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Dioxin binding activities of polymorphic forms of mouse and human arylhydrocarbon receptors
1Department of Chemistry, Faculty of Science, Tohoku University, Sendai, Japan.
Abstract:
The genetic difference in the susceptibility of mice to environmental toxicities induced by dioxin and related chemicals is governed by polymorphism of the arylhydrocarbon receptor (AhR) (Poland, A., and Knutson, C. (1982) Annu. Rev. Pharmacol. Toxicol. 22, 517-554). cDNA cloning of AhR cDNA from responder (C57BL/6) and non-responder (DBA/2J) mice allowed us to analyze the structure and function of these AhRs. Both of the AhRs, which were expressed in COS-7 cells transfected with their expression plasmids, showed a clear 9 S complex with 2,3,7,8-[3H]tetrachlorodibenzo-p-dioxin (TCDD) in a linear glycerol gradient centrifugation, consistent with the result of the endogenously expressed AhR in Hepa-1 cells. This result provides the first direct evidence that the cDNA-encoded protein binds the ligand specifically. Scatchard plot analysis revealed that the dissociation constant (Kd) of C57BL AhR for TCDD is 0.27 nM, while that of DBA AhR is elevated up to six times that high. Chimeric plasmids between the two cDNAs and site-directed mutagenesis revealed two critical alterations responsible for the reduced ligand binding activity: an Ala375 to Val alteration and an elongated carboxyl-terminal sequence due to a T to C mutation at the first letter of the termination codon of C57BL AhR. Two variants with reduced and intermediate ligand binding activity were also found in human AhRs with amino acid alterations equivalent to those of the DBA AhR. Importance of the amino acid at position 381 of human AhR (equivalent to position 375 of mouse AhR) in the ligand binding was confirmed by the fact that the mutation of Val381 to Asp completely abolished the ligand binding activity of human AhR.
Insights
Genetic variations in the aryl hydrocarbon receptor (AhR) influence dioxin toxicity. Researchers identified specific AhR gene mutations in mice and humans that affect dioxin binding and toxicity, impacting environmental health.
Area of Science:
- Biochemistry
- Toxicology
- Genetics
Background:
- Genetic differences in susceptibility to dioxin toxicity are linked to aryl hydrocarbon receptor (AhR) gene variations.
- Understanding AhR polymorphism is crucial for assessing environmental toxicity risks.
Purpose of the Study:
- To analyze the structural and functional differences of AhR in mice with varying dioxin sensitivities.
- To identify specific genetic alterations responsible for differential ligand binding in AhR.
Main Methods:
- cDNA cloning of AhR from responder (C57BL/6) and non-responder (DBA/2J) mice.
- Expression of AhR in COS-7 cells and analysis of ligand binding using glycerol gradient centrifugation and Scatchard plot analysis.
- Site-directed mutagenesis and chimeric plasmid construction to pinpoint critical amino acid changes.
Main Results:
- Expressed AhRs from both mouse strains specifically bound 2,3,7,8-[3H]tetrachlorodibenzo-p-dioxin (TCDD).
- DBA/2J mouse AhR exhibited a six-fold higher dissociation constant (Kd) for TCDD compared to C57BL/6 mouse AhR.
- Two key alterations in DBA/2J AhR (Ala375 to Val and an elongated carboxyl-terminus) were identified as responsible for reduced TCDD binding.
- Human AhR variants with similar alterations showed reduced or intermediate ligand binding activity.
- Mutation of Val381 (human AhR) to Asp abolished ligand binding, confirming its importance.
Conclusions:
- The study provides direct evidence that cDNA-encoded AhR proteins bind ligands.
- Specific amino acid substitutions and carboxyl-terminal modifications in AhR significantly impact TCDD binding affinity.
- These findings elucidate the molecular basis of differential dioxin toxicity and have implications for human health risk assessment.