Human Retinoic Acid Receptor Research Articles

Biomineralized Nuclear Receptor Protein-Selection Mass Spectrometry for the Discovery of Drugs and Toxics.

Protein-selection mass spectrometry is cost-effective for the discovery of drugs and toxics. Nuclear receptors (NRs) are major targets for pharmaceuticals and endocrine-disrupting chemicals and are, thus, widely used as "bait" proteins. However, their application is limited due to the tendency to lose protein activity during cold storage. To address this problem, we introduced a novel biomineralization-based approach to preserve activity in NRs, exemplified by human retinoic acid receptor alpha (hRARα), a target for cancer and leucocythemia therapy. Since information on the coordination chemistry of metal ion and NR protein complexes is almost unavailable, we applied peptide mapping analysis for the first time for the rational design of his-hRARα-Co phosphate nanobiomaterial with high bioactivity. This nanobiomaterial successfully captured hRARα bioactive chemicals from a Chinese herb and environmental water and discovered an unsaturated fatty acid, (±)-(9Z,11E)-13-hydroxy-9,11-octadecadienoic acid ((±)13-HODE), which exhibited strong hRARα antagonistic activity.

Urine Excretion, Organ Distribution, and Placental Transfer of 6PPD and 6PPD-Quinone in Mice and Potential Developmental Toxicity through Nuclear Receptor Pathways.

The rubber antioxidant 6PPD has gained significant attention due to its highly toxic transformation product, 6PPD-quinone (6PPDQ). Despite their detection in urines of pregnant women, the placental transfer and developmental toxicity of 6PPD and 6PPDQ are unknown. Here, we treated C57Bl/6 mice with 4 mg/kg 6PPD or 6PPDQ to investigate their urine excretion and placental transfer. Female and male mice exhibited sex difference in excretion profiles of 6PPD and 6PPDQ. Urine concentrations of 6PPDQ were one order of magnitude lower than those of 6PPD, suggesting lower excretion and higher bioaccumulation of 6PPDQ. In pregnant mice treated with 6PPD or 6PPDQ from embryonic day 11.5 to 15.5, 6PPDQ showed ∼1.5-8 times higher concentrations than 6PPD in placenta, embryo body, and embryo brain, suggesting higher placental transfer of 6PPDQ. Using in vitro dual-luciferase reporter assays, we revealed that 6PPDQ activated the human retinoic acid receptor α (RARα) and retinoid X receptor α (RXRα) at concentrations as low as 0.3 μM, which was ∼10-fold higher than the concentrations detected in human urines. 6PPD activated the RXRα at concentrations as low as 1.2 μM. These results demonstrate the exposure risks of 6PPD and 6PPDQ during pregnancy and emphasize the need for further toxicological and epidemiological investigations.

Detection of Retinoic Acid-Active Chemicals in Diverse Sample Matrices Via a Quantum Dots-Based Nuclear Receptor Fluorescence Probe-Mediated Biosensor.

Developing a sensitive and reliable method for the screening of various endocrine-disrupting chemicals (EDCs) is in high demand and yet remains a significant challenge. Herein, we developed a CdSe/ZnS QDs-based nuclear receptor fluorescence probe (QDs-NRFP)-mediated biosensor for the screening of retinoic acid (RA)-active chemicals (a class of EDCs). The QDs-NRFP can be prepared on the spot via an antigen-antibody immunobinding interaction between the GST tag of the human retinoic acid receptor β ligand-binding domain (GST-hRARβ-LBD) and the CdSe/ZnS QDs-labeled anti-GST tag antibody. It can not only maintain the high binding activity of GST-hRARβ-LBD but also improve the sensitivity due to the high quantum yield of CdSe/ZnS QDs. Based on the indirect competition bioassay, the developed biosensor showed a detection limit of 1.8 ng/L all-trans-retinoic acid binding activity equivalent (atRA-BAE) with a linear range of 7.5-1183.6 ng/L. Compared with many cell-dependent in vitro assays, the QDs-NRFP-mediated biosensor is cell-free and unaffected by the cytotoxic substances in matrices and exhibited obvious superiority in detection time (within 40 min) and accuracy. As a case study, the biosensor was applied to detect RA binding activities in various sample matrices obtained from a wastewater treatment plant (WWTP) and physiological samples and showed satisfactory accuracy and reliability. The developed QDs-NRFP-mediated biosensor is expected to be capable of screening various EDCs with universality based on different nuclear receptor signaling pathways, which will substantially accelerate the assessment of global EDCs.

