Ligand and coactivator identity determines the requirement of the charge clamp for coactivation of the peroxisome proliferator-activated receptor gamma

The activation function 2 (AF-2)-dependent recruitment of coactivator is essential for gene activation by nuclear receptors. We show that the peroxisome proliferator-activated receptor gamma (PPARgamma) (NR1C3) coactivator-1 (PGC-1) requires both the intact AF-2 domain of PPARgamma and the LXXLL domain of PGC-1 for ligand-dependent and ligand-independent interaction and coactivation. Although the AF-2 domain of PPARgamma is absolutely required for PGC-1-mediated coactivation, this coactivator displayed a unique lack of requirement for the charge clamp of the ligand-binding domain of the receptor that is thought to be essential for LXXLL motif recognition. The mutation of a single serine residue adjacent to the core LXXLL motif of PGC-1 led to restoration of the typical charge clamp requirement. Thus, the unique structural features of the PGC-1 LXXLL motif appear to mediate an atypical mode of interaction with PPARgamma. Unexpectedly, we discovered that various ligands display variability in terms of their requirement for the charge clamp of PPARgamma for coactivation by PGC-1. This ligand-selective variable requirement for the charge clamp was coactivator-specific. Thus, distinct structural determinants, which may be unique for a particular ligand, are utilized by the receptor to recognize the coactivator. Our data suggest that even subtle differences in ligand structure are perceived by the receptor and translated into a unique display of the coactivator-binding surface of the ligand-binding domain, allowing for differential recognition of coactivators that may underlie distinct pharmacological profiles observed for ligands of a particular nuclear receptor.     

A pan-PPAR ligand induces hepatic fatty acid oxidation in PPARα−/− mice possibly through PGC-1 mediated PPARδ coactivation

Tetradecylthioacetic acid (TTA) is a hypolipidemic modified fatty acid and a peroxisome proliferator-activated receptor (PPAR) ligand. The mechanisms of TTA-mediated effects seem to involve the PPARs, but the effects have not been assigned to any specific PPAR subtype. PPARα−/− mice were employed to study the role of PPARα after TTA treatment. We also performed in vitro transfection assays to obtain mechanistic knowledge of how TTA affected PPAR activation in the presence of PPARγ coactivator (PGC)-1 and steroid receptor coactivators (SRC)-1 and SRC-2, which are associated with energy balance and mitochondrial biogenesis. We show that TTA increases hepatic fatty acid β-oxidation in PPARα−/− mice. TTA acts as a pan-PPAR ligand in vitro, and PGC-1, SRC-1 and SRC-2 have cell type and PPAR-specific effects together with TTA. In the absence of exogenous ligands, SRC-1 did not induce PPAR activity, while PGC-1 was the most potent PPAR coactivator. When the coactivators were overexpressed, pronounced effects of TTA were observed especially for PPARδ and PPARγ. We conclude that PPARα is involved in, but not required for, the hypolipidemic mechanisms of TTA. It appears that the activity of PPARδ, with substantial contribution of nuclear receptor coactivators, PGC-1 in special, is conducive to TTA's mechanism of action.     

Structural and biochemical basis for the binding selectivity of peroxisome proliferator-activated receptor gamma to PGC-1alpha

The functional interaction between the peroxisome proliferator-activated receptor gamma (PPARgamma) and its coactivator PGC-1alpha is crucial for the normal physiology of PPARgamma and its pharmacological response to antidiabetic treatment with rosiglitazone. Here we report the crystal structure of the PPARgamma ligand-binding domain bound to rosiglitazone and to a large PGC-1alpha fragment that contains two LXXLL-related motifs. The structure reveals critical contacts mediated through the first LXXLL motif of PGC-1alpha and the PPARgamma coactivator binding site. Through a combination of biochemical and structural studies, we demonstrate that the first LXXLL motif is the most potent among all nuclear receptor coactivator motifs tested, and only this motif of the two LXXLL-related motifs in PGC-1alpha is capable of binding to PPARgamma. Our studies reveal that the strong interaction of PGC-1alpha and PPARgamma is mediated through both hydrophobic and specific polar interactions. Mutations within the context of the full-length PGC-1alpha indicate that the first PGC-1alpha motif is necessary and sufficient for PGC-1alpha to coactivate PPARgamma in the presence or absence of rosiglitazone. These results provide a molecular basis for specific recruitment and functional interplay between PPARgamma and PGC-1alpha in glucose homeostasis and adipocyte differentiation.     

Multiplexed molecular interactions of nuclear receptors using fluorescent microspheres

BACKGROUND: We describe a novel microsphere-based system to identify and characterize multiplexed interactions of nuclear receptors with peptides that represent the LXXLL binding region of coactivator proteins. METHODS: In this system, individual microsphere populations with unique red and orange fluorescent profiles are coupled to specific coactivator peptides. The coactivator peptide-coupled microsphere populations are combined and incubated with a nuclear receptor that has been coupled to a green fluorochrome. Flow cytometric analysis of the microspheres simultaneously decodes each population and detects the binding of receptor to respective coactivator peptides by the acquisition of green fluorescence. RESULTS: We have used this system to determine the binding affinities of human estrogen receptor beta ligand binding domain (ERbeta LBD) and human peroxisome proliferator activated receptor gamma ligand binding domain (PPARgamma LBD) to a set of 34 coactivator peptides. Binding of ERbeta LBD to a coactivator peptide sequence containing the second LXXLL motif of steroid receptor coactivator-1 (SRC-1(2) (676-700) is shown to be specific and saturable. Analysis of receptor binding to a multiplexed set of coactivator peptides shows PPARgamma LBD binds with high affinity to cAMP response element binding protein (CBP) peptides and to the related P300 peptide while ERbeta LBD exibits little binding to these peptides. Using the microsphere-based assay we demonstrate that ERbeta LBD and PPARgamma LBD binding affinities for the coactivator peptides are increased in the presence of agonist (estradiol or GW1929, respectively) and that ERbeta LBD binding is decreased in the presence of antagonist (raloxifene or tamoxifen). CONCLUSIONS: This unique microsphere-based system is a sensitive and efficient method to simultaneously evaluate many receptor-coactivator interactions in a single assay volume. In addition, the system offers a powerful approach to study small molecule modulation of nuclear receptor binding.     

Ligands differentially modulate the protein interactions of the human estrogen receptors alpha and beta

The interactions of human estrogen receptor subtypes ERalpha and ERbeta with DNA and a 210 amino acid residue fragment of the coactivator protein SRC-1 bearing three nuclear receptor interaction motifs were investigated quantitatively using fluorescence anisotropy in the presence of agonist and antagonist ligands. ERalpha and ERbeta were found to bind in a similar manner to DNA, and both salt and temperature affected the affinity and/or stoichiometry of these interactions. The agonist ligands estradiol, estrone and estriol did not modify the binding of ERalpha to the fluorescein-labeled target estrogen response element. However, in the case of ERbeta, these ligands led to the formation of some higher-order protein-DNA complexes and a small decrease in affinity. The partial agonist 4-hydroxytamoxifen had little effect on either ER subtype, whereas the pure antagonist ICI 182,780 led to the cooperative formation of protein-DNA complexes of higher order than dimer, as further demonstrated by competition experiments and gel mobility-shift assays. In addition to DNA binding, the interaction of both ER subtypes with the Alexa488-labeled SRC-1 coactivator fragment was investigated by fluorescence anisotropy. The agonist ligands estrone, estradiol, estriol, genistein and ethynyl estradiol exhibited distinct capacities for inducing the recruitment of SRC-1 that were not correlated with their affinity for the receptor. Moreover, estrone and genistein exhibited subtype specificity in that they induced SRC-1 recruitment to ERbeta with much higher efficiency than in the case of ERalpha. The differential coactivator recruitment capacities of the ER agonists and their receptor subtype coactivator recruitment specificity may be linked to the molecular structure of the agonists with respect to their interactions with a specific histidine residue located at the back of the ligand-binding pocket. Altogether, these quantitative in vitro studies of ER interactions reveal the complex energetic and stoichiometric consequences of changes in the chemical structures of these proteins and their ligands.     

Steroid receptor coactivator 1 links the steroid and interferon gamma response pathways

We show here that steroid receptor coactivator 1 (SRC-1) is a coactivator of MHC class II genes that stimulates their interferon gamma (IFNgamma) and class II transactivator (CIITA)-mediated expression. SRC-1 interacts physically with the N-terminal activation domain of CIITA through two regions: one central [extending from amino acids (aa) 360-839] that contains the nuclear receptors binding region and one C-terminal (aa 1138-1441) that contains the activation domain 2. Using chromatin immunoprecipitation assays we show that SRC-1 recruitment on the class II promoter is enhanced upon IFNgamma stimulation. Most importantly, SRC-1 relieves the inhibitory action of estrogens on the IFNgamma-mediated induction of class II genes in transient transfection assays. We provide evidence that inhibition by estradiol is due to multiple events such as slightly reduced recruitment of CIITA and SRC-1 and severely inhibited assembly of the preinitiation complex.