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G H Perdew

Publications and source records attributed to G H Perdew.

At least 37 records · Page 2Linked to original sources
PubMed1997

Characterization of a subset of the basic-helix-loop-helix-PAS superfamily that interacts with components of the dioxin signaling pathway.

In an effort to better understand the mechanism of toxicity of 2,3,7, 8-tetrachlorodibenzo-p-dioxin, we employed an iterative search of human expressed sequence tags to identify novel basic-helix-loop-helix-PAS (bHLH-PAS) proteins that interact with either the Ah receptor (AHR) or the Ah receptor nuclear translocator (ARNT). We characterized five new "members of the PAS superfamily," or MOPs 1-5, that are similar in size and structural organization to the AHR and ARNT. MOPs 1-4 have N-terminal bHLH and PAS domains and C-terminal variable regions. MOP5 contained the characteristic PAS domain and a variable C terminus; it is possible that the cDNA contains a bHLH domain, but the entire open reading frame has yet to be completed. Coimmunoprecipitation studies, yeast two-hybrid analysis, and transient transfection experiments demonstrated that MOP1 and MOP2 dimerize with ARNT and that these complexes are transcriptionally active at defined DNA enhancer sequences in vivo. MOP3 was found to associate with the AHR in vitro but not in vivo. This observation, coupled with the fact that MOP3 formed tighter associations with the 90-kDa heat shock protein than the human AHR, suggests that MOP3 may be a conditionally active bHLH-PAS protein that requires activation by an unknown ligand. The expression profiles of the AHR, MOP1, and MOP2 mRNAs, coupled with the observation that they all share ARNT as a common dimeric partner, suggests that the cellular pathways mediated by MOP1 and MOP2 may influence or respond to the dioxin signaling pathway.

Amino Acid Sequence↗
PubMed1997

A 50 kilodalton protein associated with raf and pp60(v-src) protein kinases is a mammalian homolog of the cell cycle control protein cdc37.

Several oncogenic protein kinases including c-raf-1 and pp60(v-src) are known to directly interact with the 90 kDa heat shock protein (hsp90)/p50 complexes. Using a monoclonal antibody to detect p50 during a purification scheme, p50 was purified to homogeneity. Internal amino acid sequence information was obtained and used to clone a partial cDNA. Comparison of the p50 sequence to other cloned proteins revealed 89% homology with a glycosaminoglycan-binding protein and 54% homology with Drosophila cell cycle control protein (cdc) 37. Monoclonal and polyclonal antibodies were produced against a cleaved fusion protein that recognizes p50 with a high level of specificity. These antibodies recognize the 50 kDa protein present in c-raf-1 and pp60(v-src) complexes. No other proteins were recognized with these antibodies suggesting that p50 is a unique protein. Immunocytochemical visualization of p50 in NIH 3T3 cells indicates a primarily cytoplasmic localization around the nuclear membrane. A survey of p50 expression in murine tissues on a protein blot revealed the following relative levels of expression; thymus > spleen > brain > heart > kidney > liver > lung > skeletal muscle. These results link studies demonstrating complexation of certain kinases with hsp90/p50 in mammalian cells and a number of reports in yeast and Drosophila, demonstrating the importance of cdc37 in cell cycle and kinase function.

3T3 Cells↗
PubMed1996

A model of protein targeting mediated by immunophilins and other proteins that bind to hsp90 via tetratricopeptide repeat domains.

We have shown recently that the immunophilins CyP-40 and FKBP52/hsp56 bind to a common site on hsp90 and that they exist in separate heterocomplexes with the glucocorticoid receptor (GR). FKBP52/hsp56 binds to hsp90 via its tetratricopeptide repeat (TPR) domains, it is not required for GR.hsp90 heterocomplex assembly, and it is thought to play a role in targeted movement of the GR. In this work we examine the hsp90 binding of four proteins (FKBP52/hsp56, CyP-40, p50, Mas70p) thought to be involved in targeted protein trafficking. FKBP52/hsp56 and CyP-40 (each with three TPRs), localize to the nucleus and nucleoli, respectively, and form relatively weak complexes with hsp90 that are competed by a CyP-40 fragment containing its three TPRs. The p50 component of the Src.hsp90 and Raf.hsp90 heterocomplexes localizes to cytoskeletal fibers extending from the perinuclear region to the plasma membrane and forming a rim under the plasma membrane of endothelial cells. p50, Mas70p (seven TPRs), which is a receptor for mitochondrial import, and the p60 (six to eight TPRs) component of the steroid receptor.hsp90 heterocomplex assembly system bind very tightly to hsp90 in a manner that is not competed by the CyP-40 fragment. However, bacterially expressed p60 blocks the binding of p50, Mas70p, FKBP52/hsp56, and CyP-40 to purified hsp90. The data are consistent with binding of all of these proteins to a site on hsp90 that is a general TPR domain acceptor. Our localization and binding data are used to develop a model in which proteins that are chaperoned by hsp90 move as dynamic complexes to their cellular sites of action, with the TPR-containing protein participating in targeting the movement of the complexes.

