help button home button Endocrine Society Molecular Endocrinology ENDO 08 Sessions Library
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit a related Letter to the Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Reprints, Permissions and Rights
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Willson, T. M.
Right arrow Articles by Moore, J. T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Willson, T. M.
Right arrow Articles by Moore, J. T.
Molecular Endocrinology 16 (6): 1135-1144
Copyright © 2002 by The Endocrine Society

Minireview: Genomics Versus Orphan Nuclear Receptors—A Half-Time Report

Timothy M. Willson and John T. Moore

GlaxoSmithKline, Discovery Research, Research Triangle Park, North Carolina 27709

Address all correspondence and requests for reprints to: Timothy M. Willson, GlaxoSmithKline, Discovery Research, NTH-M.1421.1A, Research Triangle Park, North Carolina 27709-3398. E-mail: tmw20653{at}gsk.com.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 THE NUCLEAR RECEPTOR GENE...
 CHEMICAL GENOMICS
 STRUCTURAL GENOMICS
 FUNCTIONAL GENOMICS
 CONCLUSIONS AND FUTURE...
 REFERENCES
 
Following the successful cloning of the orphan nuclear receptors during the 1990s we entered the 21st century with knowledge of the full complement of human nuclear receptors. Many of these proteins are ligand-activated transcription factors that act as the cognate receptors for steroid, retinoid, and thyroid hormones. In addition to these well characterized endocrine hormone receptors, there are a large number of orphan receptors of which less is known about the nature and function of their ligands. The task of deciphering the physiological function of these orphan receptors has been aided by a new generation of genomic technologies. Through application of chemical, structural, and functional genomics, several orphan nuclear receptors have emerged as pharmaceutical drug targets for the treatment of important human diseases. The significant progress that has been made in the functional analysis of more than half of the nuclear receptor gene family provides an opportunity to review the impact of genomics in this endeavor.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
THE NUCLEAR RECEPTOR GENE...
 CHEMICAL GENOMICS
 STRUCTURAL GENOMICS
 FUNCTIONAL GENOMICS
 CONCLUSIONS AND FUTURE...
 REFERENCES
 
HORMONES FUNCTION AS chemical messengers within the body to control almost every aspect of mammalian physiology from development to the regulation of cellular metabolism. Hormones mediate their biological activity through specific cell surface and intracellular proteins, known as receptors, which have proved to be powerful targets for pharmaceutical intervention in disease processes. Currently, one third of all common prescription drugs act through cell surface seven-transmembrane receptors or intracellular nuclear receptors. For this reason, characterization of mammalian hormone receptors in normal physiology and abnormal disease processes is a major goal of biomedical research.

The nuclear receptors are transcription factors that regulate expression of specific target genes (1). The ability of cell-permeable hormones to regulate the activity of these transcription factors provides a mechanism for small organic molecules to directly regulate pathways of gene expression within cells. So there has been great interest in the identification of nuclear receptors and the characterization of the biochemical pathways that they regulate. Pursuit of this knowledge has invariably encompassed a marriage of chemistry and biology from the early days of endocrine hormone isolation to the current era of genomics (2). The purpose of this review is to illustrate how chemical, structural, and functional genomics have expanded our understanding of nuclear receptors and led to the identification of new pharmaceutical drug targets.


   THE NUCLEAR RECEPTOR GENE FAMILY
TOP
 ABSTRACT
 INTRODUCTION
 THE NUCLEAR RECEPTOR GENE...
CHEMICAL GENOMICS
 STRUCTURAL GENOMICS
 FUNCTIONAL GENOMICS
 CONCLUSIONS AND FUTURE...
 REFERENCES
 
The history of nuclear receptor gene family can be divided into two eras (Fig. 1Go). The biochemical era in the first half of the 20th century saw the identification of the steroid, retinoid, and thyroid hormones, a feat that led to the award of four Nobel prizes in chemistry and medicine (Fig. 1Go). These high-affinity hormones served as reagents to track their cognate receptors during purification and subsequent biochemical analysis (3). The advent of molecular biology in the second half of the 20th century switched the emphasis of research from proteins to genes. The genomic era began with the challenge of identifying the full complement of nuclear receptors, a goal that has recently been achieved in multiple organisms.



View larger version (26K):
[in this window]
[in a new window]
  		Figure 1. Two Eras of Nuclear Receptor Research

The figure represents the conserved domains found in members of the nuclear receptor supergene family.

 
The molecular cloning of the nuclear receptors started in the 1980s with the steroid hormone receptors. The cloning of the GR relied heavily on reagents available from the purification and biochemical characterization of the protein. Selective GR antibodies were used to immunoprecipitate protein-encoding mRNA for cDNA synthesis (4) or to screen bacterial expression libraries (5, 6). The resulting cDNA representing the full-length coding region of GR provided the first glimpse at the amino acid sequence of a nuclear receptor. The full-length ER{alpha} cDNA was isolated around the same time through three independent strategies. In one case, antibodies were used to isolate human ER{alpha} from expression libraries (7). Alternatively, partial sequencing of purified human ER protein allowed design of ER-specific oligonucleotides to screen {lambda}-phage cDNA libraries (8), and in the third case CHO cells transformed with human cDNA were screened using radiolabeled E2 (9). These studies emphasize how early advances of nuclear receptor molecular biology relied on the reagents and expertise of the past biochemical era.

Comparison of the newly emerging nuclear receptor sequences gave rise to the exciting observation that significant regional homology existed between the steroid hormone receptors as well as the cellular receptors for thyroid hormone (10, 11) and RA (12, 13). Two hallmark domains were identified; a central DNA-binding domain (DBD) and a C-terminal ligand-binding domain (LBD) (Fig. 1Go). Drosophila nuclear receptors that were identified through genetic screens (14) also shared these conserved domains. These observations implied that the human and Drosophila nuclear receptors were members of a gene family (now termed the nuclear receptor superfamily) that arose from a common primordial gene. Importantly, this knowledge allowed the identification of additional members of the nuclear receptor superfamily using sequence data alone, without the requirement for biochemical characterization (e.g. hormone binding).

Additional nuclear receptor genes were rapidly identified using a variety of genetic, biochemical, and molecular biology techniques. Initially, oligonucleotides representing conserved nuclear receptor motifs (such as the DBD) were employed as molecular probes to perform low-stringency hybridizations to cDNA libraries. A large number of mammalian (15) and Drosophila nuclear receptors (16, 17) were identified in this manner. Another powerful approach employed the use of degenerate PCR primers representing the highly conserved P box and Gly-Met junction within the DBD (18, 19). Through application of these cloning methods, the number of orphan nuclear receptors quickly surpassed the number of classical nuclear hormone receptors (20, 21, 22) (Tables 1Go and 2Go).


