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Department of Biochemistry and Physiology (G.Z., Y.L., S.M., H.A.W., A.E., J.M.S., R.G.S., D.E.M.) Department of Molecular Design & Diversity (R.C., J.D.H.) Merck Research Laboratories Rahway, New Jersey 07065
Ligand-dependent interactions between nuclear
receptors and members of a family of nuclear receptor coactivators are
associated with transcriptional activation. Here we used fluorescence
resonance energy transfer (FRET) as an approach for detecting and
quantitating such interactions. Using the ligand binding domain
(LBD) of peroxisome proliferator-activated receptor (PPAR
) as a
model, known agonists (thiazolidinediones and 
12, 14-PGJ2) induced a
specific interaction resulting in FRET between the fluorescently
labeled LBD and fluorescently labeled coactivators [CREB-binding
protein (CBP) or steroid receptor coactivator-1 (SRC-1)]. Specific
energy transfer was dose dependent; individual ligands displayed
distinct potency and maximal FRET profiles that were identical when
results obtained using CBP vs. SRC-1 were compared. In
addition, half-maximally effective agonist concentrations
(EC50s) correlated well with reported results
using cell-based assays. A site-directed AF2 mutant of PPAR (E471A)
that abrogated ligand-stimulated transcription in transfected cells
also failed to induce ligand-mediated FRET between PPAR LBD and CBP
or SRC-1. Using estrogen receptor (ER
) as an alternative system,
known agonists induced an interaction between ER LBD and SRC-1,
whereas ER antagonists disrupted agonist-induced interaction of ER
with SRC-1. In the presence of saturating agonist concentrations,
unlabeled CBP or SRC-1 was used to compete with fluorescently labeled
coactivators with saturation kinetics. Relative affinities for the
individual receptor-coactivator pairs were determined as follows:
PPAR-CBP = ER-SRC-1 > PPAR-SRC-1 >> ER-CBP.
Conclusions: 1) FRET-based coactivator association is a novel approach
for characterizing nuclear receptor agonists or antagonists; individual
ligands display potencies that are predictive of in vivo
effects and distinct profiles of maximal activity that are suggestive
of alternative receptor conformations. 2) PPAR interacts with both
CBP and SRC-1; transcriptional activation and coactivator association
are AF2 dependent. 3) Nuclear receptor LBDs have distinct affinities
for individual coactivators; thus, PPAR has a greater apparent
affinity for CBP than for SRC-1, whereas ER interacts preferentially
with SRC-1 but very weakly with CBP.
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