Herbert H Samuels, M.D.

A Novel Transcription Complex That Selectively Modulates Apoptosis of Breast Cancer Cells through Regulation of FASTKD2
Yeung, Kay T; Das, Sharmistha; Zhang, Jin; Lomniczi, Alejandro; Ojeda, Sergio R; Xu, Chong-Feng; Neubert, Thomas A; Samuels, Herbert H
2011 Jun;31(11):2287-2298, Molecular & cellular biology
We previously reported that expression of NRIF3 (nuclear receptor interacting factor-3) rapidly and selectively leads to apoptosis of breast cancer cells. DIF-1 (also known as interferon regulatory factor-2 binding protein 2 [IRF-2BP2]), the cellular target of NRIF3, was identified as a transcriptional repressor, and DIF-1 knockdown leads to apoptosis of breast cancer cells but not other cell types. Here, we identify IRF-2BP1 and EAP1 (enhanced at puberty 1) as important components of the DIF-1 complex mediating both complex stability and transcriptional repression. This interaction of DIF-1, IRF-2BP1, and EAP1 occurs through the conserved C4 zinc fingers of these proteins. Microarray studies were carried out in breast cancer cell lines engineered to conditionally and rapidly increase the levels of the death domain (DD1) region of NRIF3. The DIF-1 complex was found to repress FASTKD2, a putative proapoptotic gene, in breast cancer cells and to bind to the FASTKD2 gene by chromatin immunoprecipitation. FASTKD2 knockdown prevents apoptosis of breast cancer cells from NRIF3 expression or DIF-1 knockdown, while expression of FASTKD2 leads to apoptosis of both breast and nonbreast cancer cells. Thus, regulation of FASTKD2 by NRIF3 and the DIF-1 complex acts as a novel death switch that selectively modulates apoptosis in breast cancer
— id: 132312, year: 2011, vol: 31, page: 2287, stat: Journal Article,

Nuclear receptor engineering based on novel structure activity relationships revealed by farnesyl pyrophosphate
Goyanka, Ritu; Das, Sharmistha; Samuels, Herbert H; Cardozo, Timothy
2010 Nov;23(11):809-815, Protein engineering, design & selection : PEDS
Nuclear receptors (NRs) comprise the second largest protein family targeted by currently available drugs, acting via specific ligand interactions within the ligand binding domain (LBD). Recently, farnesyl pyrophosphate (FPP) was shown to be a unique promiscuous NR ligand, activating a subset of NR family members and inhibiting wound healing in skin. The current study aimed at visualizing the unique basis of FPP interaction with multiple receptors in order to identify general structure-activity relationships that operate across the NR family. Docking of FPP to the 3D structures of the LBDs of a diverse set of NRs consistently revealed an electrostatic FPP pyrophosphate contact with an NR arginine conserved in the NR family, a hydrophobic farnesyl contact with NR helix-12 and a ligand binding pocket volume between 300 and 430 A(3) as the minimal requirements for FPP activation of any NR. Lack of any of these structural features appears to render a given NR resistant to FPP activation. We used these structure-activity relationships to rationally design and successfully engineer several mutant human estrogen receptors that retain responsiveness to estradiol but no longer respond to FPP
— id: 113803, year: 2010, vol: 23, page: 809, stat: Journal Article,

Farnesyl pyrophosphate inhibits epithelialization and wound healing through the glucocorticoid receptor
Vukelic, Sasa; Stojadinovic, Olivera; Pastar, Irena; Vouthounis, Constantinos; Krzyzanowska, Agata; Das, Sharmistha; Samuels, Herbert H; Tomic-Canic, Marjana
2010 Jan 15;285(3):1980-1988, Journal of biological chemistry
Farnesyl pyrophosphate (FPP), a key intermediate in the mevalonate pathway and protein farnesylation, can act as an agonist for several nuclear hormone receptors. Here we show a novel mechanism by which FPP inhibits wound healing acting as an agonist for glucocorticoid receptor (GR). Elevation of endogenous FPP by the squalene synthetase inhibitor zaragozic acid A (ZGA) or addition of FPP to the cell culture medium results in activation and nuclear translocation of the GR, a known wound healing inhibitor. We used functional studies to evaluate the effects of FPP on wound healing. Both FPP and ZGA inhibited keratinocyte migration and epithelialization in vitro and ex vivo. These effects were independent of farnesylation and indicate that modulation of FPP levels in skin may be beneficial for wound healing. FPP inhibition of keratinocyte migration and wound healing proceeds, in part, by repression of the keratin 6 gene. Furthermore, we show that the 3-hydroxy-3-methylglutaryl-CoA-reductase inhibitor mevastatin, which blocks FPP formation, not only promotes epithelialization in acute wounds but also reverses the effect of ZGA on activation of the GR and inhibition of epithelialization. We conclude that FPP inhibits wound healing by acting as a GR agonist. Of special interest is that FPP is naturally present in cells prior to glucocorticoid synthesis and that FPP levels can be further altered by the statins. Therefore, our findings may provide a better understanding of the pleiotropic effects of statins as well as molecular mechanisms by which they may accelerate wound healing
— id: 114360, year: 2010, vol: 285, page: 1980, stat: Journal Article,

Identification and characterization of a novel nuclear protein complex Involved In nuclear hormone receptor-mediated gene regulation
Garapaty, Shivani; Xu, Chong-Feng; Trojer, Patrick; Mahajan, Muktar A; Neubert, Thomas A; Samuels, Herbert H
2009 Mar 20;284(12):7542-7552, Journal of biological chemistry
NRC/NCoA6 plays an important role in mediating the effects of ligand-bound nuclear hormone receptors as well as other transcription factors. NRC interacting factor 1 (NIF-1) was cloned as a novel factor that interacts in vivo with NRC. Although NIF-1 does not directly interact with nuclear hormone receptors, it enhances activation by nuclear hormone receptors presumably through its interaction with NRC. To further understand the cellular and biological function of NIF-1, we identified NIF-1 associated proteins by in-solution proteolysis followed by mass spectrometry. The identified components revealed factors involved in histone methylation and cell cycle control and include Ash2L, RbBP5, WDR5, HCF-1, DBC-1, and EMSY. Although the NIF-1 complex contains Ash2L, RbBP5, and WDR5 suggesting that the complex might methylate histone H3-Lys4, we found that the complex contains a H3 methyltransferase activity that modifies a residue other than H3-Lys 4. The identified components form at least two distinct sized NIF-1 complexes. DBC-1 and EMSY were identified as integral components of a ~1.5 MDa NIF-1 complex and were found to play an important role in the regulation of nuclear receptor-mediated transcription. Stimulation of the Sox9 and HoxA1 genes by retinoic acid receptor-a was found to require both DBC-1 and EMSY in addition to NIF-1 for maximal transcriptional activation. Interestingly, NRC was not identified as a component of the NIF-1 complex, suggesting that NIF-1 and NRC do not exist as stable in vitro purified complexes although the separate NIF-1 and NRC complexes appear to functionally interact in the cell
— id: 95312, year: 2009, vol: 284, page: 7542, stat: Journal Article,

Identification of a novel pathway that selectively modulates apoptosis of breast cancer cells
Tinnikov, Alexander A; Yeung, Kay T; Das, Sharmistha; Samuels, Herbert H
2009 Feb 15;69(4):1375-1382, Cancer research
Expression of the nuclear receptor interacting factor 3 (NRIF3) coregulator in a wide variety of breast cancer cells selectively leads to rapid caspase-2-dependent apoptotic cell death. A novel death domain (DD1) was mapped to a 30-amino acid region of NRIF3. Because the cytotoxicity of NRIF3 and DD1 seems to be cell type-specific, these studies suggest that breast cancer cells contain a novel 'death switch' that can be specifically modulated by NRIF3 or DD1. Using an MCF-7 cell cDNA library in a yeast two-hybrid screen, we cloned a factor that mediates apoptosis by DD1 and refer to this factor as DD1-interacting factor-1 (DIF-1). DIF-1 is a transcriptional repressor that mediates its effect through SirT1, and this repression is attenuated by the binding of NRIF3/DD1. DIF-1 expression rescues breast cancer cells from NRIF3/DD1-induced apoptosis. Small interfering RNA (siRNA) knockdown of DIF-1 selectively leads to apoptosis of breast cancer cells, further suggesting that DIF-1 plays a key role in NRIF3/DD1-mediated apoptosis. A protein kinase A inhibitor (H89) also elicits apoptosis of breast cancer cells but not of the other cell types examined, and DIF-1 also protects these cells from H89-mediated apoptosis. In addition, H89 incubation results in a rapid increase in NRIF3 levels and siRNA knockdown of NRIF3 protects breast cancer cells from H89-mediated apoptosis. Our results indicate that DIF-1 plays a key role in breast cancer cell survival and further characterizing this pathway may provide important insights into developing novel therapies to selectively target breast cancer cells for apoptosis
— id: 97576, year: 2009, vol: 69, page: 1375, stat: Journal Article,

Statins, inhibitors of cholesterol synthesis, accelerate wound healing via farnesyl pyrophosphate
Vukelic, S; Stojadinovic, O; Pastar, I; Das, S; Samuels, H; Krzyzanowska, A; Brem, H; Tomic-Canic, M
2009 APR ;129(5):S51-S51, Journal of investigative dermatology
— id: 97875, year: 2009, vol: 129, page: S51, stat: Journal Article,

INHIBITORS OF CHOLESTEROL SYNTHESIS, STATINS, ACCELERATE WOUND HEALING
Vukelic, S; Stojadinovic, O; Pastar, I; Das, S; Samuels, H; Krzyzanowska, A; Ragupathi, M; Brem, H; Tomic-Canic, M
2009 MAR-APR ;17(2):A21-A21, Wound repair & regeneration
— id: 97662, year: 2009, vol: 17, page: A21, stat: Journal Article,

Components of the CCR4-NOT Complex Function as Nuclear Hormone Receptor Coactivators via Association with the NRC-interacting Factor NIF-1
Garapaty, Shivani; Mahajan, Muktar A; Samuels, Herbert H
2008 Mar 14;283(11):6806-6816, Journal of biological chemistry
CCR4-NOT is an evolutionarily conserved, multicomponent complex known to be involved in transcription as well as mRNA degradation. Various subunits (e.g. CNOT1 and CNOT7/CAF1) have been reported to be involved in influencing nuclear hormone receptor activities. Here, we show that CCR4/CNOT6 and RCD1/CNOT9, members of the CCR4-NOT complex, potentiate nuclear receptor activity. RCD1 interacts in vivo and in vitro with NIF-1 (NRC-interacting factor), a previously characterized nuclear receptor cotransducer that activates nuclear receptors via its interaction with NRC. As with NIF-1, RCD1 and CCR4 do not directly associate with nuclear receptors; however, they enhance ligand-dependent transcriptional activation by nuclear hormone receptors. CCR4 mediates its effect through the ligand binding domain of nuclear receptors and small interference RNA-mediated silencing of endogenous CCR4 results in a marked decrease in nuclear receptor activation. Furthermore, knockdown of CCR4 results in an attenuated stimulation of RARalpha target genes (e.g. Sox9 and HoxA1) as shown by quantitative PCR assays. The silencing of endogenous NIF-1 also resulted in a comparable decrease in the RAR-mediated induction of both Sox9 and HoxA1. Furthermore, CCR4 associates in vivo with NIF-1. In addition, the CCR4-enhanced transcriptional activation by nuclear receptors is dependent on NIF-1. The small interference RNA-mediated knockdown of NIF-1 blocks the ligand-dependent potentiating effect of CCR4. Our results suggest that CCR4 plays a role in the regulation of certain endogenous RARalpha target genes and that RCD1 and CCR4 might mediate their function through their interaction with NIF-1
— id: 76777, year: 2008, vol: 283, page: 6806, stat: Journal Article,

Nuclear receptor coactivator/coregulator NCoA6(NRC) is a pleiotropic coregulator involved in transcription, cell survival, growth and development
Mahajan, Muktar A; Samuels, Herbert H
2008 ;6:e002-e002, Nuclear receptor signaling
NCoA6 (also referred to as NRC, ASC-2, TRBP, PRIP and RAP250) was originally isolated as a ligand-dependent nuclear receptor interacting protein. However, NCoA6 is a multifunctional coregulator or coactivator necessary for transcriptional activation of a wide spectrum of target genes. The NCoA6 gene is amplified and overexpressed in breast, colon and lung cancers. NCoA6 is a 250 kDa protein which harbors a potent N-terminal activation domain, AD1; and a second, centrally-located activation domain, AD2, which is necessary for nuclear receptor signaling. The intrinsic activation potential of NCoA6 is regulated by its C-terminal STL regulatory domain. Near AD2 is an LxxLL-1 motif which interacts with a wide spectrum of ligand-bound NRs with high-affinity. A second LxxLL motif (LxxLL-2) located towards the C-terminal region is more restricted in its NR specificity. The potential role of NCoA6 as a co-integrator is suggested by its ability to enhance transcriptional activation of a wide variety of transcription factors and from its in vivo association with a number of known cofactors including CBP/p300. NCoA6 has been shown to associate with at least three distinct coactivator complexes containing Set methyltransferases as core polypeptides. The composition of these complexes suggests that NCoA6 may play a fundamental role in transcriptional activation by modulating chromatin structure through histone methylation. Knockout studies in mice suggest that NCoA6 is an essential coactivator. NCoA6-/- embryos die between 8.5-12.5 dpc from general growth retardation coupled with developmental defects in the heart, liver, brain and placenta. NCoA6-/- MEFs grow at a reduced rate compared to WT MEFs and spontaneously undergo apoptosis, indicating the importance of NCoA6 as a prosurvival and anti-apoptotic gene. Studies with NCoA6+/- and conditional knockout mice suggest that NCoA6 is a pleiotropic coregulator involved in growth, development, wound healing and maintenance of energy homeostasis
— id: 76769, year: 2008, vol: 6, page: e002, stat: Journal Article,

The nuclear receptor interacting factor-3 transcriptional coregulator mediates rapid apoptosis in breast cancer cells through direct and bystander-mediated events
Das, Sharmistha; Nwachukwu, Jerome C; Li, Dangsheng; Vulin, Anthony I; Martinez-Caballero, Sonia; Kinnally, Kathleen W; Samuels, Herbert H
2007 Feb 15;67(4):1775-1782, Cancer research
We previously reported that amino acids 20 to 50 of nuclear receptor interacting factor-3 mediates rapid apoptosis in breast cancer cell lines but not in cells derived from other tissues. We refer to this short region as death domain-1 (DD1). Small interfering RNA studies indicated that DD1-mediated apoptosis is caspase-2 dependent. In this study, we examined DD1-mediated apoptosis in more detail and generated stable caspase-2 knockdown breast cancer cells. These cells are resistant to DD1-mediated apoptosis. Time-lapse movies suggested that DD1-mediated apoptosis also leads to a 'bystander effect.' We found that within 5 h of DD1 expression, breast cancer cells release a factor(s) into the medium that leads to apoptosis of naive breast cancer cells or DD1-resistant cells (e.g., HeLa). The DD1-expressing caspase-2 knockdown cells also release a factor(s) that kills other cells, indicating that this effect is not dependent on the apoptogenic process. The bystander effect seems dependent on the production of reactive oxygen species (ROS). These and other studies indicate that DD1 expression in breast cancer cells leads to at least two death signals: one involving the rapid production of ROS and/or other soluble factors that directly or indirectly leads to a bystander effect and a second caspase-2-dependent process that leads to apoptosis in cells in which DD1 is expressed
— id: 70733, year: 2007, vol: 67, page: 1775, stat: Journal Article,

Nuclear receptor coregulator (NRC): mapping of the dimerization domain, activation of p53 and STAT-2, and identification of the activation domain AD2 necessary for nuclear receptor signaling
Mahajan, Muktar A; Murray, Audrey; Levy, David; Samuels, Herbert H
2007 Aug;21(8):1822-1834, Molecular endocrinology
Nuclear receptor coregulator (NRC) is a 250-kDa nuclear protein involved in transcriptional activation of nuclear hormone receptors, nuclear factor-kappaB, c-Jun, c-Fos, and cAMP response element-binding protein. NRC is organized into a modular structure consisting of two activation domains (AD1 and AD2), two nuclear hormone receptor-interacting motifs, LxxLL-1 and LxxLL-2, and a C-terminal regulatory region rich in serines, threonines, and leucines. The LxxLL-1 motif of NRC binds to a broad spectrum of nuclear hormone receptors with high affinity whereas LxxLL-2 interacts with a very limited number of receptors. In this study we present further evidence that NRC can act as a dimer and have identified a dimerization region of 146 amino acids including LxxLL-1. Mutation of the core LxxLL-1 motif, however, indicates that it is not involved in the dimerization of NRC. AD2, just C-terminal of LxxLL-1, was found to play a central role in ligand-dependent activation by nuclear receptors even though AD1 exhibits more potent intrinsic activity. Thus, a short region of approximately 300 amino acids including and flanking LxxLL-1 plays an important role in NRC dimerization and nuclear receptor binding and transcriptional activation. In addition, consistent with its role as a cointegrator for transcriptional activation, NRC also functions as a coactivator for signal transducer and activator of transcription 2 (STAT-2) and p53. Activation of p53 by NRC appears to involve a novel mechanism where NRC interacts indirectly with p53 through Trap80, a member of the mediator complex, which binds NRC interacting factor-1 (NIF-1), which interacts with and potentiates the effect of NRC
— id: 73864, year: 2007, vol: 21, page: 1822, stat: Journal Article,

Studies of the bystander effect associated with intrinsic apoptosis
Martinez-Caballero, S; Lue, J; Huang, E; Das, S; Samuels, HH; Kinnally, KW
2007 ;282(6):567A-567A, Biophysical journal
— id: 103312, year: 2007, vol: 282, page: 567A, stat: Journal Article,

Role of PSF-TFE3 oncoprotein in the development of papillary renal cell carcinomas
Mathur, M; Samuels, H H
2007 Jan 11;26(2):277-283, Oncogene
A subset of papillary renal cell carcinomas (RCC) is characterized by the expression of a TFE3 fusion protein, where the fusion partner can be any of the several proteins identified so far such as PSF (PTB associated splicing factor), NonO, PRCC, CLTC and ASPL. These proteins result from chromosomal translocations involving the TFE3 gene located on the X chromosome. Our present study documents the central role of PSF-TFE3 in oncogenic transformation. We show that the inhibition of PSF-TFE3 expression through siRNA or shRNA leads to impaired growth, proliferation, invasion potential and long-term survival of UOK-145 papillary renal carcinoma-derived cells, which endogenously express PSF-TFE3. The oncogenic potential of PSF-TFE3 became evident by stable expression of PSF-TFE3 in NIH-3T3 mouse fibroblast cells, which leads to the acquisition of anchorage-independent growth as revealed by soft agar assay. In addition, the expression of PSF-TFE3 in normal renal proximal tubular epithelial cells from where such tumors originate leads to dedifferentiation and loss of some key functional proteins, which may reflect an initial step in the multistep process of tumor development. This suggests that the expression of PSF-TFE3 in renal epithelial cells plays an important role in the initiation and maintenance of oncogenic phenotype in papillary RCC
— id: 70300, year: 2007, vol: 26, page: 277, stat: Journal Article,

