Tfm mice exhibit complete infertility.¹⁶⁶

Steroid hormone receptors are present in relatively low numbers in target tissues. Their instability when isolated from cells and tendency to adhere to the surfaces of test tubes require special consideration in handling under experimental and clinical conditions. The basic principles of measurement of nuclear hormone receptors are the same as for plasma membrane receptors.¹⁸⁵ Multipoint titration and sucrose density gradient analyses are commonly used for measurement of steroid hormone receptors in tissue samples.¹⁸⁶ Tissue homogenates containing receptors are incubated with increasing amounts of radioactively labeled steroids of high specific activity in the presence and absence of corresponding excess unlabeled steroid or incubating with a fixed concentration of labeled steroid and increasing amount of corresponding unlabeled steroid.¹⁸⁷ Tritiated or radioiodinated steroids are most commonly used, because they are available in high specific activities. The binding in the presence of corresponding excess unlabeled steroid hormone is nonspecific binding, which is unsaturable and generally represents binding to nonreceptor sites. Nonspecific binding is subtracted from total binding (the amount bound in the absence of unlabeled steroid) to obtain saturable and high-affinity specific binding. By appropriately varying the incubation conditions, kinetic and equilibrium binding constants, specificity of binding, and other properties can be determined by titration analysis. Receptor-bound and free hormones are separated by dextran-coated charcoal¹⁸⁷ or precipitation of steroid-receptor complex with protamine sulfate¹⁸⁸ or absorption to hydroxyapatite¹⁸⁹ followed by centrifugation and scintillation counting. For clinical samples, ER and PR status in breast tumor samples are quantitated by monoclonal antibody-based methods using a commercially available kit from Abbott Laboratories.¹⁹⁰ One important clinical application of this method is the use of ERα and PR status as an indication for antiestrogen therapy.¹⁹¹

Scatchard analysis is the most universal method for calculating binding affinity (K_d) and number (binding capacity) of steroid hormone receptors.¹⁹² The shape of Scatchard plots can be linear or curvilinear with upward or downward concavity. The interpretation and calculation of binding constants is the same as for membrane receptors.¹⁸⁵

The apparent dissociation constants for steroid hormones binding to their cognate receptors is in the range of 10–11 to 10–9 M, which is close to circulating steroid hormone concentrations. The affinity of nonrelated steroid hormones to the receptor is generally about 100 times lower than the appropriate steroid hormone. However, if the levels of these nonrelated hormones are greatly elevated because of some physiologic or pathologic process, a significant binding and biologic response to them can be expected.

Ligand binding specificity is also influenced by interaction with coactivators. In the presence of coactivator ARA70, E₂ bound to the AR and induced AR-responsive transcriptional activity more than 30-fold in DU145 human prostate cancer cells.¹⁹³ In contrast, the synthetic estrogen diethylstilbestrol (DES) had no effect. The significance of this ability of E₂ to activate AR is strengthened by finding patients with Reifenstein partial androgen-insensitive syndrome with a single mutation in the AR that makes the receptor nonresponsive to E₂-stimulation in the presence of ARA70. These data suggest that testosterone and dihydrotestosterone are not the only ligands for the AR.¹⁹³

Before the development of cloning techniques, steroid hormone receptor structures were analyzed by painstaking methods of classic protein chemistry. Protein purification required processing of large amounts of tissues and sequencing was laborious and slow. Ligand-binding characteristics were described by Scatchard analysis and binding displacement curves.

The advent of molecular biology has revolutionized the study of hormone receptor biology, providing a wealth of powerful techniques that are indispensable to elucidating the structure and mechanism of action of the steroid and nuclear hormone receptors. Common techniques used by molecular biologists include Southern, Northern, and Western blotting, used to detect the presence and size of specific DNA, RNA, and protein sequences, respectively.¹⁹⁴ Molecular cloning of DNA provides a technique whereby a single segment of DNA can be isolated from a large population of genes, purified to homogeneity and amplified to produce sufficient quantities for structural and functional analysis. Receptors are routinely cloned from RNA extracted from small amounts of hormone-responsive tissues or cells. When it is possible to predict a certain degree of homology of DNA sequence of the hormone receptor with other known proteins, specific DNA segments can be amplified using the polymerase chain reaction (PCR), with the cDNA as template and highly conserved sequences from related proteins as primers.¹⁹⁵ Alternatively, specific enzymes may be used to copy, cut, and splice together pieces of DNA that are inserted into circular plasmid DNA or bacteriophage viruses, introduced into bacteria and allowed to divide many times. The greatly amplified DNA is then isolated and purified, and the nucleotide sequence of the cloned DNA segment is determined. The cDNA clone can be transcribed into mRNA and used to confirm biologic activity in in vivo and in vitro functional expression systems, and the amino acid sequence of the encoded protein can be readily determined. The cDNA can be tagged with radioactivity or a fluorescent marker and used to detect the presence or describe the distribution of receptor mRNA in various tissues and endocrine states using solution hybridization and in situ hybridization methods.

Once the molecular structure of the receptor protein is known, the structure-function relationships of certain amino acid sequences relative to those of other receptors in the steroid hormone receptor family are investigated using domain-swapping and site-directed mutagenesis methods. In domain-swapping experiments, hybrid mRNA transcripts can be produced from a receptor cDNA in which a key functional element, such as the LBD, is replaced with the nucleotide sequence from the same region of another closely related receptor.¹⁹⁶ With this technique, it has been possible to determine the functional significance and specificity of the various regions of the steroid receptor proteins.¹⁹⁶ Site-directed mutagenesis has been used to determine the identity of amino acid residues that are important for DNA and ligand binding in steroid receptors.

The electrophoretic mobility shift assay (EMSA), also called gel mobility shift assay, is the most common technique to examine the binding of a given steroid/nuclear receptor to DNA. For EMSA, a preparation of a particular steroid/nuclear receptor is incubated with a short (20 to 400 bp) fragment of [32P]-labeled double-stranded DNA. The DNA may be a synthetic construct of a particular HRE or a fragment from the promoter of a particular gene that is thought to contain a HRE. The protein-DNA reaction mixture is then separated by a nondenaturing polyacrylamide gel electrophoresis. The free [32P]-labeled DNA migrates to the bottom of the gel, and the migration of the [32P]-labeled DNA that is bound by the receptor is slowed. The gel is dried and exposed to x-ray film or placed in a Phosphorimager cassette to detect where the 32P is located. The free, unbound [32P]-labeled DNA appears at the bottom and the receptor-bound DNA is located toward the middle or top of the gel. Addition of a 50- to 200-fold molar excess of cold competitor DNA is used to determine the specificity of the receptor-DNA complex. Addition of an antibody to a particular steroid/nuclear receptor is used to confirm the identity of the protein in the protein-DNA complex. The antibody may block the receptor-DNA binding, leaving an empty spot on the autoradiograph; "supershift" the receptor-DNA complex by binding stably to it and further retarding its mobility in the electrophoretic field; or have no effect, indicating that the epitope recognized by the antibody is not available under the assay conditions. EMSA is useful to compare the relative binding affinities of a given receptor for various HREs in vitro.¹⁹⁷

To examine how a particular ligand, steroid receptor, and HRE affect transcription, transient transfection experiment are performed using cultured mammalian cells.¹²⁹ For these experiments, the cell must contain the steroid receptor of interest or the receptor must be introduced into the cell by means of an expression vector encoding the cDNA of the receptor with a strong viral promoter, such as CMV or RSV, to ensure its expression. The reporter vector contains a reporter gene not expressed in normal mammalian cells. Examples of reporter genes are chloramphenicol acetyltransferase (CAT)¹⁹⁸ and luciferase.¹²⁹ The reporter and hormone receptor expression plasmids are transfected into the mammalian cells using calcium phosphate coprecipitation,¹⁹⁹ electroporation,²⁰⁰ or lipid-mediated transfer (e.g. lipofectamine).¹²⁹ After a recovery period, the cells are treated with hormone for 12 to 48 hours. The cells are then lysed, and reporter gene activity is measured. As a control, cells are usually co-transfected with a β-galactosidase reporter as an indication of the percent efficiency with which the cells have taken up the plasmid DNA.¹²⁹ This technique allows quantitation of how different ligands and DNA sequences affect the transcriptional response of a given steroid receptor. Cotransfection of the cells with expression plasmids for coactivators or corepressors allows determination of the functional consequence of these proteins.

