Reduction in cytochrome P-450 enzyme expression is associated with repression of CAR (constitutive androstane receptor) and PXR (pregnane X receptor) in mouse liver during the acute phase response

Expression of P-450 (Cyp) enzymes is reduced in liver during the acute phase response, contributing to the decrease in bile acid levels and drug metabolism during infection. Nuclear hormone receptors CAR and PXR are key transactivators of Cyp2b and Cyp3a genes, respectively. Injection of bacterial lipopolysaccharide (LPS) induced the expected reduction in Cyp2b10 and Cyp3a mRNA levels in mouse liver. These decreases were associated with a marked reduction in CAR and PXR mRNA levels within 4 h following treatment. LPS-induced CAR and PXR repression were dose-dependent and sustained for at least 16 h. LPS treatment also reversed the up-regulation of Cyp3a in mice pre-treated with PXR ligand RU486. In addition, we observed a concomitant decrease in RXR (retinoid X receptor) mRNA levels, the obligatory partner of both CAR and PXR for high affinity binding to DNA. These findings represent one possible molecular mechanism underlying sepsis-induced repression of Cyp enzymes.     

Competitive cofactor recruitment by orphan receptor hepatocyte nuclear factor 4alpha1: modulation by the F domain

For most ligand-dependent nuclear receptors, the status of endogenous ligand modulates the relative affinities for corepressor and coactivator complexes. It is less clear what parameters modulate the switch between corepressor and coactivator for the orphan receptors. Our previous work demonstrated that hepatocyte nuclear factor 4alpha1 (HNF4alpha1, NR2A1) interacts with the p160 coactivator GRIP1 and the cointegrators CBP and p300 in the absence of exogenously added ligand and that removal of the F domain enhances these interactions. Here, we utilized transient-transfection analysis to demonstrate repression of HNF4alpha1 activity by the corepressor silencing mediator of retinoid and thyroid receptors (SMRT) in several cell lines and on several HNF4alpha-responsive promoter elements. Glutathione S-transferase pulldown assays confirmed a direct interaction between HNF4alpha1 and receptor interaction domain 2 of SMRT. Loss of the F domain resulted in marked reduction of the ability of SMRT to interact with HNF4alpha1 in vitro and repress HNF4alpha1 activity in vivo, although the isolated F domain itself failed to interact with SMRT. Surprisingly, loss of both the A/B and F domains restored full repression by SMRT, suggesting involvement of both domains in the SMRT interaction. Finally, we show that when coexpressed along with HNF4alpha1 and GRIP1, CBP, or p300, SMRT can titer out HNF4alpha1-mediated transactivation in a dose-dependent manner and that this competition derives from mutually exclusive binding. Collectively, these results suggest that HNF4alpha can functionally interact with both a coactivator and a corepressor without altering the status of any putative ligand and that the presence of the F domain may play a role in discriminating between the different coregulators.     

A common mechanism for posttranslational activation of plasma membrane receptors?

The process for posttranslational acquisition of ligand binding function is remarkably similar for three receptors with dissimilar structures, namely, the insulin, epidermal growth factor, and acetylcholine receptors. These receptors lack the ability to bind ligand immediately after translation, but slowly (t1/2 = 30-45 min) acquire this capacity while in the endoplasmic reticulum. This activation step occurs with similar kinetics for all three receptors and, in each case, required N-linked glycosylation. Several lines of evidence suggest a common mechanism for the acquisition of ligand binding function that involves the rearrangement of metastable disulfide bonds formed during or immediately after translation. This process precedes subunit assembly of both insulin and acetylcholine receptors, which also occurs in the endoplasmic reticulum. The posttranslational processing steps leading to the acquisition of ligand binding function may be an example of a more general process affecting cell surface proteins.     

Analysis of intracellular receptor/ligand sorting in endosomes

After binding to specific cell surface receptors, many extracellular ligand molecules are internalized via the process termed receptor-mediated endocytosis. Within the cell, in endosomes, a sorting process occurs: receptors and ligands are directed along various intracellular pathways. The extent of this intracellular separation of receptors from ligands has been shown experimentally to vary with receptor and ligand properties such as binding affinity and valency. In this paper, we propose and analyze a simple model mechanism for the sorting process based on binding and dissociation kinetics along with diffusive molecular transport. We show that the outcome of the sorting process can be directly linked to measurable parameters such as the intrinsic rate constants for the binding to, dissociation from, and crosslinking of receptors by ligands. We further show that this mechanism is able to account for the wide range of reported experimental observations. Manipulation of ligand and receptor properties guided by the results presented here may enable the outcome of the sorting process to be controlled.