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Review
Volume 7 | 2009
Cite this Article: Nuclear Receptor Signaling (2009) 7, e005.
Dynamic and combinatorial control of gene expression by nuclear retinoic acid receptors (RARs)
Cécile Rochette-Egly and Pierre Germain
Institut de Génétique et de Biologie Moléculaire et Cellulaire, Department of Functional Genomics, INSERM U596, CNRS UMR7104, Université Louis Pasteur de Strasbourg, Strasbourg, France (CRE) and Centre de Biochimie Structurale, Department “Structure, Cancer et Virulence”, CNRS U5048 - INSERM U554, Montpellier, France (PG)

Received: February 5, 2009; Accepted: April 17, 2009; Published: May 8, 2009

Copyright © 2009, Rochette-Egly and Germain. This is an open-access article distributed under the terms of the Creative Commons Non-Commercial Attribution License, which permits unrestricted non-commercial use, distribution and reproduction in any medium, provided the original work is properly cited.

Article DOI: 10.1621/nrs.07005

Abstract

Nuclear retinoic acid receptors (RARs) are transcriptional regulators controlling the expression of specific subsets of genes in a ligand-dependent manner. The basic mechanism for switching on transcription of cognate target genes involves RAR binding at specific response elements and a network of interactions with coregulatory protein complexes, the assembly of which is directed by the C-terminal ligand-binding domain of RARs. In addition to this scenario, new roles for the N-terminal domain and the ubiquitin-proteasome system recently emerged. Moreover, the functions of RARs are not limited to the regulation of cognate target genes, as they can transrepress other gene pathways. Finally, RARs are also involved in nongenomic biological activities such as the activation of translation and of kinase cascades. Here we will review these mechanisms, focusing on how kinase signaling and the proteasome pathway cooperate to influence the dynamics of RAR transcriptional activity.

Abbreviations
AF-1: activation function 1; AF-2: activation function 2; AP-1: activating protein-1; CAK: Cdk activating complex; CAMK: calmodulin kinase; CARM1: coactivator-associated arginine methyltransferase 1; CBP: CREB (cyclic AMP response element binding protein) binding protein; Cdk: cyclin-dependent kinase; CRABP: cellular retinoic acid binding protein; CRBP: cellular retinol binding protein; CTE: C-terminal extension; Cyp26A1: cytochrome 450, family 26, subfamily a, polypeptide 1; DBD: DNA-binding domain; DR: direct repeat; DRIP: vitamin D receptor interacting protein; GluR1: glutamate receptor 1; HAT: histone acetyl transferase; HDAC: histone deacetyl transferase; HMT: histone methyl transferase; HNF: hepatocyte nuclear factor; Hox: homeobox gene; JNK: c-jun N-terminal kinase; LBD: ligand binding domain; LBP: ligand binding pocket; MAPK: mitogen-activated protein kinase; MSK: mitogen and stress-activated kinase; NCoR: nuclear receptor corepressor; NTD: N-terminal domain; p/CAF: p300/CBP-associated factor; PcG: polycomb group; PI3K: phosphoinositide 3-kinase; PIN1: protein interacting with NIMA (never in mitosis A); PKC: protein kinase C; PRAME: preferentially expressed antigen in melanoma; PRMT1: protein arginine methyl transferase; RA: retinoic acid; RAR: nuclear retinoic acid receptor; RXR: nuclear retinoid X receptor; RARE: RA response element; RIP140: receptor interacting protein of 140kDa; SAFB2: scaffold attachment factor B2 protein; SAGA: Spt-Ada-Gcn5-acetyl-transferase; SH3: Src-homology 3; SMRT: silencing mediator for retinoid and thyroid hormone receptors; SRC: steroid receptor coactivator; SWI/SNF: switch/sucrose non-fermenting; TBL1: transducin β-like 1; TBLR1: TBL1-related protein 1; TIF1α: transcription intermediary factor-1 α; TRAP: thyroid receptor associated protein; TRIP-1: thyroid receptor interacting protein-1; WW: trytophan-tryptophan
Introduction

Nuclear retinoic acid (RA) receptors (RARs) consist of three subtypes, α (NR1B1), β (NR1B2) and γ (NR1B3) encoded by separate genes [Germain et al., 2006a; Germain et al., 2006c], which function as ligand-dependent transcriptional regulators heterodimerized with retinoid X receptors (RXRs). For each subtype, there are at least 2 isoforms, which are generated by differential promoter usage and alternative splicing and differ only in their N-terminal regions. Activation of RARs by cognate ligands triggers transcriptional events leading to the activation or repression of subsets of target genes involved in cellular differentiation, proliferation and apoptosis ([Bour et al., 2006], and references therein).