Steroid hormones exert profound effects on mood, mental state, behavior, and memory by acting by interaction with steroid receptors in the brain. Estrogens and progestins have been shown to regulate the synthesis and release of brain neurotransmitters, including norepinephrine, dopamine, serotonin, GnRH, β-endorphin, corticotropin-releasing factor, and prolactin.²⁰¹ Intracerebroventricular injection of antisense oligonucleotides to PR completely suppressed sexual behavior.²⁰² Estrogens¹⁶⁶ and progestins play a key role in female rodent sexual receptivity.²⁰³ Female ERKO and PRKO mice exhibited reduced¹⁷⁰ or no lordosis,²⁰³ respectively. PR regulation of dopamine synthesis is thought to be responsible for the lack of responsiveness to male advances.²⁰³ PRKO male mice showed reduced mount frequencies compared with wild-type mice, indicating a role for progestins in regulating male sexual response as well.²⁰⁴ These reports indicate that steroid hormones acting through their receptors and genomic mechanisms initiate events that regulate neuronal networks involved in sexual behavior.

Clinical manifestations of abnormal steroid hormone receptor function involve variations in receptor numbers and the ability to stimulate transcription of certain genes. Hormone resistance, the failure of tissues to respond to normal circulating hormone levels, has been shown in a variety of endocrine systems to be caused by the genetic lack or functional defect in steroid hormone receptors. Deletions in all or part of the AR gene have been shown in several individuals with complete androgen insensitivity.^205,206 The lack of functional ARs results in testicular feminization.

Sex steroid hormones have been implicated in the pathogenesis of breast, uterine, ovarian, prostate, thyroid, and other cancers. Expression of mutant forms of ERα may be an important factor in some cases of breast cancer.²⁰⁷ Determination of steroid hormone receptor expression (i.e. ERα and PR) in breast tumors is critical to the selection of appropriate therapy. Hormone-dependent tumors tend to have elevated levels of steroid hormone receptors.²⁰⁸ One study found that 50% of the sixty breast tumors sampled expressed ERα and ERβ and that the ERα+ /ERβ+ phenotype was associated with node-positive status.²⁰⁹ Elevated levels of ERα and ERβ are highly associated with poor histologic differentiation in breast cancer and may serve as a predictor of responsiveness to endocrine therapy and a prognostic indicator of metastatic disease.²⁰⁸ The combined presence of ERα and PR in a breast tumor provides an additional indication of the probability of prolonged survival after antiestrogen therapy.¹⁹¹

Steroid hormone receptor analysis has limited value in the selection of patients with endometrial cancer for endocrine therapy, because normal and malignant uterine tissues contain significant levels of ERs and PRs.²¹⁰ Although the results of different studies disagree, PR expression appears a better prognostic indicator than ERα expression in ovarian cancer.²¹¹ The relation between steroid receptor status and response to endocrine therapy in ovarian cancer remains to be established.

Glucocorticoid resistance results from an inability of glucocorticoids to exert their effects in their target tissues.²¹² Glucocorticoid resistance is associated with elevated ACTH and cortisol, with an attendant increase in adrenal androgens and steroids, and with salt-retaining activity. Clinical manifestations of glucocorticoid resistance vary from asymptotic to different degrees of hypertension or hypokalemic alkalosis or hyperandrogenism. In women, the excess androgens can result in acne, hirsutism, male-type baldness, menstrual irregularities, oligoanovulation, and infertility; in men, it may lead to infertility; and in children, it may cause precocious puberty. Different molecular defects in the GR gene, resulting in various defects in the receptor protein alter the functional characteristics or concentrations of GR and cause glucocorticoid resistance.²¹² It is postulated that acquired tissue-specific glucocorticoid resistance may play a role in the origin and pathogenesis of depression, steroid-resistant asthma, autoimmune disorders, and acquired immunodeficiency syndrome (AIDS). GR mutations have been suggested to play a role in the pathogenesis of leukemia, hereditary glucocorticoid resistance, and Nelson's syndrome.²¹³ Corticosteroid-resistant asthma has been associated with decreased GR and increased AP-1-DNA binding in peripheral blood mononuclear cells compared with corticosteroid-sensitive asthma.²¹⁴ These findings indicate that variations in the GR may play a central role in a wide variety of diseases.²¹³ Hypertension and pseudohypoaldosteronism are associated with diminished or complete loss of high-affinity aldosterone receptors in select target tissues in some patients.²¹⁵ MR induces the transcription of specific genes that regulate apical amiloride-sensitive epithelial sodium channels located in the apical membrane of epithelial cells from the distal colon and kidney collecting duct.²¹⁶

Estrogens play a central role in the immune response and immune-mediated diseases, and cells involved in the immune response—thymocytes, macrophages, and endothelial cells—express ERα.²¹⁷ Certain pathologic states are characterized by the production of abnormal levels of antibodies to steroid hormone receptors. Although the autoimmune disorder systemic lupus erythematosus (SLE) was postulated to result from antiestrogen receptor action,²¹⁸ a later report showed that peripheral blood monocytes of SLE patients showed a similar K_d and number of specific [³H]E₂ binding sites compared with normal values.²¹⁹ Similarly, ERα variants do not appear to play a role in SLE.²²⁰

Endocrine disruptors are chemicals in the environment that are thought to disrupt endogenous hormone action by acting as hormone agonists or antagonists in vivo. Endocrine disruptors have been postulated to play a role in the increased incidence of breast cancer in the United States since 1940,²²¹ decreased sperm counts,²²² and reproductive tract abnormalities in wildlife species.²²³

Among the chemicals thought to be xenoestrogens are various organochloride pesticides (e.g. dieldrin; polychlorinated biphenyls [PCBs], a family of 209 related compounds widely used as industrial coolants); alkylphenolic polyethoxylates (APEOs: 4-nonylphenol [NP] and 4-octylphenol [OP]), used as nonionic surfactants in detergents, paints, herbicides, and pesticides; bisphenol A, used in manufacturing plastics; and the heavy metal cadmium.²²² Endocrine disruptors are also natural dietary components (e.g. the phytoestrogen coumestrol).²²⁴ Because they are lipophilic, endocrine disruptors can enter the human body by ingestion or adsorption through the skin and mucosal membranes. Certain Endocrine disruptors, such as OP and NP,²²⁵ are present in water from sewage treatment plants and bioaccumulate in aquatic species and in animal fat.²²¹ Bisphenol A is a contaminant in canned foods²²⁶ and was found in human saliva after dental work.²²⁷ The level of human exposure to these agents and whether these levels are sufficient to cause harmful effects remain controversial.²²² Long-term exposure to exogenous estrogens is thought to increase the relative risk of breast, liver, ovarian, and uterine tumors.²²⁸

Because endocrine disruptors lack structural similarity to natural estrogens (i.e. E₂), it is not immediately obvious which compounds have estrogenic or antiestrogenic activity. A single endocrine disruptor may exhibit agonist activity with one steroid receptor and antagonist activity with a different receptor (e.g. DDE with ERα and AR).²²⁹ The U.S. Environmental Protection Agency (EPA) is funding considerable research effort on endocrine disruptors.

The past 15 years have seen remarkable progress in our understanding of the mechanism of action of steroid hormones and receptor function. The development of molecular cloning techniques and targeted gene disruption has greatly facilitated the molecular understanding of steroid receptor action. One major advance that arose through the application of these techniques is the concept of a large superfamily of related receptors that share a common modular structure. The steroid receptor superfamily includes receptors for steroid hormones and thyroid hormones and a diverse set of other gene regulators, some of which are called orphan receptors and whose ligands, if necessary, are unknown. Newly identified orphan receptor ligands include metabolic intermediates, indicating an important role for these compounds in regulating lipid metabolism and steroidogenesis.⁵ Tremendous progress has been made in understanding the way hormone-liganded steroid receptors interact with specific binding sites in hormone-regulated genes and the role of receptor interaction with other nuclear transcription factors in the regulation of hormone target gene transcription. Another significant advance is the identification of coactivators and corepressors as proteins that interact directly with steroid/nuclear receptors, but not with DNA, and that aid receptors in modulating target gene expression. The nucleosome remodeling activity of these coactivators and corepressors and their associated proteins has revealed the importance of chromatin structure in hormone-induced gene transcription. The importance of multiple levels of crosstalk between cell membrane-bound receptors, acting through second messenger phosphorylation cascades, and nuclear hormone receptors and between different classes of transcriptional enhancer proteins indicates the overall complexity involved in specific gene regulation. Developments in the analysis of abnormal receptor structure and function have enhanced our potential for clinical diagnosis and treatment of numerous endocrine disorders.