Quantitative Imaging of Retinoic Acid Activities in Living Mammalian Cells.

Retinoic acid (RA) is an intriguing metabolite that is necessary for embryonic development and differentiation in vertebrates. The present protocol demonstrates how to image RA activities indirectly in mammalian cells with ligand-activatable single-chain bioluminescence (BL) probes. We introduce 13 different molecular designs for characterizing an efficient single-chain probe that quantitatively visualizes RA activities with significant sensitivity. The key components included in the probes are (i) the N- and C-terminal fragments of artificial luciferase 16 (ALuc16), (ii) the ligand-binding domain of human retinoic acid receptor α (RAR LBD), and (iii) an LXXLL motif derived from common coactivators of nuclear receptors. The probe is highly selective and sensitive to all-trans-RA (at-RA) in animal cells. This protocol exemplifies quantitative imaging of the RA levels in serum and cerebrospinal fluid with a linear range in two orders. The present protocol is an important addition to conventional techniques on quantitative imaging of endogenous at-RA levels in live mammalian cells.

Peptide-Carbon Quantum Dots conjugate, Derived from Human Retinoic Acid Receptor Responder Protein 2, against Antibiotic-Resistant Gram Positive and Gram Negative Pathogenic Bacteria.

Antibiotic-resistant bacterial infections have become global issues for public health, which increases the utter need to develop alternatives to antibiotics. Here, the HSER (Homo sapiens retinoic acid receptor) peptide was designed from retinoic acid receptor responder protein 2 of Homo sapiens, and was conjugated with synthesized CQDs (carbon quantum dots) for enhanced antibacterial activity in combination, as individually they are not highly effective. The HSER–CQDs were characterized using spectrophotometer, HPLC coupled with electrospray-ionization quadrupole time-of-flight mass spectrometer (ESI–qTOF) mass spectrometer, zeta potential, zeta size, and FTIR. Thereafter, the antibacterial activity against Vancomycin-Resistant Staphylococcus aureus (VRSA) and Escherichia coli (carbapenem resistant) was studied using growth curve analysis, further supported by microscopic images showing the presence of cell debris and dead bacterial cells. The antibacterial mechanism of HSER–CQDs was observed to be via cell wall disruption and also interaction with gDNA (genomic DNA). Finally, toxicity test against normal human epithelial cells showed no toxicity, confirmed by microscopic analysis. Thus, the HSER–CQDs conjugate, having high stability and low toxicity with prominent antibacterial activity, can be used as a potential antibacterial agent.

Self-binding peptides: Binding-upon-folding versus folding-upon-binding

Self-binding peptide (SBP) represents a novel biomolecular phenomenon spanning between folding and binding. It is a structurally independent, short peptide segment within a monomeric protein and fulfills biological function by dynamically binding to/unbinding from its target domain in the same monomer. Here, four representative SBP systems, including mouse proto-oncogene Vav, human retinoic acid receptor RARγ, fruit fly scaffold module INAD and crypto 14-3-3 protein Cp14b, are investigated systematically by using atomistic molecular dynamics (MD) simulations and post binding energetics analyses. The native bound structure, artificial unbound state and isolated peptide segment of SBP moieties in the four systems were constructed, analyzed and compared in detail. It is revealed that the SBP interaction with their targets is almost a binding phenomenon at single-molecule level, but presence of a polypeptide linker between the SBP and target can promote the binding efficiency since the linker restriction largely increases the probability of SBP-target encounters in a statistical physics point of view. In this respect, unlike classical peptide-mediated interactions where the intrinsically disordered peptides are folded into an ordered structure upon binding to their protein partners (folding-upon-binding), we herein propose SBPs as a new and reversed biological event that is naturally a folding phenomenon but exhibits a typical binding behavior (binding-upon-folding).