The compounds that bind RARs and modulate their activity are referred to as retinoids. This generic term covers molecules that include natural vitamin A (retinol) metabolites and active synthetic analogs. Retinoids are hydrophobic, lipid-soluble, and of small size, so that they can easily cross the lipid bi-layer of cell membranes. Natural retinoids, exemplified by all-trans RA, are produced in vivo from the oxidation of vitamin A [Chambon, 2005; Sporn et al., 1994] (Figure 1). An isomerization product of RA, 9-cis RA, also binds RARs with high affinity, but whether this compound is a natural bioactive retinoid remains controversial [Germain et al., 2006b]. Beyond the natural compounds, major research efforts in retinoid chemistry have been directed towards the identification of potent synthetic molecules and led to the generation of several classes of compounds with a panel of activities ranging from agonists to antagonists, selective or not to RAR subtypes [de Lera et al., 2007] (Figure 1).

Figure 1: Chemical structure, transcriptional activity, and selectivity of main retinoids.

See text for details.


Note that for (B) and (D) in Figure 1, a given ligand may be considered as selective for a certain RAR subtype when it exhibits an affinity difference greater than 100-fold between its primary target and other receptors (see the recommended usage of terms in the field of nuclear receptors [Germain et al., 2006c]).

RARs have a well-defined domain organization and structure, consisting mainly of a central DNA-binding domain (DBD) linked to a C-terminal ligand-binding domain (LBD) (Figure 2). In the past 20 years, it has been established that the basic mechanism for transcriptional regulation by RARs relies on DNA binding to specific sequence elements located in the promoters of target genes and on ligand-induced conformational changes in the LBD that direct the dissociation/association of several coregulator complexes [Chambon, 1996; Germain et al., 2003; Laudet and Gronemeyer, 2001; Lefebvre et al., 2005]. The description of the crystallographic structures of these domains and the characterization of the multiprotein complexes that specify the transcriptional activity of RARs provided a wealth of information on how these receptors regulate transcription. However, recent years have witnessed the importance of the ubiquitin-proteasome system and that of the N-terminal domain (NTD), which also interacts with specific coregulators, despite its native disordered structure [Bour et al., 2007]. Moreover, according to recent studies, RARs are involved in other nongenomic biological activities such as the activation of translation and of kinase cascades. These kinases target RARs and their coregulators, adding more complexity to the understanding of RAR-mediated transcription. In this review we will focus, in addition to the basic scenario (DNA and ligand binding, dynamics of coregulator exchanges at the LBD), on recent advances in the nongenomic effects of RA, and on how phosphorylation cascades, the NTD and the ubiquitin-proteasome system cooperate for fine-tuning RAR activity.

Figure 2: Schematic representation of the RAR proteins with the functional domains and the main phosphorylation sites.

RARs have a modular structure composed of six conserved regions designated A to F. The C region contains the DBD. The E region contains several domains, the LBD, the AF-2 domain, the cyclin H binding domain and the dimerisation domain. It also contains a phosphorylation site for several kinases (PKA and MSK1). The N-terminal domain (NTD) corresponds to regions A and B and contains a proline-rich motif with phosphorylation sites for Cdks and MAPKs.


Structure/function analysis

As with most nuclear receptors, RARs exhibit a modular structure composed of 6 regions of homology (designated A to F, from the N-terminal to the C-terminal end) (Figure 2) harboring specific functions [Chambon, 1996; Laudet and Gronemeyer, 2001]. Regions C and E, which encompass the DBD and the LBD, respectively, are the most conserved and important domains and govern the classical model of RAR transcriptional activity. In contrast, the A/B, D, and F regions are poorly conserved.

The DNA binding domain (DBD)

The DBD, which confers sequence-specific DNA recognition, is composed of two zinc-nucleated modules, two α-helices and a COOH-terminal extension (CTE) [Zechel et al., 1994a; Zechel et al., 1994b]. Helix 1 and helix 2 cross at right angles to form the core of the DBD folding into a single globular domain that has been determined by nuclear magnetic resonance and crystallographic studies [Lee et al., 1993]. The DBD includes several highly-conserved sequence elements, referred to as P, D, T and A boxes, that have been shown to define or contribute to the response element’s specificity, to a dimerization interface within the DBDs and to contacts with the DNA backbone and residues flanking the DNA core recognition sequence [Germain et al., 2003; Germain et al., 2006c].