1. Mangelsdorf DJ, Thummel C, Beato M et al: The nuclear receptor superfamily: the second decade. Cell 83: 835– 839, 1995

2. Tsai M-J, O'Malley BW: Molecular mechanisms of action of steroid/thyroid receptor superfamily members. Annu Rev Biochem 63: 451– 483, 1994

3. Laudet V: Evolution of the nuclear receptor superfamily: early diversification from an ancestral orphan receptor. J Mol Endocrinol 19: 207– 226, 1997

4. Mangelsdorf DJ, Evans RM: The RXR heterodimers and orphan receptors. Cell 83: 841– 850, 1995

5. Blumberg B, Evans RM: Orphan nuclear receptors—New ligands and new possibilities. Genes Dev 12: 3149– 3155, 1998

6. Kuiper GGJM, Enmark E, Pelto-Huikko M, Nilsson S, Gustafsson J-A: Cloning of a novel estrogen receptor expressed in rat prostate and ovary. Proc Natl Acad Sci USA 93: 5925– 5930, 1996

7. Mosselman S, Polman J, Dijkrms R: ERβ: Identification and characterization of a novel human estrogen receptor. FEBS Lett 392: 49– 53, 1996

8. Ogawa S, Inoue S, Watanabe T et al: The complete primary structure of human estrogen receptor beta (hER beta) and its heterodimerization with ER alpha in vivo and in vitro. Biochem Biophys Res Commun 243: 122– 126, 1998

9. Harding HP, Lazar MA: The monomer-binding orphan receptor Rev-Erb represses transcription as a dimer on a novel direct repeat. Mol Cell Biol 15: 4791– 4802, 1995

10. Seol W, Chung M, Moore DD: Novel receptor interaction and repression domains in the orphan receptor SHP. Mol Cell Biol 17: 7126– 7131, 1997

11. Evans RM: The steroid and thyroid hormone receptor superfamily. Science 240: 889– 895, 1988

12. Montano MM, Muller V, Trogaugh A, Katzenellenbogen BS: The carboxy-terminal F domain of the human estrogen receptor: Role in the transcriptional activity of the receptor and the effectiveness of antiestrogens as estrogen antagonists. Mol Endocrinol 9: 814– 825, 1995

13. Green S, Chambon P: Oestradiol induction of a glucocorticoid-responsive gene by a chimaeric receptor. Nature 325: 75– 78, 1987

14. O'Malley BW: The steroid receptor superfamily: More excitement predicted for the future. Mol Endocrinol 4: 363– 369, 1990

15. Godowski PJ, Rusconi S, Miesfeld R, Yamamoto KR: Glucocorticoid receptor mutants that are constitutive activators of transcriptional enhancement. Nature 325: 365– 368, 1987

16. Richer JK, Lange CA, Wierman AM et al: Progesterone receptor variants found in breast cells repress transcription by wild-type receptors. Breast Cancer Res Treat 48: 231– 241, 1998

17. Gao T, McPhaul MJ: Functional activities of the A and B forms of the human androgen receptor in response to androgen receptor agonists and antagonists. Mol Endocrinol 12: 654– 663, 1998

18. Klug A, Schwabe JWR: Zinc fingers. FASEB J 9: 597– 604, 1995

19. Picard D, Kumar V, Chambon P, Yamamoto KR: Signal transduction by steroid hormones: Nuclear localization is differentially regulated in estrogen and glucocorticoid receptors. Cell Regul 1: 291– 299, 1990

20. Safer JD, Cohen RN, Hollenberg AN, Wondisford FE: Defective release of corepressor by hinge mutants of the thyroid hormone receptor found in patients with resistance to thyroid hormone. J Biol Chem 273: 30175– 3082, 1998

21. Jackson TA, Richer JK, Bain DL et al: The partial agonist activity of antagonist-occupied steroid receptors is controlled by a novel hinge domain-binding coactivator L7/SPA and the corepressors N-CoR or SMRT. Mol Endocrinol 11: 693– 705, 1997

22. Bourguet W, Ruff M, Chambon P et al: Crystal structure of the ligand-binding domain of the human nuclear receptor RXRα. Nature 375: 377– 382, 1995

23. Renaud J-P, Rochel N, Ruff M et al: Crystal structure of the RAR-γ ligand-binding domain bound to all-trans retinoic acid. Nature 378: 681– 689, 1995

24. Wagner RL, Apriletti JW, McGrath ME et al: A structural role for hormone in the thyroid hormone receptor: A structural role for hormone in the thyroid hormone receptor. Nature 378: 690– 697, 1995

25. Brzozowski AM, Pike AC, Dauter Z et al: Molecular basis of agonism and antagonism in the oestrogen receptor. Nature 389: 753– 758, 1997

26. Hollenberg SM, Weinberger C, Ong ES et al: Primary structure and expression of a functional human glucocorticoid receptor cDNA. Nature 318: 635– 641, 1985

27. Oakley RH, Webster JC, Sar M et al: Expression and subcellular distribution of the beta-isoform of the human glucocorticoid receptor. Endocrinology 138: 5028– 5038, 1997

28. Hanstein B, Liu H, Yancisin MC, Brown M: Functional analysis of a novel estrogen receptor-β isoform. Mol Endocrinol 13: 129– 137, 1999

29. Denis M, Gustafsson JA, Wikstrom AC: Interaction of the Mr = 90,000 heat shock protein with the steroid-binding domain of the glucocorticoid receptor. J Biol Chem 263: 18520– 18523, 1988

30. Tsai SY, Tsai M-J: Chick ovalbumin upstream promoter-transcription factors (COUP-TFs): Coming of age. Endocr Rev 18: 229– 240, 1997

31. Danielian PS, White R, Lees JA, Parker MG: Identification of a conserved region required for hormone dependent transcriptional activation by steroid hormone receptors. EMBO J 11: 1025– 1033, 1992

32. Horwitz K, Jackson T, Bain D et al: Nuclear receptor coactivators and corepressors. Mol Endocrinol 10: 1167– 1177, 1996

33. Beato M, Candau R, Chavez S et al: Interaction of steroid hormone receptors with transcription factors involves chromatin remodelling. J Steroid Biochem Mol Biol 56: 47– 59, 1996

34. Hollenberg SM, Evans RM: Multiple and cooperative trans-activation domains of the human glucocorticoid receptor. Cell 55: 899– 906, 1988

35. Tang Y, Getzenberg RH, Vietmeier BN et al: The DNA-binding and tau2 transactivation domains of the rat glucocorticoid receptor constitute a nuclear matrix-targeting signal. Mol Endocrinol 12: 1420– 1431, 1998

36. Stein GS, van Wijnen AJ, Stein JL et al: Interrelationships of nuclear structure and transcriptional control: Functional consequences of being in the right place at the right time. J Cell Biochem 70: 200– 212, 1998

37. Kraus WL, McInerney EM, Katzenellenbogen BS: Ligand-dependent, transcriptionally productive association of the amino- and carboxy-terminal regions of a steroid hormone nuclear receptor. Proc Natl Acad Sci USA 92: 12314– 12318, 1995

38. Xu M, Chakraborti PK, Garabedian MJ et al: Modular structure of glucocorticoid receptor domains is not equivalent to functional independence: Stability and activity of the steroid binding domain are controlled by sequences in separate domains. J Biol Chem 271: 21430– 21438, 1996

39. Greenblatt J: RNA polymerase II holoenzyme and transcriptional regulation. Curr Opin Cell Biol 9: 310– 319, 1997

40. Sauer F, Tjian R: Mechanisms of transcriptional activation: Differences and similarities between yeast, Drosophila, and man. Curr Opin Genet Dev 7: 176– 181, 1997

41. Beato M, Sanchez-Pacheco A: Interaction of steroid hormone receptors with the transcription initiation complex. Endocr Rev 17: 587– 609, 1996

42. Beato M, Chalepakis G, Schauer M, EPS: DNA regulatory elements for steroid hormones. J Steroid Biochem 32: 737– 47, 1989

43. Mymryk JS, Archer TK: Influence of hormone antagonists on chromatin remodeling and transcription factor binding to the mouse mammary tumor virus promoter in vivo. Mol Endocrinol 9: 1825– 1834, 1995

44. Jantzen HM, Strahle U, Gloss B et al: Cooperativity of glucocorticoid response elements located far upstream of the tyrosine aminotransferase gene. Cell 49: 29– 38, 1987