View this table:
[in this window]
[in a new window]
  		Table 1. Human Nuclear Hormone Receptors

 

View this table:
[in this window]
[in a new window]
  		Table 2. Human Orphan Nuclear Receptors

 
Approximately 3 yr ago, the chosen method for identification of new nuclear receptors shifted from the laboratory to the computer. The in silico search for nuclear receptors was made possible by the availability of large databases of randomly generated partial cDNA sequence, known as expressed sequence tags (ESTs), and the development of bioinformatic search and query tools such as BLAST. Two new mammalian nuclear receptors were successfully identified through automated searches of EST databases. The pregnane X receptor (PXR) was uncovered in a public mouse EST database by a high throughput in silico screen for nuclear receptor-like sequences (23), and the photoreceptor cell-specific receptor was found in a human EST database (24). However, after an initial burst of activity in the late 1990s, the appearance of new nuclear receptor sequences in EST databases soon declined. One possible reason for this decline is the bias of random ESTs toward the more abundant mRNA in a cell or tissue, so that receptors expressed at low levels or in rare tissues are less likely to be identified.

Recently, complete genome sequences have become available for three organisms. In contrast to EST databases, genome sequence allows identification of all gene family members without being limited by temporal, spatial, or quantitative aspects of mRNA expression. So, the mining of genome sequence databases has the potential to dramatically expand the nuclear receptor set for a given organism by identifying genes expressed at low levels or restricted to specific tissues. When the Caenorhabditis elegans genome sequence was reported, more than 200 orphan nuclear receptors were quickly identified (25). Furthermore, analysis of the C. elegans genome revealed multiple new subfamilies of nuclear receptors with divergent P box motifs in their DBDs that would not have been cloned using degenerate PCR primers. This dramatic increase in the number of known C. elegans nuclear receptors led to speculation that the human nuclear receptor set would be dramatically expanded through genome sequencing (26). Surprisingly, however, when the Drosophila genome was reported, only 21 nuclear receptor sequences were found (27, 28). With this puzzling backdrop, the nuclear receptor community was keen to learn whether the total set of human receptors would reflect the diversity seen in C. elegans or parallel the limited number found in Drosophila.

In 2000, the complete human genome sequence became available from both the International Human Genome Sequencing Consortium and Celera Genomics. Large numbers of novel ion channel, protease, and seven-transmembrane receptor genes were identified through analysis of the sequence. In contrast, analysis of the complete human genome identified only three new candidate nuclear receptor sequences (27, 28, 29). The sequences were most closely related to the hepatocyte nuclear factor 4 (HNF4), the farnesoid X receptor (FXR), and the chicken ovalbumin upstream promoter transcription factor, but unfortunately all three contained multiple stop codons within their coding regions. The three sequences are likely to be nonfunctional pseudogenes (27), even though the FXR-related gene is expressed in a limited number of tissues (27, 29). Therefore, the complete human nuclear receptor set comprises only the 48 genes that were identified using methods that predated the completion of the human genome project.

The total number of nuclear receptor genes reflects only one level of complexity within an organism. The diversity of nuclear receptors at the protein level (the proteome) is also a product of differential transcriptional and posttranscriptional processing events. Undoubtedly, additional characterization of the nuclear receptor superfamily will encompass functional variants of each receptor that are present at the protein level. Because the complement of nuclear receptor genes provides the foundation to tackle these proteomic questions, the tools of the genomic era will provide the springboard for a new wave of biochemical research.


   CHEMICAL GENOMICS
TOP
 ABSTRACT
 INTRODUCTION
 THE NUCLEAR RECEPTOR GENE...
 CHEMICAL GENOMICS
STRUCTURAL GENOMICS
 FUNCTIONAL GENOMICS
 CONCLUSIONS AND FUTURE...
 REFERENCES
 
The gene family of 48 human nuclear receptors can be reduced to a chemical proteome of 28 ligand-binding sites, if we assume that the subtypes of a particular receptor bind to the same natural ligand. This set of 28 ligand-binding sites is composed of eight classical hormone receptors (Table 1Go) and 20 orphan receptors (Table 2Go). The classical steroid, retinoid, and thyroid hormones are high-affinity ligands for their cognate receptors (30). It has transpired that many of the orphan receptors identified during the genomic era do not bind to endocrine hormones, but bind with low affinity to small molecules found in the body or environment. To date, naturally occurring ligands have been identified for nearly half of the mammalian orphan receptors, so that there are now more human nuclear receptors with known ligands than without (Tables 1Go and 2Go). Knowledge of the natural ligand for an orphan nuclear receptor has proved to be one of the most powerful ways to define its physiological function (2). Thus, the orphan nuclear receptors provide a test case by which to study the impact of chemical genomics in the characterization of gene function.

Three approaches have proved successful in the hunt for orphan receptor ligands. The first approach employed focused screening of carefully selected sets of candidate ligands (20). Many investigators have assembled collections of test ligands by reasoning that orphan receptors are likely to bind to small lipophilic molecules. By this approach the RXR, PPAR, liver X receptor (LXR), and FXR were shown to bind to naturally occurring retinoids (31), fatty acids (32), oxysterols (33), and bile acids (34, 35), respectively. Because retinoids have traditionally been employed for chemotherapy (36), identification of RXR as a retinoid receptor led to the use of RXR ligands in treatment of various cancers. For the PPARs, their role as receptors for dietary and endogenous fatty acids accounts for the diverse therapeutic potential of synthetic PPAR ligands. Fatty acids have been shown to affect insulin sensitivity, lipid metabolism, and skin development (37). Likewise, PPAR ligands have shown clinical utility in the treatment of type 2 diabetes, dyslipidemia, and psoriasis (38). In the case of LXR, the discovery that it functions as a nuclear oxysterol receptor was pivotal in uncovering its role in the regulation of cholesterol metabolism. Synthetic LXR ligands increase the efflux of cholesterol from macrophages and raise high-density lipoprotein cholesterol, which suggests that they will be powerful drugs for combating atherosclerosis (39). Finally, the identification of endogenous bile acids as FXR ligands led to the study of the role of this receptor in bile acid and lipid metabolism. Synthetic FXR ligands suppress bile acid biosynthesis, lower serum triglycerides, and may have utility in the treatment of diseases of abnormal bile acid metabolism (40).

The second successful approach for identifying orphan receptor ligands has employed random screening of known drug molecules. Synthetic drugs have been shown to bind and activate PXR, the constitutive androstane receptor (CAR), and the estrogen related receptor (ERR) (2, 39). Remarkably, the biological activity attributed to each of these orphan receptors was previously considered to be an unexplained side effect of the drug in question. For example, a series of structurally unrelated drugs share the common property of inducing cytochrome P450 (CYP)3A levels in the liver and binding to PXR (41), implicating the orphan receptor as the unifying factor responsible for transcriptional regulation of this class of CYP. The connection is further supported by the species specificity of PXR ligands. The human-specific PXR agonist rifampicin (Fig. 2Go) induces CYP3A in humans but not rodents; conversely the rodent-specific PXR agonist pregnenolone 16{alpha}-carbonitrile induces CYP3A in rodent but not human hepatocytes (42). A similar line of reasoning led to the association of CAR with the regulation of the CYP2B class of CYP. The barbiturate phenobarbital, which is known to induce expression of CYP2B, was shown to cause translocation of CAR from the cytoplasm to the nucleus of hepatocytes (43). Although phenobarbital activates CAR by an indirect mechanism, TCPOBOP (Fig. 2Go), a structurally unrelated CYP2B inducer, has been shown to bind and activate the receptor. Thus, CAR activators share a common property of CYP2B induction, thereby linking this orphan receptor with transcriptional regulation of this class of cytochrome P450 (44). In the case of ERR{gamma}, diethylstilbestrol and 4-hydroxytamoxifen (Fig. 2Go) were recently reported to bind to the receptor and repress its constitutive transcriptional activity (45). Both of these drug molecules have a long history of use in women’s health, through their opposing effects on the activity of the classical ER{alpha}. Although the full implication of these findings is still unknown, it has been proposed that ERR ligands may have utility in the treatment of breast cancer (46).



