General Receptor for Phosphoinositides 1, a Novel Repressor of Thyroid Hormone Receptor Action that Prevents Deoxyribonucleic Acid Binding
Marie-Belle Poirier1,
Genevieve Hamann1,
Marie-Eve Domingue,
Melanie Roy,
Tayna Bardati and
Marie-France Langlois
Department of Medicine and Physiology, Division of Endocrinology, Faculté de médecine et des sciences de la santé, Université de Sherbrooke, Sherbrooke, Quebec, Canada J1H 5N4
Address all correspondence and requests for reprints to: Marie-France Langlois, M.D., Department of Medicine, Division of Endocrinology, Faculté de médecine et des sciences de la santé, Université de Sherbrooke, Centre Hospitalier Universitaire de Sherbrooke, 3001 12th Avenue North, Sherbrooke, Quebec, Canada J1H 5N4. E-mail: Marie-France.Langlois{at}USherbrooke.ca.
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ABSTRACT
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Thyroid hormone receptors (TRs) bind to response elements (TREs) located in the promoter region of target genes and modulate their transcription. The effects of TRs require the presence of coregulators that act as adaptor molecules between TRs and complexes that are involved in chromatin remodeling or that directly contact the basal transcription machinery. Using the yeast two-hybrid system, we identified a new interacting partner for TRs: GRP1 (general receptor for phosphoinositides-1), a nucleotide exchange factor, which had never been shown to interact with nuclear receptors. We reconfirmed the interaction between TRs and GRP1 in yeast and glutathione-S-transferase pull-down assays, and determined the areas of TRs and GRP1 involved in the interaction. Coimmunoprecipitation studies demonstrated that the interaction between GRP1 and TRs takes place in the cytoplasm and the nucleus of mammalian cells. To assess functional consequences of the interaction, we used transient transfection of CV-1 cells with TR and GRP1 expression vectors and luciferase reporter genes. On positive TREs, GRP1 decreased activation by 4560%. On the negative TREs it increased repression by blunting the activation in the absence of T3, except for TRß2, which was not affected. Using EMSA, we have determined that addition of GRP1 diminishes the formation of TR/TR homodimers and TR/retinoid X receptor heterodimers on TREs, which could explain the effect of GRP1 on transcription. Furthermore, protein interaction assays using increasing concentrations of double-stranded TREs show a dose-dependent decrease of the interaction between GRP1 and TRs. The homo/heterodimers formed by TRs and retinoic X receptor-
were not influenced by the presence of GRP1, also suggesting that GRP1 interferes directly with DNA binding. Taken together, these data provide evidence that GRP1 is a new corepressor for TRs, which modulates both positive and negative regulation by T3 by decreasing TR-complex formation on TREs.
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INTRODUCTION
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THYROID HORMONES produce important physiological effects in development, growth, and homeostasis. Cellular effects of thyroid hormones are principally mediated by the binding of T3 to thyroid hormone receptors (TRs), which are part of the nuclear receptor superfamily of ligand-dependent transcription factors (1). It is well described that most effects of thyroid hormones are due to the regulation of target gene expression by TRs. In addition, there is evidence that thyroid hormones also have rapid nongenomic pathways involving cytoskeletal remodeling, mitochondrial respiration, and several signalization pathways (2).
In vertebrates, two genes code for TRs: c-erbA
and c-erbAß, from which three active isoforms are derived: TR
1, TRß1, and TRß2. TRs share domain organization similar to other nuclear receptors (Fig. 1A
). The N-terminal transactivation region (domain A/B) is the least conserved between TR isoforms and responsible for some of their functional differences (3, 4). The DNA-binding domain (DBD, domain C) of TRs is composed of two zinc fingers and is mainly responsible for the recognition of the thyroid hormone response element (TRE), a consensus DNA sequence located in the promoter region of target genes. The hinge region (domain D) contains the nuclear localization signal and interacts with corepressors. Finally, the carboxyl-terminal ligand-binding domain (LBD, domain E/F) is responsible for the binding of T3, dimerization, and recruitment of some coactivators.

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Fig. 1. Schematic Representation of the Constructions Used in This Study
A, TRß2wt, TRß2 bait used for yeast two-hybrid screen, and TRß2 mutants. B, Wild-type GRP1 (GRP1wt) and deletion mutants.
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Recent years have yielded prolific results in the discovery of partners for nuclear receptors, including TRs, which are important for the mediation of their transcriptional effects. These proteins, corepressors and coactivators, are crucial in thyroid hormone action and generally act as adaptor molecules between TRs and complexes that are either involved in chromatin remodeling or recruitment of the basal transcription machinery (5, 6). Most known coregulators interact with the hinge region or LBD of nuclear receptors; this may be due, in part, to the receptor domains that have been used as bait to screen libraries for new partners. However, other binding sites for coregulators have been described. For example, the N terminus of TRß2 interacts with the silencing domain of the corepressor SMRT (silencing mediator of retinoic and thyroid hormone receptor), and this interaction blocks the recruitment of other components of the corepressor complex (7). Recently, some coregulators have been described to interact with the DBD of nuclear receptors: the histone acetyltransferase p300/CREB-binding protein-associated factor (8); GT198, which acts like a tissue-specific coactivator (9); and PTB (polypyrimidine tract-binding protein 1)-associated splicing factor (PSF), a corepressor that recruits Sin3A to TRs (10).
To identify new interacting partners for TRs, we screened a human fetal brain cDNA library in a yeast two-hybrid system, using the DBD and a small region of TRß2 N terminus as bait. We identified GRP1 (general receptor for phosphoinositides 1), a nucleotide exchange factor for ARF (ADP-ribosylation factor) (11), as a new TR interacting-partner. GRP1, also known as ARNO3, is a member of the cytohesin family. Although its functions are not yet well defined, GRP1 has been found to participate, with ARF6, in the formation of membrane ruffles (12, 13). Overexpression of GRP1 induces the disassembly of the Golgi complex, suggesting that ARF1 is also a potential substrate of GRP1 (11).
In this report, we describe and characterize the interaction between GRP1 and TRs, for the first time, and explore a novel function of GRP1 as a repressor of TR-mediated transcription.
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RESULTS
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Cloning of a Novel Protein Interacting with TRs
To identify new interacting partners for TRs, we performed a yeast two-hybrid screen of a human fetal brain cDNA library using amino acids 89220 of TRß2 as bait. This region comprises the DBD and a section of the unique N terminus of TRß2 (Fig. 1A
). Positive clones were isolated, some of which were known proteins but not previously described as TR partners. A BLAST (National Center for Biotechnology Information, Bethesda, MD) sequence analysis revealed one clone to be a partial nucleotide sequence of GRP1, also known as ARNO3. GRP1, a nucleotide exchange factor for ARF (11, 13, 14, 15), which is schematically represented in Fig. 1B
, possesses three well-described regions: a coiled coil domain that recognizes scaffold proteins like GRP1 signaling partner 1 and Tamalin (GRP1-associated scaffold protein) (16, 17); a Sec7 domain that is responsible for the nucleotide exchange (11, 15); and a pleckstrin homology (PH) domain, which recognizes membrane phosphoinositides with a specificity for phosphoinositide-3,4,5-trisphosphate (PIP3) (18, 19, 20). The partial cDNA clone we identified encompassed the PH domain and polybasic tail of GRP1 [100% homolog to amino acids (a.a.) 285399] and the 3'-untranslated region. The interaction between TRs and this initial clone was reconfirmed, in yeast cotransformed with the bait, using ß-galactosidase assays. We then amplified and cloned the complete cDNA sequence of GRP1 using specific primers and cDNA derived from RNA of JEG-3 cells.
Expression of GRP1 in Human Tissues and Cell Lines
To determine the expression level of GRP1 in human tissues and cell lines, a commercial dot blot membrane was hybridized with a radiolabeled probe representing the complete cDNA of GRP1 (Fig. 2A
). GRP1 was expressed in all of the adult and fetal human tissues and cell lines tested. Results reported per ng of RNA per dot revealed that the highest expression was found in placenta (dot blot position B8), testis (F8), and the SW480 cell line derived from a colorectal adenocarcinoma (G10). Patterns of GRP1 transcript found by the dot blot assay are in accordance with previous studies that have shown ubiquitous expression of GRP1 mRNA in mouse tissues (21). The presence of GRP1 protein has also been reported in HIRcB cells and 3T3-L1 adipocytes (12).

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Fig. 2. Ubiquitous Expression of GRP1 in Human Tissues and Cell Lines
A, A commercial multiple-tissue dot blot membrane was hybridized with a radiolabeled probe representing the complete cDNA of GRP1. GRP1 is ubiquitously expressed in adult and fetal human tissues and cell lines. Highest expression is found in the placenta (B8), testis (F8), and SW480 cell line (position G10). After stripping of the membrane, a ubiquitin probe was used as loading control (data not shown). B, Representative Western blot analysis of GRP1 protein expression in different cell lines: HeLa, JEG-3, CV-1, and GH3 cells. Whole-cell extracts (50 µg) were loaded into each lane. The immunoblot was revealed using an antibody raised against GRP1 (GL02). The GRP1 protein is expressed in every carcinoma cell line tested.
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We have also assessed the expression profiles of the GRP1 protein by Western blotting in different cell lines: HeLa, JEG-3, CV-1, and GH3 cells (Fig. 2B
). All these carcinoma cell lines endogenously express GRP1.
GRP1 Interacts with TRs in Their DBD
We next assessed in vitro interactions between TRs and GRP1 using pull-down analysis of recombinant glutathione S-transferase (GST) fusion proteins and 35S-labeled in vitro-translated proteins. As shown in Fig. 3A
, full-length TR
1, TRß1, and TRß2 interact with GRP1. Because GRP1 was isolated after a yeast two-hybrid experiment using TRß2, we used deletion mutants of this TR isoform to determine the regions implicated in the interaction (Fig. 1A
). TRß2 1120
, which lacks the a.a. 89116 of TRß2 conferring the specificity of this isoform in the regulation of the hypothalamo-pituitary axis (3), interacts with GRP1 (Fig. 3B
). The construction bearing only the DBD of TRs (DBD-only) is sufficient to precipitate GRP1, and deletion of the DBD (ØDBD) results in a loss of the interaction. From these results we conclude that the DBD of TRs is sufficient and essential for the interaction to occur.

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Fig. 3. TRs and GRP1 Interact in Vitro
Representative pull-down analysis of GST fusion proteins and 35S-labeled in vitro-translated proteins. Percentage of input is included for reference (lane 1), and GST alone was used as a negative control (lane 2). A, TR 1, TRß1, and TRß2 interact with GRP1-GST. B, GST-TRß2 and TRß2 deletion mutant 1120 interact similarly with GRP1; the DBD of TRs is sufficient to mediate this interaction (DBD-only). The deletion of the TR-DBD (ØDBD) results in loss of the interaction, suggesting that the DBD is essential and sufficient for the interaction to occur. C, In vitro interaction between TRs and GRP1 was not affected by the addition of increasing levels of T3 or the vehicle alone. The percentage of binding for each protein was obtained using the ImageQuant 5.0 program; results are from at least three independent experiments.
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Because the interaction between TRs and coregulators is often influenced by the presence of hormone, we assessed the influence of T3 in GST pull-down assays. We tested physiological (10 nM) and supraphysiological T3 concentrations (100 nM). In Fig. 3C
the GST pull-down assay and the gel analysis show that the interaction between GRP1 and TRs is not affected by increasing concentrations of T3.