Amino Acid Sequence↗
PubMed1996

Characterization of the activated form of the aryl hydrocarbon receptor in the nucleus of HeLa cells in the absence of exogenous ligand.

The aryl hydrocarbon receptor (AhR) is known to mediate 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced toxic effects. Immunocytochemical studies revealed that AhR in HeLa cells is localized throughout the cell. Upon TCDD treatment most of the cytoplasmic receptor is translocated into the nucleus in a time-dependent manner. A significant amount of AhR was found to be tightly associated with the nuclear fraction of untreated HeLa cells. The level of receptor in the nuclear fraction was approximately 16% of the total cellular receptor pool. Further characterization of AhR heterocomplex from the HeLa nuclear fraction by sucrose density gradient analysis revealed that the AhR was present in the 6 S form, and that the nuclear AhR could be coimmunoprecipitated using anti-Arnt mAb. The ability of the AhR to specifically interact with dioxin-responsive elements (DRE) was demonstrated utilizing wild-type and two mutant DREs in gel shift assays. These results would suggest that, in HeLa cells, the AhR-Arnt heterodimer is associated with the nuclear fraction under normal culture conditions. Therefore, HeLa cells can be used as a model system to study the biochemical and molecular function of the Ah receptor and the process that leads to activation of the AhR in the absence of exogenous ligand.

Aryl Hydrocarbon Receptor Nuclear Translocator↗
PubMed1996

Mapping the 90 kDa heat shock protein binding region of the Ah receptor.

Expression of a series of Ah receptor (AhR) deletion mutants in an in vitro translation system has been previously used to map several functional domains of the murine AhR (Dolwick et al. (1993) Proc. Natl. Acad. Sci. USA 90, 8566-8570). In this report, quantitative immunoprecipitation of 90-kDa heat shock protein (hsp90) from reticulocyte lysate allowed us to measure the level of the AhR and AhR deletion mutants complexed with hsp90. After translation of a series of deletion mutants it was determined that there are two distinct domains important in forming a stable AhR/hsp90 complex, corresponding to amino acid sequences 1-166 and 289-347 of the AhR. Neither ARNT, nor Per were able to stably interact with hsp90. Thus, the AhR appears to be a unique member of the PAS domain family of proteins that binds a known ligand and stably interacts with hsp90.

Animals↗
PubMed1995

Comparative properties of the nuclear aryl hydrocarbon (Ah) receptor complex from several human cell lines.

The aryl hydrocarbon (Ah) responsiveness of the T-47D, Hep G2, LS180, MCF-7, A431, C-4II and MDA-MB-231 human cancer cell lines was determined by the induction of CYP1A1 mRNA levels and ethoxyresorufin O-deethylase activity. With the exception of teh MDA-MB-231 breast cancer cell line, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) significantly induced CYP1A1 mRNA levels and ethoxyresorufin O-deethylase activity in the remaining six cell lines and, based on their EC50 values, for ethoxyresorufin O-deethylase induction, their Ah responsiveness followed the order T-47D > C-4II > MCF-7 > LS180 > HEP G2 > A431. In contrast, all the cell lines expressed the nuclear Ah receptor complex (167.1-24.5 fmol/mg protein) which bound to a 32P-labeled consensus dioxin responsive element (DRE) in a gel mobility shift assay. The results of gel permeation chromatography a sucrose density gradient centrifugation studies showed that the calculated Mr values for the nuclear Ah receptor complex varied from 175 kDa (MDA-MB-231 cells) to 221 kDa and the apparent molecular weight of the nuclear Ah receptor complex cross-linked to a bromodeoxyuridine-substituted DRE was 200 kDa. The data show that the molecular properties and levels of the nuclear Ah receptor complex from seven different human cancer cell lines do not predict Ah responsiveness.

