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Raku Sonoo,2,
Yu-ichi Sekiya,1
Nanae Sunahara,1
Miwako Kawano,1
Mitsutoshi Wayama,1
Ryuichi Hirota,2
Yoh-ichi Kawabe,1
Akiko Murayama,1
Shigeaki Kato,3
Keiji Kimura,1 and
Junn Yanagisawa1,2*
Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba Science City, Ibaraki 305-8572, Japan,1 Ankhs Incorporated, Tsukuba Industrial Liaison and Cooperative Research Center, University of Tsukuba, Tsukuba Science City, Ibaraki 305-8577, Japan,2 Institute of Molecular and Cellular Biosciences, University of Tokyo, Bunkyo-ku, Tokyo 113-0032, Japan3
Received 25 April 2006/ Returned for modification 12 June 2006/ Accepted 17 August 2006
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ABSTRACT |
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INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and ERß, which function as ligand-induced transcription factors and belong to the nuclear receptor (NR) superfamily (2, 5, 12, 20, 31, 33, 41, 49). When the estrogen level is high, estrogen binds to ERs to activate the transcription of target genes. This induces the ligand-binding domain (LBD) to undergo a characteristic conformational change, whereupon the receptor dimerizes, binds to DNA, and subsequently stimulates gene expression (10). ER is stimulated by two distinct activation regions, activation function 1 (AF-1) and AF-2, which are located in the LBD and exert ligand-dependent transcriptional activity. Crystal structure analysis of ERs and other NRs has revealed the presence of 12 conserved helices in their LBDs (46). The LBD forms a structure described as a sandwich of 12 -helices (helices 1 to 12) with a central hydrophobic ligand-binding pocket. Helix 12, the most C-terminal of these helices, has been identified as the critical core (AD core) of the AF-2 function of the receptor and plays an important role in coactivator binding to the ligand-bound receptor (6, 19, 24, 25, 33, 35, 39, 43, 45, 55, 56). When the level of estrogen is decreased, receptor-mediated transcription should be downregulated. Because the response to estrogen is tightly controlled, the downregulation of ER-mediated transcription should also be tightly regulated. Although such regulation of the NRs has generated intense interest, the molecular mechanisms regulating these processes are not understood (38). To switch off transcription, there are at least two possible mechanisms. The first involves the dissociation of ligands from the hormone-binding pocket of the receptor. However, the dissociation rate of the ligand from its receptor is very low, because the receptor locks the hormone within its activation pocket and covers it with coactivators (16). Another possible mechanism is the rapid degradation of active receptors. Several nuclear receptors, such as the ER and those for progesterone, glucocorticoid, thyroid hormone receptor, retinoid X, and retinoic acid, are ubiquitinated and degraded in the course of their nuclear activities (8, 11, 13, 26, 29, 32, 36, 47, 51, 57). These degradation steps might be involved in turning off NR-mediated transcription.
Evidence now suggests that proteasome-dependent degradation of receptors is necessary for the activation of these receptors (38). Moreover, this requires NR-mediated transcriptional activity, implying that the degradation and the transactivation of NRs are mutually independent processes. In the ubiquitin-proteasome pathway, proteins destined for degradation are conjugated by polyubiquitin chains, in which one ubiquitin molecule is conjugated by another through one of its seven lysine residues, typically K48. These polyubiquitin chains are then recognized by the regulatory complex of the 26S proteasome.
Here we show that there is a ubiquitin-proteasome pathway for ERß that is not coupled to transactivation. To investigate this, we purified the ubiquitin ligase complex for ERß and identified a protein complex containing the carboxyl terminus of heat shock cognate 70 (HSC70)-interacting protein (CHIP) (1). CHIP binds directly to the N-terminal 37-amino-acid region of ERß and ubiquitinates it to induce ERß degradation. In contrast, the C-terminal F domain inhibits the binding of proteasome to the receptor and protects it from proteolysis in the absence of estrogen. Suppression of CHIP by interfering RNA (RNAi) inhibited the switching off of receptor-mediated transcription that occurs when the ligand dose is reduced. Our results suggest that ligand-dependent degradation selectively and rapidly eliminates the active form of a nuclear receptor to downregulate receptor-mediated transcription after ligand withdrawal.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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/ß and CHIP expression plasmids and their deletion mutants have been described previously (48). ERE-TATA-Luc and ERE-TATA-LucCP were constructed by inserting three consensus estrogen response elements into a luciferase reporter plasmid encoding a TATA box, pGL3-Basic or phRG(R2.2)-Basic. pGL3-Basic, phRG(R2.2)-Basic, and pGL3-Control vectors were purchased from Promega (Madison, WI).