Triphenyl phosphate-induced developmental toxicity in zebrafish: Potential role of the retinoic acid receptor

Using zebrafish as a model, we previously reported that developmental exposure to triphenyl phosphate (TPP) - a high-production volume organophosphate-based flame retardant - results in dioxin-like cardiac looping impairments that are independent of the aryl hydrocarbon receptor. Using a pharmacologic approach, the objective of this study was to investigate the potential role of retinoic acid receptor (RAR) - a nuclear receptor that regulates vertebrate heart morphogenesis - in mediating TPP-induced developmental toxicity in zebrafish. We first revealed that static exposure of zebrafish from 5-72h post-fertilization (hpf) to TPP in the presence of non-toxic concentrations of an RAR antagonist (BMS493) significantly enhanced TPP-induced toxicity (relative to TPP alone), even though identical non-toxic BMS493 concentrations mitigated retinoic acid (RA)-induced toxicity. BMS493-mediated enhancement of TPP toxicity was not a result of differential TPP uptake or metabolism, as internal embryonic doses of TPP and diphenyl phosphate (DPP) - a primary TPP metabolite - were not different in the presence or absence of BMS493. Using real-time PCR, we then quantified the relative change in expression of cytochrome P450 26a1 (cyp26a1) - a major target gene for RA-induced RAR activation in zebrafish - and found that RA and TPP exposure resulted in a ∼5-fold increase and decrease in cyp26a1 expression, respectively, relative to vehicle-exposed embryos. To address whether TPP may interact with human RARs, we then exposed Chinese hamster ovary cells stably transfected with chimeric human RARα-, RARβ-, or RARγ to TPP in the presence of RA, and found that TPP significantly inhibited RA-induced luciferase activity in a concentration-dependent manner. Overall, our findings suggest that zebrafish RARs may be involved in mediating TPP-induced developmental toxicity, a mechanism of action that may have relevance to humans.

Development of yeast reporter assay for screening specific ligands of retinoic acid and retinoid X receptor subtypes

Retinoic acids are essential for embryonic development, tissue organization, and homeostasis and act via retinoic acid receptors (RARs) that form heterodimers with retinoid X receptors (RXRs). Human RARs and RXRs include the three subtypes α, β, and γ, which have varying distributions and physiological functions among human tissues. Recent reports show that subtype-specific binding of several chemicals to RARs or RXRs may lead to endocrine disruption. To evaluate these ligand-like chemicals, convenient assay systems for each receptor subtype are required. We developed reporter assay yeasts to screen ligands for RXR subtype receptor homodimers. To screen RAR ligands, yeasts were engineered to express RAR subtypes with defective RXRα, which fails to bind to coactivators because of its shortened c-terminus. These assay yeasts were validated using known RXR- and RAR-specific ligands and subtype-specific responses were clearly shown. Subtype-specific ligand activities of the suspected chemical RAR or RXR ligands o-t-butylphenol, triphenyltin chloride, tributyltin chloride, and 4-nonylphenol were determined. The present assay yeasts may be valuable tools for subtype-specific assessments of unidentified environmental ligand chemicals and receptor-specific pharmaceuticals.

Expression of a Mutant Retinoic Acid Receptor β Alters Lineage Differentiation in Mouse Embryonic Stem Cells

We have introduced 1 to 2 copies of a deletion mutant (betaDeltaC) of the human retinoic acid receptor beta into mouse embryonic stem (ES) cells. The betaDeltaC-expressing cells were 10 to 100 times less sensitive to RA-induced differentiation in comparison with their parental cells. In the presence of 10(-7) M RA in monolayer culture, they showed no growth arrest or differentiation, but remained pluripotent. Embryoid bodies (EBs) derived from betaDeltaC-expressing cells differentiated into cardiomyocytes rather than neurons after treatment with 10(-6) M RA, and became neurons upon exposure to 10(-5) or 10(-4) M RA. Remarkably, after 10 passages of continuous culture in the presence of 10(-7) M RA, they still were able to form chimeras after injection into blastocysts. These data suggest that appropriate levels of normal retinoid receptors are crucial for lineage-specific differentiation of mouse ES cells in vitro. The betaDeltaC mutant protein may prove to be useful in promoting "stemness" of ES cells in culture.

