Helix 12 stabilization contributes to basal transcriptional activity of PXR

Abstract

Pregnane X receptor (PXR) plays an important role in xenobiotic metabolism. While ligand binding induces PXR-dependent gene transcription, PXR shows constitutive transcriptional activity in the absence of ligands when expressed in cultured cells. This constitutive activity sometimes hampers investigation of PXR activation by compounds of interest. In this study, we investigated the molecular mechanism of PXR activation. In the reported crystal structures of unliganded PXR, helix 12 (H12), including a coactivator binding motif, was stabilized, while it is destabilized in the unliganded structures of other nuclear receptors, suggesting a role for H12 stabilization in the basal activity of PXR. Since Phe420, located in the loop between H11 and H12, is thought to interact with Leu411 and Ile414 to stabilize H12, we substituted alanine at Phe420 (PXR-F420A) and separately inserted three alanine residues directly after Phe420 (PXR-3A) and investigated their influence on PXR-mediated transcription. Reporter gene assays demonstrated that the mutants showed drastically reduced basal activity and enhanced responses to various ligands, which was further enhanced by coexpression of the coactivator peroxisome proliferator-activated receptor gamma coactivator 1α. Mutations of both Leu411 and Ile414 to alanine also suppressed basal activity. Mammalian two-hybrid assays showed that PXR-F420A and PXR-3A bound to corepressors and coactivators in the absence and presence of ligands, respectively. We conclude that the intramolecular interactions of Phe420 with Leu411 and Ile414 stabilize H12 to recruit coactivators even in the absence of ligands, contributing to the basal transcriptional activity of PXR. We propose that the generated mutants might be useful for PXR ligand screening.

Regulation of PXR Function by Coactivator and Corepressor Proteins: Ligand Binding Is Just the Beginning

“…The stimulation of PXR-SRC1 interaction by rifampicin was reported in several studies [76,77]. These findings were confirmed using other cellular scaffolds such as Mz-Hep1 cells [78] and COS1 cells [19]. FOXO1 PCN ↑ GST pull-down assay [111] Compounds and stimuli modifying the interaction between PXR and different coactivators are presented.…”

“…On the contrary, the mutations F420A and F420-3A in PXR's LBD increased the ligand-independent binding of PXR to NCoR, whereby rifampicin resulted in the dissociation of the interaction. Altogether, these findings point to the involvement of specific amino acids in the interaction PXR-corepressor [19]. In this regard, activation of PKC stimulated the interaction PXR-NCoR [139], while activation of PKA decreased the interaction [84].…”

“…Besides the above-described mechanisms, PXR activation in the absence of a ligand has also been described. Mutagenesis experiments identified a critical role of the Phe420 residue in the AF2 region in PXR-ligand independent activity [19]. The same study also described the stimulation of this novel ligand-independent PXR activity by coactivators.…”

“…Unexpectedly, addition of rifampicin had an inhibitory effect. On the contrary, when two variants of PXR were used (PXR-F420A and PXR-3A) bearing mutations in the coactivator binding site, the interaction PXR-PGC1α was stimulated by rifampicin [19]. The cellular scaffold (i.e., COS1 vs. HepG2) may explain the partially contradicting outcomes [20,97].…”

“…Different independent studies pointed to a nuclear localization of PXR, even in its transcriptionally-inactive form [17][18][19]. In the absence of a ligand, PXR interacts with the nuclear receptor corepressor 2 (NCoR2), also known as SMRT (silencing mediator of retinoid and thyroid hormone receptors) [20][21][22].…”