Affinity Labels↗
PubMed1995

Developmental expression of two members of a new class of transcription factors: I. Expression of aryl hydrocarbon receptor in the C57BL/6N mouse embryo.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor with a basic region/helix-loop-helix (bHLH) motif. AhR has been sequenced and the functional domains defined and there is information on the formation of complexes with other peptides and interactions with DNA, although these areas continue to be investigated. AhR mediates many biological effects such as developmental toxicity, including induction of cleft palate and hydronephrosis. This regulatory protein is expressed in embryonic liver and has been immunohistochemically localized in cells of human and mouse secondary palate. The expression of AhR in embryonic tissues and its ability to disrupt development suggests a significant role for this protein in development. The present study examines the pattern of AhR expression in the C57BL/6N mouse embryo from gestation days (GD) 10-16, using in situ hybridization and immunohistochemical analysis. AhR mRNA was localized with 35S-RNA antisense riboprobe (cAh1 probe, 1.8 Kb amino terminal DNA). AhR protein was localized with purified monoclonal antibody (RPT-9) raised against the N-terminal peptide sequence. AhR mRNA and protein were expressed in GD 10-13 neuroepithelium, and as development progressed the levels in brain decreased. GD 10-12 embryos also showed AhR in branchial arches, heart, somites, and liver. AhR protein and mRNA in heart were highest at GD 10-11 and decreased with age. In liver, AhR mRNA and protein levels increased and nuclear localization became more pronounced with gestational age. In GD 14-16 embryos levels in liver and adrenal were highest, but AhR was present in ectoderm, bone, and muscle. AhR expression was specific for both cell type, organ/tissue, and developmental stage, suggesting that this novel ligand-activated transcriptional regulator may be important in normal embryonic development.

Animals↗
PubMed1995

Evidence for two functionally distinct forms of the human Ah receptor.

The Ah receptor (AhR) was visualized using monoclonal antibody Rpt 1 on protein blots of HeLa cell cytosol; two bands were detected at 104 and 106 kDa. The photoaffinity ligand, 2-azido-3-[125I]iodo-7,8-dibromodibenzo-p-dioxin, was added to HeLa cells in culture, and after 1 hour the cells were UV irradiated. Cytosolic and high salt nuclear preparations were isolated and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by transfer of the protein to membrane. The AhR was visualized on the membrane, revealing two bands. Alignment of an autoradiogram with the membrane revealed that only the 106 kDa (upper) band was photoaffinity labeled. The nuclear fraction contained only the photoaffinity-labeled 106 kDa form of the AhR. The 104 kDa AhR does not appear to be a proteolytic product of the 106 kDa form. Cyanogen bromide fragmentation revealed that both forms contain the same size N-terminal fragment. Sucrose density gradient analysis of HeLa cell cytosol indicated that both forms cosedimented at 9 S. Both the 106 and 104 kDa AhR bands were detected in four different human cell lines. Together, these results would indicate that the AhR in human cell lines exists in two distinct forms.

Affinity Labels↗
PubMed1995

Production and characterization of monoclonal antibodies directed against the Ah receptor.

Six hybridomas secreting monoclonal antibodies that are specific for the N-terminal peptide sequence of the murine Ah receptor were isolated. These antibodies bind with high specificity to the Ah receptor on protein blots of Hepa 1c1c7 cytosol. Three IgG1 antibodies (Rpt 1, 2, and 3) were capable of detecting 2 ng of receptor using peroxidase-goat anti-mouse IgG antibody conjugate on a protein blot. Monoclonal antibody Rpt 9 exhibited the greatest ability to immunoprecipitate the nondenatured 9S form of the Ah receptor and to visualize the AhR on liver tissue sections using immunohistochemical techniques. All of the monoclonal antibodies produced were able to bind to the mouse, rat, and human Ah receptor. These monoclonal antibodies should be useful in a wide number of applications in the study of Ah receptor biochemistry.

Amino Acid Sequence↗
PubMed1994

Subunit composition of the heteromeric cytosolic aryl hydrocarbon receptor complex.