View larger version (23K):
[in this window]
[in a new window]
  		Figure 2. Synthetic Orphan Nuclear Receptor Ligands

 
The third approach that has recently identified orphan receptor ligands is x-ray crystallography, as illustrated in the following section with HNF4 and the retinoid-related orphan receptor (ROR). It is notable that these two receptors have been resistant to both focused and random high-throughput screening, which raises the possibility that identification of ligands for the remaining orphan receptors may become progressively more difficult by traditional screening methods.


   STRUCTURAL GENOMICS
TOP
 ABSTRACT
 INTRODUCTION
 THE NUCLEAR RECEPTOR GENE...
 CHEMICAL GENOMICS
 STRUCTURAL GENOMICS
FUNCTIONAL GENOMICS
 CONCLUSIONS AND FUTURE...
 REFERENCES
 
The activation of gene transcription by nuclear receptors is a complex multistep process that is orchestrated by the LBD. The LBD, located at the C terminus of the receptor, contains a binding site for cofactors, known as coactivators and corepressors, which couple ligand-induced conformational changes to the regulation of gene transcription (reviewed in Ref. 47). In 1995, Moras and co-workers (48) published the first x-ray crystal structure of a nuclear receptor LBD, a homodimer of RXR{alpha}. In the following 6 yr, the structures of an additional 16 nuclear receptor LBDs have been disclosed. Many of these structures have been obtained as complexes with agonist and antagonist ligands, as well as with fragments of coactivator and corepressor proteins (49, 50). With LBD structures solved for more than a third of the mammalian nuclear receptors, it is now possible to conduct an analysis of the impact of structural genomics on our understanding of this gene family.

The nuclear receptor LBDs have a common fold composed of a three-layer sandwich of {alpha}-helices. This fold became apparent from comparison of the first two retinoid receptor structures and has been seen in all subsequent LBD structures (Fig. 3Go). The domain has four functional regions (49). The top half of the LBD (Fig. 3Go) forms the structural core of the protein and is relatively invariant between receptors. In contrast, the ligand-binding site, in the bottom half of the LBD (Fig. 3Go), shows differences between receptors commensurate with their recognition of unique hormones and ligands. In particular, the number of ß-strands in this region coupled with the presence or absence of helix 2 contributes to the variability in the ligand-binding site. The third functional region is a hydrophobic cleft on the surface of the LBD that serves as the docking site for coactivator and corepressor proteins (50). This cleft is bounded by a charge clamp (51) formed between a Lys at the C-terminal end of helix 3 and a Glu on the activation function 2 (AF2) helix. The precise positioning of the AF2 helix is controlled by the binding of ligands to the LBD, which allows small molecules to regulate the transcriptional activity of the receptor. The fourth key region of the LBD comprises a dimerization interface along the outside of helix 10. Homodimers show C2 symmetry around this interface, whereas RXR heterodimers form an asymmetric interface with additional contributions from amino acids in helix 9 (52).



View larger version (35K):
[in this window]
[in a new window]
  		Figure 3. X-ray Crystal Structures of Orphan Nuclear Receptor LBDs

The protein is represented as red tubes ({alpha}-helices), yellow arrows (ß-sheets), or blue worms (loop sections). The bound molecules are shown as atomic space-filling representations. The major {alpha}-helices and the N and C termini of the protein referred to in the text are labeled. The presence or absence of helix 2 and the number of ß-sheets affect the size and shape of the ligand-binding pocket. A, PPAR{delta} with eicosapentaenoic acid (54 ). The carboxylic acid head group contacts the AF2 helix. The lipophilic tail was observed in a tail-up (green) and tail-down (magenta) conformation. B, HNF4{gamma} with palmitic acid (Williams, S. P., personal communication). The carboxylic acid head group is buried in the core of the protein. The lipophilic tail fills the small ligand-binding site and does not contact the AF2 helix, C, PXR with SR12813 (61 ). All three orientations of the drug molecule that were found within the ligand binding site are shown.

 
Comparison of the structures of the LBDs shows that they have evolved to serve a remarkable variety of functions within their capacity as a ligand sensors. The Y-shaped PPAR ligand-binding site (Fig. 3AGo) is the largest seen to date at 1,400Å3 (53). The large pocket allows the receptor to bind a range of fatty acid and fatty acid metabolites (54). One arm of the pocket contains three polar tyrosine and histidine residues that bind to the negatively charged carboxylate of the fatty acid through an intricate network of hydrogen bonds. One of the tyrosine residues lies within the AF2 helix, allowing acidic ligands to directly regulate the formation of the coactivator binding cleft. The other two arms of the binding site are lined with hydrophobic amino acids and are arranged in a manner that permits the lipophilic tails of fatty acids to bind in multiple orientations (54). Thus, the PPAR LBD is ideally suited for its role as a general lipid sensor.

The PPAR ligand-binding site stands in stark contrast with the structure of HNF4, a member of the nuclear receptor gene family that was originally cloned in a screen for liver-enriched transcription factors (55). The crystal structure of the HNF4{gamma} LBD (Fig. 3BGo) reveals a smaller ligand-binding site of only 625Å3, which is occupied entirely by a fatty acid that copurifies with the protein (Wisely, B., and S. P. Williams, personal communication). The fatty acid is bound into this site with its acidic head group buried within the core of the protein, in the opposite orientation to that seen in PPAR (56). In HNF4, the fatty acid does not contact the AF2 helix and there is no obvious way for it to modulate the activity of the receptor. Notably, HNF4 is a constitutively active transcription factor in cells. One explanation for this phenomenon is that the fatty acid is not a true ligand, but plays a permanent role in maintaining the structural fold of the LBD in an active conformation. Heterozygous mutations in HNF4{alpha} that decrease receptor activity cause the genetic disorder of maturity onset diabetes of the young, in which patients show defects in glucose-stimulated insulin secretion. Therefore, there is intense pharmaceutical interest in modulating the activity of HNF4 as a potential approach to treating type 2 diabetes. Unfortunately, the collected structural and biochemical data suggest that HNF4 may be a constitutively active transcription factor that is not regulated by conventional small molecule ligands (56, 57). Instead, HNF4 may function as a coactivator-docking site (58) or may be regulated by posttranslational modification of the protein (59, 60), which will require new approaches to regulate its activity with synthetic drug molecules.

