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Differential interaction of steroid hormone receptors with LXXLL motifs in SRC-1a depends on residues flanking the motif
Abstract
Steroid hormones induce the transcriptional activity of their cognate receptors by recruiting a variety of cofactors. One of these, steroid receptor co-activator-1 (SRC-1) interacts with the ligand binding domains of a number of different receptors by means of LXXLL motifs. We have investigated the relative interaction of four such motifs in SRC-1a using a yeast two-hybrid assay. We demonstrate that ERalpha, ERbeta and ERbeta2 preferentially interact with motif 2 while GR, AR, PPARalpha and PPARgamma preferentially interact with motif 4. We show that the interactions depend not only on the LXXLL motif itself but also on residues flanking the motif.
... In M2H assays, SIRT1 NR-box interacted with both ER subtypes; mutating the conserved leucines diminished but did not eliminate receptor binding ( Figure 3A,B). These results suggest that although the NR-box is necessary, flanking residues (see Figure 1A,B) may also be involved in ER binding consistent with other observations [41,[48][49][50]. ER-binding specificity was further assured by a modified M2H assay using an ER instead of the Gal4 reporter gene. ...
... Interestingly, the NR-box was necessary for ligand-dependent ER binding as its mutagenesis markedly reduced SIRT1 NRB interaction in the presence of E2. However the mutations did not completely eliminate SIRT1 NRB binding, further suggesting that other residues at the NR interface may also bind to the receptors [41,[48][49][50]. As expected, the STACs-AD was sufficient for interaction with both ER subtypes and E2 further increased binding ( Figure 5C,D). ...
SIRT1 and orthologous sirtuins regulate a universal mechanism of ageing and thus determine lifespan across taxa; however, the precise mechanism remains vexingly polemical. They also protect against many metabolic and ageing-related diseases by dynamically integrating several processes including autophagy, proteostasis, calorie restriction, circadian rhythmicity and metabolism. These sirtuins are therefore important drug targets particularly because they also transduce allosteric signals from sirtuin-activating compounds such as resveratrol into increased healthspan in evolutionarily diverse organisms. While many of these functions are apparently regulated by deacetylation, that mechanism may not be all-encompassing. Since gonadal signals have been shown to regulate ageing/lifespan in worms and flies, the present study hypothesized that these sirtuins may act as intermediary factors for steroid hormone signal transduction. Accordingly, SIRT1 and its orthologues, Sir2 and Sir-2.1, are shown to be veritable nuclear receptor coregulators that classically coactivate the oestrogen receptor in the absence of ligand; coactivation was further increased by 17β-oestradiol. Remarkably in response to the worm steroid hormone dafachronic acid, SIRT1 reciprocally coactivates DAF-12, the steroid receptor that regulates nematode lifespan. These results suggest that steroid hormones may co-opt and modulate a phyletically conserved mechanism of sirtuin signalling through steroid receptors. Hence, it is interesting to speculate that certain sirtuin functions including prolongevity and metabolic regulation may be mechanistically linked to this endocrine signalling pathway; this may also have implications for understanding the determinative role of gonadal steroids such as oestradiol in human ageing. At its simplest, this report shows evidence for a hitherto unknown deacetylation-independent mechanism of sirtuin signalling.
... A phage library was constructed in the peptide library format of X7-LXXLL-X7, where L is leucine and X is any amino acid from I, L, V, F, C, A, G, P, T, S, Y, N, H, D, or R. The library was designed with 7 amino acids flexible motifs at the N-and C-terminus since it is known that the flanking region of LXXLL motifs influences the binding to ERs. 44,45 The phage T7 display system was chosen since recent studies showed that this system is less codon biased than phage M13 systems, 46 which means that more diverse peptides can be selected. The phage library was amplified by infecting E. coli BLT5403 after packing. ...
... Previously, systematic studies have demonstrated that the sequences right outside the LXXLL motif are important determinants of the binding specificity and affinity of coactivators to their NR partners; and based on immediate flanking residues these LXXLL-peptides have been divided into four different classes, mainly focussing on the peptide N-terminus. 32,44,45,47 However, determinants for preferential ER targets of the LXXLL peptides still lack full molecular insight. In addition, the role of ER phosphorylation on Y537 (ERa) and Y488 (ERb) in modulating the LXXLL-peptide-ER interaction is completely underexplored. ...
The Estrogen Receptors ERα and ERβ bind cofactor proteins via short LXXLL motifs. The exact regulation and selectivity of these interactions remains an open question and the role of post-translational modifications (PTMs) is virtually unexplored. Here, we designed an X(7)-LXXLL-X(7) T7 phage display library and screened this against four ER protein constructs: the 'naked' ERα and ERβ Ligand Binding Domains (LBDs) and the tyrosine phosphorylated ERα (pY537) and ERβ (pY488) LBDs. The site-selective tyrosine phosphorylated protein constructs were obtained via a protein semi-synthesis approach. Phage display screening yielded preferential sets of peptides. LXXLL peptides with a low pI/acidic C-terminus prefer binding to the naked ERβ over the phosphorylated ERβ analogue and ERα constructs. Peptides with a high pI/basic C-terminus show the opposite behaviour. These findings not only show regulation of the ERβ-cofactor interaction via tyrosine phosphorylation, but also suggest that ERβ and its tyrosine 488 phosphorylation play crucial roles in modulating interactions of coactivators to ERα since the natural Steroid Receptor Coactivators (SRCs) feature LXXLL motifs with acidic C-termini, while the repressor protein RIP140 features LXXLL motifs with basic C-termini. This insight provides explanation for ER transcriptional activity and can lead to more focussed targeting of the ER-coactivator interaction.
... . Tubulin tyrosine ligase-like protein 5 (TTLL5) is one of 13 members of the TTLL superfamily, all of which contain a core tubulin tyrosine ligase (TTL) domain of about 190 amino acids (1)(2)(3). For STAMP/TTLL5, along with TTLL1, 2,4,6,7,9,11, and 13, the TTL domain plus about 150 amino acids on either side is involved in the progressive addition of glutamic acid residues to the γ-carboxyl group of one or more glutamic acids in the vicinity of the C-terminus of polymerized tubulin (1)(2)(3)(4). TTLL3, 8 and 10 are also considered to be polyglycylases (3,5). ...
... STAMP also specifically interacts with, and modifies the transcriptional activity parameters of, androgen and progesterone receptors but not either estrogen receptor alpha, another member of the classical steroid receptor family, or any other of the five nuclear receptors examined (7,9). Thus, STAMP is a member of those cofactors that display preferential activity and binding with a sub-set of related transcription factors (10)(11)(12)(13). The Cterminal extension of STAMP/TTLL5 contains not only the cofactor interaction domain (CID) needed for binding the coactivators TIF2 and SRC-1 but also the receptor interaction domains (RIDs) for glucocorticoid and progesterone receptor binding (7,9). ...
TTLL5/STAMP (tubulin tyrosine ligase-like family member 5) has multiple activities in cells. TTLL5 is one of 13 TTLLs, has
polyglutamylation activity, augments the activity of p160 coactivators (SRC-1 and TIF2) in glucocorticoid receptor-regulated
gene induction and repression, and displays steroid-independent growth activity with several cell types. To examine TTLL5/STAMP
functions in whole animals, mice were prepared with an internal deletion that eliminated several activities of the Stamp gene. This mutation causes both reduced levels of STAMP mRNA and C-terminal truncation of STAMP protein. Homozygous targeted
mutant (Stamptm/tm) mice appear normal except for marked decreases in male fertility associated with defects in progressive sperm motility.
Abnormal axonemal structures with loss of tubulin doublets occur in most Stamptm/tm sperm tails in conjunction with substantial reduction in α-tubulin polyglutamylation, which closely correlates with the reduction
in mutant STAMP mRNA. The axonemes in other structures appear unaffected. There is no obvious change in the organs for sperm
development of WT versus Stamptm/tm males despite the levels of WT STAMP mRNA in testes being 20-fold higher than in any other organ examined. This defect in
male fertility is unrelated to other Ttll genes or 24 genes previously identified as important for sperm function. Thus, STAMP appears to participate in a unique,
tissue-selective TTLL-mediated pathway for α-tubulin polyglutamylation that is required for sperm maturation and motility
and may be relevant for male fertility.
... Coactivator proteins, such as steroid receptor coactivator-1 (SRC-1) (6) transcriptional intermediary factor (TIF2) (7), and cAMP response element binding protein (CBP) (8), bind to nuclear receptors through small amphipathic alpha-helical motifs with an LXXLL sequence (9 -11). Res-idues adjacent to the LXXLL motif have also been shown to influence the specificity of LBD-coactivator interactions (12)(13)(14). Fragments of coactivator proteins or short peptides containing LXXLL motifs are sufficient to determine interactions with nuclear receptor LBDs (9 -11). Scanning mutagenesis and co-crystallization studies with LXXLL peptides have demonstrated that the amphipathic peptide helix docks into a ligand-induced hydrophobic trough in the receptor (11,15,16). ...
... The magnitude of the ligand-induced effect on ER LBD binding was often much greater than that seen for PPAR␥ LBD. The SRC-1 protein has been reported to be an important interaction partner for both ER and PPAR␥ (6,14,30). Both ER LBD and PPAR␥ LBD show ligand-induced binding to the peptides SRC-1(2) (676 -700) and SRC-1(3) (735-759) in the microsphere-based assay. ...
We describe a novel microsphere-based system to identify and characterize multiplexed interactions of nuclear receptors with peptides that represent the LXXLL binding region of coactivator proteins.
In this system, individual microsphere populations with unique red and orange fluorescent profiles are coupled to specific coactivator peptides. The coactivator peptide-coupled microsphere populations are combined and incubated with a nuclear receptor that has been coupled to a green fluorochrome. Flow cytometric analysis of the microspheres simultaneously decodes each population and detects the binding of receptor to respective coactivator peptides by the acquisition of green fluorescence.
We have used this system to determine the binding affinities of human estrogen receptor beta ligand binding domain (ERbeta LBD) and human peroxisome proliferator activated receptor gamma ligand binding domain (PPARgamma LBD) to a set of 34 coactivator peptides. Binding of ERbeta LBD to a coactivator peptide sequence containing the second LXXLL motif of steroid receptor coactivator-1 (SRC-1(2) (676-700) is shown to be specific and saturable. Analysis of receptor binding to a multiplexed set of coactivator peptides shows PPARgamma LBD binds with high affinity to cAMP response element binding protein (CBP) peptides and to the related P300 peptide while ERbeta LBD exibits little binding to these peptides. Using the microsphere-based assay we demonstrate that ERbeta LBD and PPARgamma LBD binding affinities for the coactivator peptides are increased in the presence of agonist (estradiol or GW1929, respectively) and that ERbeta LBD binding is decreased in the presence of antagonist (raloxifene or tamoxifen).
This unique microsphere-based system is a sensitive and efficient method to simultaneously evaluate many receptor-coactivator interactions in a single assay volume. In addition, the system offers a powerful approach to study small molecule modulation of nuclear receptor binding.
... The presence of three of these motifs suggests that a certain measure of specificity is involved when the RIDs interact with the liganded receptors. For example, studies on the differential requirements of each LXXLL motif of SRC-1 demonstrated that ERα preferentially interacted with the second motif and that only one motif was sufficient to support stable interactions (McInerney et al., 1998; Needham et al., 2000). A fourth LXXLL motif located at the extreme Cterminus of SRC-1a, an isoform that probably arises from differential splicing (Kalkhoven et al., 1998), also binds strongly to ERα and ERβ as well as other nuclear receptors (Needham et al., 2000). ...
... For example, studies on the differential requirements of each LXXLL motif of SRC-1 demonstrated that ERα preferentially interacted with the second motif and that only one motif was sufficient to support stable interactions (McInerney et al., 1998; Needham et al., 2000). A fourth LXXLL motif located at the extreme Cterminus of SRC-1a, an isoform that probably arises from differential splicing (Kalkhoven et al., 1998), also binds strongly to ERα and ERβ as well as other nuclear receptors (Needham et al., 2000). Although the presence of the LXXLL motif is essential to ER interactions, specificity is also determined by amino acid sequences flanking the motif. ...
The estrogen receptors are members of a large family of transcription factors, which include receptors for steroid hormones, retinoids, vitamin D and thyroid hormone, as well as several orphan receptors with yet undiscovered ligands. The process by which small lipophilic molecules signal in target tissues is complex and involves nuclear receptor associations with a multitude of coregulator proteins. These proteins participate in and recruit many enzymatic and structural activities that allow modulation of chromatin structure to facilitate stimulation or repression of gene expression. This review will present an overview of those regulatory factors that interact with the estrogen receptors and participate in the transmission of the estrogenic signal.
... This domain contains three LXXLL motifs (named NR boxes), which are involved in receptor-SRC-1 interaction (54 -57). Residues flanking NR boxes on both sides make significant contacts with the ligand binding domain of receptors (58). These residues have also been shown to be important for the preferential binding of various NR boxes to different nuclear receptors and thus to determine a preference of the various receptors for specific p160 coactivators (58). ...
... Residues flanking NR boxes on both sides make significant contacts with the ligand binding domain of receptors (58). These residues have also been shown to be important for the preferential binding of various NR boxes to different nuclear receptors and thus to determine a preference of the various receptors for specific p160 coactivators (58). The sumoylation sites perfectly flank NR box 3. ...
SUMO-1 (small ubiquitin-like modifier) conjugation regulates the subcellular localization, stability, and activity of a variety of proteins. We show here that SUMO-1 overexpression markedly enhances progesterone receptor (PR)-mediated gene transcription. PR undergoes a sumoylation at lysine 388 located in its N-terminal domain. However, sumoylation of the receptor is not responsible for enhanced transcription because substitution of its target lysine did not abolish the effect of SUMO-1 and even converted the receptor into a slightly more active transactivator. Furthermore estrogen receptor alpha (ERalpha)-driven transcription is also enhanced by SUMO-1 overexpression contrasting with the absence of sumoylation of this receptor. We thus analyzed SUMO-1 conjugation to the steroid receptor coactivator SRC-1. We showed that this protein contains two major sites of conjugation at Lys-732 and Lys-774. Sumoylation was shown to increase PR-SRC-1 interaction and to prolong SRC-1 retention in the nucleus. It did not prevent SRC-1 ubiquitinylation and did not exert a clear effect on the stability of the protein. Overexpression of SUMO-1 enhanced PR-mediated gene transcription even in the presence of non-sumoylated mutants of SRC-1. This observation suggests that among the many protein partners involved in steroid hormone-mediated gene regulation several are probably targets of SUMO-1 modification.
... Specifically, HIV-1 Vpr-mediated transactivation is shown to occur through steroid receptors and other coactivators (28,29). Steroid receptors belong to a super family of liganddependent transcription factors that are known to interact with proteins containing the signature motif, LxxLL (30,31). Upon binding to their ligands, steroid receptors translocate into the nucleus and activate/repress transcription depending on the signal (32,33). ...
Human Immunodeficiency Virus-1 Vpr, a nonstructural protein incorporated into virus particles, possesses several features contributing to virus replication and cytopathic effects. Vpr induced effects are mediated through its interactions with viral and/or host cellular proteins, in particular, the host protein, Glucocorticoid Receptor (GR). Though GR is known to increase Vpr-mediated HIV-1 transactivation, Vpr-GR interaction and its subcellular localization have not been studied in cells. Towards this, we evaluated Vpr interaction with GR in cells using Bimolecular Fluorescence Complementation analysis by generating chimeric Vpr or GR with the N- and C-terminal fragments of Venus protein. Our results showed that interaction between Vpr and GR requires certain Vpr and GR domains. Specifically, leucine residues in the third helical domain of Vpr and N-terminal domain of GR is involved in Vpr-GR interaction. Altering these residues not only interferes with Vpr-GR interaction, but also prevents translocation of this complex into the nucleus. Further, utilizing a mutant Vpr unable to oligomerize, we show that Vpr oligomerization is essential for optimal interaction with GR. In conclusion, by taking advantage of BiFC system, specific residues in Vpr have been found to be associated with binding GR and the subcellular distribution of Vpr-GR complex.
... More than 400 NR coregulators have been identified and characterized (14), the first being members of the steroid receptor coactivator (SRC) gene family (15). For many NRs, transcriptional activity derives from the AF2 region within the LBD and involves the interaction between a conserved hydrophobic cleft on the surface of the LBD and short, leucine-rich hydrophobic motifs (NR boxes, consensus LxxLL motif) reiterated within each coactivator (16). In addition, NR-interacting coactivators themselves have, or recruit other nuclear proteins that have, enzymatic activities crucial for efficient gene expression (17). ...
A homozygous missense mutation in the gene encoding the estrogen receptor α (ERα) was previously identified in a female patient with estrogen insensitivity syndrome. We investigated the molecular features underlying the impaired transcriptional response of this mutant (ERα-Q375H) and four other missense mutations at this position designed to query alternative mechanisms. The identity of residue 375 greatly affected the sensitivity of the receptor to agonists without changing the ligand binding affinity. Instead, the mutations caused changes in the affinity of coactivator binding and alterations in the balance of coactivator and corepressor recruitment. Comparisons among the transcriptional regulatory responses of these six ERα genotypes to a set of ER agonists showed that both steric and electrostatic factors contributed to the functional deficits in gene regulatory activity of the mutant ERα proteins. ERα-coregulator peptide binding in vitro and RIME (rapid immunoprecipitation mass spectrometry of endogenous) analysis in cells showed that the degree of functional impairment paralleled changes in receptor-coregulator binding interactions. These findings uncover coupling between ligand binding and coregulator recruitment that affects the potency rather than the efficacy of the receptor response without substantially altering ligand binding affinity. This highlights a molecular mechanism for estrogen insensitivity syndrome involving mutations that perturb a bidirectional allosteric coupling between ligand binding and coregulator binding that determines receptor transcriptional output.
... They showed in a series of yeast two hybrid experiments the second LXXLL motif of SRC-1 preferentially interacts with ERa and ERp, while the fourth LXXLL motif (interestingly only found in the SRC-la isoform) preferentially interacts with peroxisome proliferator activated receptors alpha and gamma (PPARa and y), AR, and GR. These interactions depend not only on the LXXLL motifs, but also on residues 77-terminal to the motif (Needham et aL, 2000). ...
Brn-3a and Brn-3b are closely related members of the POU domain-containing transcription factors. While Brn-3a is associated with neuronal differentiation and Brn-3b with neuronal proliferation, their expression is not strictly limited to the nervous system. It has been shown that these Brn-3 proteins are expressed in non-neuronal tissues including cervical epithelium, testis and breast. Moreover, these Brn-3 proteins functionally interact with the estrogen receptor and in association with the estrogen receptor have differing transactivation potentials. The p160 steroid receptor coactivators (Srcs) are also able to interact with the estrogen receptor in a ligand dependent manner and are able to enhance estrogen dependent transcription. This work describes a functional interaction between Brn-3 proteins and members of the steroid receptor coactivator family. In affinity chromatography and coimminoprecipitation experiments, Src-1 proteins were shown to physically interact with Brn-3a and Brn-3b. In addition, the transactivation potential of the Brn-3/Src complexes was tested on two promoters in three different cell lines. The steroid receptor coactivators potentiated the transcriptional effects of Brn-3 a and Brn-3b. The Src proteins, however, differed in their ability to potentiate the transcriptional activity of Brn-3 proteins on different promoters in transiently transfected cells. In addition, maximal activation of the Brn-3/Src complex differed between cell lines, indicating that additional cell-type specific proteins are important for maximal activation by the complex. It has recently been shown that Brn-3b may play a role in regulating BRCA-1 in mammary tumors as its expression is enhanced in primary breast tumors with reduced BRCA-1 expression. Moreover, this elevated Brn-3b expression is not seen in normal mammary cells, benign tumors, or malignant tumors which do not have reduced levels of BRCA-1. This work also describes the effects of stable Brn-3b overexpression and reduction in the estrogen receptor positive breast adenocarcinoma cell line MCF7. Overexpression of Brn-3b resulted in increased growth rate, saturation density, proliferation, and ability to form anchorage independent colonies when compared to mock transfected cells. MCF7 cells with reduced Brn-3b levels exhibited a significant decrease in growth rate, saturation density, proliferation, and ability to form anchorage independent colonies when compared to mock transfected controls. Differential gene expression in these cell lines was examined using both specific western immunodetection as well as comprehensive DNA array technology. Immunodetection revealed increases in ER, HSP-27, and Brn-3a in Brn-3b overexpressing cells as well as a decrease in these proteins in the Brn-3b reduced cell lines. The DNA array including 1200 cancer related genes revealed several genes differentially expressed when the Brn-3b overexpressing and reduced cell line mRNAs were compared. Finally, the results of three of the genes shown to be differentially regulated in the cDNA array differential display were corroborated by semi-quantitative RT-PCR.
... The E R a has been demonstrated to interact preferentially with LXXLL motif 2 in SRCl and TIF2 (Kalkhoven et al., 1998;Voegel et al., 1998). This motif was also shown to bind with highest affinity to TRp (Darimont et al., 1998), while alternative motifs are preferentially utilised by other receptors (Ding et al., 1998;Mcinerney et al., 1998;Needham et al., 2000). Sequence alignment indicates that conservation of a particular LXXLL motif in the three p i 60 coactivators is greater than the conservation between individual motifs (Figure 3.11). ...
Oestrogens regulate the transcription of target genes by binding to the oestrogen receptors (ERα and ERβ) which function as ligand inducible transcription factors. Both ERα and ERβ are members of the nuclear receptor superfamily which is characterised by a highly conserved DNA-binding domain. There are two distinct transcriptional activation domains: the ligand independent API at the N-terminus and the ligand dependent AF2 at the C-terminus which is encompassed by the ligand binding domain (LBD). Following sequence specific binding of oestrogen receptors to the promoter of target genes, additional co-factors are recruited in order to remodel the chromatin structure or to aid the docking of the RNA polymerase II holoenzyme. The AF2 activity of ERα is mediated through interaction between the LBD and coactivator proteins upon ligand binding. In order to define the ERα-coactivator interface at a molecular level, systematic mutagenesis was carried out. This led to the identification of a group of conserved hydrophobic residues in the LBD that are required for binding the p160 family of coactivators. Together with helix 12 and lysine 366 at the C-terminal end of helix 3, they form a hydrophobic groove that accommodates an LXXLL motif, which is essential for coactivator binding to the receptor. The presence of endogenous coactivators is a major impediment for studying designated receptor-coactivator pairs in mammalian cells. To circumvent this problem, a yeast genetic screen was conducted to identify suppressor mutant coactivators for a transcriptionally defective ERα. The V380H mutant receptor fails to interact with wild-type p160 coactivators such as SRC1e. However, an altered specificity mutant SRC1e recovered from the screen is able to interact with the mutant receptor, and fully rescues its transcriptional activity in transfected mammalian cells. Remarkably, introduction of the analogous mutation into other p160 coactivator family members confers the ability to suppress the V380H mutation. This suggests that at least in the assays employed, recruitment of a p160 coactivator by ERα is sufficient to activate transcription.
... Cependant, cette conformation est très favorable à la liaison avec le ligand(Fang et al. 1996;Housley et al. 1990).Une fois le ligand fixé dans la poche du LBD de l'AR, le complexe AR-HSPs se dissocie. La fixation du ligand rend également l'AR beaucoup plus stable notamment grâce à des interactions intramoléculaires qui se produisent entre le motif Tau-1 du NTD et l'AF2 du LBDLangley et al. 1995;Doesburg et al. 1997;Wilson 2011).Ces interactions intramoléculaires (appelées interactions N/C) entrainent un changementconformationnel allostérique qui amorce la fixation de co-activateurs de l'AR.Ce recrutement a lieu entre les motifs LxxLL des protéines co-régulatrices et la région AF2 du LBD de l'AR(Needham et al. 2000). Les sites de dimérisation (vraisemblablement présents dans le LBD)(Nemoto et al. 1994) ainsi que le NLS sont également mis à jour ce qui permet la translocation de l'homodimère AR-AR dans le noyau au travers des pores nucléaires. ...
La déficience intellectuelle (DI) et les troubles du spectre autistique (ASD) sont deux troubles neurodéveloppementaux (NDD) présentant de nombreux chevauchements génétiques et phénotypiques ainsi qu’un biais de sexe important, avec plus de garçons atteints (1,4x plus pour la DI et 4x plus pour l'ASD). Au sein de notre laboratoire, le taux de diagnostic des patients souffrant de DI et/ou d’ASD est significativement plus élevé chez les filles que chez les garçons. De façon surprenante, nous n’avons pas observé de différence significative entre filles et garçons au niveau de la proportion de mutations pathogènes sur le chromosome X (5,3% versus 7,6%), confirmant ainsi que les mutations causales totalement pénétrantes sur ce chromosome ne peuvent pas expliquer la totalité de l’excès de garçons atteints de DI ou d'ASD. Nous avons donc choisi d’étudier une autre des hypothèses, plus environnementale, qui pourrait rendre le cerveau masculin plus susceptible au développement de NDD : le rôle des androgènes au cours du développement du cerveau. J'ai étudié l’effet de ces hormones masculines dans des précurseurs neuronaux humains (hNSCs) et observé que les androgènes augmentent la prolifération des hNSCs et les protègent contre la mort cellulaire en conditions stressantes. J'ai également mis en évidence que les androgènes, via leur récepteur (le récepteur aux androgènes), régulent une centaine de gènes dans les hNSCs avec, parmi eux, un enrichissement en gènes connus pour être différentiellement exprimés chez les individus avec ASD (dont NRCAM et FAM107A). La régulation de ces gènes par les androgènes pendant le développement du cerveau pourrait ainsi participer à la sensibilité accrue du cerveau masculin, exposé à d'autres facteurs génétiques et environnementaux, à développer une NDD.
... 124 In addition, the intrinsic dipole moment of the co-activator α-helix is matched on the AF-2 by a negatively charged residue E897, on H12, at the N-terminus and a positively charged K720, on H3, at the C-terminus, which form a charge clamp. 125 The LXXLL motif, named the NR box, is present in several coactivators such as proteins of the p160 family. 126 Mainly formed by residues from helices 3, 4, 5, and 12, the AF-2 surface on the AR-LBD is unique among NRs in preferring to interact with the more bulky hydrophobic motifs F/WXXLF respect to the LXXLL. ...
Investigations on cellular protein interaction networks (PINs) reveal that proteins that constitute hubs in a PIN are notably enriched in Intrinsically Disordered Proteins (IDPs) compared to proteins that constitute edges, highlighting the role of IDPs in signaling pathways. Most IDPs rapidly undergo disorder-to-order transitions upon binding to their biological targets to perform their function. Conformational dynamics enables IDPs to be versatile and to interact with a broad range of interactors under normal physiological conditions where their expression is tightly modulated. IDPs are involved in many cellular processes such as cellular signaling, transcriptional regulation, and splicing; thus, their high-specificity/low-affinity interactions play crucial roles in many human diseases including cancer. Prostate cancer (PCa) is one of the leading causes of cancer-related mortality in men worldwide. Therefore, identifying molecular mechanisms of the oncogenic signaling pathways that are involved in prostate carcinogenesis is crucial. In this review, we focus on the aspects of cellular pathways leading to PCa in which IDPs exert a primary role.
... Although three LXXLL motifs are clustered in the central region of the p160 protein, each LXXLL motif can individually bind NRs with varying affinity. The residues immediately flanking the LXXLL motifs contribute to the specificity of these interactions (Needham et al., 2000). ...
A large variety of mechanisms are utilized in the regulation of gene expression by the glucocorticoid receptor. The receptor is subject to complex subcellular-trafficking processes that modulate the distribution of the receptor in living cells. Although DNA sequence is the primary recognition element in genome regulatory sites, access of the receptor to these sites is strongly modulated by chromatin structure, and the subsequent reorganization of local nucleoprotein domains is a key component of receptor function. The receptor-response elements can be located at large distances from gene targets, indicating that long-range interactions within the nuclear architecture contribute to receptor action. Furthermore, the receptor is subject to many post-translational modifications, and these alterations can affect receptor function at several levels. Finally, interactions between receptors and the genome are surprisingly rapid, indicating a dynamic aspect of receptor movement only recently appreciated.