Contamination with retinoic acid receptor agonists in two rivers in the Kinki region of Japan

This study was conducted to investigate the agonistic activity against human retinoic acid receptor (RAR) alpha in the Lake Biwa-Yodo River and the Ina River in the Kinki region of Japan. To accomplish this, a yeast two-hybrid assay was used to elucidate the spatial and temporal variations and potential sources of RARalpha agonist contamination in the river basins. RARalpha agonistic activity was commonly detected in the surface water samples collected along two rivers at different periods, with maximum all-trans retinoic acid (atRA) equivalents of 47.6 ng-atRA/L and 23.5 ng-atRA/L being observed in Lake Biwa-Yodo River and Ina River, respectively. The results indicated that RARalpha agonists are always present and widespread in the rivers. Comparative investigation of RARalpha and estrogen receptor alpha agonistic activities at 20 stations along each river revealed that the spatial variation pattern of RARalpha agonist contamination was entirely different from that of the estrogenic compound contamination. This suggests that the effluent from municipal wastewater treatment plants, a primary source of estrogenic compounds, seemed not to be the cause of RARalpha agonist contamination in the rivers. Fractionation using high performance liquid chromatography (HPLC) directed by the bioassay found two bioactive fractions from river water samples, suggesting the presence of at least two RARalpha agonists in the rivers. Although a trial conducted to identify RARalpha agonists in the major bioactive fraction was not completed as part of this study, comparison of retention times in HPLC analysis and quantification with liquid chromatography-mass spectrometry analysis revealed that the major causative contaminants responsible for the RARalpha agonistic activity were not RAs (natural RAR ligands) and 4-oxo-RAs, while 4-oxo-RAs were identified as the major RAR agonists in sewage in Beijing, China. These findings suggest that there are unknown RARalpha agonists with high activity in the rivers.

各種受容体導入酵母アッセイを用いた河川水の曝露モニタリング：茨城県と東京都を例として

Although the existence of chemicals having binding affinities for cellular receptors is becoming known and some of them are detected in environmental samples, their multiple abilities cannot be ascertained by individual measurements of chemicals. To enable monitoring of river water quality from effects on receptors, we attempted to measure the receptor activities of river water samples by multiple yeast assays transfected with a cellular receptor and co-activator. Water samples from rivers subjected substantially to treated sewage (such as Tama River located in Tokyo), rivers flowing in overpopulated centers (Sumida River, Kanda River and Meguro River in Tokyo) and rivers with luxuriant algae (Sakura River in Ibaraki) had higher activities to the human and medaka -estrogen receptors, the human aryl hydrocarbon receptor and the human retinoic acid receptor, respectively. Because these receptor activities were not consistent with the results of commonly used methods for water quality monitoring, the yeast assays are expected to provide new perspectives on estimation of influences by receptor effects to quality surveys of environmental waters.

A2E, a Pigment of the Lipofuscin of Retinal Pigment Epithelial Cells, Is an Endogenous Ligand for Retinoic Acid Receptor

Lipofuscin contains fluorophores, which represent a biomarker for cellular aging. Although it remains unsubstantiated clinically, experimental results support that the accumulation of lipofuscin is related to an increased risk of choroidal neovascularization due to age-related macular degeneration, a leading cause of legal blindness. Here, we report that a major lipofuscin component, A2E, activates the retinoic acid receptor (RAR). In vitro experiments using luciferase reporter assay, competitional binding assay, analysis of target genes, and chromatin immunoprecipitation (ChIP) assay strongly suggest that A2E is a bona fide ligand for RAR and induces sustained activation of RAR target genes. A2E-induced vascular endothelial growth factor (VEGF) expression in a human retinal pigment epithelial cell line (ARPE-19) and RAR antagonist blocked the up-regulation of VEGF. The conditioned medium of A2E-treated ARPE-19 cells induced tube formation in human umbilical vascular endothelial cells, which was blocked by the RAR antagonist and anti-VEGF antibody. These results suggest that A2E accumulation results in the phenotypic alteration of retinal pigment epithelial cells, predisposing the environment to choroidal neovascularization development. This is mediated through the agonistic function of A2E, at least in part. The results of this study provide a novel potential therapeutic target for this incurable condition.