45. Cole TJ, Blendy JA, Schmid W et al: Expression of the mouse glucocorticoid receptor and its role during development. J Steroid Biochem Mol Biol 47: 49– 53, 1993

46. Drouin J, Sun YL, Chamberland M et al: Novel glucocorticoid receptor complex with DNA element of the hormone-repressed POMC gene. EMBO J 12: 145– 156, 1993

47. Chandran UR, Attardi B, Friedman R et al: Glucocorticoid repression of the mouse gonadotropin-releasing hormone gene is mediated by promoter elements that are recognized by heteromeric complexes containing glucocorticoid receptor. J Biol Chem 271: 20412– 20420, 1996

48. Subramaniam N, Cairns W, Okret S: Studies on the mechanism of glucocorticoid-mediated repression from a negative glucocorticoid response element from the bovine prolactin gene. DNA Cell Biol 16: 153– 163, 1997

49. Klein-Hitpass L, Ryffel GU, Heitlinger E, Cato AC: A 13 bp palindrome is a functional estrogen responsive element and interacts specifically with estrogen receptor. Nucleic Acids Res 16: 647– 663, 1988

50. Anolik JH, Klinge CM, Brolly CL et al: Stability of the ligand of estrogen response element-bound estrogen receptor depends on flanking sequences and cellular factors. J Steroid Biochem Mol Biol 59: 413– 429, 1996

51. Walker P, Germond JE, Brown-Luedi M et al: Sequence homologies in the region preceding the transcription initiation site of the liver estrogen-responsive vitellogenin and apo-VLDL II genes. Nucleic Acids Res 12: 8611– 8626, 1984

52. Jeltsch JM, Roberts M, Schatz C et al: Structure of the human oestrogen-responsive gene pS2. Nucleic Acids Res 15: 1401– 1414, 1987

53. Weisz A, Bresciani F: Estrogen induces expression of c-fos and c-myc protooncogenes in rat uterus. Mol Endocrinol 2: 816– 824, 1988

54. Kraus WL, Montano MM, Katzenellenbogen BS: Identification of multiple, widely spaced estrogen-responsive regions in the rat progesterone receptor gene. Mol Endocrinol 8: 952– 969, 1994

55. Umesono K, Murakami KK, Thompson CC, Evans RM: Direct repeats as selective response elements for thyroid hormone, retinoic acid, and vitamin D₃ receptors. Cell 65: 1255– 1266, 1991

56. Thompson EB: Steroid hormones: Membrane transporters of steroid hormones. Curr Biol 5: 730– 732, 1995

57. Pugeat M, Moulin P, Cousin P et al: Interrelations between sex hormone-binding globulin (SHBG), plasma lipoproteins and cardiovascular risk. J Steroid Biochem Mol Biol 53: 567– 572, 1995

58. Joseph DR: Structure, function, and regulation of androgen-binding protein/sex hormone-binding globulin. Vitam Horm 49: 197– 280, 1994

59. Danzo BJ, Joseph DR: Structure-function relationships of rat androgen—binding protein/human sex hormone binding globulin: the effect of mutagenesis on steroid-binding parameters. Endocrinology 135: 157– 167, 1994

60. Westphal U: Steroid-protein interactions II. Monogr Endocrinol 27: 1– 603, 1986

61. Porto CS, Lazari MF, Abreu LC, Bardin CW, Gunsalus GL: Receptors for androgen-binding proteins: Internalization and intracellular signalling. J Steroid Biochem Mol Biol 53: 561– 565, 1995

62. Rosner W, Hryb DJ, Khan MS et al: Sex hormone-binding globulin: anatomy and physiology of a new regulatory system. J Steroid Biochem Mol Biol 40: 813– 80, 1991

63. Jensen EV, DeSombre ER: Estrogen-receptor interaction. Science 182: 126– 14, 1973

64. Gorski J, Hansen JC, Welshons WV: Estrogen receptors as nuclear proteins. Adv Exp Med Biol 230: 13– 29, 1987

65. Dittmar KD, Demady DR, Stancato LF et al: Folding of the glucocorticoid receptor by the heat shock protein (hsp) 90-based chaperone machinery: The role of p23 is to stabilize receptor·hsp90 heterocomplexes formed by hsp90·p60·hsp70. J Biol Chem 272: 21213– 21220, 1997

66. Gehring U: Steroid hormone receptors and heat shock proteins. Vitam Horm 54: 167– 205, 1998

67. Pratt WB, Toft DO: Steroid receptor interactions with heat shock protein and immunophilin chaperones. Endocr Rev 18: 306– 360, 1997

68. Pratt WB, Scherrer LC, Hutchison KA, Dalman FC: A model of glucocorticoid receptor unfolding and stabilization by a heat shock protein complex. J Steroid Biochem Mol Biol 41: 223– 229, 1992

69. King WJ, Greene GL: Monoclonal antibodies localize oestrogen receptor in the nuclei of target cells. Nature 307: 745– 747, 1984

70. Perrot-Applanat M, Groyer-Picard MT, Logeat F, Milgrom E: Ultrastructural localization of the progesterone receptor by an immunogold method: Effect of hormone administration. J Cell Biol 102: 1191– 1199, 1986

71. Husmann DA, Wilson CM, McPhaul MJ et al: Antipeptide antibodies to two distinct regions of the androgen receptor localize the receptor protein to the nuclei of target cells in the rat and human prostate. Endocrinology 126: 2359– 2368, 1990

72. Segnitz B, Gehring U: Subunit structure of the nonactivated human estrogen receptor. Proc Natl Acad Sci USA 92: 2179– 2183, 1995

73. Lee HS, Aumais J, White JH: Hormone-dependent transactivation by estrogen receptor chimeras that do not interact with hsp90. Evidence for transcriptional repressors. J Biol Chem 271: 25727– 25730, 1996

74. Landel CC, Kushner PJ, Greene GL: The interaction of human estrogen receptor with DNA is modulated by receptor-associated proteins. Mol Endocrinol 8: 1407– 1419, 1994

75. Xiao N, DeFranco DB: Overexpression of unliganded steroid receptors activates endogenous heat shock factor. Mol Endocrinol 11: 1365– 1374, 1997

76. Klinge CM, Brolly CL, Bambara RA, Hilf R: Hsp70 is not required for high affinity binding of purified calf uterine estrogen receptor to estrogen response element DNA in vitro. J Steroid Biochem Mol Biol 63: 283– 301, 1997

77. Guiochon-Mantel A, Delabre K, Lescop P, Milgrom E: Intracellular traffic of steroid hormone receptors. J Steroid Biochem Mol Biol 56: 1– 6, 1996

78. DeFranco DB: Subnuclear trafficking of steroid receptors. Biochem Soc Trans 25: 592– 7, 1997

79. Ruh MF, Cox LK, Ruh TS: Estrogen receptor interaction with specific histones. Biochem Pharmacol 52: 869– 878, 1996

80. Zhu XG, Hanover JA, Hager GL, Cheng SY: Hormone-induced translocation of thyroid hormone receptors in living cells visualized using a receptor green fluorescent protein chimera. J Biol Chem 273: 27058– 27063, 1998

81. Kurokawa R, Soderstrom M, Horlein A et al: Polarity-specific activities of retinoid acid receptors determined by a co-repressor. Nature 377: 451– 454, 1995

82. Horlein AJ, Naar AM, Heinzel T et al: Ligand-independent repression by the thyroid hormone receptor mediated by a nuclear receptor co-repressor. Nature 377: 397– 404, 1995

83. Carson MA, Tsai MJ, Conneely OM et al: Structure-function properties of the chicken progesterone receptor A synthesized from complementary deoxyribonucleic acid. Mol Endocrinol 1: 791– 801, 1987

84. Hollenberg SM, Giguere V, Segui P, Evans RM: Colocalization of DNA-binding and transcriptional activation functions in the human glucocorticoid receptor. Cell 49: 39– 46, 1987

85. Chen JD, Evans RM: A transcriptional co-repressor that interacts with nuclear hormone receptors. Nature 377: 454– 457, 1995

86. Alland L, Muhle R, Hou HJ et al: Role for N-CoR and histone deacetylase in Sin3-mediated transcriptional repression. Nature 387: 49– 55, 1997

87. Heinzel T, Lavinsky RM, Mullen T-M et al: A complex containing N-CoR, mSin3 and histone deacetylase mediates transcriptional repression. Nature 387: 43– 48, 1997

