Determination of Aryl Hydrocarbon Receptor Nuclear Translocator Protein Concentration and Subcellular Localization in Hepatic and Nonhepatic Cell Culture Lines: Development of Quantitative Western Blotting Protocols for Calculation of Aryl Hydrocarbon Receptor and Aryl Hydrocarbon Receptor Nuclear Translocator Protein in Total Cell Lysates
- Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, South Carolina 29425
Abstract
Western blot analysis was used to determine the concentration of the aryl hydrocarbon receptor nuclear translocator (ARNT) protein and aryl hydrocarbon receptor (AHR) in 11 mammalian cell culture lines derived from hepatic and nonhepatic tissues. The strategy was to first use Western blot analysis to determine the expression of ARNT or AHR in each cell line relative to its concentration in murine wild-type Hepa-1c1c7 (Hepa-1) cells. Actual ARNT and AHR concentrations in known amounts of total cell lysates were then determined by generating a standard curve with defined amounts of a highly purified ARNT or AHR protein and performing regression analysis. The results show that the level of ARNT expression in each of the cell lines is similar and represents ∼0.001–0.002% of total cellular protein. The range of expression was only ∼3-fold with wild-type Hepa-1 cells expressing the highest level of ARNT (33,000/cell) and canine kidney cells (MDCK line) expressing 14,000 ARNT molecules/cell. In contrast, the concentration of AHR varied by 65-fold over the different cell lines with the wild-type Hepa-1 expressing 323,000 AHR/cell and rat hepatoma cells (H4IIE) expressing 4700. The ratio of AHR to ARNT ranged from 0.3 in H4IIE cells to 10 in the Hepa-1 line with the majority of cells expressing 1–5 times more AHR than ARNT protein. Immunocytochemical staining of each cell line showed that ARNT was exclusively localized to the nuclear compartment and that a conserved nuclear localization signal mapped to the NH-terminal portion of the protein.
Footnotes
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Send reprint requests to: Dr. Richard S. Pollenz, Dept. of Biochemistry and Molecular Biology, Medical University of South Carolina, 171 Ashley Avenue, Charleston, SC 29425-2211. E-mail:pollenzr{at}musc.edu
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↵1 J. Holmes, unpublished observations.
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↵2 The term “conventional” ARNT refers to all ARNT isoforms except ARNT2.
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↵3 R. S. Pollenz, unpublished observations.
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↵4 Preliminary results show that AHR but not ARNT protein is depleted for ≤7 days in numerous tissues of the rat after a single oral dose of TCDD.
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This project was funded in part by the Medical University of South Carolina Institutional Research Funds for 1995–1996.
- Abbreviations:
- ARNT
- aryl hydrocarbon nuclear translocator
- AHR
- aryl hydrocarbon receptor
- bHLH
- basic helix-loop-helix
- BCA
- bicinchoninic acid
- BEAR-2
- bacterial expressed aryl hydrocarbon receptor
- BEARNT
- bacterial expressed aryl hydrocarbon nuclear translocator
- hARNT
- human aryl hydrocarbon nuclear translocator
- rARNT
- rat aryl hydrocarbon nuclear translocator
- mARNT
- mouse aryl hydrocarbon nuclear translocator
- TCDD
- 2,3,7,8-tetrachlorodibenzo-p-dioxin
- PAS
- periodicity-aryl hydrocarbon nuclear translocator-simple-minded, GAR-HRP, goat anti-rabbit horseradish peroxidase
- GAR-TR
- goat anti-rabbit Texas red
- TTBS
- Tris-buffered saline/Tween 20
- BSA
- bovine serum albumen
- DMEM
- Dulbecco’s minimum Eagle’s medium
- FBS
- fetal bovine serum, SDS, sodium dodecyl sulfate
- NLS
- nuclear localization signal
- PAGE
- polyacrylamide gel electrophoresis
- EGTA
- ethylene glycol bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid
- HEPES
- 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
- P450
- cytochrome P450
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- Received January 31, 1997.
- Accepted April 30, 1997.
- The American Society for Pharmacology and Experimental Therapeutics
