Identification of a Critical Amino Acid in the Aryl Hydrocarbon Receptor

Abstract

Two aryl hydrocarbon receptors (rtAHR2␣ and rtAHR2␤) have been identified in the rainbow trout (Oncorhynchus mykiss). These receptors share 98% amino acid identity, yet their functional properties differ. Both rtAHR2␣ and rtAHR2␤ bind 2,3,7,8-tetrachlorodibenzop-dioxin (TCDD), dimerize with rainbow trout ARNTb (rtARNTb), and recognize dioxin response elements in vitro. However, in a transient transfection assay the two proteins show differential ability to recognize enhancers, produce transactivation, and respond to TCDD. To identify the sequence differences that confer the functional differences between rtAHR2␣ and rtAHR2␤, we constructed chimeric rtAHRs, in which segments of one receptor form was replaced with the corresponding part from the other isoform. This approach progressively narrowed the region being examined to a single residue, corresponding to position 111 in rtAHR2␤. Altering this residue in rtAHR2␤ from the lysine to glutamate found in rtAHR2␣ produced an rtAHR2␤ with the properties of rtAHR2␣. All other known AHRs resemble rtAHR2␣ and carry glutamate at this position, located at the N terminus of the PAS-A domain. We tested the effect of altering this glutamate in the human and zebrafish AHRs to lysine. This lysine substitution produced AHRs with transactivation properties that were similar to rtAHR2␤. These results identify a critical residue in AHR proteins that has an important impact on transactivation, enhancer site recognition, and regulation by ligand.

Functional properties of the four Atlantic salmon (Salmo salar) aryl hydrocarbon receptor type 2 (AHR2) isoforms

“…The EC 50 values for induction of reporter gene expression also suggest that AHR2α is the most sensitive of the four AHR2s, and that AHR2β is the least sensitive. The observed difference in concentration response relationships between these two Atlantic salmon AHR2s is consistent with analyses of the two rainbow trout AHR2 orthologs, which previously have been demonstrated to differ in responsiveness to TCDD (Abnet et al 1999a;Andreasen et al 2002c;Pollenz et al 2002). EC 50 values for the rtAHR2s are ~0.1 nM (α) and ~1 nM (β), comparable to the observed values for the Atlantic salmon AHR2α (~0.1 nM) and AHR2β (~0.4 nM).…”

A Dominant Negative Zebrafish Ahr2 Partially Protects Developing Zebrafish from Dioxin Toxicity

“…Light bars indicate activity in the absence of TCDD; dark bars indicate activity in the presence of TCDD (1 ng/ml). Units are Relative Luciferase Units, a dimensionless ratio of luciferase activity from the luminometer divided by the β-galactosidase activity from the control reporter used for transformation normalization [5], [26]. Single asterisks: different from control no TCDD value, p<0.05.…”

“…This is not an accurate representation of the normal induction of the CYP1a1 promoter by TCDD-activated AHR, in which basal activity is low, and fold induction is high. The high COS-7 basal activity requires expression of both AHR and ARNT [5], [6], [7], [8], [25], [26], [31], [34].…”

“…COS-7 cells were seeded on 24-well plates at a density of 5×10 4 cells/well 1 day before transfection [5], [26]. Transfections were performed using SuperFect (QIAGEN).…”

“…Luciferase activity was normalized to β-galactosidase activity for each sample [26]. Vector controls with no AHR or ARNT expression produced no luciferase activity.…”