Receptor Interaction Domain Research Articles

Adaptive sequence evolution in a color gene involved in the formation of the characteristic egg-dummies of male haplochromine cichlid fishes

BackgroundThe exceptionally diverse species flocks of cichlid fishes in East Africa are prime examples of parallel adaptive radiations. About 80% of East Africa's more than 1 800 endemic cichlid species, and all species of the flocks of Lakes Victoria and Malawi, belong to a particularly rapidly evolving lineage, the haplochromines. One characteristic feature of the haplochromines is their possession of egg-dummies on the males' anal fins. These egg-spots mimic real eggs and play an important role in the mating system of these maternal mouthbrooding fish.ResultsHere, we show that the egg-spots of haplochromines are made up of yellow pigment cells, xanthophores, and that a gene coding for a type III receptor tyrosine kinase, colony-stimulating factor 1 receptor a (csf1ra), is expressed in egg-spot tissue. Molecular evolutionary analyses reveal that the extracellular ligand-binding and receptor-interacting domain of csf1ra underwent adaptive sequence evolution in the ancestral lineage of the haplochromines, coinciding with the emergence of egg-dummies. We also find that csf1ra is expressed in the egg-dummies of a distantly related cichlid species, the ectodine cichlid Ophthalmotilapia ventralis, in which markings with similar functions evolved on the pelvic fin in convergence to those of the haplochromines.ConclusionWe conclude that modifications of existing signal transduction mechanisms might have evolved in the haplochromine lineage in association with the origination of anal fin egg-dummies. That positive selection has acted during the evolution of a color gene that seems to be involved in the morphogenesis of a sexually selected trait, the egg-dummies, highlights the importance of further investigations of the comparative genomic basis of the phenotypic diversification of cichlid fishes.

Gustducin and transducin: a tale of two G proteins.

In the vertebrate taste cell, heterotrimeric guanine nucleotide-binding proteins (G proteins) are involved in the transduction of both bitter and sweet taste stimulants. The bitter compound denatonium raises the intracellular Ca2+ concentration in rat taste cells, apparently via G protein-mediated increases in inositol trisphosphate. Sucrose causes a G protein-dependent generation of cAMP in rat taste bud membranes; elevation of cAMP levels leads to taste cell depolarization. To identify and characterize those proteins involved in the taste transduction process, we have cloned G protein alpha subunit (G alpha) cDNAs from rat taste cells. Using degenerate primers corresponding to conserved regions of G proteins, we used the polymerase chain reaction to amplify and clone taste cell G alpha cDNAs. Eight distinct G alpha cDNAs were isolated, cloned and sequenced from a taste cell library. Among these clones was alpha gustducin, a novel taste G alpha closely related to the transducins. In addition to alpha gustducin, we cloned rod and cone transducins from taste cells. This is the first identification of transducin expression outside photoreceptor cells. The primary sequence of alpha gustducin shows similarities to the transducins in the receptor interaction domain and the phosphodiesterase activation site. These sequence similarities suggest that gustducin and transducin regulate taste cell phosphodiesterase, probably in bitter taste transduction.

The Silencing Mediator of Retinoic Acid and Thyroid Hormone Receptor (SMRT) Corepressor Is Required for Full Estrogen Receptor α Transcriptional Activity

Multiple factors influence estrogen receptor alpha (ERalpha) transcriptional activity. Current models suggest that the silencing mediator of retinoic acid and thyroid hormone receptor (SMRT) corepressor functions within a histone deactylase-containing protein complex that binds to antiestrogen-bound ERalpha and contributes to negative regulation of gene expression. In this report, we demonstrate that SMRT is required for full agonist-dependent ERalpha activation. Chromatin immunoprecipitation assays demonstrate that SMRT, like ERalpha and the SRC-3 coactivator, is recruited to an estrogen-responsive promoter in estrogen-treated MCF-7 cells. Depletion of SMRT, but not histone deacetylases 1 or 3, negatively impacts estradiol-stimulated ERalpha transcriptional activity, while exogenous expression of SMRT's receptor interaction domains blocks ERalpha activity, indicating a functional interaction between this corepressor and agonist-bound ERalpha. Stimulation of estradiol-induced ERalpha activity by SMRT overexpression occurred in HeLa and MCF-7 cells, but not HepG2 cells, indicating that these positive effects are cell type specific. Similarly, the ability of SMRT depletion to promote the agonist activity of tamoxifen was observed for HeLa but not MCF-7 cells. Furthermore, impairment of agonist-stimulated activity by SMRT depletion is specific to ERalpha and not observed for receptors for vitamin D, androgen, or thyroid hormone. Nuclear receptor corepressor (N-CoR) depletion increased the transcriptional activity of all four tested receptors. SMRT is required for full expression of the ERalpha target genes cyclin D1, BCL-2, and progesterone receptor but not pS2, and its depletion significantly attenuated estrogen-dependent proliferation of MCF-7 cells. Taken together, these data indicate that SMRT, in conjunction with gene-specific and cell-dependent factors, is required for positively regulating agonist-dependent ERalpha transcriptional activity.