A third contrast in LBD structure is shown by PXR (Fig. 3CGo), a nuclear receptor that functions as a promiscuous xenobiotic sensor to regulate the expression of CYP3A in the liver and intestine. Although the PXR ligand-binding site, at 1150Å3, is not the largest, it has features that are ideally suited to its role as a xenosensor (61). The receptor has five strands of ß-sheet instead of the usual two or three strands, and a large insert of 45 amino acids that replaces helix 2. These elements lead to a reshaping of the protein such that helix 6 is unwound, and part of the 45-amino acid insert is used as the floor of a spherical ligand-binding site that is lined with predominantly hydrophobic amino acids. In addition, a flexible loop that replaces helix 6 may expand and contract to accommodate ligands of various sizes, including the macrocyclic antibiotic rifampicin—probably the largest known nuclear receptor ligand (Fig. 2Go). The cocrystal structure of PXR with the cholesterol-lowering drug SR12813 (Fig. 2Go) revealed another surprise (61). The drug was bound in multiple orientations, in which each unique orientation employed a different set of interactions with the protein. Apparently, the spherical pocket does not require ligands to satisfy a single shape or arrangement of hydrogen bonding interactions. These molecular features may give PXR the ability to recognize a wide range of structurally unrelated xenobiotics.

The crystal structures of the remaining orphan nuclear receptor LBDs have the potential to uncover important clues about their physiological function. The recent structure of RORß (62), which has a large ligand-binding site and shows partial occupancy by stearic acid, suggests that the RORs may be another family of fatty acid-sensing receptors akin to the PPARs. In contrast, receptors that crystallize with tightly bound lipids, as is seen with HNF4 (56), or lack a ligand-binding pocket, as is predicted for RevErbA (63), may be examples of constitutively active transcription factors. Interestingly, many of the remaining orphan nuclear receptors show constitutive activation or repression of transcription in cells. The act of systematically solving the LBD structures of the remaining orphan nuclear receptors will undoubtedly provide important insight into which ones are likely to be good targets for small molecule pharmaceutical drugs and, conversely, which ones may be less tractable using traditional chemical technologies.


   FUNCTIONAL GENOMICS
TOP
 ABSTRACT
 INTRODUCTION
 THE NUCLEAR RECEPTOR GENE...
 CHEMICAL GENOMICS
 STRUCTURAL GENOMICS
 FUNCTIONAL GENOMICS
CONCLUSIONS AND FUTURE...
 REFERENCES
 
An overarching goal of functional genomics is to define the biological role of each expressed gene. For the orphan nuclear receptors, discovery of their normal physiological function is extremely important because it provides a rationale for determining the therapeutic utility of their ligands. Several new functional genomic technologies are being applied to this task.

Reverse endocrinology exploits the availability of potent and selective small-molecule ligands to identify nuclear receptor target genes by differential gene expression (DGE) (2). This approach is especially pertinent for nuclear receptors because they are transcription factors that directly regulate gene expression. In cases where clues about the biology of the receptor are available, the differential effects of ligands on candidate genes can be examined on an individual basis. However, the advent of high-throughput DGE technologies [e.g. Affymetrix (Santa Clara, CA) microarrays and Curagen Corp. (New Haven, CT) Gene Calling] has enabled comprehensive, unbiased analysis of gene expression (64). Global DGE studies in the liver using the selective FXR ligand GW4064 (Fig. 2Go) have uncovered an elaborate nuclear receptor-regulatory cascade for the maintenance of bile acid homeostasis (65). Among the mRNA whose expression was induced by the FXR ligand was the short heterodimer partner (SHP), an atypical nuclear receptor that lacks a DBD. SHP, a constitutive repressor of gene expression, mediates the effects of FXR in the suppression of bile acid biosynthesis. Based on these data it has been proposed that cholestasis, a serious and life-threatening disease that is characterized by accumulation of bile acids in the liver, could be treated with potent FXR agonists (40). Other recently reported global DGE studies include the identification of PPAR{gamma} target genes that led to a model for PPAR{gamma} action in insulin sensitization (66), and differentiation of CAR-dependent and CAR-independent effects of phenobarbital in the liver (67). As DGE technologies become widely accessible, the use of reverse endocrinology in functional analysis of orphan nuclear receptors is likely to remain popular. Future improvements in efficiency will depend less on the speed of DGE data generation and more on the development of improved bioinformatic tools to discern key pathways of gene regulation within complex data sets.

Traditional molecular genetics continues to play an important role in nuclear receptor functional analysis. Importantly, genetic analysis of receptor function does not require the availability of a ligand. Key insights into the physiological function of several orphan nuclear receptors without known ligands have been derived from analysis of knockout mice generated using homologous recombination (68). For example, nerve growth factor immediate early gene B (NGFI-B) ß knockout mice fail to develop midbrain dopaminergic neurons (69). The knockout phenotype led to the suggestion that NGFI-Bß ligands might be useful for the treatment of disorders of dopamine signaling such as Parkinson’s disease. One general drawback of conventional nuclear receptor gene knockouts is that about half of them result in embryonic lethality. To circumvent this problem, new genetic strategies such as the conditional site-specific recombination using the Cre/lox system are being used to introduce temporal or tissue-specific somatic mutations of genes (70, 71). Recently, an inducible Cre/lox system was used to knock out steroidogenic factor 1 (SF1) in the anterior pituitary of the mouse, limiting the severe phenotype of the conventional SF1 knockout. The phenotype of the conditional knockout mice established an essential role of SF1 in pituitary production of the gonadotropes LH and FSH (72).

Nuclear receptor knockouts can now be accomplished in a broader range of cells using two new genetic techniques. Antisense oligonucleotides (73) and small RNA interference technologies (74) introduce sequence-specific, posttranscriptional gene silencing, allowing the suppression of nuclear receptor expression in cell culture. The effect of HNF4{alpha} suppression on gene expression in human hepatocytes has recently been evaluated using antisense RNA (75). In these experiments, an adenoviral vector was used to efficiently deliver an HNF4{alpha} antisense RNA expression construct to hepatocytes in culture, resulting in nearly undetectable levels of HNF4{alpha} protein. Specific ablation of HNF4{alpha} led to dysregulation of multiple drug-metabolizing CYPs.

In the postgenome era, human genetic studies will be increasingly used as a source of new information on orphan nuclear receptor function. One of the fruits of the human genome project is a wealth of information on human DNA sequence variation. Although the overall difference between any two human genomes is less than 0.1%, there are still several million single nucleotide polymorphisms (SNPs) within each individual (76, 77). The availability of a dense set of SNPs increases the probability of finding nuclear receptor variants that can be associated with human diseases. Recently, SNP analysis has led to association of NGFI-Bß with schizophrenia and manic depressive disorder (78). Another study based on a SNP analysis of patients with maturity onset diabetes of the young associated mutations in SHP with mild obesity (79). This field is burgeoning with new information and is one of the most exciting avenues for future exploration into orphan nuclear receptor function in humans.