Screening and detection of the in vitro agonistic activity of xenobiotics on the retinoic acid receptor

The retinoic acid receptors (RARs) play key roles in various biological processes in response to endogenous retinoic acids. However, excessive embryonic exposure to specific ligands for each subtype of the RAR was reported to induce specific developmental abnormalities. We measured the RAR agonistic activity of 543 chemicals using an assay system adopting yeast cells transfected with the human RAR gamma and a coactivator. Eighty-five of the 543 chemicals, including 16 organochlorine pesticides, 14 styrene dimers, 9 monoalkylphenols and 6 parabens, exhibited RAR gamma agonistic effects in this assay. In particular, monoalkylphenols having a 6-9 carbon alkyl group para to the phenolic hydroxyl group possessed high affinity for the RAR gamma, and their activities were 1.363-0.446% of that of all-trans RA. para-Alkylphenols chlorinated at the ortho position also were about as active or more active than their unchlorinated analogs. In addition, all tested styrene dimers showed positive effects, and the activity of 1-phenyltetralin, the strongest in this category, was 1.169% that of all-trans RA. A number of chemicals having binding affinity for the RAR gamma were revealed in this study (both newly identified and confirmed), further comprehensive studies of in vitro and in vivo effects via the RARs are required for the reliable risk assessment of chemicals. In vitro receptor binding studies represent an important step in hazard identification and suggest a potential mechanism of action, which can be an important step in risk assessment and in particular for screening studies to identify potential toxicity and inform mechanistic studies.

Receptor‐specific scoring functions derived from quantum chemical models improve affinity estimates for in‐silico drug discovery

The adaptation of forcefield-based scoring function to specific receptors remains an important challenge for in-silico drug discovery. Here we compare binding energies of forcefield-based scoring functions with models that are reparameterized on the basis of large-scale quantum calculations of the receptor. We compute binding energies of eleven ligands to the human estrogen receptor subtype alpha (ERalpha) and four ligands to the human retinoic acid receptor of isotype gamma (RARgamma). Using the FlexScreen all-atom receptor-ligand docking approach, we compare docking simulations parameterized by quantum-mechanical calculation of a large protein fragment with purely forcefield-based models. The use of receptor flexibility in the FlexScreen permits the treatment of all ligands in the same receptor model. We find a high correlation between the classical binding energy obtained in the docking simulation and quantum mechanical binding energies and a good correlation with experimental affinities R=0.81 for ERalpha and R=0.95 for RARgamma using the quantum derived scoring functions. A significant part of this improvement is retained, when only the receptor is treated with quantum-based parameters, while the ligands are parameterized with a purely classical model.

A Novel Role for the Glucocorticoid Receptor in the Regulation of Monocyte Chemoattractant Protein-1 mRNA Stability

Monocyte chemoattractant protein-1 (MCP-1) plays an important role in attracting monocytes to sites of inflammation and is the dominant mediator of macrophage accumulation in atherosclerotic plaques. We have previously shown that glucocorticoids inhibit the secretion of MCP-1 in arterial smooth muscle cells (SMC) by markedly decreasing MCP-1 mRNA stability. We now report that the destabilization of MCP-1 mRNA is mediated by the glucocorticoid receptor (GR). The GR antagonist, RU486, blocked the effect of the glucocorticoid dexamethasone (Dex) on MCP-1 mRNA stability in SMC culture. Using a previously reported in vitro mRNA gel shift and stability assay, antibodies to the GR blocked the ability of cytoplasmic extracts from Dex-treated SMC to decay MCP-1 mRNA. Recombinant human GR (rhGR) bound in a concentration-dependent manner to in vitro transcribed MCP-1 mRNA, whereas other members of the steroid hormone receptor family did not. Binding of GR to MCP-1 mRNA was specific as it was not found to bind other mRNAs. Immunoprecipitation of GR in extracts from Dex-treated SMC followed by real-time reverse transcription-PCR demonstrated that endogenous GR was bound specifically to MCP-1 mRNA. The addition of exogenous rhGR blocked the ability of extracts from Dex-treated SMC to degrade MCP-1 mRNA, suggesting that exogenous rhGR can compete with an endogenous GR-containing degradative complex. These data suggest a novel role for the GR in binding to and facilitating mRNA degradation. These results provide novel insights into GR function and may provide a new approach to attenuate the inflammatory response mediated by MCP-1.

