Overexpression of ARNT2b does not reverse the repression of AHR2-dependent transactivation by AHRR1 or AHRR2. COS-7 cells were transfected with pGudLuc6.1 and pRL-TK. A, Western blot analysis confirmed the presence of increasing amounts of ARNT2b protein in cells transfected with increasing amounts of ARNT2b expression construct. The mouse monoclonal antibody to human ARNT, MA1-515 (Affinity BioReagents) was used. ARNT2b synthesized by in vitro transcription and translation was run as a positive control. GR corresponds to cells transfected only with the two luciferase constructs and an empty expression construct. B and C, zebrafish AHR2 (5 ng), pcDNA 3.1-AHRR1 (5 ng) (B) or pcDNA 3.1-AHRR2 (5 ng) (C) and increasing amounts of ARNT2b expression constructs were cotransfected into cells as indicated in the figure. Cells were treated with 0.5% DMSO or 10 nM TCDD. Firefly luciferase activity was measured and normalized to the transfection control R. reniformis luciferase. Results are representative of at least two independent experiments. [The apparent low responsiveness (-fold induction) of the reporter gene is commonly seen in transient transfection assays in which AHR is transfected along with a reporter gene; it may reflect some constitutive activation of AHR when overexpressed from a plasmid, leading to higher basal reporter gene expression and thus lower -fold inducibility; Fukunaga and Hankinson, 1996.].

AHRR1-Y9F is a functional repressor of AHR-dependent transactivation. A, inhibition of AHR2-dependent transactivation by AHRR1 and AHRR1-Y9F. COS-7 cells were transfected with pGudLuc6.1, and pRL-TK. AHR2 (5 ng), ARNT2b (5 ng), and AHRR1-EGFP or AHRR1-Y9F-EGFP expression constructs were also cotransfected into cells as indicated in the figure. Cells were dosed with 0.5% DMSO or 10 nM TCDD. Firefly luciferase activity was measured and normalized to the transfection control R. reniformis luciferase. B, both the wild-type and the mutant Y9F repressor inhibit AHR2-dependent transactivation in a concentration-dependent manner. COS-7 cells were transfected with expression constructs and exposed to DMSO or TCDD, and luciferase activity was measured, as described in text. Results are representative of at least two independent experiments. C, inhibition of luciferase activity as a function of amount of transfected AHRR1-EGFP or AHRR1-Y9F-EGFP DNA. Firefly luciferase activity was expressed as percentage of control, where control equals luciferase activity in the absence of AHRR construct, after treatment with DMSO or TCDD (e.g., lane 2 in B). Results of two experiments, each done in triplicate, were averaged. Curves were fitted, and IC₅₀ values determined using Prism (GraphPad Software, San Diego, CA). IC₅₀ values (and 95% confidence intervals) were as follows: AHRR1/DMSO, 0.47 ng (0.30–0.73); AHRR1/TCDD, 0.85 ng (0.65–1.12); AHRR1-Y9F/DMSO, 1.22 ng (0.81–1.81); and AHRR1-Y9F/TCDD, 1.74 (1.12–2.70). Lines represent the fitted curves for wild-type AHRR1 (solid lines) and AHRR1-Y9F (dashed lines). D, expression of transfected AHRR1-EGFP and AHRR1-Y9F-EGFP constructs in COS-7 cells. Cells were transfected with the indicated amounts of AHRR1 or AHRR1-Y9F expression plasmid, along with expression constructs for AHR2 (5 ng), ARNT2 (25 ng), pGudLuc6.1 (20 ng), and pRL-TK (3 ng). Cell lysates were analyzed by Western blot using affinity-purified antiserum against AHRR1.

AHRR1 does not reduce AHR2 protein levels or block nuclear translocation. A, COS-7 cells were transiently transfected with the indicated constructs and dosed with DMSO or TCDD. The transfected cell lysates were analyzed by a Western blot using an AHR2 antibody. An actin antibody was used as a control on the same lysates. B, COS-7 cells were cotransfected with mouse AHR-YFP, human ARNT, with or without AHRR1 and mouse ARA9 constructs. Cells were prepared as described under Materials and Methods, and they were visualized using an Axio Imager.Z1 fluorescence microscope (Carl Zeiss).