   CONCLUSIONS AND FUTURE DIRECTIONS
TOP
 ABSTRACT
 INTRODUCTION
 THE NUCLEAR RECEPTOR GENE...
 CHEMICAL GENOMICS
 STRUCTURAL GENOMICS
 FUNCTIONAL GENOMICS
 CONCLUSIONS AND FUTURE...
REFERENCES
 
Several important themes have emerged from chemical, structural, and functional genomic analysis of the orphan nuclear receptors. Unlike the classical endocrine hormones, all of the naturally occurring ligands identified for the orphan nuclear receptors have relatively low affinity, and often no single ligand has emerged as a true hormone. For example, the PPARs bind to a range of fatty acids and fatty acid metabolites, whereas the LXRs bind to a range of oxysterols that are found in different tissues. The physiological function of these orphan nuclear receptors is consistent with their roles as low-affinity sensors for relatively abundant metabolites (2, 39). It would appear unlikely, therefore, that a unique high-affinity hormone will be identified for these or many of the other orphan nuclear receptors.

To date, almost all of the natural ligands that have been identified are agonists that increase receptor activity. These compounds have proven to be powerful tools in analysis of receptor function by reverse endocrinology (2). However, the development of antagonist ligands would provide an additional capacity to perform chemical knockouts of receptor function in mature animals or in animal models of disease. Increased understanding of the molecular mechanism of receptor activation combined with the availability of cocrystal structures with coactivators and corepressors may soon permit the rational design of potent antagonist ligands. This combination of chemical and structural genomics will be an important undertaking for future medicinal chemists.

Perhaps the most controversial concept that has emerged from structural analysis of the orphan nuclear receptors is the possibility that some of the remaining family members may not be small molecule receptors. Several of the orphans that may fall into this category are highly validated as pharmaceutical drug targets because of their genetic association with human diseases (e.g. HNF4{alpha} with diabetes, SHP with obesity, and NGFI-Bß with schizophrenia) (22). If it transpires that the activity of these transcription factors cannot be modulated by conventional small-molecule ligands, researchers may need to consider alternative approaches by which to develop therapeutic agents. Early indications are that short peptides can be developed that disrupt the action coactivator proteins through competition with the binding cleft on the surface of the receptor (80, 81). These peptides are able to block receptor function when introduced into cells. Thus, it may be possible to directly modulate cofactor binding to these transcription factors if issues of cell penetration can be overcome through the design and synthesis of peptidomimetic molecules (82).

Less than two decades after the cloning of the GR cDNA, we now know that there are a remarkably small number of human nuclear receptors. Each of these transcription factors appears to play an important role in mammalian physiology, and many of them have emerged as important drug targets. The physiological functions of at least half of the mammalian nuclear receptors are now known, and increasingly rapid progress is being made to uncover the biology of the remaining members of the gene family. At the current pace, it now seems possible that we will complete the functional analysis of the orphan nuclear receptors within the next decade. This achievement will undoubtedly lead to new opportunities for the development of human therapeutic agents.

The half-time score: all tied with everything to play for!


   ACKNOWLEDGMENTS
 
We thank Mill Lambert for assistance in generating Fig. 3Go.


   FOOTNOTES
 
Abbreviations: AF2, Activation function 2; CAR, constitutive androstane receptor; CYP, cytochrome P450; DBD, DNA-binding domain; DGE, differential gene expression; ERR, estrogen-related receptor; EST, expressed sequence tag; FXR, farnesoid X receptor; HNF4, hepatocyte nuclear receptor 4; LBD, ligand-binding domain; LXR, liver X receptor; NGFI-B, nerve growth factor immediate early gene B; PXR, pregnane X receptor; ROR, retinoid-related orphan receptor; SF1, steroidogenic factor 1; SHP, short heterodimer partner; SNP, single-nucleotide polymorphism.

Received for publication January 22, 2002. Accepted for publication March 1, 2002.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 THE NUCLEAR RECEPTOR GENE...
 CHEMICAL GENOMICS
 STRUCTURAL GENOMICS
 FUNCTIONAL GENOMICS
 CONCLUSIONS AND FUTURE...
 REFERENCES
 

