Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

To better understand structure-activity relationships in the human estrogen receptor (ER), we examined the role of tyrosine 537 in the transcriptional response of the receptor, since this residue is close to a region of the hormone-binding domain shown previously to be important in hormone-dependent transcriptional activity and because this amino acid has been proposed to be a tyrosine kinase phosphorylation site important in the activity of the ER. We substituted five amino acids at this position (alanine, phenylalanine, glutamic acid, lysine, or serine) and screened these mutants for their biological activities in the presence and absence of estradiol. Two of the ER mutants, Y537A and Y537S, displayed estrogen-independent constitutive activity that was approximately 20% or 100%, respectively, of the activity of the wild type receptor with estradiol, when assessed in two different cell backgrounds using three different estrogen-responsive promoters. In some circumstances, the Y537E and Y537K proteins also exhibited some low level of constitutive activity. The constitutive activity of the mutants, as well as their activity in the presence of E2, was fully suppressed by antiestrogen. The extent of interaction of the constitutively active ERs with the steroid receptor coactivator-1 (SRC-1) closely parallel the magnitude of transcriptional activity of the receptor. Whereas wild type ER showed interaction with SRC-1 only in the presence of estrogen, Y537A and Y537S ER showed moderate or full interaction in the absence of ligand, an interaction that was blocked by antiestrogen, and the magnitude of interaction was increased to or remained at 100% upon estradiol treatment, implying that the ability of an ER to associate with SRC-1 is a good indicator of a transcriptionally active conformational state of the receptor. Our findings indicate that tyrosine 537 is in a region important in the ligand regulation of ER transcriptional activity and that the presence of certain amino acids at this position can shift ER into a conformation that is active even without ligand. However, tyrosine is not required at this site for estrogen binding or transcriptional response to estrogen in the systems investigated. Our findings, interpreted in light of the recently published x-ray crystal structure of the ligand-binding domains of three related receptors of the nuclear receptor superfamily, suggest that some of the amino acid substitutions introduced at position 537 may facilitate the shift of helix 12 of the ER into an active conformation and/or allow for differential stabilization of the receptor in its active form.
Mol Endocrinol 1996 Nov
PMID:Constitutively active human estrogen receptors containing amino acid substitutions for tyrosine 537 in the receptor protein. 892 65

Estrogen receptor beta (ER beta) is a novel steroid receptor that is expressed in rat prostate and ovary. We have cloned the mouse homolog of ER beta and mapped the gene, designated Estrb, to the central region of chromosome 12. The cDNA encodes a protein of 485 amino acids that shares, respectively, 97% and 60% identity with the DNA- and ligand-binding domains of mouse (m) ER alpha. Mouse ER beta bind to an inverted repeat spaced by three nucleotides in a gel mobility shift assay and transactivates promoters containing synthetic or natural estrogen response elements in an estradiol (E2)-dependent manner. Scatchard analysis indicates that mER beta has slightly lower affinity for E2 [dissociation constant (Kd) = 0.5 nM] when compared with mER alpha (Kd = 0.2 nM). Antiestrogens, including 4-hydroxytamoxifen (OHT), ICI 182,780, and a novel compound, EM-800, inhibit E2-dependent transactivation efficiently. However, while OHT displays partial agonistic activity with ER alpha on a basal promoter linked to estrogen response elements in Cos-1 cells, this effect is not observed with mER beta. Cotransfection of mER beta and H-RasV12 causes enhanced activation in the presence of E2. Mutagenesis of a serine residue (position 60), located within a mitogen-activated protein kinase consensus phosphorylation site abolishes the stimulatory effect of Ras, suggesting that the activity of mER beta is also regulated by the mitogen-activated protein kinase pathway. Surprisingly, the coactivator SRC-1 up-regulates mER beta transactivation both in the absence and presence of E2, and in vitro interaction between SRC-1 and the ER beta ligand-binding domain is enhanced by E2. Moreover, the ligand-independent stimulatory effect of SRC-1 on ER beta transcriptional activity is abolished by ICI 182,780, but not by OHT. Our results demonstrate that while ER beta shares many of the functional characteristics of ER alpha, the molecular mechanisms regulating the transcriptional activity of mER beta may be distinct from those of ER alpha.
Mol Endocrinol 1997 Mar
PMID:Cloning, chromosomal localization, and functional analysis of the murine estrogen receptor beta. 905 81