88. Nagy L, Kao H-Y, Chakravarti D et al: Nuclear receptor repression mediated by a complex containing SMRT, mSin3A, and histone deacetylase. Cell 89: 373– 380, 1997

89. Weigel NL, Zhang Y: Ligand-independent activation of steroid hormone receptors. J Mol Med 76: 469– 479, 1998

90. Eickelberg O, Roth M, Lorx R et al: Ligand-independent activation of the glucocorticoid receptor by beta 2-adrenergic receptor agonists in primary human lung fibroblasts and vascular smooth muscle cells. J Biol Chem 274: 1005– 1010, 1999

91. Massaad C, Houard N, Lombes M, Barouki R: Modulation of human mineralocorticoid receptor function by protein kinase A. Mol Endocrinol 13: 57– 65, 1999

92. Starr D, Hoopes B, Hawley D: DNA bending is an important component of site-specific recognition by the TATA binding protein. J Mol Biol 250: 434– 446, 1995

93. Nardulli AM, Greene GL, Shapiro DJ: Human estrogen receptor bound to an estrogen response element bends DNA. Mol Endocrinol 7: 331– 340, 1993

94. Petz LN, Nardulli AM, Kim J et al: DNA bending is induced by binding of the glucocorticoid receptor DNA-binding domain and progesterone receptors to their response element. J Steroid Biochem Mol Biol 60: 31– 41, 1997

95. Prendergast P, Pan Z, Edwards DP: Progesterone receptor-induced bending of its target DNA: Distinct effects of the A and B receptor forms. Mol Endocrinol 10: 393– 407, 1996

96. Shulemovich K, Dimaculangan DD, Katz D, Lazar MA: DNA bending by thyroid hormone receptor: Influence of half-site spacing and RXR. Nucleic Acids Res 23: 811– 818, 1995

97. Boonyaratanakornkit V, Melvin V, Prendergast P et al: High-mobility group chromatin proteins 1 and 2 functionally interact with steroid hormone receptors to enhance their DNA binding in vitro and transcriptional activity in mammalian cells. Mol Cell Biol 18: 4471– 4487, 1998

98. Verrijzer CP, Tjian R: TAFs mediate transcriptional activation and promoter selectivity. Trends Biochem Sci 21: 338– 342, 1996

99. Pfahl M: Nuclear receptor/AP-1 interaction. Endocr Rev 14: 651– 658, 1993

100. Scholz A, Truss M, Beato M: Hormone-induced recruitment of Sp1 mediates estrogen activation of the rabbit uteroglobin gene in endometrial epithelium. J Biol Chem 273: 4360– 4366, 1998

101. Klein-Hitpass L, Schwerk C, Kahmann S, Vassen L: Targets of activated steroid hormone receptors: Basal transcription factors and receptor interacting proteins. J Mol Med 76: 490– 496, 1998

102. Meyer ME, Gronemeyer H, Turcotte B et al: Steroid hormone receptors compete for factors that mediate their enhancer function. Cell 57: 433– 442, 1989

103. Yoshinaga SK, Peterson CL, Herskowitz I, Yamamoto KR: Roles of SWI1, SWI2, and SWI3 proteins for transcriptional enhancement by steroid receptors. Science 258: 1598– 1604, 1992

104. Ding XF, Anderson CM, Ma H et al: Nuclear receptor-binding sites of coactivators glucocorticoid receptor interacting protein 1 (GRIP1) and steroid receptor coactivator 1 (SRC-1): Multiple motifs with different binding specificities. Mol Endocrinol 12: 302– 313, 1998

105. Eng FCS, Barsalou A, Akutsu N et al: Different classes of coactivators recognize distinct but overlapping binding sites on the estrogen receptor ligand binding domain. J Biol Chem 273: 28371– 28377, 1998

106. Dallas PB, Cheney IW, Liao D-W et al: p300/CREB binding protein-related protein p270 Is a component of mammalian SWI/SNF complexes. Mol Cell Biol 18: 3596– 3603, 1998

107. Spencer TE, Jenster G, Burcin MM et al: Steroid receptor coactivator-1 is a histone acetyltransferase. Nature 389: 194– 198, 1997

108. Struhl K: Histone acetylation and transcriptional regulatory mechanisms. Genes Dev 12: 599– 606, 1998

109. Tsukiyama T, Wu C: Chromain remodeling and transcription. Curr Opin Gen Dev 7: 182– 191, 1997

110. Shibata H, Spencer TE, Onate SA et al: Role of co-activators and co-repressors in the mechanism of steroid/thyroid receptor action. Recent Prog Horm Res 52: 141– 164, 1997

111. Zhou G, Cummings R, Li Y et al: Nuclear receptors have distinct affinities for coactivators: Characterization by fluorescence resonance energy transfer. Mol Endocrinol 12: 1594– 604, 1998

112. Misiti S, Schomburg L, Yen PM, Chin WW: Expression and hormonal regulation of coactivator and corepressor genes. Endocrinology 139: 2493– 500, 1998

113. Folkers GE, van der Burg B, van der Saag PT: Promoter architecture, cofactors, and orphan receptors contribute to cell-specific activation of the retinoic acid receptor beta2 promoter. J Biol Chem 273: 32200– 12, 1998

114. Gottlicher M, Heck S, Herrlich P: Transcriptional cross-talk, the second mode of steroid hormone receptor action. J Mol Med 76: 480– 489, 1998

115. Maroder M, Farina AR, Vacca A et al: Cell-specific bifunctional role of Jun oncogene family members on glucocorticoid receptor-dependent transcription. Mol Endocrinol 7: 570– 584, 1993

116. Diamond MI, Miner JN, Yoshinaga SK, Yamamoto KR: Transcription factor interactions: Selectors of positive or negative regulation from a single DNA element. Science 249: 1266– 72, 1990

117. Webb P, Lopez GN, Uht RM, Kushner PJ: Tamoxifen activation of the estrogen receptor/AP-1 pathway. Mol Endocrinol 9: 443– 456, 1995

118. Tzukerman M, Zhang XK, Pfahl M: Inhibition of estrogen receptor activity by the tumor promoter 12-O-tetradeconylphorbol-13-acetate: A molecular analysis. Mol Endocrinol 5: 1983– 1992, 1991

119. Paech K, Webb P, Kuiper GG et al: Differential ligand activation of estrogen receptors ERalpha and ERbeta at AP1 sites. Science 277: 1508– 1510, 1997

120. Krishnan V, Wang X, Safe S: Estrogen receptor-SP1 complexes mediate estrogen-induced cathepsin D gene expression in MCF-7 human breast cancer cells. J Biol Chem 269: 15912– 15917, 1994

121. Rishi AK, Shao ZM, Baumann RG et al: Estradiol regulation of the human retinoic acid receptor alpha gene in human breast carcinoma cells is mediated via an imperfect half-palindromic estrogen response element and Sp1 motifs. Cancer Res 55: 4999– 5006, 1995

122. Duan R, Porter W, Safe S: Estrogen-induced c-fos protooncogene expression in MCF-7 human breast cancer Cells: Role of estrogen receptor Sp1 complex formation. Endocrinology 139: 1981– 1990, 1998

123. Porter W, Saville B, Hoivik D, Safe S: Functional synergy between the transcription factor Sp1 and the estrogen receptor. Mol Endocrinol 11: 1569– 1580, 1997

124. Fronsdal K, Engedal N, Slagsvold T, Saatcioglu F: CREB binding protein is a coactivator for the androgen receptor and mediates cross-talk with AP-1. J Biol Chem 273: 31853– 9, 1998

125. Sheppard KA, Phelps KM, Williams AJ et al: Nuclear integration of glucocorticoid receptor and nuclear factor-kappa B signaling by CREB-binding protein and steroid receptor coactivator-1. J Biol Chem 273: 29291– 29294, 1998

126. Pazin MJ, Kadonaga JT: What's up and down with histone deacetylation and transcription? Cell 89: 325– 8, 1997

127. Gelmetti C, Zhang J, Minucci S et al: Aberrant recruitment of the nuclear receptor corepressor-histone deacetylase complex by the acute myeloid leukemia fusion partner ETO. Mol Cell Biol 18: 7185– 7191, 1998

128. Klinge CM, Bambara RA, Hilf R: Antiestrogen-liganded estrogen receptor interaction with estrogen responsive element DNA in vitro. J Steroid Biochem Mol Biol 43: 249– 262, 1992

129. Klinge CM, Silver BF, Driscoll MD et al: COUP-TF interacts with estrogen receptor, binds to estrogen response elements and half-sites, and modulates estrogen-induced gene expression. J Biol Chem 272: 31465– 31474, 1997

130. Somjen D, Waisman A, Kaye AM: Tissue selective action of tamoxifen methiodide, raloxifene and tamoxifene on creatinine kinase B activity in vitro and in vivo. J Steroid Biochem Mol Biol 59: 389– 396, 1996

131. Onate SA, Boonyaratanakornkit V, Spencer TE et al: The steroid receptor coactivator-1 contains multiple receptor interacting and activation domains that cooperatively enhance the activation function 1 (AF1) and AF2 domains of steroid receptors. J Biol Chem 273: 12101– 12108, 1998

132. Le Douarin B, You J, Nielsen AL, Chambon P, Losson R: TIF1α: A possible link between KRAB zinc finger proteins and nuclear receptors. J Steroid Biochem Mol Biol 65: 43– 50, 1998

133. Smith CL, Nawaz Z, O'Malley BW: Coactivator and corepressor regulation of the agonist/antagonist activity of the mixed antiestrogen, 4-hydroxytamoxifen. Mol Endocrinol 11: 657– 666, 1997

134. Lavinsky R, Jepsen K, Heinzel T et al: Diverse signaling pathways modulate nuclear receptor recruitment of N-CoR and SMRT complexes. Proc Natl Acad Sci USA 95: 2920– 2925, 1998

135. Wehling M: Specific, nongenomic actions of steroid hormones. Annu Rev Physiol 59: 365– 93, 1997

136. Gametchu B, Watson CS, Pasko D: Size and steroid-binding characterization of membrane-associated glucocorticoid receptor in S-49 lymphoma cells. Steroids 56: 402– 10, 1991

137. Pappas TC, Gametchu B, Watson CS: Membrane estrogen receptors identified by multiple antibody labeling and impeded-ligand binding. FASEB J 9: 404– 10, 1995

138. Watson CS, Pappas TC, Gametchu B: The other estrogen receptor in the plasma membrane: Implications for the actions of environmental estrogens. Environ Health Perspect 103 (Suppl 7):41– 50, 1995

139. Steinsapir J: Microsomal steroid receptors in target tissues. Receptor 2: 45– 76, 1992

140. Fink G, Sumner BE, McQueen JK et al: Sex steroid control of mood, mental state and memory. Clin Exp Pharmacol Physiol 25: 764– 775, 1998

141. Farhat MY, Abi-Younes S, Dingaan B et al: Estradiol increases cyclic adenosine monophosphate in rat pulmonary vascular smooth muscle cells by a nongenomic mechanism. J Pharmacol Exp Ther 276: 652– 657, 1996

142. Mueck AO, Seeger H, Lippert TH: Calciumantagonistic effect of natural and synthetic estrogens—investigations on a nongenomic mechanism of direct vascular action. Int J Clin Pharmacol Ther 34: 424– 426, 1996

143. Kauser K, Rubanyi GM: Potential cellular signaling mechanisms mediating upregulation of endothelial nitric oxide production by estrogen. J Vasc Res 34: 229– 236, 1997

144. Tesarik J, Mendoza C: Direct non-genomic effects of follicular steroids on maturing human oocytes: Oestrogen versus androgen antagonism. Hum Reprod Update 3: 95– 100, 1997

145. Razandi M, Pedram A, Greene GL, Levin ER: Cell membrane and nuclear estrogen receptors (ERs) originate from a single transcript: Studies of ERα and ERβ expressed in Chinese hamster ovary cells. Mol Endocrinol 13: 307– 319, 1999

146. Scherrer LC, Pratt WB: Association of the transformed glucocorticoid receptor with a cytoskeletal protein complex. J Steroid Biochem Mol Biol 41: 719– 721, 1992

147. Koukouritaki SB, Margioris AN, Gravanis A et al: Dexamethasone induces rapid actin assembly in human endometrial cells without affecting its synthesis. J Cell Biochem 65: 492– 500, 1997

148. Demonacos CV, Karayanni N, Hatzoglou E et al: Mitochondrial genes as sites of primary action of steroid hormones. Steroids 61: 226– 232, 1996

149. Orchinik M, Hastings N, Witt D, McEwen BS: High-affinity binding of corticosterone to mammalian neuronal membranes: Possible role of corticosteroid binding globulin. J Steroid Biochem Mol Biol 60: 229– 236, 1997

150. Baulieu EE, Robel P: Non-genomic mechanisms of action of steroid hormones. Ciba Found Symp 191: 24– 37, 1995

151. Revelli A, Massobrio M, Tesarik J: Nongenomic actions of steroid hormones in reproductive tissues. Endocr Rev 19: 3– 17, 1998

152. Meyer C, Schmid R, Schmieding K et al: Characterization of high affinity progesterone-binding membrane proteins by anti-peptide antiserum. Steroids 63: 111– 116, 1998

153. Hawkinson JE, Kimbrough CL, Belelli D et al: Correlation of neuroactive steroid modulation of [35S]t-butylbicyclophosphorothionate and [3H]flunitrazepam binding and gamma-aminobutyric acidA receptor function. Mol Pharmacol 46: 977– 985, 1994

154. Grazzini E, Guillon G, Mouillac B, Zingg HH: Inhibition of oxytocin receptor function by direct binding of progesterone. Nature 392: 509– 512, 1998

155. Kastner P, Krust A, Turcotte B et al: Two distinct estrogen-regulated promoters generate transcripts encoding the two functionally different human progesterone receptor forms A and B. EMBO J 9: 1603– 1614, 1990

156. Lazar MA: Thyroid hormone receptors: multiple forms, multiple possibilities. Endocr Rev 14: 184– 193, 1993

157. Syms AJ, Norris JS, Panko WB, Smith RG: Mechanism of androgen-receptor augmentation: Analysis of receptor synthesis and degradation by the density-shift technique. J Biol Chem 260: 455– 461, 1985

158. Nardulli AM, Greene GL, BW OM, Katzenellenbogen BS: Regulation of progesterone receptor messenger ribonucleic acid and protein levels in MCF-7 cells by estradiol: analysis of estrogen's effect on progesterone receptor synthesis and degradation. Endocrinology 122: 935– 944, 1988

159. McIntyre WR, Samuels HH: Triamcinolone acetonide regulates glucocorticoid-receptor levels by decreasing the half-life of the activated nuclear-receptor form. J Biol Chem 260: 418– 47, 1985

160. Hiipakka RA, Liao S: Steroid receptor recycling and interaction of receptor with RNA. Am J Clin Oncol 11 (Suppl 2):S18- S22, 1988

161. Horwitz KB, McGuire WL: Nuclear mechanisms of estrogen action. Effects of estradiol and anti-estrogens on estrogen receptors and nuclear receptor processing. J Biol Chem 253: 8185– 8191, 1978

162. Lee JH, Kim J, Shapiro DJ: Regulation of Xenopus laevis estrogen receptor gene expression is mediated by an estrogen response element in the protein coding region. DNA Cell Biol 14: 419– 430, 1995

163. Okulicz WC, Evans RW, Leavitt WW: Progesterone regulation of estrogen receptor in the rat uterus: A primary inhibitory influence on the nuclear fraction. Steroids 37: 463– 470, 1981

164. Dotzlaw H, Leygue E, Watson PH, Murphy LC: Estrogen receptor-β messenger RNA expression in human breast tumor biopsies: Relationship to steroid receptor status and regulation by progestins. Cancer Res 59: 529– 532, 1999

165. Okret S, Dong Y, Bronnegard M, Gustafsson JA: Regulation of glucocorticoid receptor expression. Biochimie 73: 51– 59, 1991

166. Couse JF, Korach KS: Exploring the role of sex steroids through studies of receptor deficient mice. J Mol Med 75: 497– 511, 1998

167. Couse JF, Curtis SW, Washburn TF et al: Analysis of transcription and estrogen insensitivity in the female mouse after targeted disruption of the estrogen receptor gene. Mol Endocrinol 9: 1441– 1454, 1995

168. Lubahn DB, Moyer JS, Golding TS et al: Alteration of reproductive function but not prenatal sexual development after insertional disruption of the mouse estrogen receptor gene. Proc Natl Acad Sci USA 90: 11162– 11166, 1993

169. Korach KS, Couse JF, Curtis SW et al: Estrogen receptor gene disruption: molecular characterization and experimental and clinical phenotypes. Recent Prog Horm Res 51:159–186; discussion 186–188, 1996