Amino-Terminal Domain of TIF2 Is Involved in Competing for Corepressor Binding to Glucocorticoid and Progesterone Receptors

Both agonist- and antagonist-bound glucocorticoid receptors (GRs) and progesterone receptors (PRs) regulate gene transcription with the assistance of corepressors (NCoR and SMRT) and coactivators (TIF2/GRIP1, SRC1, and AIB1). Receptor binding of these cofactors is competitive and is considered to involve interactions between the C-terminal ligand binding domain of receptors and receptor interaction domains (RIDs) in the middle and C-terminus of coactivators and corepressors, respectively. Therefore, our recent finding that an amino terminal fragment of TIF2 (TIF2.0 = amino acids 1-627) competed for GR and PR interactions with corepressors in mammalian two-hybrid assays was unexpected. Here, we use biochemical approaches (mammalian two-hybrid, pull-down, and coimmunoprecipitation assays) to locate an N-terminal GR region that is sufficient to bind TIF2.0. In contrast, an N-terminal sequence of PR-B that is largely missing in the shorter PR-A is necessary but not sufficient for TIF2.0 binding. Mutagenesis studies of NCoR establish that the more amino-terminal RID#1, but not RID#2, is necessary for binding to both GR and PR agonist and antagonist complexes. ChIP assays indicate that PR and NCoR each selectively localize to the enhancer element (PRE) of a transiently transfected PREtkLUC reporter in the presence of antagonist steroid, whereas exogenous TIF2.0 reduces the amount of PRE-associated NCoR. Importantly, exogenous TIF2.0 also inhibits the biological responses to added NCoR under the same conditions as those used in the ChIP assays. These results suggest that both N-terminal and middle sequences of TIF2 participate in competing with corepressor for regulating the gene transcriptional responses of GRs and PRs.

Aberrant Association of Promyelocytic Leukemia Protein-Retinoic Acid Receptor-α with Coactivators Contributes to Its Ability to Regulate Gene Expression

The aberrant association of promyelocytic leukemia protein-retinoic acid receptor-alpha (PML-RARalpha) with corepressor complexes is generally thought to contribute to the ability of PML-RARalpha to regulate transcription. We report here that PML-RARalpha acquires aberrant association with coactivators. We show that endogenous PML-RARalpha interacts with the histone acetyltransferases CBP, p300, and SRC-1 in a hormoneindependent manner, an association not seen for RARalpha. This hormone-independent coactivator binding activity requires an intact ligand-binding domain and the NR box of the coactivators. Confocal microscopy studies demonstrate that exogenous PML-RARalpha sequesters and colocalizes with coactivators. These observations correlate with the ability of PML-RARalpha to attenuate the transcription activation of the Notch signaling downstream effector, CBF1, and of the glucocorticoid receptor. This includes attenuation of the glucocorticoid-induced leucine zipper (GILZ) and FLJ25390 target genes of the endogenous glucocorticoid receptor. Furthermore, treatment of NB4 cells with all-trans-retinoic acid, which promotes PML-RARalpha degradation, resulted in increased activation of GILZ. On the basis of these findings, we propose a model in which the hormone-independent association between PML-RARalpha and coactivators contributes to its ability to regulate gene expression.