In a previous cross-linking study we have shown that the cytosolic aryl hydrocarbon receptor (AhR) complex has a heterotetrameric structure (Perdew, G. H. (1992) Biochem. Biophys. Res. Commun. 182, 55-62). In this report, both cross-linked and [35S]methionine-labeled Hepa 1c1c7 cytosol were used to characterize the subunit composition of the AhR complex by immunoprecipitation with an AhR polyclonal antibody followed by immunochemical analysis using antibodies against the AhR and 90-kDa heat shock protein (hsp90). Results indicated that the four subunits found in cross-linking experiments were composed of three species: the AhR ligand binding subunit, hsp90, and an unknown 43-kDa protein. The stoichiometry of hsp90 present in each AhR complex was determined in two separate experiments: 1) from cross-linking experiments, stoichiometry was determined by quantitative immunoblotting with anti-AhR and anti-hsp90 antibodies followed by quantitation with 125I-counterantibody on protein blots; 2) using 35S-labeled Hepa 1 cytosol, the hsp90/AhR stoichiometry was determined by immunopurifying receptor complexes, and the amount of 35S-labeled AhR and hsp90 was assessed. The stoichiometry values obtained were 2.4 and 1.72 mol of hsp90/mol of AhR using each experimental approach, respectively.

Cell Line↗
PubMed1994

Immunohistochemical double-staining for Ah receptor and ARNT in human embryonic palatal shelves.

The aryl hydrocarbon receptor (AhR) and the AhR nuclear translocator protein (ARNT) are basic-helix-loop-helix-PAS (HLH) proteins involved in transcriptional regulation. Polycyclic aromatic halogenated chemicals, of which 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is the most potent, bind to the AhR. In the cellular cytoplasm, the AhR exists as a complex with the heat shock protein HSP90 and other small peptides. This complex dissociates following ligand binding and then the ligand-bound AhR binds ARNT. The ligand-AhR-ARNT complex interacts with a specific, nuclear DNA sequence, the dioxin response element (DRE), altering transcription of a regulated gene. Studies in hepatoma cell lines indicate that both proteins are required for regulation of transcription. In this study, AhR and ARNT were localized immunohistochemically in human embryonic palatal cells and specific patterns of expression were seen for each protein. A double-staining protocol revealed that epithelial cells expressed both AhR and ARNT, but in mesenchyme and nasal spine cartilage individual cells were identified which expressed either AhR or ARNT. This heterogeneous pattern may be a means of suppressing transcriptional regulation and also suggests the existence of other, unidentified basic-helix-loop-helix partner(s). The heterogeneous expression pattern may also reflect a complex role for these HLH proteins as transcriptional regulators of embryonic development.

Aryl Hydrocarbon Receptor Nuclear Translocator↗
PubMed1994

Ah receptor in embryonic mouse palate and effects of TCDD on receptor expression.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is the most potent member of a family of halogenated aromatic hydrocarbons which are widespread environmental contaminants. In animals the adverse biological effects of TCDD include carcinogenesis, reproductive toxicity, immune function alteration, hyperkeratosis, hepatotoxicity, thymic involution, and teratogenesis. In the mouse embryo, TCDD induces cleft palate through a mechanism which involves altered differentiation and proliferation of the palatal cells, resulting in the failure of opposing shelves to fuse. Cleft palate induction by TCDD requires the Ah receptor. This study examines the expression of the Ah receptor in secondary palate of control and TCDD-exposed C57BL/6N embryos using in situ hybridization, Northern blots, and immunohistochemistry. Ah receptor protein expression was significantly higher in epithelial versus mesenchymal cells, and regional differences in expression within the epithelium were statistically significant. TCDD exposure was shown to downregulate Ah receptor mRNA and protein throughout the palatal shelf and this occurred at both the teratogenic dose and the dose which was not sufficient to produce cleft palate. This study represents the first demonstration of the tissue and cellular localization of the Ah receptor, raising questions about the extrapolation of results from cultured tumor cells to those observed in vivo.