170. Ogawa S, Taylor JA, Lubahn DB et al: Reversal of sex roles in genetic female mice by disruption of estrogen receptor gene. Neuroendocrinology 64: 467– 470, 1996

171. Hess RA, Bunick D, Lee KH et al: A role for oestrogens in the male reproductive system. Nature 390: 509– 512, 1997

172. Ogawa S, Lubahn DB, Korach KS, Pfaff DW: Behavioral effects of estrogen receptor gene disruption in male mice. Proc Natl Acad Sci USA 94: 1476– 1481, 1997

173. Iafrati MD, Karas RH, Aronovitz M et al: Estrogen inhibits the vascular injury response in estrogen receptor alpha-deficient mice. Nat Med 3: 545– 548, 1997

174. Smith EP, Boyd J, Frank GR et al: Estrogen resistance caused by a mutation in the estrogen-receptor gene in a man. N Engl J Med 331: 1056– 1061, 1994

175. Krege JH, Hodgin JB, Couse JF et al: Generation and reproductive phenotypes of mice lacking estrogen receptor beta. Proc Natl Acad Sci USA 95: 15677– 15682, 1998

176. Lydon JP, DeMayo FJ, Funk CR et al: Mice lacking progesterone receptor exhibit pleiotropic reproductive abnormalities. Genes Dev 9: 2266– 2278, 1995

177. Chappell PE, Lydon JP, Conneely OM et al: Endocrine defects in mice carrying a null mutation for the progesterone receptor gene. Endocrinology 138: 4147– 4152, 1997

178. Reichardt HM, Kaestner KH, Wessely O et al: Analysis of glucocorticoid signalling by gene targeting. J Steroid Biochem Mol Biol 65: 111– 115, 1998

179. Tronche F, Kellendonk C, Reichardt HM, Schutz G: Genetic dissection of glucocorticoid receptor function in mice. Curr Opin Genet Dev 8: 532– 538, 1998

180. Reichardt HM, Kaestner KH, Tuckermann J et al: DNA binding of the glucocorticoid receptor is not essential for survival. Cell 93 1: 531– 554, 1998

181. Berger S, Bleich M, Schmid W et al: Mineralocorticoid receptor knockout mice: Pathophysiology of Na+ metabolism. Proc Natl Acad Sci USA 95: 9424– 9429, 1998

182. Hubert C, Gasc J-M, Berger S et al: Effects of mineralocorticoid receptor gene disruption on the components of the renin-angiotensin system in 8-day-old mice. Mol Endocrinol 13: 297– 306, 1999

183. He WW, Lindzey JK, Prescott JL, Kumar MV, Tindall DJ: The androgen receptor in the testicular feminized (Tfm) mouse may be a product of internal translation initiation. Receptor 4: 121– 134, 1994

184. Lindzey J, Kumar MV, Grossman M et al: Molecular mechanisms of androgen action. Vitam Horm 49: 383– 432, 1994

185. Rao CV: Cell membrane receptors. In: Sciarra J, ed. Gynecology and Obstetrics. Philadelphia: Harper & Row, 1988

186. Clark GM, McGuire WL: Steroid receptors and other prognostic factors in primary breast cancer. Semin Oncol 15: 20– 25, 1988

187. Chamness GC, Zava DT, McGuire WL: Methods for assessing the binding of steroid hormones in nuclei. Methods Cell Biol 17: 325– 338, 1978

188. Syne JS, Markaverich BM, Clark JH, Panko WB: Estrogen binding sites in the nucleus of normal and malignant human tissue: Optimization of an exchange assay for the measurement of specific binding. Cancer Res 42: 4443– 4448, 1982

189. Pavlik EJ, Coulson PB: Hydroxylapatite "batch" assay for estrogen receptor: Increased sensitivity over present receptor assays. J Steroid Biochem 7: 357– 368, 1976

190. Pasic R, Djulbegovic B, Wittliff JL: Comparison of sex steroid receptor determinations in human breast cancer by enzyme immunoassay and radioligand binding. J Clin Lab Anal 4: 430– 436, 1990

191. Jordan VC: Tamoxifen treatment for breast cancer: Concept to gold standard. Oncology 11 (Suppl 1):7– 13, 1997

192. Scatchard G: The attraction of proteins for small molecules and ions. Ann N Y Acad Sci 51: 660– 672, 1949

193. Yeh S, Miyamoto H, Shima H, Chang C: From estrogen to androgen receptor: a new pathway for sex hormones in prostate. Proc Natl Acad Sci USA 95: 5527– 5532, 1998

194. Sambrook J, Fritsch EF, Maniatis T: Molecular Cloning. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press, 1989

195. He WW, Fischer LM, Sun S et al: Molecular cloning of androgen receptors from divergent species with a polymerase chain reaction technique: Complete cDNA sequence of the mouse androgen receptor and isolation of androgen receptor cDNA probes from dog, guinea pig and clawed frog. Biochem Biophys Res Commun 171: 697– 704, 1990

196. Kumar V, Green S, Stack G et al: Functional domains of the human estrogen receptor. Cell 51: 941– 951, 1987

197. Driscoll MD, Sathya G, Muyan M et al: Sequence requirements for estrogen receptor binding to estrogen response elements. J Biol Chem 273: 29321– 29330, 1998

198. Theulaz I, Hipskind R, ten Heggeler-Bordier B et al: Expression of human estrogen receptor mutants in Xenopus oocytes: correlation between transcriptional activity and ability to form protein-DNA complexes. EMBO J 7: 1653– 60, 1988

199. Montano MM, Jaiswal AK, Katzenellenbogen BS: Transcriptional regulation of the human quinone reductase gene by antiestrogen-liganded estrogen receptor-alpha and estrogen receptor-beta. J Biol Chem 273: 25443– 25449, 1998

200. Van Tendeloo VF, Snoeck HW, Lardon F et al: Nonviral transfection of distinct types of human dendritic cells: High-efficiency gene transfer by electroporation into hematopoietic progenitor- but not monocyte-derived dendritic cells. Gene Ther 5: 700– 707, 1998

201. Genazzani AR, Lucchesi A, Stomati M et al: Effects of sex steroid hormones on the neuroendocrine system. Eur J Contracept Reprod Health Care 2: 63– 69, 1997

202. Mani SK, Blaustein JD, Allen JM et al: Inhibition of rat sexual behavior by antisense oligonucleotides to the progesterone receptor. Endocrinology 135: 1409– 1414, 1994

203. Mani SK, Blaustein JD, BW OM: Progesterone receptor function from a behavioral perspective. Horm Behav 31: 244– 255, 1997

204. Phelps SM, Lydon JP, BW Om, Crews D: Regulation of male sexual behavior by progesterone receptor, sexual experience, and androgen. Horm Behav 34:294–302, 1998

205. Murono K, Mendonca BB, Arnhold IJ et al: Human androgen insensitivity due to point mutations encoding amino acid substitutions in the androgen receptor steroid-binding domain. Hum Mutat 6: 152– 162, 1995

206. Shkolny DL, Brown TR, Punnett HH et al: Characterization of alternative amino acid substitutions at arginine 830 of the androgen receptor that cause complete androgen insensitivity in three families. Hum Mol Genet 4: 515– 521, 1995

207. Murphy LC, Dotzlaw H, Leygue E et al: The pathophysiological role of estrogen receptor variants in human breast cancer. J Steroid Biochem Mol Biol 65: 175– 180, 1998

208. McGuire WL, Clark GM, Dressler LG et al: Role of steroid hormone receptors as prognostic factors in primary breast cancer. NCL Monogr 19–23, 1986

209. Speirs V, Parkes AT, Kerin MJ et al: Coexpression of estrogen receptor alpha and beta: Poor prognostic factos in human breast cancers? Cancer Res 59: 525– 528, 1999

210. Fukuda K, Mori M, Uchiyama M et al: Prognostic significance of progesterone receptor immunohistochemistry in endometrial carcinoma. Gynecol Oncol 69: 220– 225, 1998

211. Hempling RE, Piver MS, Eltabbakh GH, Recio FO: Progesterone receptor status is a significant prognostic variable of progression-free survival in advanced epithelial ovarian cancer. Am J Clin Oncol 21: 447– 451, 1998

212. Arai K, Chrousos GP: Syndromes of glucocorticoid and mineralocorticoid resistance. Steroids 60: 173– 179, 1995

213. DeRijk R, Sternberg EM: Corticosteroid resistance and disease. Ann Med 29: 79– 82, 1997

214. Lane SJ, Lee TH: Mechanisms of corticosteroid resistance in asthmatic patients. Int Arch Allergy Immunol 113: 193– 195, 1997