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.
Mol Cell Biol 1997 May
PMID:GRIP1, a transcriptional coactivator for the AF-2 transactivation domain of steroid, thyroid, retinoid, and vitamin D receptors. 911 44

Hormone-dependent transcriptional activation by nuclear receptors depends on the presence of a conserved C-terminal amphipathic alpha-helix (helix 12) in the ligand-binding domain. Here we show that a lysine residue, which is conserved in most nuclear receptors in the predicted helix 3, is also required for estrogen-dependent transactivation. The replacement of lysine 366 with alanine appreciably reduced activation function 2 (AF-2) activity without affecting steroid- or DNA-binding activity in the mouse estrogen receptor. The mutation dramatically reduced the ability of the receptor to bind steroid receptor coactivator 1 (SRC-1) but had no effect on receptor-interacting protein 140 (RIP-140) binding, indicating that while their sites of interaction overlap, they are not entirely consistent and in keeping with the proposal that the recruitment of coactivators, such as SRC-1, is required for AF-2 activity. Although the function of RIP-140 remains to be established, RIP-140 appears to be capable of recruiting the basal transcription machinery, since overexpression of the protein markedly increased the transcriptional activity of the mutant receptor. Since the lysine residue is conserved, we propose that it is required, together with residues in helix 12, to form the surface by which members of the nuclear receptor family interact with coactivators.
Mol Cell Biol 1997 Apr
PMID:AF-2 activity and recruitment of steroid receptor coactivator 1 to the estrogen receptor depend on a lysine residue conserved in nuclear receptors. 912 31

As the obligate member of most nuclear receptor heterodimers, retinoid X receptors (RXRs) can potentially perform two functions: cooperative binding to hormone response elements and coordinate regulation of target genes by RXR ligands. In this paper we describe allosteric interactions between RXR and two heterodimeric partners, retinoic acid receptors (RARs) and peroxisome proliferator-activated receptors (PPARs); RARs and PPARs prevent and permit activation by RXR-specific ligands, respectively. By competing for dimerization with RXR on response elements consisting of direct-repeat half-sites spaced by 1 bp (DR1 elements), the relative abundance of RAR and PPAR determines whether the RXR signaling pathway will be functional. In contrast to RAR, which prevents the binding of RXR ligands and recruits the nuclear receptor corepressor N-CoR, PPAR permits the binding of SRC-1 in response to both RXR and PPAR ligands. Overexpression of SRC-1 markedly potentiates ligand-dependent transcription by PPARgamma, suggesting that SRC-1 serves as a coactivator in vivo. Remarkably, the ability of RAR to both block the binding of ligands to RXR and interact with corepressors requires the CoR box, a structural motif residing in the N-terminal region of the RAR ligand binding domain. Mutations in the CoR box convert RAR from a nonpermissive to a permissive partner of RXR signaling on DR1 elements. We suggest that the differential recruitment of coactivators and corepressors by RAR-RXR and PPAR-RXR heterodimers provides the basis for a transcriptional switch that may be important in controlling complex programs of gene expression, such as adipocyte differentiation.
Mol Cell Biol 1997 Apr
PMID:Peroxisome proliferator-activated receptors and retinoic acid receptors differentially control the interactions of retinoid X receptor heterodimers with ligands, coactivators, and corepressors. 912 66

Mixed antiestrogens, such as 4-hydroxytamoxifen (4HT), act as either partial agonists or antagonists of estrogen receptor (ER) function in a tissue-, cell-, and promoter-specific manner, suggesting that intracellular factors modulate their ability to regulate transcription. To determine whether coactivators and corepressors have the capacity to modulate the relative agonist/antagonist activity of 4HT, ER-dependent gene expression was measured in the absence or presence of expression vectors for SRC-1 (steroid receptor coactivator-1) or SMRT (silencing mediator of retinoic acid and thyroid hormone receptors). In Hep G2 cells in which 4HT is an agonist, exogenous SRC-1 enhanced estradiol (E2)- and 4HT-stimulated transcription in a dose-dependent manner, while SMRT overexpression strongly reduced basal and 4HT-stimulated gene expression with no effect on E2 activity. These observations were not cell- or promoter-specific inasmuch as similar results were obtained in HeLa cells under conditions in which 4HT is an antagonist. A protein-protein interaction assay indicated that the full-length ER binds to SMRT in vitro. To assess whether relative coactivator and corepressor expression within a given cell could modulate the balance of 4HT agonist/antagonist activity, SRC-1 and SMRT were coexpressed. SMRT overexpression blocked SRC-1 coactivation of 4HT-stimulated gene expression and preferentially inhibited 4HT agonist activity whether or not exogenous SRC-1 was present. The cumulative data in this model system indicate that the relative expression of coactivators and corepressors can modulate 4HT regulation of ER transcriptional activity and suggest they could contribute to the tissue-specific ability of mixed antiestrogens to activate or inhibit ER-mediated gene expression.
Mol Endocrinol 1997 Jun
PMID:Coactivator and corepressor regulation of the agonist/antagonist activity of the mixed antiestrogen, 4-hydroxytamoxifen. 917 Dec 29