RARs bind as asymmetrically-oriented heterodimers with RXRs, to specific DNA sequences or RA response elements (RAREs), composed typically of two direct repeats of a core hexameric motif, PuG (G/T) TCA [Germain et al., 2003; Leid et al., 1992; Mangelsdorf and Evans, 1995] and located in the regulatory sequences of target genes. The classical RARE is a 5bp-spaced direct repeat (referred to as DR5). However, the heterodimers also bind to direct repeats separated by 1bp (DR1) or 2bp (DR2). Note that RXR homodimers also bind to DR1.

RAREs have been identified in the promoters of a large number of RA target genes implicated in a wide variety of functions. For instance, the classical DR5 elements are found in the promoters of the RARβ2 gene itself [de The et al., 1990], of the CYP26A1 (cytochrome 450, family 26, subfamily a, polypeptide 1) gene [Loudig et al., 2000] and of several Homeobox (Hox) and hepatocyte nuclear factor (HNF) genes [Dupe et al., 1997; Qian et al., 2000]. DR2 elements were identified in the CRBPI (Cellular retinol binding protein I) and CRABPII (Cellular retinoic acid binding protein II) gene promoters [Durand et al., 1992; Smith et al., 1991]. The only natural DR1 element has been found in the rat CRBPII gene promoter [Mangelsdorf et al., 1991].

On DR2 and DR5 elements, in vitro, RXR occupies the 5’ hexameric motif, whereas the RAR partner occupies the 3’ motif (5’-RXR-RAR-3’) [Chambon, 1996; Laudet and Gronemeyer, 2001]. In contrast, on DR1 elements, the polarity is reversed, with the RAR DBD binding upstream and the RXR DBD downstream (5’-RAR-RXR-3’), switching the activity of the heterodimer from an activator to a repressor of target genes in the presence of RA.

So far, it has proved difficult to visualize the full-length RXR-RAR heterodimer in complex with DNA. However, the crystal structure of the DBDs in complex with DNA has been solved [Khorasanizadeh and Rastinejad, 2001; Rastinejad, 2001; Rastinejad et al., 2000]. Each DBD interacts with the DNA major groove at the level of a half-site through the P box of the first helix containing three exposed residues responsible for discrimination between different half-sites’ sequences. Then, they arrange head-to-tail, with cooperative contacts between the DBDs, leading to a mutual reinforcement of protein-protein and protein-DNA interactions. Depending on the DR spacing, different regions of the DBD of each receptor are used to create the dimerization interface, in order to achieve the required binding to the response elements. The heterodimeric DBD interface that is responsible for the binding of RXR-RAR heterodimers to DR5 elements involves the D box of the RXR second zinc-finger, and the tip of the RAR first zinc finger. However, when the heterodimers bind with reverse polarity to DR1 elements, they associate through the second zinc finger of RAR and the so-called T box (within the CTE- of RXR). This implies that the DBDs must be rotationally flexible with respect to the LBD dimerization interface and that the DNA curvature is different (for review, see [Renaud and Moras, 2000] and references therein). In conclusion, the DBDs of each heterodimerization partner dictate the specificity of RARE recognition and contribute through their dimerization to increase DNA binding efficiency.

The ligand binding domain (LBD)

The structures of the RAR LBDs are rather similar, as demonstrated by crystallographic studies [Moras and Gronemeyer, 1998; Renaud and Moras, 2000; Wurtz et al., 1996]. The LBD is formed by 12 conserved α helices and a β-turn (situated between H5 and H6). Helices 1-11 are folded into a three-layered, and parallel helical sandwich with H4, H5, H8, H9 and H11 sandwiched between H1, H2 and H3 on one side and H6, H7 and H10 on the other. In contrast, the C-terminal helix, H12, is more flexible and adopts conformations that may differ from one RAR subtype to the other (see below). The LBD is functionally complex, as it contains the ligand-binding pocket (LBP), the main dimerization domain and the ligand-dependent activation function-2 (AF-2).

The ligand-binding pocket (LBP). The ligand-binding pocket (LBP) comprises hydrophobic residues mainly from helices H3, H5, H11 and the β-sheet, and crystallographic studies revealed the structural basis of ligand recognition [Bourguet et al., 2000a; Li et al., 2003; Renaud et al., 1995]. The shape of the LBP matches the volume of the ligand, maximizing the hydrophobic contacts and contributing to the selectivity of ligand binding [Gehin et al., 1999; Klaholz et al., 2000; Klaholz et al., 1998].

