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EVOLUTIONARY HOMOLOGS part 2/3

Ah receptor/Arnt heterodimers

The human aryl hydrocarbon receptor (AhR) and aryl hydrocarbon receptor nuclear translocatorprotein (Arnt) were coexpressed in the yeast Saccharomyces cerevisiae to create a system for thestudy of the Ahr/Arnt heterodimeric transcription factor. Specific transcriptional activation mediated byAhR/Arnt heterodimer (which is a functional indicator of receptor expression) was assessed bybeta-galactosidase activity produced from a reporter plasmid. Yeast expressing AhR and Arntdisplay constitutive transcriptional activity that is not augmented by the addition of AhR agonists instrains that required exogenous tryptophan for viability. In contrast, strains with an intact pathway fortryptophan biosynthesis do respond to AhR agonists and have lower levels of backgroundbeta-galactosidase activity. In the yeast system, hexachlorobenzene, benzo(a)pyrene, and beta-naphthoflavone are effective AhR agonists for beta-galactosidase activity induction. Tryptophan, indole, indole acetic acid, and tryptamineactivate transcription in yeast coexpressing AhR and Arnt. Indole-3-carbinol is an exceptionally potent AhR agonist in yeast. This yeast system is useful for the study of AhR/Arnt protein complexes, andmay prove to be generally applicable to the investigation of other multiprotein complexes (Miller, 1997).

In mouse hepatoma cells, the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, ordioxin) induces Cyp1A1 gene transcription, a process that requires two basic helix-loop-helix regulatoryproteins: the aromatic hydrocarbon receptor (AhR) and the aromatic hydrocarbon receptor nucleartranslocator (Arnt). Ligation-mediated PCR technique was used to analyze dioxin-inducedchanges in protein-DNA interactions and chromatin structure of the Cyp1A1 enhancer-promoter in itsnative chromosomal setting. Dioxin-induced binding of the AhR/Arnt heterodimer to enhancer chromatinis associated with a localized (about 200 bp) alteration in chromatin structure that is manifested byincreased accessibility of the DNA; these changes probably reflect direct disruption of a nucleosomeby AhR/Arnt. Dioxin induces analogous AhR/Arnt-dependent changes in chromatin structure andaccessibility at the Cyp1A1 promoter. However, the changes at the promoter must occur by adifferent, more indirect mechanism, because they are induced from a distance and do not reflect a localeffect of AhR/Arnt binding. Dose-response experiments indicate that the changes in chromatinstructure at the enhancer and promoter are graded, mirroring the graded induction of Cyp1A1transcription by dioxin (Okino, 1995).

Function of Ah receptor/Arnt heterodimers: a role for phosphorylation

The Ah receptor binds aryl hydrocarbons such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) withhigh affinity. After binding aryl hydrocarbons, the receptor releases the 90-kDa heat shock protein andforms a heterodimer with the Arnt protein capable of binding at xenobiotic-responsive elements (XREs) andstimulating the transcription of genes involved in the metabolism of aryl hydrocarbons. The activity ofthe Ah receptor/Arnt dimer can be decreased by treatments that cause the down-regulation of proteinkinase C and decrease the nuclear accumulation of the receptor. Incubation with acid phosphatase orwith alkaline phosphatase has been reported to block XRE binding. Thus the literature suggests thatphosphorylation regulates Ah receptor activity by affecting DNA binding and/or nuclear transport. Areporter plasmid containing two XREs was used to investigate the effects of phosphatase inhibitors onTCDD-dependent transcription carried out by the Hepa-1 mouse liver cell line. The inhibitors calyculin A andokadaic acid cause two- to threefold increases in TCDD-dependent transcription, at concentrationscapable of selectively inhibiting protein phosphatase 1 and protein phosphatase 2A. The inhibitorcyclosporin A doubles TCDD-dependent transcription at a concentration capable of selectivelyinhibiting protein phosphatase 2B. All three of the phosphatase inhibitors increase TCDD-dependenttranscription without affecting transcription in the absence of TCDD. Nuclear extracts were preparedfrom cells treated with concentrations of either okadaic acid or cyclosporin A, both of which substantially stimulateTCDD-dependent transcription. Neither of the inhibitors significantly increase the level ofTCDD-dependent XRE binding in the extracts. GAL4-Arnt fusion proteins were used to furtherinvestigate whether the phosphatase inhibitors affected a step other than DNA binding. Okadaic acidtreatment specifically increases the ability of a GAL4 fusion protein containing the Arnt PAS andtransactivation domains to stimulate transcription. These results suggest that serine/threonine-specificprotein phosphatases can act at a level subsequent to XRE binding to inhibit the ability of the Ahreceptor/Arnt dimer to stimulate transcription (Li, 1997).

Interaction of Ahr with HSP90

Functional domains of the mouse aryl hydrocarbon receptor (Ahr) were investigated by deletionanalysis. Ligand binding is localized to a region encompassing the PAS B repeat. Theligand-mediated dissociation of Ahr from the 90-kDa heat shock protein (HSP90) does not require thearyl hydrocarbon receptor nuclear translocator (Arnt), but it is slightly enhanced by this protein. OneHSP90 molecule appears to bind within the PAS region. The other molecule of HSP90 appears torequire interaction at two sites: one over the basic helix-loop-helix region, and the other located withinthe PAS region. Each mutant was analyzed for dimerization with full-length mouse Arnt andsubsequent binding of the dimer to the xenobiotic responsive element (XRE). In order to minimize anyartificial steric hindrances to dimerization and XRE binding, each Ahr mutant was also tested with anequivalently deleted Arnt mutant. The basic region of Ahr is required for XRE binding but not fordimerization. Both the first and second helices of the basic helix-loop-helix motif and the PAS regionare required for dimerization. These last results are analogous to those previously obtained for Arnt,compatible with the notion that equivalent regions of Ahr and Arnt associate with eachother. Deletion of the carboxyl-terminal half of Ahr does not affect dimerization or XRE binding but, incontrast to an equivalent deletion of Arnt, eliminates biological activity, as assessed by an in vivotranscriptional activation assay, suggesting that this region of Ahr plays a more prominent role intranscriptional activation of the cyp1a1 gene than does the corresponding region of Arnt (Fukunaga, 1995).