... In contrast, Y537 (the human equivalent of mouse Y541) does not appear to be in close contact with the LXXLL core motif sequence. However, sequences flanking the core motifs are known to be important for differential interactions of coactivators with steroid receptors and other NRs (Needham et al. 2000, Coulthard et al. 2003, although such sequences have yet to be observed in LBD/coactivator peptide crystal structures. Thus, it remains to be established how replacement of Y541 with alanine or acidic residues enhances ligand-independent binding of coactivators in mammalian cells. ...
Steroid receptors activate transcription in yeast cells via interactions with endogenous coactivators and/or basal factors. We examined the effects of mutations in the ligand binding domain on the transcriptional activity of ERα in yeast. Our results show that mutations in Helix 3 (K366A) and Helix 12 (M547A, L548A) disrupt transcriptional activity of ERα in yeast, as previously observed in mammalian cells. However, replacement of a conserved tyrosine residue in Helix 12 with alanine or aspartate (Y541A and Y541D), which renders ERα constitutively active in mammalian cells, had only a weak stimulatory effect on ligand-independent reporter activation by ERα in yeast. Two-hybrid interaction experiments revealed that a Y541A mutant expressed in yeast was capable of ligand-independent binding to a mammalian coactivator, suggesting that there is a subtle difference in how this mutant interacts with mammalian and yeast cofactors. We also show that the ligand-dependent activities of ERα and progesterone receptor (PR) in yeast cells were strongly enhanced by the human p160 protein steroid receptor coactivator (SRC1), but not by CREB-Binding Protein (CBP) or the p300/CBP associated factor (P/CAF). Although the SRC1 activation domains AD1 and AD2 are functional in yeast, deletion of these sequences only partially impaired SRC1 coactivator function in this organism; this is in contrast to similar experiments in mammalian cells. Thus SRC1 sequences involved in recruitment of CBP/p300 and Co-ActivatorAssociated Arginine Methyltransferase (CARM-1) in mammalian cells are not essential for its function in yeast, suggesting that SRC1 operates via distinct mechanisms in yeast and mammalian cells.
... TFIIB and TFIIF [32]; (ii) functionally different proteins with AR co-activating or co-repressing properties (e.g. histone acetyl transferases (HATs), co-activators such as NCOA1 (SRC1, AIB1), NCOA2 (TIF2, SRC2), NCOA3 (SRC3), and co-repressors like SIRT1 and NCOR1 [33][34][35][36] and (iii) specific transcription factors that differ from general transcription factors. Some of them interact directly with AR (DAX-1 with the AR Ligand Binding Domain -LBD-) [37] and affect its ability to be recruited at ARE sites without binding directly to the DNA. ...
Androgen Receptor (AR) and Estrogen Receptors (ERs) are key nuclear receptors that can cooperate in orchestrating gene expression programs in multiple tissues and diseases, targeting binding elements in promoters and distant enhancers. We report the unbiased identification of enhancer elements bound by AR and ER-α whose activity can be allele-specific depending on the status of nearby Single Nucleotide Polymorphisms (SNP). ENCODE data were computationally mined to nominate genomic loci with: (i) chromatin signature of enhancer activity from activation histone marks, (ii) binding evidence by AR and ER-α, (iii) presence of a SNP. Forty-one loci were identified and two, on 1q21.3 and 13q34, selected for characterization by gene reporter, Chromatin immunoprecipitation (ChIP) and RT-qPCR assays in breast (MCF7) and prostate (PC-3) cancer-derived cell lines. We observed allele-specific enhancer activity, responsiveness to ligand-bound AR, and potentially influence on the transcription of closely located genes (RAB20, ING1, ARHGEF7, ADAM15). The 1q21.3 variant, rs2242193, showed impact on AR binding in MCF7 cells that are heterozygous for the SNP. Our unbiased genome-wide search proved to be an efficient methodology to discover new functional polymorphic regulatory regions (PRR) potentially acting as risk modifiers in hormone-driven cancers and overall nominated SNPs in PRR across 136 transcription factors.
... Indeed, the information available indicates that Tai proteins may have different partners, which allows the possibility to perform a variety of tasks, and the data reported herein suggest that Tai isoforms may be influential in determining which partners are chosen and which roles are played. In mammals, SRC1 has at least 5 isoforms that differ in the C-terminal region [30] and which affect the interaction with nuclear receptors in different hormone signalling pathways [31][32][33]. In light of such versatile interaction capabilities and actions, vertebrate SRCs have been qualified, admittedly rather pompously, as masters of systems biology [34]. ...
Recent studies in vitro have reported that the Methoprene-tolerant (Met) and Taiman (Tai) complex is the functional receptor of juvenile hormone (JH). Experiments in vivo of Met depletion have confirmed this factor's role in JH signal transduction, however, there is no equivalent data regarding Tai because its depletion in larval or nymphal stages of the beetle Tribolium castaneum and the bug Pyrrhocoris apterus results in 100% mortality. We have discovered that the cockroach Blattella germanica possesses four Tai isoforms resulting from the combination of two indels in the C-terminal region of the sequence. The presence of one equivalent indel-1 in Tai sequences in T. castaneum and other species suggests that Tai isoforms may be common in insects. Concomitant depletion of all four Tai isoforms in B. germanica resulted in 100% mortality, but when only the insertion 1 (IN-1) isoforms were depleted, mortality was significantly reduced and about half of the specimens experienced precocious adult development. This shows that Tai isoforms containing IN-1 are involved in transducing the JH signal that represses metamorphosis. Reporter assays indicated that both T. castaneum Tai isoforms, one that contains the IN-1 and another that does not (DEL-1) activated a JH response element (kJHRE) in Krüppel homolog 1 in conjunction with Met and JH. The results indicate that Tai is involved in the molecular mechanisms that repress metamorphosis, at least in B. germanica, and highlight the importance of distinguishing Tai isoforms when studying the functions of this transcription factor in development and other processes.
... However, as different nuclear receptors have evolved to bind hydrophobic LxxLL consensus motifs through similar binding mechanisms, it remains important that mimetics are designed in a way that conveys greater selectivity to the AR. Improving mimetics by including specific flanking sequences is expected to improve selectivity, as studies have shown that the sequences immediately flanking the consensus motif confer specificity in vivo [83,[186][187][188][189] . Further characterization of the unique requirements for androgen receptor-specific coactivator binding may be useful for the design of peptide antagonists. ...
Androgens and androgen receptors (AR) play a pivotal role in expression of the male phenotype. Several diseases, such as androgen insensitivity syndrome (AIS) and prostate cancer, are associated with alterations in AR functions. Indeed, androgen blockade by drugs that prevent the production of androgens and/or block the action of the AR inhibits prostate cancer growth. However, resistance to these drugs often occurs after 2-3 years as the patients develop castration-resistant prostate cancer (CRPC). In CRPC, a functional AR remains a key regulator. Early studies focused on the functional domains of the AR and its crucial role in the pathology. The elucidation of the structures of the AR DNA binding domain (DBD) and ligand binding domain (LBD) provides a new framework for understanding the functions of this receptor and leads to the development of rational drug design for the treatment of prostate cancer. An overview of androgen receptor structure and activity, its actions in prostate cancer, and how structural information and high-throughput screening have been or can be used for drug discovery are provided herein.
... doi:10.1371/journal.pone.0059726.g007 activation, other mechanisms may also be involved, such as interaction with nuclear coactivators shared by PPAR transcription factors and the GR [66][67][68]. ...
FOXO1 is involved in glucocorticoid- and sepsis-induced muscle wasting, in part reflecting regulation of atrogin-1 and MuRF1. Mechanisms influencing FOXO1 expression in muscle wasting are poorly understood. We hypothesized that the transcription factor peroxisome proliferator-activated receptor β/δ (PPARβ/δ) upregulates muscle FOXO1 expression and activity with a downstream upregulation of atrogin-1 and MuRF1 expression during sepsis and glucocorticoid treatment and that inhibition of PPARβ/δ activity can prevent muscle wasting. We found that activation of PPARβ/δ in cultured myotubes increased FOXO1 activity, atrogin-1 and MuRF1 expression, protein degradation and myotube atrophy. Treatment of myotubes with dexamethasone increased PPARβ/δ expression and activity. Dexamethasone-induced FOXO1 activation and atrogin-1 and MuRF1 expression, protein degradation, and myotube atrophy were inhibited by PPARβ/δ blocker or siRNA. Importantly, muscle wasting induced in rats by dexamethasone or sepsis was prevented by treatment with a PPARβ/δ inhibitor. The present results suggest that PPARβ/δ regulates FOXO1 activation in glucocorticoid- and sepsis-induced muscle wasting and that treatment with a PPARβ/δ inhibitor may ameliorate loss of muscle mass in these conditions.
... These results demonstrate that the LXXLL motif in HADHB does not play a predominant role in determining the ERα-HADHB interaction. Binding between LXXLL motifs in co-activators/co-repressors and the AF2 domain of ERα/ERβ is known to be E2-dependent (34). We then performed the in-vitro binding assay in the presence or absence of E2 to test whether E2 affects the binding of HADHB to ERα. ...
Estrogen receptors are localized in mitochondria, but their functions in this organelle remain unclear. We previously found that ERα interacted with mitochondrial protein HADHB and affected the thiolytic cleavage activity of HADHB in β-oxidation. It is known that ERβ binds to ERα. In addition, ERβ is predominately located in mitochondria. These facts led us to speculate that ERβ may also be associated with HADHB in mitochondria. In order to test this hypothesis, we performed co-immunoprecipitation and confocal microscopy analyses with human breast cancer MCF7 cells. The results demonstrated that ERβ was indeed associated and colocalized with HADHB within mitochondria. Interestingly, in contrast to the stimulatory effect of ERα on HADHB enzyme activity observed in the previous study, silencing of ERβ enhanced the enzyme activity of HADHB in the present study, suggesting that ERβ plays an inhibitory role in HADHB enzyme activity in the breast cancer cells. Our results imply that ERα and ERβ may differentially affect cellular oxidative stress through influencing the rate of β-oxidation of fatty acids in breast cancer cells.
... RXRa- LBD or PPARc-LBD is reported to exhibit notably conformational changes upon agonist binding, subsequently recruiting its co-activator such as the steroid receptor coinducer-1 (SRC1) [25]. To study whether Rut is able to enhance the interaction between RXRa-LBD or PPARc-LBD and the co-activator, the binding affinity of RXRa-LBD or PPARc-LBD against SRC1 613–773 containing the LXXLL motifs [26] in the presence of different concentrations of Rut is measured. As indicated, RXRa-LBD dose-dependently interacts with SRC1 (K D = 1.16 mM, Supple- mentaryFig. ...
Glucose transporter 4 (GLUT4) is a principal glucose transporter in response to insulin, and impaired translocation or decreased expression of GLUT4 is believed to be one of the major pathological features of type 2 diabetes mellitus (T2DM). Therefore, induction of GLUT4 translocation or/and expression is a promising strategy for anti-T2DM drug discovery. Here we report that the natural product (+)-Rutamarin (Rut) functions as an efficient dual inducer on both insulin-induced GLUT4 translocation and expression. Rut-treated 3T3-L1 adipocytes exhibit efficiently enhanced insulin-induced glucose uptake, while diet-induced obese (DIO) mice based assays further confirm the Rut-induced improvement of glucose homeostasis and insulin sensitivity in vivo. Subsequent investigation of Rut acting targets indicates that as a specific protein tyrosine phosphatase 1B (PTP1B) inhibitor Rut induces basal GLUT4 translocation to some extent and largely enhances insulin-induced GLUT4 translocation through PI3 kinase-AKT/PKB pathway, while as an agonist of retinoid X receptor α (RXRα), Rut potently increases GLUT4 expression. Furthermore, by using molecular modeling and crystallographic approaches, the possible binding modes of Rut to these two targets have been also determined at atomic levels. All our results have thus highlighted the potential of Rut as both a valuable lead compound for anti-T2DM drug discovery and a promising chemical probe for GLUT4 associated pathways exploration.
... In addition to these LXD motif requirements, we also incorporated the flanking regional properties of amino acid charge and hydrophobicity common to known nuclear receptor cofactors. By using these characteristics of known cofactor LXD's as pre-filtering parameters [37,38,40,[42][43][44][45][46][47], we identified 130 genes which matched the requisite motif primary sequence, protein sub-structure and amino acid composition. This list of putative cofactors yielded a Gene Ontology enrichment for "positive regulation of transcription", with a Benjamini corrected p-value of 1.5 E-6 which further supported the impact of our proteome pre-screen. ...
During Drosophila development, titers of the steroid ecdysone trigger and maintain temporal and tissue specific biological transitions. Decades of evidence reveal that the ecdysone response is both unique to specific tissues and distinct among developmental timepoints. To achieve this diversity in response, the several isoforms of the Ecdysone Receptor, which transduce the hormone signal to the genome level, are believed to interact with tissue specific cofactors. To date, little is known about the identity of these cofactor interactions; therefore, we conducted a bioinformatics informed, RNAi luciferase reporter screen against a subset of putative candidate cofactors identified through an in silico proteome screen. Candidates were chosen based on criteria obtained from bioinformatic consensus of known nuclear receptor cofactors and homologs, including amino acid sequence motif content and context.
The bioinformatics pre-screen of the Drosophila melanogaster proteome was successful in identifying an enriched putative candidate gene cohort. Over 80% of the genes tested yielded a positive hit in our reporter screen. We have identified both cell type specific and common cofactors which appear to be necessary for proper ecdysone induced gene regulation. We have determined that certain cofactors act as co-repressors to reduce target gene expression, while others act as co-activators to increase target gene expression. Interestingly, we find that a few of the cofactors shared among cell types have a reversible roles to function as co-repressors in certain cell types while in other cell types they serve as co-activators. Lastly, these proteins are highly conserved, with higher order organism homologs also harboring the LXXLL steroid receptor interaction domains, suggesting a highly conserved mode of steroid cell target specificity.
