Specific structural motifs determine TRAP220 interactions with nuclear hormone receptors

The TRAP coactivator complex is a large, multisubunit complex of nuclear pr oteins which associates with nuclear hormone receptors (NRs) in the presenc e of cognate ligand and stimulates NR-mediated transcription. A single subu nit, TRAP220, is thought to target the entire complex to a liganded recepto r through a domain containing two of the signature LXXLL motifs shown previ ously in other types of coactivator proteins to be essential for mediating NR binding. In this work, we demonstrate that each of the two LXXLL-contain ing regions, termed receptor binding domains 1 and 2 (RBD-1 and RBD-2), is differentially preferred by specific NRs. The retinoid X receptor (RXR) dis plays a weak yet specific activation function 2 (AF2)-dependent preference for RBD-1, while the thyroid hormone receptor (TR), vitamin D-3 receptor (V DR), and peroxisome proliferator-activated receptor all exhibit a strong AF 2-dependent preference for RBD-2. Using site-directed mutagenesis, we show that preference for RBD-2 is due to the presence of basic-polar residues on the amino-terminal end of the core LXXLL motif. Furthermore, we show that the presence and proper spacing of both RBD-1 and RBD-2 are required for an optimal association of TRAP220 with RXR-TR or RXR-VDR heterodimers bound t o DNA and for TRAP220 coactivator function. On the basis of these results, we suggest that a single molecule of TRAP220 can interact with both subunit s of a DNA-bound NR heterodimer.