Animals↗
PubMed1994

Interactive regulation of Ah and glucocorticoid receptors in the synergistic induction of cleft palate by 2,3,7,8-tetrachlorodibenzo-p-dioxin and hydrocortisone.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a wide-spread environmental contaminant that produces adverse biological effects including carcinogenesis, reproductive toxicity, immune dysfunction, hyperkeratosis, hepatotoxicity, thymic involution and teratogenesis. In the mouse embryo, TCDD induces cleft palate and hydronephrosis. Glucocorticoids are endogenous steroid compounds that have an important role in development, but are teratogenic at pharmacological doses. The synthetic glycocorticoid, hydrocortisone (HC), induces cleft plate and a potent, synergistic interaction has been observed between TCDD and HC. Both TCDD and HC act through receptor-mediated mechanisms and each compound has its own receptor, the Ah receptor (AhR) and the glucocorticoid receptor (GR), respectively. The morphology and etiology of TCDD- and HC-induced clefts are distinctly different, as HC clefting is due to formation of small palatal shelves, while TCDD-treated shelves fail to fuse due to effects on epithelial cell proliferation and differentiation. The present study examines the expression of AhR and GR in the embryonic palate following exposure to TCDD, HC, and HC + TCDD. C57BL/6N pregnant mice were treated with HC (25 or 100 mg/kg/day GD10-13, sc), TCDD (3 micrograms/kg/day GD10-13, or 24 micrograms/kg GD10, orally), or HC + TCDD (25 mg/kg/day sc and 3 micrograms/kg/day orally, GD10-13). Craniofacial tissues were collected from the embryos on GD14 and examined for AhR and GR expression using in situ hybridization. Northern blots, and immunohistochemistry. We found that in the embryonic palate exposed to TCDD, the AhR was downregulated and the GR expression increased. Conversely, following HC exposure, the GR was downregulated and AhR levels were elevated. HC + TCDD produced increased expression of both receptors. Effects on AhR appeared to be regulated at the transcriptional level, as both protein and mRNA were altered in similar directions. The observed cross-regulation of the receptors is believed to be important in the synergistic interaction between TCDD and HC for the induction of cleft palate.

Animals↗
PubMed1994

Physicochemical and immunocytochemical analysis of the aryl hydrocarbon receptor nuclear translocator: characterization of two monoclonal antibodies to the aryl hydrocarbon receptor nuclear translocator.

The aryl hydrocarbon receptor nuclear translocator (Arnt) is a basic helix-loop-helix transcription factor that heterodimerizes with the aryl hydrocarbon receptor to mediate signal transduction pathways inducible by 2,3,7,8-tetrachlorodibenzo-p-dioxin and other planar aromatic hydrocarbons. Monoclonal antibodies (MAbs) have been raised against a carboxyl-terminal 19-amino acid peptide hapten (MAb 2B10) and against a carboxyl-terminal 378-amino acid polypeptide-staphylococcal Protein A fusion protein (MAb 4G9) of Arnt and their characterization is described. Western blot experiments show that both MAbs specifically cross-react with an approximately 85-kDa band in cytosol prepared from COS-7 cells transfected with the full length human Arnt cDNA pBMSNeo-D24-1 and in Hepa 1c1c7 cytosol but not in Arnt-deficient Hepa 1-C4 mutant cytosol. Velocity sedimentation of Hepa 1c1c7 cytosol on sucrose gradients and Superose 6 gel permeation chromatography were used to estimate the sedimentation coefficient. Stokes radius, and relative molecular mass of Arnt as approximately 3.6-4.1 S, 6.8 nm, and 101-115 kDa, respectively. These results indicate that Arnt probably exists in monomeric form in Hepa 1c1c7 cytosolic extracts. Laser scanning confocal microscopy and indirect immunofluorescence microscopy revealed Arnt to be distributed throughout the non-nucleolar portion of the nucleus of Hepa 1c1c7, VT(2) (Hepa 1-C4T mutant cell line deficient in Arnt function and stably transfected with pBMSNeo D24-1, expressing the full length human Arnt cDNA), and HeLa cells. The establishment of the nuclear localization of Arnt in human and murine cell lines shown here indicates that its nuclear localization may be conserved across species. Immunofluorescence analysis of Arnt in three cell lines using two MAbs (to distinct epitopes) provides evidence that suggests that the aryl hydrocarbon receptor heterodimerizes with Arnt in the nucleus.

Amino Acid Sequence↗
PubMed1993

Raf exists in a native heterocomplex with hsp90 and p50 that can be reconstituted in a cell-free system.