In conclusion, we submit these cofactors as novel components of the ecdysone signaling pathway in order to further elucidate the dynamics of steroid specificity.
... This small family consists of three 160 kDa proteins, namely SRC-1, SRC-2 (TIF2/GRIP1) and SRC-3 (AIB1/NCoA3) [23,[36][37][38][39]. The p160 coactivators have been shown to bind to TAU-5 of the AR NTD and to the AF-2 surface [40][41][42]. They influence AR transactivation capacity directly via intrinsic histone acetyltransferase (HAT) activity [43], and indirectly by acting as platforms for the recruitment of secondary coactivators such as cAMP responsive element binding protein (CREB) binding protein (CBP) [44], p300 [44] and p300/CBP-associated factor [45], coactivator-associated arginine methyltransferase 1 (CARM1) and protein arginine methyltransferase 1 (PRMT1) [46,47]. ...
Androgens, acting through the androgen receptor (AR), are responsible for many male reproductive and nonreproductive functions. Moreover, aberrant androgen/AR signaling plays a critical role in androgen-dependent prostate cancer (PCa) as well as castration-resistant prostate cancer (CRPC). The formation of a productive AR transcriptional complex requires AR cofactors that interact functionally and structurally with the AR. Since the discovery of the first such cofactor in 1995, an ever increasing number of proteins have been identified as AR coactivators or corepressors. The expression and function of several AR cofactors have been investigated in PCa, and a clear link between AR cofactors and the development and progression of PCa has been identified. Recently, AR splice variants in CRPC were reported, which display significant constitutive activity in the absence of ligand. Then, this discovery revolutionized the concept of AR cofactors in CRPC. The current review aims to provide an overview of AR cofactor proteins in the context of PCa. In addition, we discuss the potential of AR cofactors as novel therapeutic targets for PCa, particularly for CRPC.
... Interactions between activated ERa bound to DNA and coactivators such as steroid receptor coactivator 1 (SRC-1) and CREB-binding protein (CBP) regulate the transcription of estrogen target genes (Robyr et al. 2000). SRC-1 functions primarily as coactivator for nuclear receptors and binds directly to liganded ERa through helical LXXLL motifs (Needham et al. 2000). The SRC-1 contributes to transcriptional activation by interacting with other coactivators such as CBP (Smith et al. 1996, Sheppard et al. 2001). ...
Steroid hormones such as 17beta-estradiol (E2) are critical to diverse cellular processes including tumorigenesis. A number of cofactors such as nuclear receptor corepressor (NCoR), CREB-binding protein (CBP), and steroid receptor coactivator 1 (SRC-1) interact with estrogen receptors (ERs) to regulate transcriptional repression or activation of target genes. Estrogen signaling in non-reproductive tract tissues such as skin is less well characterized and the effectiveness of anti-estrogen therapy for cancer arising from these tissues is unknown. We show that tamoxifen (TAM) treatment inhibited cell cycle progression and proliferation of human cancer lines derived from stratified squamous epithelium squamous cell carcinoma (SCC). E2 had no effect on proliferation of these lines despite low levels of ERalpha expression. The E2 treatment promoted displacement of the NCoR from ERalpha and recruitment of CBP to the receptor. SRC-1 expression was not detected in these SCC lines; however, transient transfection of SRC-1, CBP, or both coactivators enhanced transactivation of an estrogen responsive promoter in cancer cells treated with E2 or TAM. In stable clones expressing SRC-1, the coactivator was recruited to ERalpha along with CBP in E2 but not in TAM-treated cells. SRC-1 expression restored the E2-mediated proliferative response to human SCC lines. This increased proliferation correlated with increased extracellular signal regulated kinase 1 (ERK1) expression. SRC-1 and CBP were recruited to the proximal ERK1 promoter region in E2 but not in TAM-treated cells. We concluded that SRC-1 was a key molecular determinant of estrogen-mediated proliferation in human SCC lines.
... They contain, an identical centrally located nuclear receptor box with a triplet of α-helical 'LXXLL' motifs for binding to the nuclear receptors, two activation domains, AD1 (interacts with CBP/p300), and AD2 (interacts with CARM1) and differ only in their C-terminal sequences. The C-terminal part of SRC1a contains an additional LXXLL motif which was found to exhibit a strong interaction with the ligand binding domain (LBD) of the GR in a yeast two-hybrid system (29). Additionally, it also has been shown to possess a repression domain in the context of ER and GR-mediated gene transactivation on a simple reporter (14;28). ...
Currently, the raising awareness of the role of glucocorticoids in the onset of numerous (neuro)-pathologies constitutes the increasing necessity of understanding the mechanisms of action of glucocorticoids in bodily processes and brain functioning. Glucocorticoids mediate their effects by binding to intracellular receptors which act as transcription factors. A remarkable and yet unexplained phenomenon described more than two decades ago, is the cell-specific effects glucocorticoids bring about on gene expression in brain. For example, while glucocorticoids suppress corticotrophin-releasing hormone (CRH) synthesis in the hypothalamus, production of CRH in the central nucleus of the amygdala (CeA) is stimulated by increased hormone levels. Inasmuch as the neuroanatomical distribution of the corticosteroid receptors does not satisfactorily explain these effects, it is of interest to decipher the role of recently discovered coregu lator proteins that modulate the direction and the magnitude of steroid receptor-driven transcription. Therefore, in the current thesis the expression and function of central coregulators was studied: the coactivators SRC1a and SRC1e along with the corepressors N-CoR and SMRT were found to be expressed in brain and involved in regulation of CRH gene expression. Finally, a method that allows detection of coregulator recruitment by steroid receptors in brain tissue was developed.
... Many nuclear receptor coactivators, including SRC-1, bind the receptor in a ligand-dependent manner via LXXLL nuclear receptor binding motifs. Although SRC-1a contains four LXXLL motifs, published data suggests that it preferentially uses motif four for binding to the GR [18]. We have established that our isolated cDNA library clone of SRC-1a encodes this motif as shown in Fig. 7. ...
We report the development of a yeast strain designed for assaying compound-protein interactions through activation of reporter gene expression. Activation of lacZ expression, driven by the GAL4 promoter, has been demonstrated for precedented compound-protein interactions between FK506 and FK506 binding protein 12 (FKBP12) and also between methotrexate (MTX) and dihydrofolate reductase (DHFR). Reporter gene expression was completely abrogated in a competitive manner by the presence of excess FK506 or MTX, respectively. In addition, a strain expressing a mutated DHFR clone with decreased binding affinity for MTX was not capable of activating reporter gene expression. While strain sensitivity is compound-dependent, the minimum compound concentration necessary to drive reporter gene expression was 20 nM for the FK506-FKBP12 interaction. The utility of this strain as a tool for identifying unknown compound-binding proteins has been demonstrated by screening a mouse cDNA library for clones that encode proteins capable of binding MTX. Four library clones of mouse DHFR were identified after screening 5 x 10(6) clones. The screen background was low and false positives were easily identified, making this yeast system particularly amenable for use in a screening context for novel compound-protein interactions.
... A more detailed analysis of SRC-1 versus SRC-1/1139 -1441 (Fig. 3B) confirms that this activity of SRC-1 is largely contained in the COOH-terminal 300 amino acids. This region is known to contain a RID that shows very strong binding to GRs (47). ...
Coactivators such as TIF2 and SRC-1 modulate the positioning of the dose-response curve for agonist-bound glucocorticoid receptors (GRs) and the partial agonist activity of antiglucocorticoid complexes. These properties of coactivators differ from their initially defined activities of binding to, and increasing the total levels of transactivation by, agonist-bound steroid receptors. We now report that constructs of TIF2 and SRC-1 lacking the two activation domains (AD1 and AD2) have significantly less ability to increase transactivation but retain most of the activity for modulating the dose-response curve and partial agonist activity. Mammalian two-hybrid experiments show that the minimum TIF2 segment with modulatory activity (TIF2.4) does not interact with p300, CREB-binding protein, or PCAF, which also modulates GR activities. DRIP150 and DRIP205 have been implicated in coactivator actions but are unable to modulate GR activities. The absence of synergism by PCAF or DRIP150 with SRC-1 or TIF2, respectively, further suggests that these other factors are not involved. The ability of a TIF2.4 fragment (i.e. TIF2.37), which is not known to interact with proteins, to block the actions of TIF2.4 suggests that an unidentified binder mediates the modulatory activity of TIF2. Pull-down experiments with GST/TIF2.4 demonstrate a direct interaction of TIF2 with GR in a hormone-dependent fashion that requires the receptor interaction domains of TIF2 and is equally robust with agonists and most antiglucocorticoids. These observations, which are confirmed in mammalian two-hybrid assays, suggest that the capacity of coactivators such as TIF2 to modulate the partial agonist activity of antisteroids is mediated by the binding of coactivators to GR-antagonist complexes. In conclusion, the modulatory activity of coactivators with GR-agonist and -antagonist complexes is mechanistically distinct from the ability of coactivators to augment the total levels of transactivation and appears to involve the binding to both GR-steroid complexes and an unidentified TIF2-associated factor(s).
Peptide-based inducers of estrogen receptor (ER) α and androgen receptor (AR) degradations via the ubiquitin-proteasome system (UPS) were developed. The designated inducers were composed of two biologically active scaffolds: the helical peptide PERM3, which is an LXXLL-like mimic of the coactivator SRC-1, and various small molecules (MV1, LCL161, VH032, and POM) that bind to E3 ligases (IAPs, VHL, and cereblon, respectively), to induce ubiquitylation of nuclear receptors that bind to SRC-1. All of the synthesized chimeric E3 ligand-containing molecules induced the UPS-mediated degradation of ERα and AR. The PERM3 peptide was applicable for the development of the ERα and AR degraders using these E3 ligands.
Cytochromes P450 (CYPs) are a large superfamily of haemoproteins involved in the metabolism of numerous endogenous and exogenous compounds. CYP2B1 and CYP2B2 expression is increased by a variety of xenobiotics including the anti-epileptic drug phenobarbital. The expression of CYP2B genes declines during hepatocyte culture. The profiles of liver-enriched and stress-induced transcription factors were examined during cell culture. No absolute correlation was found between the profiles of transcription factors (C/EBP, SP1, NFκB, AP1) and CYP2B mRNA amounts. The binding activity of SP1 varied in a manner similar to CYP2B mRNA expression up to 24 hours of cell culture. This led to the identification of two putative SP1 elements in the CYP2B1 promoter. Co-transfection studies of primary hepatocytes with an SP1 expression vector and a luciferase reporter gene linked to the CYP2B1 promoter (-2/-179) indicates that SP1 has a functional role in the regulation of CYP2B1 gene expression. Phenobarbital and other CYP2B inducers, such as the plant product picrotoxin, stimulate constitutive androstane receptor (CAR) activation of a reporter gene linked to the CYP2B1 gene PBRE (phenobarbital response element) in transfected, primary hepatocytes and liver, but not in the transfected, transformed cell lines, HEPG2, CV-1 and HeLa, which support only constitutive transactivation by CAR. Co-transfection experiments in primary hepatocytes demonstrated that the co-activator SRC-1, mediates the action of CAR. Gel shift assays show that phenobarbital is a direct ligand of CAR. Different classes of inducers were shown to mediate induction of CYP2B genes via different receptors. Pregnenolone 16a-carbonitrile and glucocorticoids mediate their actions on the CYP2B1 gene through the Pregnane X Receptor (PXR). Both CAR and PXR bind to the same PBRE direct repeat (DR)4 element to mediate xenobiotic-regulated CYP2B1 expression. Our results suggest that CAR also binds to the DR3 PXRE of the CYP3A1 promoter to mediate the phenobarbital response of this gene.
Estrogen insensitivity syndrome (EIS) arises from rare mutations in estrogen receptor α (ERα, encoded by ESR1 gene) resulting in the inability of estrogen to exert its biological effects. Due to the rarity, mutations in ESR1 gene and the underlying molecular mechanisms of EIS have not been thoroughly studied. Here, we investigate known ESR1 mutants, Q375H and R394H, associated with EIS patients using in vitro and in vivo systems. Comparison of the transcriptome and DNA methylome from stable cell lines of both Q375H and R394H clinical mutants show a differential profile compared to WT ERα resulting in loss of estrogen-responsiveness. Molecular dynamic simulation shows that both ESR1 mutations change the ERα conformation of the ligand receptor complexes. Furthermore, we generated a mouse model Esr1-Q, harboring the human mutation using CRISPR/Cas9 genome editing. Female and male Esr1-Q mice are infertile and have similar phenotypes to αERKO mice. Overall phenotypes of the Esr1-Q mice correspond to those observed in the Q375H patient. Finally, we explore the effects of a synthetic progestogen and a GnRH inhibitor in the Esr1-Q mice for potentially reversing the impaired female reproductive tract function. These findings provide an important basis for understanding the molecular mechanistic consequences associated with EIS.
Like G protein–coupled receptors (GPCRs) and protein kinases, nuclear receptors (NRs) are a rich source of pharmaceutical targets. Over 80 NR-targeting drugs have been approved for 18 NRs. The focus of drug discovery in NRs has hitherto been on identifying ligands that bind to the canonical ligand binding pockets of the C-terminal ligand binding domains (LBD). Due to the development of drug resistance and selectivity concerns, there has been considerable interest in exploring other, non-canonical ligand binding sites. Unfortunately, the potencies of compounds binding at other sites have generally not been sufficient for clinical development. However, the situation has changed dramatically over the last three years, as compounds with sufficient potency have been reported for several NR targets. Here we review recent developments in this area from a medicinal chemistry point of view in the hope of stimulating further interest in this area of research.
Among PAS domain proteins, the p160 coactivator class may be the most perplexing. Like many PAS domain proteins, members of the p160 family sense the presence of small, lipophilic molecules. And like many eukaryotic PAS domain proteins they regulate transcription. However, p160 coactivators have not been found to bind directly either to small molecules or to DNA; rather they bind to steroid hormone receptors in a ligand-dependent manner and serve as coactivators of transcription. As such, they form a bridge between the hormone receptors, general transcription machinery, and histone acetyl transferases (HATs), which regulate chromatin conformation. These proteins are essential to the organism for normal responses to steroid hormones and for energy homeostasis. Yet to this day, the precise function of the PAS domain within the P160 coactivators remains something of a mystery.
Androgens play critical roles in male sexual differentiation, pubertal sexual maturation, the maintenance of spermatogenesis, and male gonadotropin regulation (Keller et al., 1996; McLachlan et al., 1996; Roy et al., 1999; Sheckter et al., 1989). The androgen receptor (AR), a 110-kDa ligand-inducible transcription factor, mediates androgen signaling by binding to androgen response elements (ARE) within the promoters of target genes (Chang et al., 1988a; Chang et al., 1988b; Lubahn et al., 1988; Tilley et al., 1989; Trapman et al., 1988). Mutations of AR that alter its ability to bind androgens, or alter its transcriptional activity after ligand binding, may result in male infertility, or complete or partial androgen insensitivity (De Bellis et al., 1994; Gottlieb et al, 2001; Mowszowicz et al., 1993; Quigley et al., 1995; Tyagi et al., 1998). Somatic AR mutations have also been identified in some prostate tumors (Bentel and Tilley, 1996).
The androgen receptor (AR) is a key regulator for the growth, differentiation and survival of prostate cancer cells. Identified as a primary target for the treatment of prostate cancer, many therapeutic strategies have been developed to attenuate AR signaling in prostate cancer cells. While frontline androgen-deprivation therapies targeting either the production or action of androgens usually yield favourable responses in prostate cancer patients, a significant number acquire treatment resistance. Known as the castration-resistant prostate cancer (CRPC), the treatment options are limited for this advanced stage. It has been shown that AR signaling is restored in CRPC due to many aberrant mechanisms such as AR mutations, amplification or expression of constitutively active splice-variants. Coregulator recruitment is a crucial regulatory step in AR signaling and the direct blockade of coactivator binding to AR offers the opportunity to develop therapeutic agents that would remain effective in prostate cancer cells resistant to conventional endocrine therapies. Structural analyses of the AR have identified key surfaces involved in protein-protein interaction with coregulators that have been recently used to design and develop promising AR-coactivator binding inhibitors. In this review we will discuss the design and development of small-molecule inhibitors targeting the AR-coactivator interactions for the treatment of prostate cancer.
Copyright © 2015. Published by Elsevier Ltd.
Androgens are important sex steroid hormones. The androgens testosterone and dihydrotestosterone
(DHT) are essential for normal male sexual differentiation and for the development
and maintenance of male reproductive tissues, including the prostate. Androgens mediate
their effects by binding to, and activation of, the androgen receptor (AR), which is a transcription
factor belonging to the nuclear receptor (NR) family. Upon androgen binding, the AR is
able to recognize specific DNA sequences from where it regulates the expression of its target
genes. A disregulated androgen-AR pathway is involved in several diseases, such as prostate
cancer, androgen insensitivity syndrome (AIS), and Kennedy’s disease or spinal and bulbar
muscular atrophy (SBMA).
A classical approach to treating prostate cancer uses antagonist ligands - so-called anti-androgens, such as bicalutamide - which block gene transcription through binding to a lipophilic pocket at the ligand binding domain of the androgen receptor (AR). An alternative strategy has been developed using compounds which directly target the surface charge clamp by mimicking the coactivator's highly conserved alpha-helical motif. Thus, to gain additional knowledge about the AR-coactivator interaction, the use of natural miniproteins as a source of novel AR-coactivator inhibitors incorporating the FXXLF motif was explored. Their stable well-defined alpha-helical secondary structures make miniproteins ideal candidates for development into AR-coactivator inhibitors. Therefore, starting from two potent miniprotein scaffold structures, identified from previous work, systematic point mutations aimed at improving AR affinity were introduced using solid-phase peptide synthesis (SPPS). Structure-activity relationship studies were performed, from which a number of high affinity inhibitors, typically in the low micromolar-to-high nanomolar range, with a ten-fold gain in potency compared with the reference compounds, were identified, thus highlighting the high potential for these scaffolds.
Activation of the estrogen receptor alpha (ERα) is of prime importance for the development of hormone-dependent breast cancers. Hence, drugs able to impede the emergence of an active folding of ERα have been used for a long time as a first line therapeutic strategy. Aromatase inhibitors that block estradiol synthesis and / or antiestrogens that compete with hormone binding to the receptor are routinely prescribed. Unfortunately, emergence of tumor resistance almost invariably results from currently used antihormonal approaches. One may anticipate that a "multi-target" strategy affecting key regulatory domains distinct from ligand binding pocket of ERα may help to circumvent this problem. To reach this goal, the synthesis of peptides that may specifically inhibit intra- or inter-molecular interactions has been proposed. This paper describes functional motifs potentially suitable for the design of such antagonists. Activity of available peptidic and non-peptidic mimics of these motifs is also reviewed.
The limiting factor in the survival of a patient with prostate cancer is the rate of progression to the noncurable androgen-independent
(AI) stage of disease. The androgen receptor (AR) is a critical regulator of prostate cancer development and is involved in
AI progression. Coregulators are proteins that interact directly with AR to enhance (coactivators) or reduce (corepressors)
its transcriptional activity. Currently, over 165 AR coregulators have been discovered. In this chapter, we focus on a subset
of the most well-characterized AR coregulators that are associated with prostate cancer. The first part of our review discusses
the mechanisms by which classical type I and nonclassical type II AR coactivators/corepressors regulate AR transcriptional
activity. The second section focuses on the role of coregulators in prostate cancer, including their expression profile in
prostate cancer patients, tumor cell growth effects, and potential as therapeutic targets. In view of their involvement in
prostate cancer progression, it is anticipated that further study of AR coregulators will provide more treatment options for
increasing survival of patients with AI prostate cancer.
It is known that estrogen receptors can function as nuclear receptors and transcription factors in the nucleus and as signaling molecules in the plasma membrane. In addition, the localization of the receptors in mitochondria suggests that they may play important roles in mitochondria. In order to identify novel proteins that are involved in ERα-mediated actions of estrogens, we used a proteomic method that integrated affinity purification, two-dimensional gel electrophoresis, and mass spectrometry to isolate and identify cellular proteins that interact with ERα. One of the proteins identified was trifunctional protein β-subunit (HADHB), a mitochondrial protein that is required for β-oxidation of fatty acids in mitochondria. We have verified the interaction between ERα and HADHB by coimmunoprecipitation and established that ERα directly binds to HADHB by performing an in-vitro binding assay. In addition, we have shown that ERα colocalizes with HADHB in the mitochondria by confocal microscopy, and the two proteins interact with each other within mitochondria by performing coimmunoprecipitation using purified mitochondria as starting materials. We have demonstrated that the expression of ERα affects HADHB activity, and a combination of 17β-estrodiol and tamoxifen affects the activity of HADHB prepared from human breast cancer cells that express ERα but not from the cells that are ERα deficient. Furthermore, we have demonstrated that 17β-estrodiol plus tamoxifen affects the association of ERα with HADHB in human cell extract. Our results suggest that HADHB is a functional molecular target of ERα in the mitochondria, and the interaction may play an important role in the estrogen-mediated lipid metabolism in animals and humans.
Androgens are key regulators of male sexual differentiation and essential for development and maintenance of male reproductive tissues. The androgens testosterone and dihydrotestosterone mediate their effect by binding to, and activation of the androgen receptor (AR). Upon activation, the AR is able to recognize specific DNA sequences in gene promoters and enhancers from where it recruits coregulators to orchestrate chromatin remodeling and transcription regulation. The number of proteins that bind to the AR has surpassed 200 and many of them enhance (coactivator) or repress (corepressor) its transactivating capacity. For most of these coregulators, their AR binding interface and their exact mode of action still needs to be elucidated, but for some of the more classical coactivators and corepressors, we gained insight in their working mechanisms. Of particular interest are specific sequences (LxxLL and FxxLF-like motifs) in a subset of coactivators that interact with the AR via a coactivator binding groove in the ligand-binding domain. As compared to other steroid receptors, the conformation of the AR coactivator binding pocket is unique and preferentially binds FxxLF-like motifs. This predisposition is expected to contribute to the regulation of specific sets of target genes via recruitment of selected coregulators. This review provides an overview of these (inter)actions with a focus on the unique characteristics of the AR coactivator binding groove.
Synthetic peptides that specifically bind nuclear hormone receptors offer an alternative approach to small molecules for the modulation of receptor signaling and subsequent gene expression. Here we describe the design of a series of novel stapled peptides that bind the coactivator peptide site of estrogen receptors. Using a number of biophysical techniques, including crystal structure analysis of receptor-stapled peptide complexes, we describe in detail the molecular interactions and demonstrate that all-hydrocarbon staples modulate molecular recognition events. The findings have implications for the design of stapled peptides in general.
Ligand-bound nuclear receptors (NRs) recruit coactivators such as members of the p160 steroid receptor coactivator (SRC) family and cyclic AMP responsive element binding protein (CREB)-binding protein (CBP) to specific enhancer elements and activate target gene transcription. In the present study, we isolated a novel SRC from the sea urchin Strongylocentrotus nudus (SnSRC) by using the ligand-binding domain of retinoid X receptor as a bait in a yeast two-hybrid screening. The SnSRC and vertebrate SRCs are different in size but share the overall characteristic domains, such as NR interacting domain (NID), CBP-binding and glutamine-rich regions. SnSRC mRNA showed highest expression levels at the 32-cell, 64-cell and pluteus larval stages. Full-length SnSRC (1992 amino acids) interacted with several NRs, including sea urchin estrogen receptor-related receptor (ERR), human and masu salmon estrogen receptors (ERα), mouse ERRγ, rat glucocorticoid receptor α, and rat thyroid receptor β. The SnSRC possesses two functional NIDs, both of which are dependent on their core LxxLL motifs. Furthermore, preferential interacting domains for ERα in the SnSRC are located in the central LxxLL motifs, revealed by the truncation and mutagenesis studies. Strikingly, the SnSRC has a single transcription activation domain, which interacts with CBP, a transcriptional integrator. In addition, transient knockdown of the SnSRC gene in the sea urchin embryo using morpholino antisense RNA induced abnormal phenotypes at gastrulation stage such as the lack of primary invagitation and exogastrulation. These results suggest that the SnSRC is a new member of the SRC family and plays an important role during early embryonic development.
Hepatocyte nuclear factor 4alpha (HNF4alpha) is a novel nuclear receptor that participates in a hierarchical network of transcription factors regulating the development and physiology of such vital organs as the liver, pancreas, and kidney. Among the various transcriptional coregulators with which HNF4alpha interacts, peroxisome proliferation-activated receptor gamma (PPARgamma) coactivator 1alpha (PGC-1alpha) represents a novel coactivator whose activation is unusually robust and whose binding mode appears to be distinct from that of canonical coactivators such as NCoA/SRC/p160 family members. To elucidate the potentially unique molecular mechanism of PGC-1alpha recruitment, we have determined the crystal structure of HNF4alpha in complex with a fragment of PGC-1alpha containing all three of its LXXLL motifs. Despite the presence of all three LXXLL motifs available for interactions, only one is bound at the canonical binding site, with no additional contacts observed between the two proteins. However, a close inspection of the electron density map indicates that the bound LXXLL motif is not a selected one but an averaged structure of more than one LXXLL motif. Further biochemical and functional studies show that the individual LXXLL motifs can bind but drive only minimal transactivation. Only when more than one LXXLL motif is involved can significant transcriptional activity be measured, and full activation requires all three LXXLL motifs. These findings led us to propose a model wherein each LXXLL motif has an additive effect, and the multiple binding modes by HNF4alpha toward the LXXLL motifs of PGC-1alpha could account for the apparent robust activation by providing a flexible mechanism for combinatorial recruitment of additional coactivators and mediators.
Androgen receptor (AR) transcriptional activity is tightly regulated by interacting cofactors and cofactor complexes. The best described cofactor interaction site in the AR is the hormone-induced coactivator binding groove in the ligand-binding domain, which serves as a high-affinity docking site for FxxLF-like motifs. This study aimed at identifying novel AR cofactors by in silico selection and functional screening of FxxLF-like peptide motifs. Candidate interacting motifs were selected from a proteome-wide screening and from a supervised screening focusing on components of protein complexes involved in transcriptional regulation. Of the 104 peptides tested, 12 displayed moderate to strong in vivo hormone-dependent interactions with AR. For three of these, ZBTB16/PLZF, SMARCA4/BRG1, and SMARCD1/BAF60a, the full-length protein was tested for interaction with AR. Of these, BAF60a, a subunit of the SWI/SNF chromatin remodeling complex, displayed hormone-dependent interactions with AR through its FxxFF motif. Vice versa, recruitment of BAF60a by the AR required an intact coactivator groove. BAF60a depletion by small interfering RNA in LNCaP cells demonstrated differential effects on expression of endogenous AR target genes. AR-driven expression of TMPRSS2 was almost completely blocked by BAF60a small interfering RNA. In summary, our data demonstrate that BAF60a directly interacts with the coactivator groove in the AR ligand-binding domain via its FxxFF motif, thereby selectively activating specific AR-driven promoters.
