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Application Note - Nuclear Receptor; Profiling Ligands
Detection of multiple nuclear receptor–coregulator interactions in a single
sample using MARCoNI PamChip technology
René Houtman, Dirk Pijnenburg, Diana Melchers & Rob Ruijtenbeek
PamGene International B. V., ’s Hertogenbosch, The Netherlands
Study Design
Here, we present our next generation of high-content
MARCoNI chips with a set of 155 immobilised peptides
on each PamChip® array, which represent coregulatorderived motifs. Per array, the nuclear receptor/ligand
conditions can be varied and in vitro interaction data for
all 155 motifs are obtained within one hour. The principle
and an example of nuclear receptor binding onto
coregulator peptides immobilized on the PamChip® arrays
are shown in figure 1. In figure 2 we show examples of
the modulation of 15 different nuclear receptor ligand
binding domains (Invitrogen) with their natural or synthetic
ligands.
Key Findings
NR-coregulator interaction profiles were performed on 15
different NRs on 155 known NR coregulator proteins
harboring either LXXLL (in coactivators) or LXXXIXXXL
(in corepressors) motifs. Each of the NRs shows a
different and specific modulation by its natural/synthetic
ligand (figure 2).
“Author Quote”
An obvious application of this NR-coregulator interaction
profiling platform is the screening at high definition of
novel NR and their NR (ant)agonists.
Background
Cofactor recruitment is a crucial regulatory step in nuclear
receptor (NR) signal transduction. The pathways towards
gene expression involve ligand-dependent and
independent interactions between NR and coregulator
(CoR) proteins. In view of drug development, profiling
these NR-CoR interactions is of importance to understand
mechanisms of drug action, to steer drug specificity, to
understand putative adverse drug effects (on- versus off
target), and to tailor the pharmacotherapies.
NR + CoReg
NR + CoReg
NR-CoReg
NR-CoReg
nd
ligad
n
liga
n
ligad
n
liga
1
1
NR-ligand + CoReg
NR-ligand + CoReg
2
NR-ligand-CoReg
2
NR-ligand-CoReg
NR
control
control
2
d
NR
+ Ligand
+ ligand
+ ligand
1
2
1
2
Figure 1: Schematic
overview of1 Nuclear 2Receptor (NR)1and Coregulator
proteins binding as function of the ligand. The blue arrow (1) shows the
effect of the ligand as an antagonist on a coregulator protein whereas the
red arrow (2) shows the effect of the ligand as an agonist on a coregulator
protein.
ER
ERα +17--estradiol
ERβ +17--estradiol
AR- T877A
Dihydroxytestosterone
GR
Dexamethasone
PR
Progesterone
LXR
LXRβ +T0901317
LXRα +T0901317
PPAR
PPAR +WY14643
PPAR + rosiglitazone
PPAR + GW501516
RXRα
Methoprene acid
FXR
GW4064
ERR
ER +4-OHT
ER+4-OHT
ER+XCT79
Figure 2: The modulation of 15 different nuclear receptors are shows as
function of their synthetic of natural ligands across 155 coregulator
proteins on the PamChip array. The modulation is the difference in binding
of the ligand binding domain (LBD) of the nuclear receptor and the LBDligand complex.
References:
Houtman R., 2nd Benelux Nuclear Receptor Meeting, Netherlands 11-2009
Conclusion
MARCoNI technology, using dynamic PamChip® peptide microarrays, provides a powerful method for profiling of endogenous
and ligand-dependent nuclear receptor-coregulator interactions.
©2013 PamGene International B.V.
All rights reserved
Application note: #201015 (10-2010)
Contact us:
Wolvenhoek 10, 5211 HH Den Bosch, the Netherlands
+31 73 615 80 80,  [email protected]