Both coactivator LXXLL motif-dependent and -independent interactions are required for peroxisome proliferator-activated receptor γ (PPARγ) function

Nuclear receptor activation is dependent on recruitment of coactivators, including CREB-binding protein (GBP/p300) and steroid receptor coactivator-1 (SRC-1), A three-dimensional NMR approach was used to probe the coactivator binding interface in the peroxisome proliferator-activated receptor gamma (PPAR gamma) ligand binding domain (LBD), In the presence of a CBP peptide, peaks corresponding to 20 residues in helices 3, 4, 5, and 12 of the LED were attenuated. Alanine mutants revealed that M301A, V315A, Y320A, L468A, and E471A were required for binding of both CBP and SRC-1 and for cell-based transcription. Several additional amino acids in helix 4 of the PPAR gamma LBD were defective with respect to CBP recruitment, but retained relatively normal SRC-1 recruitment. Thus these amino acid residues may be important determinants of specificity for nuclear receptor LED interactions with discrete coactivator molecules.