  1. Mangelsdorf DJ, Thummel C, Beato M, Herrlich P, Schuetz G, Umesono K, Blumberg B, Kastner P, Mark M, Chambon P, Evans RM 1995 The nuclear receptor superfamily: the second decade. Cell 83:835–839[CrossRef][Medline]
  2. Kliewer SA, Lehmann JM, Willson TM 1999 Orphan nuclear receptors: shifting endocrinology into reverse. Science 284:757–760[Abstract/Free Full Text]
  3. Jensen EV, Greene GL, Closs LE, DeSombre ER, Nadji M 1982 Receptors reconsidered: a 20-year perspective. Recent Prog Horm Res 38:1–40
  4. Miesfeld R, Okret S, Wikstrom AC, Wrange O, Gustafsson JA, Yamamoto KR 1984 Characterization of a steroid hormone receptor gene and mRNA in wild-type and mutant cells. Nature 312:779–781[CrossRef][Medline]
  5. Weinberger C, Hollenberg SM, Ong ES, Harmon JM, Brower ST, Cidlowski J, Thompson EB, Rosenfeld MG, Evans RM 1985 Identification of human glucocorticoid receptor complementary DNA clones by epitope selection. Science 228:740–742[Abstract/Free Full Text]
  6. Govindan MV, Devic M, Green S, Gronemeyer H, Chambon P 1985 Cloning of the human glucocorticoid receptor cDNA. Nucleic Acids Res 13:8293–8304[Abstract/Free Full Text]
  7. Walter P, Green S, Greene G, Krust A, Bornert JM, Jeltsch JM, Staub A, Jensen E, Scrace G, Waterfield M 1985 Cloning of the human estrogen receptor cDNA. Proc Natl Acad Sci USA 82:7889–7893[Abstract/Free Full Text]
  8. Green S, Walter P, Kumar V, Krust A, Bornert JM, Argos P, Chambon P 1986 Human oestrogen receptor cDNA: sequence, expression and homology to v-erb-A. Nature 320:134–139[CrossRef][Medline]
  9. Greene GL, Gilna P, Waterfield M, Baker A, Hort Y, Shine J 1986 Sequence and expression of human estrogen receptor complementary DNA. Science 231:1150–1154[Abstract/Free Full Text]
  10. Sap J, Munoz A, Damm K, Goldberg Y, Ghysdael J, Leutz A, Beug H, Vennstroem B 1986 The c-erb-A protein is a high-affinity receptor for thyroid hormone. Nature 324:635–640[CrossRef][Medline]
  11. Weinberger C, Thompson CC, Ong ES, Lebo R, Gruol DJ, Evans RM 1986 The c-erb-A gene encodes a thyroid hormone receptor. Nature 324:641–646[CrossRef][Medline]
  12. Petkovich M, Brand NJ, Krust A, Chambon P 1987 A human retinoic acid receptor which belongs to the family of nuclear receptors. Nature 330:444–450[CrossRef][Medline]
  13. Giguere V, Ong ES, Segui P, Evans RM 1987 Identification of a receptor for the morphogen retinoic acid. Nature 330:624–629[CrossRef][Medline]
  14. Nauber U, Pankratz MJ, Kienlin A, Seifert E, Klemm U, Jackle H 1988 Abdominal segmentation of the Drosophila embryo requires a hormone receptor-like protein encoded by the gap gene knirps. Nature 336:489–492[CrossRef][Medline]
  15. Willy PJ, Umesono K, Ong ES, Evans RM, Heyman RA, Mangelsdorf DJ 1995 LXR, a nuclear receptor that defines a distinct retinoid response pathway. Genes Dev 9:1033–1045[Abstract/Free Full Text]
  16. Oro AE, Ong ES, Margolis JS, Posakony JW, McKeown M, Evans RM 1988 The Drosophila gene knirps-related is a member of the steroid-receptor gene superfamily. Nature 336:493–496[CrossRef][Medline]
  17. Koelle MR, Talbot WS, Segraves WA, Bender MT, Cherbas P, Hogness DS 1991 The Drosophila EcR gene encodes an ecdysone receptor, a new member of the steroid receptor superfamily. Cell 67:59–77[CrossRef][Medline]
  18. Becker-Andre M, Andre E, DeLamarter JF 1993 Identification of nuclear receptor mRNAs by RT-PCR amplification of conserved zinc-finger motif sequences. Biochem Biophys Res Commun 194:1371–1379[CrossRef][Medline]
  19. Mosselman S, Polman J, Dijkema R 1996 ERß: identification and characterization of a novel human estrogen receptor. FEBS Lett 392:49–53[CrossRef][Medline]
  20. Mangelsdorf DJ, Evans RM 1995 The RXR heterodimers and orphan receptors. Cell 83:841–850[CrossRef][Medline]
  21. Blumberg B, Evans RM 1998 Orphan nuclear receptors—new ligands and new possibilities. Genes Dev 12:3149–3155[Free Full Text]
  22. Giguere V 1999 Orphan nuclear receptors: from gene to function. Endocr Rev 20:689–725[Abstract/Free Full Text]
  23. Kliewer SA, Moore JT, Wade L, Staudinger JL, Watson MA, Jones SA, McKee DD, Oliver BB, Willson TM, Zetterstrom RH, Perlmann T, Lehmann JM 1998 An orphan nuclear receptor activated by pregnanes defines a novel steroid signaling pathway. Cell 92:73–82[CrossRef][Medline]
  24. Kobayashi M, Takezawa S, Hara K, Yu RT, Umesono Y, Agata K, Taniwaki M, Yasuda K, Umesono K 1999 Identification of a photoreceptor cell-specific nuclear receptor. Proc Natl Acad Sci USA 96:4814–4819[Abstract/Free Full Text]
  25. Sluder AE, Mathews SW, Hough D, Yin VP, Maina CV 1999 The nuclear receptor superfamily has undergone extensive proliferation and diversification in nematodes. Genome Res 9:103–120[Abstract/Free Full Text]
  26. Enmark E, Gustafsson JA 2000 Nematode genome sequence dramatically extends the nuclear receptor superfamily. Trends Pharmacol Sci 21:85–87[CrossRef][Medline]
  27. Maglich JM, Sluder A, Guan X, Shi Y, McKee DD, Carrick K, Kamdar K, Willson TM, Moore JT 2001 Comparison of complete nuclear receptor sets from the human, Caenorhabditis elegans and Drosophila genomes. Genome Biol 2:research0029.1–0029.7
  28. Enmark E, Gustafsson J-A 2001 Comparing nuclear receptors in worms, flies and humans. Trends Pharmacol Sci 22:611–615[CrossRef][Medline]
  29. Robinson-Rechavi M, Carpentier A, Duffraisse M, Laudet V 2001 How many nuclear hormone receptors are there in the human genome? Trends Genet 17:554–556[CrossRef][Medline]
  30. O’Malley BW, Schrader WT 1976 The receptors of steroid hormones. Sci Am 234:32–43[Medline]
  31. Heyman RA, Mangelsdorf DJ, Dyck JA, Stein RB, Eichele G, Evans RM, Thaller C 1992 9-cis Retinoic acid is a high affinity ligand for the retinoid X receptor. Cell 68:397–406[CrossRef][Medline]
  32. Göttlicher M, Widmark E, Li Q, Gustafsson JA 1992 Fatty acids activate a chimera of the clofibric acid-activated receptor and the glucocorticoid receptor. Proc Natl Acad Sci USA 89:4653–4657[Abstract/Free Full Text]
  33. Janowski BA, Willy PJ, Devi TR, Falck JR, Mangelsdorf DJ 1996 An oxysterol signalling pathway mediated by the nuclear receptor LXR{alpha}. Nature 383:728–731[CrossRef][Medline]
  34. Makishima M, Okamoto AY, Repa JJ, Tu H, Learned RM, Luk A, Hull MV, Lustig KD, Mangelsdorf DJ, Shanz B 1999 Identification of a nuclear receptor for bile acids. Science 284:1362–1365[Abstract/Free Full Text]
  35. Parks DJ, Blanchard SG, Bledsoe RK, Chandra G, Consler TG, Kliewer SA, Stimmel JB, Willson TM, Zavacki AM, Moore DD, Lehmann JM 1999 Bile acids: natural ligands for an orphan nuclear receptor. Science 284:1365–1368[Abstract/Free Full Text]
  36. Thacher SM, Vasudevan J, Chandraratna RAS 2000 Therapeutic applications for ligands of retinoid receptors. Curr Pharm Des 6:25–58[CrossRef][Medline]
  37. Simopoulos AP 1999 Essential fatty acids in health and chronic disease. Am J Clin Nutr 70:560S–569S
  38. Willson TM, Brown PJ, Sternbach DD, Henke BR 2000 The PPARs: from orphan receptors to drug discovery. J Med Chem 43:527–550[CrossRef][Medline]
  39. Chawla A, Repa JJ, Evans RM, Mangelsdorf DJ 2001 Nuclear receptors and lipid physiology: opening the X-files. Science 294:1866–1870[Abstract/Free Full Text]
  40. Willson TM, Jones SA, Moore JT, Kliewer SA 2001 Chemical genomics: functional analysis of orphan nuclear receptors in the regulation of bile acid metabolism. Med Res Rev 21:513–522[CrossRef][Medline]
  41. Lehmann JM, McKee DD, Watson MA, Willson TM, Moore JT, Kliewer SA 1998 The human orphan nuclear receptor PXR is activated by compounds that regulate CYP3A4 gene expression and cause drug interactions. J Clin Invest 102:1016–1023[Medline]
  42. Jones SA, Moore LB, Shenk JL, Wisely GB, Hamilton GA, McKee DD, Tomkinson NCO, LeCluyse EL, Lambert MH, Willson TM, Kliewer SA, Moore JT 2000 The pregnane X receptor: a promiscuous xenobiotic receptor that has diverged during evolution. Mol Endocrinol 14:27–39[Abstract/Free Full Text]
  43. Kawamoto T, Sueyoshi T, Zelko I, Moore R, Washburn K, Negishi M 1999 Phenobarbital-responsive nuclear translocation of the receptor CAR in induction of the CYP2B gene. Mol Cell Biol 19:6318–6322[Abstract/Free Full Text]
  44. Tzameli I, Moore DD 2001 Role reversal: new insights from new ligands for the xenobiotic receptor CAR. Trends Endocrinol Metab 12:7–10[CrossRef][Medline]
  45. Shiau AK, Coward P, Schwarz M, Lehmann JM 2001 Orphan nuclear receptors: from new ligand discovery technologies to novel signaling pathways. Curr Opin Drug Discovery Dev 4:575–590[Medline]