Receptor-coupling properties of the invertebrate visual guanine nucleotide binding protein iGqα

Invertebrate visual iGqα is homologous to mammalian mGqα in two of the three domains important for G protein interaction with receptors; the C-terminus and the linker regions that connect the helical and ras-like domains of the α subunit. The third receptor-interacting domain, the N-terminus, contains a six amino acid extension MTLESI in mGqα that is not present in iGqα. In co-expression studies we assessed the promiscuity and efficacy of receptor coupling to phospholipase C (PLC) by iGqα, a non-palmitoylated mutant iGqα(C3,4A), mGqα and G15α. The invertebrate G proteins and mGqα only coupled to Gq-coupled receptors (m1 muscarinic acetylcholine receptor (mChR1), α1A-adrenergic receptor (α1-AR)) and not to the Gi/s-coupled receptors (CCR1 receptor, β2-adrenergic receptor or dopamine D1 receptor) while G15α coupled to all receptors. iGqα and iGqα(C3,4A) both had double the efficacy for PLC activation compared to the mammalian G proteins when co-expressed with mChR1 and α1-AR. The increased efficacy of iGqα compared to mGqα was also seen downstream of PLC with carbachol stimulation of the mitogen-activated protein kinase, ERK1/2. Addition of the MTLESI extension onto the N-terminus of iGqα decreased its efficacy by 35% whereas deletion of this sequence from mGqα increased its efficacy by 60% in the PLC and ERK1/2 assays. iGqα, iGqα(C3,4A) and mGqα all displayed similar receptor-independent AlF4–activation of PLC and guanosine triphosphate hydrolysis (GTPase) activity. iGqα, and iGqα(C3,4A) both had increased receptor-activated guanosine 5′-[γ-[35S]thio]triphosphate ([35S]GTPγS) binding when compared to mGqα when co-expressed with the mChR1. These results demonstrate that Gq protein efficacy is at least partially determined by the presence of the amino-terminal MTLESI extension. Comparison of [35S]GTPγS binding rates helps explain the increased efficacy of the invertebrate G proteins.

Phosphorylation of Thyroid Hormone Receptor-associated Nuclear Receptor Corepressor Holocomplex by the DNA-dependent Protein Kinase Enhances Its Histone Deacetylase Activity

It is well documented that unliganded thyroid hormone receptor (TR) functions as a transcriptional repressor of specific cellular target genes by acting in concert with a corepressor complex harboring histone deacetylase (HDAC) activity. To fully explore the cofactors that interact with the transcriptionally repressive form of TR, we biochemically isolated a multiprotein complex that assembles on a TR.retinoid X receptor (RXR) heterodimer in HeLa nuclear extracts and identified its polypeptide components by mass spectrometry. A subset of TR.RXR-associated polypeptides included NCoR, SMRT, TBL1, and HDAC3, which represent the core components of a previously described NCoR/SMRT corepressor complex. We also identified several polypeptides that constitute a DNA-dependent protein kinase (DNA-PK) enzyme complex, a regulator of DNA repair, recombination, and transcription. These polypeptides included the catalytic subunit DNA-PKcs, the regulatory subunits Ku70 and Ku86, and the poly(ADP-ribose) polymerase 1. Density gradient fractionation and immunoprecipitation analyses provided evidence for the existence of a high molecular weight TR.RXR.corepressor holocomplex containing both NCoR/SMRT and DNA-PK complexes. Chromatin immunoprecipitation studies confirmed that unliganded TR.RXR recruits both complexes to the triiodothyronine-responsive region of growth hormone gene in vivo. Interestingly, DNA-PKcs, a member of the phosphatidylinositol 3-kinase family, was found to phosphorylate HDAC3 when the purified TR.RXR.corepressor holocomplex was incubated with ATP. This phosphorylation was accompanied by a significant enhancement of the HDAC activity of this complex. Collectively, our results indicated that DNA-PK promotes the establishment of a repressive chromatin at a TR target promoter by enhancing the HDAC activity of the receptor-bound NCoR/SMRT corepressor complex.

In Vitro Fluorescence Anisotropy Analysis of the Interaction of Full-length SRC1a with Estrogen Receptors α and β Supports an Active Displacement Model for Coregulator Utilization