215. Kuhnle U, Hinkel GK, Akkurt HI, Krozowski Z: Familial pseudohypoaldosteronism: A review on the heterogeneity of the syndrome. Steroids 60: 157– 160, 1995

216. Fuller PJ, Lim-Tio S: Aldosterone action, sodium channels and inherited disease. J Endocrinol 148: 387– 390, 1996

217. Cutolo M, Sulli A, Seriolo B et al: Estrogens, the immune response and autoimmunity. Clin Exp Rheumatol 13: 217– 226, 1995

218. Kelly RH, Vertosick FT Jr: Sytemic lupus erythematosus: a role for anti-receptor antibodies? Med Hypotheses 20: 95– 101, 1986

219. Suenaga R, Mitamura K, Evans MJ, Abdou NI: Binding affinity and quantity of estrogen receptor in peripheral blood monocytes of patients with systemic lupus erythematosus. Lupus 5: 227– 231, 1996

220. Suenaga R, Evans MJ, Mitamura K et al: Peripheral blood T cells and monocytes and B cell lines derived from patients with lupus express estrogen receptor transcripts similar to those of normal cells. J Rheumatol 25: 1305– 1312, 1998

221. Davis DL, Bradlow HL, Wolff M et al: Medical hypothesis: Xenoestrogens as preventable causes of breast cancer. Environ Health Persect 101: 372– 377, 1993

222. Feldman D: Estrogens from plastic—Are we being exposed? [Editorial] Endocrinology 138: 1777– 1779, 1997

223. Wolff MS, Collman GW, Barrett JC et al: Breast cancer and environmental risk factors: Epidemiological and experimental findings. Annu Rev Pharmacol Toxicol 36: 573– 596, 1996

224. Miksicek RJ: Commonly occurring plant flavonoids have estrogenic activity. Mol Pharm 44: 37– 43, 1993

225. Giger W, Brunner PH, Schaffner C: 4-Nonylphenol in sewage sludge: Accumulation of toxic metabolites from nonionic surfactants. Science 225: 623– 625, 1984

226. Brotons JA, Olea-Serrano MF, Villalobos M et al: Xenoestrogens released from lacquer coatings in food cans. Environ Health Perspect 104: 608– 612, 1995

227. Olea N, Pulgar R, Perez P et al: Estrogenicity of resin-based composites and sealants used in dentistry. Environ Health Perspect 104: 298– 305, 1996

228. Lupulescu A: Estrogen use and cancer risk: A review. Exp Clin Endocrinol 101: 204– 214, 1993

229. Sonnenschein C, Soto AM: An updated review of environmental estrogen and androgen mimics and antagonists. J Steroid Biochem Mol Biol 65: 143– 150, 1998

230. Wahli W, Martinez E: Superfamily of steroid nucleara receptors: Positive and negative regulators of gene expression. FASEB J 5: 2243– 2249, 1991

231. Gronemeyer H: Transcription activation by estrogen and progesterone receptors. Annu Rev Genet 25: 89– 123, 1991

232. Lekstrom-Himes J, Xanthopoulos KG: Biological role of the CCAAT/enhancer-binding protein family of transcription factors. J Biol Chem 273: 28545– 28548, 1998

233. Kadonaga JT, Carner KR, Masiarz FR, Tijian R: Isolation of cDNA encoding transcription factor Sp1 and functional analysis of the DNA binding domain. Cell 51: 1079– 1090, 1987

234. Naar AM, Beaurang PA, Robinson KM et al: Chromatin, TAFs, and a novel multiprotein coactivator are required for synergistic activation by Sp1 and SREBP-1a in vitro. Genes Dev 12: 3020– 3031, 1998

235. Zwijsen RML, Buckle RS, Hijmans EM et al: Ligand-independent recruitment of steroid receptor coactivators to estrogen receptor by cyclin D. Genes Dev 12: 3488– 3498, 1998

236. Haussler MR, Whitfield GK, Haussler CA et al: The nuclear vitamin D receptor: Biological and molecular regulatory properties revealed. J Bone Miner Res 13: 325– 349, 1998

237. Giguere V: Retinoic acid receptors and cellular retinoid binding proteins: Complex interplay in retinoid signaling. Endocr Rev 15: 61– 79, 1994

238. Anzick SL, Kononen J, Walker RL et al: AIB1, a steroid receptor coactivator amplified in breast and ovarian cancer. Science 277: 965– 967, 1997

239. Chen H, Lin RJ, Schiltz L et al: Nuclear receptor coactivator ACTR is a novel histone acetyltransferase and forms a multimeric activation complex with P/CAF and CBP/p300. Cell 90: 569– 580, 1997

240. Kim HJ, Lee SK, Na SY et al: Molecular cloning of xSRC-3, a novel transcription coactivator from Xenopus, that is related to AIB1, p/CIP, and TIF2. Mol Endocrinol 12: 1038– 1047, 1998

241. Miyamoto H, Yeh S, Wilding G, Chang C: Promotion of agonist activity of antiandrogens by the androgen receptor coactivator, ARA70, in human prostate cancer DU145 cells. Proc Natl Acad Sci USA 95: 7379– 7384, 1998

242. Yeh S, Chang C: Cloning and characterization of a specific coactivator, ARA70, for the androgen receptor in human prostate cells. Proc Natl Acad Sci USA 93: 5517– 5521, 1997

243. Alen P, Claessens F, Schoenmakers E et al: Interaction of the putative androgen receptor-specific coactivator ARA70/ELE1alpha with multiple steroid receptors and identification of an internally deleted ELE1beta isoform. Mol Endocrinol 13: 117– 128, 1999

244. Bannister AJ, Kouzarides T: The CBP co-activator is a histone acetyltransferase. Nature 384: 641– 643, 1996

245. Kurokawa R, Kalafus D, Ogliastro MH et al: Differential use of CREB binding protein-coactivator complexes. Science 279: 700– 703, 1998

246. Hanstein B, Eckner R, DiRenzo J et al: P300 is a component of an estrogen receptor coactivator complex. Proc Natl Acad Sci USA 93: 11540– 11545, 1996

247. Kamei Y, Xu L, Heinzel T et al: A CBP integrator complex mediates transcriptional activation and AP-1 inhibition by nuclear receptors. Cell 85: 403– 414, 1996

248. Ogryzko VV, Kotani T, Zhang X et al: Histone-like TAFs within the PCAF histone acetylase complex. Cell 94: 35– 44, 1998

249. Halachmi S, Marden E, Martin G et al: Estrogen receptor-associated proteins: Possible mediators of hormone-induced transcription. Science 264: 1455– 1458, 1994

250. Treuter E, Albrektsen T, Johansson L et al: A regulatory role for RIP140 in nuclear receptor activation. Mol Endocrinol 12: 864– 881, 1998

251. Takeshita A, Yen PM, Misiti S et al: Molecular cloning and properties of a full-length putative thyroid hormone receptor coactivator. Endocrinology 137: 3594– 3597, 1996

252. Yao T-P, Ku G, Shou B et al: The nuclear hormone receptor coactivator SRC-1 is a specific target of p300. Proc Natl Acad Sci USA 93: 10026– 10031, 1996

253. vom Baur E, Zechel C, Heery D et al: Differential ligand-dependent interactions between the AF-2 activating domain of nuclear receptors and the putative transcriptional intermediary factors mSUG1 and TIF1. EMBO J 15: 110– 124, 1996

254. Thénot S, Henriquet C, Rochefort H, Cavaills V: Differential interaction of nuclear receptors with the putative human transcriptional coactivator hTIF1. J Biol Chem 272: 12062– 12068, 1997

255. Voegel JJ, Heine MJ, Tini M et al: The coactivator TIF2 contains three nuclear receptor-binding motifs and mediates transactivation through CBP binding-dependent and -independent pathways. EMBO J 17: 507– 519, 1998

256. Wagner BL, Norris JD: The nuclear corepressors NCoR and SMRT are key regulators of both ligand- and 8-bromo-cyclic AMP-dependent transcriptional activity of the human progesterone receptor. Mol Cell Biol 18: 1369– 1378, 1998

257. Soderstrom M, Vo A, Heinzel T et al: Differential effects of nuclear receptor corepressor (N-CoR) expression levels on retinoic acid receptor-mediated repression support the existence of dynamically regulated corepressor complexes. Mol Endocrinol 11: 682– 692, 1997