Evidences suggest Peroxisome Proliferator Activated Receptor-gamma (PPAR-gamma) ligand, pioglitazone results in the attenuation of gastric mucosal injury. But the molecular mechanism through which these agonists actually elicit gastroprotection through modulating inflammatory responses has not yet been established. Chronic gastric ulcer induced in rats by intraluminal application of acetic acid resulted in elevation of proinflammatory cytokines gene expression, such as, TNF-alpha (Tumor Necrosis Factor-alpha), IL-1beta (Interleukin-1beta) and the protein levels of nuclear p65 subunit of NF-kappaB (Nuclear Factor-kappaB) but decreased levels of PPAR-gamma gene expression. Pioglitazone treatment reduced the severity of ulceration, repressed levels of TNF-alpha, IL-1beta and nuclear p65 subunit as well as increased the abundance of PPAR-gamma in gastric mucosa. Moreover, it significantly upregulated protein levels of glucocorticoid receptor demonstrating its possible involvement in pioglitazone mediated ulcer healing along with PPAR-gamma. Administration of pioglitazone reverted back the decreased levels of both PPAR-gamma and glucocorticoid receptor, resulting in their redistribution to the nucleus from the cytosol in course of ulcer healing. Moreover, pharmacological inhibition of glucocorticoid receptor function by its antagonist (RU486) inhibited pioglitazone mediated downregulation of TNF-alpha and IL-1beta gene expression confirming involvement of glucocorticoid receptor in pioglitazone mediated ulcer healing. Co-immunoprecipitation studies further established association of PPAR-gamma with glucocorticoid receptor during ulcer healing which was enhanced following pioglitazone administration. Thus, the present study is first of its kind bearing direct relevance to the participation of both PPAR-gamma and glucocorticoid receptor and their physical association in influencing amelioration of inflammatory responses during pioglitazone mediated gastric ulcer healing.
An α-helical motif containing the sequence LXXLL is required for the ligand-dependent binding of transcriptional co-activators to nuclear receptors. By using a peptide
inhibition assay, we have defined the minimal “core” LXXLL motif as an 8-amino acid sequence spanning positions −2 to +6 relative to the primary conserved leucine residue. In yeast
two-hybrid assays, core LXXLL motif sequences derived from steroid receptor co-activator (SRC1), the 140-kDa receptor interacting protein (RIP140), and
CREB-binding protein (CBP) displayed differences in selectivity and affinity for nuclear receptor ligand binding domains.
Although core LXXLL motifs from SRC1 and RIP140 mediated strong interactions with steroid and retinoid receptors, three LXXLL motifs present in the global co-activator CBP were found to have very weak affinity for these proteins. Core motifs with
high affinity for steroid and retinoid receptors were generally found to contain a hydrophobic residue at position −1 relative
to the first conserved leucine and a nonhydrophobic residue at position +2. Our results indicate that variant residues in
LXXLL core motifs influence the affinity and selectivity of co-activators for nuclear receptors.
This review summarises the correlations between testosterone levels and male physical appearance and behaviour. Methodological shortcomings concerning the measurement of testosterone could limit the value of these findings. In addition, testosterone measured in body fluids represents only one step in the cascade of action from production to biological effect, and could therefore provide only a limited view of the complexity of physiological events. Testosterone levels are influenced by conditions that are partly controlled or initiated by the hormone itself, but also by circumstances beyond hormonal or individual control. Different kinds of behaviour are not only subject to influence by environment, but also androgens can reinforce the particular kind of conduct and the behavioural impact can wield negative or positive feedback on testosterone secretion. Therefore, both generalisation and individualisation of study results will lead to doubtful conclusions and prejudices. Results of such studies must be viewed with caution, and over-simplification as well as over-interpretation should be avoided.
Nuclear receptors (NRs) require coactivators to efficiently activate transcription of their target genes. Many coactivators including the p160 proteins utilize a short NR box motif to recognize the ligand-binding domain of the NR when it is activated by ligand. To investigate the ability of various ligands to specify the affinity of NR boxes for a ligand-bound NR, we compared the capacity of p160 NR boxes to be recruited to estrogen receptor (ERalpha) and ERbeta in the presence of 17beta-estradiol, diethylstilbestrol, and genestein. A time-resolved fluorescence-based binding assay was used to determine the dissociation constants for the 10 NR boxes derived from the three p160 coactivators for both ER subtypes in the presence of the each of the agonists. While the affinity of some NR boxes for ER was independent of the agonist, we identified several NR boxes that had significantly different affinities for ER depending on which agonist was bound to the receptor. Therefore, an agonist may specify the affinity of an NR for various NR boxes and thus regulate the coactivator selectivity of the receptor.
SHP (short heterodimer partner) is an orphan nuclear receptor, first described for its interaction with nuclear receptors. This study explores a new way of inhibiting the androgen-signaling pathway. We demonstrated that SHP inhibited up to 97% of AR-induced activity. Characterization of AR/SHP interaction provided evidence of a clear ligand dependency. We also showed that the LXXI/LL motifs previously found on SHP mediated the interaction with the AR ligand-binding domain (AR-LBD), the motif responsible for the interaction being slightly different from that found with ER. The AR N-terminal domain (AR-NTD), in contrast to that of other nuclear receptors, accounts for most of the entire receptor transactivation potential. SHP also interacted with AR-NTD, thus stabilizing the interaction with AR. We demonstrated that SHP inhibited both AR-LBD and NTD-dependent transactivation, which evidenced for the first time a protein capable of inhibiting a steroid receptor amino-terminal-dependent transactivation. We further characterized the SHP mechanism of action by showing that SHP reversed AR coactivator-mediated activation. Conversely, FHL2 and TIF2 counteracted SHP-mediated inhibition of AR. SHP evidences a new way of inhibiting AR activity by competing with AR coactivators. This new type of inhibitor could dictate the activity of nuclear receptors, depending on the equilibrium between activators and inhibitors.
The androgen receptor (AR) activation function 2 region of the ligand binding domain binds the LXXLL motifs of p160 coactivators weakly, engaging instead in an androgen-dependent, interdomain interaction with an FXXLF motif in the AR NH2 terminus. Here we show that FXXLF motifs are present in previously reported AR coactivators ARA70/RFG, ARA55/Hic-5, and ARA54, which account for their selection
in yeast two-hybrid screens. Mammalian two-hybrid assays, ligand dissociation rate studies, and glutathioneS-transferase adsorption assays indicate androgen-dependent selective interactions of these FXXLF motifs with the AR ligand binding domain. Mutagenesis of residues within activation function 2 indicates distinct but overlapping
binding sites where specificity depends on sequences within and flanking the FXXLF motif. Mutagenesis of the FXXLF motifs eliminated interaction with the ligand binding domain but only modestly reduced AR coactivation in transcription
assays. The studies indicate that the FXXLF binding motif is specific for the AR and mediates interactions both within the AR and with coregulatory proteins.
The biological action of androgens is mediated through the androgen receptor (AR). Androgen-bound AR functions as a transcription factor to regulate genes involved in an array of physiological processes, most notably male sexual differentiation and maturation, and the maintenance of spermatogenesis. The transcriptional activity of AR is affected by coregulators that influence a number of functional properties of AR, including ligand selectivity and DNA binding capacity. As the promoter of target genes, coregulators participate in DNA modification, either directly through modification of histones or indirectly by the recruitment of chromatin-modifying complexes, as well as functioning in the recruitment of the basal transcriptional machinery. Aberrant coregulator activity due to mutation or altered expression levels may be a contributing factor in the progression of diseases related to AR activity, such as prostate cancer. AR demonstrates distinct differences in its interaction with coregulators from other steroid receptors due to differences in the functional interaction between AR domains, possibly resulting in alterations in the dynamic interactions between coregulator complexes.
Nuclear receptors (NRs) act as ligand‐inducible transcription factors which regulate the expression of target genes upon binding to cognate response elements. The ligand‐dependent activity of the NR activation function AF‐2 is believed to be mediated to the transcription machinery through transcriptional mediators/intermediary factors (TIFs). We report here the cloning of the 160 kDa human nuclear protein TIF2, which exhibits all properties expected for a mediator of AF‐2: (i) it interacts in vivo with NRs in an agonist‐dependent manner; (ii) it binds directly to the ligand‐binding domains (LBDs) of NRs in an agonist‐ and AF‐2‐integrity‐dependent manner in vitro; (iii) it harbours an autonomous transcriptional activation function; (iv) it relieves nuclear receptor autosquelching; and (v) it enhances the activity of some nuclear receptor AF‐2s when overexpressed in mammalian cells. TIF2 exhibits partial sequence homology with the recently isolated steroid receptor coactivator SRC‐1, indicating the existence of a novel gene family of nuclear receptor transcriptional mediators.
Oestrogens are involved in the growth, development and homeostasis of a number of tissues1. The physiological effects of these steroids are mediated by a ligand-inducible nuclear transcription factor, the oestrogen receptor (ER)2. Hormone binding to the ligand-binding domain (LBD) of the ER initiates a series of molecular events culminating in the activation or repression of target genes. Transcriptional regulation arises from the direct interaction of the ER with components of the cellular transcription machinery3,4. Here we report the crystal structures of the LBD of ER in complex with the endogenous oestrogen, 17-oestradiol, and the selective antagonist raloxifene5, at resolutions of 3.1 and 2.6 Å, respectively. The structures provide a molecular basis for the distinctive pharmacophore of the ER and its catholic binding properties. Agonist and antagonist bind at the same site within the core of the LBD but demonstrate different binding modes. In addition, each class of ligand induces a distinct conformation in the transactivation domain of the LBD, providing structural evidence of the mechanism of antagonism.
Ligand-dependent activation of gene transcription by nuclear receptors is dependent on the recruitment of coactivators, including a family of related NCoA/SRC factors, via a region containing three helical domains sharing an LXXLL core consensus sequence, referred to as LXDs. In this manuscript, we report receptor-specific differential utilization of LXXLL-containing motifs of the NCoA-1/SRC-1 coactivator. Whereas a single LXD is sufficient for activation by the estrogen receptor, different combinations of two, appropriately spaced, LXDs are required for actions of the thyroid hormone, retinoic acid, peroxisome proliferator-activated, or progesterone receptors. The specificity of LXD usage in the cell appears to be dictated, at least in part, by specific amino acids carboxy-terminal to the core LXXLL motif that may make differential contacts with helices 1 and 3 (or 3′) in receptor ligand-binding domains. Intriguingly, distinct carboxy-terminal amino acids are required for PPARγ activation in response to different ligands. Related LXXLL-containing motifs in NCoA-1/SRC-1 are also required for a functional interaction with CBP, potentially interacting with a hydrophobic binding pocket. Together, these data suggest that the LXXLL-containing motifs have evolved to serve overlapping roles that are likely to permit both receptor-specific and ligand-specific assembly of a coactivator complex, and that these recognition motifs underlie the recruitment of coactivator complexes required for nuclear receptor function.
We have previously reported the presence of a hormone-inducible transcriptional activation function (TAF-2) within the region of the estrogen receptor (ER) that contains the hormone binding domain. We show here that the N-terminal A/B region of the ER contains an independent constitutive activation function (TAF-1) that exhibits cell type specificity since it activates transcription efficiently in chicken embryo fibroblasts, but only poorly in HeLa cells. By analyzing the ability of TAF-1, TAF-2, and the GAL4 and VP16 acidic activating domains (AADs) to homosynergize and heterosynergize with one another and with the factor binding to the upstream element (UE) of the adenovirus 2 major late promoter, we show that the activation properties of TAF-1 and TAF-2 are different and distinct from those of AADs, in agreement with the absence of acidic amino acid stretches in TAF-1 and TAF-2.
We present the DNA sequence of a 914-base pair fragment from Saccharomyces cerevisiae that contains the GAL1-GAL10 divergent
promoter, 140 base pairs of GAL10 coding sequence, and 87 base pairs of GAL1 coding sequence. From this fragment, we constructed
four pairs of GAL1-lacZ and GAL10-lacZ fusions on various types of yeast plasmid vectors. On each type of vector, the fused
genes were induced by galactose and repressed by glucose. The response of a GAL1-lacZ fusion to gal4 and gal80 regulatory
mutations was similar to the response of intact chromosomal GAL1 and GAL10 genes. A set of deletions that removed various
portions of the GAL10 regulatory sequences from a GAL10-CYC1-lacZ fusion was constructed in vitro. These deletions defined
a relatively guanine-cytosine-rich region of 45 base pairs that contained sequences necessary for full-strength galactose
induction and an adjacent guanine-cytosine rich 55 base pairs that contained sequences sufficient for weak induction.
The ability of nuclear receptors (NRs) to activate transcription of target genes requires the binding of cognate ligands to their ligand-binding domains (LBDs). Information provided by the three-dimensional structures of the unliganded RXR alpha and the liganded RAR gamma LBDs has been incorporated into a general alignment of the LBDs of all NRs. A twenty amino-acid region constitutes a NR-specific signature and contains most of the conserved residues that stabilize the core of the canonical fold of NR LBDs. A common ligand-binding pocket, involving predominantly hydrophobic residues, is inferred by homology modelling of the human RXR alpha and glucocorticoid receptor ligand-binding sites according to the RAR gamma holo-LBD structure. Mutant studies support these models, as well as a general mechanism for ligand-induced activation deduced from the comparison of the transcriptionally active RAR gamma holo- and inactive RXR alpha apo-LBD structures.
Nuclear receptors (NRs) act as ligand-inducible transcription factors which regulate the expression of target genes upon binding to cognate response elements. The ligand-dependent activity of the NR activation function AF-2 is believed to be mediated to the transcription machinery through transcriptional mediators/intermediary factors (TIFs). We report here the cloning of the 160 kDa human nuclear protein TIF2, which exhibits all properties expected for a mediator of AF-2: (i) it interacts in vivo with NRs in an agonist-dependent manner; (ii) it binds directly to the ligand-binding domains (LBDs) of NRs in an agonist- and AF-2-integrity-dependent manner in vitro; (iii) it harbours an autonomous transcriptional activation function; (iv) it relieves nuclear receptor autosquelching; and (v) it enhances the activity of some nuclear receptor AF-2s when overexpressed in mammalian cells. TIF2 exhibits partial sequence homology with the recently isolated steroid receptor coactivator SRC-1, indicating the existence of a novel gene family of nuclear receptor transcriptional mediators.
The estrogen receptor (ER) is a ligand-dependent transcription factor that regulates expression of target genes in response to estrogen in concert with other cellular signaling pathways. This suggests that the mechanism by which ER transmits an activating signal to the general transcription machinery may include factors that integrate these diverse signals. We have previously characterized the estrogen receptor-associated protein, ERAP160, as a factor that complexes with ER in an agonist-dependent manner. We have now found that the transcriptional coactivator p300 associates with agonist bound ER and augments ligand-dependent activation by ER. Our studies show that an ER coactivator complex involves a direct hormone-dependent interaction between ER and ERAP160, resulting in the recruitment of p300. In addition, antibodies directed against the cloned steroid receptor coactivator 1 (SRC1) recognize ERAP160. The known role of p300 in multiple signal transduction pathways, including those involving the second messenger cAMP, suggests p300 functions as a point of integration between ER and these other pathways.
Nuclear receptors (NRs) are ligand-inducible transcription factors that mediate complex effects on development, differentiation and homeostasis. They regulate the transcription of their target genes through binding to cognate DNA sequences as homodimers or heterodimers. The molecular mechanisms underlying transcriptional activation by NRs are still poorly understood, although intermediary factors (mediators) appear to be involved in mediating the transactivation functions of NRs. TIF1 has been identified previously as a protein that interacts specifically with the ligand binding domain of several nuclear receptors, both in yeast and in vitro. The characteristics of these interactions have led us to suggest that TIF1 might be a mediator of the NR ligand-inducible activation function AF-2. Using a two-hybrid screening in yeast, we have now identified two TIF1-binding proteins, mHP1 alpha and mMOD1, that are mouse homologues of the Drosophila heterochromatinic protein 1. Using mHP1 alpha as a bait in a second two-hybrid screening, we have isolated cDNAs encoding proteins that are also very likely to be involved in chromatin structure and function, as well as a protein structurally and functionally related to TIF1 (renamed TIF1 alpha), which was named TIF1 beta. Here we discuss how the function of members of the TIF1 family in the control of transcription could be exerted at the level of the structure of the chromatin template.
The binding of lipophilic hormones, retinoids and vitamins to members of the nuclear-receptor superfamily modifies the DNA-binding and transcriptional properties of these receptors, resulting in the activation or repression of target genes. Ligand binding induces conformational changes in nuclear receptors and promotes their association with a diverse group of nuclear proteins, including SRC-1/p160, TIF-2/GRIP-1 and CBP/p300 which function as co-activators of transcription, and RIP-140, TIF-1 and TRIP-1/SUG-1 whose functions are unclear. Here we report that a short sequence motif LXXLL (where L is leucine and X is any amino acid) present in RIP-140, SRC-1 and CBP is necessary and sufficient to mediate the binding of these proteins to liganded nuclear receptors. We show that the ability of SRC-1 to bind the oestrogen receptor and enhance its transcriptional activity is dependent upon the integrity of the LXXLL motifs and on key hydrophobic residues in a conserved helix (helix 12) of the oestrogen receptor that are required for its ligand-induced activation function. We propose that the LXXLL motif is a signature sequence that facilitates the interaction of different proteins with nuclear receptors, and is thus a defining feature of a new family of nuclear proteins.
Steroid receptor co-activator (SRC1) is one of a number of transcriptional co-activators that are capable of potentiating the activity of nuclear receptors including the oestrogen receptor (ER). Here we report that two isoforms, SRC1a and SRC1e, which diverge at their C-termini, are functionally distinct as they differ in their abilities to enhance the activity of the ER in intact cells. SRC1e enhanced the ability of the ER to stimulate transcription to a greater extent than SRC1a, which had negligible effects on certain promoters. To elucidate the basis of this functional difference, we compared the nuclear receptor-binding properties and mapped the transcriptional activation domains in the two SRC1 isoforms. Both isoforms share a triplet of nuclear receptor-binding motifs (LXXLL motifs) for binding to functional ER dimers, and an activation domain which co-localizes with the CBP-binding domain, while SRC1a contains a unique LXXLL motif in its C-terminus. Although this LXXLL motif increases the affinity for the ER in vitro, it does not appear to be responsible for the functional difference between the two isoforms. This difference is due to a second activation domain that is CBP independent and is suppressed in the SRC1a isoform. Thus, SRC1 exists as functionally distinct isoforms which are likely to play different roles in ER-mediated transcription.
The nuclear receptor (NR) coactivator TIF2 possesses a single NR interaction domain (NID) and two autonomous activation domains, AD1 and AD2. The TIF2 NID is composed of three NR-interacting modules each containing the NR box motif LxxLL. Mutation of boxes I, II and III abrogates TIF2-NR interaction and stimulation, in transfected cells, of the ligand-induced activation function-2 (AF-2) present in the ligand-binding domains (LBDs) of several NRs. The presence of an intact NR interaction module II in the NID is sufficient for both efficient interaction with NR holo-LBDs and stimulation of AF-2 activity. Modules I and III are poorly efficient on their own, but synergistically can promote interaction with NR holo-LBDs and AF-2 stimulation. TIF2 AD1 activity appears to be mediated through CBP, as AD1 could not be separated mutationally from the CBP interaction domain. In contrast, TIF2 AD2 activity apparently does not involve interaction with CBP. TIF2 exhibited the characteristics expected for a bona fide NR coactivator, in both mammalian and yeast cells. Moreover, in mammalian cells, a peptide encompassing the TIF2 NID inhibited the ligand-induced AF-2 activity of several NRs, indicating that NR AF-2 activity is either mediated by endogenous TIF2 or by coactivators recognizing a similar surface on NR holo-LBDs.
The activity of the AF-2 transcriptional activation function of nuclear receptors (NR) is mediated by the partially homologous transcriptional coactivators, glucocorticoid receptor interacting protein 1 (GRIP1)/transcriptional intermediary factor 2 (TIF2) and steroid receptor coactivator 1 (SRC-1). GRIP1 and SRC-1 bound nine different NRs and exhibited similar, but not identical, NR binding preferences. The most striking difference was seen with the androgen receptor, which bound well to GRIP1 but poorly to SRC-1. GRIP1 and SRC-1 contain three copies of the NR binding motif LXXLL (called an NR Box) in their central regions. Mutation of both NR Box II and NR Box III in GRIP1 almost completely eliminated functional and binding interactions with NRs, indicating that these two sites are crucial for most of GRIP1's NR binding activity. Interactions of GRIP1 with the estrogen receptor were more strongly affected by mutations in NR Box II, whereas interactions with the androgen receptor and glucocorticoid receptor were more strongly affected by NR Box III mutations. One isoform of SRC-1 has an additional NR Box (NR Box IV) at its extreme C terminus with an NR-binding preference somewhat different from that of the central NR-binding domain of SRC-1. GRIP1 has no NR Box in its C-terminal region and therefore no C-terminal NR-binding function. In summary, GRIP1 and SRC-1 have overlapping NR-binding preferences, but specific NRs display both coactivator and NR Box preferences that may contribute to the specificity of hormonal responses.
Nuclear receptors transduce hormonal signals by binding directly to DNA target sites in promoters and modulating the transcription of linked genes. Receptor-mediated transactivation appears to be potentiated in response to ligand by a number of coactivators that may provide key interactions with components of the transcription preinitiation complex and/or alter chromatin structure. Here, we use the vitamin D3 receptor ligand-binding domain (VDR LBD) as an affinity matrix to identify components of a transcriptionally active nuclear extract that interact with VDR in response to ligand. We describe the purification of a complex of at least 10 VDR interacting proteins (DRIPs) ranging from 65 to 250 kD that associate with the receptor in a strictly 1,25-dihydroxyvitamin D3-dependent manner. These proteins also appear to interact with other, but not all, nuclear receptors, such as the thyroid hormone receptor. The DRIPs are distinct from known nuclear receptor coactivators, although like these coactivators, their interaction also requires the AF-2 transactivation motif of VDR. In addition, the DRIP complex contains histone acetyltransferase activity, indicating that at least one or more of the DRIPs may function at the level of nucleosomal modification. However, we show that the DRIPs selectively enhance the transcriptional activity of VDR on a naked DNA template utilizing a cell-free, ligand-dependent transcription assay. Moreover, this activity can be specifically depleted from the extract by liganded, but not unliganded, VDR-LBD. Overexpression of DRIP100 in vivo resulted in a strong squelching of VDR transactivation, suggesting the sequestration of other limiting factors, including components of the DRIP complex. These results demonstrate the existence of a new complex of novel functional nuclear receptor coactivators.
The peroxisome proliferator-activated receptor-gamma (PPAR-gamma) is a ligand-dependent transcription factor that is important in adipocyte differentiation and glucose homeostasis and which depends on interactions with co-activators, including steroid receptor co-activating factor-1 (SRC-1). Here we present the X-ray crystal structure of the human apo-PPAR-gamma ligand-binding domain (LBD), at 2.2 A resolution; this structure reveals a large binding pocket, which may explain the diversity of ligands for PPAR-gamma. We also describe the ternary complex containing the PPAR-gamma LBD, the antidiabetic ligand rosiglitazone (BRL49653), and 88 amino acids of human SRC-1 at 2.3 A resolution. Glutamate and lysine residues that are highly conserved in LBDs of nuclear receptors form a 'charge clamp' that contacts backbone atoms of the LXXLL helices of SRC-1. These results, together with the observation that two consecutive LXXLL motifs of SRC-1 make identical contacts with both subunits of a PPAR-gamma homodimer, suggest a general mechanism for the assembly of nuclear receptors with co-activators.
Combinatorial regulation of transcription implies flexible yet precise assembly of multiprotein regulatory complexes in response to signals. Biochemical and crystallographic analyses revealed that hormone binding leads to the formation of a hydrophobic groove within the ligand binding domain (LBD) of the thyroid hormone receptor that interacts with an LxxLL motif-containing alpha-helix from GRIP1, a coactivator. Residues immediately adjacent to the motif modulate the affinity of the interaction; the motif and the adjacent sequences are employed to different extents in binding to different receptors. Such interactions of amphipathic alpha-helices with hydrophobic grooves define protein interfaces in other regulatory complexes as well. We suggest that these common structural elements impart flexibility to combinatorial regulation, whereas side chains at the interface impart specificity.
Nuclear receptors modulate the transcription of genes in direct response to small lipophilic ligands. Binding to ligands induces conformational changes in the nuclear receptors that enable the receptors to interact with several types of cofactor that are critical for transcription activation (transactivation). We previously described a distinct set of ligand-dependent proteins called DRIPs, which interact with the vitamin D receptor (VDR); together, these proteins constitute a new cofactor complex. DRIPs bind to several nuclear receptors and mediate ligand-dependent enhancement of transcription by VDR and the thyroid-hormone receptor in cell-free transcription assays. Here we report the identities of thirteen DRIPs that constitute this complex, and show that the complex has a central function in hormone-dependent transactivation by VDR on chromatin templates. The DRIPs are almost indistinguishable from components of another new cofactor complex called ARC, which is recruited by other types of transcription activators to mediate transactivation on chromatin-assembled templates. Several DRIP/ARC subunits are also components of other potentially related cofactors, such as CRSP, NAT, SMCC and the mouse Mediator, indicating that unique classes of activators may share common sets or subsets of cofactors. The role of nuclear-receptor ligands may, in part, be to recruit such a cofactor complex to the receptor and, in doing so, to enhance transcription of target genes.
The functionally conserved proteins CBP and p300 act in conjunction with other factors to activate transcription of DNA. A new factor, p/CIP, has been discovered that is present in the cell as a complex with CBP and is required for transcriptional activity of nuclear receptors and other CBP/p300-dependent transcription factors. The highly related nuclear-receptor co-activator protein NCoA-1 is also specifically required for ligand-dependent activation of genes by nuclear receptors. p/CIP, NCoA-1 and CBP all contain related leucine-rich charged helical interaction motifs that are required for receptor-specific mechanisms of gene activation, and allow the selective inhibition of distinct signal-transduction pathways.
The oestrogen receptor stimulates transcription by means of at least two distinct transcriptional activation domains, TAF-1 in the N-terminal domain and TAF-2 in the hormone binding domain. Here we show that TAF-2 activity requires a region in the C-terminus of the hormone binding domain between residues 538 and 552 in the mouse oestrogen receptor which is conserved among many nuclear hormone receptors. Point mutagenesis of conserved hydrophobic and charged residues significantly reduced ligand dependent transcriptional activation but had no effect on steroid or DNA binding. Mutation of the corresponding residues in the glucocorticoid receptor also abolished transcriptional activation. We therefore propose that the conserved region may be essential for ligand dependent transcriptional activation by other members of the nuclear receptor family.
A rat brain cDNA library has been constructed in a Saccharomyces cerevisiae expression vector and used to isolate genes that can function in yeast to suppress the phenotypic effects of RAS2val19, a mutant form of the RAS2 gene analogous to an oncogenic mutant of the human HRAS gene. One cDNA, DPD, was cloned and its genetic and biochemical properties were characterized. A DPD product would share 80% amino acid sequence identity with the Drosophila melanogaster dunce-encoded protein over an extended region. We have shown that the DPD protein is a high-affinity cAMP-specific phosphodiesterase.
A series of yeast shuttle vectors and host strains has been created to allow more efficient manipulation of DNA in Saccharomyces cerevisiae. Transplacement vectors were constructed and used to derive yeast strains containing nonreverting his3, trp1, leu2 and ura3 mutations. A set of YCp and YIp vectors (pRS series) was then made based on the backbone of the multipurpose plasmid pBLUESCRIPT. These pRS vectors are all uniform in structure and differ only in the yeast selectable marker gene used (HIS3, TRP1, LEU2 and URA3). They possess all of the attributes of pBLUESCRIPT and several yeast-specific features as well. Using a pRS vector, one can perform most standard DNA manipulations in the same plasmid that is introduced into yeast.
We have identified two discrete transactivation domains within the mouse oestrogen receptor whose relative activities vary
according to the target promoter. One domain lies within the N-terminal region and is active in the absence of oestradiol.
The second domain is contained within the C-terminal portion of the protein and depends upon oestrogen binding for its activity.
The location and oestrogen dependence of this domain has been confirmed using chimaeric receptors containing the Lex A DNA
binding domain. Although transactivation by the C-terminal domain is dependent upon ligand binding the analysis of receptor
deletion mutants has demonstrated that these two functions are not entirely coincident.