Regulation of RARbeta1 expression in head and neck cancer cells by cell density-dependent chromatin remodeling

Retinoids have shown siginificant activities in cancer prevention and therapy. Many of their effects are mediated by nuclear retinoid receptors including retinoic acid receptors (RARs ?, ?, and ?) and retinoid X receptors (RXRs ?, ?, and ?. Human retinoic acid receptor ? (RAR?) has three different isoforms: ?1, ?2, and ?4. The tumor suppressive characteristics of RAR?2, its silencing by promoter hypermethylation, and its re-expression following demethylation have been reported. In contrast, RAR?1, an embryonic isoform with restricted expression in adult tissues has been linked to carcinogenesis. However, factors regulating RAR?1 expression have not yet been clarified. During studies on the head and neck squamous cell carcinoma cells, we found that the expression of RAR? increased in cells grown to high density. Real-time reverse-transcriptase polymerase chain reaction revealed that the isoform increased in these cells was RAR?1. Epigenetic modifications of this isoform were tested using combined bisulfite restriction analysis and chromatin immunoprecipitation assays. The UMSCC38 cell line showed significant RAR?1 expression (P < 0.001), which was dependent on cell density and culture duration. The increased expression of RARβ1 was not due to demethylation of its promoter. However, higher cell densities were associated with increased acetylation of histone 3 at lysine 9 in RARβ1 but not in RARβ2. These findings reveal that the expression of RARβ1 is regulated by cell density through changes in histone acetylation.

Activation of retinoic acid receptor-dependent transcription by organochlorine pesticides

Five organochlorine pesticides, namely, chlordane, dieldrin, aldrin, endrin, and endosulfan, activate human retinoic acid receptor (RAR)-mediated gene transcription via a retinoic acid response element (RARE). Transactivation studies were performed with stable RARalpha, beta, or gamma reporter cell lines in which the RAR DNA-binding domain (DBD) was replaced by that of estrogen receptor alpha (ERalpha)? Five of the organochlorine pesticides tested activated RARbeta and RARgamma but not RARalpha; their half-maximal luciferase activity (EC(50)) was determined. Furthermore, that activity was RAR-specific and organochlorine pesticides did not activate the retinoid X receptor (RXR) pathway. However, competitive binding experiments with [(3)H]-CD367, a pan-RAR agonist, showed that only chlordane could bind RARbeta and RARgamma, albeit with low affinity. In addition, organochlorine pesticides strongly induce cytochrome P450RAI1 (P450RAI1), a key factor of retinoic acid level regulation in many tissues and whose expression and activity are strongly induced by retinoic acid. This study shows that organochlorine pesticides can activate two RAR homologues, with low-binding affinity. Although the agonistic potential of organochlorine pesticides is lower than that of (E)-4-[2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propenyl] benzoic acid (TTNPB), they are able to induce RAR-mediated gene transcription as P450RAI1 and may disrupt the retinoid signaling pathway. Because these chemicals are extremely persistent and tend to accumulate in biological tissues, these results support the hypothesis that the increase in teratogenicity observed in some developing countries could be due to prolonged exposure to organochlorine pesticides ubiquitously present in the environment.