Given that the DBD is the most conserved region among nuclear receptors and is essential and sufficient for the interaction between GRP1 and TRs, we tested the possible interaction of GRP1 and other members of the nuclear receptor superfamily. As demonstrated in Fig. 4
, GRP1 interacts well with human androgen receptor and human estrogen receptor and weakly with human retinoic X receptor (hRXR); no detectable interaction was found with human retinoic acid receptor (hRAR). Therefore, GRP1 could also have an effect on the action of other nuclear receptors.
TRs Interact with the PH Domain of GRP1
To identify the region of interaction with TRs, we designed constructions of GRP1 (Fig. 1B
). As shown in Fig. 5
, GST pull-down assays conducted with these deletion mutants of GRP1 show that the coiled-coil and the sec7 domain do not interact with TRß2. The PH domain of GRP1 strongly binds the receptor with 2-fold higher binding compared with wild-type GRP1. These results confirming the interaction of GRP1 through its PH domain or polybasic tail are not surprising because the initial GRP1 clone isolated in the yeast two-hybrid screen contained only this region of GRP1.

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Fig. 5. TR Interacts with the PH Domain of GRP1
Representative pull-down analysis of GST fusion proteins and 35S-labeled in vitro-translated proteins. Percentage of input is included for reference (lane 1), and GST alone was used as a negative control (lane 2). A, GST-wild-type GRP1 (GRP1wt) and PH domain interact with 35S-labeled TRß2. B, GST-TRß2 does not interact with 35S-labeled GRP1 mutant CC-sec7. The percentage of binding for each protein was obtained using the ImageQuant 5.0 program; results are from at least three independent experiments.
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GRP1 Interacts with TRs in Intact Mammalian Cells
After identifying the interaction in yeast and studying the regions involved in vitro, we wanted to examine whether native GRP1 and TRs are able to interact in mammalian cells. We have shown that GRP1 mRNA and protein are expressed in HeLa cells (Fig. 2
, A and B). It has also been described that HeLa cells contain functional TRs (22, 23). Thus, using coimmunoprecipitation of HeLa whole-cell extracts, we could demonstrate the interaction of the endogenous proteins in a mammalian cell environment (Fig. 6A
). We also wanted to examine whether the interaction between TRs and GRP1 was taking place in the cytoplasm and/or nucleus. Although TRs shuttle rapidly between the cytoplasm and nucleus, the majority of TRs are found in the nucleus (24, 25, 26). On the other hand, the majority of GRP1 is located in the cytoplasm and translocates to the cytoplasmic membrane after PIP3 production (12, 27, 28). However, endogenous and green fluorescent protein (GFP)-tagged GRP1 can be observed in the nucleus of cells in many reports (although not always mentioned in the text) (12, 13, 27, 28). We thus proceeded to cell fractionation studies with immunoblot analysis. Immunoblot of cellular fractions confirmed that GRP1 is present predominantly in the cytoplasm; however, it also shows that the GRP1 protein can be found in significant levels in the nuclear fraction of cells (Fig. 6B
). Coimmunoprecipitation studies performed in cell fractions suggest that GRP1 and TR interact both in the cytoplasm and nucleus (Fig. 6A
). We used an anti-I
B-
antibody to show that there was no cross-contamination between the cytoplasmic and nuclear fractions (Fig. 6C
). The I
B-
family of proteins is strictly found in the cytoplasm where it inhibits the binding of p50-p65 nuclear factor-
B complexes (29). Also, TRs can be detected in whole-cell extracts of HeLa cells and after coimmunoprecipitation with GRP1-specific antibody (N17) (Fig. 6D
). As demonstrated previously in vitro in Fig. 3C
, the TR-GRP1 interaction is not influenced significantly by the presence of the ligand in coimmunoprecipitation studies (Fig. 6E
).

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Fig. 6. TRs and GRP1 Protein Interact in Intact HeLa Cells
A, HeLa cell extracts were immunoprecipitated with TR-specific antibody FL408 (IP TR) or with preimmune rabbit sera (IP Pi) and subjected to immunoblot using GRP1-specific antibody GL02. No signal is found when cellular extracts are immunoprecipitated with the pre-immune (Pi) serum, showing specificity of the immunoprecipitation reaction. In vitro-translated GRP1 (GRP1 i.v.) was used as a positive control. Data show that GRP1 interacts with TRs in whole-cell, cytoplasmic, and nuclear fractions. B, Representative Western blot analysis of HeLa whole-cell, cytoplasmic, or nuclear extracts (50 µg per lane). Extracts and in vitro-translated GRP1 (GRP1 i.v.) were resolved by 10% SDS-PAGE. Immunoblots were conducted with the GRP1-specific antibody GL02. GRP1 is localized mainly in the cytoplasm but is also present in the nucleus. C, Immunoblot analysis of HeLa whole-cell, cytoplasmic, and nuclear extracts (50 µg per lane) with an I B- antibody shows that nuclear extracts are not cross-contaminated with cytoplasmic proteins. D, Whole-cell extracts of HeLa cells were immunoprecipitated with GRP1-specific antibody (N17, IP GRP1) or with an antibody against histidine probe (H-3, sc-8036, IP His-probe) as a negative control and subjected to immunoblot using TR-specific antibody FL408. GST-tagged TRß2 (92 kDa) and whole-cell extract of HeLa (60 µg) were used as positive controls for the immunoblot. This result shows the presence of endogenous TRs and the interaction with GRP1 in HeLa cells. E, Coimmunoprecipitation study of HeLa whole-cell extracts (300 µg). Proteins were immunoprecipitated using a TR-specific antibody FL408 in the presence of 100 nM T3 or the vehicle alone followed by immunoblotting with GRP1-specific antibody (N17). The interaction between TRs and GRP1 is not significantly influenced by T3. Gel-Pro Analyser program, version 4, was used for gel analysis.
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From these experiments, we conclude that in vitro and in vivo interaction assays confirm the presence of a complex between GRP1 and TRs in the cytoplasm and nucleus of cells.