Antibodies.
Anti-FLAG-M2 mouse monoclonal antibody (Sigma, St. Louis, MO), anti-hemagglutinin (HA) mouse monoclonal antibody (Roche), anti-human ER antibody (Chemicon, Temecula, CA), anti-human ERß antibody (PPMX), and anti-glyceraldehyde-3-phosphate-dehydrogenase mouse monoclonal antibody (American Research Products, Inc.) were used at appropriate dilutions, according to the manufacturers' instructions. A rabbit polyclonal antibody directed against CHIP was provided by T. Chiba (Tokyo Metropolitan Institute of Medical Science).
Cell culture and transfection. MDA-MB231 breast cancer cells and human embryonic kidney 293 cells were routinely maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS). Twenty-four hours before transfection, the medium was changed to phenol red-free DMEM containing 4% charcoal-stripped FBS. Transfection was performed using PerFectin transfection reagent (Gene Therapy Systems) or TransFast transfection reagent (Promega) according to the manufacturers' protocols. The cells were treated with or without MG132 (10–6 M) and estrogen (10–8 M). Twenty-four hours after the addition of estrogen, the cells were harvested and analyzed by Western blotting using the appropriate antibodies.
Coimmunoprecipitation and Western blotting. The 293 cells were transfected with the appropriate plasmids and lysed in TNE buffer (10 mM Tris-HCl [pH 7.8], 0.5% Nonidet P-40 [NP-40], 0.15 M NaCl, 1 mM EDTA, 1 µM phenylmethylsulfonyl fluoride [PMSF], 1 µg/ml aprotinin). Extracted proteins were immunoprecipitated with antibody-coated protein A/G-Sepharose (Amersham) or anti-FLAG M2 agarose (Sigma). The bound proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred onto polyvinylidene difluoride membranes (Millipore), and detected with the appropriate antibodies and secondary antibodies conjugated with horseradish peroxidase. Specific proteins were detected using an enhanced chemiluminescence Western blot detection system (Amersham).
Luciferase assay. For luciferase assays, ERE-TATA-Luc plasmids were cotransfected with expression vector encoding ERß or its mutants. As a reference plasmid with which to normalize transfection efficiency, the pRSVßGAL vector or the pRL-CMV vector was cotransfected in all experiments. Twenty-four hours after transfection, the culture medium was replaced with fresh medium containing 0.2% FBS, either estrogen (10–8 M) or ethanol vehicle was added, and the cells were incubated for an additional 24 h. Cell extracts were prepared, and luciferase assays were performed following the manufacturer's protocol (Promega). ß-Galactosidase activity was measured to control for the efficiency of each transfection. Individual transfections, each consisting of triplicate wells, were repeated at least three times.
Protein purification. Glutathione S-transferase (GST) protein or an immobilized 37-amino-acid region that we designated the "domain required for degradation" (DRD) fused to GST (GST-DRD) was preincubated for 1 h at 4°C in GST-binding buffer (20 mM Tris-HCl [pH 7.9], 180 mM KCl, 0.2 mM EDTA, 0.5 mM PMSF, and 1 mM dithiothreitol) containing bovine serum albumin (BSA; 1 mg/ml). Bead-immobilized proteins were then incubated at 4°C for 6 to 10 h with 293 cell extracts. After the beads had been washed three times with GST buffer (GST-binding buffer with 0.1% NP-40), they were washed again with GST buffer containing 0.2% N-lauroyl sarcosine. Proteins bound to the DRD were eluted with 15 mM reduced glutathione in elution buffer (50 mM Tris-HCl [pH 8.3], 150 mM KCl, 0.5 mM EDTA, 0.5 mM PMSF, 5 mM NaF, 0.08% NP-40, 0.5 mg/ml BSA, and 10% glycerol). Recombinant CHIP was expressed in Escherichia coli and purified using Ni-agarose.
Immunofluorescence. The 293 cells were grown on poly-L-lysine-coated 12-well culture dishes and transfected with plasmids. Twenty-four hours after transfection, the cells were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 10 min and permeabilized with Triton buffer (50 mM Tris-HCl [pH 7.5], 0.5% Triton X-100, 150 mM NaCl, 2 mM EDTA) for 15 min. The cells in each well were blocked with PBS containing 1% BSA and 0.5% goat serum for 3 h at 37°C. The cells were incubated with the appropriate antibody in PBS containing 1% BSA for 2 h at 37°C. After the cells had been washed with PBS, they were incubated with Alexa-Fluor-488-conjugated goat anti-rat immunoglobulin G or Alexa-Fluor-594-conjugated goat anti-mouse immunoglobulin G (Molecular Probes) for 1 h at 37°C and washed with PBS. The samples were mounted with Vectashield mounting medium (Vector Laboratories) and analyzed using a Keyence Biozero BZ-8000.
Pulse-chasing. The 293 cells were transfected with ERß, and 48 h after transfection the cells were labeled for 30 min at 37°C with [35S]methionine (50 µCi/ml) in methionine-free DMEM. The cells were then washed twice and incubated in DMEM containing 0.2% FBS for the indicated periods (chase). At each time point, cell lysates were immunoprecipitated with anti-ERß antibody. The immunoprecipitates were resolved by SDS-PAGE and visualized by autoradiography. A phosphorimager was used to quantify the metabolically labeled ERß present at each time point.
RNAi. The 293gp cells were transfected with pSINsi-hU6 vector (Takara) containing the target sequence of CHIP or LacZ (control) and with vesicular stomatitis virus-encoded envelope protein G to generate the small interfering RNA (siRNA) retroviral supernatant. MDA-MB-231 cells were transfected with retroviral supernatant in the presence of Polybrene (8 µg/ml). Twenty-four hours after transfection, the viral supernatant was replaced with fresh DMEM containing 10% FBS. The transfected cells were selected with G418 (1 mg/ml). The target sequences were 5'-GCACGACAAGTACATGGCGGA-3' for CHIP and 5'-GCTACACAAATCAGCGATT-3' for LacZ.
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RESULTS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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or ERß were transfected into 293 cells, and the protein levels were examined by Western blot analysis. ER(
181), which does not contain the A/B domain, still underwent ligand-dependent degradation (Fig. 1E). In contrast, truncation of the A/B domain of ERß, forming ERß(140), produced no ligand-dependent degradation, suggesting that the A/B domain of ERß is necessary for its ligand-dependent degradation (Fig. 1E).
We then investigated the region responsible for the degradation of ERß. The levels of truncated ERß protein in the presence and the absence of estrogen were examined by Western blotting. As shown in Fig. 2A, ERß(20), in which the 20 N-terminal amino acids are deleted, was elevated in the presence of estrogen compared with wild-type ERß. ERß(37) exhibited almost no degradation, indicating that the N-terminal 37 amino acids are essential for the ligand-dependent degradation of ERß (Fig. 2A). To assess whether this region is sufficient for the ligand-dependent degradation of ERß, it was fused to ERß(140), which does not contain the A/B domain and shows little ligand-dependent degradation. In contrast to that of ERß(140), the level of ERß(38-140) protein, in which the 37-amino-acid region is fused to ERß(140), was reduced in an estrogen-dependent manner (Fig. 2A). This reduction was abrogated by the addition of MG132, indicating that ERß(38-140) is degraded by proteasomal pathways. Interestingly, the degradation of ERß(mC), which has three amino acid substitutions in the DNA-binding domain (C domain) and has almost no ability to bind DNA (30), was similar to that of wild-type ERß (Fig. 2A), indicating that binding to the DNA element is not necessary for ligand-dependent receptor degradation. Similar results were obtained when other cell lines, such as MCF-7 or MDA-MB-231, were used (data not shown). From these observations, we concluded that the N-terminal 37-amino-acid region of ERß is involved in its ligand-dependent degradation and designated this region the DRD. We next determined the ubiquitination status of the truncated forms of ERß. The DRD deletion accumulated a monoubiquitinated form and showed a reduced level of the polyubiquitinated form (Fig. 2B), suggesting that the DRD is required for the polyubiquitination of ERß.
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To test the first possibility, we introduced an amino acid substitution at the lysine residue in the DRD. ERß(K4A), in which the lysine residue in the DRD is replaced by alanine, showed ligand-dependent degradation similar to that of the wild type (Fig. 2A), implying that the lysine residue in the DRD is not a target for ubiquitination. Therefore, we investigated the other possibility.
We generated a GST-fused 37-amino-acid region (GST-DRD) with which to purify proteins that specifically bind to the DRD. Whole-cell extracts prepared from 293 cells were incubated with either GST-DRD or GST. The bound proteins were precipitated with glutathione beads and separated using SDS-PAGE (Fig. 3A). Mass fingerprinting methods revealed that the proteins that specifically bound to the DRD were the carboxyl terminus of CHIP, HSP70, and HSP90 (Fig. 3A).
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DRD showed no ability to bind CHIP (Fig. 3D). Therefore, we tested whether CHIP enhances ERß ubiquitination. When ERß was coexpressed with CHIP, smeary bands of ubiquitin-conjugated ERß products were observed (Fig. 3E). In contrast, CHIP expression had no effect on the ubiquitination status of ERß(DRD). Thus, CHIP appears to mediate the ubiquitination of ERß. CHIP participates in the degradation of ERß. ERß is known to localize predominantly in the nucleus. Although a large proportion of the CHIP protein was localized in the cytoplasm, a small amount was observed in the nucleus by immunostaining (Fig. 4A) and Western blot analysis (Fig. 4B, left panel). To test whether CHIP-mediated ubiquitination induces the degradation of ERß, DNA encoding ERß was transfected into 293 cells with or without DNA encoding CHIP. Western blot analysis revealed that the steady-state level of ERß decreased in both the nucleus and the cytoplasm when CHIP was produced (Fig. 4B, left panel). ERß in MDA-MB-231 cells also decreased with the production of CHIP (Fig. 4B, right panel). We further determined CHIP's function by producing MDA-MB-231 cells in which endogenous CHIP expression was suppressed by the introduction of a siRNA complementary to sequences present in the CHIP mRNA. The introduction of the siRNA vector into MDA-MB-231 cells suppressed the expression of CHIP mRNA (data not shown) and protein (Fig. 4C, left panel) and the accumulation of ERß protein (Fig. 4C, right panel). In contrast, a control vector failed to alter the CHIP or ERß protein levels (Fig. 4C, right panel). Thus, CHIP appears to be involved in estrogen-dependent ERß degradation. To confirm this, pulse-chase experiments were performed. In the presence of estrogen, the half-life of ERß exceeded 8 h, but it had a half-life of about 4 h when CHIP was expressed (Fig. 4D).
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140), and ERß(AF-1), all of which contain the DRD, but did not enhance the degradation of ERß(DRD) or ERß(140), neither of which contains the DRD (Fig. 5A). To test the specificity of this effect, we created constructs in which the TPR and U-box domains of CHIP were deleted (TPR and U-box, respectively). CHIP binds to HSP/HSC70 via its TPR motif and displays E3 ubiquitin ligase activity mediated by its U-box domain. Although the expression of these truncated proteins was similar to that of full-length CHIP (data not shown), deletion of either of these domains abolished the effects of CHIP on ERß (Fig. 5B). Such a requirement for a TPR motif indicates that CHIP may have to interact with HSP/HSC70 to promote ERß degradation. The functional requirement for the U-box confirms that CHIP regulates ERß ubiquitination.
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in the absence of estrogen (48). Since ER and ERß heterodimerize readily in the presence of each other, we examined the effect of cointroducing ER on CHIP-dependent degradation of ERß. As shown in Fig. 5C, the degradation of ERß was not affected by coexpression of ER.
The F domain regulates ERß degradation negatively.
Our results showed that whereas CHIP binds and ubiquitinates ERß in both the presence and the absence of estrogen, the degradation of ERß is estrogen dependent. To investigate this discrepancy, DNA sequences encoding truncated forms of ERs were transfected into 293 cells and the protein levels were examined. The AD core domain is necessary for estrogen-dependent degradation of ER (29, 48). Consistent with this observation, ER(AD) was stabilized by ligand binding and thus accumulated in response to estrogen (Fig. 6A). Interestingly, the deletion of the AD core of ERß resulted in a dramatic reduction in ERß in the absence of estrogen (Fig. 6A). This observation raises the possibility that the C-terminal region of ERß is necessary for the stabilization of ERß. To test this possibility, DNA sequences encoding deletion mutants of the C terminus of ERß were transfected into 293 cells and levels of expressed protein were determined by Western blot analysis. When the F domain was deleted [ERß(F)], a dramatic reduction in ERß protein was observed in the absence of estrogen (Fig. 6B). This reduction in ERß was inhibited by MG132, indicating that the F domain suppresses the proteasome-dependent degradation of ERß. The degradation of ERß(F) required the DRD, because ERß(DRD;F) was not degraded in the absence of estrogen. In contrast, the degradation of ERß(140;F) was enhanced by fusion with the DRD [ERß(DRD;140;F)] in both the presence and the absence of estrogen (Fig. 6B). These results suggest that the F domain suppresses the DRD-dependent degradation of ERß in the absence of estrogen.
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did not affect its degradation. To study this, we generated a chimeric ERß, ERß (F), in which the F domain was substituted with an ER F domain. However, there is almost no homology between the ER F and ERß F domains, and the F domain of ER was able to protect ERß from proteolysis in the absence of estrogen (Fig. 6B).
Next, we determined the ubiquitination statuses of ERß and ERß(F). That of ERß(F) was similar to that of full-length ERß (Fig. 7A), indicating that the F domain does not regulate the ubiquitination step of ERß. Thus, the F domain may inhibit the accession of 26S proteasome to ERß structurally. Therefore, we evaluated the binding between ERß and proteasomes. FLAG-tagged ERß or ERß(F) was transfected into 293 cells with SUG-1, which is one of the components of regulatory subunit of 26S proteasome. A coimmunoprecipitation experiment revealed that deletion of the F domain enhanced association between ERß and SUG-1 (Fig. 7B). In addition, SUG-1 efficiently reduced ERß(F) rather than ERß (Fig. 7C). Thus, in the absence of estrogen, the F domain appears to abrogate the binding of the 26S proteasome to ERß to protect the receptor from proteolysis.
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is abrogated by the addition of MG132. The transcriptional activity of ERß was also reduced by MG132 (Fig. 8A). Thus, it is likely that receptor degradation is necessary for the ligand-dependent transactivation of the ERß gene. To investigate the association between CHIP-mediated receptor degradation and transactivation, we evaluated the transcriptional activities of ERß deletion mutants by use of a transient reporter assay. As shown in Fig. 8B, ERß(DRD) exhibited a transcriptional activity higher than that of full-length ERß. However, ERß(140) still showed transcriptional activity. When the DRD was fused to ERß(140) to form ERß(DRD;140), its transcriptional activity was reduced (Fig. 8B). Similarly, ERß(140; F) exhibited higher transcriptional activity than ERß(F), but fusion of DRD decreased its transcriptional activity (Fig. 8B). In agreement with these results, CHIP expression downregulated the transcriptional activity of the ERß gene (Fig. 8C). In contrast, CHIP siRNA enhanced transcriptional activity (Fig. 8C). The transcriptional activity of ERß(DRD) was not affected by expression of CHIP (Fig. 8C). Overall, our results indicate that CHIP-mediated degradation is not necessary for receptor-mediated transactivation. In addition, CHIP is able to ubiquitinate and to degrade ERß(mC) (Fig. 5A), indicating that binding to the DNA element is not necessary for CHIP-mediated receptor degradation. These results show that large amounts of liganded ERß protein are selectively and rapidly degraded independently of receptor-mediated transcription by CHIP.
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DRD) after estrogen withdrawal. DNA sequences encoding either ERß or ERß(DRD) were transfected into 293 cells together with a plasmid in which the PEST sequence was fused to the luciferase gene (ERE-TATA-LucCP), and the luciferase activity was measured at various time points after estrogen withdrawal. As shown in Fig. 9A, the transcriptional activity of the ERß sequence was reduced much faster than that of ERß(DRD). Using MDA-MB-231 cells that stably express either control siRNA or CHIP siRNA, we tested whether CHIP is involved in the downregulation of ERß-mediated transcription after reduction of the ligand dose. Plasmids containing the PEST-fused luciferase gene were transfected into MDA-MB-231 cells expressing either control siRNA or CHIP siRNA. ERß gene-mediated transcription was downregulated faster in the MDA-MB-231 cells expressing control siRNA than in the cells expressing CHIP siRNA (Fig. 9A).
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Overall, these results suggest that CHIP-mediated degradation selectively and rapidly eliminates the active form of the NR to downregulate receptor-mediated transcription after ligand withdrawal.
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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F) was similar to that of full-length ERß. In addition, coimmunoprecipitation experiments revealed that the F domain inhibits the binding of unliganded ERß to the regulatory subunit of 26S proteasome. Thus, the F domain may regulate not ubiquitination itself but the step involving proteasomal degradation. When estrogen binds to ERß, the conformation of the C-terminal region, including the F domain, is changed, and the protective effect of the F domain is abolished to induce degradation. Some members of the NR superfamily have an F domain, but its function is unknown. Our observations provide the first evidence that the F domain of ERß is involved in the regulation of its proteasome-dependent degradation. The activation of NRs appears to be coupled to their degradation by the ubiquitin-proteasome pathway (3, 4, 8). Furthermore, several components of the ubiquitin-proteasome pathway, including SUG1/TRIP1 (17, 27, 32), RSP5/RPF1 (23), E6-AP (37), and UBC9 (42), have been identified as NR-interacting proteins. TBL1/TBLR1, which are components of the NR corepressor complex, also mediate corepressor/coactivator exchange by the ligand-dependent recruitment of the ubiquitin-19S proteasome complex (40). Thus, the ubiquitin-proteasome pathway appears to play an important role in regulating NR levels and restricting the duration and magnitude of receptor activity in response to ligands. Furthermore, proteasome-dependent degradation of the steroid receptor requires transcriptional activity, suggesting that receptor degradation and receptor transactivation are mutually interdependent.
Several recent studies have focused on the involvement of the ubiquitin-proteasome pathway in the estrogen-dependent transactivation of ER (14, 29, 36, 44, 48, 50). Moreover, ER and its coactivators cycle onto and off of estrogen-responsive promoters in ligand-dependent manners (35, 44, 45). In this process, ER is reportedly ubiquitinated after each round of transcription, facilitating its release from the promoter, which may be essential for subsequent ER-mediated transcription. We have shown that there is a degradation pathway for ERß that is not coupled to transcription. Using ERß(DRD), we also have shown that transcription-uncoupled receptor degradation is necessary to abolish transcription via the proteolysis of the liganded receptor when the ligand dose is reduced. ERß(DRD) still exhibited slight ligand-dependent degradation, indicating that there may be other degradation pathways for ERß. These pathways could be coupled to the transactivation of ERß.
To investigate the ubiquitin-proteasome pathway for ERß, we purified the ubiquitin ligase complex that specifically binds to the DRD and identified a protein complex containing CHIP. CHIP was first reported to induce the ubiquitination of GR bound to HSP90 for proteasomal degradation (7). Recent observations have indicated that CHIP targets a number of HSP70/90-associated proteins for ubiquitination and degradation (15, 18, 21, 22, 28, 34, 48, 52-54). We (48) and others (9) have previously shown that CHIP binds unliganded ER as a protein complex containing HSP90, HSC70, HSP70, HSP40, BAG-1, and BAG-2, all of which possess or assist chaperone functions, and a DnaJ-like protein, KIAA0678. CHIP preferentially induces the hydrolysis of abnormal or mutant forms of ER, acting as an E3 ubiquitin ligase capable of distinguishing the nonnative states from the native states of the receptor in vivo.
Our results obtained from the analysis of ERß suggest that CHIP is involved both in the "quality control" of receptor proteins and in switching off receptor-mediated transcription (Fig. 10). The response of endocrine target tissues to hormones is tightly regulated and is dependent upon circulating levels of available hormonal ligands. When the dose of circulating ligand is reduced, receptor-mediated transcription should be downregulated. The dissociation rate of estrogen from its receptor is very low because a coactivator complex contacts the protein surface above the ligand pocket (16). Thus, it is difficult to cause rapid downregulation by ligand dissociation from its receptor. Our results provide the first evidence that receptor degradation is involved in the cessation of transcription when the ligand dose is reduced. Several nuclear receptors are reported to exhibit agonist-dependent degradation. Therefore, it is possible that other forms of receptor-mediated transcription are downregulated by the same mechanism as ERß when the agonist dose is reduced.
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FOOTNOTES |
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Published ahead of print on 28 August 2006.
These authors contributed equally to this work.
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