Dataset Information

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

ABSTRACT: Estrogen receptor alpha (ER?) is an estrogenic ligand-dependent transcription regulator. The sequence of ER? protein is highly conserved among species. It has been thought that the function of human and mouse ER?s is identical. We demonstrate the differential 4-hydroxy-tamoxifen (4OHT) effect on mouse and human ER? 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? LBD fusion protein expression plasmids than in the human ER? LBD fusion protein expression plasmid transfected cells. This result suggests that the efficacy of the 4OHT-dependent mouse ER? LBD homodimerization activity is higher than human ER? 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? 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? 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.

SUBMITTER: Arao Y

PROVIDER: S-EPMC6953383 | biostudies-literature | 2018 Dec

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

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

Arao Yukitomo Y Korach Kenneth S KS

Journal of visualized experiments : JoVE 20181219 142

Estrogen receptor alpha (ERα) is an estrogenic ligand-dependent transcription regulator. The sequence of ERα protein is highly conserved among species. It has been thought that the function of human and mouse ERαs is identical. We demonstrate the differential 4-hydroxy-tamoxifen (4OHT) effect on mouse and human ERα 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 mammalia ...[more]

PMID: 30614492

Similar Datasets

Project description:The protein encoded by GmRLK18-1 (Glyma_18_02680 on chromosome 18) was a receptor like kinase (RLK) encoded within the soybean (Glycine max L. Merr.) Rhg1/Rfs2 locus. The locus underlies resistance to the soybean cyst nematode (SCN) Heterodera glycines (I.) and causal agent of sudden death syndrome (SDS) Fusarium virguliforme (Aoki). Previously the leucine rich repeat (LRR) domain was expressed in Escherichia coli.The aims here were to evaluate the LRRs ability to; homo-dimerize; bind larger proteins; and bind to small peptides. Western analysis suggested homo-dimers could form after protein extraction from roots. The purified LRR domain, from residue 131-485, was seen to form a mixture of monomers and homo-dimers in vitro. Cross-linking experiments in vitro showed the H274N region was close (<11.1 A) to the highly conserved cysteine residue C196 on the second homo-dimer subunit. Binding constants of 20-142 nM for peptides found in plant and nematode secretions were found. Effects on plant phenotypes including wilting, stem bending and resistance to infection by SCN were observed when roots were treated with 50 pM of the peptides. Far-Western analyses followed by MS showed methionine synthase and cyclophilin bound strongly to the LRR domain. A second LRR from GmRLK08-1 (Glyma_08_g11350) did not show these strong interactions.The LRR domain of the GmRLK18-1 protein formed both a monomer and a homo-dimer. The LRR domain bound avidly to 4 different CLE peptides, a cyclophilin and a methionine synthase. The CLE peptides GmTGIF, GmCLE34, GmCLE3 and HgCLE were previously reported to be involved in root growth inhibition but here GmTGIF and HgCLE were shown to alter stem morphology and resistance to SCN. One of several models from homology and ab-initio modeling was partially validated by cross-linking. The effect of the 3 amino acid replacements present among RLK allotypes, A87V, Q115K and H274N were predicted to alter domain stability and function. Therefore, the LRR domain of GmRLK18-1 might underlie both root development and disease resistance in soybean and provide an avenue to develop new variants and ligands that might promote reduced losses to SCN.

Dataset Information

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

Publications

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

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets