Ligand Promiscuity of Aryl Hydrocarbon Receptor Agonists and Antagonists Revealed by Site-Directed Mutagenesis

Abstract

The aryl hydrocarbon receptor (AhR) is a ligand-dependent transcription factor that can be activated by structurally diverse chemicals. To examine the mechanisms responsible for the promiscuity in AhR ligand binding, we determined the effects of mutations within the AhR ligand-binding domain (LBD) on the activity of diverse AhR ligands. Site-directed mutagenesis identified Ile319 of the mouse AhR and, to a lesser extent, Phe318 as residues involved in ligand-selective modulation of AhR transformation using a panel of 12 AhR ligands. These ligands could be categorized into four distinct structurally related groups based on their ability to activate AhR mutants at position 319 in vitro. The mutation I319K was selectively activated by FICZ and not by other examined ligands in vitro and in cell culture. F318L and F318A mutations resulted in the conversion of AhR agonists ␤-naphthoflavone and 3-methylcholanthrene, respectively, into partial agonists/antagonists. Hsp90 binding to the AhR was decreased with several mutations and was inversely correlated with AhR ligand-binding promiscuity. Together, these data define overlapping amino acid residues within the AhR LBD involved in the selectivity of ligand binding, the agonist or antagonist mode of ligand binding, and hsp90 binding and provide insights into the ligand diversity of AhR activators.

Highly potent visnagin derivatives inhibit Cyp1 and prevent doxorubicin cardiotoxicity

“…This effect may be related to inhibition of the AHR but does not seem to explain C23's increased potency in our initial in vitro studies using the well-characterized AHR agonist TCDD. The functional activity of the AHR has been reported to vary based on the specific ligand to which it is bound (25), and it is possible that C23 is a more potent inhibitor of the AHR when activated by DOX rather than by TCDD. The effect of C23 on inhibition of DOX-mediated CYP1 induction may also occur through a mechanism that is independent of direct AHR antagonism, for instance through degradation of the AHR, inhibition of the AHR nuclear translocator, or direct effects on CYP1 transcription.…”

The Aryl Hydrocarbon Receptor in Barrier Organ Physiology, Immunology, and Toxicology

“…The first one claims that the AhR is promiscuous and AhR signaling can be activated by structurally very diverse chemicals even at low affinity (Denison et al, 2011;Soshilov and Denison, 2014). The second one claims that seemingly genuine AhR activators inhibit the transcription and/or activity of CYP1A1 and thereby inhibit the metabolic degradation of the endogenous ligand FICZ .…”

“…The second one claims that seemingly genuine AhR activators inhibit the transcription and/or activity of CYP1A1 and thereby inhibit the metabolic degradation of the endogenous ligand FICZ . Soshilov and Denison (2014) have challenged this latter indirect mechanism. They employed site-directed mutagenesis of the LBD of AhR and describe one particular mutant, I319K, which was activated only by FICZ in a luciferase reporter system.…”

“…However, the approximate dimensions of the ligand binding pocket and several critical amino acid residues in the binding pocket are known. This knowledge has enabled docking studies of proposed AhR ligands (Goryo et al, 2007;Soshilov and Denison, 2014).…”

Aryl Hydrocarbon Receptor Activation in Chronic Kidney Disease: Role of Uremic Toxins

“…In the nucleus, this complex releases the protein so that it is able to bind to the AhR nuclear translocator (ARNT) through its PAS domain, resulting in the ligand-bound AhR-ARNT dimer. This heterodimer binds to specific DNA sequences (referred to as DRE or XRE for dioxin-or xenobiotic-responsive element) located within the promoters of target genes, such as cytochrome P450, family 1, member 1A ( Cyp1A1 ); cytochrome P450, family 1, member 2A; cytochrome P450, family 1, sub family B (Cyp1B1); AhR repressor; and cyclooxygenase-2 ( COX-2 ) [6,13,14] .…”