Nuclear Receptor Assay Applications Presented Fall 2009

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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.