A yeast two-hybrid system was used to identify a protein that interacts with and enhances the human progesterone receptor (hPR) transcriptional activity without altering the basal activity of the promoter. Because the protein stimulated transactivation of all the steroid receptors tested, it has been termed steroid receptor coactivator-1 (SRC-1). Coexpression of SRC-1 reversed the ability of the estrogen receptor to squelch activation by hPR. Also, the amino terminal truncated form of SRC-1 acted as a dominant-negative repressor. Together, these results indicate that SRC-1 encodes a coactivator that is required for full transcriptional activity of the steroid receptor superfamily.
The ligand binding domain of the estrogen receptor contains a hormone-dependent transcriptional activation function. To investigate the mechanism by which it stimulates transcription, we have expressed fusion proteins containing either the wild-type or a transcriptionally defective form of this domain fused to glutathione-S-transferase and searched for proteins that specifically interact in vitro. By far-Western blotting, three proteins of 160, 140, and 80 kDa expressed in different mammalian cells (HeLa, ZR75-1, and COS-1) were shown to associate directly with the wild-type receptor in the presence of estrogen. Two additional proteins appeared to interact indirectly with the hormone binding domain since they were detected only by a pull-down assay. All of these interactions were abolished by antiestrogens, such as 4-hydroxytamoxifen, ICI 164384, or ICI 182780, which inhibit hormone-dependent transcription. Moreover, they were not observed with the transcriptionally defective form of the receptor even in the presence of estrogen. Thus, since the ability of these proteins to interact with the hormone binding domain correlates with its transcriptional activity, one or more of them may contribute to hormone-dependent transcriptional activation by the estrogen receptor.
The estrogen receptor is a transcription factor which, when bound to estradiol, binds DNA and regulates expression of estrogen-responsive
genes. A 160-kilodalton estrogen receptor-associated protein, ERAP160, was identified that exhibits estradiol-dependent binding
to the receptor. Mutational analysis of the receptor shows that its ability to activate transcription parallels its ability
to bind ERAP160. Antiestrogens are unable to promote ERAP160 binding and can block the estrogen-dependent interaction of the
receptor and ERAP160 in a dose-dependent manner. This evidence suggests that ERAP160 may mediate estradiol-dependent transcriptional
activation by the estrogen receptor. Furthermore, the ability of antiestrogens to block estrogen receptor-ERAP160 complex
formation could account for their therapeutic effects in breast cancer.
The ligand-binding domain of nuclear receptors appears to contain a common fold that generates a conserved ligand-binding pocket. Their transcriptional activity is induced by ligand through realignment of a C-terminal helix to form a novel interacting surface.
Nuclear receptors regulate gene expression by direct activation of target genes and inhibition of AP-1. Here we report that, unexpectedly, activation by nuclear receptors requires the actions of CREB-binding protein (CBP) and that inhibition of AP-1 activity is the apparent result of competition for limiting amounts of CBP/p300 in cells. Utilizing distinct domains, CBP directly interacts with the ligand-binding domain of multiple nuclear receptors and with the p160 nuclear receptor coactivators, which upon cloning have proven to be variants of the SRC-1 protein. Because CBP represents a common factor, required in addition to distinct coactivators for function of nuclear receptors, CREB, and AP-1, we suggest that CBP/p300 serves as an integrator of multiple signal transduction pathways within the nucleus.
Transcriptional regulation by nuclear hormone receptors is thought to involve interactions with putative cofactors that may potentiate receptor function. Here we show that human thyroid hormone receptor alpha purified from HeLa cells grown in the presence of thyroid hormone (T3) is associated with a group of distinct nuclear proteins termed thyroid hormone receptor-associated proteins (TRAPs). In an in vitro system reconstituted with general initiation factors and cofactors (and in the absence of added T3), the "liganded" thyroid hormone receptor (TR)/TRAP complex markedly activates transcription from a promoter template containing T3-response elements. Moreover, whereas the retinoid X receptor is not detected in the TR/TRAP complex, its presence is required for the function of the complex. In contrast, human thyroid hormone receptor alpha purified from cells grown in the absence of T3 lacks the TRAPs and effects only a low level of activation that is dependent on added ligand. These findings demonstrate the ligand-dependent in vivo formation of a transcriptionally active TR-multisubunit protein complex and suggest a role for TRAPs as positive coactivators for gene-specific transcriptional activation.
The nuclear receptor superfamily includes receptors for steroids, retinoids, thyroid hormone and vitamin D, as well as many related proteins. An important feature of the action of the lipophilic hormones and vitamins is that the maintenance of homeostatic function requires both intrinsic positive and negative regulation. Here we provide in vitro and in vivo evidence that identifies the CREB-binding protein (CBP) and its homologue P300 (refs 6,7) as cofactors mediating nuclear-receptor-activated gene transcription. The role of CBP/P300 in the transcriptional response to cyclic AMP, phorbol esters, serum, the lipophilic hormones and as the target of the E1A oncoprotein suggests they may serve as integrators of extracellular and intracellular signalling pathways.
Retinoic acid, steroid and thyroid hormones regulate complex programs of gene expression by binding to intracellular receptors that are members of the nuclear receptor superfamily of ligand-dependent transcription factors. Recent studies have led to the identification and cloning of genes encoding coactivator molecules that appear to play important roles in mediating ligand-dependent transcription by members of this family. The identification of these coactivator molecules suggests a point of entry into the general transcriptional machinery that is common to several other classes of regulated transcription factors.
After binding to enhancer elements, transcription factors require transcriptional coactivator proteins to mediate their stimulation of transcription initiation. A search for possible coactivators for steroid hormone receptors resulted in identification of glucocorticoid receptor interacting protein 1 (GRIP1). The complete coding sequence for GRIP1, isolated from a mouse brain cDNA library, contains an open reading frame of 1,462 codons. GRIP1 is the probable ortholog of the subsequently identified human protein transcription intermediary factor 2 (TIF2) and is also partially homologous to steroid receptor coactivator 1 (SRC-1). The full-length GRIP1 interacted with the hormone binding domains (HBDs) of all five steroid receptors in a hormone-dependent manner and also with HBDs of class II nuclear receptors, including thyroid receptor alpha, vitamin D receptor, retinoic acid receptor alpha, and retinoid X receptor alpha. In contrast to agonists, glucocorticoid antagonists did not promote interaction between the glucocorticoid receptor and GRIP1. In yeast cells, GRIP1 dramatically enhanced the transcriptional activation function of proteins containing the HBDs of any of the above-named receptors fused to the GAL4 DNA binding domain and thus served as a transcriptional coactivator for them. This finding contrasts with previous reports of TIF2 and SRC-1, which in mammalian cells enhanced the transactivation activities of only a subset of the steroid and nuclear receptors that they physically interacted with. GRIP1 also enhanced the hormone-dependent transactivation activity of intact glucocorticoid receptor, estrogen receptor, and mineralocorticoid receptor. Experiments with glucocorticoid receptor truncation and point mutants indicated that GRIP1 interacted with and enhanced the activity of the C-terminal AF-2 but not the N-terminal AF-1 transactivation domain of the glucocorticoid receptor. These results demonstrate directly that AF-1 and AF-2 domains accomplish their transactivation activities through different mechanisms: AF-2 requires GRIP1 as a coactivator, but AF-1 does not.
Members of the recently recognized SRC-1 family of transcriptional coactivators interact with steroid hormone receptors to
enhance ligand-dependent transcription. AIB1, a member of the SRC-1 family, was cloned during a search on the long arm of
chromosome 20 for genes whose expression and copy number were elevated in human breast cancers. AIB1 amplification and overexpression
were observed in four of five estrogen receptor–positive breast and ovarian cancer cell lines. Subsequent evaluation of 105
unselected specimens of primary breast cancer found AIB1 amplification in approximately 10 percent and high expression in
64 percent of the primary tumors analyzed. AIB1 protein interacted with estrogen receptors in a ligand-dependent fashion,
and transfection of AIB1 resulted in enhancement of estrogen-dependent transcription. These observations identify AIB1 as
a nuclear receptor coactivator whose altered expression may contribute to development of steroid-dependent cancers.
We report here the identification of a novel cofactor, ACTR, that directly binds nuclear receptors and stimulates their transcriptional activities in a hormone-dependent fashion. ACTR also recruits two other nuclear factors, CBP and P/CAF, and thus plays a central role in creating a multisubunit coactivator complex. In addition, and unexpectedly, we show that purified ACTR is a potent histone acetyltransferase and appears to define a distinct evolutionary branch to this recently described family. Thus, hormonal activation by nuclear receptors involves the mutual recruitment of at least three classes of histone acetyltransferases that may act cooperatively as an enzymatic unit to reverse the effects of histone deacetylase shown to be part of the nuclear receptor corepressor complex.
The ligand-binding domain of nuclear receptors contains a transcriptional activation function (AF-2) that mediates hormone-dependent
binding of coactivator proteins. Scanning surface mutagenesis on the human thyroid hormone receptor was performed to define
the site that binds the coactivators, glucocorticoid receptor–interacting protein 1 (GRIP1) and steroid receptor coactivator
1 (SRC-1). The residues involved encircle a small surface that contains a hydrophobic cleft. Ligand activation of transcription
involves formation of this surface by folding the carboxyl-terminal α helix against a scaffold of three other helices. These
features may represent general ones for nuclear receptors.
Ligand-dependent activation of transcription by nuclear receptors (NRs) is mediated by interactions with coactivators. Receptor agonists promote coactivator binding, and antagonists block coactivator binding. Here we report the crystal structure of the human estrogen receptor alpha (hER alpha) ligand-binding domain (LBD) bound to both the agonist diethylstilbestrol (DES) and a peptide derived from the NR box II region of the coactivator GRIP1 and the crystal structure of the hER alpha LBD bound to the selective antagonist 4-hydroxytamoxifen (OHT). In the DES-LBD-peptide complex, the peptide binds as a short alpha helix to a hydrophobic groove on the surface of the LBD. In the OHT-LBD complex, helix 12 occludes the coactivator recognition groove by mimicking the interactions of the NR box peptide with the LBD. These structures reveal the two distinct mechanisms by which structural features of OHT promote this "autoinhibitory" helix 12 conformation.
Coactivators previously implicated in ligand-dependent activation functions by thyroid hormone receptor (TR) include p300 and CREB-binding protein (CBP), the steroid receptor coactivator-1 (SRC-1)-related family of proteins, and the multicomponent TR-associated protein (TRAP) complex. Here we show that two positive cofactors (PC2 and PC4) derived from the upstream stimulatory activity (USA) cofactor fraction act synergistically to mediate thyroid hormone (T3)-dependent activation either by TR or by a TR-TRAP complex in an in vitro system reconstituted with purified factors and DNA templates. Significantly, the TRAP-mediated enhancement of activation by TR does not require the TATA box-binding protein-associated factors of TFIID. Furthermore, neither the pleiotropic coactivators CBP and p300 nor members of the SRC-1 family were detected in either the TR-TRAP complex or the other components of the in vitro assay system. These results show that activation by TR at the level of naked DNA templates is enhanced by cooperative functions of the TRAP coactivators and the general coactivators PC2 and PC4, and they further indicate a potential functional redundancy between TRAPs and TATA box-binding protein-associated factors in TFIID. In conjunction with earlier studies on other nuclear receptor-interacting cofactors, the present study also suggests a multistep pathway, involving distinct sets of cofactors, for activation of hormone responsive genes.
The human thyroid hormone receptor-associated protein (TRAP) complex, an earlier described coactivator for nuclear receptors, and an SRB- and MED-containing cofactor complex (SMCC) that mediates activation by Gal4-p53 are shown to be virtually the same with respect to specific polypeptide subunits, coactivator functions, and mechanisms of action (activator interactions). In parallel with ligand-dependent interactions of nuclear receptors with the TRAP220 subunit, p53 and VP16 activation domains interact directly with a newly cloned TRAP80 subunit. These results indicate novel pathways for the function of nuclear receptors and other activators (p53 and VP16) through a common coactivator complex that is likely to target RNA polymerase II. Identification of the TRAP230 subunit as a previously predicted gene product also suggests a coactivator-related transcription defect in certain disease states.
Transcriptional activation by the estrogen receptor is mediated through its interaction with coactivator proteins upon ligand
binding. By systematic mutagenesis, we have identified a group of conserved hydrophobic residues in the ligand binding domain
that are required for binding the p160 family of coactivators. Together with helix 12 and lysine 366 at the C-terminal end
of helix 3, they form a hydrophobic groove that accommodates an LXXLL motif, which is essential for mediating coactivator
binding to the receptor. Furthermore, we demonstrated that the high-affinity binding of motif 2, conserved in the p160 family,
is due to the presence of three basic residues N terminal to the core LXXLL motif. The recruitment of p160 coactivators to
the estrogen receptor is therefore likely to depend not only on the LXXLL motif making hydrophobic interactions with the docking
surface on the receptor, but also on adjacent basic residues, which may be involved in the recognition of charged residues
on the receptor to allow the initial docking of the motif.
Determinants of co-activator LXXLL motif speci®city in nuclear receptor transcriptional activation
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Mullen, A. Krones, J. Inostroza, J. Torchia, R.T. Nolte, N. Assa-Munt, M.V. Milburn, C.K. Glass, M.G. Rosenfeld, Determinants of co-activator LXXLL motif speci®city in nuclear receptor transcriptional activation, Genes Dev. 12 (1998) 3357±3368. M. Needham et al. / Journal of Steroid Biochemistry & Molecular Biology 72 (2000) 35±46
PPARa (aa 281±468) and PPARg (aa 176± 479) were also tested for their interaction with the SRC-1a motifs in the presence and absence of syn-Fig
- Finally
Finally, PPARa (aa 281±468) and PPARg (aa 176± 479) were also tested for their interaction with the SRC-1a motifs in the presence and absence of syn-Fig.
Sequence and characterization of a co-activator for the steroid hormone receptor superfamily
- Onate
Estrogen receptor-associated proteins — possible mediators of hormone-induced transcription
- Halachmi
The structural basis of estrogen receptor/co-activator recognition and the antagonism of this interaction by tamoxifen
- Shiau