Both Coactivator LXXLL Motif-dependent and -independent Interactions Are Required for Peroxisome Proliferator-activated Receptor γ (PPARγ) Function

Shiying Chen,Bruce A Johnson,Ying Li,Susan Aster,Brian Mckeever,Ralph Mosley,David E Moller,Gaochao Zhou

doi:10.1074/jbc.275.6.3733

Abstract

Nuclear receptor activation is dependent on recruitment of coactivators, including CREB-binding protein (CBP/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 (PPARgamma) 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 LBD were attenuated. Alanine mutants revealed that K301A, 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 PPARgammaLBD 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 LBD interactions with discrete coactivator molecules.

Highlights

From the ‡Department of Metabolic Disorders, the §Department of Endocrinology and Chemical Biology, the ¶Department of Medicinal Chemistry, and the ʈDepartment of Molecular Systems, Merck Research Laboratories, Rahway, New Jersey 07065
We identified the ligand binding domain (LBD) residues of PPAR␥ that are required for both CBP and steroid receptor coactivator-1 (SRC-1) binding; in addition, we identified several amino acids that differentially affect binding to CBP and SRC-1
These results suggest that certain residues within the LBD of nuclear receptors have an important role in mediating functional specificity by allowing for differential interactions with individual coactivators

Summary

Accelerated Publication

Both Coactivator LXXLL Motif-dependent and -independent Interactions Are Required for Peroxisome Proliferator-activated Receptor ␥ (PPAR␥) Function*. Several additional amino acids in helix 4 of the PPAR␥LBD were defective with respect to CBP recruitment, but retained relatively normal SRC-1 recruitment These amino acid residues may be important determinants of specificity for nuclear receptor LBD interactions with discrete coactivator molecules. We identified the LBD residues of PPAR␥ that are required for both CBP and SRC-1 binding; in addition, we identified several amino acids that differentially affect binding to CBP and SRC-1 These results suggest that certain residues within the LBD of nuclear receptors have an important role in mediating functional specificity by allowing for differential interactions with individual coactivators

EXPERIMENTAL PROCEDURES

RESULTS AND DISCUSSION

Wild type ϩ