GRP1 Represses TR-Induced Transcriptional Activation
To explore the nuclear functions for this newly discovered interaction, we analyzed the role of GRP1 in the transactivation of TR in mammalian cells, using transient transfections with luciferase reporter genes. In TR-deficient CV-1 cells (3, 30, 31), we studied negative and positive TREs. In Fig. 7
, A and B, using TRETK, a positive TRE composed of two idealized palindromes arranged in tandem, we can observe repression of transcription by all isoforms in the absence of T3, and activation of transcription in its presence. TR
1 is more potent and TRß2 is less effective in this action, as previously described (1). When GRP1 is cotransfected, we observe a decrease of the maximal activation for all TR isoforms, which results in a 4560% decrease in fold activation of this reporter gene. On positive TREs, the repression in the absence of ligand is not affected by GRP1. Decrease in TR-mediated transcriptional activation is also seen for other positive TREs (pTREs) like the inverted palindromes found in the chicken lysozyme gene (LYSX2, Fig. 7
, C and D) and direct repeats (DR+4, data not shown).

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Fig. 7. GRP1 Decreases Ligand-Dependent Activation on pTREs
Effect of TR isoforms and GRP1 cotransfected in CV-1 cells on the regulation of positive response elements TRETK (A and B) and LYSX2 (C and D) showing a decrease in T3-dependent activation levels and fold activation. The data are expressed in relative luciferase units (RLU) compared with pSG5 vector alone without T3 (A and C), or fold activation (B and D) (RLU in the presence of T3/RLU in the absence of T3). Data are compiled from at least three independent experiments and presented as mean ± SEM.
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Unlike the majority of genes reported to be influenced by thyroid hormone, genes from the hypothalamo-pituitary axis are negatively regulated by T3 (1). These genes show transcriptional activation in the absence of thyroid hormone and repression in the presence of T3, as shown in Fig. 8
. These negative TREs were studied with contransfection of RXR, because CV-1 cells are RXR deficient (32). We have previously shown that RXR is a necessary partner for negative regulation, especially by the TR
1 and TRß1 isoforms (32).

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Fig. 8. GRP1 Decreases Ligand-Independent Activation on nTREs
Effect of TR isoforms and GRP1 cotransfected in CV-1 cells on the regulation of negative responsive element TRH (A and B) and TSH (C and D) showing a decrease in the activation in the absence of T3 and fold repression. The data are expressed in relative luciferase units (RLU) compared with pSG5 vector alone without T3 (A and C), or fold repression (B and D) (RLU in the absence of T3/RLU in the presence of T3). Data are compiled from at least three independent experiments and presented as mean ± SEM.
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Cotransfection of GRP1 almost completely abolishes the activation in the absence of ligand of TR
1 and TRß1, on both the TRH (Fig. 8A
) and TSH
promoters (Fig. 8C
). This translates in a decrease in fold repression (Fig. 8
, B and D). GRP1 does not affect overall repression induced by the TRß2 isoform; TRß2 has been shown previously to be more potent in the regulation of the hypothalamo-pituitary axis (3) and to interact differently with coregulator proteins (7, 33). Also, the heterodimeric partner RXR, which is important for negative regulation by TR
1 and ß1, was previously shown not to be critical for TRß2 (32). It is thus not surprising to find a different behavior of TRß2 for genes that are negatively regulated by T3.
We next studied the dose response of GRP1 (50150-350-500-1000 ng per six-well plate) on the TRETK reporter to assure that the effect seen was specific and not due to squelching. Compared with the transfection of the empty pSG5 vector, cotransfection of GRP1 with TRß1 results in a decreased activation that is seen starting at 50 ng and leveling off at 350 ng of GRP1 (Fig. 9
).

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Fig. 9. Dose-Response Studies of the Effect of GRP1 on the TRETK Reporter
Effect of TRß1 and increasing doses (501000 ng) of GRP1 or empty pSG5 vector cotransfected in CV-1 cells, on the TRETK reporter gene. The repressor effect of GRP1 is visible at low doses and plateaus at 350 ng. The data are expressed in relative luciferase units, compared with pSG5 vector alone without T3, compiled from at least three independent experiments and presented as mean ± SEM.
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GRP1 Decreases DNA Binding of TR Complexes
Because the interaction with GRP1 takes place in the DBD of TRs, the next hypothesis we wanted to verify was that the observed repression of transcriptional activity in the presence of GRP1 could be secondary to an inhibition of receptor binding to TREs. We thus performed EMSAs to assess the formation of TR/TR homodimers and TR/RXR heterodimers on TREs. As shown in Fig. 10
, the addition of GRP1 decreases the binding of TR/TR homodimers by 2535% and decreases the binding of TR/RXR heterodimers by 4550% on direct repeat (Fig. 10
, A and B) and palindromic (Fig. 10
, C and D) pTREs. Interestingly, no additional complex is formed when GRP1 is added, suggesting that it is not able to interact with TRs that are bound on DNA. The composition of TRE-bound complexes was confirmed by supershifts with anti-TR and anti-RXR antibodies (data not shown).

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Fig. 10. GRP1 Decreases Homodimer and Heterodimer Formation on TREs
EMSA showing the effect of increasing amounts of in vitro-translated GRP1 protein on the formation of TRß1-homodimer or TRß1/RXR heterodimer on pTREs. 32P-radiolabeled probes representing pTREs are incubated with in vitro-translated TRß1, RXR , and GRP1 (1,3, 5, and 7 µl) as indicated. Unprogrammed lysate (UPL) is used to adjust for the amount of protein in each reaction. Diagrams accompanying the representative autoradiography show the quantification of binding intensity of complexes using the ImageQuant 5.0 program, from at least three independent experiments, expressed as mean ± SEM. A, EMSA using a direct repeat (DR+4) pTRE showing that GRP1 decreases the TRß1-homodimer formation. B, GRP1 inhibits the TRß1/RXR -heterodimer formation on the DR+4 pTRE. C, EMSA using a palindromic (PAL) pTRE showing that GRP1 decreases TRß1-homodimer formation. D, GRP1 inhibits the TRß1/RXR -heterodimer formation on the palindromic (PAL) pTRE.
