Name: detecting the ligand-binding domain dimerization activity of estrogen receptor alpha using the mammalian two-hybrid assay
Uploaded: 2018-12-19T13:00:00
Description: Watch this Scientific Journal Video about Detecting the Ligand-binding Domain Dimerization Activity of Estrogen Receptor Alpha Using the Mammalian Two-Hybrid Assay at JoVE.com

The authors thank Drs. Sueyoshi and Wang at the National Institute of Environmental Health Sciences (NIEHS) for their critical reading of the manuscript, and&#160;Tanner Jefferson for performing procedures on the video.&#160;This work was supported by the National Institutes of Health Grant 1ZIAES070065 (to K.S.K.) from the Division of Intramural Research of the NIEHS.

1. Preparation of Plasmids for the Mammalian Two-hybrid Assay
<ol>
	<li>Use the following plasmids: pG5-Luc, pBIND-ER&#945;EF, and pACT-ER&#945;EF. 
	NOTE: The plasmids are available from the authors upon request and are sent on filter paper. pG5-Luc is the reporter plasmid that contains five repeats of GAL4 responsive elements fused with the luciferase expression unit (Figure 1A). pBIND-ER&#945;EF is the protein expression plasmid for GAL4DBD-fused ER&#945; LBD prote...

<ol>
	<li>Arao, Y., Korach, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+F+domain+of+estrogen+receptor+&#945;+is+involved+in+species-specific,+tamoxifen-mediated+transactivation.">The F domain of estrogen receptor &#945; is involved in species-specific, tamoxifen-mediated transactivation.</a> Journal of Biological Chemistry. 293 (22), 8495-8507 (2018).</li><li>Brzozowski, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+basis+of+agonism+and+antagonism+in+the+oestrogen+receptor.">Molecular basis of agonism and antagonism in the oestrogen receptor.</a> Nature. 389 (6652), 753-758 (1997).</li><li>Shiau, A. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+structural+basis+of+estrogen+receptor/coactivator+recognition+and+the+antagonism+of+this+interaction+by+tamoxifen.">The structural basis of estrogen receptor/coactivator recognition and the antagonism of this interaction by tamoxifen.</a> Cell. 95 (7), 927-937 (1998).</li><li>Yang, J., Singleton, D. W., Shaughnessy, E. A., Khan, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+F-domain+of+estrogen+receptor-alpha+inhibits+ligand+induced+receptor+dimerization.">The F-domain of estrogen receptor-alpha inhibits ligand induced receptor dimerization.</a> Molecular and Cellular Endocrinology. 295 (1-2), 94-100 (2008).</li><li>Montano, M. M., M&#252;ller, V., Trobaugh, A., Katzenellenbogen, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+carboxy-terminal+F+domain+of+the+human+estrogen+receptor:+role+in+the+transcriptional+activity+of+the+receptor+and+the+effectiveness+of+antiestrogens+as+estrogen+antagonists.">The carboxy-terminal F domain of the human estrogen receptor: role in the transcriptional activity of the receptor and the effectiveness of antiestrogens as estrogen antagonists.</a> Molecular Endocrinology. 9 (7), 814-825 (1995).</li><li>Koide, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+Regions+within+the+F+Domain+of+the+Human+Estrogen+Receptor+&#945;+that+Are+Important+for+Modulating+Transactivation+and+Protein-Protein+Interactions.">Identification of Regions within the F Domain of the Human Estrogen Receptor &#945; that Are Important for Modulating Transactivation and Protein-Protein Interactions.</a> Molecular Endocrinology. 21 (4), 829-842 (2011).</li><li>Mitry, R. R., Hughes, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Human Cell Culture Protocols. , (2011).</li><li>Arao, Y., Coons, L. A., Zuercher, W. J., Korach, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transactivation+Function-2+of+Estrogen+Receptor+&#945;+Contains+Transactivation+Function-1-regulating+Element.">Transactivation Function-2 of Estrogen Receptor &#945; Contains Transactivation Function-1-regulating Element.</a> Journal of Biological Chemistry. 290 (28), 17611-17627 (2015).</li><li>Huang, H. -. J., Norris, J. D., McDonnell, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+negative+regulatory+surface+within+estrogen+receptor+alpha+provides+evidence+in+support+of+a+role+for+corepressors+in+regulating+cellular+responses+to+agonists+and+antagonists.">Identification of a negative regulatory surface within estrogen receptor alpha provides evidence in support of a role for corepressors in regulating cellular responses to agonists and antagonists.</a> Molecular Endocrinology. 16 (8), 1778-1792 (2002).</li><li>Chang, C. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissection+of+the+LXXLL+nuclear+receptor-coactivator+interaction+motif+using+combinatorial+peptide+libraries:+discovery+of+peptide+antagonists+of+estrogen+receptors+alpha+and+beta.">Dissection of the LXXLL nuclear receptor-coactivator interaction motif using combinatorial peptide libraries: discovery of peptide antagonists of estrogen receptors alpha and beta.</a> Molecular and Cellular Biology. 19 (12), 8226-8239 (1999).</li><li>Arao, Y., Hamilton, K. J., Coons, L. A., Korach, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estrogen+receptor+&#945;+L543A,+L544A+mutation+changes+antagonists+to+agonists,+correlating+with+the+ligand+binding+domain+dimerization+associated+with+DNA+binding+activity.">Estrogen receptor &#945; L543A, L544A mutation changes antagonists to agonists, correlating with the ligand binding domain dimerization associated with DNA binding activity.</a> The Journal of Biological Chemistry. 288 (29), 21105-21116 (2013).</li></ol>

