Amino Acid Substitutions in the Aryl Hydrocarbon Receptor Ligand Binding Domain Reveal YH439 As an Atypical AhR Activator

Abstract

The aryl hydrocarbon receptor (AhR) is traditionally defined as a transcription factor activated by exogenous polyaromatic and halogenated aromatic hydrocarbon (PAH/HAH) ligands. Active AhR induces genes involved in xenobiotic metabolism, including cytochrome P4501A1, which function to metabolize activating ligands. However, recent studies implicate AhR in biological events that are apparently unrelated to the xenobiotic response, implying that endogenous activation mechanisms exist. Three AhR genes in zebrafish (Danio rerio) encode proteins that demonstrate differential activation in response to PAH/HAHs, with the nonresponsive drAhR1a having some sequence divergence from the PAH/HAH-responsive AhRs in the ligand binding domain (LBD). We used these differences to guide the mutagenesis of mouse AhR (mAhR), aiming to generate variants that functionally discriminate between activation mechanisms. We found substitution of histidine 285 in the LBD with tyrosine gave a receptor that could be activated by iso-, a potential AhR ligand chemically distinct from classic PAH/HAH-type ligands, but prevented activation by both exogenous PAH/HAH ligands and the endogenous activation mimics of suspension culture and application of shear-stressed serum. The differential response of H285Y mAhR to YH439 suggests that this activator has a novel mode of interaction that tolerates tyrosine at position 285 in the LBD and is distinct from the binding mode of the well characterized PAH/HAH ligands. In support of this, the PAHtype antagonist 3Ј,4Ј-dimethoxyflavone blocked mAhR activation by 2,3,7,8-tetrachlorodibenzo-p-dioxin but not YH439. Furthermore, the strict correlation between response to exogenous PAH/HAH ligands and mimics of endogenous activation suggests that a PAH-type ligand may underpin endogenous mechanisms of activation.The aryl hydrocarbon receptor (AhR), also known as the dioxin receptor, is a basic helix-loop-helix-Per-aryl hydrocarbon receptor nuclear translocator (ARNT)-Sim transcription factor best characterized by activation in response to exogenous polyaromatic and halogenated aromatic hydrocarbon (PAH/HAH) type ligands. Latent AhR is bound by chaperones, which serve to present a ligand-accessible conformation of the ligand binding domain (LBD) and maintain cytoplasmic localization (Mitchell and Elferink, 2009). Xenobiotic PAH ligands such as 3-methylcholanthrene (3MC) and benzo[a]pyrene (B[a]P), and the prototypical HAH, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) bind to the AhR within the LBD, causing a conformational change that allows nuclear import and exchange of chaperones for the AhR partner ARNT. The AhR/ARNT heterodimer binds xenobiotic-response elements (XREs) in regulatory regions of target genes and associates with coactivators to initiate transcription (Kobayashi et al., 1997;Kumar et al., 1999). PAH activation

Xenobiotics and Loss of Cell Adhesion Drive Distinct Transcriptional Outcomes by Aryl Hydrocarbon Receptor Signaling

“…A previous study by our laboratory suggested that YH439 has a novel mode of activation, which does not require full access to the AhR ligand-binding pocket (Whelan et al, 2010). As a result, the possibility that YH439 may regulate some AhR target genes differentially from TCDD exists.…”

“…The pLV501 lentiviral expression vector was derived from pLV410G (gift from A/Professor Simon Barry, Women's and Children's Health Research Institute) digested with ClaI and EcoRV, replacing the gateway cassette with that of pLenti4/V5-DEST (Invitrogen, Carlsbad, CA) excised with ClaI and PmlI. To generate the pLV501_HisMyc_mAhR construct, HisMyc epitope-tagged full-length mouse AhR cDNA was recombined from the pENTR1A_ HisMyc_mAhR donor vector (Whelan et al, 2010) into the pLV501 destination vector by standard Gateway cloning (Invitrogen).…”

“…Structure-activity relationship analyses suggest that the ligand-binding pocket of AhR is promiscuous. Both polycyclic aromatic hydrocarbons and halogenated aromatic hydrocarbons (HAHs) are classic activators that bind to AhR with high affinity (Denison and Nagy, 2003), whereas other compounds such as omeprazole (Dzeletovic et al, 1997) and isopropyl-2-(1,3-dithietane-2-ylidene)-2-[N-(4-methylthiazol-2-yl)carbamoyl]acetate (YH439) (Lee et al, 1996) that fall outside aromatic classification have also been shown to activate the AhR, with YH439 functioning as an atypical AhR ligand (Whelan et al, 2010).…”

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

“…For a given cell type, they may even depend on the tissue milieu, such as an ongoing immune response (Sun et al, 2004;Frericks et al, 2007;Beischlag et al, 2008;Tappenden et al, 2013). There appears to be only one ligand binding pocket in the AhR [in the Per-ARNT-Sim-B domain], and the binding affinity of the best characterized highaffinity ligand, TCDD (2,3,7,8-tetrachlorodibenzo-pdioxin), depends on only a few amino acids in the binding pocket of the AhR, as demonstrated by modeling and mutation studies (Whelan et al, 2010;DeGroot et al, 2012;Wu et al, 2013). The AhR can also be activated by numerous stress factors and substances that may not fit into the binding pocket, such as hyperoxia, oxidized low-density lipoproteins, hydrogen peroxide, ozone, or metals (references in Wincent et al, 2012).…”

Development and characterization of monoclonal antibodies against human aryl hydrocarbon receptor

“…It was reported that there were preferences for different ligands to bind AhR (Xing et al, 2012). Site-directed mutation and ligand binding analysis suggested that structurally diverse ligands, such as 3-methylcholanthrene (MC), beta-naphthoflavone (BNF) and dibenzo-p-dioxins (TCDD), might interact with AhR through different sites, resulting in different transactivation activities and nuclear localizations (Goryo et al, 2007;Backlund and Ingelman-Sundberg, 2004;Whelan et al, 2010). In line with this notion, Zhao et al suggested that the different consequences following the AhR activations triggered by different types of ligands, like TCDD and BNF, may be due to the distinct AhR transactivation process occurring before its translocation (Zhao et al, 2010).…”