C terminus of AHRR1 is not required for repression. A, Both pcDNA3.1-AHRR1 deletion mutants repress AHR2. COS-7 cells were transiently transfected as described in Fig. 3 with pcDNA3.1-AHRR1Δ270–550 and pcDNA3.1-AHRR1Δ189–550. B, addition of exogenous ARNT enhances repression by pcDNA3.1-AHRR1Δ270–550. Results are representative of two independent experiments. Expression levels of AHRR1Δ189–550 and AHRR1-Y9FΔ189–550 proteins in the cells have not been measured because the peptide against which the AHRR1 antibody is directed is not present in these proteins. However, AHRR1, AHRR1-Y9F, AHRRΔ270–550, and AHRR1Δ189–550 all express at similar levels by in vitro transcription and translation reactions, suggesting that levels of expression in COS-7 cells are unlikely to differ dramatically.

Effects of Y9F mutation on AHRE binding and nuclear localization of AHRR1. A, alignment showing that the tyrosine at position 9 is conserved among AHRs and AHRRs. B, Y9F mutation disrupts DNA binding in AHRR1. Wild-type pcDNA 3.1-AHRR1, pcDNA 3.1-AHRR1-Y9F, and mAHRR were in vitro translated using the TnT rabbit reticulocyte system and incubated with similarly expressed ARNT2b in the presence of ³²P-labeled mouse AHRE probe. Mixtures were then run on a nondenaturing gel. C, Y9F mutation does not affect the nuclear localization of AHRR1. Hepa1c1c7 cells were transiently transfected and visualized 12 h after transfection by fluorescence microscopy. Results are representative of two independent experiments. UPL, unprogrammed lysate. Gen-Bank accession numbers for the sequences used are as follows: zebrafish AHRR1 (AY928203), zebrafish AHRR2 (AY928204), killifish AHRR (AF443441), mouse AHRR (AB015140), rat AHRR (NP_001019456), human AHRR (AB033060), human AHR (L19872), mouse AHR (M94623), rat AHR (NM_013149), killifish AHR1*1 (AF024591), killifish AHR2 (U29679), and zebrafish AHR2 (AF063446). Prefixes used are as follows: Dr, Danio rerio; Fh, F. heteroclitus; Mm, Mus musculus; Rn, Rattus norvegicus; and Hs, Homo sapiens.

Overexpression of ARNT fails to reverse repression by AHRR1-Y9F. COS-7 cells were transiently transfected as described in Fig. 1. In brief, 5 ng of AHRR1-EGFP or AHRR1-Y9F-EGFP was cotransfected with AHR2 and increasing amounts of ARNT2b. Cells were dosed with 0.5% DMSO or 10 nM TCDD, and firefly luciferase activity was measured and normalized to transfection control. Results are representative of three independent experiments.

Effect of AHRR1 C-terminal deletions on AHRE binding and dimerization with ARNT2. A, schematic representation of AHRR1 C-terminal deletion mutants used in this study. Functional domains are listed. B, electrophoretic mobility shift assay. Wild-type pcDNA3.1-AHRR1, pcDNA3.1-AHRR1Δ270–550, pcDNA3.1-AHRR1Δ189–550, and pcDNA3.1-AHRR1-Y9FΔ189–550 were in vitro translated using the TnT rabbit reticulocyte system and incubated with similarly expressed ARNT2b in the presence of ³²P-labeled mouse AHRE probe. Mixtures were then run on a nondenaturing gel. C, full-length or truncated mutant constructs of AHRR1 were synthesized in the presence of [³⁵S]methionine, incubated with unlabeled zebrafish ARNT2, and then coimmunoprecipitated using an ARNT antibody (αA) or normal mouse IgG (Ig). Immunoprecipitated products were separated on a 12% acrylamide gel and visualized by fluorography. Arrows indicate the position of wild-type AHRR1 (a, 61.2 kDa) and its deletion variants (b, AHRR1Δ270–550, 30.0 kDa; c, AHRR1Δ189–550, 21.3 kDa). Lanes 1 to 8 are products immunoprecipitated with anti-ARNT (lanes 1, 3, 5, and 7) or IgG (lanes 2, 4, 6, and 8). Lanes 9 to 12 are aliquots of the supernatant after immunoprecipitation. Results are representative of two independent experiments. The anti-ARNT antibody did not immunoprecipitate AHRR1 in the absence of ARNT2 (data not shown).

Repression of AHR2-dependent transactivation by an AHRR1 double mutant. A, pcDNA3.1-AHRR1-Y9FΔ189–550 behaves similarly to pcDNA3.1-AHRR1Δ189–550. Transient transfections were performed as described in Fig. 3, with the addition of pcDNA3.1-AHRR1-Y9FΔ189–550. B, overexpression of ARNT2b does not reverse repression of AHR by the double mutant AHRR1. COS-7 cells were transiently transfected as described in Fig. 1 but with the addition of pcDNA3.1-AHRR1-Y9FΔ189–550. Results are representative of at least two independent experiments.