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Abnormal liver development and resistance to 2,3,7,8-tetrachlorodibenzo-p-dioxin toxicity in mice carrying a mutation in the DNA-binding domain of the aryl hydrocarbon receptor.

Bunger MK, Glover E, Moran SM, Walisser JA, Lahvis GP, Hsu EL, Bradfield CA - Toxicol. Sci. (2008)

Bottom Line: The classical AHR pathway involves ligand binding, nuclear translocation, heterodimerization with the AHR nuclear translocator (ARNT), and binding of the heterodimer to dioxin response elements (DREs), thereby modulating the transcription of an array of genes.Here, we report the generation of a mouse model that expresses an AHR protein capable of ligand binding, interactions with chaperone proteins, functional heterodimerization with ARNT, and nuclear translocation, but is unable to bind DREs.These data suggest that DNA binding is necessary for AHR-mediated developmental and toxic signaling.

View Article: PubMed Central - PubMed

Affiliation: McArdle Laboratory for Cancer Research, University of Wisconsin School of Medicine and Public Health, Wisconsin 53706, USA.

ABSTRACT
The aryl hydrocarbon receptor (AHR) is known for its role in the adaptive and toxic responses to a large number of environmental contaminants, as well as its role in hepatovascular development. The classical AHR pathway involves ligand binding, nuclear translocation, heterodimerization with the AHR nuclear translocator (ARNT), and binding of the heterodimer to dioxin response elements (DREs), thereby modulating the transcription of an array of genes. The AHR has also been implicated in signaling events independent of nuclear localization and DNA binding, and it has been suggested that such pathways may play important roles in the toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Here, we report the generation of a mouse model that expresses an AHR protein capable of ligand binding, interactions with chaperone proteins, functional heterodimerization with ARNT, and nuclear translocation, but is unable to bind DREs. Using this model, we provide evidence that DNA binding is required AHR-mediated liver development, as Ahr(dbd/dbd) mice exhibit a patent ductus venosus, similar to what is seen in Ahr(-/-) mice. Furthermore, Ahr(dbd/dbd) mice are resistant to TCDD-induced toxicity for all endpoints tested. These data suggest that DNA binding is necessary for AHR-mediated developmental and toxic signaling.

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Schematic of the functional domains of the AHR protein. The enlarged portion depicts the GS insertion between the last residue of the basic domain and the first residue of the HLH domain, thereby introducing a BamHI restriction site.
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fig1: Schematic of the functional domains of the AHR protein. The enlarged portion depicts the GS insertion between the last residue of the basic domain and the first residue of the HLH domain, thereby introducing a BamHI restriction site.

Mentions: A mutant AHR cDNA, designated “AHRdbd,” was generated by the insertion of nucleotides GGTACC, coding for amino acids glycine and serine, between arginine-39 (R39) and aspartate-40 (D40) of the wild-type AHR cDNA (Fig. 1). Expression of the recombinant AHRdbd protein in rabbit reticulocyte lysate produced a protein approximately 95 kDa, in accordance with the known size of the wild-type protein. Photoaffinity labeling experiments indicated that the AHRdbd protein bound ligand with a capacity and affinity that was similar to its wild-type counterpart (data not shown). The DRE binding properties of AHRdbd were analyzed by a gel-shift protocol. Neither AHR nor AHRdbd proteins interacted significantly with a 32P-labeled DRE oligonucleotide in the absence of ARNT or in the presence of ARNT when an agonist was not present. The addition of the agonist, β-napthoflavone (BNF), induced formation of the AHR/ARNT/DRE complex, but not the AHRdbd/ARNT/DRE complex (Fig. 2A).


Abnormal liver development and resistance to 2,3,7,8-tetrachlorodibenzo-p-dioxin toxicity in mice carrying a mutation in the DNA-binding domain of the aryl hydrocarbon receptor.

Bunger MK, Glover E, Moran SM, Walisser JA, Lahvis GP, Hsu EL, Bradfield CA - Toxicol. Sci. (2008)

Schematic of the functional domains of the AHR protein. The enlarged portion depicts the GS insertion between the last residue of the basic domain and the first residue of the HLH domain, thereby introducing a BamHI restriction site.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2563146&req=5

fig1: Schematic of the functional domains of the AHR protein. The enlarged portion depicts the GS insertion between the last residue of the basic domain and the first residue of the HLH domain, thereby introducing a BamHI restriction site.
Mentions: A mutant AHR cDNA, designated “AHRdbd,” was generated by the insertion of nucleotides GGTACC, coding for amino acids glycine and serine, between arginine-39 (R39) and aspartate-40 (D40) of the wild-type AHR cDNA (Fig. 1). Expression of the recombinant AHRdbd protein in rabbit reticulocyte lysate produced a protein approximately 95 kDa, in accordance with the known size of the wild-type protein. Photoaffinity labeling experiments indicated that the AHRdbd protein bound ligand with a capacity and affinity that was similar to its wild-type counterpart (data not shown). The DRE binding properties of AHRdbd were analyzed by a gel-shift protocol. Neither AHR nor AHRdbd proteins interacted significantly with a 32P-labeled DRE oligonucleotide in the absence of ARNT or in the presence of ARNT when an agonist was not present. The addition of the agonist, β-napthoflavone (BNF), induced formation of the AHR/ARNT/DRE complex, but not the AHRdbd/ARNT/DRE complex (Fig. 2A).

Bottom Line: The classical AHR pathway involves ligand binding, nuclear translocation, heterodimerization with the AHR nuclear translocator (ARNT), and binding of the heterodimer to dioxin response elements (DREs), thereby modulating the transcription of an array of genes.Here, we report the generation of a mouse model that expresses an AHR protein capable of ligand binding, interactions with chaperone proteins, functional heterodimerization with ARNT, and nuclear translocation, but is unable to bind DREs.These data suggest that DNA binding is necessary for AHR-mediated developmental and toxic signaling.

View Article: PubMed Central - PubMed

Affiliation: McArdle Laboratory for Cancer Research, University of Wisconsin School of Medicine and Public Health, Wisconsin 53706, USA.

ABSTRACT
The aryl hydrocarbon receptor (AHR) is known for its role in the adaptive and toxic responses to a large number of environmental contaminants, as well as its role in hepatovascular development. The classical AHR pathway involves ligand binding, nuclear translocation, heterodimerization with the AHR nuclear translocator (ARNT), and binding of the heterodimer to dioxin response elements (DREs), thereby modulating the transcription of an array of genes. The AHR has also been implicated in signaling events independent of nuclear localization and DNA binding, and it has been suggested that such pathways may play important roles in the toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Here, we report the generation of a mouse model that expresses an AHR protein capable of ligand binding, interactions with chaperone proteins, functional heterodimerization with ARNT, and nuclear translocation, but is unable to bind DREs. Using this model, we provide evidence that DNA binding is required AHR-mediated liver development, as Ahr(dbd/dbd) mice exhibit a patent ductus venosus, similar to what is seen in Ahr(-/-) mice. Furthermore, Ahr(dbd/dbd) mice are resistant to TCDD-induced toxicity for all endpoints tested. These data suggest that DNA binding is necessary for AHR-mediated developmental and toxic signaling.

Show MeSH
Related in: MedlinePlus