A Tetratricopeptide Repeat Half-Site in the Aryl Hydrocarbon Receptor Is Important for DNA Binding andtrans-Activation Potential
- Center for Molecular Toxicology and Carcinogenesis, Department of Veterinary Science, The Pennsylvania State University, University Park, Pennsylvania
Abstract
Similar to certain unliganded steroid hormone receptor complexes, the unliganded aryl hydrocarbon receptor has been shown to consist of a multimeric core complex that includes the 90-kDa heat shock protein (hsp90) and the immunophilin-like hepatitis B X-associated protein 2 (XAP2). Immunophilins and XAP2 associated with these complexes bind to the carboxyl-terminal end of hsp90 through an interaction with their tetratricopeptide repeat (TPR) domains. The consensus TPR binding motif contains two domains, A and B. Recently, the carboxyl terminus of XAP2 has been shown to contain a highly conserved TPR domain that is required for the assembly of XAP2 with both hsp90 and AhR. A search of the murine AhR sequence identified domain B (A-F-A-P) of the consensus TPR sequence directly adjacent to the carboxyl-terminal side of the helix-loop-helix region of the murine and human AhR. We hypothesized that this conserved domain B region may be involved with mediating interactions between either AhR-hsp90, AhR-XAP2, and/or AhR-AhR nuclear translocator protein. Site-directed mutagenesis of the amino-terminal alanine residue of this region to an aspartic acid (A78D) completely inhibited 2,3,7,8-tetrachloro-p-dioxin (TCDD) -dependent activation of a xenobiotic response element (XRE) driven gene expression construct in transfected COS-1 and BP8 cells. The A82F mutation caused a 40 to 50% decrease in TCDD-dependent activation. The inability of A78D and the reduction of A82F totrans-activate XRE-driven reporter activity did not result from impaired AhR-XAP2-hsp90 interactions, TCDD-dependent AhR translocation to the nucleus, or AhR-AhR nuclear translocator protein interactions. In vitro DNA binding analysis demonstrated that loss oftrans-activation potential by the A78D mutation resulted from impaired XRE binding. This study underscores the potential importance of AhR mutations that occur naturally outside of known functional domains.
Footnotes
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Send reprint requests to: Gary H. Perdew, Center for Molecular Toxicology and Carcinogenesis, Department of Veterinary Science, Pennsylvania State University, 226 Fenske Lab, University Park, Pennsylvania 16802
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This work was supported by the National Institute of Environmental Health Sciences (NIEHS) Grant ES04869 (G.P.) and an NIEHS Postdoctoral Fellowship (S.L.).
- Abbreviations:
- HAHs
- halogenated aromatic hydrocarbons
- PAHs
- polycyclic aromatic hydrocarbons
- AhR
- aryl hydrocarbon receptor
- hsp90
- 90-kDa heat-shock protein
- XAP2
- hepatitis B virus X-associated protein 2
- ARNT
- aryl hydrocarbon receptor nuclear translocator
- XRE
- xenobiotic response element
- HLH
- helix-loop-helix
- PAS
- PER-ARNT-SIM (periodicity/aryl hydrocarbon nuclear translocator/simple-minded)
- TPR
- tetratricopeptide repeat
- mAhR
- murine aryl hydrocarbon receptor
- CHAPS
- 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate
- PVDF
- polyvinyllidine difluoride
- TBE
- Tris-borate-EDTA
- MOPS
- 4-morpholinepropanesulfonic acid
- MENG
- MOPS/EDTA/NaN3/glycerol
- Tricine
- N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine
- PAGE
- polyacrylamide gel electrophoresis
- mAb
- monoclonal antibody
- RLUs
- relative light units
- DMSO
- dimethyl sulfoxide
- TCDD
- 2,3,7,8-tetrachlorodibenzo-p-dioxin
- CDTA
- trans-1,2-diaminocyclohexane-N,N,N′,N′-tetraacetic acid
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- Received July 11, 2000.
- Accepted September 12, 2000.
- The American Society for Pharmacology and Experimental Therapeutics