The nuclear hormone receptors belonging to the steroid/thyroid/retinoid receptor superfamily are ligand-inducible transcription factors. These receptors modulate transcription of specific cellular genes, either positively or negatively, by interacting with specific hormone response elements located near the target promoters. Recent studies indicated that the hormone- occupied, DNA-bound receptor acts in concert with a cellular coregulatory factor, termed coactivator, and the basal transcription machinery to mediate gene activation. Consistent with this scenario, a number of nuclear proteins with potential coactivator function have been isolated. In the present study, we demonstrate that steroid receptor coactivator-1 (SRC-1), a recently isolated candidate coactivator, functions as a positive regulator of the thyroid hormone receptor (TR)-mediated transactivation pathway. In transient transfection experiments, coexpression of SRC-1 significantly enhanced ligand-dependent transactivation of a thyroid hormone response element (TRE)-linked promoter by human TRbeta. Our studies revealed that deletion of six amino acids (451-456) in the extreme COOH-terminal region of TRbeta resulted in a receptor that retained the ability to bind T3 but failed to be stimulated by SRC-1. These six amino acids are part of an amphipathic helix that is highly conserved among nuclear hormone receptors and contains the core domain of the ligand-dependent transactivation function, AF-2. In agreement with this observation, in vitro protein binding studies showed that SRC-1 interacted with a ligand binding domain peptide (145-456) of TRbeta in a T3-dependent manner, whereas it failed to interact with a mutant ligand binding domain lacking the amino acids (451-456). We demonstrated that a synthetic peptide containing the COOH-terminal amino acids (437-456) of TRbeta efficiently blocked the ligand-induced binding of SRC-1 to the receptor. These results suggest that the conserved amphipathic helix that constitutes the AF-2 core domain of TRbeta is critical for interaction with SRC-1 and thereby plays a central role in coactivator-mediated transactivation. We further observed that a heterodimer of TRbeta and retinoid X receptor-alpha (RXR alpha), either in solution or bound to a DR+4 TRE, recruited SRC-1 in a T3-dependent manner. The AF-2 of TR was clearly involved in this process because a TR-RXR heterodimer containing a mutant TRbeta (1-450) with impaired AF-2 failed to bind to SRC-1. Surprisingly, the RXR-specific ligand 9-cis-retinoic acid induced binding of SRC-1 to the RXR component of the TRE-bound heterodimer. This novel finding suggests that RXR, as a heterodimeric partner of TR, has the potential to play an active role in transcriptional regulation. Our results raise the interesting possibility that a RXR-specific ligand may modulate T3-mediated signaling by inducing additional interactions between TRE-bound TR-RXR heterodimer and the coactivator.
Mol Endocrinol 1997 Jun
PMID:Analysis of the functional role of steroid receptor coactivator-1 in ligand-induced transactivation by thyroid hormone receptor. 917 Dec 39

The estrogen receptor (ER) belongs to a large family of nuclear receptors, many of whose members function as ligand-dependent transcriptional activators. The mechanism by which the receptor is converted from an inactive into an activated state is not yet completely understood. To investigate the kind of changes in receptor conformation and interactions that are involved in this activation, we have used the wild type ER and a set of constitutively active ER point mutants that show from 20% to nearly 100% activity in the absence of estrogen. These mutants are of particular interest as they could mimic, in the absence of ligand, the activated state of the wild type receptor. We have analyzed several transcriptional steps that could be involved in the activation: the ability of these receptors 1) to interact with several coactivators (steroid receptor coactivator-1, SRC-1; transcription intermediary factor-1, TIF-1; and estrogen receptor-associated protein 140, ERAP 140) and with members of the preinitiation complex [TATA box-binding protein (TBP), transcription factor IIB (TFIIB)]; 2) to exhibit conformational changes revealed by proteolytic digest patterns similar to those observed for the wild type hormone-occupied ER; and 3) to bend estrogen response element-containing DNA, which is thought to be one of the important phenomena triggering transcriptional activation. Our results demonstrate that the interaction of these mutant receptors with coactivators is likely to be one of the features of the activated step, as the mutant receptors interacted with some coactivators in a ligand-independent manner in proportion to their extent of constitutive activity. However, the different degrees of ligand-independent interaction of the mutant ERs with the three coactivators suggest that SRC-1, TIF-1, and ERAP 140 may play different roles in receptor activity. Limited proteolytic digest experiments reveal that the activated state of the receptor corresponds to a particular conformation of the receptor, which is fully observed with the mutant ER showing the highest activity in the absence of estrogen. Finally, it appears that in inactive or active states, the receptor exhibits distinctly different DNA-bending abilities. Addition of estradiol is able to modify the bending ability of only the wild type receptor, whereas estradiol has no influence on the constitutive receptors, which exhibited the same bending ability as that observed for the ligand-occupied wild type receptor. These data document that the ER undergoes major changes in its conformation and also in its functional properties when it is turned from an inactive into an active state and that mutational changes in the ER protein that result in constitutive, hormone-independent activation mimic many of the changes in ER properties that are normally under hormone regulation.
Mol Endocrinol 1997 Aug
PMID:Mechanistic aspects of estrogen receptor activation probed with constitutively active estrogen receptors: correlations with DNA and coregulator interactions and receptor conformational changes. 925 27

The orphan receptor steroidogenic factor 1 (SF-1) plays a central role in development and differentiation of the adrenal gland and gonads. It also regulates the expression of several pivotal steroidogenic enzymes and other proteins that are essential for reproductive function. Its mechanism of target gene activation that directs these intricate processes has not been previously established. We demonstrate here that the activation function-2 (AF-2) activation hexamer (AF-2-AH) of SF-1, located within its carboxy-terminal region, is required for reporter gene activation by SF-1, as well as for SF-1-mediated induction of a steroidogenic phenotype in embryonic stem cells. We further demonstrate that SF-1's AF-2-AH is not sufficient for gene activation, requiring an additional, proximally located domain of SF-1, positioned between residues 187-245. Correspondingly, we show that the coactivator SRC-1 potentiates the activity of SF-1 and that the interaction between SF-1 and SRC-1 requires both AF-2-AH and the proximal activation domain. We conclude that SF-1 harbors at least two activation domains within its carboxy terminus and that both are required for its transcriptional activation function and for direct interaction with SRC-1. It is likely that SRC-1 plays a key role in gene regulation by SF-1.
Mol Endocrinol 1997 Oct
PMID:The activation function-2 hexamer of steroidogenic factor-1 is required, but not sufficient for potentiation by SRC-1. 932 45

The vitamin D receptor (VDR) binds to the vitamin D response element (VDRE) and mediates the effects of the biologically active form of vitamin D, 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3], on gene expression. The VDR binds to the VDRE as a heterodimeric complex with retinoid X receptor. In the present study, we have used a yeast two-hybrid system to clone complementary DNA that codes for VDR-interacting protein(s). We found that the human steroid receptor coactivator-1 (SRC-1) interacts with the VDR in a ligand-dependent manner, as demonstrated by beta-galactosidase production. The interaction of the VDR and the SRC-1 takes place at physiological concentrations of 1,25(OH)2D3. A 48.2-fold stimulation of beta-galactosidase activity was observed in the presence of 10(-10) M 1,25-(OH)2D3. In addition, a direct interaction between the ligand-activated glutathione-S-transferase-VDR and 35S-labeled SRC-1 was observed in vitro. Deletion-mutation analysis of the VDR established that the ligand-dependent activation domain (AF-2) of the VDR is required for the interaction with SRC-1. One deletion mutant, pGVDR-(1-418), bound the ligand but failed to interact with the SRC-1, whereas another deletion mutant, pGVDR-(1-423), bound the ligand and interacted with the SRC-1. We demonstrated that all the deletion mutants were expressed as analyzed by a Gal4 DNA-binding domain antibody. Deletion mutation analysis of the SRC-1 demonstrated that 27 amino acids (DPCNTNPTPMTKATPEEIKLEAQSQFT) of the SRC-1 are essential for interaction with the AF-2 motif of the VDR.
Mol Endocrinol 1998 Jan
PMID:Mapping the domains of the interaction of the vitamin D receptor and steroid receptor coactivator-1. 944 Aug 10


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