Binding of full-length P160 coactivators to hormone response element-steroid receptor complexes has been difficult to investigate in vitro. Here, we report a new application of our recently described fluorescence anisotropy microplate assay to investigate binding and dissociation of full-length steroid receptor coactivator-1a (SRC1a) from full-length estrogen receptor alpha (ERalpha) or estrogen receptor beta (ERbeta) bound to a fluorescein-labeled (fl) estrogen response element (ERE). SRC1a exhibited slightly higher affinity binding to flERE.ERbeta than to flERE.ERalpha. Binding of SRC1a to flERE.ERalpha and to flERE.ERbeta was 17beta-estradiol (E2)-dependent and was nearly absent when ICI 182,780, raloxifene, or 4-hydroxytamoxifen were bound to the ERs. SRC1a binds to flERE.E2-ERalpha and flERE.E2-ERbeta complexes with a t1/2 of 15-20 s. Short LXXLL-containing nuclear receptor (NR) box peptides from P160 coactivators competed much better for SRC1a binding to flERE.E2-ER than an NR box peptide from TRAP220. However, approximately 40-250-fold molar excess of the P160 NR box peptides was required to inhibit SRC1a binding by 50%. This suggests that whereas the NR box region is a primary site of interaction between SRC1a and ERE.E2-ER, additional contacts between the coactivator and the ligand-receptor-DNA complex make substantial contributions to overall affinity. Increasing amounts of NR box peptides greatly enhanced the rate of dissociation of SRC1a from preformed flERE.E2-ER complexes. The data support a model in which coactivator exchange is facilitated by active displacement and is not simply the result of passive dissociation and replacement. It also shows that an isolated coactivator exhibits an inherent capacity for rapid exchange.

Thermodynamic Characterization of the Interaction between CAR−RXR and SRC-1 Peptide by Isothermal Titration Calorimetry

The constitutive androstane receptor (CAR) enhances transcription of specific target genes that regulate several metabolic pathways. CAR functions as an obligate heterodimer (CAR-RXR) with the retinoid X receptor (RXR). Also part of the active receptor complex is the steroid receptor coactivator-1 (SRC-1) which interacts with the receptor complex via specific receptor interaction domains (RIDs). A peptide derived from SRC-1 RID2 is used to study the thermodynamic properties of the interaction with the CAR-RXR ligand binding domain (LBD) complex. In the absence of ligands for both CAR and RXR, binding of coactivator peptide to the CAR-RXR heterodimer is characterized by a favorable enthalpy change and an unfavorable entropy change. The addition of the CAR agonist, TCPOBOP, increases the affinity for coactivator by decreasing the unfavorable entropy and increasing the favorable intrinsic enthalpy of the interaction. The RXR ligand, 9-cis-RA, generates a second SRC-1 site and increases the affinity by improving the entropic component of binding. There is an additional increase in affinity for one of the two sites in the presence of both ligands. The change in heat capacity (deltaCp) is also investigated. A 2-fold difference in deltaCp is observed between liganded and unliganded CAR-RXR. The observed thermodynamic parameters for binding of SRC-1 peptide to liganded and apo CAR-RXR as well as the difference in the deltaCp data provide evidence that the apo CAR-RXR heterodimer is conformationally mobile. The more favorable enthalpic contribution for TCPOBOP-bound CAR-RXR indicates that preformation of the binding site improves the complementarity of the coactivator-receptor interaction.

Identification of Regions within the F Domain of the Human Estrogen Receptor α that Are Important for Modulating Transactivation and Protein-Protein Interactions

The estrogen receptor (ER)alpha is a biologically and clinically important ligand-modulated transcription factor. The F domain of the ERalpha modulates its functions in a ligand-, promoter-, and cell-specific manner. To identify the region(s) responsible for these functions, we characterized the effects of serial truncations within the F domain. We found that truncating the last 16 residues of the F domain altered the activity of the human ERalpha (hERalpha) on an estrogen response element-driven promoter in response to estradiol or 4-hydroxytamoxifen (4-OHT), its sensitivity to overexpression of the coactivator steroid receptor coactivator-1 in mammalian cells, and its interaction with a receptor-interacting domain of the coactivator steroid receptor coactivator-1 or engineered proteins ("monobodies") that specifically bind to ERalpha/ligand complexes in a yeast two-hybrid system. Most importantly, the ability of the ER to induce pS2 was reduced in MDA-MB-231 cells stably expressing this truncated ER vs. the wild-type ER. The region includes a distinctive segment (residues 579-584; LQKYYIT) having a high content of bulky and/or hydrophobic amino acids that was previously predicted to adopt a beta-strand-like structure. As previously reported, removal of the entire F domain was necessary to eliminate the agonist activity of 4-OHT. In addition, mutation of the vicinal glycine residues between the ligand-binding domain and F domains specifically reduced the 4-OHT-dependent interactions of the hERalpha ligand-binding domain and F domains with monobodies. These results show that regions within the F domain of the hERalpha selectively modulate its activity and its interactions with other proteins.

A Novel Fusion between ETV6 and NCOA2 Defines a New Entity of Acute Leukemia with Co-Expression of T-Cell and Myeloid Markers.

