Ecdysone receptor

Evolutionary homologs

Table of contents

Retinoic acid receptor RAR: a model for Ecdysone receptor in vertebrates - RAR-RXR interaction and binding to DNA

Retinoic acid, a pleiotropic regulator of development and homeostasis, controls the expression of specific gene networks via direct interactions with nuclear receptors. The retinoic acid receptor (RAR), as a heterodimer with the retinoid-x receptor (RXR), binds to DNA recognition sites, referred to as retinoic acid response elements (RAREs), that are generally composed of a direct repeat of the half-site core motif PuGGTCA spaced by 2 (DR-2) or 5 (DR-5) basepairs. The asymmetric nature of direct repeat RAREs suggests that RAR and RXR bind preferentially to one of the two half-site core motifs. RXR occupies the 5'-up-stream half-site, and RAR the 3'-down-stream half-site of the direct repeat in both DR-2 and DR-5 RAREs. A region adjacent to the zinc finger region of RAR and RXR is essential for specific and cooperative binding of DNA-binding domain peptides to RAREs. However, differential utilization of these determinants mediates RAR-RXR heterodimer binding to DR-2 and DR-5 RAREs. The demonstration of ordered but nonequivalent binding of RAR-RXR complexes to DR-2 and DR-5 RAREs sets a precedent for the generation of sequence specificities in heterodimeric DNA-binding proteins (Predki, 1994).

The biological activities of the retinoids are mediated by two nuclear hormone receptors: the retinoic acid receptor (RAR) and the retinoid-X receptor (RXR). RXR (and its insect homolog Ultraspiracle) is a common heterodimeric partner for many other nuclear receptors, including the insect ecdysone receptor. Whereas RXR can be readily expressed in Escherichia coli to produce soluble protein, this is not the case for many of its heterodimeric partners. For example, overexpression of RAR results mostly in inclusion bodies with the residual soluble component unable to interact with RXR or ligand efficiently. Similar results are seen with other RXR/ultraspiracle partners. To overcome these problems, a novel double cistronic vector was designed to coexpress RXR and its partner ligand-binding domains in the same bacterial cell. This results in a dramatic increase in production of soluble and apparently stable heterodimer. Hormone-binding studies using the purified RXR-RAR heterodimer reveal increased ligand-binding capacity of both components of 5- to 10-fold, resulting in virtually complete functionality. Bacterially expressed receptors can exist in one of three distinct states: insoluble, soluble but unable to bind ligand, or soluble with full ligand-binding capacity. These results suggest that coexpression may represent a general strategy for biophysical and structural analysis of receptor complexes (Li, 1997).

Heterodimerization is a common paradigm among eukaryotic transcription factors. The 9-cis retinoic acid receptor (RXR) serves as a common heterodimerization partner for several nuclear receptors, including the thyroid hormone receptor (T3R) and retinoic acid receptor (RAR). This raises the question as to whether these complexes possess dual hormonal responsiveness. A strategy was devised to examine the transcriptional properties of each receptor, either individually or when tethered to a heterodimeric partner. The intrinsic binding properties of RXR are masked in T3R-RXR and RAR-RXR heterodimers. In contrast, RXR is active as a non-DNA-binding cofactor with the NGFI-B/Nurr1 orphan receptors. Heterodimerization of RXR with constitutively active NGFI-B/Nurr1 creates a novel hormone-dependent complex. These findings suggest that allosteric interactions among heterodimers create complexes with unique properties. It is suggested that allostery is a critical feature underlying the generation of diversity in hormone response networks (Forman, 1995).

The receptor for 9-cis-retinoic acid, retinoid X receptor (RXR), forms heterodimers with several nuclear receptors, including the receptor for all-trans-retinoic acid, RAR. Previous studies have shown that retinoic acid receptor can be activated in RAR/RXR heterodimers, whereas RXR is believed to be a silent co-factor. Efficient growth arrest and differentiation of the human monocytic cell line U-937 require activation of both RAR and RXR. The allosteric inhibition of RXR is not obligatory; RXR can be activated in the RAR/RXR heterodimer in the presence of RAR ligands. Remarkably, RXR inhibition by RAR can also be relieved by an RAR antagonist. Moreover, the dose response of RXR agonists differs between RXR homodimers and RAR/RXR heterodimers, indicating that these complexes are pharmacologically distinct. The AF2 activation domain of both subunits contributes to activation even if only one of the receptors is associated with ligand. These data emphasize the importance of signaling through both subunits of a heterodimer in the physiological response to retinoids and show that the activity of RXR is dependent on both the identity and the ligand binding state of its partner (Botling, 1997).