Herein, we described the protocol for the M2H assay, focusing on the assay conditions for detecting the homodimerization activity of ER&#945; LBD as an example. In general, the M2H assay is not popular for the assessment of ligand-dependent ER&#945; LBD dimerization activity. This is due to the ER&#945; LBD possessing a transcriptional activation function; the activity of which disturbs, in some cases, the results of the M2H assay. However, as we demonstrate here, the M2H assay can be used for analyzing the LBD dimerizat...

Estrogen receptor alpha (ER&#945;) is an estrogenic ligand-dependent transcription regulator. The amino acid sequenceof ER&#945; is highly conserved among species. Because of the higher homology between human and mouse ER&#945;, the function of these receptors is thought of as identical, and the differential activity of estrogenic substances (e.g., tamoxifen) in those species is caused by the species&#39; differences in chemical metabolisms rather than by the structural differences of ER&#945;. ER&#945; has highly conserved domain structures that are common among the nuclear receptor (NR) superfamily, designated A to F domains. The E domain or ligand-binding domain (LBD) includes the ligand-binding pocket and the transcriptional activation function 2, named AF-2. The F domain is localized immediately adjacent to the E domain and is the most variable domain among the NRs. Even between human and mouse ER&#945;, the homology of the F domain is significantly lower than that of the other domains1. The ligand-bound LBD of ER&#945; enhances the homodimerization of the ER&#945; protein to bind the specific estrogen-responsive DNA element directly for regulating the ligand-dependent gene transcription (classical action of ER&#945;). Crystallographic studies have revealed the differential positioning of helix 12 (AF-2 core domain) with estradiol (E2)- or 4-hydroxy-tamoxifen (4OHT)-bound LBD dimers2,3. The ER&#945; F domain (45 amino acids) connect helix 12 directly. However, there is no information regarding the effect of this extension of 45 amino acids (F domain) from the helix 12 on the ER&#945; LBD dimerization. In this study, we demonstrate the contribution of the F domain to the species-specific 4OHT-dependent ER&#945; LBD homodimerization using a mammalian two-hybrid (M2H) assay.
The M2H assay is a method to demonstrate protein-protein interactions in mammalian cells introducing the three different plasmid DNAs: two protein expression plasmids, which express GAL4 DNA-binding domain (DBD) fusion ER&#945; LBD and VP16AD fusion ER&#945; LBD, and a GAL4RE-fused luciferase expression reporter plasmid. When the GAL4DBD fusion ER&#945; LBD and the VP16AD fusion ER&#945; LBD interact (make a dimer) in the cells, this protein complex binds to the GAL4RE and, then, activates luciferase gene expression through the VP16AD-dependent transactivation function. The level of LBD homodimerization can be evaluated by the luciferase activity.
The yeast two-hybrid (Y2H) assay is an alternative method based on the same principle that uses yeast as the host environment. Previous reports using the Y2H system demonstrated that F-domain-truncated human ER&#945; LBD increases E2-dependent coactivator recruitment, concluding that the F domain prevents E2-mediated transcription4. This result is inconsistent with other reports which have demonstrated the attenuated transcriptional activity of F-domain-truncated human ER&#945; in the mammalian cells5,6. Recently, our study, using the M2H system, demonstrated that the E2-dependent coactivator recruitment activity of human ER&#945; LBD is decreased by F domain truncation in mammalian cells and is consistent with transcription activity1. These observations suggest that the physiological role of protein-protein interaction differs in a cell type-specific manner and context. The M2H assay can demonstrate protein-protein interaction activity in the same cellular context that is used for determining the transcriptional activity. This provides an advantage of the M2H assay compared to the Y2H or other in vitro protein-protein interaction analyses.
There remain questions regarding the molecular mechanisms of the partial agonist activity of selective estrogen receptor modulators (SERMs) (e.g., 4OHT) to regulate ER&#945;-mediated transcription. ER&#945; LBD-based M2H assays may be applied to study the mechanism of the partial agonist activity of SERMs in various mammalian cell types.

<table border="0" cellpadding="0" cellspacing="0" style="width:891px;" width="891">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">pG5-Luc</td>
			<td style="width:112px;">Promega</td>
			<td style="width:119px;">E249A</td>
			<td style="width:360px;">Component of CheckMate Mammalian Two-Hybrid System</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">pBIND</td>
			<td style="width:112px;">Promega</td>
			<td>E245A</td>
			<td>Component of CheckMate Mammalian Two-Hybrid System</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">pACT</td>
			<td style="width:112px;">Promega</td>
			<td>E246A</td>
			<td>Component of CheckMate Mammalian Two-Hybrid System</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">One Shot TOP10 Chemically Competent E. coli</td>
			<td>Invitrogen</td>
			<td>C404010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Centrifuge Rotor</td>
			<td>SORVALL</td>
			<td>75006445</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Swing Buckets</td>
			<td>SORVALL</td>
			<td>75006441</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cell Resuspension Solution (CRA)</td>
			<td style="width:112px;">Promega</td>
			<td>A7112</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cell Lysis Solution (CLA)</td>
			<td style="width:112px;">Promega</td>
			<td>A7122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Neutralization Solution (NSA)</td>
			<td style="width:112px;">Promega</td>
			<td>A7131</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Column Wash Solution (CWB)</td>
			<td style="width:112px;">Promega</td>
			<td>A8102</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Wizard Midipreps DNA Purification Resin</td>
			<td style="width:112px;">Promega</td>
			<td>A7701</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Wizard Midicolumns</td>
			<td style="width:112px;">Promega</td>
			<td>A7651</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MEM, no glutamine, no phenol red</td>
			<td>Gibco</td>
			<td style="width:119px;">51200038</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-glutamine (200 mM)</td>
			<td>Gibco</td>
			<td>A2916801</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">0.5% Trypsin-EDTA (10x)</td>
			<td>Gibco</td>
			<td style="width:119px;">15400054</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">Penicillin-Streptomycin (100x)</td>
			<td style="width:112px;">Sigma-Aldrich</td>
			<td style="width:119px;">P0781</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BenchMark fetal bovine serum (FBS)</td>
			<td>Gemini-Bio</td>
			<td>100-106</td>
			<td>Heat inactivated</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Charcoal:dextran stripping fetal bovine serum</td>
			<td>Gemini-Bio</td>
			<td>100-119</td>
			<td>Heat inactivated</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM, high glucose, no glutamine, no phenol red</td>
			<td>Gibco</td>
			<td>31053028</td>
			<td>for transfection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lipofectamine 2000</td>
			<td>Invitrogen</td>
			<td style="width:119px;">11668027</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Passive Lysis 5X Buffer</td>
			<td style="width:112px;">Promega</td>
			<td>E1941</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dual-Luciferase Reporter Assay System</td>
			<td style="width:112px;">Promega</td>
			<td>E1980</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SpectraMax L microplate reader</td>
			<td>Molecular Devices</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">SoftMax Pro Software</td>
			<td>Molecular Devices</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

detecting the ligand-binding domain dimerization activity of estrogen receptor alpha using the mammalian two-hybrid assay

Estrogen receptor alpha (ER&#945;) is an estrogenic ligand-dependent transcription regulator. The sequence of ER&#945; protein is highly conserved among species. It has been thought that the function of human and mouse ER&#945;s is identical. We demonstrate the differential 4-hydroxy-tamoxifen (4OHT) effect on mouse and human ER&#945; ligand-binding domain (LBD) dimerization activity using the mammalian two-hybrid (M2H) assay. The M2H assay can demonstrate the efficiency of LBD homodimerization activity in mammalian cells, utilizing the transfection of two protein expression plasmids (GAL4 DNA-binding domain [DBD] fusion LBD and VP16 transactivation domain [VP16AD] fusion LBD) and a GAL4-responsive element (GAL4RE) fused luciferase reporter plasmid. When the GAL4DBD fusion LBD and the VP16AD fusion LBD make a dimer in the cells, this protein complex binds to the GAL4RE and, then, activates a luciferase gene expression through the VP16AD dependent transcription activity. The 4OHT-mediated luciferase activation is higher in the HepG2 cells that were transfected with the mouse ER&#945; LBD fusion protein expression plasmids than in the human ER&#945; LBD fusion protein expression plasmid transfected cells. This result suggests that the efficacy of the 4OHT-dependent mouse ER&#945; LBD homodimerization activity is higher than human ER&#945; LBD. In general, the utilization of the M2H assay is not ideal for the evaluation of nuclear receptor LBD dimerization activity, because agonistic ligands enhance the basal level of the LBD activity and that impedes the detection of LBD dimerization activity. We found that 4OHT does not enhance ER&#945; LBD basal activity. That is a key factor for being able to determine and detect the 4OHT-dependent LBD dimerization activity for successfully using the M2H assay. ER&#945; LBD-based M2H assays may be applied to study the partial agonist activity of selective estrogen receptor modulators (e.g., 4OHT) in various mammalian cell types.

Figure 3&#160;displays the scheme of possible responses in the combination (i) and combination (ii) plasmid-transfected cells. The experimental results are shown in Figure 4. The activity of combination (i) (pG5-Luc + pBIND-mER&#945;EF + pACT) shows stimulation by 10 nM E2 (Figure 4A), because the ER&#945; LBD contains the ligand-dependent transactivation functional domain, AF-2. Ago...

Watch this Scientific Journal Video about Detecting the Ligand-binding Domain Dimerization Activity of Estrogen Receptor Alpha Using the Mammalian Two-Hybrid Assay at JoVE.com

Detecting the Ligand-binding Domain Dimerization Activity of Estrogen Receptor Alpha Using the Mammalian Two-Hybrid Assay

We present a method for analyzing the 4-hydroxy-tamoxifen-dependent estrogen receptor alpha ligand-binding domain dimerization activity using the mammalian two-hybrid assay.

Estrogen receptor alpha (ER&#945;) is an estrogenic ligand-dependent transcription regulator. The sequence of ER&#945; protein is highly conserved among ...

Research

JoVE Journal

Cancer Research

A General Method for Detecting Nitrosamide Formation in the In Vitro Metabolism of Nitrosamines by Cytochrome P450s

A General Method for Detecting Nitrosamide Formation in the In Vitro Metabolism of Nitrosamines by Cytochrome P450s

N-nitrosamines are a well-established group of environmental carcinogens, which require cytochrome P450 oxidation to exhibit activity. The accepted ...

Evaluation of the Cell Invasion and Migration Process: A Comparison of the Video Microscope-based Scratch Wound Assay and the Boyden Chamber Assay

The invasion and migration abilities of tumor cells are main contributors to cancer progression and recurrence. Many studies have explored the migration ...

Using the Dot Assay to Analyze Migration of Cell Sheets

Although complex organisms appear static, their tissues are under a continuous turnover. As cells age, die, and are replaced by new cells, cells move ...

Using CRISPR/Cas9 Gene Editing to Investigate the Oncogenic Activity of Mutant Calreticulin in Cytokine Dependent Hematopoietic Cells

Clustered regularly interspaced short palindromic repeats (CRISPR) is an adaptive immunity system in prokaryotes that has been repurposed by scientists to ...

A Comprehensive Procedure to Evaluate the In Vitro Performance of the Putative Hemangioblastoma Neovascularization Using the Spheroid Sprouting Assay

A Comprehensive Procedure to Evaluate the In Vitro Performance of the Putative Hemangioblastoma Neovascularization Using the Spheroid Sprouting Assay

The inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene plays a crucial role in the development of hemangioblastomas (HBs) within the human ...

Modeling Osteosarcoma Using Li-Fraumeni Syndrome Patient-derived Induced Pluripotent Stem Cells

Li-Fraumeni syndrome (LFS) is an autosomal dominant hereditary cancer disorder. Patients with LFS are predisposed to a various type of tumors, including ...

Surface-enhanced Resonance Raman Scattering Nanoprobe Ratiometry for Detecting Microscopic Ovarian Cancer via Folate Receptor Targeting

Ovarian cancer represents the deadliest gynecologic malignancy. Most patients present at an advanced stage (FIGO stage III or IV), when local metastatic ...

Manufacturing Chimeric Antigen Receptor (CAR) T Cells for Adoptive Immunotherapy

Manufacturing Chimeric Antigen Receptor CAR T Cells for Adoptive Immunotherapy

Adoptive immunotherapy holds promise for the treatment of cancer and infectious disease. We describe a simple approach to transduce primary human T cells ...

In Vitro Establishment of a Genetically Engineered Murine Head and Neck Cancer Cell Line using an Adeno-Associated Virus-Cas9 System

The use of primary normal epithelial cells makes it possible to reproducibly induce genomic alterations required for cellular transformation by ...

Mapping the Structure-Function Relationships of Disordered Oncogenic Transcription Factors Using Transcriptomic Analysis

Many cancers are characterized by chromosomal translocations which result in the expression of oncogenic fusion transcription factors. Typically, these ...