We have identified a novel recurrent translocation and the genes involved therein, which is associated with both lymphoid and myeloid acute leukemia. Fluorescence in situ hybridization (FISH) and molecular analyses revealed an in-frame fusion of ETV6 (alias TEL) and NCOA2 (alias TIF2) in six cases of childhood acute leukemia. ETV6 encodes an ets transcription repressor and is frequently involved in chromosome rearrangements with a multitude of partners in lymphoid and myeloid leukemia, and to a lesser extent also in solid tumors. NCOA2 is a member of the p160 family of nuclear receptor transcriptional coactivators (NRCoAs), which have a common domain structure with a conserved N-terminal bHLH-PAS domain, a centrally located (nuclear) receptor interaction domain (RID/NID), and a C-terminal transcriptional activation domain (AD2). NRCoAs typically display HAT activity and/or directly interact with CBP. In this context, NCOA2 also contains a C-terminal CBP-interaction domain (CID), and thus is a transcription factor that mediates transcriptional activation in a ligand-dependent fashion by a mechanism involving chromatin remodeling. As a result of the translocation the pointed (PNT) protein interaction domain of ETV6 is fused to the C-terminus of NCOA2 including the CBP and AD2 motifs. The same C-terminal domains of NCOA2 are also involved in the previously identified MOZ-NCOA2 fusion, which is generated by a inv(8)(p11q13). The absence of the reciprocal NCOA2-ETV6 transcript in one of the cases, and the facts that MOZ-NCOA2 is transforming and that the reciprocal NCOA2-MOZ is not expressed, strongly suggest that the ETV6-NCOA2 chimeric protein and not the reciprocal NCOA2-ETV6 is responsible for leukemogenesis. Unlike in cases with a ETV6-RUNX1 fusion, which is frequently associated with a concurrent secondary deletion of the non-rearranged ETV6 allele, in all cases the second ETV6 and also one NCOA2 allele were retained and both genes were readily expressed. With respect to the immunophenotype, according to the European Group for the Immunological Characterization of Leukemias (EGIL), three of the cases were classified as T-ALL (one as T-I My+), two as biphenotypic leukemia (BAL), and one as AML with M1 phenotype. Nevertheless, all leukemias displayed aberrant co-expression of various T-cell and myeloid/NK markers, and were double-negative for CD4/CD8, suggesting transformation of early progenitors. In contrast to the MOZ-NCOA2 fusion, which is exclusively associated with AML, mostly of M4 and M5 subtype, the ETV6-NCOA2 fusion seems to define a new entity of acute leukemia with aberrant co-presence of T lymphoid and myeloid antigens.

Kinase-Specific Phosphorylation of the Estrogen Receptor Changes Receptor Interactions with Ligand, Deoxyribonucleic Acid, and Coregulators Associated with Alterations in Estrogen and Tamoxifen Activity

Posttranslational modifications of the estrogen receptor (ER) are emerging as important regulatory elements of cross talk between different signaling pathways. ER phosphorylation, in particular, has been implicated in the ligand-independent effects of ER and in tamoxifen resistance of breast tumors. In our studies, Western immunoblot analysis of endogenous ER in parental MCF-7 cells reveals specific, ligand-dependent phosphorylations at S118 and S167, with this ligand dependence being lost in tamoxifen-resistant, MCF-7 Her2/neu cells. Using highly purified components and sensitive fluorescence methods in an in vitro system, we show that phosphorylation by different kinases alters ER action through distinct mechanisms. Phosphorylation by Src and protein kinase A increases affinity for estradiol (E2), whereas ER phosphorylation by MAPK decreases trans-hydroxytamoxifen (TOT) binding. Affinity of ER for the consensus estrogen response element is also altered by phosphorylation in a ligand-specific manner, with decrease in affinity of MAPK- and Src-phosphorylated ER in the presence of TOT. ER phosphorylation by MAPK, AKT, or protein kinase A increases recruitment of steroid receptor coactivator 3 receptor interaction domain to the DNA-bound receptor in the presence of E2. Taken together, these results suggest that ER phosphorylation alters receptor functions (ligand, DNA, and coactivator binding), effecting changes that could lead to an increase in E2 agonism and a decrease in TOT antagonistic activity, reflecting changes encountered in tamoxifen resistance in endocrine therapy of breast cancer.

