Agonist and Antagonist Activity in a GFP Yeast Based Estrogen Receptor Functional Assay

AGONIST AND ANTAGONIST ACTIVITY IN A GFP YEAST BASED ESTROGEN RECEPTOR FUNCTIONAL ASSAY ABSTRACT RESULTS AND LIMITATIONS Estrogen receptors (ERα and ERβ) are ligand-binding transcription factors activated by the hormone 17-β estradiol. Ligand binding triggers ER dimerization, translocation of the receptor from the cytosol into the nucleus and eventually activation of the genes under control of ER. Studies have revealed a role for estrogen receptors in male and female sexual development, reproductive functions, bone metabolism and regulation of neuroendocrine and cardiovascular systems. ER is also known to bind to other nonnative ligands known in pharmacology as receptor agonists or antagonists. Agonists provoke a biological response when bound to the receptor; antagonists inhibit a biological response when bound. Our lab is interested in the promiscuous binding of the estrogen receptor and its ability to activate different genes in different tissues. Fluorescence polarization assays have previously been performed using ER to study ligand binding affinities for the receptor. However, this technique is unable to determine whether these ligands are agonists or antagonists and allow ER dimerization and gene activation. To investigate these phenomena, an activity assay that measures ER controlled gene expression has been developed which provides the opportunity to gain further insight into the functional activity in living systems. Recombinant yeast cells that express ERα use the green fluorescent protein (GFP) reporter to determine whether ERα, in the presence of a particular ligand, has activated gene of expression. We have correlated the binding to agonist and antagonist behavior of several xeno- and phyto- estrogens. INTRODUCTION COMPETITIVE LIGAND BINDING ASSAY METHOD AND RESULTS STORAGE CONDITIONS • Inoculate agar plate containing selective medium with yeast from frozen -80 0C glycerol stock (20% glycerol v/v). Incubate plate at 300C for 24-48 hours and store at 40C for 2 weeks CULTURE CONDITIONS • Selective minimal medium containsYNB (-AA/-AS), dextrose, ammonium sulfate and L-leucine (MML ) YEGFP ASSAY • Use one colony to incolulate 10 mL MML. Leave culture to grow overnight at 300C and 200 rpm • Dilute overnight culture in fresh MML such that the OD630=0.05 ± 0.01. Data courtesy of Suzannah Luft, Eric Glustrom, Elias Aba-Miliki, Richmond AmpiahBonney and Patricia O’Hara • Competitive ligand binding assays allow us to measure the relative binding affinities of several non-native ligands to ER • Add 200 μL of diluted culture and 2 μL of test compounds dissolved in DMSO to 96 well microplate. Incubate for 4-24 hours at 300C and 200 rpm • Measure fluorescence using The SpectraMax® M5 Multi-Detection Microplate Reader. Excite at 488 nm and measure emission at 530 nm DOSE-RESPONSE CURVES • For example, some DDT derivatives such as HPTE, VF77-1 and Vf72-1 were found to bind strongly to ERα and ERβ 2 • However, these binding assays do not allow us to determine ER-mediated gene activation and therefore do not distinguish between agonists and antagonists SOLUTION The yeast estrogen bioassay developed by Dr. Toine Bovee et al. in the Netherlands1 provides a solution to this problem. In the bioassay, mutant S. cerevisiae express human ERα (hERα) and use the yeast enhanced green fluorescent protein (yEGFP) reporter construct. yEGFP exhibits bright green fluorescence (λem=530 nm) when exposed to violet light (λex=488 nm) yEGFP MOLECULAR MECHANISM CHALLENGES AND FUTURE WORK • Somewhat difficult to obtain suitable dose-response curves using the native estradiol ligand Invitrogen fluorescence polarization assays use fluormoneTM, a fluorescent estrogen ligand with low polarization • Optimize S. cerevisiae bioassay • Addition of ER to fluormoneTM results in a bound complex and high polarization • Use yEGFP bioassay to identify the agonist and antagonist behavior of previously characterized ligands • Addition of ER/fluormone complex to competitor molecules results in low polarization if the competitor displaces the fluormoneTM TM • The change in polarization in the presence of a competitor molecule is used to determine the binding affinities of test compounds of interest 1 • Estradiol enters the yeast cell, binds ERα and inhibitory chaperone proteins dissociate • ERα dimerizes and translocates into the nucleus where the ERα homodimer binds EREs within the yEGFP promoter. Cofactors are recruited to activate transcription of yEGFP • yEGFP fluorescence can be used to determine whether a ligand of interest has activated yEGFP transcription • Fluorescence intensity can also be used to determine the extent of ERα activation LITERATURE CITED 1 2 http://www.invitrogen.com/site/us/en/home/References/Molecular-Probes-The-Handbook/Technical-Notes-and-Product-Highlights/Fluorescence-Polarization-FP.html Bovee, Toine F. et al., “Development of A Yeast Estrogen Bioassay, Based on the Expression of Green Fluorescent Protein,” Gene 325 (Feb 2004):187-200