Several nuclear receptors including the all-trans retinoic acid receptor RAR, form heterodimers with the 9-cis retinoic acid receptor, RXR. RXR-RAR heterodimers show an impressive flexibility in DNA binding and can recognize palindromic, inverted palindromes and direct repeats of the core half-site sequence AGGTCA. Dimerization interfaces in the DNA-binding domains of RXR, RAR, and thyroid hormone receptor (TR) that promote selective binding to strictly spaced direct repeats have previously been identified. However, an additional dimerization domain is present within the ligand-binding domains (LBDs) of these receptors. A transferable 40-amino acid region is located within the LBDs of RXR, RAR, TR, and chicken ovalbumin upstream promoter transcription factor that is critical for determining identity in the heterodimeric interaction and for high-affinity DNA binding. This region overlaps almost perfectly with a helical segment in the RXR LBD crystal structure that is part of the dimer interface. These data suggest a sequential pathway for nuclear receptor dimerization whereby the LBD dimerization interface initiates the formation of solution heterodimers that, in turn, acquire the capacity to bind to a number of differently organized repeats. Formation of a second dimer interface within the DNA-binding domain (DBD) restricts receptors to direct repeat targets. Accordingly, the combination of an obligatory (LBD) and an optional (DBD) dimerization domain imparts a dynamic DNA-binding potential to the heterodimerizing receptors that both increases the diversity of the hormonal response as well as providing a restricted set of target sequences in direct repeat elements that ensures physiological specificity (Perlmann, 1996).

A mouse mutation has been engineered that specifically deletes the C-terminal 18 amino acid sequence of the RXRalpha protein. This deletion (RXRalphaaf2o) corresponds to the last helical alpha structure (H12) of the ligand-binding domain (LBD), and includes the core of the Activating Domain of the Activation Function 2 (AF-2 AD core), which is thought to be crucial in mediating ligand-dependent transactivation by RXRalpha. The homozygous mutants, which die during the late fetal period or at birth, exhibit a subset of the abnormalities previously observed in RXRalpha-/- mutants, often with incomplete penetrance. In marked contrast, compound mutants bearing mutations in RXRalpha and RXRbeta or RXRalpha, RXRbeta and RXR gamma display a large array of malformations, which nearly recapitulate the full spectrum of the defects that characterize the fetal vitamin A-deficiency (VAD) syndrome and were previously found in RAR single and compound mutants, as well as in RXRalpha/RAR(alpha, beta or gamma) compound mutants. Analysis of RXRalphaaf2o/RAR(alpha, beta or gamma) compound mutants also reveals that they exhibit many of the defects observed in the corresponding RXRalpha/RAR compound mutants. Together, these results demonstrate the importance of the integrity of RXR AF-2 for the developmental functions mediated by RAR/RXR heterodimers, and hence suggest that RXR ligand-dependent transactivation is instrumental in retinoid signaling during development. In the presence of both RXR and RAR ligands, RXRalphaAF2o/RAR heterodimers are less efficient than wild-type heterodimers at providing stable occupancy of RA response elements. Since RXRalphaAF2o/RAR heterodimers bind RARE in vitro as efficiently as WT heterodimers, irrespective of ligand presence, this RXR AF2- and ligand-dependent enhancement of promoter occupancy most probably involves events occurring at the chromatin level (Mascrez, 1998).

A subset of nuclear receptors, including those for thyroid hormone (TR), retinoic acid, vitamin D3, and eicosanoids, can form heterodimers with the retinoid X receptor (RXR) on DNA regulatory elements in the absence of their cognate ligands. In a mammalian two-hybrid assay, recruitment of a VP16-RXR chimera by a Gal4-TRbeta ligand-binding domain fusion is enhanced up to 50-fold by thyroid hormone (T3). This is also observed with a mutant fusion, Gal4-TR(L454A), lacking ligand-inducible activation function (AF-2) and unable to interact with putative coactivators, suggesting that the AF-2 activity of TR or intermediary cofactors is not involved in this effect. The wild-type and mutant Gal4-TR fusions also exhibit hormone-dependent recruitment of RXR in yeast. Hormone-dependent recruitment of RXR is also evident with another Gal4-TR mutant, AHTm, which does not interact with the nuclear receptor corepressor N-CoR, suggesting that ligand-enhanced dimerization is not a result of T3-induced corepressor release. The interaction between RXR and TR is augmented by T3 in vitro, arguing against altered expression of either partner in vivo mediating this effect. It is proposed that ligand-dependent heterodimerization of TR and RXR in solution may provide a further level of control in nuclear receptor signaling (Collingwood, 1997).

All-trans-retinoic acid (trans-RA) and other retinoids exert anticancer effects through two types of retinoid receptors, the RA receptors (RARs) and retinoid X receptors (RXRs). Previous studies have demonstrated that the growth-inhibitory effects of trans-RA and related retinoids are impaired in certain estrogen-independent breast cancer cell lines due to their lower levels of RAR alpha and RARbeta. In this study, several synthetic retinoids were evaluated for their ability to induce growth inhibition and apoptosis in both trans-RA-sensitive and trans-RA-resistant breast cancer cell lines. RXR-selective retinoids, particularly in combination with RAR-selective retinoids, can significantly induce RARbeta and inhibit the growth and induce the apoptosis of trans-RA-resistant, RAR alpha-deficient MDA-MB-231 cells but have low activity against trans-RA-sensitive ZR-75-1 cells that express high levels of RAR alpha. The effects of RXR-selective retinoids on MDA-MB-231 cells are most likely mediated by RXR-nur77 heterodimers that bind to the RA response element in the RARbeta promoter and activate the RARbeta promoter in response to RXR-selective retinoids. In contrast, growth inhibition by RAR-selective retinoids in trans-RA-sensitive, RAR alpha-expressing cells most probably occurs through RXR-RAR alpha heterodimers that also bind to and activate the RARbeta promoter. In MDA-MB-231 clones stably expressing RAR alpha, both RARbeta induction and growth inhibition by RXR-selective retinoids are suppressed, while the effects of RAR-selective retinoids are enhanced. Together, these results demonstrate that activation of RXR can inhibit the growth of trans-RA-resistant MDA-MB-231 breast cancer cells and suggest that low cellular RAR alpha may regulate the signaling switch from RAR-mediated to RXR-mediated growth inhibition in breast cancer cells (Wu, 1997).

The 9-cis retinoic acid receptor (retinoid X receptor, RXR) forms heterodimers with the all-trans retinoic acid receptor (RAR) and other nuclear receptors on DNA regulatory sites composed of tandem binding elements. The 1.70Å resolution structure of the ternary complex of RXR and RAR DNA-binding regions in complex with the retinoic acid response element DR1 is described. The receptors recognize identical half-sites through extensive base-specific contacts; however, RXR binds exclusively to the 3' site to form an asymmetric complex with the reverse polarity of other RXR heterodimers. The subunits associate in a strictly DNA-dependent manner using the T-box of RXR and the Zn-II region of RAR, both of which are reshaped in forming the complex. The protein-DNA contacts as well as the dimerization interface and the DNA curvature in the RXR-RAR complex are all distinct from those of the RXR homodimer, which also binds DR1. Together, these structures illustrate how the nuclear receptor superfamily exploits conformational flexibility and locally induced structures to generate combinatorial transcription factors (Rastinejad, 2000).

The crystal structure of a heterodimer between the ligand-binding domains (LBDs) of the human RARalpha bound to a selective antagonist and the constitutively active mouse RXRalphaF318A mutant shows that, pushed by a bulky extension of the ligand, RAR alpha helix H12 adopts an antagonist position. The unexpected presence of a fatty acid in the ligand-binding pocket of RXRalphaF318A is likely to account for its apparent 'constitutivity'. Specific conformational changes suggest the structural basis of pure and partial antagonism. The RAR-RXR heterodimer interface is similar to that observed in most nuclear receptor (NR) homodimers. A correlative analysis of 3D structures and sequences provides a novel view on dimerization among members of the nuclear receptor superfamily (Bourguet, 2000).

Circadian clock genes are expressed in the suprachiasmatic nucleus and in peripheral tissues to regulate cyclically physiological processes. Synchronization of peripheral oscillators is thought to involve humoral signals, but the mechanisms by which these are mediated and integrated are poorly understood. A hormone-dependent physical interaction of the nuclear receptors, RARalpha and RXRalpha, with CLOCK and the Cycle homolog MOP4 is reported. These interactions negatively regulate CLOCK/MOP4:BMAL1-mediated transcriptional activation of clock gene expression in vascular cells. MOP4 exhibits a robust rhythm in the vasculature, and retinoic acid can phase shift Per2 mRNA rhythmicity in vivo and in serum-induced smooth muscle cells in vitro, providing a molecular mechanism for hormonal control of clock gene expression. It is proposed that circadian or periodic availability of nuclear hormones may play a critical role in resetting a peripheral vascular clock (McNamara, 2001).

Degradation of RAR

The nuclear retinoic acid receptor RARgamma2 undergoes proteasome-dependent degradation upon ligand binding. Evidence is provided that the domains that signal proteasome-mediated degradation overlap with those that activate transcription, i.e. the activation domains AF-1 and AF-2. The AF-1 domain signals RARgamma2 degradation through its phosphorylation by p38MAPK in response to RA. The AF-2 domain acts via the recruitment of SUG-1, which belongs to the 19S regulatory subunit of the 26S proteasome. Blocking RARgamma2 degradation through inhibition of either the p38MAPK pathway or the 26S proteasome function impairs its RA-induced transactivation activity. Thus, the turnover of RARgamma2 is linked to transactivation (Gianni, 2002).

Table of contents

Ecdysone receptor: Biological Overview | Regulation | Targets of Activity | Protein interactions | Developmental Biology | Effects of mutation | References

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