Interference with nuclear factor kappa B and c-Jun NH2-terminal kinase signaling by TRAF6C small interfering RNA inhibits myeloma cell proliferation and enhances apoptosis

The tumor necrosis factor receptor (TNFR)-associated factor (TRAF) family of six adaptor proteins (TRAF1-6) links the TNFR superfamily to the nuclear factor kappa B (NF-kappaB) and activator protein-1 (AP-1) transcriptional activators. Unlike other TRAFs, TRAF6 is also involved in Toll-like/interleukin (IL)-1 receptor (TIR) signal transduction. Thus, inhibition of TRAF6 function could interrupt both CD40 (TNFR family) and IL-1 growth signals, pathways critical to myeloma proliferation. To block TRAF6-mediated IL-1 signaling, we constructed small interfering RNA (siRNA) against TRAF6. We found that siRNA targeting the TRAF6 C-terminal (siTRAF6C) receptor interaction domain specifically reduced only TRAF6 protein expression, without affecting TRAF2 or 5 levels, and substantially interfered with IL-1-induced NF-kappaB and c-Jun/AP-1 activation. Inhibition by siTRAF6C was concentration-dependent. SiTRAF6C also significantly reduced myeloma proliferation and enhanced apoptosis in a similar dose-dependent fashion in vitro. More importantly, marked siTRAF6C growth inhibition was detected in vivo when these cells were implanted into the bone marrow of irradiated normal mice. In contrast, introduction of siRNA derived from the TRAF6 Zn-finger domain or an irrelevant siRNA construct failed to alter cell growth or cell death. These studies suggest that TRAF6 may be a new molecular target to block cell signal transduction important for the survival and proliferation of multiple myeloma cells.

Rho Family Guanine Nucleotide Exchange Factor Brx Couples Extracellular Signals to the Glucocorticoid Signaling System

Glucocorticoids regulate many crucial biologic functions through their cytoplasmic/nuclear glucocorticoid receptors (GR). Excess, deficiency, or alteration in tissue sensitivity to glucocorticoids has been associated with major causes of human morbidity and mortality. Brx, a cytoplasmic Rho family guanine nucleotide exchange factor, binds to and influences the activity of several nuclear hormone receptors. We examined the functional and molecular interactions between GR and Brx. The glucocorticoid sensitivity of lymphocytes obtained from mice haplo-insufficient for Brx was significantly decreased. Conversely, GR-mediated transcriptional activity of a glucocorticoid response element (GRE)-mediated glucocorticoid-responsive promoter was enhanced by Brx in a guanine nucleotide exchange factor domain-dependent fashion. Brx interacted with GR, forming a ternary complex with RhoA. In a chromatin immunoprecipitation assay, Brx and RhoA were co-precipitated with GREs only in the presence of ligand-activated GR. Extracellularly administered lysophosphatidic acid, which activates its signaling cascade through a specific membrane GTP-binding protein (G-protein)-coupled receptor in a G-protein alpha(13)-, Brx-, and RhoA-dependent fashion, enhanced GR transcriptional activity, whereas depletion of endogenous Brx attenuated this effect. These findings suggest that glucocorticoid signaling and, hence, the tissue sensitivity to glucocorticoids, may be coupled to extracellular signals via Brx and small G-proteins. Nuclear Brx might act as a local GRE-GR-transcriptosome activator by mediating the effect of small G-proteins on glucocorticoid-regulated genes.

Ligand-specific allosteric regulation of coactivator functions of androgen receptor in prostate cancer cells

The androgen receptor not only mediates prostate development but also serves as a key regulator of primary prostatic cancer growth. Although initially responsive to selective androgen receptor modulators (SARMs), which cause recruitment of the nuclear receptor-corepressor (N-CoR) complex, resistance invariably occurs, perhaps in response to inflammatory signals. Here we report that dismissal of nuclear receptor-corepressor complexes by specific signals or androgen receptor overexpression results in recruitment of many of the cohorts of coactivator complexes that permits SARMs and natural ligands to function as agonists. SARM-bound androgen receptors appear to exhibit failure to recruit specific components of the coactivators generally bound by liganded nuclear receptors, including cAMP response element-binding protein (CBP)/p300 or coactivator-associated arginine methyltransferase 1 (CARM1) to the SARM-bound androgen receptor, although still causing transcriptional activation of androgen receptor target genes. SARM-bound androgen receptors use distinct LXXLL (L, leucine; X, any amino acid) helices in the p160 nuclear receptor interaction domains that may impose selective allosteric effects, providing a component of the molecular basis of differential responses to different classes of ligands by androgen receptor.

Lipid−Protein Interactions of Growth Factor Receptor-Bound Protein 14 in Insulin Receptor Signaling

Many retinal degenerative diseases show an early loss of rod cells followed by cone cells. In these degenerations the pathological phenotype is apoptosis. We have previously demonstrated the light-dependent tyrosine phosphorylation of the insulin receptor in the retina, which leads to the activation of anti-apoptotic signaling molecules. The mechanism of the regulation of the insulin receptor in the retina is not known. Yeast two-hybrid screening of a bovine retinal cDNA library with the cytoplasmic domain of the retinal insulin receptor (IRbeta) identified a member of the Grb7 (growth factor receptor-bound protein 7) gene family, Grb14. In this report, we describe the unique features of Grb14. Grb14 forms a specific complex with the cytoplasmic domain of IRbeta when both are expressed as hybrid proteins in yeast cells. This interaction is strictly dependent upon receptor tyrosine kinase activity. Deletion mutagenesis on Grb14 indicated a phosphorylated insulin receptor interacting (PIR) domain between the PH (pleckstrin homology) and SH2 (Src homology) domains that binds to IRbeta. Nuclear import assays in yeast indicated the presence of a functional nuclear localization signal in Grb14 between amino acids 63 and 68 (RRKKD). Subcellular localization of isolated retinas probed with anti-Grb14 antibody further confirmed the presence of Grb14 in nuclear fractions. Analysis using a protein-lipid overlay assay indicated binding of Grb14 and its PH domain to D3 phosphoinositides. In addition, Grb14-phosphoinositide 3,4,5-trisphosphate complexes are detected in lysates prepared from insulin-stimulated retina tissues, whereas Grb14-phosphoinositide 4,5-bisphosphate interactions are observed under non-insulin stimulated conditions. These findings suggest that Grb14 could be a diverse regulator of insulin receptor mediated pathways in the retina.

Regulation and Binding of Pregnane X Receptor by Nuclear Receptor Corepressor Silencing Mediator of Retinoid and Thyroid Hormone Receptors (SMRT)

The pregnane X receptor (PXR) is an orphan nuclear receptor predominantly expressed in liver and intestine. PXR coordinates hepatic responses to prevent liver injury induced by environmental toxins. PXR activates cytochrome P450 3A4 gene expression upon binding to rifampicin (Rif) and clotrimazole (CTZ) by recruiting transcriptional coactivators. It remains unclear whether and how PXR regulates gene expression in the absence of ligand. In this study, we analyzed interactions between PXR and the silencing mediator of retinoid and thyroid hormone receptors (SMRT) and determined the role of SMRT in regulating PXR activity. We show that SMRT interacts with PXR in glutathione S-transferase pull-down, yeast two-hybrid, and mammalian two-hybrid assays. The interaction is mediated through the ligand-binding domain of PXR and the SMRTs' nuclear receptor-interacting domain 2. The PXR-SMRT interaction is sensitive to species-specific ligands, and Rif causes an exchange of the corepressor SMRT with the p160 coactivator known as receptor-associated coactivator 3 (RAC3). Deletion of the PXR's activation function 2 helix enhances SMRT binding and abolishes ligand-dependent dissociation of SMRT. Coexpression of PXR with SMRT results in colocalization at discrete nuclear foci. Finally, transient transfection assays show that overexpression of SMRT inhibits PXR's transactivation of the Cyp3A4 promoter, whereas silencing of SMRT enhances the reporter expression. Taken together, our results suggest that the corepressor SMRT may bind to and regulate the transcriptional activity of PXR.

Differential Recruitment of p160 Coactivators by Glucocorticoid Receptor between Schwann Cells and Astrocytes