  46. Lu D, Kiriyama Y, Lee KY, Giguere V 2001 Transcriptional regulation of the estrogen-inducible pS2 breast cancer marker gene by the ERR family of orphan nuclear receptors. Cancer Res 61:6755–6761[Abstract/Free Full Text]
  47. Hermanson O, Glass CK, Rosenfeld MG 2002 Nuclear receptor coregulators: multiple modes of modification. Trends Endocrinol Metab 13:55–60[CrossRef][Medline]
  48. Bourguet W, Ruff M, Chambon P, Gronemeyer H, Moras D 1995 Crystal structure of the ligand-binding domain of the human nuclear receptor RXR-{alpha}. Nature 375:377–382[CrossRef][Medline]
  49. Weatherman RV, Fletterick RJ, Scanlan TS 1999 Nuclear-receptor ligands and ligand-binding domains. Annu Rev Biochem 68:559–581[CrossRef][Medline]
  50. Xu HE, Stanley TB, Montana VG, Lambert MH, Shearer BG, Cobb JE, McKee DD, Galardi CM, Plunket KD, Nolte RT, Parks DJ, Moore JT, Kliewer SA, Willson TM, Stimmel JB 2002 Structural basis for antagonist-mediated recruitment of nuclear co-repressors by PPAR{alpha}. Nature 415:813–816[Medline]
  51. Nolte RT, Wisely GB, Westin S, Cobb JE, Lambert MH, Kurokawa R, Rosenfeld MG, Willson TM, Glass CK, Milburn MV 1998 Ligand binding and co-activator assembly of the peroxisome proliferator-activated receptor-{gamma}. Nature 395:137–143[CrossRef][Medline]
  52. Steinmetz ACU, Renaud J-P, Moras D 2001 Binding of ligands and activation of transcription by nuclear receptors. Annu Rev Biophys Biomol Struct 30:329–359[CrossRef][Medline]
  53. Xu HE, Lambert MH, Montana VG, Plunket KD, Moore LB, Collins JL, Oplinger JA, Kliewer SA, Gampe Jr RT, McKee DD, Moore JT, Willson TM 2001 Structural determinants of ligand binding selectivity between the peroxisome proliferator-activated receptors. Proc Natl Acad Sci USA 98:13919–13924[Abstract/Free Full Text]
  54. Xu HE, Lambert MH, Montana VG, Parks DJ, Blanchard SG, Brown PJ, Sternbach DD, Lehmann JM, Wisely GB, Willson TM, Kliewer SA, Milburn MV 1999 Molecular recognition of fatty acids by peroxisome proliferator-activated receptors. Mol Cell 3:397–403[CrossRef][Medline]
  55. Herbst RS, Nielsch U, Sladek F, Lai E, Babiss LE, Darnell Jr JE 1991 Differential regulation of hepatocyte-enriched transcription factors explains changes in albumin and transthyretin gene expression among hepatoma cells. New Biol 3:289–296[Medline]
  56. Willson TM, Structure and function of the orphan nuclear receptors. Program of the 83rd Meeting of The Endocrine Society, Denver, CO, 2001, p 30 (Abstract S8-3)
  57. Bogan AA, Dallas-Yang Q, Ruse Jr MD, Maeda Y, Jiang G, Nepomuceno L, Scanlan TS, Cohen FE, Sladek FM 2000 Analysis of protein dimerization and ligand binding of orphan receptor HNF4{alpha}. J Mol Biol 302:831–851[CrossRef][Medline]
  58. Yoon JC, Puigserver P, Chen G, Donovan J, Wu Z, Rhee J, Adelmant G, Stafford J, Kahn CR, Granner DK, Newgard CB, Spiegelman BM 2001 Control of hepatic gluconeogenesis through the transcriptional coactivator PGC-1. Nature 413:131–138[CrossRef][Medline]
  59. Viollet B, Kahn A, Raymondjean M 1997 Protein kinase A-dependent phosphorylation modulates DNA-binding activity of hepatocyte nuclear factor 4. Mol Cell Biol 17:4208–4219[Abstract/Free Full Text]
  60. Ruse MD, Pivalsky ML, Sladek FM 2002 Competitive cofactor recruitment by orphan receptor hepatocyte nuclear factor 4{alpha}1: modulation by the F domain. Mol Cell Biol 22:1626–1638[Abstract/Free Full Text]
  61. Watkins RE, Wisely GB, Moore LB, Collins JL, Lambert MH, Williams SP, Willson TM, Kliewer SA, Redinbo MR 2001 The human nuclear xenobiotic receptor PXR: structural determinants of directed promiscuity. Science 292:2329–2333[Abstract/Free Full Text]
  62. Stehlin C, Wurtz J-M, Steinmetz A, Greiner E, Schule R, Moras D, Renaud J-P 2001 X-ray structure of the orphan nuclear receptor RORß ligand-binding domain in the active conformation. EMBO J 20:5822–5831[CrossRef][Medline]
  63. Renaud J-P, Harris JM, Downes M, Burke LJ, Muscat GEO 2000 Structure-function analysis of the Rev-erbA and RVR ligand-binding domains reveals a large hydrophobic surface that mediates corepressor binding and a ligand cavity occupied by side chains. Mol Endocrinol 14:700–717[Abstract/Free Full Text]
  64. Rininger JA, DiPippo VA, Gould-Rothberg BE 2000 Differential gene expression technologies for identifying surrogate markers of drug efficacy and toxicity. Drug Disc Today 5:560–568[CrossRef][Medline]
  65. Goodwin B, Jones SA, Price RR, Watson MA, McKee DD, Moore LB, Galardi C, Wilson JG, Lewis MC, Roth ME, Maloney PR, Willson TM, Kliewer SA 2000 A regulatory cascade of the nuclear receptors FXR, SHP-1, and LRH-1 represses bile acid biosynthesis. Mol Cell 6:517–526[CrossRef][Medline]
  66. Way JM, Harrington WW, Brown KK, Gottschalk WK, Sundseth SS, Mansfield TA, Ramachandran RK, Willson TM, Kliewer SA 2001 Comprehensive messenger ribonucleic acid profiling reveals that peroxisome proliferator-activated receptor {gamma} activation has coordinate effects on gene expression in multiple insulin-sensitive tissues. Endocrinology 142:1269–1277[Abstract/Free Full Text]
  67. Ueda A, Hamadeh HK, Webb HK, Yamamoto Y, Sueyoshi T, Afshari CA, Lehmann JM, Negishi M 2002 Diverse roles of the nuclear orphan receptor CAR in regulating hepatic genes in response to phenobarbital. Mol Pharmacol 61:1–6[Abstract/Free Full Text]
  68. Kastner P, Mark M, Chambon P 1995 Nonsteroid nuclear receptors: what are genetic studies telling us about their role in real life? Cell 83:859–869[CrossRef][Medline]
  69. Zetterstrom RH, Solomin L, Jansson L, Hoffer BJ, Olson L, Perlmann T 1997 Dopamine neuron agenesis in Nurr1-deficient mice. Science 276:248–250[Abstract/Free Full Text]
  70. Abuin A, Holt KH, Platt KA, Sands AT, Zambrowicz BP 2002 Full-speed mammalian genetics: in vivo target validation in the drug discovery process. Trends Biotechnol 20:36–42[CrossRef][Medline]
  71. Metzger D, Chambon P 2001 Site- and time-specific gene targeting in the mouse. Methods 24:71–80[CrossRef][Medline]
  72. Zhao L, Bakke M, Krimkevich Y, Cushman LJ, Parlow AF, Camper SA, Parker KL 2001 Steroidogenic factor 1 (SF1) is essential for pituitary gonadotrope function. Development 128:147–154[Abstract]
  73. Bennett CF, Cowsert LM 1999 Application of antisense oligonucleotides for gene functionalization and target validation. Curr Opin Mol Ther 1:359–371[Medline]
  74. Elbashir SM, Harborth J, Lendeckel W, Yalcin A, Weber K, Tuschl T 2001 Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411:494–498[CrossRef][Medline]
  75. Jover R, Bort R, Gomez-Lechon MJ, Castell JV 2001 Cytochrome P450 regulation by hepatocyte nuclear factor 4 in human hepatocytes: a study using adenovirus-mediated antisense targeting. Hepatology 33:668–675[CrossRef][Medline]
  76. Taylor JG, Choi EH, Foster CB, Chanock SJ 2001 Using genetic variation to study human disease. Trends Mol Med 7:507–512[CrossRef][Medline]
  77. Nowotny P, Kwon JM, Goate AM 2001 SNP analysis to dissect human traits. Curr Opin Neurobiol 11:637–641[CrossRef][Medline]
  78. Buervenich S, Carmine A, Arvidsson M, Xiang F, Zhang Z, Sydow O, Jonsson EG, Sedvall GC, Leonard S, Ross RG, Freedman R, Chowdari KV, Nimgaonkar VL, Perlmann T, Anvret M, Olson L 2000 NURR1 mutations in cases of schizophrenia and manic-depressive disorder. Am J Med Genet 96:808–813[CrossRef][Medline]
  79. Nishigori H, Tomura H, Tonooka N, Kanamori M, Yamada S, Sho K, Inoue I, Kikuchi N, Onigata K, Kojima I, Kohama T, Yamagata K, Yang Q, Matsuzawa Y, Miki T, Seino S, Kim MY, Choi HS, Lee YK, Moore DD, Takeda J 2001 Mutations in the small heterodimer partner gene are associated with mild obesity in Japanese subjects. Proc Natl Acad Sci USA 98:575–580[Abstract/Free Full Text]
  80. Hall JM, Chang C-Y, McDonnell DP 2000 Development of peptide antagonists that target estrogen receptor ß-coactivator interactions. Mol Endocrinol 14:2010–2023[Abstract/Free Full Text]
  81. McDonnell DP, Chang C-Y, Norris JD 2000 Development of peptide antagonists that target estrogen receptor-cofactor interactions. J Steroid Biochem Mol Biol 74:327–335[CrossRef][Medline]
  82. Orner BP, Ernst JT, Hamilton AD 2001 Toward proteomimetics: terphenyl derivatives as structural and functional mimics of extented regions of an {alpha}-helix. J Am Chem Soc 123:5382–5383[CrossRef][Medline]
  83. Nuclear Receptors Committee 1999 A unified nomenclature system for the nuclear receptor superfamily. Cell 97:161–163[CrossRef][Medline]