Direct identification of proteins from T47D cells and murine brain tissue by matrix-assisted laser desorption/ionization post-source decay/collision-induced dissociation
Pevsner, Paul H; Naftolin, Frederick; Hillman, Dean E; Miller, Douglas C; Fadiel, Ahmed; Kogus, Alexander; Stern, Arnold; Samuels, Herbert H
2007 ;21(3):429-436, Rapid communications in mass spectrometry
The purpose of this study is to determine the feasibility of the direct matrix-assisted laser desorption/ionization (MALDI) identification of proteins in fixed T47D breast cancer cells and murine brain tissues. The ability to identify proteins from cells and tissue may lead to biomarkers that effectively predict the onset of defined disease states, and their dynamic behavior could be an important hint for drug target discoveries. Direct tissue application of trypsin allows protein identification in cells and tissues, while maintaining spatial integrity and intracellular organization. Using a chemical printer, matrix was co-registered on trypsinized human T47D breast cancer cells and cryo-preserved sections of murine brain tissue, followed by MALDI post-source decay (PSD) or MALDI collision-induced dissociation (CID), respectively. Mass-to-charge (m/z) data from the cells and brain tissues were processed using Mascot software interrogation of the National Center for Biotechnology Information (NCBI) database. Histone H2B was identified from cultured T47D human breast cancer cells. Tubulin beta2 was identified from mouse brain cortex following an induced stroke. These results suggest that MALDI PSD/CID, combined with bioinformatics, can be used for the direct identification of proteins from cells and tissues. Refinements in preparation techniques may improve this approach to provide a tool for quantitative proteomics and clinical analysis
— id: 70734, year: 2007, vol: 21, page: 429, stat: Journal Article,

International Union of Pharmacology. LIX. The pharmacology and classification of the nuclear receptor superfamily: thyroid hormone receptors
Flamant, Frederic; Baxter, John D; Forrest, Douglas; Refetoff, Samuel; Samuels, Herbert; Scanlan, Tom S; Vennstrom, Bjorn; Samarut, Jacques
2006 Dec;58(4):705-711, Pharmacological reviews
— id: 69534, year: 2006, vol: 58, page: 705, stat: Journal Article,

The N-Terminal A/B domain of the thyroid hormone receptor-beta2 isoform influences ligand-dependent recruitment of coactivators to the ligand-binding domain
Tian, Henghe; Mahajan, Muktar A; Wong, Chun Tung; Habeos, Ioanis; Samuels, Herbert H
2006 Sep;20(9):2036-2051, Molecular endocrinology
Thyroid hormone receptors (TRs), expressed as TRalpha1, TRbeta1, and TRbeta2 isoforms, are members of the steroid hormone nuclear receptor gene superfamily, which comprises ligand-dependent transcription factors. The TR isoforms differ primarily in their N-terminal (A/B) domains, suggesting that the A/B regions mediate distinct transcriptional activation functions in a cell type-dependent or promoter-specific fashion. The nuclear receptor ligand-binding domain (LBD) undergoes a conformational change upon ligand binding that results in the recruitment of coactivators to the LBD. For glucocorticoid receptor and estrogen receptor-alpha, the same coactivator can contact both the LBD and A/B domains, thus leading to enhanced transcriptional activation. Very little is known regarding the role of the A/B domains of the TR isoforms. The A/B domain of TRbeta2 exhibits higher ligand-independent transcriptional activity than the A/B regions of TRalpha1 or TRbeta1. Thus, we examined the role of the A/B domain and the LBD of rat TRbeta2 in integrating the transcriptional activation function of the A/B and LBD domains by different coactivators. Both domains are essential for a productive functional interaction with cAMP response element-binding protein (CREB)-binding protein (CBP), and we found that CBP binds to the A/B domain of TRbeta2 in vitro. In contrast, steroid receptor coactivator-1a (SRC-1a) interacts strongly with the LBD but not the A/B domain. The coactivator NRC (nuclear receptor coactivator) interacts primarily with the LBD, although a weak interaction with the A/B domain further enhances ligand-dependent binding with TRbeta2. Our studies document the interplay between the A/B domain and the LBD of TRbeta2 in recruiting different coactivators to the receptor. Because NRC and SRC-1a bind CBP, and CBP enhances ligand-dependent activity, our studies suggest a model in which coactivator recruitment of NRC (or SRC-1a) occurs primarily through the LBD whereas the complex is further stabilized through an interaction of CBP with the N terminus of TRbeta2
— id: 68746, year: 2006, vol: 20, page: 2036, stat: Journal Article,

Functional consequences of interactions between thyroid hormone receptors and retinoid X receptor
Li D; Mahajan MA; Samuels HH
2005 ;12(5):356-362, Current opinion in endocrinology & diabetes
Purpose of review: Thyroid hormone receptors mediate a wide variety of biologic processes in cells. They are members of the nuclear hormone receptor gene family, which includes the receptors that mediate the effects of steroid hormones, vitamin D, and the retinoids. These receptors are DNA binding proteins, which act as ligand-dependent transcription factors. One of the retinoid receptors, retinoid X receptor, acts as a dimerization partner for the thyroid hormone receptors and several other members of the nuclear receptor family. This review gives a historical perspective and overview of the thyroid hormone receptors and reviews the role of the retinoid X receptor in influencing the action of the thyroid hormones. Recent findings: Retinoid X receptor forms heterodimers with the thyroid hormone receptors and several other nuclear receptors and enhances the binding of these receptors to DNA. In addition, for thyroid hormone receptor/retinoid X receptor heterodimers. the general consensus has been that activation occurs through the thyroid hormone receptor component of the heterodimer and that retinoid X receptor does not bind its ligand or mediate cross-talk with the thyroid hormone receptor component of the heterodimer. Several recent studies, however, indicate that retinoid X receptor can bind its cognate ligand and modulate the activity of the thyroid hormone receptor/retinoid X receptor heterodimer through a variety of novel mechanisms. Summary: The retinoid X receptors play an important role in mediating the action of the thyroid hormones and in part mediate these effects through cross-talk with thyroid hormone receptor in the heterodimer. Future studies that expand on recent findings in the field should help provide new insights into transcriptional regulation by thyroid hormone nuclear receptors. copyright 2005 Lippincott Williams & Wilkins
— id: 58899, year: 2005, vol: 12, page: 356, stat: Journal Article,

Nuclear hormone receptor coregulator: role in hormone action, metabolism, growth, and development
Mahajan, Muktar A; Samuels, Herbert H
2005 Jun;26(4):583-597, Endocrine reviews
Nuclear hormone receptor coregulator (NRC) (also referred to as activating signal cointegrator-2, thyroid hormone receptor-binding protein, peroxisome proliferator activating receptor-interacting protein, and 250-kDa receptor associated protein) belongs to a growing class of nuclear cofactors widely known as coregulators or coactivators that are necessary for transcriptional activation of target genes. The NRC gene is also amplified and overexpressed in breast, colon, and lung cancers. NRC is a 2063-amino acid protein that harbors a potent N-terminal activation domain (AD1) and a second more centrally located activation domain (AD2) that is rich in Glu and Pro. Near AD2 is a receptor-interacting domain containing an LxxLL motif (LxxLL-1), which interacts with a wide variety of ligand-bound nuclear hormone receptors with high affinity. A second LxxLL motif (LxxLL-2) located in the C-terminal region of NRC is more restricted in its nuclear hormone receptor specificity. The intrinsic activation potential of NRC is regulated by a C-terminal serine, threonine, leucine-regulatory domain. The potential role of NRC as a cointegrator is suggested by its ability to enhance transcriptional activation of a wide variety of transcription factors and from its in vivo association with a number of known transcriptional regulators including CBP/p300. Recent studies in mice indicate that deletion of both NRC alleles leads to embryonic lethality resulting from general growth retardation coupled with developmental defects in the heart, liver, brain, and placenta. NRC(-/-) mouse embryo fibroblasts spontaneously undergo apoptosis, indicating the importance of NRC as a prosurvival and antiapoptotic gene. Studies with 129S6 NRC(+/-) mice indicate that NRC is a pleiotropic regulator that is involved in growth, development, reproduction, metabolism, and wound healing
— id: 56108, year: 2005, vol: 26, page: 583, stat: Journal Article,

Induction of PDCD4 tumor suppressor gene expression by RAR agonists, antiestrogen and HER-2/neu antagonist in breast cancer cells. Evidence for a role in apoptosis
Afonja, Olubunmi; Juste, Dominique; Das, Sharmistha; Matsuhashi, Sachiko; Samuels, Herbert H
2004 Oct 21;23(49):8135-8145, Oncogene
The growth of human breast tumor cells is regulated through signaling involving cell surface growth factor receptors and nuclear receptors of the steroid/thyroid/retinoid receptor gene family. Retinoic acid receptors (RARs), members of the steroid/thyroid hormone receptor gene family, are ligand-dependent transcription factors, which have in vitro and in vivo growth inhibitory activity against breast cancer cells. RAR-agonists inhibit the proliferation of many human breast cancer cell lines, particularly those whose growth is stimulated by estradiol (E2) or growth factors. Additionally, RAR-agonists and synthetic retinoids such as Ferentinide have been shown to induce apoptosis in malignant breast cells but not normal breast cells. To better define the genes involved in RAR-mediated growth inhibition of breast cancer cells, we used oligonucleotide microarray analysis to create a database of genes that are potentially regulated by RAR-agonists in breast cancer cells. We found that PDCD4 (programmed cell death 4), a tumor suppressor gene presently being evaluated as a target for chemoprevention, was induced about three-fold by the RARalpha-selective agonist Am580, in T-47D breast cancer cells. RAR pan-agonists and Am580, but not retinoid X receptors (RXR)-agonists, stimulate the expression of PDCD4 in a wide variety of retinoid-inhibited breast cancer cell lines. RAR-agonists did not induce PDCD4 expression in breast cancer cell lines, which were not growth inhibited by retinoids. We also observed that antiestrogen and the HER-2/neu antagonist, Herceptin (Trastuzumab), also induced PDCD4 expression in T-47D cells, suggesting that PDCD4 may play a central role in growth inhibition in breast cancer cells. Transient overexpression of PDCD4 in T-47D (ER+, RAR+) and MDA-MB-231 (ER-, RAR-) cells resulted in apoptotic death, suggesting a role for PDCD4 in mediating apoptosis in breast cancer cells. PDCD4 protein expression has previously been reported in small ductal epithelium of normal breast. To date, there has been no report of induction of PDCD4 expression by RAR-agonists, antiestrogen or HER2/neu antagonist in breast cancer cells and its potential role in apoptosis in these cells
— id: 46457, year: 2004, vol: 23, page: 8135, stat: Journal Article,

The NRIF3 family of transcriptional coregulators induces rapid and profound apoptosis in breast cancer cells
Li, Dangsheng; Das, Sharmistha; Yamada, Tatsuya; Samuels, Herbert H
2004 May;24(9):3838-3848, Molecular & cellular biology
Many anticancer drugs kill cancer cells by inducing apoptosis. Despite the progress in understanding apoptosis, how to harness the cellular death machinery to selectively deliver tumor-specific cytotoxicity (while minimizing damage to other cells) remains an important challenge. We report here that expression of the NRIF3 family of transcriptional coregulators in a variety of breast cancer cell lines induces rapid and profound apoptosis (nearly 100% cell death within 24 h). A novel death domain (DD1) was mapped to a short 30-amino-acid region common to all members of the NRIF3 family. Mechanistic studies showed that DD1-induced apoptosis occurs through a novel caspase 2-mediated pathway that involves mitochondrial membrane permeabilization but does not require other caspases. Interestingly, the cytotoxicity of NRIF3 and DD1 appears to be cell type specific, as they selectively kill breast cancer or related cells but not other examined cells of different origins. Our study demonstrates the feasibility of selectively inducing cytotoxicity in a specific cancer and suggests that breast cancer cells contain a novel 'death switch' that can be specifically triggered by NRIF3 or DD1. Strategies utilizing NRIF3 and/or DD1 and/or targeting this death switch may lead to the development of novel and more selective therapeutics against breast cancer
— id: 43267, year: 2004, vol: 24, page: 3838, stat: Journal Article,

Novel roles of retinoid X receptor (RXR) and RXR ligand in dynamically modulating the activity of the thyroid hormone receptor/RXR heterodimer
Li, Dangsheng; Yamada, Tatsuya; Wang, Fang; Vulin, A Igor; Samuels, Herbert H
2004 Feb 27;279(9):7427-7437, Journal of biological chemistry
Many members of the type II nuclear receptor subfamily function as heterodimers with the retinoid X receptor (RXR). A permissive heterodimer (e.g. peroxisome proliferator-activated receptor/RXR) allows for ligand binding by both partners of the receptor complex. In contrast, RXR has been thought to be incapable of ligand binding in a nonpermissive heterodimer, such as that of thyroid hormone receptor (TR)/RXR, where it has been referred to as a silent partner. However, we recently presented functional evidence suggesting that RXR in the TR/RXR heterodimer can bind its natural ligand 9-cis-RA in cells. Here we extended our study of the interrelationship of TR and RXR. We examined the potential modulatory effect of RXR and its ligand on the activity of TR, primarily using a Gal4-TR chimera. This study led to several novel and unexpected findings: 1) heterodimerization of apo-RXRalpha (in the absence of 9-cis-RA) with Gal4-TR inhibits T3-mediated transactivation; 2) the inhibition of Gal4-TR activity by RXRalpha is further enhanced by 9-cis-RA; 3) two different RXR subtypes (alpha and beta) differentially modulate the activity of Gal4-TR; 4) the N-terminal A/B domains of RXR alpha and beta are largely responsible for their differential modulation of TR activity; and 5) the RXR ligand 9-cis-RA appears to differentially affect T3-mediated transactivation from the Gal4-TR/RXRalpha (which is inhibited by 9-cis-RA) and TRE-bound TR/RXRalpha (which is further activated by 9-cis-RA) heterodimers. Taken together, these results further support our recent proposal that the RXR component in a TR/RXR heterodimer is not silent and, more importantly, reveal novel aspects of regulation of the activity of the TR/RXR heterodimer by RXR and RXR ligand
— id: 42149, year: 2004, vol: 279, page: 7427, stat: Journal Article,

The nuclear hormone receptor coactivator NRC is a pleiotropic modulator affecting growth, development, apoptosis, reproduction, and wound repair
Mahajan, Muktar A; Das, Sharmistha; Zhu, Hong; Tomic-Canic, Marjana; Samuels, Herbert H
2004 Jun;24(11):4994-5004, Molecular & cellular biology
Nuclear hormone receptor coregulator (NRC) is a 2,063-amino-acid coregulator of nuclear hormone receptors and other transcription factors (e.g., c-Fos, c-Jun, and NF-kappaB). We and others have generated C57BL/6-129S6 hybrid (C57/129) NRC(+/-) mice that appear outwardly normal and grow and reproduce. In contrast, homozygous deletion of the NRC gene is embryonic lethal. NRC(-/-) embryos are always smaller than NRC(+/+) embryos, and NRC(-/-) embryos die between 8.5 and 12.5 days postcoitus (dpc), suggesting that NRC has a pleotrophic effect on growth. To study this, we derived mouse embryonic fibroblasts (MEFs) from 12.5-dpc embryos, which revealed that NRC(-/-) MEFs exhibit a high rate of apoptosis. Furthermore, a small interfering RNA that targets mouse NRC leads to enhanced apoptosis of wild-type MEFs. The finding that C57/129 NRC(+/-) mice exhibit no apparent phenotype prompted us to develop 129S6 NRC(+/-) mice, since the phenotype(s) of certain gene deletions may be strain dependent. In contrast with C57/129 NRC(+/-) females, 20% of 129S6 NRC(+/-) females are infertile while 80% are hypofertile. The 129S6 NRC(+/-) males produce offspring when crossed with wild-type 129S6 females, although fertility is reduced. The 129S6 NRC(+/-) mice tend to be stunted in their growth compared with their wild-type littermates and exhibit increased postnatal mortality. Lastly, both C57/129 NRC(+/-) and 129S6 NRC(+/-) mice exhibit a spontaneous wound healing defect, indicating that NRC plays an important role in that process. Our findings reveal that NRC is a coregulator that controls many cellular and physiologic processes ranging from growth and development to reproduction and wound repair
— id: 43266, year: 2004, vol: 24, page: 4994, stat: Journal Article,

PSF-TFE3 oncoprotein in papillary renal cell carcinoma inactivates TFE3 and p53 through cytoplasmic sequestration
Mathur, Mukul; Das, Sharmistha; Samuels, Herbert H
2003 Aug 7;22(32):5031-5044, Oncogene
Papillary renal cell carcinomas are associated with chromosomal translocations involving the helix-loop-helix leucine-zipper region of the TFE3 gene on the X chromosome. These translocations lead to the expression of TFE3 chimeras of PRCC, RCC17, NonO and PSF (PTB-associated splicing factor). In this study, we explored the role of PSF-TFE3 fusion protein in mediating cell transformation. Unlike wild-type TFE3 or PSF, which are nuclear proteins, PSF-TFE3 is not a nuclear protein and is targeted to the endosomal compartment. Although PSF-TFE3 has no effect on the nuclear localization of wild-type PSF, it sequesters wild-type TFE3 as well as p53 in the extranuclear compartment leading to functionally null p53 and TFE3 cells. In UOK-145 papillary renal carcinoma cells, which endogenously express PSF-TFE3, siRNA complementary to the PSF-TFE3 fusion junction leads to a reduction in PSF-TFE3 and redistribution of endogenous TFE3 and p53 from the cytoplasmic compartment to the nucleus. Our results indicate that PSF-TFE3 acts through a novel mechanism, and exports TFE3, p53 and possibly other factors from the nucleus to the cytoplasm for degradation leading to the transformed phenotype. Thus, PSF-TFE3 is a promising target for the treatment for a subset of renal cell carcinomas
— id: 38103, year: 2003, vol: 22, page: 5031, stat: Journal Article,

Discovery of diverse thyroid hormone receptor antagonists by high-throughput docking
Schapira, Matthieu; Raaka, Bruce M; Das, Sharmistha; Fan, Li; Totrov, Maxim; Zhou, Zhiguo; Wilson, Stephen R; Abagyan, Ruben; Samuels, Herbert H
2003 Jun 10;100(12):7354-7359, Proceedings of the National Academy of Sciences of the United States of America
Treatment of hyperthyroidism, a common clinical condition that can have serious manifestations in the elderly, has remained essentially unchanged for >30 years. Directly antagonizing the effect of the thyroid hormone at the receptor level may be a significant improvement for the treatment of hyperthyroid patients. We built a computer model of the thyroid hormone receptor (TR) ligand-binding domain in its predicted antagonist-bound conformation and used a virtual screening algorithm to select 100 TR antagonist candidates out of a library of >250,000 compounds. We were able to obtain 75 of the compounds selected in silico and studied their ability to act as antagonists by using cultured cells that express TR. Fourteen of these compounds were found to antagonize the effect of T3 on TR with IC50s ranging from 1.5 to 30 microM. A small virtual library of compounds, derived from the highest affinity antagonist (1-850) that could be rapidly synthesized, was generated. A second round of virtual screening identified new compounds with predicted increased antagonist activity. These second generation compounds were synthesized, and their ability to act as TR antagonists was confirmed by transfection and receptor binding experiments. The extreme structural diversity of the antagonist compounds shows how receptor-based virtual screening can identify diverse chemistries that comply with the structural rules of TR antagonism
— id: 63078, year: 2003, vol: 100, page: 7354, stat: Journal Article,