In the nervous system, glucocorticoids can exert beneficial or noxious effects, depending on their concentration and the duration of hormonal stimulation. They exert their effects on neuronal and glial cells by means of their cognate receptor, the glucocorticoid receptor (GR), which recruits the p160 coactivator family members SRC-1 (steroid receptor coactivator 1), SRC-2, and SRC-3 after hormone binding. In this study, we investigated the molecular pathways used by the GR in cultured glial cells of the central and the peripheral nervous systems, astrocytes and Schwann cells (MSC80 cells), respectively. We performed functional studies based on transient transfection of a minimal glucocorticoid-sensitive reporter gene into the glial cells to test the influence of overexpression or selective inhibition by short interfering RNA of the three p160 coactivator family members on GR transactivation. We demonstrate that, depending on the glial cell type, GR differentially recruits p160 family members: in Schwann cells, GR recruited SRC-1a, SRC-1e, or SRC-3, whereas in astrocytes, SRC-1e and SRC-2, and to a lesser extent SRC-3, were active toward GR signaling. The C-terminal nuclear receptor-interacting domain of SRC-1a participates in its exclusion from the GR transcriptional complex in astrocytes. Immunolocalization experiments revealed a cell-specific intracellular distribution of the p160s, which was dependent on the duration of the hormonal induction. For example, within astrocytes, SRC-1 and SRC-2 were mainly nuclear, whereas SRC-3 unexpectedly localized to the lumen of the Golgi apparatus. In contrast, in Schwann cells, SRC-1 showed a nucleocytoplasmic shuttling depending on hormonal stimulation, whereas SRC-2 remained strictly nuclear and SRC-3 remained predominantly cytoplasmic. Altogether, these results highlight the cell specificity and the time dependence of p160s recruitment by the activated GR in glial cells, revealing the complexity of GR-p160 assembly in the nervous system.

Rapid estrogen-induced phosphorylation of the SRC-3 coactivator occurs in an extranuclear complex containing estrogen receptor.

SRC-3/AIB1/ACTR/pCIP/RAC3/TRAM1 is a primary transcriptional coregulator for estrogen receptor (ER). Six SRC-3 phosphorylation sites have been identified, and these can be induced by steroids, cytokines, and growth factors, involving multiple kinase signaling pathways. Using phosphospecific antibodies for six phosphorylation sites, we investigated the mechanisms involved in estradiol (E2)-induced SRC-3 phosphorylation and found that this occurs only when either activated estrogen receptor alpha (ERalpha) or activated ERbeta is present. Both the activation function 1 and the ligand binding domains of ERalpha are required for maximal induction. Mutations in the coactivator binding groove of the ERalpha ligand binding domain inhibit E2-stimulated SRC-3 phosphorylation, as do mutations in the nuclear receptor-interacting domain of SRC-3, suggesting that ERalpha must directly contact SRC-3 for this posttranslational modification to take place. A transcriptionally inactive ERalpha mutant which localizes to the cytoplasm supports E2-induced SRC-3 phosphorylation. Mutations of the ERalpha DNA binding domain did not block this rapid E2-dependent SRC-3 phosphorylation. Together these data demonstrate that E2-induced SRC-3 phosphorylation is dependent on a direct interaction between SRC-3 and ERalpha and can occur outside of the nucleus. Our results provide evidence for an early nongenomic action of ER on SRC-3 that supports the well-established downstream genomic roles of estrogen and coactivators.

Retinoid X Receptor-α-Dependent Transactivation by a Naturally Occurring Structural Variant of Human Constitutive Androstane Receptor (NR1I3)

The constitutive androstane receptor (CAR) mediates the hepatic induction of various xenobiotic metabolizing enzymes and transporters after specific chemical exposures. Recent reports have established the existence of several human CAR mRNA splice variants, including a prominently expressed form termed CAR3, a receptor that possesses a 5 amino acid insertion within its ligand binding domain. In this study, we demonstrate that, in contrast to the constitutively active reference form of the receptor, CAR3 is ligand-activated, transactivating an optimized DR-4 x 3 reporter in response to the human CAR ligand 6-(4-chlorophenyl)imidazo[2,1-b]thiazole-5-carbaldehyde O-(3, 4-dichlorobenzyl)oxime (CITCO). The transactivation response requires the DNA binding domain and AF-2 motif of CAR3 and is markedly enhanced by retinoid X receptor-alpha (RXR) cotransfection. The stimulatory effects of RXR involve a unique mechanism, because they were completely dependent on the RXR AF-2 function but independent of both the RXR A/B domain and its C domain/heterodimerization region. Mammalian two-hybrid results demonstrated that RXR enhanced CITCO-dependent interaction of CAR3 with the receptor interaction domain of SRC-1, indicating that RXR augments CAR3 activity by facilitating coactivator recruitment. It is noteworthy that clotrimazole also functions as a ligand activator of CAR3, in contrast to the inverse agonist activity exhibited by this agent on the reference form of the receptor. Furthermore, results of transfection assays reveal that CAR3 is capable of transactivating the natural CYP2B6 and CYP3A4 gene enhancers, exhibiting both ligand- and RXR-dependence. These results demonstrate that CAR3, unlike CAR1, is a ligand-activated receptor and that CAR3 may regulate gene expression in vivo in a manner distinct from the reference form of the receptor.