Recently, we have demonstrated that the tyrosine kinase pp60v-src can undergo cell-free assembly into a heterocomplex with rabbit hsp90 and p50 when the immunoadsorbed protein is incubated with rabbit reticulocyte lysate (Hutchison, K. A., Brott, B. K., De Leon, J. H., Perdew, G. H., Jove, R., and Pratt, W. B. (1992) J. Biol. Chem 267, 2902-2908). Using a baculovirus system to express a high level of human c-Raf serine/threonine kinase in Sf9 insect cells, we show here that immunoadsorbed c-Raf undergoes similar lysate-mediated assembly into a heterocomplex with hsp90 and p50. As with pp60v-src and steroid receptors, binding of c-Raf to hsp90 occurs in an ATP-dependent and K(+)-dependent manner and the resulting heterocomplex is stabilized by molybdate. With a very rapid and gentle procedure of Sf9 cell cytosol preparation and c-Raf immunoadsorption, we show coimmunoadsorption of the insect homologue of hsp90. The same procedures permit detection of a native complex of v-Raf with rat hsp90 and p50 in stably transfected rat 3Y1 fibroblasts, and v-Raf is also assembled into a heterocomplex with rabbit hsp90 and p50 by reticulocyte lysate. Using the 22W mutant of c-Raf in which the NH2-terminal half has been deleted, we show that the catalytic domain of the kinase is sufficient for both formation of the native heterocomplex in mouse NIH 3T3 cells and cell-free reconstitution of the heterocomplex by rabbit reticulocyte lysate. Although the native Raf-heat shock protein heterocomplex is less stable than native pp60v-src and glucocorticoid receptor heterocomplexes, by analogy with these proteins its detection may have important implications regarding the mechanism of Raf trafficking through the cytoplasm.

3T3 Cells↗
PubMed1993

Alterations in the Ah receptor level after staurosporine treatment.

Toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is known to be mediated by the aryl hydrocarbon receptor (AhR). The ligand-receptor complex mediates the induction of CYP1A1 (cytochrome P(1)450) gene transcription through interaction with an Ah-responsive element. Staurosporine, a potent inhibitor of protein kinases inactivates the TCDD-induced CYP1A1 gene transcription. This study was initiated to study binding capacity, capability to translocate, and levels of the AhR in Hepa 1 cells after staurosporine treatment. Levels of the AhR were quantitated in Hepa 1 cells by sandwich type radioimmunochemical method using Rpt 1 (AhR monoclonal antibody) as the primary antibody and [125I]goat anti-mouse as the secondary antibody. Staurosporine treatment caused a time- and concentration-dependent decrease in the intracellular concentration of AhR. At 8 h, the EC50 for the staurosporine-dependent decrease in AhR was 75 nM. Although the receptor levels decreased significantly, it did not affect the properties of the receptor as judged by its ligand binding capacity and functional nuclear translocation of the existing AhR. The isoelectric point of the receptor was essentially unaltered after staurosporine treatment, an indirect indication that the degree of AhR phosphorylation did not change. There could be a variety of explanations for these results such as the decrease in the AhR levels may be due to either an increase in proteolytic activity caused by the imbalance of kinase/phosphatase levels or a decrease in de novo receptor synthesis.

Alkaloids↗
PubMed1993

Half-life of aryl hydrocarbon receptor in Hepa 1 cells: evidence for ligand-dependent alterations in cytosolic receptor levels.

The rate of turnover of the Ah receptor (AhR) was determined using the density shift method in Hepa 1 and in a Hepa 1 mutant line, c4, which fails to accumulate AhR complexes in the nucleus. The half-life of the AhR was found to be 7.7 and 9.7 h in Hepa 1 and c4 cells, respectively. The effect of AhR occupation with either an agonist, beta-naphthoflavone (beta NF), or a partial antagonist, alpha-naphthoflavone (alpha NF), on AhR half-life and concentration in the cytosolic fraction was examined. In Hepa 1 cells, a 12-h exposure to beta NF resulted in a 62% decrease in AhR concentration. The same treatment, using alpha NF as the ligand, resulted in a 14% decrease. The half-life of the AhR increased from 7.7 to 9.3 h during beta NF treatment and was essentially the same during alpha NF treatment in Hepa 1 cells. In c4 cells, a 12-h exposure to beta NF resulted in a 44% decrease in AhR concentrations, whereas exposure to alpha NF resulted in an 8% decrease. The half-life of the AhR in c4 cells during beta NF exposure increased from 9.7 to 14.6 h, and alpha NF exposure increased half-life to 17.6 h. These results indicate: (a) cytosolic AhR concentrations are modulated by ligand occupation, (b) exposure to AhR ligands, after an initial decrease in AhR levels, resulted in an increase in AhR half-life, and (c) similar results were obtained in Hepa 1 and c4 cells, this would indicate that AhR occupation with ligand and subsequent AhR-ligand nuclear translocation does not appear to play a significant role in regulation of AhR half-life in Hepa 1 cells.

Animals↗