Role of arginine residues 14 and 15 in dictating DNA binding stability and transactivation of the aryl hydrocarbon receptor/aryl hydrocarbon receptor nuclear translocator heterodimer

Abstract

The aryl hydrocarbon receptor (AHR) and its DNA binding partner, the aryl hydrocarbon receptor nuclear translocator (ARNT) are basic helix-loop-helix/PAS proteins. The goal of the current study was to determine the extent to which residues R14 and R15 contained within the basic region of the AHR contribute to the DNA binding affinity and stability of the AHR/ARNT heterodimer. Towards this end, we first performed equilibrium binding and dissociation rate analyses using a single dioxin response element (DRE-1). While the K(D) and Bmax values obtained from the equilibrium binding analysis were similar for the wild-type AHR (wt AHR) and that containing the substitutions of R14 and R15 with Q residues (Q14Q15 AHR), dissociation rate analyses revealed that the stability of the Q14Q15 AHR DNA binding complex was approximately 10-fold less. Using a two-site DNA binding model, we also found that AHR/ARNT heterodimer does not participate in cooperative binding, as binding of the second dimer appears to be prohibited by occupation of the first. This property was similar regardless of the composition of the amino acids at positions 14 and 15. Finally, reporter assays revealed that the Q14Q15 substitutions severely compromised the ability of the AHR to activate gene expression despite appropriate nuclear localization. The present results revealed that DNA binding stability of the AHR/ARNT heterodimer is an important requirement for its transactivation capabilities and that this stability is governed, in part, by residues R14 and R15 that lie within the basic region of the AHR.

Figure 1

Analysis of DNA binding of AHR variants using DRE-1. Association equilibrium analysis (A, B). The AHR/ARNT complexes were formed following the incubation of approximately 1 fmol of ARNT with either the wt AHR (A) or Q14Q15 AHR (B) proteins that were generated using in vitro transcription/translation reactions. Increasing concentrations of the ³²P-labeled probe (DRE-1) were added and the gel shift reactions were performed as described in Materials and Methods. (C) Graphical representation of the gel shift analyses. The K _D values were obtained from nonlinear regression analysis. The concentrations of DNA contained within the AHR/ARNT complex (specific binding) were determined using phosphorImager analysis and plotted as a function of the input ³²P-labeled probe. The 95% CI of the K _D of the wt AHR = 0.63 to 6.27 and that of the B _max = 1.44 × 10⁷ to 4.88 × 10⁷. The 95% CI of the K _D of the Q14Q15 AHR = 2.58 × 10⁷ to 7.83 × 10⁷ and that of the B _max = 2.85 × 10⁷ to 6.23 × 10⁷. The R ² values were > 0.90. The data are representative of at least three experiments performed in duplicate.

Figure 2

Dissociation analysis of the wt AHR/ARNT and Q14Q15 AHR/ARNT complexes. The AHR/ARNT DNA binding complexes were formed as described in Figure 1 except using 1 ng of ³²P-labeled probe(DRE-1). After equilibrium binding had been reached (10 min), excess of unlabeled oligonucleotide was added to the mixture and aliquots were removed at the indicated time points. Pre, the unlabeled oligonucleotide was added prior to the addition of the ³²P-labeled probe. A representation of the gel shift analyses of the wt AHR (A) and Q14Q15 AHR (B) are shown. (C) Graphical representation of the gel shift analyses. Each value represents the average of two independent experiments. The 95% confidence intervals of the linear regression slopes are depicted as follows: wt AHR −0.0147 to −0.0067 and Q14Q15 AHR −0.109 to −0.051.

Figure 3

Use of an oligonucleotide containing two DREs (2DRE-11) requires dimerization of the AHR and ARNT to allow formation of two AHR/ARNT complexes. Gel shift reactions were performed as described in Materials and Methods using either the wt AHR or Q14Q15 AHR and the ³²P-labeled DRE-2 as a probe. (A) The gel shift reactions were performed with the wild-type AHR and contained either the anti-ARNT immunoglobulins (lane 2), the anti-AHR immunoglobulins (lane 3), control immunoglobulins (nonspecific rabbit IgG, lane 4, or preimmune serum, lane 5), or excess concentrations of the unlabeled, wt 2DRE-11 (lane 6) or unlabeled, mutated 2DRE-11 (lane 7). I indicates complex I whereas II indicates complex II. (B) The gel shift reactions were performed using the Q14Q15 AHR in the absence (lane 5) or presence of the anti-ARNT immunoglobulin (lane 1), the anti-AHR immunoglobulin (lane 2), or excess concentrations of unlabeled, mutated 2DRE-11 (lane 3) or unlabeled, wild-type 2DRE-11 (lane 5). (C) The gel shift reactions were performed using either the wt 2DRE-11 (lane 1) or wt 1DRE (lane 2) as the probe.

Figure 4

Association equilibrium analysis of either the wt AHR or Q14Q15 AHR using either wt 2DRE-11 or wt 2DRE-22. The gel shift analyses were performed as described in Figure 1 and Materials and Methods using increasing concentrations of either wt AHR (A, D) or Q14Q15 AHR (B, E). The gels shown in (A) and (B) were performed using the wt 2DRE-11 as a probe whereas those shown in (D) and (E) were performed using the wt 2DRE-22 as a probe. The graphical representations of these analyses are shown in (C) and (F), respectively. The complexes that contained the AHR and the free probe were quantitated using PhosphorImager analysis to obtain total binding (i.e., that present in complex I + complex II + free probe = total binding). Each AHR-containing complex is expressed as percent of the total. (C) Depiction of the analyses performed using wt2 DRE-11. (D) Depiction of the analyses using wt2 DRE-22. The data are representative of two experiments performed in duplicate.

Figure 4

Association equilibrium analysis of either the wt AHR or Q14Q15 AHR using either wt 2DRE-11 or wt 2DRE-22. The gel shift analyses were performed as described in Figure 1 and Materials and Methods using increasing concentrations of either wt AHR (A, D) or Q14Q15 AHR (B, E). The gels shown in (A) and (B) were performed using the wt 2DRE-11 as a probe whereas those shown in (D) and (E) were performed using the wt 2DRE-22 as a probe. The graphical representations of these analyses are shown in (C) and (F), respectively. The complexes that contained the AHR and the free probe were quantitated using PhosphorImager analysis to obtain total binding (i.e., that present in complex I + complex II + free probe = total binding). Each AHR-containing complex is expressed as percent of the total. (C) Depiction of the analyses performed using wt2 DRE-11. (D) Depiction of the analyses using wt2 DRE-22. The data are representative of two experiments performed in duplicate.

Figure 5

Impact of the Q14Q15 substitutions on nuclear translocation and transactivation of the AHR. Full-length AHR constructs containing the Q14Q15 substitutions as well as an additional nuclear localization sequence and GFP at their C-termini were transiently transfected into CV-1 cells. After a 1-h treatment with either DMSO (A) or TCDD (B), the cells were visualized using fluorescent microscopy. (C) CV-1 cells were transiently transfected with the indicated expression plasmids and luciferase reporter plasmids that contained either the wild-type DRE (wtDRE) or mutated DRE (mtDRE). After an overnight incubation, the cells were cultured with either 0.1% DMSO or 1 nM TCDD for 4 h, harvested, and the renilla and luciferase values were determined. The graph is representative of two separate experiments.