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To further investigate the hypothesis that GRP1 competes with DNA for TR binding, we performed a GST pull-down experiment using 35S-radiolabeled GRP1 and TRß2-GST where we added increasing amounts of double-stranded oligos corresponding to the direct repeat (DR)+4 TRE or a nonspecific sequence corresponding to one CCAAT enhancer binding protein-binding site of the haptoglobin A protein promoter region (34). In Fig. 11A
(upper panel) is a representative GST pull-down experiment in which increasing amounts of DR+4 oligos (1, 3, and 5 pmol) caused a diminution of the complex formed by TRß2 and GRP1, also represented graphically after gel quantification. The amount of complexes formed gradually decreased to 75% when 5 pmol of DR+4 was added. No change in the interaction between TRß2 and GRP1 was found when increasing amounts of the nonspecific sequence oligos (Fig. 11B
) were added. Therefore, GRP1 competes with DNA for TR binding, and the GST pull-down results are in concordance with our EMSA experiments.
To further assess the hypothesis that GRP1 could influence directly the binding of homo/heterodimers on TREs or dimer formation, we designed GST pull-down assays. TR-GST fusion protein fixed on glutathione (GSH) beads was incubated with radiolabeled TRß2 or RXR
in the presence of increasing amounts of GRP1. Our results show that GRP1 did not influence TR-TR or TR-RXR dimer formation (Fig. 11C
). Taken together, these results clearly suggest that GRP1 directly decreases DNA binding rather than dimer formation. From these data, we conclude that competition between TREs and GRP1 for TR binding could thus explain the mechanism of action of this novel corepressor.
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DISCUSSION
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We have identified a novel TR-interacting protein, GRP1, which interacts with the receptor in both in vitro pull-down assays and in the cytoplasm and nucleus of mammalian cells. More importantly, we have demonstrated that GRP1 inhibits TR-mediated activation of transcription and decreases TR homodimer and heterodimer binding to TREs and that GRP1 competes with TRE for TR binding by GST pull-down assays. This is a newly recognized mechanism of inhibition of thyroid hormone action.
GRP1, also known as ARNO3, is a guanine nucleotide exchange factor for ARF, which is a small G protein implicated in vesicle-trafficking pathways and actin cytoskeleton assembly (35, 36). This protein is composed of three distinct regions: an N-terminal coiled-coil domain, a central Sec7 domain responsible for ARF binding and exchange activity, and a C-terminal PH domain that recognizes phosphoinositides (Fig. 1B
). GRP1 was first identified as a putative PIP3 receptor (15). After signaling-mediated induction of phosphatidylinositol 3-kinase, it is translocated to the cell membrane where it binds PIP3 and uses mainly ARF6 as a substrate for its guanine nucleotide exchange factor activity (13, 28). Overexpression of GRP1 results in fragmentation of the Golgi apparatus (11) and induces ARF6-dependent actin rearrangements (12). However, the exact physiological functions of GRP1 remain unknown, and we are the first to report an interaction with TRs and members of the nuclear receptor superfamily. In addition, it is novel for GRP1 to have a role in transcriptional regulation by decreasing the availability of the receptors for their DNA-binding sites.
Although data analysis from previous publications demonstrates the presence of GRP1 in the nucleus of cells, we are the first to explore nuclear roles of GRP1. GRP1 is found predominantly in the cytoplasm of cells, but it is also seen in the nucleus, especially after PIP3 formation and translocation of cytoplasmic GRP1 to the cell membrane (12, 13, 27, 28). A nuclear localization of GRP1 is thus compatible with the functions of GRP1 as a repressor of transcription, which we describe here.
Considering the cellular localization of GRP1, it is also possible that the interaction with TRs might take place in the cytoplasm before TRs enter the nucleus. TRs are synthesized in the cytoplasm, and the first step in their nuclear action is transport to the nucleus. GFP fluorescent probes have allowed the study of TR distribution in living cells: it was shown by Baumann et al. (24) that in the absence of T3, the nuclear/cytoplasm (n/c) ratio for TRß was 5.5 or 8590% nuclear. Zhu et al. (25) have found that the n/c ratio of TRß1-GFP in the absence of the ligand was 1.5, corresponding to approximately 60% nuclear. Although there seems to be some variability in the results reported regarding the subcellular localization of TRß in hormone-free cells, both reports stated that there is only 1015% of TRß present in the cytoplasm in T3-treated cells. They also have shown rapid changes in the intracellular distribution of TRs in living cells, and both agreed that, in the presence of the hormone, most of the receptors enter the nucleus (24, 25). The fact that TRs interact with GRP1 both in the nucleus and cytoplasm may also suggest that, in addition to the transcriptional effects of GRP1, it might be implicated in nongenomic actions of thyroid hormone that take place in the cytoplasm or in the detergent-insoluble regions of the cellular membrane where GRP1 is also found. In fact, thyroid hormone has been implicated in the activation of phosphatidylinositol 3-kinase (37), an important player in GRP1 subcellular localization. In addition, it was recently described that other members of the nuclear receptor superfamily such as ER, which was also shown to interact with GRP1 in this report, are located in part near the cell membrane in membrane ruffles and caveolae, and important functions of nuclear receptors are now being attributed to their extranuclear fractions (38, 39). GRP1 might thus be implicated in membrane localization and actions of these nuclear receptors, and further studies are needed to accurately describe the roles of GRP1 in thyroid hormone action.
Numerous coregulator proteins associate with the LBD of TRs to modulate transcription of target genes. NCoR (nuclear receptor corepressor) and SMRT were the first corepressors identified (40, 41). These corepressor proteins are composed of receptor-binding domains and repressor domains, which recruit Sin3A and histone deacetylase to repress transcriptional activity. GRP1 is a repressor of a novel class with no sequence homology with NCoR and SMRT. However, like NCoR and SMRT (42), GRP1 influences the regulation of both pTREs and negative TREs (nTREs): on pTREs, it decreases T3-dependent activation, whereas on nTREs it diminishes ligand-independent activation in an isoform-specific manner.
