Download Nuclear Receptor Assay Applications

Nuclear Receptor Assay
Applications
Presented Fall 2009
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Application Overview
POST-TRANSCRIPTION
MIRNA CONTROL
PROTEIN
In vivo
applications
INTERACTIONS
NUCLEAR
RECEPTORS
SIGNALING
PATHWAY
PROMOTER
DISSECTION
2
expression level
varies with treatment
Experimental
firefly luciferase
construct
expression level
varies little with
treatment
Control
Renilla luciferase
construct
Traditional Nuclear Receptor Assays
A good assay
Works with endogenous receptors
Steroid
Steroid
Receptor
RNA
Pol II
NRE
3
luciferase
Your expression analysis found an uncharacterized NR…
N-Terminal
Domain
A/B
Hinge
Region
C
DNA Binding
Domain
D
C-Terminal
Domain
E
Ligand Binding
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
F
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
UAS
GAL4
UAS
GAL4
UAS
GAL4
UAS
GAL4
UAS
GAL4
UAS
GAL4
BD
GAL4
UAS
GAL4
UAS
GAL4
UAS
TATA
Box
luc2P
pGL4.35[luc2P/9XGAL4UAS/Hygro] Vector
5
What you need…
GAL4
UAS
GAL4
UAS
GAL4
UAS
GAL4
UAS
GAL4
UAS
GAL4
UAS
GAL4
UAS
GAL4
UAS
GAL4
UAS
TATA
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
4
DUAL-LUCIFERASE®
ASSAY
pGL4.35
pBIND
Screen for
agonists,
antagonists,
co-activators,
co-repressors
7
Make your own
Nuclear Receptor
Luciferase Reporter
Cell line
• pGL4.35 HygR
Perform mutagenesis on
the ligand binding domain
and assay in your
responsive cell.
• No interference from
endogenous receptor
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 (PGC1α) 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
AF-2
(Helix 12)
Hinge
Region
GAL4 Binding Domain
D
E
F
Vitamin D3 Binding
Domain
Constructs
VitD3
+
–
1
–
+
5
+
–
1
–
+
+
–
1
–
+
1
Gal4BD-VDR + pG5-luc
pG5-luc
PGC-1α
DMSO
Fold
Increase
Luc
activity
Gal4BDVDR
Gal4BD-VDR + PGC-1α
+ pG5-luc
Gal4BD-VDR(ΔAF-2)
+ PGC-1α + pG5-luc
up to
500
Conclusions supported by work with full receptor & mammalian two-hybrid work
10
More Information…
Go to
pGL4.35
Product
Protocol
Go to
pBIND-ERα
Product
Protocol
Go to
pBIND-GR
Product
Protocol
Go to
cell line
Technical
Manual
Paguio, A., et al. (2008) Improvements to luciferase
reporter assays for nuclear receptor function. PS065
presented at Society for Biomolecular Sciences
meeting.
11