Remodeling of Gap Junctions in Mouse Hearts Hypertrophied by Forced Retinoic Acid Signaling

Beta-MHC-hRARalpha transgenic mice express a constitutively active (truncated) form of the human retinoic acid receptor which triggers development of dilated cardiomyopathy. In those hearts, we studied expression of gap junction proteins in relation to electrical impulse propagation. As compared to wildtype mice, hearts of 4-6 month old mice with 7-12 inserted hRARalpha copies are marked by an increased heart weight/body weight- and heart weight/tibia length ratio. 3-extremity lead ECGs revealed prolongation of the Q-j interval suggesting delayed ventricular activation. Mapping of electrical activity of epi- and endocardial left ventricular free wall revealed activation delay, increased heterogeneity in conduction and regional conduction block. Ventricular tachycardias did not occur spontaneously nor could be induced by ventricular pacing. Immunohistochemical analysis showed profound and heterogeneous redistribution and down-regulation of the gap junction protein connexin43 (Cx43) in the left ventricular free wall. Here, hRARalpha expression induced re-expression of the hypertrophic markers alpha-skeletal actin and beta-MHC, and in 3 out of 10 severely affected mice, re-expression of Cx40. Concomitant with changes in expression/distribution of Cx43, changes in expression and distribution of beta-catenin and N-cadherin (two other intercalated disk associated proteins) were observed. Beta-MHC-hRARalpha transgenic hearts show heterogeneous re-expression of (early) sarcomeric genes while expression of connexin43, N-cadherin and beta-catenin is down-regulated. We postulate that the resulting aberrant ventricular activation does not trigger development of lethal arrhythmias due to the small size of remaining healthy ventricular tissue where the transgene is not expressed.

Retinoblastoma-binding Protein 2 (Rbp2) Potentiates Nuclear Hormone Receptor-mediated Transcription

Retinoblastoma-binding protein 2 (Rbp2) was originally identified as a retinoblastoma protein (RB) pocket domain-binding protein. Although Rbp2 has been shown to interact with RB, p107, TATA-binding protein, and T-cell oncogene rhombotin-2, the physiological function of Rbp2 remains unclear. Here we demonstrate that Rbp2 not only binds to nuclear receptors (NRs) but also enhances the transcription mediated by them. Rbp2 interacts with the DNA-binding domains of NRs and potentiates NR-mediated transcription in an AF-2-dependent manner. Both the N-terminal and C-terminal domains of Rbp2 are critical for the transactivation activity of Rbp2 on NRs. The C terminus is the NR-interacting region. In addition, RB functions in maximizing the effect of Rbp2 on the transcription by NRs. These results suggest that Rbp2 is a coregulator of NRs and define a potential role for Rbp2 in NR-mediated transcription.

Structural basis for isotype selectivity of the human retinoic acid nuclear receptor

The human retinoic acid receptor (hRAR) belongs to the family of nuclear receptors that regulate transcription in a ligand-dependent way. The isotypes RARalpha,beta and gamma are distinct pharmacological targets for retinoids that are involved in the treatment of various skin diseases and cancers, in particular breast cancer and acute promyelocytic leukemia. Therefore, synthetic retinoids have been developed aiming at isotype selectivity and reduced side-effects. We report the crystal structures of three complexes of the hRARgamma ligand-binding domain (LBD) bound to agonist retinoids that possess selectivity either for RARgamma (BMS184394) or for RARbeta/gamma (CD564), or that are potent for all RAR-isotypes (panagonist BMS181156). The high resolution data (1.3-1. 5 A) provide a description at the atomic level of the ligand pocket revealing the molecular determinants for the different degrees of ligand selectivity. The comparison of the complexes of the chemically closely related retinoids BMS184394 and CD564 shows that the side-chain of Met272 adopts different conformations depending on the presence of a hydrogen bond between its sulfur atom and the ligand. This accounts for their different isotype selectivity. On the other hand, the difference between the pan- and the RARbeta, gamma-selective agonist is probably due to a steric discrimination at the level of the 2-naphthoic acid moiety of CD564. Based on this study, we propose a model for a complex with the RARgamma-specific agonist CD666 that shows the possible applications for structure-based drug design of RAR isotype-selective retinoids.