NUCLEAR RECEPTORS HAVE DISTINCT AFFINITIES FOR COACTIVATORS - CHARACTERIZATION BY FLUORESCENCE RESONANCE ENERGY-TRANSFER

Ligand-dependent interactions between nuclear receptors and members of a family of nuclear receptor coactivators are associated with transcr iptional activation. Here we used fluorescence resonance energy transf er (FRET) as an approach for detecting and quantitating such interacti ons. Using the ligand binding domain (LBD) of peroxisome proliferator- activated receptor (PPAR gamma) as a model, known agonists (thiazolidi nediones and Delta 12, 14-PGJ2) induced a specific interaction resulti ng in FRET between the fluorescently labeled LBD and fluorescently lab eled coactivators [CREB-binding protein (CBP) or steroid receptor coac tivator-1 (SRC-1)]. Specific energy transfer was dose dependent; indiv idual ligands displayed distinct potency and maximal FRET profiles tha t were identical when results obtained using CBP vs. SRC-1 were compar ed. In addition, half-maximally effective agonist concentrations (EC(5 0)s) correlated well with reported results using cell-based assays. A site-directed AF2 mutant of PPAR gamma (E471A) that abrogated ligand-s timulated transcription in transfected cells also failed to induce lig and-mediated FRET between PPAR gamma LBD and CBP or SRC-1. Using estro gen receptor (ER alpha) as an alternative system, known agonists induc ed an interaction between ER alpha LBD and SRC-1, whereas ER antagonis ts disrupted agonist-induced interaction of ER alpha with SRC-1. In th e presence of saturating agonist concentrations, unlabeled CBP or SRC- 1 was used to compete with fluorescently labeled coactivators with sat uration kinetics. Relative affinities for the individual receptor-coac tivator pairs were determined as follows: PPAR gamma-CBP = ER alpha-SR C-1 > PPAR gamma-SRC-1 much greater than ER alpha-CBP. Conclusions: 1) FRET-based coactivator association is a novel approach for characteri zing nuclear receptor agonists or antagonists; individual ligands disp lay potencies that are predictive of in vivo effects and distinct prof iles of maximal activity that are suggestive of alternative receptor c onformations. 2) PPAR gamma interacts with both CBP and SRC-1; transcr iptional activation and coactivator association are AF2 dependent. 3) Nuclear receptor LBDs have distinct affinities for individual coactiva tors; thus, PPAR gamma has a greater apparent affinity for CBP than fo r SRC-1, whereas ER alpha interacts preferentially with SRC-1 but very weakly with CBP.