RAR agonists stimulate SOX9 gene expression in breast cancer cell lines: evidence for a role in retinoid-mediated growth inhibition
Afonja, Olubunmi; Raaka, Bruce M; Huang, Ambrose; Das, Sharmistha; Zhao, Xinyu; Helmer, Elizabeth; Juste, Dominique; Samuels, Herbert H
2002 Nov 7;21(51):7850-7860, Oncogene
Retinoic acid receptors (RARs) are ligand-dependent transcription factors which are members of the steroid/thyroid hormone receptor gene family. RAR-agonists inhibit the proliferation of many human breast cancer cell lines, particularly those whose growth is stimulated by estradiol (E2) or growth factors. PCR-amplified subtractive hybridization was used to identify candidate retinoid-regulated genes that may be involved in growth inhibition. One candidate gene identified was SOX9, a member of the high mobility group (HMG) box gene family of transcription factors. SOX9 gene expression is rapidly stimulated by RAR-agonists in T-47D cells and other retinoid-inhibited breast cancer cell lines. In support of this finding, a database search indicates that SOX9 is expressed as an EST in breast tumor cells. SOX9 is known to be expressed in chondrocytes where it regulates the transcription of type II collagen and in testes where it plays a role in male sexual differentiation. RAR pan-agonists and the RARalpha-selective agonist Am580, but not RXR agonists, stimulate the expression of SOX9 in a wide variety of retinoid-inhibited breast cancer cell lines. RAR-agonists did not stimulate SOX9 in breast cancer cell lines which were not growth inhibited by retinoids. Expression of SOX9 in T-47D cells leads to cycle changes similar to those found with RAR-agonists while expression of a dominant negative form of SOX9 blocks RA-mediated cell cycle changes, suggesting a role for SOX9 in retinoid-mediated growth inhibition
— id: 39566, year: 2002, vol: 21, page: 7850, stat: Journal Article,

Functional evidence for retinoid X receptor (RXR) as a nonsilent partner in the thyroid hormone receptor/RXR heterodimer
Li, Dangsheng; Li, Tong; Wang, Fang; Tian, Heather; Samuels, Herbert H
2002 Aug;22(16):5782-5792, Molecular & cellular biology
Many members of the thyroid hormone/retinoid receptor subfamily (type II nuclear receptors) function as heterodimers with the retinoid X receptor (RXR). In heterodimers which are referred to as permissive, such as peroxisome proliferator activated receptor/RXR, both partners can bind cognate ligands and elicit ligand-dependent transactivation. In contrast, the thyroid hormone receptor (TR)/RXR heterodimer is believed to be nonpermissive, where RXR is thought to be incapable of ligand binding and is often referred to as a silent partner. In this report, we used a sensitive derepression assay system that we developed previously to reexamine the TR/RXR interrelationship. We provide functional evidence suggesting that in a TR/RXR heterodimer, the RXR component can bind its ligand in vivo. Ligand binding by RXR does not appear to directly activate the TR/RXR heterodimer; instead, it leads to a (at least transient or dynamic) dissociation of a cellular inhibitor(s)/corepressor(s) from its TR partner and thus may serve to modulate unliganded TR-mediated repression and/or liganded TR-mediated activation. Our results argue against the current silent-partner model for RXR in the TR/RXR heterodimer and reveal an unexpected aspect of cross regulation between TR and RXR
— id: 32316, year: 2002, vol: 22, page: 5782, stat: Journal Article,

NRC-Interacting Factor 1 Is a Novel Cotransducer That Interacts with and Regulates the Activity of the Nuclear Hormone Receptor Coactivator NRC
Mahajan, Muktar A; Murray, Audrey; Samuels, Herbert H
2002 Oct;22(19):6883-6894, Molecular & cellular biology
We previously reported the cloning and characterization of a novel nuclear hormone receptor transcriptional coactivator, which we refer to as NRC. NRC is a 2,063-amino-acid nuclear protein which contains a potent N-terminal activation domain and several C-terminal modules which interact with CBP and ligand-bound nuclear hormone receptors as well as c-Fos and c-Jun. In this study we sought to clone and identify novel factors that interact with NRC to modulate its transcriptional activity. Here we describe the cloning and characterization of a novel protein we refer to as NIF-1 (NRC-interacting factor 1). NIF-1 was cloned from rat pituitary and human cell lines and was found to interact in vivo and in vitro with NRC. NIF-1 is a 1,342-amino-acid nuclear protein containing a number of conserved domains, including six Cys-2/His-2 zinc fingers, an N-terminal stretch of acidic amino acids, and a C-terminal leucine zipper-like motif. Zinc fingers 1 to 3 are potential DNA-binding BED finger domains recently proposed to play a role in altering local chromatin architecture. We mapped the interaction domains of NRC and NIF-1. Although NIF-1 does not directly interact with nuclear receptors, it markedly enhances ligand-dependent transcriptional activation by nuclear hormone receptors in vivo as well as activation by c-Fos and c-Jun. These results, and the finding that NIF-1 interacts with NRC in vivo, suggest that NIF-1 functions to regulate transcriptional activation through NRC. We suggest that NIF-1, and factors which associate with coactivators but not receptors, be referred to as cotransducers, which act in vivo either as part of a coactivator complex or downstream of a coactivator complex to modulate transcriptional activity. Our findings suggest that NIF-1 may be a functional component of an NRC complex and acts as a regulator or cotransducer of NRC function
— id: 32315, year: 2002, vol: 22, page: 6883, stat: Journal Article,

Domain structure of the NRIF3 family of coregulators suggests potential dual roles in transcriptional regulation
Li D; Wang F; Samuels HH
2001 Dec;21(24):8371-8384, Molecular & cellular biology
The identification of a novel coregulator for nuclear hormone receptors, designated NRIF3, was recently reported (D. Li et al., Mol. Cell. Biol. 19:7191-7202, 1999). Unlike most known coactivators, NRIF3 exhibits a distinct receptor specificity in interacting with and potentiating the activity of only TRs and RXRs but not other examined nuclear receptors. However, the molecular basis underlying such specificity is unclear. In this report, we extended our study of NRIF3-receptor interactions. Our results suggest a bivalent interaction model, where a single NRIF3 molecule utilizes both the C-terminal LXXIL (receptor-interacting domain 1 [RID1]) and the N-terminal LXXLL (RID2) modules to cooperatively interact with TR or RXR (presumably a receptor dimer), with the spacing between RID1 and RID2 playing an important role in influencing the affinity of the interactions. During the course of these studies, we also uncovered an NRIF3-NRIF3 interaction domain. Deletion and mutagenesis analyses mapped the dimerization domain to a region in the middle of NRIF3 (residues 84 to 112), which is predicted to form a coiled-coil structure and contains a putative leucine zipper-like motif. By using Gal4 fusion constructs, we identified an autonomous transactivation domain (AD1) at the C terminus of NRIF3. Somewhat surprisingly, full-length NRIF3 fused to the DNA-binding domain of Gal4 was found to repress transcription of a Gal4 reporter. Further analyses mapped a novel repression domain (RepD1) to a small region at the N-terminal portion of NRIF3 (residues 20 to 50). The NRIF3 gene encodes at least two additional isoforms due to alternative splicing. These two isoforms contain the same RepD1 region as NRIF3. Consistent with this, Gal4 fusions of these two isoforms were also found to repress transcription. Cotransfection of NRIF3 or its two isoforms did not relieve the transrepression function mediated by their corresponding Gal4 fusion proteins, suggesting that the repression involves a mechanism(s) other than the recruitment of a titratable corepressor. Interestingly, a single amino acid residue change of a potential phosphorylation site in RepD1 (Ser(28) to Ala) abolishes its transrepression function, suggesting that the coregulatory property of NRIF3 (or its isoforms) might be subjected to regulation by cellular signaling. Taken together, our results identify NRIF3 as an interesting coregulator that possesses both transactivation and transrepression domains and/or functions. Collectively, the NRIF3 family of coregulators (which includes NRIF3 and its other isoforms) may play dual roles in mediating both positive and negative regulatory effects on gene expression
— id: 26563, year: 2001, vol: 21, page: 8371, stat: Journal Article,

PSF is a novel corepressor that mediates its effect through Sin3A and the DNA binding domain of nuclear hormone receptors
Mathur M; Tucker PW; Samuels HH
2001 Apr;21(7):2298-2311, Molecular & cellular biology
Members of the type II nuclear hormone receptor subfamily (e.g., thyroid hormone receptors [TRs], retinoic acid receptors, retinoid X receptors [RXRs], vitamin D receptor, and the peroxisome proliferator-activated receptors) bind to their response sequences with or without ligand. In the absence of ligand, these DNA-bound receptors mediate different degrees of repression or silencing of gene expression which is thought to result from the association of their ligand binding domains (LBDs) with corepressors. Two related corepressors, N-CoR and SMRT, interact to various degrees with the LBDs of these type II receptors in the absence of their cognate ligands. N-CoR and SMRT have been proposed to act by recruiting class I histone deacetylases (HDAC I) through an association with Sin3, although they have also been shown to recruit class II HDACs through a Sin3-independent mechanism. In this study, we used a biochemical approach to identify novel nuclear factors that interact with unliganded full-length TR and RXR. We found that the DNA binding domains (DBDs) of TR and RXR associate with two proteins which we identified as PSF (polypyrimidine tract-binding protein-associated splicing factor) and NonO/p54(nrb). Our studies indicate that PSF is a novel repressor which interacts with Sin3A and mediates silencing through the recruitment of HDACs to the receptor DBD. In vivo studies with TR showed that although N-CoR fully dissociates in the presence of ligand, the levels of TR-bound PSF and Sin3A appear to remain unchanged, indicating that Sin3A can be recruited to the receptor independent of N-CoR or SMRT. RXR was not detected to bind N-CoR although it bound PSF and Sin3A as effectively as TR, and this association with RXR did not change with ligand. Our studies point to a novel PSF/Sin3-mediated pathway for nuclear hormone receptors, and possibly other transcription factors, which may fine-tune the transcriptional response as well as play an important role in mediating the repressive effects of those type II receptors which only weakly interact with N-CoR and SMRT
— id: 20740, year: 2001, vol: 21, page: 2298, stat: Journal Article,

In silico discovery of novel Retinoic Acid Receptor agonist structures
Schapira M; Raaka BM; Samuels HH; Abagyan R
2001 ;1(1):1-1, BioMedCentral structural biology
BACKGROUND: Several Retinoic Acid Receptors (RAR) agonists have therapeutic activity against a variety of cancer types; however, unacceptable toxicity profiles have hindered the development of drugs. RAR agonists presenting novel structural and chemical features could therefore open new avenues for the discovery of leads against breast, lung and prostate cancer or leukemia. RESULTS: We have analysed the induced fit of the active site residues upon binding of a known ligand. The derived binding site models were used to dock over 150,000 molecules in silico (or virtually) to the structure of the receptor with the Internal Coordinates Mechanics (ICM) program. Thirty ligand candidates were tested in vitro. CONCLUSIONS: Two novel agonists resulting from the predicted receptor model were active at 50 nM. One of them displays novel structural features which may translate into the development of new ligands for cancer therapy
— id: 20742, year: 2001, vol: 1, page: 1, stat: Journal Article,

A new family of nuclear receptor coregulators that integrate nuclear receptor signaling through CREB-binding protein
Mahajan MA; Samuels HH
2000 Jul;20(14):5048-5063, Molecular & cellular biology
We describe the cloning and characterization of a new family of nuclear receptor coregulators (NRCs) which modulate the function of nuclear hormone receptors in a ligand-dependent manner. NRCs are expressed as alternatively spliced isoforms which may exhibit different intrinsic activities and receptor specificities. The NRCs are organized into several modular structures and contain a single functional LXXLL motif which associates with members of the steroid hormone and thyroid hormone/retinoid receptor subfamilies with high affinity. Human NRC (hNRC) harbors a potent N-terminal activation domain (AD1), which is as active as the herpesvirus VP16 activation domain, and a second activation domain (AD2) which overlaps with the receptor-interacting LXXLL region. The C-terminal region of hNRC appears to function as an inhibitory domain which influences the overall transcriptional activity of the protein. Our results suggest that NRC binds to liganded receptors as a dimer and this association leads to a structural change in NRC resulting in activation. hNRC binds CREB-binding protein (CBP) with high affinity in vivo, suggesting that hNRC may be an important functional component of a CBP complex involved in mediating the transcriptional effects of nuclear hormone receptors
— id: 11637, year: 2000, vol: 20, page: 5048, stat: Journal Article,

Rational discovery of novel nuclear hormone receptor antagonists
Schapira M; Raaka BM; Samuels HH; Abagyan R
2000 Feb 1;97(3):1008-1013, Proceedings of the National Academy of Sciences of the United States of America
Nuclear hormone receptors (NRs) are potential targets for therapeutic approaches to many clinical conditions, including cancer, diabetes, and neurological diseases. The crystal structure of the ligand binding domain of agonist-bound NRs enables the design of compounds with agonist activity. However, with the exception of the human estrogen receptor-alpha, the lack of antagonist-bound 'inactive' receptor structures hinders the rational design of receptor antagonists. In this study, we present a strategy for designing such antagonists. We constructed a model of the inactive conformation of human retinoic acid receptor-alpha by using information derived from antagonist-bound estrogen receptor-alpha and applied a computer-based virtual screening algorithm to identify retinoic acid receptor antagonists. Thus, the currently available crystal structures of NRs may be used for the rational design of antagonists, which could lead to the development of novel drugs for a variety of diseases
— id: 8562, year: 2000, vol: 97, page: 1008, stat: Journal Article,

A novel multifunctional motif in the amino-terminal A/B domain of T3Ralpha modulates DNA binding and receptor dimerization
Hadzic E; Habeos I; Raaka BM; Samuels HH
1998 Apr 24;273(17):10270-10278, Journal of biological chemistry
We reported previously that deletion of the 50-amino acid NH2-terminal A/B domain of the chicken (c) or rat thyroid hormone (T3) receptor-alpha (T3Ralpha) decreased the T3-dependent stimulation of genes regulated by native thyroid hormone response elements (TREs). This requirement of the NH2-terminal A/B domain for transcriptional activation was mapped to amino acids 21-30 of cT3Ralpha. Expression of transcription factor IIB (TFIIB) in cells was shown to enhance T3-dependent transcriptional activation by cT3Ralpha, and this enhancement by TFIIB was dependent on the same 10-amino acid sequence. In vitro binding studies indicated that cT3Ralpha interacts efficiently with TFIIB, and this interaction requires amino acids 23KRKRK27 in the A/B domain. In this study we document the functional importance of these five basic residues in transcriptional activation by cT3Ralpha, further supporting the biological significance of these residues and their interaction with TFIIB. Interestingly, we also find that the same amino acids also affect DNA binding and dimerization of cT3Ralpha. Gel mobility shift assays reveal that a cT3Ralpha mutant that has all five basic amino acids changed from 23KRKRK27 to 23TITIT27 binds to a palindromic TRE predominantly as a homodimer, whereas cT3Ralpha with the wild-type 23KRKRK27 sequence binds predominantly as a monomer. This results from both a marked decrease in the ability of the cT3Ralpha mutant to bind as a monomer and from an enhanced ability to dimerize as reflected by an increase in DNA-bound T3R-retinoic X receptor heterodimers. These effects of 23KRKRK27 on DNA binding, dimerization, transcriptional activation, and the association of T3Ralpha with TFIIB support the notion that this basic amino acid motif may influence the overall structure and function of T3Ralpha and, thus, play a role in determining the distinct context-dependent transactivation potentials of the individual T3R isoforms
— id: 8033, year: 1998, vol: 273, page: 10270, stat: Journal Article,

TLS (translocated-in-liposarcoma) is a high-affinity interactor for steroid, thyroid hormone, and retinoid receptors
Powers CA; Mathur M; Raaka BM; Ron D; Samuels HH
1998 Jan;12(1):4-18, Molecular endocrinology
Nuclear receptors for steroid hormones, thyroid hormone, retinoids, and vitamin D are thought to mediate their transcriptional effects in concert with coregulator proteins that modulate receptor interactions with components of the basal transcription complex. In an effort to identify potential coregulators, receptor fusions with glutathione-S-transferase were used to isolate proteins in nuclear extracts capable of binding nuclear hormone receptors. Glutathione-S-transferase fusions with mouse retinoid X receptor-alpha enabled the selective isolation of a 65-kDa protein (p65) from nuclear extracts of rat and human cells. Binding of p65 to mouse retinoid X receptor-alpha was centered around the DNA-binding domain. p65 also bound regions encompassing the DNA-binding domain in estrogen, thyroid hormone, and glucocorticoid receptors. p65 was identified as TLS (translocated-in-liposarcoma), a recently identified member of the RNP family of nuclear RNA-binding proteins whose members are thought to function in RNA processing. The N-terminal half of TLS bound to thyroid hormone receptor with high affinity while the receptor was bound to appropriate DNA target sites. Functional studies indicated that the N-terminal half of TLS can interact with thyroid hormone receptor in vivo. TLS was originally discovered as part of a fusion protein arising from a chromosomal translocation causing human myxoid liposarcomas. TLS contains a potent transactivation domain whose translocation-induced fusion with a DNA-binding protein (CHOP) yields a powerful transforming oncogene and transcription factor. The transactivation and RNA-binding properties of TLS and the nature of its interaction with nuclear receptors suggest a novel role in nuclear receptor function
— id: 7748, year: 1998, vol: 12, page: 4, stat: Journal Article,

Role of the conserved C-terminal region of thyroid hormone receptor-alpha in ligand-dependent transcriptional activation
Selmi-Ruby S; Casanova J; Malhotra S; Roussett B; Raaka BM; Samuels HH
1998 Mar 16;138(1-2):105-114, Molecular & cellular endocrinology
The ligand binding domain (LBD) of thyroid hormone (T3) receptors contains subdomains that participate in transcriptional activation, hormone-relieved repression and dimerization. A sequence conserved within the nuclear receptor superfamily is found at positions 397-405 of the 408-amino acid chicken T3 receptor-alpha (cTR alpha) and is deleted in the related avian v-erbA. Since v-erbA exhibits compromised ligand binding and transcriptional activation, this conserved region may play a role in ligand-dependent transcriptional activation. Transfections reveal that cTR alpha(1-392) and site-directed mutants cTR alpha(L398R) and cTR alpha(F399E) are inactive, while cTR alpha(1-403) displays reduced ligand-dependent transcriptional activity. The loss of transcriptional activity in cTR alpha(1-392) is not caused by impaired DNA binding or receptor dimer formation. Proteolytic protection assays reveal that both transcriptionally active and inactive cTR alpha derivatives undergo T3-mediated conformational changes. Gal4 chimeras containing the final 16, 35 or 44 amino acids of cTR alpha indicate that the conserved C-terminal region does not function as an independent transactivation domain. Our results are consistent with a model in which ligand plays a structural role to position the conserved C-terminal regions of cTR alpha and related receptors in a transcriptionally active conformation
— id: 7788, year: 1998, vol: 138, page: 105, stat: Journal Article,

Constitutive activation of gene expression by thyroid hormone receptor results from reversal of p53-mediated repression
Qi JS; Desai-Yajnik V; Yuan Y; Samuels HH
1997 Dec;17(12):7195-7207, Molecular & cellular biology
Thyroid hormone receptor (T3R) is a member of the steroid hormone receptor gene family of nuclear hormone receptors. In most cells T3R activates gene expression only in the presence of its ligand, L-triiodothyronine (T3). However, in certain cell types (e.g., GH4C1 cells) expression of T3R leads to hormone-independent constitutive activation. This activation by unliganded T3R occurs with a variety of gene promoters and appears to be independent of the binding of T3R to specific thyroid hormone response elements (TREs). Previous studies indicate that this constitutive activation results from the titration of an inhibitor of transcription. Since the tumor suppresser p53 is capable of repressing a wide variety of gene promoters, we considered the possibility that the inhibitor is p53. Evidence to support this comes from studies indicating that expression of p53 blocks T3R-mediated constitutive activation in GH4C1 cells. In contrast with hormone-independent activation by T3R, p53 had little or no effect on T3-dependent stimulation which requires TREs. In addition, p53 mutants which oligomerize with wild-type p53 and interfere with its function also increase promoter activity. This enhancement is of similar magnitude to but is not additive with the stimulation mediated by unliganded T3R, suggesting that they target the same factor. Since p53 mutants are known to target wild-type p53 in the cell, this suggests that T3R also interacts with p53 in vivo and that endogenous levels of p53 act to suppress promoter activity. Evidence supporting both functional and physical interactions of T3R and p53 in the cell is presented. The DNA binding domain (DBD) of T3R is important in mediating constitutive activation, and the receptor DBD appears to functionally interact with the N terminus of p53 in the cell. In vitro binding studies indicate that the T3R DBD is important for interaction of T3R with p53 and that this interaction is reduced by T3. These findings are consistent with the in vivo studies indicating that p53 blocks constitutive activation but not ligand-dependent stimulation. These studies provide insight into mechanisms by which unliganded nuclear hormone receptors can modulate gene expression and may provide an explanation for the mechanism of action of the v-erbA oncoprotein, a retroviral homolog of chicken T3R alpha
— id: 12194, year: 1997, vol: 17, page: 7195, stat: Journal Article,

Interactions of the thyroid hormone receptor with the human immunodeficiency virus type I (HIV-1) long terminal repeat and the HIV-1 tat transactivator
DesaiYajnik, V; Samuels, HH
1996 MAR ;44(3):A237-A237, Journal of investigative medicine
— id: 52949, year: 1996, vol: 44, page: A237, stat: Journal Article,

Differential transactivation potentials of the thyroid hormone receptor alpha and beta isoforms
Hadzic, E; Samuels, HH
1996 MAR ;44(3):A237-A237, Journal of investigative medicine
— id: 52950, year: 1996, vol: 44, page: A237, stat: Journal Article,

Hormone-dependent and -independent transcriptional activation by thyroid hormone receptors are mediated by different mechanisms [see comments]
Helmer EB; Raaka BM; Samuels HH
1996 Feb;137(2):390-399, Endocrinology
Transcriptional activation by thyroid hormone (T3) receptor (T3R) generally requires the binding of its high affinity ligand. However, we reported previously that chicken T3R alpha (cTaR alpha) and human T3R beta 1 (hT3R beta 1) could activate transcription from several promoters containing T3R response elements (TREs) in a hormone-independent fashion when expressed in rat anterior pituitary GH4C1 cells. In this study we show that rat T3R alpha 1 also activates transcription without T3 in GH4C1 cells and that the oncoprotein v-erbA that is derived from cT3R alpha but does bind T3 is not a constitutive activator in these cells. Increased expression of T3R results in transcriptional activation of both native and minimal promoters, and this activation does not appear to require a defined TRE in the promoter. Because hormone-independent activity is not observed in several other cell lines, this activity may involve specific factors present in GH4C1 cells. Three mutants with single amino acid changes in a 20-amino acid region of the ligand-binding domain of cT3R alpha do not mediate hormone-independent activity. This region is highly conserved within the nuclear receptor family and has been implicated in interactions with other proteins, suggesting participation of other transcription or accessory factors in the hormone-independent activity of T3R. Two of these mutants mediate hormone-dependent transcriptional activation similar to wild-type cT3R alpha. All three mutants interact in vitro with retinoid X receptor beta similar to wild-type cT3R alpha. Our findings suggest that hormone-dependent and hormone-independent transactivation proceed by distinct mechanisms
— id: 6914, year: 1996, vol: 137, page: 390, stat: Journal Article,

Molecular interactions of p53 with thyroid hormone nuclear receptors
Qi, JS; DesaiYajnik, V; Samuels, HH
1996 MAR ;44(3):A234-A234, Journal of investigative medicine
— id: 52945, year: 1996, vol: 44, page: A234, stat: Journal Article,

Novel regulation of keratin gene expression by thyroid hormone and retinoid receptors
Tomic-Canic M; Day D; Samuels HH; Freedberg IM; Blumenberg M
1996 Jan 19;271(3):1416-1423, Journal of biological chemistry
Expression of keratin proteins, markers of epidermal differentiation and pathology, is uniquely regulated by the nuclear receptors for retinoic acid (RAR) and thyroid hormone (T3R) and their ligands: it is constitutively activated by unliganded T3R, but it is suppressed by ligand-occupied T3R or RAR. This regulation was studied using gel mobility shift assays with purified receptors and transient transfection assays with vectors expressing various receptor mutants. Regulation of keratin gene expression by RAR and T3R occurs through direct binding of these receptors to receptor response elements of the keratin gene promoters. The DNA binding 'C' domain of these receptors is essential for both ligand-dependent and -independent regulation. However, the NH2-terminal 'A/B' domain of T3R is not required for either mode of regulation of keratin gene expression. Furthermore, v-ErbA, an oncogenic derivative of cT3R, also activates keratin gene expression. In contrast to the previously described mechanism of gene regulation by T3R, heterodimerization with the retinoid X receptor is not essential for activation of keratin gene expression by unliganded T3R. These findings indicate that the mechanism of regulation of keratin genes by RAR and T3R differs significantly from the mechanisms described for other genes modulated by these receptors
— id: 8045, year: 1996, vol: 271, page: 1416, stat: Journal Article,

Interactions of thyroid hormone receptor with the human immunodeficiency virus type 1 (HIV-1) long terminal repeat and the HIV-1 Tat transactivator
Desai-Yajnik V; Hadzic E; Modlinger P; Malhotra S; Gechlik G; Samuels HH
1995 Aug;69(8):5103-5112, Journal of virology
Thyroid hormone (T3) receptor (T3R) regulates the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) by binding to and activating thyroid hormone response elements (TREs) embedded within the viral NF-kappa B and Sp1 motifs. The TREs within the NF-kappa B sites are necessary for activation by T3 in the absence of Tat, while those in the Sp1 motifs function as TREs only when Tat is expressed, suggesting that Tat and T3R interact in the cell. Transactivation of the HIV-1 LTR by T3R alpha and several receptor mutants revealed that the 50-amino-acid N-terminal A/B region of T3R alpha, known to interact with the basal transcription factor TFIIB, is critical for activation of both Tat-dependent and Tat-independent responsive sequences of the LTR. A single amino acid change in the highly conserved tau 1 region in the ligand-binding domain of T3R alpha eliminates Tat-independent but not Tat-dependent activation of the HIV-1 LTR by T3. Ro 5-3335 [7-chloro-5-(2-pyrryl)-3H-1,4-benzodiazepin-2(H)-one], which inhibits Tat-mediated transactivation of HIV-1, also inhibits the functional interaction between Tat and T3R alpha. Binding studies with glutathione-S-transferase fusion proteins and Western (immunoblot) analysis indicate that T3R alpha interacts with Tat through amino acids within the DNA-binding domain of T3R alpha. Mutational analysis revealed that amino acid residues in the basic and C-terminal regions of Tat are required for the binding of Tat to T3R alpha, while the N terminus of Tat is not required. These studies provide functional and physical evidence that stimulation of the HIV-1 LTR by T3 involves an interaction between T3R alpha and Tat. Our results also suggest a model in which multiple domains of T3R alpha interact with Tat and other factors to form transcriptionally important complexes
— id: 6817, year: 1995, vol: 69, page: 5103, stat: Journal Article,

A 10-amino-acid sequence in the N-terminal A/B domain of thyroid hormone receptor alpha is essential for transcriptional activation and interaction with the general transcription factor TFIIB
Hadzic E; Desai-Yajnik V; Helmer E; Guo S; Wu S; Koudinova N; Casanova J; Raaka BM; Samuels HH
1995 Aug;15(8):4507-4517, Molecular & cellular biology
The effects of the thyroid hormone (3,5,3'-triiodo-L-thyronine [T3]) on gene transcription are mediated by nuclear T3 receptors (T3Rs). alpha- and beta-isoform T3Rs (T3R alpha and -beta) are expressed from different genes and are members of a superfamily of ligand-dependent transcription factors that also includes the receptors for steroid hormones, vitamin D, and retinoids. Although T3 activates transcription by mediating a conformational change in the C-terminal approximately 220-amino-acid ligand-binding domain (LBD), the fundamental mechanisms of T3R-mediated transcriptional activation remain to be determined. We found that deletion of the 50-amino-acid N-terminal A/B domain of chicken T3R alpha (cT3R alpha) decreases T3-dependent stimulation of genes regulated by native thyroid hormone response elements about 10- to 20-fold. The requirement of the A/B region for transcriptional activation was mapped to amino acids 21 to 30, which contain a cluster of five basic amino acids. The A/B region of cT3R alpha is not required for T3 binding or for DNA binding of the receptor as a heterodimer with retinoid X receptor. In vitro binding studies indicate that the N-terminal region of cT3R alpha interacts efficiently with TFIIB and that this interaction requires amino acids 21 to 30 of the A/B region. In contrast, the LBD interacts poorly with TFIIB. The region of TFIIB primarily involved in the binding of cT3R alpha includes an amphipathic alpha helix contained within residues 178 to 201. Analysis using a fusion protein containing the DNA-binding domain of GAL4 and the entire A/B region of cT3R alpha suggests that this region does not contain an intrinsic activation domain. These and other studies indicate that cT3R alpha mediates at least some of its effects through TFIIB in vivo and that the N-terminal region of DNA-bound cT3R alpha acts to recruit and/or stabilize the binding of TFIIB to the transcription complex. T3 stimulation could then result from ligand-mediated changes in the LBD which may lead to the interaction of other factors with cT3R alpha, TFIIB, and/or other components involved in the initiation of transcription
— id: 6831, year: 1995, vol: 15, page: 4507, stat: Journal Article,

The ligand-binding domains of the thyroid hormone/retinoid receptor gene subfamily function in vivo to mediate heterodimerization, gene silencing, and transactivation
Qi JS; Desai-Yajnik V; Greene ME; Raaka BM; Samuels HH
1995 Mar;15(3):1817-1825, Molecular & cellular biology
The ligand-binding domains (LBDs) of the thyroid/retinoid receptor gene subfamily contain a series of heptad motifs important for dimeric interactions. This subfamily includes thyroid hormone receptors (T3Rs), all-trans retinoic acid (RA) receptors (RARs), 9-cis RA receptors (RARs and retinoid X receptors [RXRs]), the 1,25-dihydroxyvitamin D3 receptor (VDR), and the receptors that modulate the peroxisomal beta-oxidation pathway (PPARs). These receptors bind to their DNA response elements in vitro as heterodimers with the RXRs. Unliganded receptors in vivo, in particular the T3Rs, can mediate gene silencing and ligand converts these receptors into a transcriptionally active form. The in vivo interactions of these receptors with RXR were studied by using a GAL4-RXR chimera containing the yeast GAL4 DNA-binding domain and the LBD of RXR beta. GAL4-RXR activates transcription from GAL4 response elements in the presence of 9-cis RA. Unliganded T3R, which does not bind or activate GAL4 elements, represses the activation of GAL4-RXR by 9-cis RA in HeLa cells. However, addition of T3 alone leads to transcriptional activation. These findings suggest that T3R can repress or activate transcription while tethered to the LBD of GAL4-RXR and that heterodimerization can occur in vivo without stabilization by hormone response elements. Similar ligand-dependent activation was observed in HeLa cells expressing RAR, VDR, or PPAR and in GH4C1 cells from endogenous receptors.(ABSTRACT TRUNCATED AT 250 WORDS)
— id: 6718, year: 1995, vol: 15, page: 1817, stat: Journal Article,

Functional evidence for ligand-dependent dissociation of thyroid hormone and retinoic acid receptors from an inhibitory cellular factor
Casanova J; Helmer E; Selmi-Ruby S; Qi JS; Au-Fliegner M; Desai-Yajnik V; Koudinova N; Yarm F; Raaka BM; Samuels HH
1994 Sep;14(9):5756-5765, Molecular & cellular biology
The ligand-binding domains of thyroid hormone (L-triiodothyronine [T3]) receptors (T3Rs), all-trans retinoic acid (RA) receptors (RARs), and 9-cis RA receptors (RARs and RXRs) contain a series of heptad motifs thought to be important for dimeric interactions. Using a chimera containing amino acids 120 to 392 of chicken T3R alpha (cT3R alpha) positioned between the DNA-binding domain of the yeast GAL4 protein and the potent 90-amino-acid transactivating domain of the herpes simplex virus VP16 protein (GAL4-T3R-VP16), we provide functional evidence that binding of ligand releases T3Rs and RARs from an inhibitory cellular factor. GAL4-T3R-VP16 does not bind T3 and does not activate transcription from a GAL4 reporter when expressed alone but is able to activate transcription when coexpressed with unliganded T3R or RAR. This activation is reversed by T3 or RA, suggesting that these receptors compete with GAL4-T3R-VP16 for a cellular inhibitor and that ligand reverses this effect by dissociating T3R or RAR from the inhibitor. A chimera containing the entire ligand-binding domain of cT3R alpha (amino acids 120 to 408) linked to VP16 [GAL4-T3R(408)-VP16] is activated by unliganded receptor as well as by T3. In contrast, GAL4-T3R containing the amino acid 120 to 408 ligand-binding region without the VP16 domain is activated only by T3. The highly conserved ninth heptad, which is involved in heterodimerization, appears to participate in the receptor-inhibitor interaction, suggesting that the inhibitor is a related member of the receptor gene family. In striking contrast to T3R and RAR, RXR activates GAL4-T3R-VP16 only with its ligand, 9-cis RA, but unliganded RXR does not appear to be the inhibitor suggested by these studies. Further evidence that an orphan receptor may be the inhibitor comes from our finding that COUP-TF inhibits activation of GAL4-T3R-VP16 by unliganded T3R and the activation of GAL4-T3R by T3. These and other results suggest that an inhibitory factor suppresses transactivation by the T3Rs and RARs while these receptors are bound to DNA and that ligands act, in part, by inactivating or promoting dissociation of a receptor-inhibitor complex
— id: 8470, year: 1994, vol: 14, page: 5756, stat: Journal Article,

Divergent dimerization properties of mutant beta 1 thyroid hormone receptors are associated with different dominant negative activities
Hao E; Menke JB; Smith AM; Jones C; Geffner ME; Hershman JM; Wuerth JP; Samuels HH; Ways DK; Usala SJ
1994 Jul;8(7):841-851, Molecular endocrinology
Syndromes of resistance to thyroid hormones are caused by mutations in the T3-binding domain of the c-erbA beta thyroid hormone receptor gene. The S receptor (deletion of THR332) is a potent dominant negative protein cloned from a kindred with generalized resistance to thyroid hormones. The G-H receptor (ARG311HIS) has compromised dominant negative function and was found in both normal individuals and in a patient with severe pituitary resistance to thyroid hormones. We have investigated the mechanism responsible for the difference in receptor phenotypes by analyzing the binding of S and G-H receptors to thyroid hormone response elements with electrophoretic mobility shift analysis. Wild-type human c-erbA beta 1 (WT), S, and G-H receptors were synthesized in reticulocyte lysate, reacted with a thyroid hormone response element consisting of a direct repeat with 4 base pairs (DR+4; AGGTCA CAGG AGGTCA), and the products analyzed by gel shift. G-H receptor homodimerization was greatly impaired; G-H formed predominantly monomeric complex compared with monomeric and homodimeric WT complexes. The G-H receptor was able to form heterodimeric complexes with cellular thyroid hormone receptor auxiliary protein (TRAP) factors including the human retinoid X receptor-alpha. When TRAP was limiting, the levels of G-H heterodimeric complex were 2- to 3-fold reduced compared with WT receptor. In contrast to the WT and G-H receptors, the S receptor formed almost exclusively homodimeric complex with DR+4; the approximate ratio of S:WT:G-H homodimeric complexes at equivalent concentrations of receptors was 60:20:1. A measurable increase (1.2- to 2.6-fold) in heterodimeric complex formation was observed with the S receptor relative to WT when TRAP was at limiting concentration. As reported previously by others, thyroid hormone significantly reduced the WT homodimeric complex with DR+4. There was no effect on the S homodimeric complex. Finally, the WT, S, and G-H receptors formed different complexes with the element consisting of an inverted repeat with 5 base pairs (IR+5; AGGTCA ACAGT TGACCT) and the IR element (AGGTCA TGACCT), which were differently regulated by thyroid hormone. The S receptor bound as a homodimer with IR+5, whereas the WT receptor bound as a homodimer only with thyroid hormone. No homodimeric complex formed with IR+5 and the G-H receptor. Qualitatively similar results were observed with the IR element. We conclude that the ARG311HIS mutation severely perturbs the homodimerization and, to a much less degree, heterodimerization functions of the c-erbA beta 1 receptor. Furthermore, the THR332 deletion mutation augments homodimerization of the c-erbA beta 1 receptor. These results indicate that different mutations in the c-erbA beta 1 thyroid hormone receptor have divergently affected dimerization activities which seem to influence the level of dominant negative activity in man
— id: 20741, year: 1994, vol: 8, page: 841, stat: Journal Article,

A sequence in the rat Pit-1 gene promoter confers synergistic activation by glucocorticoids and protein kinase-C
Jong MT; Raaka BM; Samuels HH
1994 Oct;8(10):1320-1327, Molecular endocrinology
The 5'-flanking region of the gene for Pit-1, a pituitary-specific transcription factor, was isolated from a rat liver genomic library and sequenced. Expression of a reporter construct containing Pit-1 promoter sequences linked to the bacterial chloramphenicol acetyltransferase (CAT) gene was assessed by transient transfection in rat pituitary GH4C1 cells. Treatment of transfected cells with either dexamethasone (DEX) for 48 h or the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) for the final 20 h of the 48-h posttransfection period had minimal effects on CAT expression. However, CAT activity was elevated about 20-fold when transfected cells were treated with both DEX and TPA. This apparent synergistic activation was lost when DEX treatment was also limited to the final 20 h of the 48-h posttransfection period, suggesting that a time-dependent accumulation of a DEX-induced gene product might be involved. This putative DEX-induced product appeared to be relatively stable, because synergistic activation was observed in cells treated with DEX alone for 36 h, followed by a 10-h incubation without DEX before the addition of TPA. The Pit-1 gene promoter region between -210 and -142 from the transcription start site conferred synergistic regulation by DEX and TPA when placed upstream of position -105 in the herpes viral thymidine kinase promoter.(ABSTRACT TRUNCATED AT 250 WORDS)
— id: 7901, year: 1994, vol: 8, page: 1320, stat: Journal Article,

The conserved ninth C-terminal heptad in thyroid hormone and retinoic acid receptors mediates diverse responses by affecting heterodimer but not homodimer formation
Au-Fliegner M; Helmer E; Casanova J; Raaka BM; Samuels HH
1993 Sep;13(9):5725-5737, Molecular & cellular biology
The receptors for thyroid hormone (T3R), all-trans-retinoic acid (RAR), and 9-cis-retinoic acid (RXR) bind DNA response elements as homo- and heterodimers. The ligand-binding domains of these receptors contain nine conserved heptads proposed to play a role in dimerization. Mutant receptors with changes in the first or last hydrophobic amino acids in the highly conserved ninth heptad of chick T3R alpha [cT3R alpha(L365R) and cT3R(L372R)] and human RAR alpha (hRAR alpha) [hRAR(M377R) and hRAR(L384R)] reveal that this heptad is essential for certain heterodimeric interactions and for diverse functional activities. Without ligands, wild-type receptors form both homodimers and heterodimers, while these mutants form only homodimers. Surprisingly, the cognate ligand for each mutant enables heterodimer formation between cT3R(L365R) and RAR or RXR and between hRAR(M377R) and T3R or RXR. Both cT3R(L365R) and hRAR(M377R) mediate ligand-dependent transcriptional regulation. However, unlike the wild-type receptor, non-ligand-associated cT3R(L365R) does not suppress the basal activity of certain promoters containing thyroid hormone response elements, suggesting that this silencing effect of T3R is mediated by unliganded heterodimers of T3R and endogenous RXR or related factors. Heterodimerization is also necessary for the strong ligand-independent inhibition between T3R and RAR on a common response element, since the ninth-heptad mutants function as poor inhibitors. However, with a T3R-specific response element, hRAR(M377R) acts as a retinoic acid-dependent inhibitor of cT3R, indicating the importance of heterodimerization for this inhibition.(ABSTRACT TRUNCATED AT 250 WORDS)
— id: 8475, year: 1993, vol: 13, page: 5725, stat: Journal Article,

DIVERSE ACTIVITIES OF THE RETINOIC ACID AND THYROID-HORMONE RECEPTORS ARE MEDIATED BY THE CONSERVED 9TH HEPTAD REPEAT OF THE LIGAND-BINDING DOMAIN
AUFLIEGNER, M; HELMER, E; CASANOVA, J; RAAKA, BM; SAMUELS, HH
1993 APR ;41(2):A188-A188, Clinical research
— id: 54262, year: 1993, vol: 41, page: A188, stat: Journal Article,

Regulation of the human immunodeficiency virus type 1 long terminal repeat: interactions of thyroid hormone receptor with retinoid-X receptor, nuclear factor kappa B, Sp1, and Tat
Desai-Yajnik V; Samuels HH
1993 ;106:13-32, Transactions of the Association of American Physicians
— id: 13334, year: 1993, vol: 106, page: 13, stat: Journal Article,

The NF-kappa B and Sp1 motifs of the human immunodeficiency virus type 1 long terminal repeat function as novel thyroid hormone response elements
Desai-Yajnik V; Samuels HH
1993 Aug;13(8):5057-5069, Molecular & cellular biology
We report that thyroid hormone (T3) receptor (T3R) can activate the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR). Purified chick T3R-alpha 1 (cT3R-alpha 1) binds as monomers and homodimers to a region in the LTR (nucleotides -104 to -75 [-104/-75]) which contains two tandem NF-kappa B binding sites and to a region (-80/-45) which contains three Sp1 binding sites. In contrast, human retinoic acid receptor alpha (RAR-alpha) and mouse retinoid X receptor beta (RXR-beta) do not bind to these elements. However, RXR-beta binds to these elements as heterodimers with cT3R-alpha 1 and to a lesser extent with RAR-alpha. Gel mobility shift assays also revealed that purified NF-kappa B p50/65 or p50/50 can bind to one but not both NF-kappa B sites simultaneously. Although the binding sites for p50/65, p50/50, and T3R, or Sp1 and T3R, overlap, their binding is mutually exclusive, and with the inclusion of RXR-beta, the major complex is the RXR-beta-cT3R-alpha 1 heterodimer. The NF-kappa B region of the LTR and the NF-kappa B elements from the kappa light chain enhancer both function as T3 response elements (TREs) when linked to a heterologous promoter. The TREs in the HIV-1 NF-kappa B sites appear to be organized as a direct repeat with an 8- or 10-bp gap between the half-sites. Mutations within the NF-kappa B motifs which eliminate binding of cT3R-alpha 1 also abolish stimulation by T3, indicating that cT3R-alpha 1 binding to the Sp1 region does not independently mediate activation by T3. The Sp1 region, however, is converted to a functionally strong TRE by the viral tat factor. These studies indicate that the HIV-1 LTR contains both tat-dependent and tat-independent TREs and reveal the potential for T3R to modulate other genes containing NF-kappa B- and Sp1-like elements. Furthermore, they indicate the importance of other transcription factors in determining whether certain T3R DNA binding sequences can function as an active TRE
— id: 13095, year: 1993, vol: 13, page: 5057, stat: Journal Article,

Interaction of thyroid hormone and retinoic acid receptors on the regulation of the rat growth hormone gene promoter
Garcia-Villalba P; Au-Fliegner M; Samuels HH; Aranda A
1993 Mar 15;191(2):580-586, Biochemical & biophysical research communications
Retinoic acid transcriptionally regulate growth hormone (GH) gene expression through sequences located in the 5'-flanking region of the gene. A partial induction by retinoic acid was obtained with the -181 bp of the rat GH promoter, and sequences up to -209 were required for a full response. These sequences contain the previously identified thyroid hormone responsive element. The retinoid X receptor RXR increased transactivation by T3 and RA. The retinoid was relatively more effective in stimulating the native GH promoter than an heterologous promoter which contains the response element, thus showing the importance of the promoter context on transactivation by the nuclear receptors
— id: 62149, year: 1993, vol: 191, page: 580, stat: Journal Article,

An arginine to histidine mutation in codon 311 of the C-erbA beta gene results in a mutant thyroid hormone receptor that does not mediate a dominant negative phenotype
Geffner ME; Su F; Ross NS; Hershman JM; Van Dop C; Menke JB; Hao E; Stanzak RK; Eaton T; Samuels HH; et al.
1993 Feb;91(2):538-546, Journal of clinical investigation
We have examined the c-erbA beta thyroid hormone receptor gene in a kindred, G.H., with a member, patient G.H., who had a severe form of selective pituitary resistance to thyroid hormones (PRTH). This patient manifested inappropriately normal thyrotropin-stimulating hormone, markedly elevated serum free thyroxine (T4) and total triiodothyronine (T3), and clinical hyperthyroidism. The complete c-erbA beta 1 coding sequence was examined by a combination of genomic and cDNA cloning for patient G.H. and her unaffected father. A single mutation, a guanine to adenine transition at nucleotide 1,232, was found in one allele of both these members, altering codon 311 from arginine to histidine. In addition, a half-sister of patient G.H. also harbored this mutant allele and, like the father, was clinically normal. The G.H. receptor, synthesized with reticulocyte lysate, had significantly defective T3-binding activity with a Ka of approximately 5 x 10(8) M-1. RNA phenotyping using leukocytes and fibroblasts demonstrated an equal level of expression of wild-type and mutant alleles in patient G.H. and her unaffected father. Finally, the G.H. receptor had no detectable dominant negative activity in a transfection assay. Thus, in contrast to the many other beta-receptor mutants responsible for the generalized form of thyroid hormone resistance, the G.H. receptor appeared unable to antagonize normal receptor function. These results suggest that the arginine at codon 311 in c-erbA beta is crucial for the structural integrity required for dominant negative function. The ARG-311-HIS mutation may contribute to PRTH in patient G.H. by inactivating a beta-receptor allele, but it cannot be the sole cause of the disease
— id: 62210, year: 1993, vol: 91, page: 538, stat: Journal Article,

The herpes simplex virus thymidine kinase gene promoter contains a novel thyroid hormone response element
Park HY; Davidson D; Raaka BM; Samuels HH
1993 Mar;7(3):319-330, Molecular endocrinology
Thyroid hormone (T3) receptors (T3Rs) regulate transcription by binding to T3 response elements (TREs) located within promoter regions of T3-regulated genes. In rat pituitary GH4C1 cells, expression of a reporter containing herpes simplex virus thymidine kinase (TK) gene sequences (-105/+51) linked to the chloramphenicol acetyltransferase gene was stimulated 4- to 5-fold by T3. Linker scanning mutants of the TK promoter revealed that regions around -80 containing a CTF/NF-1 recognition sequence and around -10 are both required for regulation by T3. Endogenous T3Rs from GH4C1 cells labeled with [125I]T3 bound only to TK promoter DNA fragments containing the -10 region. The -22/-2 sequence (TK-TRE) contains half-sites oriented as an inverted repeat separated by 6 basepairs that are identical to and similar to an optimized TRE half-site. Purified chicken T3R alpha 1 forms apparent monomeric and dimeric complexes on the 32P-labeled TK-TRE, as found previously with an inverted repeat of the optimized TRE (TREp) with no basepair gap. T3 enhances the formation and alters the mobility of these complexes on both elements. When positioned up-stream of a heterologous promoter-chloramphenicol acetyltransferase reporter, the TK-TRE conferred T3 regulation by endogenous T3R in GH4C1 cells and by cotransfected chicken T3R alpha 1 in HeLa cells. The TK-TRE does not bind and is not activated by retinoic acid receptor. T3Rs and nuclear proteins from GH4C1, HeLa, and COS1 cells form heterodimers on the TK-TRE which differ in abundance and mobility from heterodimers formed on the TREp. The identification of a TRE in the TK promoter raises the possibility that T3R or related proteins may play important roles in regulating the life cycle of herpes simplex virus
— id: 13239, year: 1993, vol: 7, page: 319, stat: Journal Article,

Conformational changes in chicken thyroid hormone receptor alpha 1 induced by binding to ligand or to DNA
Toney JH; Wu L; Summerfield AE; Sanyal G; Forman BM; Zhu J; Samuels HH
1993 Jan 12;32(1):2-6, Biochemistry
A classic model of steroid/thyroid hormone receptor activation postulates that a conformational change or 'transformation' occurs upon ligand binding as a first step toward regulation of gene transcription. In order to test this model, physical studies have been carried out using purified full-length chicken thyroid hormone receptor alpha 1 (cT3R-alpha 1) expressed in Escherichia coli. Circular dichroism spectroscopic studies reveal that cT3R-alpha 1 adopts a different conformation upon specific binding to a cognate ligand triiodothyroacetic acid as well as to a thyroid hormone response element, an idealized inverted repeat AGGTCA TGACCT. These results suggest that cT3R-alpha 1 may adopt distinct conformations whether free or bound to ligand or to DNA. These states may reflect the changes in the conformation of steroid/thyroid hormone receptors in the signal transduction pathway
— id: 62151, year: 1993, vol: 32, page: 2, stat: Journal Article,

REGULATION OF THE HIV-1 LTR - INTERACTIONS OF THYROID-HORMONE RECEPTOR WITH RXR, NF-KAPPA-B, SP1, AND TAT
YAJNIK, V; SAMUELS, HH
1993 APR ;41(2):A201-A201, Clinical research
— id: 54264, year: 1993, vol: 41, page: A201, stat: Journal Article,

A CONSERVED HEPTAD MEDIATES HETERODIMER BUT NOT HOMODIMER INTERACTIONS OF RETINOIC ACID AND THYROID-HORMONE RECEPTORS
AUFLIEGNER, M; SAMUELS, HH
1992 APR ;40(2):A301-A301, Clinical research
— id: 51998, year: 1992, vol: 40, page: A301, stat: Journal Article,

Capacity for cooperative binding of thyroid hormone (T3) receptor dimers defines wild type T3 response elements
Brent GA; Williams GR; Harney JW; Forman BM; Samuels HH; Moore DD; Larsen PR
1992 Apr;6(4):502-514, Molecular endocrinology
Thyroid hormone response elements (T3REs) have been identified in a variety of promoters including those directing expression of rat GH (rGH), alpha-myosin heavy chain (rMHC), and malic enzyme (rME). A detailed biochemical and genetic analysis of the rGH element has shown that it consists of three hexamers related to the consensus [(A/G)GGT(C/A)A]. We have extended this analysis to the rMHC and rME elements. Binding of highly purified thyroid hormone receptor (T3R) to T3REs was determined using the gel shift assay, and thyroid hormone (T3) induction was measured in transient tranfections. We show that the wild type version of each of the three elements binds T3R dimers cooperatively. Mutational analysis of the rMHC and rME elements identified domains important for binding T3R dimers and allowed a direct determination of the relationship between T3R binding and function. In each element two hexamers are required for dimer binding, and mutations that interfere with dimer formation significantly reduce T3 induction. Similar to the rGH element, the rMHC T3RE contains three hexameric domains arranged as a direct repeat followed by an inverted copy, although the third domain is weaker than in rGH. All three are required for full function and T3R binding. The rME T3RE is a two-hexamer direct repeat T3RE, which also binds T3R monomer and dimer. Across a series of mutant elements, there was a strong correlation between dimer binding in vitro and function in vivo for rMHC (r = 0.99, P less than 0.01) and rME (r = 0.67, P less than 0.05) T3REs. Our results demonstrate a similar pattern of T3R dimer binding to a diverse array of hexameric sequences and arrangements in three wild type T3REs. Addition of nuclear protein enhanced T3R binding but did not alter the specificity of binding to wild type or mutant elements. Binding of purified T3R to T3REs was highly correlated with function, both with and without the addition of nuclear protein. T3R dimer formation is the common feature which defines the capacity of these elements to confer T3 induction
— id: 62301, year: 1992, vol: 6, page: 502, stat: Journal Article,

EVIDENCE THAT T3 MEDIATED EFFECTS OF THYROID-HORMONE RECE
CASANOVA, J; AUFLIEGNER, M; HELMER, E; SAMUELS, HH
1992 APR ;40(2):A253-A253, Clinical research
— id: 51992, year: 1992, vol: 40, page: A253, stat: Journal Article,

Half-site spacing and orientation determines whether thyroid hormone and retinoic acid receptors and related factors bind to DNA response elements as monomers, homodimers, or heterodimers
Forman BM; Casanova J; Raaka BM; Ghysdael J; Samuels HH
1992 Mar;6(3):429-442, Molecular endocrinology
The receptors for thyroid hormone (T3R) and retinoic acid (RAR) are members of a nuclear receptor subfamily that are capable of recognizing similar DNA sequences. Native response elements for T3R and RAR consist of two or more putative half-site binding motifs organized as imperfect direct or inverted repeats separated by different sized nucleotide gaps. To clarify how T3R, RAR, and related factors recognize DNA response elements, we analyzed the interaction of purified receptors with a series of inverted and direct repeats of an idealized AGGTCA half-site separated by different sized nucleotide gaps. Our results indicate that RAR and T3R can bind to half-sites as monomers and, depending on the orientation and distance between half-sites, also bind as homodimers or T3R-RAR heterodimers. T3R also binds to certain DNA elements as a heterodimer with one or more nuclear factors from eucaryotic cells. Thus, the orientation and spacing of half-sites play a central role in determining which configuration of receptors and nuclear factors will interact with a specific DNA element. This along with the ability of these factors to participate in reversible protein-protein interactions serve to broaden and diversify the responses mediated by T3R, RAR, and related members of this nuclear receptor subfamily
— id: 13686, year: 1992, vol: 6, page: 429, stat: Journal Article,

NOVEL REGULATION OF THE PIT-1 PROMOTER BY GLUCOCORTICOIDS
JONG, MTC; RAAKA, BM; SAMUELS, HH
1992 APR ;40(2):A309-A309, Clinical research
— id: 52000, year: 1992, vol: 40, page: A309, stat: Journal Article,

THYROID-HORMONE RECEPTOR STIMULATES THE HIV-1-LTR BY BINDING TO AND ACTIVATING THE VIRAL NF-KAPPA-B AND SP1 DNA ELEMENTS
YAJNIK, V; SAMUELS, HH
1992 APR ;40(2):A301-A301, Clinical research
— id: 51997, year: 1992, vol: 40, page: A301, stat: Journal Article,

Influence of thyroid hormone on ADP-ribosylation of nuclear proteins in cultured GH1 cells
Aranda A; Copp RP; Pascual A; Samuels HH
1991 Feb 25;279(2):179-183, FEBS letters
We present evidence that T3 can alter the ADP-ribosylation of chromatin associated proteins. Nuclei from GH1 cells were incubated with [adenylate-32P]NAD and the radioactivity incorporated into histone and non-histone proteins was quantitated and analyzed by gel electrophoresis and autoradiography. Incubation of GH1 cells for 24 h with T3 lowered by 40-70% the [32P]ADP-ribose incorporated into nuclear proteins. However, incubation for 3 h with T3 resulted in a stimulation instead of a decrease of in vitro [32P]ADP-ribose incorporation. The major ADP-ribosylated component electrophoresed as a 120,000 molecular mass non-histone protein, and radiolabeled histones were also observed. The same protein species were observed for all the experimental groups and T3 affected the extent of ADP-ribosylation but did not alter the sedimentation of the [32P]ADP-ribosylated components excised from chromatin after micrococcal nuclease digestion
— id: 63082, year: 1991, vol: 279, page: 179, stat: Journal Article,

Effects of varying the position of thyroid hormone response elements within the rat growth hormone promoter: implications for positive and negative regulation by 3,5,3'-triiodothyronine
Brent GA; Williams GR; Harney JW; Forman BM; Samuels HH; Moore DD; Larsen PR
1991 Apr;5(4):542-548, Molecular endocrinology
The thyroid hormone response element (T3RE) of the rat GH (rGH) promoter is located at -188 to -165 relative to the mRNA start site (TSS). Similar sites have been identified in other genes regulated by T3. We have investigated some of these T3REs in positions within the rGH promoter to assess the relative influences of DNA-binding site and position on positive and negative regulation by T3. Synthetic oligonucleotides were used with sequences from the rGH T3RE and proposed negative T3REs (nT3RE) from the rat and human alpha-subunit and rat beta TSH genes. The nT3REs were placed in the background of the wild-type rGH promoter in two positions, at -55 and down-stream of the TSS, with up- and down-mutations of the rGH T3RE. Rat GH T3RE elements were placed 700 basepairs up-stream of a basal rGH promoter and some also at the -55 and TSS positions. Constructions were tested in a transient transfection assay in rat pituitary tumor cells. Two copies of the rGHPAL (palindromic T3RE) placed 700 basepairs up-stream of the rGH promoter conferred 10-fold T3 induction. In the -55 position, the rGHPAL increased T3 induction compared to that in controls, whereas a fragment from the rat and human alpha-subunit gene in the same position reduced induction. Negative T3REs from rat beta TSH and human alpha-subunit reduced T3 induction 50% when placed at the TSS position of a rGH promoter containing an up-mutant T3RE. The T3REPAL placed at the same site increased T3 induction.(ABSTRACT TRUNCATED AT 250 WORDS)
— id: 63080, year: 1991, vol: 5, page: 542, stat: Journal Article,

pEXPRESS: a family of expression vectors containing a single transcription unit active in prokaryotes, eukaryotes and in vitro
Forman BM; Samuels HH
1991 Aug 30;105(1):9-15, Gene
We have constructed a family of expression vectors containing a single transcription unit that is active in Escherichia coli, eukaryotic cells, and in coupled in vitro transcription-translation systems. These vectors use the Rous sarcoma virus-long terminal repeat (RSV-LTR) as the promoter/enhancer for eukaryotic cells. In vitro transcription is made possible by inclusion of a bacteriophage T7 promoter. This same promoter is actively transcribed in E. coli that produce T7 RNA polymerase. Other features of this transcription unit include a high-efficiency eukaryotic translation start codon, a phage f1 origin of DNA replication for site-directed mutagenesis and a three-frame stop codon that facilitates C-terminal deletion mutagenesis. We term this vector family, pEXPRESS
— id: 13937, year: 1991, vol: 105, page: 9, stat: Journal Article,

Cloning and partial sequence of a cDNA for rabbit prothrombin
Karpatkin M; Tang ZC; Meer J; Blei F; Samuels HH
1991 Jun 15;62(6):757-763, Thrombosis research
A 1466 base pair cDNA for rabbit prothrombin has been isolated and partially sequenced. The deduced amino acid sequence shows considerable homology with the sequences of human and bovine prothrombin. The cDNA extends from the equivalent of nucleotide 516 in the bovine sequence through the coding region and 99 nucleotides in the 3' non-coding region
— id: 13995, year: 1991, vol: 62, page: 757, stat: Journal Article,

Specificity of a retinoic acid response element in the phosphoenolpyruvate carboxykinase gene promoter: consequences of both retinoic acid and thyroid hormone receptor binding
Lucas PC; Forman BM; Samuels HH; Granner DK
1991 Oct;11(10):5164-5170, Molecular & cellular biology
The ability of a retinoic acid (RA) response element (RARE) in the phosphoenolpyruvate carboxykinase (PEPCK) gene promoter to mediate effects of either RA or thyroid hormone (T3) on gene expression was studied. Fusion gene constructs consisting of PEPCK promoter sequences ligated to the chloramphenicol acetyltransferase (CAT) reporter gene were used for this analysis. While T3 induced CAT expression to a small degree (about twofold) when such constructs were transiently transfected into H4IIE rat hepatoma cells, along with an expression vector encoding the alpha subtype of the T3 receptor (TR), this effect was mediated by promoter sequences distinct from the PEPCK RARE. Although TRs were capable of binding the PEPCK RARE in the form of putative monomers, dimers, and heterodimers with RA receptors (RARs), this element failed to mediate any positive effect of T3 on gene expression. In contrast, the PEPCK RARE mediated six- to eightfold induction of CAT expression by RA. When TRs were coexpressed along with RARs in transfected H4IIE cells, this RA induction was substantially blunted in a T3-independent manner. This inhibitory effect may be due to the binding of nonfunctional TRs or TR-RAR heterodimers to the PEPCK RARE. A model is proposed to explain the previously observed in vivo effects of T3 on PEPCK gene expression
— id: 62277, year: 1991, vol: 11, page: 5164, stat: Journal Article,

Specificity of a retinoic acid response element in the phosphoenolpyruvate carboxykinase gene promoter: consequences of both retinoic acid and thyroid hormone receptor binding
Lucas PC; Forman BM; Samuels HH; Granner DK
1991 Dec;11(12):6343-6343, Molecular & cellular biology
— id: 63079, year: 1991, vol: 11, page: 6343, stat: Journal Article,

A retinoic acid response element is part of a pleiotropic domain in the phosphoenolpyruvate carboxykinase gene
Lucas PC; O'Brien RM; Mitchell JA; Davis CM; Imai E; Forman BM; Samuels HH; Granner DK
1991 Mar 15;88(6):2184-2188, Proceedings of the National Academy of Sciences of the United States of America
Several hormones, including insulin, glucagon, and glucocorticoids, regulate the expression of the rate-limiting gluconeogenic enzyme, phosphoenolpyruvate carboxykinase [GTP: oxaloacetate carboxy-lyase (transphosphorylating); EC 4.1.1.32; PEPCK] in liver. In this report we demonstrate that retinoic acid (RA) also regulates PEPCK expression by inducing a 3-fold increase in the rate of transcription of the PEPCK gene. A RA response element located between -468 and -431 in the PEPCK promoter mediates a 7-fold increase in expression of a chimeric construct containing the basal PEPCK promoter ligated to the chloramphenicol acetyltransferase reporter gene. This element confers RA responsiveness through the heterologous thymidine kinase promoter and functions relatively independent of position and orientation. An 18-base-pair core sequence (-451 to -434) (i) mediates an effect of RA on PEPCK gene expression and contains motifs found in two other RA response elements; (ii) corresponds to AF1, an accessory factor element that is an integral component of the complex glucocorticoid response unit in the PEPCK gene promoter; (iii) is in a region involved in the developmental expression of the PEPCK gene; and (iv) shows homology to elements involved in the tissue-specific regulation of genes, including the hepatic apolipoprotein genes and the alpha 1-antitrypsin gene
— id: 63081, year: 1991, vol: 88, page: 2184, stat: Journal Article,

DOMINANT NEGATIVE POTENCY OF MUTANT T3 RECE
MEIER, CA; MCCLASKEY, JH; USALA, SJ; DICKSTEIN, BM; MUCHMORE, P; BERCU, BB; SAMUELS, HH; WEINTRAUB, BD
1991 APR ;39(2):A293-A293, Clinical research
— id: 51616, year: 1991, vol: 39, page: A293, stat: Journal Article,

Thyroid hormone receptor/and v-erbA. A single amino acid difference in the C-terminal region influences dominant negative activity and receptor dimer formation
Selmi S; Samuels HH
1991 Jun 25;266(18):11589-11593, Journal of biological chemistry
Thyroid hormone receptors are cellular homologues (c-erbAs) of the v-erbA oncoprotein of the avian erythroblastosis virus. Exclusive of the viral gag region, v-erbA differs from the chick c-erbA-alpha receptor by two amino acid changes N-terminal of the DNA binding domain, two amino acid changes in the DNA binding domain, nine amino acid changes in the C-terminal region corresponding to the ligand binding domain of c-erbA, and a nine-amino acid deletion near the C terminus. v-erbA does not bind thyroid hormone and when expressed in cells inhibits the activity of wild-type thyroid hormone receptors. We reported previously that mutants of chick c-erbA/thyroid hormone receptor which lack the DNA binding domain (DBD-) inhibit transcriptional activition by wild-type thyroid hormone and retinoic acid receptors (Forman, B. M., Yang, C.-R., Au, M., Casanova, J., Ghysdael, J., and Samuels, H. H. (1989) Mol. Endocrinol. 3, 1610-1626). This dominant negative activity mapped to a series of hydrophobic heptad motifs which are conserved in the C terminus of these receptors and have been suggested to play a role in receptor dimerization. In this study we show that unlike DBD- c-erbA, DBD- v-erbA does not block receptor activity, suggesting that v-erbA acts by competing for DNA response elements rather than by formation of nonfunctional v-erbA/c-erbA heterodimers. This difference in activity was localized to a single Pro to Ser change in v-erbA just N-terminal of the last heptad motif. Introduction of this Pro to Ser change into DBD- c-erbA resulted in a protein which was inactive both functionally and in blocking receptor dimer formation in vitro
— id: 13993, year: 1991, vol: 266, page: 11589, stat: Journal Article,

Oligomeric binding of T3 receptor is required for maximal T3 response
Williams GR; Harney JW; Forman BM; Samuels HH; Brent GA
1991 Oct 15;266(29):19636-19644, Journal of biological chemistry
Receptors in the thyroid-steroid hormone superfamily bind preferentially as dimers to palindromic response elements containing two hexameric half-sites. The 23-base pair rat growth hormone (rGH) T3 response element (T3RE), however, contains three hexameric binding domains, all of which are required for maximal T3 response. We examined the binding of purified T3 receptor alpha (T3R alpha), overexpressed in Escherichia coli, to wild-type and up and down mutations of the rGH T3RE to evaluate whether transcriptional potency correlates with changes in T3R binding. T3R binds to the rGH T3RE as a monomer, dimer, or higher order oligomer. Cooperative T3R dimer binding was demonstrated to two hexameric domains of the rGH T3RE arranged as either direct or inverted repeats. Decreased binding was seen with point mutations in each domain as well as with mutations which altered hexamer orientation and spacing within the site. These results demonstrate that all three hexamers of the rGH T3RE are involved in binding T3R. Occupancy of all three hexamers by T3R in the gel shift assay was observed with functional up mutations of the T3RE, increasing receptor concentration or addition of nuclear extract. The transcriptional response potencies of T3RE up or down mutants in a transient transfection assay correlated closely with T3R binding. These results confirm our earlier hypothesis that all three hexamers of the rGH T3RE bind T3R in a novel binding arrangement and provide a model for the interaction of T3R and other nuclear proteins with the DNA sequences of thyroid hormone-regulated genes
— id: 62185, year: 1991, vol: 266, page: 19636, stat: Journal Article,

STIMULATORY, INHIBITORY, AND SYNERGISTIC EFFECTS OF RECEPTORS FOR THYROID-HORMONE AND RETINOIC ACID - ROLE OF LIGAND AND IMPLICATIONS FOR MORPHOGENESIS AND DEVELOPMENT
Au, M; Forman, BM; Casanova, J; Samuels, HH
1990 Apr;38(2):A475-A475, Clinical research
— id: 31968, year: 1990, vol: 38, page: A475, stat: Journal Article,

Dimerization among nuclear hormone receptors
Forman BM; Samuels HH
1990 Jul;2(7):587-594, New biologist
The nuclear hormone receptors comprise a superfamily of ligand-modulated transcription factors that regulate homeostasis, reproduction, development, and differentiation. The DNA-binding domain of the nuclear hormone receptors contains two zinc finger motifs and binds to response elements composed of two-half-sites separated by a variably sized gap. DNA binding specificity is accomplished by a combination of mechanisms. First, discrimination among half-site sequences is mediated by three amino acids within the first zinc finger. Second, response elements with different half-site spacing can be discriminated by five amino acids in the second zinc finger, which may act as a dimerization interface. A second dimerization signal is embedded within the ligand-binding domain of several receptors. Ligand binds to sequences adjacent to this region and enhances dimerization. It is possible that dimerization of these receptors could account for certain physiologic and pathologic conditions observed in vivo
— id: 63084, year: 1990, vol: 2, page: 587, stat: Journal Article,

Interactions among a subfamily of nuclear hormone receptors: the regulatory zipper model
Forman BM; Samuels HH
1990 Sep;4(9):1293-1301, Molecular endocrinology
The nuclear hormone receptors comprise a superfamily of ligand-modulated transcription factors that regulate homeostasis, reproduction, development, and differentiation. Three amino acids within the zinc finger DNA binding motif determine target gene specificity. Groups of receptors exist with similar DNA binding specificity. A complex carboxy terminal region mediates ligand binding, dimerization, and hormone-relieved transcriptional inactivation. We summarize the current understanding of these phenomena and suggest a novel model that structurally and functionally links these events. This 'regulatory zipper model' may explain the mechanism by which ligand activates nuclear hormone receptors
— id: 62300, year: 1990, vol: 4, page: 1293, stat: Journal Article,

COOPERATIVE AND ANTAGONISTIC INTERACTIONS AMONG THYROID-HORMONE, VITAMIN-D AND RETINOIC ACID RECEPTOR HETERODIMERS
Forman, BM; Selmi, S; Casanova, J; Pike, JW; Samuels, HH
1990 Apr;38(2):A445-A445, Clinical research
— id: 31967, year: 1990, vol: 38, page: A445, stat: Journal Article,

THE GENERALIZED THYROID-HORMONE RESISTANCE SYNDROME - SIMULATION OF THE DOMINANT NEGATIVE PHENOTYPE INVITRO WITH LIGAND-BINDING MUTANTS OF THE THYROID-HORMONE RECEPTOR
Forman, BM; Yang, CR; Casanova, J; Samuels, HH
1990 Apr;38(2):A288-A288, Clinical research
— id: 31957, year: 1990, vol: 38, page: A288, stat: Journal Article,

The homeodomain protein, Pit-1/GHF-1, is capable of binding to and activating cell-specific elements of both the growth hormone and prolactin gene promoters
Fox SR; Jong MT; Casanova J; Ye ZS; Stanley F; Samuels HH
1990 Jul;4(7):1069-1080, Molecular endocrinology
Studies were conducted to determine whether the trans-acting protein Pit-1/GHF-1 can bind to and activate promoter elements in both the GH and PRL genes that are necessary for cell-specific expression. Four pituitary cell lines that differentially express the endogenous GH and PRL genes were examined for their ability to activate GH and PRL promoter constructs containing sequences necessary for cell-specific expression (CSEs). Plasmids containing one CSE, -96 PRL and -104 GH, were similarly expressed in each of the four cell lines. Of the plasmids containing two CSEs, -173 PRL was always activated to a greater extent than -145 GH, with this relative activation being stronger in GC and GH1 cells than in 235-1 and GH4C1 cells. Protein-DNA binding assays were used to show that the GH and PRL CSEs specifically bound two highly abundant nuclear proteins (31 and 33 kDa). The two proteins were present at similar levels in all four pituitary cell lines and were recognized by a Pit-1/GHF-1 antibody. In contrast, HeLa and Rat2 cells did not activate transfected GH or PRL plasmids and did not contain nuclear proteins that specifically bound to the GH and PRL CSEs. However, cotransfection of these cells with the expression vector RSV-Pit-1/GHF-1 resulted in the activation of -173 PRL and -145 GH (PRL greater than GH). HeLa cells transfected with RSV-Pit-1/GHF-1 also contained 31- and 33-kDa nuclear proteins that bound to the GH and PRL CSEs. These results show that Pit-1/GHF-1 is present at levels in pituitary cell lines that are sufficient to activate the minimal elements in both the GH and PRL promoters necessary for cell-specific expression of these genes
— id: 63083, year: 1990, vol: 4, page: 1069, stat: Journal Article,

THE GROWTH-HORMONE GENE PROMOTER BINDS A 38 KDA PROTEIN IN ASSOCIATION WITH THE PITUITARY-SPECIFIC TRANSCRIPTION FACTOR-PIT-1/GHF-1
Fox, SR; Copp, RP; Samuels, HH
1990 Apr;38(2):A296-A296, Clinical research
— id: 31958, year: 1990, vol: 38, page: A296, stat: Journal Article,

CELL-SPECIFIC REGULATION OF TRANSCRIPTION FROM THE BETA-2 ADRENERGIC-RECEPTOR PROMOTER BY THYROID AND GLUCOCORTICOID HORMONES
Helmer, E; Raaka, BM; Samuels, HH
1990 Apr;38(2):A475-A475, Clinical research
— id: 31969, year: 1990, vol: 38, page: A475, stat: Journal Article,

THE SYNTHETIC STEROID RU486 FUNCTIONS BOTH AS A GLUCOCORTICOID AGONIST AND ANTAGONIST
Raaka, BM; Erlich, WJ; Finnerty, M; Russo, MA; Samuels, HH
1990 Apr;38(2):A475-A475, Clinical research
— id: 31970, year: 1990, vol: 38, page: A475, stat: Journal Article,

Nuclear receptors for retinoic acid and thyroid hormone regulate transcription of keratin genes
Tomic M; Jiang CK; Epstein HS; Freedberg IM; Samuels HH; Blumenberg M
1990 Nov;1(12):965-973, Cell regulation
In the epidermis, retinoids regulate the expression of keratins, the intermediate filament proteins of epithelial cells. We have cloned the 5' regulatory regions of four human epidermal keratin genes, K#5, K#6, K#10, and K#14, and engineered constructs in which these regions drive the expression of the CAT reporter gene. By co-transfecting the constructs into epithelial cells along with the vectors expressing nuclear receptors for retinoic acid (RA) and thyroid hormone, we have demonstrated that the receptors can suppress the promoters of keratin genes. The suppression is ligand dependent; it is evident both in established cell lines and in primary cultures of epithelial cells. The three RA receptors have similar effects on keratin gene transcription. Our data indicate that the nuclear receptors for RA and thyroid hormone regulate keratin synthesis by binding to negative recognition elements in the upstream DNA sequences of the keratin genes. RA thus has a twofold effect on epidermal keratin expression: qualitatively, it regulates the regulators that effect the switch from basal cell-specific keratins to differentiation-specific ones; and quantitatively, it determines the level of keratin synthesis within the cell by direct interaction of its receptors with the keratin gene promoters
— id: 14309, year: 1990, vol: 1, page: 965, stat: Journal Article,

RETINOIC ACID AND THYROID-HORMONE FUNCTION INDEPENDENTLY AND SYNERGISTICALLY TO REGULATE GROWTH-HORMONE GENE-EXPRESSION
Au, M; Aranda, A; Samuels, HH
1989 Apr;37(2):A530-A530, Clinical research
— id: 31707, year: 1989, vol: 37, page: A530, stat: Journal Article,

Identification of an adenosine 3',5'-monophosphate (cAMP)-responsive region in the rat growth hormone gene: evidence for independent and synergistic effects of cAMP and thyroid hormone on gene expression
Copp RP; Samuels HH
1989 May;3(5):790-796, Molecular endocrinology
Rat GH gene expression is known to be stimulated by several factors, including thyroid hormone and GRF. This effect of GRF appears to be mediated by cAMP resulting from activation of adenylate cyclase by the peptide. The elements of the rat GH gene important for thyroid hormone stimulation and cell-specific expression have been previously mapped using gene transfection techniques. Cell-specific expression of the gene is mediated by two cell-specific elements located from -137 to -107 and from -95 to -65. Sequences mediating thyroid hormone stimulation are thought to be located between -208 and -160. In this study, using three different methods to elevate cAMP levels in cells [forskolin, a direct activator of the adenylate cyclase catalytic subunit; 8-(4-chlorophenylthio)cAMP, a nonmetabolizable cAMP analog; and isobutylmethylxanthine, a phosphodiesterase inhibitor], we show that 5'-flanking DNA of the rat GH gene can mediate stimulation by cAMP (10- to 20-fold). The cAMP-responsive region was mapped to sequences between -104 and +11, which contains the proximal cell-specific element (-95/-65) important for cell-specific expression. Either the -97/-65 or the -104/-47 region of the gene, cloned upstream of a heterologous promoter, conferred only minimal or no activation by cAMP. This suggests that these sequences are not the direct target of cAMP action or that they are insufficient alone to mediate the cAMP response. The cAMP regulatory element (TGACGTCA) is not found between - 104 and +11, and cAMP activation does not appear to act via putative AP-2 elements, since phorbol esters did not stimulate expression.(ABSTRACT TRUNCATED AT 250 WORDS)
— id: 10665, year: 1989, vol: 3, page: 790, stat: Journal Article,

IDENTIFICATION OF A CAMP-RESPONSIVE REGION IN THE RAT GROWTH- HORMONE GENE - EVIDENCE FOR INDEPENDENT AND SYNERGISTIC EFFECTS OF CAMP AND THYROID-HORMONE
Copp, RP; Samuels, HH
1989 Apr;37(2):A357-A357, Clinical research
— id: 31698, year: 1989, vol: 37, page: A357, stat: Journal Article,

A domain containing leucine-zipper-like motifs mediate novel in vivo interactions between the thyroid hormone and retinoic acid receptors
Forman BM; Yang CR; Au M; Casanova J; Ghysdael J; Samuels HH
1989 Oct;3(10):1610-1626, Molecular endocrinology
The thyroid hormones and retinoic acid are potent modulators of differentiation, development, and gene expression. The transcriptional activities of these ligands are mediated by closely related nuclear receptors which bind and activate identical hormone responsive DNA elements. We noticed that a region within the ligand binding or E domain is well conserved between receptors for these hormones. This region contains hydrophobic heptad repeats that are structurally similar to the leucine-zipper dimerization domain. To study the function of this conserved domain, we examined the transcriptional responses of thyroid hormone receptor/c-erbA deletion mutants which lacked the heptad repeats. We previously reported that the chick c-erbA-alpha possesses hormone-independent (constitutive) activity in cells which express endogenous rat thyroid hormone receptor. We now demonstrate that this activity is abolished upon deletion of the conserved heptad repeats. This suggests that the heptad repeats mediate in vivo interactions between chick c-erbA and rat thyroid hormone receptors. To further test this hypothesis deletion mutants of chick c-erbA were constructed which contained all eight heptad repeats but which lacked the zinc-finger DNA binding domain. Although these mutants are transcriptionally inactive, they act in a dominant-negative fashion to block trans-activation by both the chick c-erbA-alpha and the endogenous thyroid hormone and retinoic acid receptors. We suggest that the heptad repeats mediate the formation of inactive mutant/wild-type hetero-dimers. Dimer formation suggests a mechanism to account for the dominant-negative phenotypes displayed by nonhormone binding variants of c-erbA, the proto-oncoprotein v-erbA and patients with the generalized thyroid hormone resistance syndrome
— id: 10483, year: 1989, vol: 3, page: 1610, stat: Journal Article,

LOCALIZATION OF A TRANSCRIPTIONAL REGULATORY DOMAIN OF A THYROID-HORMONE RECEPTOR C-ERBA THAT IS ACTIVE IN THE ABSENCE OF HORMONE-BINDING AND DNA-BINDING
Forman, BM; Yang, CR; Casanova, J; Samuels, HH
1989 Apr;37(2):A596-A596, Clinical research
— id: 31718, year: 1989, vol: 37, page: A596, stat: Journal Article,

IDENTIFICATION OF 2 NUCLEAR PROTEINS WHICH BIND SEQUENCES NECESSARY FOR CELL-SPECIFIC EXPRESSION OF BOTH THE GROWTH- HORMONE AND PROLACTIN GENES
Fox, SR; Forman, BM; Casanova, J; Stanley, F; Samuels, HH
1989 Apr;37(2):A596-A596, Clinical research
— id: 31719, year: 1989, vol: 37, page: A596, stat: Journal Article,

Characterization of the domain structure of chick c-erbA by deletion mutation: in vitro translation and cell transfection studies
Horowitz ZD; Yang CR; Forman BM; Casanova J; Samuels HH
1989 Jan;3(1):148-156, Molecular endocrinology
Chicken c-erbA (Ck-c-erbA) cDNA (1250 base pairs), a cellular homologue of the avian erythroblastosis virus v-erbA gene, encodes a 408 amino acid protein which binds L-T3 and its analogs with affinities similar to that of endogenous thyroid hormone nuclear receptors. By analogy with steroid receptors, Ck-c-erbA(Met1-Val408) contains an A and B domain (amino acids 1-50); a putative DNA binding C domain (amino acids 51-118); a hydrophilic D domain (amino acids 119-189); and a putative ligand binding E domain (amino acids 187-408). To further characterize the ligand binding region of Ck-c-erbA, two deletion mutations were constructed: Ck-c-erbA(Met120-Val408) which encodes a 289 amino acid protein lacking regions A, B, and C; and Ck-c-erbA-(Met199-Val408) which encodes a 210 amino acid protein lacking regions A, B, C, D, and the first 12 amino acids of the E region. The in vitro translation products ([35S]methionine) of cDNA transcripts of a human placental c-erbA, Ck-c-erbA (Met1-Val408), and Ck-c-erbA(Met120-Val408) efficiently bind L-[125I]T3, whereas Ck-c-erbA(Met199-Val408) does not bind L-[125I]T3. In frame substitution of the last 14 C-terminal amino acids of Ck-c-erbA(Met1-Val408) for the last 7 C-terminal amino acids of v-erbA reduces but does not eliminate L[125I]T3 binding. These results indicate that a broad region of the E domain is important for ligand binding.(ABSTRACT TRUNCATED AT 250 WORDS)
— id: 10856, year: 1989, vol: 3, page: 148, stat: Journal Article,

THE HERPES-SIMPLEX VIRAL THYMIDINE KINASE GENE PROMOTER IS TRANSCRIPTIONALLY ACTIVATED BY THYROID-HORMONE RECEPTOR
Park, HY; Samuels, HH
1989 Apr;37(2):A535-A535, Clinical research
— id: 31710, year: 1989, vol: 37, page: A535, stat: Journal Article,

The glucocorticoid antagonist 17 alpha-methyltestosterone binds to the 10 S glucocorticoid receptor and blocks agonist-mediated dissociation of the 10 S oligomer to the 4 S deoxyribonucleic acid-binding subunit
Raaka BM; Finnerty M; Samuels HH
1989 Feb;3(2):332-341, Molecular endocrinology
The glucocorticoid antagonist 17 alpha-methyltestosterone inhibits binding of the agonist [3H]triamcinolone acetonide ot the glocucorticoid receptor in cytosol prepared from rat pituitary tumor GH1 cells. Competitive binding studies indicate that the dissociation constant for 17 alpha-methyltestosterone is about 1 microM. After incubation of intact GH1 cells with 10 nM [3H]triamcinolone acetonide at 37 C and subsequent cell fractionation at 4 C, three glucocorticoid receptor forms are observed: cytosolic 10 S receptor, cytosolic 4 S receptor, and nuclear receptor. Concurrent incubation with 17 alpha-methyltestosterone reduces the amount of [3H]triamcinolone acetonide bound to each of these receptor forms. Ligand-exchange assays performed at 0 C in intact cells using [3H]triamcinolone acetonide show that the exchangeable antagonist is associated predominantly with cytosolic 10 S receptor. Immunochemical analysis using monoclonal antibody BuGR2 indicates that 17 alpha-methyltestosterone does not cause substantial accumulation of glucocorticoid receptors in GH1 cell nuclei and, when present together with agonist, reduces nuclear accumulation of receptor seen with agonist alone. Results from dense amino acid labeling studies show that unlike [3H]triamcinolone acetonide, 17 alpha-methyltestosterone does not reduce the total amount of cellular glucocorticoid receptor and does not reduce receptor half-life. These results are consistent with a model for glucocorticoid receptor transformation in which binding of agonist promotes the dissociation of an oligomeric 10 S cytosolic receptor protein to its DNA-binding 4 S subunit. The antagonist 17 alpha-methyltestosterone competes with agonist for binding to the 10 S cytosolic receptor but does not appear to promote dissociation of the oligomer, thus inhibiting agonist-mediated nuclear actions of the glucocorticoid receptor
— id: 10729, year: 1989, vol: 3, page: 332, stat: Journal Article,

Thyroid hormone receptors and action: the 5'-flanking region of the rat growth hormone gene can mediate regulated gene expression
Samuels HH; Casanova J; Copp RP; Janocko L; Raaka BM; Sahnoun H; Yaffe BM
1989 ;15(4):495-545, Endocrine research
— id: 10779, year: 1989, vol: 15, page: 495, stat: Journal Article,

Regulation of gene expression by thyroid hormone
Samuels HH; Forman BM; Horowitz ZD; Ye ZS
1989 ;51:623-639, Annual review of physiology
— id: 10831, year: 1989, vol: 51, page: 623, stat: Journal Article,

CHOLERA-TOXIN AFFECTS NUCLEAR ADP-RIBOSYLATION IN GH1 CELLS
ARANDA, A; PASCUAL, A; COPP, R; SAMUELS, H
1988 JAN 15 ;150(1):323-328, Biochemical & biophysical research communications
— id: 41863, year: 1988, vol: 150, page: 323, stat: Journal Article,

c-erbA protooncogenes mediate thyroid hormone-dependent and independent regulation of the rat growth hormone and prolactin genes
Forman BM; Yang CR; Stanley F; Casanova J; Samuels HH
1988 Oct;2(10):902-911, Molecular endocrinology
Regulation of gene expression by the thyroid hormones is thought to be mediated by a nuclear-associated receptor found in a wide variety of cells and tissues. Cellular homologues of the avian erythroblastosis virus oncogene, v-erbA, encode proteins which bind thyroid hormone with similar affinities as thyroid hormone receptors. However, it has not been shown that any of the c-erbA proteins can function as receptor and modulate thyroid hormone responsive genes. In this study, using transient expression of chimeric reporter constructs, we document that the chick fibroblast c-erbA-alpha and the human placental c-erbA-beta modulate cis-acting regulatory sequences of two thyroid hormone responsive genes; rat GH and PRL. From these results we conclude: 1) in a receptor deficient cell line (235-1) both c-erbA subtypes act as hormone-dependent modulators of PRL gene expression and hence function as thyroid hormone receptors, 2) in two different receptor containing cell lines (GH4C1 and GH1), both c-erbA proteins act in a hormone-independent fashion to regulate PRL and GH expression. This suggests that events other than ligand binding can result in formation of a c-erbA protein that modulates transcription of thyroid hormone responsive genes, 3) no qualitative functional differences were detected between alpha- and beta-c-erbA subtypes, and 4) depending on the cell-type, L-T3 acts through its endogenous receptor to stimulate (GH4C1) or suppress (GH1) expression of a chimeric PRL construct. In these cells, c-erbA expression results in the same positive or negative response as the endogenous receptor except that the response occurs in the absence of hormone. These results suggest that the endogenous receptor and the c-erbAs act by augmenting the effect of transcription factors which can positively or negatively control gene expression
— id: 10956, year: 1988, vol: 2, page: 902, stat: Journal Article,

EXPRESSION OF THYROID-HORMONE RECEPTOR RELATED PROTO-ONCOGENE ERBA PROTEINS LEADS TO HORMONE-INDEPENDENT REGULATION OF RAT GROWTH-HORMONE AND PROLACTIN GENES
Forman, BM; Yang, CR; Stanley, F; Casanova, J; Samuels, HH
1988 Apr;36(3):A552-A552, Clinical research
— id: 31507, year: 1988, vol: 36, page: A552, stat: Journal Article,

Analysis of photoaffinity label derivatives to probe thyroid hormone receptor in human fibroblasts, GH1 cells, and soluble receptor preparations
Horowitz ZD; Sahnoun H; Pascual A; Casanova J; Samuels HH
1988 May 15;263(14):6636-6642, Journal of biological chemistry
The regulation of growth hormone gene expression by thyroid hormone in cultured GH1 cells is mediated by a chromatin-associated receptor. We have previously described a photoaffinity label derivative of 3,5,3'-triiodo-L-thyronine (L-T3) in which the alanine side chain was modified to form N-2-diazo-3,3,3-trifluoropropionyl-L-T3 (L-[125I]T3-PAL). On exposure to 254 nm UV light, L-[125I]T3-PAL generates a carbene which covalently modifies two thyroid hormone receptor forms in intact GH1 cells; an abundant 47,000 Mr species and a less abundant 57,000 Mr form. We have now synthesized similar photoaffinity label derivatives of 3,5,3',5'-tetraiodo-L-thyronine (L-T4) and 3,3',5'-triiodo-L-thyronine (L-rT3). Both compounds identify the same receptor forms in intact cells and in nuclear extracts in vitro as L-[125I]T3-PAL. Labeling by L-[125I]rT3-PAL was low and consistent with the very low occupancy of receptor by L-rT3. Underivatized L-[125I]T3 and L-[125I]T4 labeled the same receptor forms at 254 nm but at a markedly lower efficiency than their PAL derivatives. In contrast, N-bromoacetyl-L-[125I]T3, a chemical affinity labeling agent, did not derivatize either receptor form in vitro. The relative efficiency of coupling to receptor at 254 nm was L-[125I]T4-PAL greater than L-[125I]T3-PAL greater than L-[125I]T4 greater than L-[125I]T3. Although L-[125I]T4-PAL has a lower affinity for receptor than L-[125I]T3-PAL, its coupling efficiency was 5-10-fold higher. This suggests that the alanine side chain of L-[125I]T4-PAL is positioned in the ligand binding region near a residue which is efficiently modified by photoactivation. With L-[125I]T4-PAL we were able to identify three different molecular weight receptor species in human fibroblast nuclei
— id: 11091, year: 1988, vol: 263, page: 6636, stat: Journal Article,

CHARACTERIZATION OF DOMAIN-STRUCTURE OF THE CHICK C-ERBA CDNA BY DELETION MUTATION - INVITRO TRANSLATION AND TRANSFECTION STUDIES
Horowitz, Z; Forman, BM; Yang, CR; Casanova, J; Samuels, HH
1988 Apr;36(3):A385-A385, Clinical research
— id: 31496, year: 1988, vol: 36, page: A385, stat: Journal Article,

Identification of the cis-acting elements and trans-acting factors that mediate cell-specific and thyroid hormone stimulation of growth hormone gene expression
Samuels HH; Aranda A; Casanova J; Copp RP; Flug F; Forman BM; Horowitz ZD; Janocko L; Park HY; Pascual A; et al.
1988 ;44:53-114, Recent progress in hormone research
— id: 63085, year: 1988, vol: 44, page: 53, stat: Journal Article,

Regulation of gene expression by thyroid hormone
Samuels HH; Forman BM; Horowitz ZD; Ye ZS
1988 Apr;81(4):957-967, Journal of clinical investigation
— id: 11135, year: 1988, vol: 81, page: 957, stat: Journal Article,

CONTROL OF RAT PROLACTIN GENE-EXPRESSION - L-T3 HAS BOTH POSITIVE AND NEGATIVE EFFECTS IN DIFFERENT CELL-LINES
Stanley, FM; Casanova, J; Samuels, HH
1988 Apr;36(3):A391-A391, Clinical research
— id: 31497, year: 1988, vol: 36, page: A391, stat: Journal Article,

Rat growth hormone gene expression. Both cell-specific and thyroid hormone response elements are required for thyroid hormone regulation
Ye ZS; Forman BM; Aranda A; Pascual A; Park HY; Casanova J; Samuels HH
1988 Jun 5;263(16):7821-7829, Journal of biological chemistry
The elements involved in mediating cell-specific and thyroid hormone stimulation of rat growth hormone gene expression have been defined by transfection studies and by nuclease footprinting. 5'-Flanking DNA extending to -104 can mediate cell-specific expression, and this is enhanced 3- to 4-fold with DNA extending to -145. Cell-specific factors, found only in rat growth hormone producing cells, bind within the -137/-107 and -95/-65 regions, and competition studies suggest that the same factor binds to both sites. The sequence A (A or T) TAAAT is found at the center of both footprints at -80 and -122, suggesting that it is a core component of the recognition sequence of the cell-specific factor. Disruption of the spatial and/or distance relationships between the two regions eliminates the enhanced level of cell-specific expression, suggesting a cooperative interaction of the proteins which bind to these elements. Sequences located between -208 and -178 can confer thyroid hormone-regulated expression when linked in either orientation in close proximity to one or both cell-specific elements. The thyroid hormone and cell-specific elements function as an enhancer-like unit and are both required to confer regulated expression to heterologous promoters. We propose that thyroid hormone acts via its receptor to enhance the function of the cell-specific element by forming a more 'active' transcription complex which stimulates the level of gene expression
— id: 11066, year: 1988, vol: 263, page: 7821, stat: Journal Article,

Rat growth hormone gene expression: both cell-specific and thyroid hormone response elements are required for thyroid hormone regulation
Ye ZS; Forman BM; Park HY; Casanova J; Samuels HH
1988 ;101:42-53, Transactions of the Association of American Physicians
The elements that we have identified in the 5'-flanking region of the rat growth hormone gene are shown in Figure 7, and the following conclusions are drawn: 1) Cell-specific expression of the rat growth hormone gene is mediated by two CSEs, which are located from -95 to -65 and from -137 to -107.2) These CSEs bind a common cell-specific trans-acting factor (GH-CSF), which is found in growth hormone-producing cells. 3) Enhanced levels of cell-specific expression may involve a protein-protein interaction when this factor binds on the same side of the DNA helix as the two CSEs. 4) A TRE is located between -208 and -178. 5) Activation of the gene by thyroid hormone appears to require both the TRE and one of the CSEs, and both are required to confer L-T3 stimulation to several heterologous promoters. 6) Our studies support the notion that stimulation by L-T3 involves the binding of the L-T3-receptor complex to the TRE, which enhances the function of the CSEs, resulting in stimulation of growth hormone gene expression
— id: 11262, year: 1988, vol: 101, page: 42, stat: Journal Article,

RAT GROWTH-HORMONE GENE-EXPRESSION - BOTH CELL-SPECIFIC AND THYROID-HORMONE RESPONSE ELEMENTS ARE REQUIRED FOR THYROID- HORMONE REGULATION
Ye, ZS; Forman, BM; Park, HY; Casanova, J; Samuels, HH
1988 Apr;36(3):A610-A610, Clinical research
— id: 31515, year: 1988, vol: 36, page: A610, stat: Journal Article,

cis-acting elements of the rat growth hormone gene which mediate basal and regulated expression by thyroid hormone
Flug F; Copp RP; Casanova J; Horowitz ZD; Janocko L; Plotnick M; Samuels HH
1987 May 5;262(13):6373-6382, Journal of biological chemistry
In GC cells, a growth hormone-producing rat pituitary cell line, 3,5,3'-triiodo-L-thyronine (L-T3) rapidly stimulates the transcription rate of the growth hormone gene which parallels the level of chromatin-associated L-T3-receptor complexes (Yaffe, B. M., and Samuels, H. H. (1984) J. Biol. Chem. 259, 6284-6291). In this study we have functionally mapped the elements of the gene which are involved in mediating basal and hormone-regulated expression. Stable transformation studies indicate that transcriptional regulation of the gene by L-T3 is mediated by sequences in the 5'-flanking region. Transient expression studies were performed using a series of chimeric plasmids in which 5'-flanking DNA was ligated to the chloramphenicol acetyltransferase gene. Transient expression occurred only in cells which expressed the endogenous growth hormone gene. Sequences between -104 and +7 were found to be essential for basal expression. One of the most highly conserved regions (-105 to -145) contains elements which further enhance the level of basal expression but are not necessary for regulated expression by L-T3. DNA between -210 and -181 was found to be essential for stimulation by L-T3 and was shown to function most efficiently with the homologous rat growth hormone promoter (-104 to +7). Sequences from -206 to -198 show about 80% homology with a sequence in the 5'-flanking region of two other rat genes which are regulated by thyroid hormone. Glucocorticoid hormones, which also transcriptionally stimulate the rat growth hormone gene, elicited only minimal effects in both stable and transient expression studies. This suggests that the elements which mediate glucocorticoid regulation of the endogenous gene are found either upstream of the cloned 5'-flanking region (1800 base pairs) or 3' of the cap site
— id: 63086, year: 1987, vol: 262, page: 6373, stat: Journal Article,

Photoaffinity labeling of thyroid hormone receptors
Horowitz ZD; Samuels HH
1987 ;33(2-3):317-332, Pharmacology & therapeutics
Photoaffinity label probes of iodothyronines can interact with nuclear receptors in intact cells and in solubilized receptor preparations. These probes have certain advantages over a chemical affinity label in analyzing receptor structure. First, a photoaffinity label probe covalently cross-links only after photoactivation. Therefore, it is possible to demonstrate with appropriate competitive inhibition studies that the photoaffinity label probe associates with the receptor in question. Secondly, since cross-linking only occurs after photolysis, it is possible to adjust the concentration of the photoaffinity label to maximize association with 'specific' binding sites relative to 'non-specific' associations prior to covalent linkage by photoactivation. The different [125I]iodothyronine-PAL analogues may be useful as probes of the thyroid hormone receptor binding domain since PAL compounds with different affinities for receptor may photocouple to different receptor residues within or proximate to the hormone binding region. These probes may also be useful as an adjunct to receptor purification and in probing the organization of the receptor in chromatin. Lastly, they may provide insights into possible alterations of receptor structure in patients with partial end organ resistance to thyroid hormone (Refetoff et al., 1967; Eil et al., 1982)
— id: 11421, year: 1987, vol: 33, page: 317, stat: Journal Article,

CONTROL OF GROWTH-HORMONE GENE-EXPRESSION BY THYROID-HORMONE
Samuels, HH; Copp, RP; Casanova, J; Janocko, L; Plotnick, M; Ye, ZS
1987 Mar 1;46(3):791-791, Federation Proceedings (Federation of American Societies for Experimental Biology)
— id: 31266, year: 1987, vol: 46, page: 791, stat: Journal Article,

Cell- and sequence-specific binding of nuclear proteins to 5'-flanking DNA of the rat growth hormone gene
Ye ZS; Samuels HH
1987 May 5;262(13):6313-6317, Journal of biological chemistry
Stimulation of growth hormone gene transcription in several rat pituitary cell lines (e.g. GC and GH1) is mediated by a thyroid hormone nuclear receptor which is a DNA binding protein. We report that these cell lines contain nuclear proteins which selectively interact with sequences found within the first 236 base pairs of 5'-flanking DNA of the rat growth hormone gene. Sequences found between -104 and -49 base pairs, relative to the transcription initiation (cap) site, bind to nuclear protein(s) which appears to be cell type specific and generate a DNase I-resistant footprint on both strands between -95 and -68. A distinct protein component(s) selectively binds to DNA between -236 and -146 but is not cell type specific. These regions correspond to those found in gene transfer studies to be important in mediating basal expression (-104/+7) and thyroid hormone-regulated expression (-236/-146) of the gene
— id: 63087, year: 1987, vol: 262, page: 6313, stat: Journal Article,

LOCALIZATION OF HORMONE REGULATORY ELEMENTS OF THE RAT GROWTH-HORMONE GENE
FLUG, F; PLOTNIK, M; CASANOVA, J; COPP, RP; SAMUELS, HH
1986 APR ;34(2):A714-A714, Clinical research
— id: 41411, year: 1986, vol: 34, page: A714, stat: Journal Article,

Stimulation of facilitated [3H]uridine transport by thyroid hormone in GH1 cells. Evidence for regulation by the thyroid hormone nuclear receptor
Stanley F; Tsai JS; Samuels HH
1986 Jul 15;261(20):9400-9404, Journal of biological chemistry
We have previously shown that 3,5,3'-triiodo-L-thyronine (L-T3) stimulates cell growth and a 4- to 8-fold increase in growth hormone mRNA in GH1 cells. These effects appear to be mediated by a thyroid hormone nuclear receptor with an equilibrium dissociation constant for L-T3 of 0.2 nM and an abundance of about 10,000 receptors per cell nucleus. In this report, we show that L-T3 exerts a pleiotypic effect on GH1 cells to rapidly (within 2 h) stimulate [3H]uridine uptake to a maximal value of 2.5- to 3-fold after 24 h. This results from an increase in the number of functional uridine 'transport sites' as shown by studies documenting an increase in the apparent Vmax with no change in the Km, 17 microM. Although the labeling of the cellular uridine pool and pools of all phosphorylated uridine derivatives was increased by L-T3, there was no change in the relative amounts of the individual pools in cells incubated with or without hormone. The intracellular concentration of [3H]uridine was estimated to be similar to that of the medium, suggesting that facilitated transport mediates [3H]uridine uptake. That this increase in [3H]uridine transport was nuclear receptor-mediated is supported by the excellent correspondence of the L-T3 dose-response curve for [3H]uridine uptake and that for L-T3 binding to receptor. Finally, inhibition of protein synthesis by cycloheximide and RNA synthesis by actinomycin D demonstrated that the L-T3 effect required continuing protein and RNA synthesis. These results are consistent with an effect of the L-T3-nuclear receptor complex to increase uridine uptake in GH1 cells by altering the expression of gene(s) essential for the transport process
— id: 62194, year: 1986, vol: 261, page: 9400, stat: Journal Article,

5'-Flanking DNA of the rat growth hormone gene mediates regulated expression by thyroid hormone
Casanova J; Copp RP; Janocko L; Samuels HH
1985 Sep 25;260(21):11744-11748, Journal of biological chemistry
Thyroid hormone has been shown to rapidly stimulate the rate of rat growth hormone gene transcription which parallels the kinetics of binding of 3,5,3'-triiodo-L-thyronine (L-T3) to its nuclear receptor (Yaffe, B. M., and Samuels, H. H. (1984) J. Biol. Chem. 259, 6284-6291). We have constructed a chimeric gene to explore whether the 5'-flanking region of the rat growth hormone gene contains a DNA element which could mediate thyroid hormone control of growth hormone gene expression. The construct consists of 1.8 kilobase pairs of the 5'-flanking region extending 11 nucleotides downstream from the transcription initiation (cap) site ligated to Escherichia coli DNA containing the structural gene for the enzyme xanthine-guanine phosphoribosyltransferase. GC cells, a growth hormone producing rat pituitary cell line, were transfected with this chimeric gene and stable transformants in which the enzyme is regulated by L-T3 were isolated by positive selection using mycophenolic acid and xanthine. These stable transformants develop with relatively high frequency and show marked L-T3 stimulation of xanthine-guanine phosphoribosyltransferase mRNA which is initiated at the cap site of the growth hormone gene. This study provides the first evidence that the 5'-flanking region of the rat growth hormone gene contains a DNA regulatory element which can mediate control of gene expression by thyroid hormone
— id: 63089, year: 1985, vol: 260, page: 11744, stat: Journal Article,

Triamcinolone acetonide regulates glucocorticoid-receptor levels by decreasing the half-life of the activated nuclear-receptor form
McIntyre WR; Samuels HH
1985 Jan 10;260(1):418-427, Journal of biological chemistry
Glucocorticoid-receptor activation in GH1 cells results from the conversion of a 10 S oligomeric cytosolic form to a 4-5 S nuclear-binding species (Raaka, B. M., and Samuels, H. H. (1983) J. Biol. Chem. 258, 417-425). In this study, we report that triamcinolone acetonide (9 alpha-fluoro-11 beta, 16 alpha, 17 alpha, 21-tetrahydroxypregna-1,4-diene-3,20-dione 16,17-acetonide) elicits a time- and dose-dependent reduction of total-cell (nuclear + cytoplasmic) receptor. The mechanism of receptor regulation was studied by dense amino acid labeling of receptor using media containing 2H, 13C, and 15N-labeled amino acids. Total cell receptor was extracted with 0.4 M KCl and newly synthesized dense receptor was separated from pre-existing receptor of normal density by centrifugation in gradients of 15-30% sucrose (w/v) in D2O. Receptor levels in cells grown without [3H]triamcinolone acetonide was 260 +/- 19 fmol/100 micrograms of DNA (16,000 molecules/cell), and, with 10 nM [3H]triamcinolone acetonide, this decreased to 130 +/- 14 fmol/100 micrograms of DNA after 30 h. Receptor half-life was 19 +/- 1.9 h in the absence and 9.5 +/- 0.3 h in the presence of triamcinolone acetonide and accounted for the decrease in steady-state receptor levels. Receptor synthesis was 9.7 +/- 0.3 fmol/100 micrograms of DNA/h (580 molecules/cell/h) both in the presence and absence of 10 nM [3H]triamcinolone acetonide. Triamcinolone acetonide reduced the half-life in proportion to the extent of receptor occupancy and activation. During the approach to steady-state conditions, 10 nM [3H]triamcinolone acetonide shortened receptor half-life almost immediately to the value in cells grown with [3H]triamcinolone acetonide for 24 h or longer. Cycloheximide did not prevent the triamcinolone acetonide-mediated decrease in receptor half-life and the shortening of receptor half-life is rapidly reversed by removal of hormone. These studies support a model of receptor regulation in which triamcinolone acetonide converts the unactivated 10 S receptor to the activated 4-5 S form which is degraded at an increased rate by the cell
— id: 63090, year: 1985, vol: 260, page: 418, stat: Journal Article,

Effects of molybdate on steroid receptors in intact GH1 cells. Evidence for dissociation of an intracellular 10 S receptor oligomer prior to nuclear accumulation
Raaka BM; Finnerty M; Sun E; Samuels HH
1985 Nov 15;260(26):14009-14015, Journal of biological chemistry
Treatment of intact GH1 cells with sodium molybdate inhibits the subsequent rate of nuclear accumulation of hormone-occupied glucocorticoid and estrogen receptors. Cells were incubated at 23 degrees C for 1 h with 30 mM molybdate and then for up to 30 min with [3H]triamcinolone acetonide or [3H]estradiol in the continued presence of molybdate. Although molybdate did not affect the rate of receptor occupancy with either steroid, cells treated with molybdate had more occupied cytosolic and fewer occupied nuclear receptors than control cells. For the glucocorticoid receptor, cells treated with molybdate had more 10 S and fewer 4 S cytosolic receptors than control cells. In low salt cytosol molybdate inhibits the temperature-mediated subunit dissociation of occupied 10 S glucocorticoid receptor. These results suggest that a hormone-mediated dissociation of an intracellular 10 S oligomeric glucocorticoid receptor form to its 4 S subunits is required prior to accumulation of occupied receptors in the nuclear fraction. In cells incubated at 37 degrees C for 1 h or longer with [3H]triamcinolone acetonide, molybdate shifts the steady state intracellular distribution of receptor toward the 10 S cytosolic receptor form, consistent with the interpretation that molybdate affects the rapidly exchanging subunit equilibrium between the 10 S and 4 S cytosolic forms by slowing the rate of 10 S receptor dissociation. Molybdate prevents loss of glucocorticoid-occupied 10 S but not 4 S receptors in heated cytosol by stabilizing the relatively protease-resistant 10 S receptor. Since molybdate stabilizes 10 S oligomeric steroid receptors in vitro, the effects of molybdate on nuclear accumulation of occupied receptors in intact cells support the intracellular existence and physiological relevance of 10 S glucocorticoid and estrogen receptors. These results support a general model for steroid receptor activation in which binding of hormone promotes dissociation of intracellular 8-10 S oligomeric receptors to their DNA-binding subunits
— id: 63088, year: 1985, vol: 260, page: 14009, stat: Journal Article,

MOLYBDATE INHIBITS NUCLEAR ACCUMULATION AND INCREASES THE HALF-LIFE OF THE ESTROGEN-RECEPTOR IN GH CELLS
Raaka, BM; Finnerty, M; Samuels, HH
1985 ;44(5):1474-1474, Federation Proceedings (Federation of American Societies for Experimental Biology)
— id: 30952, year: 1985, vol: 44, page: 1474, stat: Journal Article,

5'-flanking DNA sequences of the growth hormone gene mediates thyroid hormone stimulation of growth hormone gene transcription
Samuels HH; Casanova J; Copp RP; Janocko L
1985 ;98:55-65, Transactions of the Association of American Physicians
— id: 63091, year: 1985, vol: 98, page: 55, stat: Journal Article,

5'-FLANKING DNA-SEQUENCES OF THE GROWTH-HORMONE GENE MEDIATE THYROID-HORMONE STIMULATION OF GROWTH-HORMONE GENE- TRANSCRI
Samuels, HH; Casanova, J; Copp, RP; Janocko, L
1985 ;33(2):A601-A601, Clinical research
— id: 30933, year: 1985, vol: 33, page: A601, stat: Journal Article,

Modulation of thyroid hormone nuclear receptors by cholera toxin in cultured GH1 cells
Aranda A; Samuels HH
1984 May 25;259(10):6110-6116, Journal of biological chemistry
The cellular actions of the thyroid hormones L-thyroxine and L-triiodothyronine are mediated by the association of hormone with a chromatin-associated receptor. In cultured GH1 cells, a hormone-responsive rat pituitary cell line, thyroid hormone decreases the concentration of its receptor at early incubation times by reducing the accumulation of newly synthesized receptor. In this study, we demonstrate that cholera toxin also reduces the amount of nuclear receptor in GH1 cells in a time- and dose-dependent fashion, without altering the affinity of the receptor for hormone. The reduction of receptor mediated by cholera toxin is not secondary to a generalized inhibition of cell protein synthesis or cell replication rates and this effect can be abolished by pretreatment of the cholera toxin with soluble ganglioside II3-alpha-N- acetylneuraminosylgangliotetraosylceramide . This effect requires an intact cholera toxin molecule and does not occur at similar concentrations of the membrane-binding B subunit of cholera toxin. In order to study the influence of cholera toxin on thyroid hormone receptor turnover, we have used a dense amino acid-labeling technique. The results indicate that cholera toxin does not change the half-life of receptor, but decreases the rate of appearance of newly synthesized receptor. This decreased rate completely accounts for the lowered steady state receptor levels. The extent of cAMP stimulation by cholera toxin does not correlate with the extent of receptor reduction and forskolin, which stimulates cAMP 25- to 500-fold, does not decrease thyroid hormone receptor abundance. These studies suggest that cholera toxin modulates receptor levels by a mechanism(s) that is not mediated by cAMP in GH1 cells
— id: 62193, year: 1984, vol: 259, page: 6110, stat: Journal Article,

Photoaffinity labeling of thyroid hormone nuclear receptors. Influence of n-butyrate and analysis of the half-lives of the 57,000 and 47,000 molecular weight receptor forms
Casanova J; Horowitz ZD; Copp RP; McIntyre WR; Pascual A; Samuels HH
1984 Oct 10;259(19):12084-12091, Journal of biological chemistry
The thyroid hormone receptor is a nuclear-associated protein which appears to mediate the actions of 3,5,3'-triiodo-L-thyronine (L-T3) and 3,5,3',5'-tetraiodo-L-thyronine (L-T4) in mammalian cells. In a previous study we reported that N-2-diazo-3,3,3-trifluoropropionyl-3,5,3'-triiodo-L-thyronine (L-T3-PAL) serves as an effective photoaffinity label probe of the receptor in GH1 cells, a growth hormone producting rat pituitary cell line. Irradiation of cells at 254 nm covalently cross-links L-[125I]T3-PAL to two molecular weight (Mr) nuclear receptor forms, an abundant 47,000 Mr component and a less abundant 57,000 Mr species (Pascual, A., Casanova, J., and Samuels, H. H. (1982) J. Biol. Chem. 257, 9640-9647). In this study we have explored a number of possible interrelationships of the different Mr receptor forms. Denaturing gel electrophoresis and autoradiography indicates that the 57,000 Mr form is a doublet species which differ in Mr by 1,000 to 2,000. The various receptor forms are not an artifact of the L-[125I]T3-PAL probe, and identical forms can be labeled at 310 nm using underivatized L-[125I]T4 with a 15-fold lower coupling efficiency. The 57,000 and 47,000 Mr receptor forms are not generated by indiscriminate proteolysis, UV peptide cleavage, or zero length protein-protein cross-linking by irradiation at 254 nm. Micrococcal nuclease excises both the 57,000 and 47,000 Mr forms, and receptor is not identified in the residual nuclear matrix fraction. Receptor is also not detected in the cytoplasmic fraction. By coupling dense amino acid labeling and photoaffinity labeling of receptor we determined a half-life of 2.4 h for the 57,000 Mr species and 5.6 h for the 47,000 Mr form while both species have similar relative synthetic rates. n-Butyrate has been previously shown to decrease receptor levels in GH1 cells. We demonstrate that n-butyrate decreases receptor levels primarily by shortening the half-life of the 47,000 Mr form
— id: 63092, year: 1984, vol: 259, page: 12084, stat: Journal Article,

PHOTOAFFINITY-LABELING OF THYROID-HORMONE RECEPTORS - ANALYSIS OF THE NUCLEAR-INTERACTIONS, SYNTHETIC RATE, AND HALF-LIFE OF THE 57K AND K-47 RECEPTOR FORMS
HOROWITZ, ZD; CASANOVA, J; SAMUELS, HH
1984 ;32(2):A546-A546, Clinical research
— id: 40982, year: 1984, vol: 32, page: A546, stat: Journal Article,

Hormonal regulation of the growth hormone gene. Relationship of the rate of transcription to the level of nuclear thyroid hormone-receptor complexes
Yaffe BM; Samuels HH
1984 May 25;259(10):6284-6291, Journal of biological chemistry
Using cultured GH1 cells, we reported that stimulation (3- to 5-fold) of growth hormone synthesis and mRNA levels by thyroid hormone is mediated by a chromatin-associated receptor. Thyroid hormone also elicits a rapid reduction of homologous receptor in GH1 cells primarily by decreasing the synthetic rate of receptor ( Raaka , B. M., and Samuels , H. H. (1981) J. Biol. Chem. 256, 6883-6889). Without 3,5,3'-triiodo-L-thyronine (L-T3), glucocorticoid agonists induced a limited and delayed effect while L-T3 + glucocorticoid synergistically stimulated the response an additional 2- to 4-fold compared to L-T3. In this study, we utilized GC cells, a related cell line, to compare the abundance of L-T3-receptor complexes to the rate of growth hormone mRNA synthesis and gene transcription. Gene transcription was assessed by in vitro labeling of nuclei with [alpha-32P]UTP which were derived from cells incubated with hormone(s), while mRNA synthesis was determined in intact cells by [3H]uridine labeling. Labeled growth hormone mRNA and gene transcripts were quantitated by filter hybridization to plasmid containing growth hormone cDNA. L-T3 rapidly decreased receptor levels in GC cells with kinetics similar to that in GH1 cells. Both the L-T3 and the synergistic L-T3 + glucocorticoid stimulation of growth hormone mRNA synthesis changed in parallel with the level of L-T3-receptor complexes. Glucocorticoid hormones alone elicited a variable response which resulted in minimal stimulation or inhibition of growth hormone mRNA synthesis or gene transcription rates. No apparent lag was identified between the kinetics of L-T3 binding to receptor and stimulation of growth hormone gene transcription. L-T3 stimulated growth hormone gene transcription rates maximally in 1 h which then progressively decreased in parallel with L-T3-receptor levels. Using [3H]uridine pulse-chase, growth hormone mRNA was found to have a half-life of approximately 50 h in agreement with the decay curve of growth hormone production of deinduced cells. Our studies suggest that regulation of the growth hormone response is predominantly determined by positive control of growth hormone gene transcription which is proportional to the concentration of thyroid hormone-receptor complexes
— id: 63094, year: 1984, vol: 259, page: 6284, stat: Journal Article,

HORMONAL-REGULATION OF GROWTH-HORMONE GENE-TRANSCRIPTION IS DIRECTLY INFLUENCED BY THE LEVEL OF NUCLEAR THYROID HORMONE-RECEPTOR COMPLEXES
YAFFE, BM; SAMUELS, HH
1984 ;32(2):A488-A488, Clinical research
— id: 40978, year: 1984, vol: 32, page: A488, stat: Journal Article,

Molecular basis of thyroid hormone action
Oppenheimer, Jack H.; Samuels, Herbert H.; Apriletti, James W
New York : Academic Press, 1983,
— id: 111, year: 1983, vol: , page: , stat: ,