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

So this method can help answer key questions about the ability of estrogenic ligands to regulate estrogen receptor alpha and the mechanisms of selective estrogen receptor modulators. The main advantage of this technique is that it is a simple method. It demonstrates ligand-dependent estrogen receptor alpha ligand binding domain dimerization activity in the cells.

Demonstrating the procedure will be Tanner Jefferson, a postdoc student from our group. This method uses three different plasmids to detect the estrogen receptor ligand binding domain dimerization activity in cells. pG5-Luc is the luciferase reporter plasmid for detecting the efficacy of dimerization activity.

pBIND mouse estrogen receptor alpha EF is a galactose-responsive transcription factor GAL4 DNA binding to a main fused mouse estrogen alpha ligand binding domain expression plasmid that binds to the GAL4 responsive element on the pG5-Luc reporter. pACT mouse estrogen receptor alpha EF is a herpes simplex virus transactivating segment protein VP16 activation domain fused mouse estrogen receptor alpha ligand binding domain expression plasmid that does not bind directly to the pG5-Luc reporter. When the proper ligand is present, the proteins derived from the pBIND mouse estrogen receptor alpha EF and pACT mouse estrogen receptor alpha EF plasmids bind through the estrogen receptor alpha ligand binding domain.

The efficiency of the dimerization is determined by the luciferase activity. The most important step is controlling the activity of the pBIND mouse estrogen receptor alpha EF derived protein. The activity of pBIND-derived estrogen receptor alpha ligand bind domain in each ligand without the pACT estrogen receptor alpha EF plasmid must be evaluated before analyzing the ligand-dependent estrogen receptor ligand binding domain dimerization activity.

Begin by adding the appropriate plasmid combinations into two 1.5-milliliter microcentrifuge tubes per combination. Prepare the two combinations of the DNA mixture. First tube contains the pG5-Luc, pBIND-estrogen receptor-alpha EF, and empty pACT plasmids.

Second tube contains the pG5-Luc, pBIND-estrogen receptor-alpha EF, and pACT-estrogen receptor-alpha EF plasmids. To precipitate the DNA, add a 1/9 of the DNA mixture volume of three molar sodium acetate and a 20X three molar sodium acetate volume of 100%ethanol to the DNA mixture in each tube and vortex the tubes. Store the mixtures at 20 degrees Celsius overnight before centrifuging at maximum speed the next morning.

Discard the supernatants and resuspend the invisible pellets in 120 microliters of 75%ethanol per tube, taking care to rinse down any DNA stuck to the insides of the tubes. After a second centrifugation under the same conditions, discard the supernatants and dry the DNA in a vacuum concentrator for 30 minutes. For the transfection, wash the prepared human hepatocellular carcinoma cells two times with 0.5 milliliters of room-temperature PBS per well per wash and feed the cells with 0.5 milliliters of fresh 37 degree Celsius phenol red-free MEM supplemented with 10%charcoal-stripped heat-inactivated fetal bovine serum and two millimolar L-glutamine.

Next, suspend dried plasmid DNA mixture and transfection reagent in the appropriate volume of DMEM per number of wells in individual 1.5-milliliter microcentrifuge tubes. Add an equal volume of transfection reagent medium to each DMEM-suspended plasmid DNA mixture tube. After a five to 10-minute incubation at room temperature, add 25 microliters of DNA mixture to the appropriate experimental wells of the 24-well human hepatocellular carcinoma cell culture plate for a six-hour transfection in the cell culture incubator.

And the end of the incubation, replace the transfection medium with 0.5 milliliters of fresh 37 degree Celsius phenol red-free MEM supplemented with 10%charcoal-stripped heat-inactivated FBS, two millimolar L-glutamine, 1%penicillin streptomycin, and ligand per well and return the cells to the cell culture incubator for another 16 to 18 hours. The next morning, wash the cells with one milliliter of PBS per well and attach the cells from each well with 100 microliters of passive lysis buffer per well and vigorous shaking with a vortex mixer. Then freeze the cell lysates in liquid nitrogen.

On the day of the dual luciferase assay, shake the lysates on a shaker until the plate reaches room temperature, and transfer 10 microliters of lysate to individual wells of a 96-well white plastic plate. Then read the luciferase activity of each cell lysate sample on a microplate reader. The treatment of pBIND mouse estrogen receptor alpha EF and pACT plasmids transfected cells with 10 nanomolar estradiol stimulates luciferase activity as the estrogen receptor alpha ligand binding domain contains the ligand-dependent transactivation functional domain, AF-2.

One and 10-nanomolar concentrations however, induce luciferase activity in the cells transfected with the combination of pBIND and pACT mouse estrogen receptor alpha EF plasmids, suggesting the feasibility of this procedure for evaluating estrogen receptor alpha ligand binding domain dimerization activity at up to one nanomolar of estradiol treatment. Treatment with 4-hydroxy-tamoxifen does not elicit luciferase activity in the pBIND mouse estrogen receptor alpha EF and pACT plasmid transfected cells, suggesting that the AF-2 function of estrogen receptor alpha is not activated by this ligand. Up to 10 nanomolar concentrations however, induces luciferase activity in the cells transfected with the combination of pBIND and pACT mouse estrogen receptor alpha EF plasmids, suggesting that this procedure is feasible for evaluating estrogen receptor alpha ligand binding domain dimerization activity at up to 10 nanomolar of 4-hydroxy-tamoxifen treatment.

The activity from the combination of pBIND and pACT mouse estrogen receptor alpha ligand binding domain expression plasmids with 4-hydroxy-tamoxifen is significantly higher than the combination of pBIND and pACT human estrogen receptor alpha ligand binding domain expression plasmids. This indicates that the 4-hydroxy-tamoxifen-dependent homodimerization activity of the mouse estrogen receptor alpha ligand binding domain is more potent than the human estrogen receptor alpha ligand binding domain. Further, the 4-hydroxy-tamoxifen-dependent homodimerization activity of the mouse estrogen receptor alpha ligand binding domain containing a human F domain is significantly lower than that of the wild type mouse estrogen receptor alpha ligand binding domain.

In contrast, the 4-hydroxy-tamoxifen-dependent homodimerization activity of the human estrogen receptor alpha ligand binding domain containing a mouse F domain is significantly higher than that of the wild type human estrogen receptor alpha ligand binding domain. This suggests that the F domain has an influence on species-specific 4-hydroxy-tamoxifen-dependent estrogen receptor alpha ligand binding domain homodimerization activity. While attempting this procedure, it is important to remember to test the basal activity of GAL4 DNA binding domain fused estrogen receptor alpha ligand bind domain activity with your ligand.

That activity should be same as vehicle treatment.