Also, the interacting domain of GRP1 is located within the DBD of TRs, which we have shown to be sufficient and essential for the interaction, confirming the importance in this region of TRs for functions other than TRE recognition and binding. It was recently found that PSF binds the DBD of nuclear receptors, and acts as a corepressor protein for TR/RXR, mediating its effect through Sin3A via chromatin remodeling (10). Like GRP1, PSF represses transcription, and its interactions with TRs are not modified by the presence of thyroid hormone, unlike most coregulators that prefer a conformation of receptors that is modified by ligand binding.
Although the majority of coregulatory proteins have been studied for their ability to remodel chromatin structure, there is a recent interest in exploring other pathways that contribute to the modification of transcriptional activity by TRs. The nuclear transport of TRs has been studied for its ability to modify transcription (25). Also, the stability and ubiquitination of TR proteins, which are influenced by phosphorylation and ligand binding, have been shown to influence reporter gene activity (30, 43). We have shown here that DNA binding of homodimers and heterodimers is decreased in the presence of increasing amounts of GRP1, and this could explain its repressive effects on transcription. Inhibition of DNA binding is a novel mechanism that will need to be further explored. Burris et al. (44) have identified a corepressor, thyroid receptor-uncoupling protein, which attenuates hormone-dependent transactivation of TR and RAR by decreasing heterodimer formation on response elements. By interacting with the DBD of TRs, GRP1 might also disturb the formation of receptor dimers, because there are dimerization interfaces in this region of nuclear receptors (45, 46). However our GST pull-down studies (Fig. 10G
) show no effect of GRP1 on TR-TR or TR-RXR dimer formation, demonstrating that the effect of GRP1 would be on the inhibition of TRs to bind to the TRE. Another possible mechanism, although not supported by our EMSA and TRE-competition studies, would be competition of GRP1 for the binding sites of other coactivators, e.g. PCAF and GT198, interacting with the DBD of TRs (8, 9).
In conclusion, we have described the interaction of TRs with a novel repressor of their transcriptional activity: GRP1. This cofactor may also be important for the activity of other nuclear receptors because we have shown it to interact with other members of the superfamily. GRP1 decreases binding of homo- and heterodimer on TREs and competes with TRE for TR binding. The exact mechanism of repression of GRP1, which probably involves decreased access of TRs to TREs, and its possible involvement in nongenomic actions of thyroid hormone need to be further studied.
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MATERIALS AND METHODS
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Yeast Two-Hybrid
We have used a portion of TRß2 composed of a.a. 89220 cloned in frame in PAS21 (Fig. 1A
) as bait to screen a human fetal brain cDNA library cloned in pACT2 (CLONTECH Laboratories, Inc., Palo Alto, CA). Plasmids were cotransformed in yeast strain Y190 according to manufacturers instructions of MATCHMAKER Two-Hybrid System 2 (CLONTECH). We initially isolated 19 clones growing on His/Trp/Leu selection media and producing LacZ. Plasmid DNA was extracted from these colonies and transformed in MC106 Escherichia coli to isolate pACT2 plasmids. These were retransformed in yeast together with the TR bait to confirm the interaction using ß-galactosidase assays. The GRP1-PH clone (Fig. 1B
), a cDNA corresponding to a.a. 285399 of GRP1, was then sequenced (Ottawa University, Ottawa, Ontario, Canada) and analyzed by sequence similarity searches (BLAST).
Plasmid Constructions
The cDNAs of human TR
1, hTRß1, hTRß2, TRß2 1120
, ØDBD, DBD-only and human androgen receptor, human estrogen receptor, hRAR, hRXR (3) were inserted into pSG5 or the pGEX-4T1 vector (Stratagene, La Jolla, CA) (47) (Fig. 1A
). The GST-ØDBD (deletion a.a. 119221) and DBD-only mutant (a.a. 116218) were produced by PCR amplification of hTRß2 and inserted into pGEX-4T1 at the EcoRI site and BamHI site, respectively.
After cloning a cDNA identical to a.a. 285399 of GRP1, we amplified the full-sized cDNA (GenBank accession no. 2909436) from a RT-PCR on JEG-3 cells RNA. The full-length GRP1 cDNA was subcloned in pSG5 expression vector at the BglII site and inserted in frame in pGEX-4T2. GRP1-PH was generated by inserting the EcoRI-XhoI fragment from the original yeast two-hybrid clone, in frame in pGEX-4T2 (Fig. 1B
). Cloning a BamHI fragment of GRP1 in frame in pGEX-4T2 produced GRP1-CC (Fig. 1B
). The GRP1-CC-Sec7 construct was obtained by PCR amplification and inserted in pSG5 (Fig. 1B
). Exactitude of all insert sequences was confirmed by DNA sequencing. The size of mutant proteins was confirmed by SDS-PAGE.
The TRETK reporter contains two copies of an idealized pTRE arranged as a palindrome, upstream of a minimal thymidine kinase promoter and fused to the luciferase gene in the PSVO vector (48). The pTREs LYSX2 (two copies of an inverted palindrome from the chicken lysozyme gene) and DR+4 (direct repeat) were incorporated in the PA3-Luc vector (49, 50). The negative thyroid response element (nTRE) reporter constructs included the 5'-flanking sequences from the human TRH (900/+55) (47), and the common glycoprotein
-subunit (TSH
, 846/+26) fused upstream of a luciferase reporter gene in the PSVO vector (51).
RNA Expression Analysis
A commercial membrane was used for dot blot analysis (Multiple Tissue Expression Array; CLONTECH; Fig. 2
). The blots were hybridized with a 32P-random-labeled cDNA probe (Rediprime II; Amersham Pharmacia Biotech Inc., Baie dUrfe, Quebec, Canada) representing the entire GRP1 cDNA obtained by digestion of the GRP1-pSG5 plasmid with BglII. After washes, the blots were revealed by autoradiography. The blots were stripped and hybridized with a probe for human ubiquitin supplied by the manufacturer and resulted in consistent signals for all poly A+ RNA dots (data not shown). Gel-Pro Analyser program, version 4 (MediaCybernetics, Carlsbad, CA) was used for gel analysis.
GST Pull-Down Assays
Fusion constructs were generated by inserting appropriate restriction fragments isolated from cloned cDNAs or PCR products in frame in pGEX-4T1 or 4T2 (Amersham Pharmacia Biotech Inc.; Fig. 1
). GST fusion proteins were prepared after optimization of manufacturers instructions. Part of the proteins were analyzed in SDS-PAGE and quantified; the remainder was kept on GSH-Sepharose beads for pull-down assays. Interacting proteins were in vitro translated and labeled with [35S]methionine (TNT T7/T3 Coupled Reticulocyte Lysate System; Promega, Nepean, Ontario, Canada). These 35S-labeled proteins were incubated with GST protein on GSH beads in HEMG buffer [40 mM HEPES (pH 7.8), 40 mM KCl, 0.2 mM EDTA, 5 mM MgCl2, 0.5% Triton X-100, 10% glycerol, 1.5 mM dithiothreitol (DTT)], supplemented with 10 mg/ml BSA and 1% protease inhibitors (Complete-Mini EDTA-free; Roche, Laval Quebec, Canada) at room temperature for 2 h. Protein-protein bead complexes were pulled down by centrifugation, washed, and separated in 10% SDS-PAGE. For the protein interaction competition experiments (Fig. 11
, A and B), unprogrammed reticulocyte lysate was added to adjust volume for each reaction. Interacting proteins were revealed by autoradiography. Data presented in the figures are representative of at least three independent experiments. To evaluate the interactions quantitatively, the autoradiograms were scanned and analyzed with ImageQuant 5.0 Build 050 software, (Molecular Dynamics, Inc., Sunnyvale, CA) or the Gel-Pro Analyser program, version 4, when stated.
The DNA competition assays were performed using the GST-GRP1 immobilized on GSH-Sepharose combined with in vitro-translated 35S-labeled hTRß2wt (52). Briefly, the protein complexes were allowed to form as described above, followed by washes to discard the unbound 35S-radiolabeled protein. The GST-complexes were incubated in the presence of 0, 1, 3, and 5 pmol of cold PCR-generated double-stranded oligos containing a nonspecific DNA sequence (haptoglobin protein A, containing a CCAAT enhancer binding protein-binding site) or the DR+4 TRE in the reaction buffer [15 mM Tris (pH 7.9), 2 mM EDTA, 20 mM KCl, 4 mM DTT, 0.5 mM ZnCl2] containing 2 µg/ml polydIdC (Amersham Pharmacia Biotech, Inc.) for 1 h at room temperature. The GSH-Sepharose was then washed thoroughly in the wash buffer [20 mM Tris (pH 7.5), 50 mM NaCl, 0.2 mM EDTA, 10% glycerol, 0.1% Nonidet P-40, 2 mM DTT]. Protein-protein bead complexes were pulled down by centrifugation, washed, and separated in 10% SDS-PAGE. To evaluate the interactions quantitatively, the autoradiograms were scanned and analyzed with ImageQuant 5.0 Build 050 software.
Cell Extracts and Immunoprecipitation Studies
CV-1 (Cercopithecus aethiops, ATCC no. CCL-70; American Type Culture Collection, Manassas, VA), GH3 (Rattus norvegicus, ATCC no. CCL-82.1), HeLa (Homo sapiens, ATCC no. CCL-105), JEG-3 (H. sapiens, ATCC no. HTB-36) were lysed in 50 mM HEPES and 1% Triton X-100 with protease inhibitors (Complete Mini, EDTA-free) on ice for 30 min. Lysates were then collected by scraping, and cells were centrifuged for 5 min at 3000 x g at 4 C. Whole-cell extracts were collected in the supernatants.
For cellular fractionation studies, cells were scraped in PBS and pelleted by centrifugation at 200 x g at 4 C, for 10 min. The pelleted cells were resuspended in homogenization buffer (0.32 M sucrose-3 mM MgCl2-0.25% Triton X-100 in a sodium-phosphate-buffered solution, pH 6.5) and lysed on ice for 10 min. After 10 min of centrifugation at 300 x g, at 4 C, the supernatant was kept as the cytoplasmic/membrane fraction. The pellet containing the nucleus was resuspended in the purification solution (1.5 M sucrose-3 mM MgCl2 in a sodium phosphate-buffered solution, pH 6.5, with protease inhibitors) and centrifuged at 12,000 rpm for 90 min at 4 C. The nucleus pellet was then incubated on ice for 20 min in lysis buffer (30 mM TrisBase pH 8.0; 2 mM EDTA; 0.4 mM NaCl; 5 mM MgCl2; 15 mM 2ß-mercaptoethanol; 10% glycerol with protease inhibitors). The nuclear extract was incubated for 45 min on ice with agitation every 15 min and recovered after centrifugation for 20 min at 12,000 rpm at 4 C. Cell extract concentrations were measured using a standard Bradford assay (Bio-Rad Laboratories, Indianapolis, IN).
In the coimmunoprecipitation experiments, TR-associated proteins were precipitated using a polyclonal antibody against the full-length TR (anti-TR
1 FL408: sc-772; Santa Cruz Biotechnology, Inc, Santa Cruz, CA) recognizing all TR isoforms of mouse, rat, human, and chicken origin. The negative controls represent complexes immunoprecipitated with preimmune rabbit sera or an antibody directed against a histidine probe (H-3, sc-8036; Santa Cruz Biotechnology, Inc.). The GRP1 protein was revealed using either the N17 goat polyclonal antibody (sc-9730; Santa Cruz Biotechnology, Inc.) or a polyclonal GRP1-antiserum produced in our laboratory after immunization of a rabbit with GST-GRP1 (GL02). Precleared cell extracts (300 µg) were incubated overnight with 35 µg of antibody at 4 C with rocking. Protein G-Sepharose was then added, and samples were rocked for 2 h at 4 C. The immunoprecipitated complexes were washed five times [first and second washes in 0.1% Triton X-1000.1% BSA prepared in trichostatin A (TSA), pH 8.0; third wash in TSA, pH 8.0; fourth wash in 0.05 M Tris-HCl, pH 6.8, prepared in TSA; fifth wash in Tris-buffered saline, pH 8.0].
For the immunoblot, proteins were resolved in 10% SDS-PAGE and transferred to a polyvinyl difluoride membrane. The membrane was blocked and incubated overnight at 4 C with constant agitation with the primary antibody: anti-TR antibody (FL408) or anti-GRP1 (GL02 or N17). Anti-I
B-
was used as control (sc-371, Santa Cruz Biotechnology, Inc.) to assure that nuclear fractions were not contaminated with cytoplasm (29). Nonradioactive in vitro-translated (GRP1-pSG5) or GST-tagged proteins (TRß2-pGEX-4T1) expressed in E. coli were used as positive controls for the immunoblot step. The membrane was washed with Tris-buffered saline/Tween 20, and detection was accomplished using horseradish peroxidase-conjugated antibody (Amersham Pharmacia Biotech, Inc.) and enhanced chemiluminescence detection system (Roche). Data presented in the figures are representative of at least three independent experiments. Gel-Pro Analyser program, version 4, was used for gel analysis.
Cell Culture and Transfection Assays
The CV-1 (Cercopithecus aethiops, ATCC no. CCL-70) cell line was maintained in DMEM supplemented with 10% fetal bovine serum, penicillin, streptomycin, and amphotericin (Life Technologies, Burlington, Ontario, Canada). Transient transfections were realized in six-well plates on subconfluent cells using the calcium-phosphate technique. Unless otherwise indicated, each experiment utilized 1.6 µg of reporter construct and 80 ng of each receptor or cofactor expression vector or vector alone per well. Sixteen hours after transfection, culture medium was replaced by medium containing fetal bovine serum stripped of hormone by anion-exchange resin and charcoal, with or without 10 nM T3. Cells were harvested and assayed 3640 h after transfection for luciferase activity. Data are from at least three independent experiments, performed in triplicate, and are displayed as mean ± SE.
EMSA
Positive TREs DR+4 (5'-TCAGGTCACAGGAGGTCAAC-3') and PAL (5'-TCAGGTCATGACCTGAC-3') double-stranded DNA probes were radiolabeled by PCR using [32P]dCTP (NEN Life Science Products, St-Laurent, Quebec, Canada). Unincorporated 32P was removed by ProbeQuant G-50 column (Amersham Biosciences). Proteins were made by in vitro transcription/translation in rabbit reticulocyte lysate (TNT T7/T3 Coupled Reticulocyte Lysate System). Protein production was verified in parallel transcription/translation reactions with 35S incorporation and visualized on SDS-PAGE followed by autoradiography. Between 1 and 7 µl of lysate proteins were incubated with radiolabeled probes (0.51 x 106 cpm depending on the experiment). Total protein volumes were adjusted with reticulocyte lysate to give equal volumes for each reaction. Reactions were performed in a 20 µl volume of binding buffer (20 mM HEPES, 50 mM KCl, 20% glycerol, 1µg/µl Poly dIdC, 1 mM DTT, 0.1 mg/ml salmon sperm DNA) for 20 min at room temperature. Supershift reactions were performed by first incubating proteins and probes in binding buffer for 20 min at room temperature, followed by an additional 30 min incubation on ice with anti-TR (FL408) or anti-RXR
(
N197; sc-774, Santa Cruz Biotechnology, Inc.) antibody to ensure complex composition. Reactions were resolved on 5.2% nondenaturing polyacrylamide gels containing 5% glycerol at 4 C for 34 h. Binding shifts were visualized by autoradiography. Data presented in the figures are representative of at least three independent experiments. Gel analysis was performed with the ImageQuant 5.0 Build 050 software.
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ACKNOWLEDGMENTS
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We thank Dr. Fredric E. Wondisford and Dr. Lenore K. Beitel for plasmids, and Dr. Claude Asselin for the haptoglobin promoter oligos.
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FOOTNOTES
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This work was supported in part by grants and scholarships from the Canadian Institutes of Health Research (MOP-15655 and MOP-67203) and Fonds de la Recherche en Santé du Québec (to M.F.L.); and by a Fonds de la Recherche en Santé du Québec scholarship (to M.E.D.).
Results from this work were presented in part at the 83rd Annual Meeting of The Endocrine Society, Denver, Colorado, 2001.
First Published Online May 5, 2005
1 M.-B.P. and G.H. contributed equally to this work. 
Abbreviations: a.a., Amino acids; ARF, ADP-ribosylation factor; DBD, DBD-binding domain; DTT, dithiothreitol; GFP, green fluorescent protein; GRP1, general receptor for phosphoinositides 1; GSH, glutathione; GST, glutathione S-transferase; LBD, ligand-binding domain; NCoR, nuclear receptor corepressor; nTRE, negative TRE; PH, pleckstrin homology; PIP3, phosphoinositide-3,4,5-trisphosphate; PSF, phosphotyrosine binding-associated splicing factor; pTRE, positive TRE; RAR, retinoic acid receptor; RXR, retinoic X receptor; SMRT, silencing mediator of retinoic and thyroid hormone receptor; TR, thyroid hormone receptor; TRE, thyroid hormone response element; TSA, trichostatin A.
Received for publication November 8, 2004.
Accepted for publication April 25, 2005.
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