This article has been cited by other articles:


Home page
 Mol. Endocrinol.Home page
S. C. Wu and Y. Zhang
Minireview: Role of Protein Methylation and Demethylation in Nuclear Hormone Signaling
Mol. Endocrinol., September 1, 2009; 23(9): 1323 - 1334.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Endocrinol.Home page
S. D. Conzen
Minireview: Nuclear Receptors and Breast Cancer
Mol. Endocrinol., October 1, 2008; 22(10): 2215 - 2228.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
D. L. Howarth, S. H. W. Law, B. Barnes, J. M. Hall, D. E. Hinton, L. Moore, J. M. Maglich, J. T. Moore, and S. W. Kullman
Paralogous Vitamin D Receptors in Teleosts: Transition of Nuclear Receptor Function
Endocrinology, May 1, 2008; 149(5): 2411 - 2422.
[Abstract] [Full Text] [PDF]

Home page
 Pharmacol. Rev.Home page
D. D. Moore, S. Kato, W. Xie, D. J. Mangelsdorf, D. R. Schmidt, R. Xiao, and S. A. Kliewer
International Union of Pharmacology. LXII. The NR1H and NR1I Receptors: Constitutive Androstane Receptor, Pregnene X Receptor, Farnesoid X Receptor {alpha}, Farnesoid X Receptor beta, Liver X Receptor {alpha}, Liver X Receptor beta, and Vitamin D Receptor
Pharmacol. Rev., December 1, 2006; 58(4): 742 - 759.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Endocrinol.Home page
H. Lempiainen, F. Molnar, M. Macias Gonzalez, M. Perakyla, and C. Carlberg
Antagonist- and Inverse Agonist-Driven Interactions of the Vitamin D Receptor and the Constitutive Androstane Receptor with Corepressor Protein
Mol. Endocrinol., September 1, 2005; 19(9): 2258 - 2272.
[Abstract] [Full Text] [PDF]

Home page
 Sci Aging Knowl EnvironHome page
K. Pardee, J. Reinking, and H. Krause
Nuclear Hormone Receptors, Metabolism, and Aging: What Goes Around Comes Around
Sci. Aging Knowl. Environ., November 24, 2004; 2004(47): re8 - re8.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
A. Pawar and D. B. Jump
Unsaturated Fatty Acid Regulation of Peroxisome Proliferator-activated Receptor {alpha} Activity in Rat Primary Hepatoctes
J. Biol. Chem., September 19, 2003; 278(38): 35931 - 35939.
[Abstract] [Full Text] [PDF]

Home page
 Nucleic Acids ResHome page
J. M. Maglich, J. A. Caravella, M. H. Lambert, T. M. Willson, J. T. Moore, and L. Ramamurthy
The first completed genome sequence from a teleost fish (Fugu rubripes) adds significant diversity to the nuclear receptor superfamily
Nucleic Acids Res., July 15, 2003; 31(14): 4051 - 4058.
[Abstract] [Full Text] [PDF]

Home page
 Endocr. Rev.Home page
P. Y. Liu, A. K. Death, and D. J. Handelsman
Androgens and Cardiovascular Disease
Endocr. Rev., June 1, 2003; 24(3): 313 - 340.
[Abstract] [Full Text] [PDF]

Home page
 Circ. Res.Home page
X. Liu, X. Huang, and C. D. Sigmund
Identification of a Nuclear Orphan Receptor (Ear2) as a Negative Regulator of Renin Gene Transcription
Circ. Res., May 16, 2003; 92(9): 1033 - 1040.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit a related Letter to the Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Reprints, Permissions and Rights
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Willson, T. M.
Right arrow Articles by Moore, J. T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Willson, T. M.
Right arrow Articles by Moore, J. T.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals