Ligand and Coactivator Identity Determines the Requirement of the Charge Clamp for Coactivation of the Peroxisome Proliferator-activated Receptor γ

Abstract

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 ␥ (PPAR␥) (NR1C3) coactivator-1 (PGC-1) requires both the intact AF-2 domain of PPAR␥ and the LXXLL domain of PGC-1 for ligand-dependent and ligand-independent interaction and coactivation. Although the AF-2 domain of PPAR␥ 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 PPAR␥. Unexpectedly, we discovered that various ligands display variability in terms of their requirement for the charge clamp of PPAR␥ 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.The peroxisome proliferator-activated receptor ␥ (PPAR␥), 1 is a ligand-activated nuclear receptor that plays an important role in adipogenesis and glucose homeostasis (1). Although originally identified as an orphan receptor, PPAR␥ as well as the closely related receptors, PPAR␣ (NR1C1) and PPAR␦ (NR1C2), are believed to bind to a variety of fatty acids and their metabolites (2). Synthetic agonists that activate PPAR␥, such as the thiazolidinediones, rosiglitazone, and pioglitazone, are effectively utilized in clinical practice as "insulin sensitizers" in type 2 diabetics (3). Like other nuclear receptors, PPAR␥ is comprised of several separable functional domains including a constitutively active transactivation domain, AF-1, in the amino-terminal region, a central highly conserved DNAbinding domain composed of two zinc fingers, and a ligandbinding domain (LBD) that contains a ligand-dependent transactivation function, AF-2, in its carboxyl-terminal region (4). PPAR␥ functions as an obligate heterodimer with the retinoid X receptor (RXR) and recognizes specific DNA sequences in the promoter regions of target genes referred to as peroxisome proliferator response elements (PPREs) (5). Once directed to the target gene, the transcriptional activation activity of the receptor is regulated by ligand. Ligand binding leads to a significant conformational change within the LBD that include...

Direct link between metabolic regulation and the heat-shock response through the transcriptional regulator PGC-1α

“…The PGC-1α WT and L2/L3 mutant proteins were equally efficient in repressing several HSF1 target genes (Fig. 5G), suggesting that these motifs and nuclear hormone receptor binding via these motifs (8,9) are dispensable for the inhibitory effects of PGC-1α on HSF1.…”

“…This domain interacts with several lysine acetyltransferase complexes that include p300, CBP, and SRC-1 (7). The ability of PGC-1α to coactivate nuclear hormone receptors depends on two N-terminal LXXLL motifs, designated L2 and L3, that mediate the interaction of PGC-1α with these transcription factors (8,9). However, PGC-1α also was described as a coactivator of several nonnuclear hormone receptor transcription factors that include NRF1 and NRF2 (3).…”

The estrogen-related receptor α (ERRα) functions in PPARγ coactivator 1α (PGC-1α)-induced mitochondrial biogenesis

“…1 D and E). The single L2A mutant, which is defective for interactions with peroxisome proliferator-activated receptors, the glucocorticoid receptor, and thyroid hormone receptors (TR), but retains interactions with ERR␣ (12,13,18,29,30), was as active as WT PGC-1␣ in inducing the expression of target genes ( Fig. 1D and data not shown).…”

Selective modulation of promoter recruitment and transcriptional activity of PPARγ

“…Thus, ligands A and B would each have ligand-specific transcriptional activity. Ligand-induced changes in PPARγ conformation and ligand-selective interaction of PPARγ and coactivators have been reported in vitro [2,13,[15][16][17]. The molecular mechanisms that regulate ligand-selective modulation of endogenous PPARγ target genes and physiological potency are not well characterized.…”