Nuclear Receptor Assay Applications Presented Fall 2009 Click the icon in the upper left hand corner to view speaker notes for slides. Have a question? Ask a Scientist GloResponse is a trademark and Dual-Luciferase is a registered trademark of Promega Corporation. HighWire Press is a registered trademark of the Board of Trustees of the Leland Stanford Junior University ©2009, Promega Corporation. All rights reserved. Application Overview POST-TRANSCRIPTION MIRNA CONTROL In vivo PROTEIN applications INTERACTIONS expression level varies with treatment NUCLEAR RECEPTORS SIGNALING Experimental firefly luciferase PATHWAY construct expression level varies little with PROMOTER treatment Control DISSECTION Renilla luciferase construct 2 Traditional Nuclear Receptor Assays Steroid A good assay Works with endogenous receptors Steroid Receptor RNA Pol II NRE luciferase 3 Your expression analysis found an uncharacterized NR… N-Terminal Hinge C-Terminal Domain Region Domain A/B C D E F DNA Binding Ligand Binding Domain Domain •Response elements are not specifically known •Agonists are not known •Coactivators are not known •How can you do work on this nuclear receptor? A One-Hybrid Luciferase Reporter Assay can help understand the nuclear receptor more fully 4 Universal Nuclear Receptor Assays Ligand binding domain responsible for: • homodimerization (class I receptors) • heterodimerization (class II receptors) • corepressor binding • coactivator binding Nuclear Receptor Ligand Binding Domain pBIND Vector GAL4 BD GAL4 GAL4 GAL4 GAL4 GAL4 GAL4 GAL4 GAL4 GAL4 TATA UAS UAS UAS UAS UAS UAS UAS UAS UAS Box luc2P pGL4.35[luc2P/9XGAL4UAS/Hygro] Vector 5 What you need… GAL4 GAL4 GAL4 GAL4 GAL4 GAL4 GAL4 GAL4 GAL4 TATA UAS UAS UAS UAS UAS UAS UAS UAS UAS Box luc2P pGL4.35[luc2P/9XGAL4UAS/Hygro] Vector Nuclear Receptor Ligand Binding Domain Both vectors ready to make stable cells • pGL4.35 Æ HygR • pFN26A Æ NeoR 6 Nuclear Receptor Assay Principle 1 TRANSFECT 2 CULTURE 2-3 DAYS 3 TREAT DUAL-LUCIFERASE® 4 ASSAY pGL4.35 Perform mutagenesis on the ligand binding domain and assay in your responsive cell. pBIND • No interference from Screen for endogenous receptor agonists, antagonists, Make your own co-activators, Nuclear Receptor co-repressors Luciferase Reporter Cell line • pGL4.35 HygR 7 Pre-designed tools available for nuclear receptor assays Pre-designed Estrogen Receptor α–& Glucocorticoid Receptor– GAL4 Binding Domain fusions available GloResponse™ 9XGAL4UAS-luc2P HEK293 Cell Line HEK 293 HygR cell line made with pGL4.35 Ready to use with the nuclear receptor or CheckMate™ Assays 8 Case Study: Coactivator of Vitamin D receptor Vitamin D receptor (VDR) is a nuclear receptor that upon ligand binding displaces a co-repressor and allows co-activators to bind, allowing transcription. Central question: • Peroxisome Proliferator-Activated Receptor γ coactivator-1 α (PGC- 1α) and the Vitamin D receptor have overlapping tissue distribution, • Does PGC1α function as a coactivator of VDR? Savkur, R.S., et al. (2005) Mol. Pharmacol. 68, 511-517. 9 Case Study: Savkur, R.S. et al. PGC-1α is a coactivator of VDR Hinge AF-2 Region (Helix 12) GAL4 Binding Domain D E F Vitamin D3 Binding Domain Fold Increase Luc Constructs DMSO VitD3 activity +– 1 Gal4BD-VDR + pG5-luc pG5-luc –+ 5 +– 1 Gal4BD-VDR + PGC-1α Gal4BD- + pG5-luc up to –+ PGC-1α VDR 500 +– 1 Gal4BD-VDR(ΔAF-2) + PGC-1α + pG5-luc –+ 1 Conclusions supported by work with full receptor & mammalian two-hybrid work 10 More Information… Go to Go to Go to Go to pGL4.35 pBIND-ERα pBIND-GR cell line Product Product Product Technical Protocol Protocol Protocol Manual Paguio, A., et al. (2008) Improvements to luciferase reporter assays for nuclear receptor function. PS065 presented at Society for Biomolecular Sciences meeting. 11.