Given that the precise contacts with ligands involve three divergent residues located in H3, H5 and H11, which are unique for each subtype receptor-cognate ligand pair, it has been possible to generate subtype-selective ligands [Germain et al., 2004] (Figure 1). For instance, the unique polar residues S232 and M272 located in the LBP of RARα and RARγ, respectively, have been exploited to develop ligands that are specific for RARα (Am580) or RARγ (BMS270394 or CD666). Via their amino group, such ligands establish hydrogen bonds with RARα S232 or RARγ M272, thereby increasing affinity and selectivity for RARα and RARγ, respectively. However, as the RARβ LBP does not contain specific polar residues, the development of RARβ-selective ligands is more challenging [Germain et al., 2004]. Nevertheless, due to differences in the volume and the shape of the LBPs of each RAR subtype, it has been possible to generate molecules with complex activities such as ligands that are RARβ agonists and RARα/RARγ antagonists, the larger size of RARβ LBP accounting for this mixed profile [Chen et al., 1995; Germain et al., 2004].

The heterodimerization surface. The heterodimerization surface involves residues from helices H7, H9, H10 and H11, as well as loops L8-9 and L9-10 [Bourguet et al., 2000b; Pogenberg et al., 2005]. Helices H9 and H10 contribute to more than 75% of the total dimerization surface and constitute the core of the dimer interface. It has been proposed that in RXR-RAR heterodimers, ligand binding affects the stability and propagation of signals across the heterodimerization surface, indicating that the LBP and the dimerization interface are in some way energetically linked [Brelivet et al., 2004].

The C-terminal helix 12, named AF-2. The C-terminal helix 12, named AF-2, controls the ability of RARs to interact with coregulators. The analysis of the crystal structures of the unliganded and ligand-bound LBDs of RXRα and RARα, respectively [Bourguet et al., 1995; Renaud et al., 1995], highlighted the crucial conformational flexibility of H12 and suggested a mechanism by which AF-2 becomes transcriptionally competent [Egea et al., 2001; Steinmetz et al., 2001]. Upon ligand binding, a series of intra-molecular interactions cause the repositioning of H11 in the continuity of H10 and the concomitant swinging of H12 which moves in a mouse trap model, sealing the “lid” of the LBP and being tightly packed against H3 and H4. Consequently, ligand binding is stabilized, and a new hydrophobic cleft is formed between H3, H4 and H12 that generates a defined interaction surface for transcriptional coactivators. In contrast, in the case of the RARβ and RARγ subtypes [Farboud et al., 2003; Farboud and Privalsky, 2004; Hauksdottir et al., 2003], biochemical studies proposed that, even in the absence of ligand, H12 would interact with H3 and adopt a constitutively closed conformation that approximates the conformation of liganded RARα. The importance of H12 in regulating coactivator and corepressor binding is detailed below.

The N-terminal AF-1 domain (NTD)

Early studies revealed the importance of the NTD of RARs, which corresponds to the A and B regions and includes the activation function AF-1, in the control of transcription of RA target genes [Nagpal et al., 1993; Nagpal et al., 1992]. However, they did not elucidate the underlying mechanism. It is interesting to note that within the NTD, the A region differs between the different subtypes and between isoforms [Chambon, 1996]. In contrast, the B region is rather conserved and depicts a proline-rich motif, which contains phosphorylation sites (Figure 2). Most importantly, proline-rich motifs can bind proteins with SH3 (Src-homology-3) or WW (tryptophan-tryptophan) domains, with phosphorylation preventing or favoring the interaction [Ball et al., 2005].

In contrast to the DBD and the LBD, there are still no high-resolution structures available for the NTD of RARs. Several biochemical and structural studies coupled to structure prediction algorithms suggested that the NTDs of RARs, as well as any member of the nuclear receptor family, are of naturally-disordered structure [Lavery and McEwan, 2005; Warnmark et al., 2003]. Most importantly, it has recently emerged that unstructured proteins or domains may be functional, undergoing transitions to more ordered states or folding into stable secondary or tertiary structures upon binding to DNA response elements or to coregulatory proteins [Dyson and Wright, 2005; Liu et al., 2006]. Moreover, disordered domains provide the flexibility that is needed for modification by enzymes such as kinases and ubiquitin-ligases [Dyson and Wright, 2005]. Such modifications may induce changes in the structural properties of the domain with profound impacts on its interactions with coregulators and/or on the dynamics of adjacent structural domains.

The D region

Poorly conserved, this region is considered to serve as a hinge between the DBD and the LBD, allowing rotation of the DBD. Therefore, it might allow the DBD and the LBD to adopt different conformations without creating steric hindrance problems. It also harbors nuclear localization signals.

The F region

This region extends C-terminal to helix 12 in RARs, but is absent in RXRs. It is highly variable in length and sequence among the different RAR subtypes and its three-dimensional structure is still unknown. Interestingly, region F is phosphorylated at multiple positions that might modify the properties of RARs [Bastien et al., 2000; Rochette-Egly et al., 1997]. Though the functions of region F are still poorly understood, it has been suggested that, in the absence of ligand, this region would stabilize H12 of the RARα subtype in an unclosed conformation, thereby enhancing corepressor binding [Farboud and Privalsky, 2004]. According to recent studies, this region would be also capable of binding to specific mRNA motifs [Poon and Chen, 2008].

The canonical RAR-mediated regulation of transcription with the LBD as the main actor

RARs are considered to be highly-regulated DNA-binding transcription factors that control transcription via several distinct mechanisms, including both repression and activation. Many years ago, it was established that after site-specific DNA binding, the final transcriptional activity of RARs depends on a set of associated proteins, the so-called corepressors and coactivators. From a molecular point of view, the discrimination between corepressors and coactivators is governed by the position of H12, which is directed by the ligand and contributes in a critical manner to the generation or removal of interaction surfaces.

The position of helix 12 governs the exposure of interaction surfaces for corepressors or coactivators

A recurring structural feature of corepressors and coactivators is the presence of highly-conserved motifs that are implicated in their recruitment at the LBD of RARs. The nuclear receptor corepressor (NCoR/NCoR1/RIP13) and silencing mediator for retinoid and thyroid hormone receptors (SMRT/NCoR2/TRAC) [Aranda and Pascual, 2001; Glass and Rosenfeld, 2000; Privalsky, 2004] contain in their C-terminal part two and three nuclear receptor interaction domains, respectively, with an LxxI/HIxxxI/L motif, which forms an extended α helix. Coactivators, which include essentially the p160 subfamily of steroid receptor coactivators (SRC), namely SRC-1 (also referred to as NCoA-1), SRC-2 (TIF-2, GRIP-1) and SRC-3 (pCIP, ACTR, AlB1, TRAM1, RAC3) [Glass and Rosenfeld, 2000; Lefebvre et al., 2005; Perissi et al., 1999], depict three copies of a highly-conserved LxxLL motif, which forms a short α helix.

In the absence of ligand, the surface of the RARα LBD presents a hydrophobic groove generated by H3, L3-4 and H4. It is worth noting that this surface is topologically related to that involved in coactivator interaction, but without H12 [Hu and Lazar, 1999] it can bind the extended LxxI/HIxxxI/L motif of corepressors. The N-terminal part of this motif extends in such a way that it masks the H12 interaction interface, thus explaining why the binding of corepressors and coactivators is mutually exclusive. Interestingly, the RARγ and RARβ subtypes interact poorly with corepressors [Farboud et al., 2003; Hauksdottir et al., 2003; Privalsky, 2004]. It has been proposed that hydrophobic interactions between H3 and H12 sequester H12 in a closed conformation, even in the absence of ligand, thus occluding the corepressor-docking site. Note that this closed conformation also prevents coactivator binding.

Upon ligand binding, RARα undergoes conformational changes and H12 becomes reoriented with a conserved glutamate residue forming a charge clamp with a lysine in H3. Such a charge clamp can specifically grip the ends of a helix of the specific length specified by the LxxLL motif of the coactivators, therefore allowing the leucine side chains to pack into the hydrophobic cavity. Because this ligand-activated charge clamp does not fit with the extended LxxI/HIxxxI/L motif of corepressors, it has been proposed that the alternative interactions of RARα with corepressors and coactivators originate from the difference in length of the interacting motifs that can be accommodated in the hydrophobic cleft in the two conformations [Germain et al., 2006c; Perissi et al., 1999].

Several crystal structures of RAR LBDs bound to synthetic retinoids revealed that ligand interactions with H11 and H12, or residues in their proximity, are primary determinants of helix 12 position, and that H12 can adopt not only the active and inactive positions, but also several intermediary positions. This implies that relatively subtle ligand modifications could significantly alter the conformation of the LBD and the H12 molecular switch, thereby generating distinct coregulator binding interfaces. Therefore, a panel of compounds comprising not only agonists, but also antagonists, inverse agonists and partia

ISSN# 1550-7629
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