Expression of a series of Ah receptor (AhR) deletion mutants in an in vitro translation system has beenpreviously used to map several functional domains of the murine AhR. In this report, quantitative immunoprecipitation of 90-kDa heatshock protein (hsp90) from reticulocyte lysate allowed a measurement of expression levels for the AhR and AhRdeletion mutants, complexed with hsp90. After translation of a series of deletion mutants it wasdetermined 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 areable to stably interact with hsp90. Thus, the AhR appears to be a unique member of the PAS domainfamily of proteins that binds a known ligand and stably interacts with hsp90 (Perdew, 1996).

CYP1A1, a target of Ah receptor/Arnt heterodimers

Introduction of a retroviral expression vector for the aryl hydrocarbon receptor (AhR) restoresCYP1A1 inducibility to a mutant derivative of the Hepa-1 cell line that is defective in induction ofCYP1A1 by ligands for the receptor. An AhR protein with normal ligand binding activity is expressedin the mutant but ligand treatment of mutant cell extract fails to induce binding of the AhR/Arnt dimer to the xenobiotic responsive element (XRE).AhR cDNAs derived from the mutant encode a protein that is unimpaired in ligand-dependentdimerization with Arnt, but the AhR.Arnt dimer so formed is severely impaired for XRE bindingactivity. The mutant cDNAs contain a C to G mutation at base 648, causing a cysteine to tryptophanalteration at amino acid 216, located between the Per-Arnt-Sim (PAS) homology region A andPAS B repeats. Introduction of the same mutation in the wild-type AhR sequence by site-directedmutagenesis similarity impairs XRE binding activity. Substitution with the conservative amino acid,serine, has no effect on XRE binding. The tryptophan mutation, but not the wild-type allele, isdetectable in the genomic DNA of the mutant. The implication that an amino acid within the PAS regionmay be involved in DNA binding indicates that the DNA binding behavior of AhR may be moreanomalous than previously suspected (Sun, 1997).

The ligand-activated aromatic hydrocarbon receptor (AhR) dimerizes with the AhR nucleartranslocator (Arnt) to form a functional complex that transactivates expression of the cytochromeP-450 CYP1A1 gene and other genes in the dioxin-inducible [Ah] gene battery. The activity of the CYP1A1 enzyme negatively regulates this process.To study the relationship between CYP1A1 activity and Ah receptor activation CYP1A1-deficient mouse hepatoma c37 cells and CYP1A1- and AhR-deficient African greenmonkey kidney CV-1 cells were used. c37 cells that have not been exposed to exogenous Ah receptor ligands already containtranscriptionally active AhR-Arnt complexes, a finding that is also observed in wild-type Hepa-1cells treated with Ellipticine, a CYP1A1 inhibitor. In CV-1 cells, transient expression of AhR andArnt leads to high levels of AhR-Arnt-dependent luciferase gene expression, even in the absenceof an agonist. Elevated reporter geneexpression correlates with constitutive nuclear localization of the AhR. Transcriptional activation ofthe luciferase reporter gene observed in CV-1 cells is significantly decreased by (1) expression of afunctional CYP1A1 enzyme, (2) competition with chimeric or truncated AhR proteins containing theAhR ligand-binding domain, and (3) treatment with the AhR antagonist alpha-naphthoflavone. Theseresults suggest that a CYP1A1 substrate, which accumulates in cells lacking CYP1A1 enzymaticactivity, is an AhR ligand responsible for endogenous activation of the Ah receptor (Chang, 1998).

A xenobiotic-responsive element (XRE)-binding factor(s) other than the AhR.Arnt complex inhibits the transcription of CYP1A1 gene in the liver from adult rabbits, known to be nonresponsiveto CYP1A1 inducers. The constitutive factor(s) in liver nuclear extracts binds to the core sequence ofXRE. The binding is eliminated by the presence of an excess amount of the AhR.Arnt complexsynthesized in vitro. To identify the constitutive factor(s), a sequence similar to rabbit XRE was sought.The sequence of rabbit XRE overlaps with that of the upstream stimulatory factor 1(USF1)-binding site in the mouse metallothionein I promoter. In fact, a super shift assay using aspecific antibody against human USF1 indicates that USF1 is capable of binding to rabbit XRE.The AhR.Arnt-mediated activation of XRE-TK/Luc reporter gene in RK13 cells isblocked by the transfection with a USF1 expression vector with the amounts of the expression vectortransfected. These results indicate that the XRE of the rabbit CYP1A1 gene is recognized by the basichelix-loop-helix proteins as a regulator of the expression of CYP1A1 in both an agonistic (AhR.Arnt) and anantagonistic (USF1) manner (Takahishi, 1997).

Transcriptional activation of the human CYP1A1 gene by halogenated and polycyclic aromatic hydrocarbons is mediated by the aryl hydrocarbon receptor (AhR) complex, a ligand-dependent transcription factor. A competent AhR comprises at least two components following nuclear translocation and DNA binding, the AhR and the AhR nuclear translocator (Arnt) protein, whose combined action on human CYP1A1 gene transcription is shown to be dependent upon functional protein kinase C (PKC). In the present study, the effects of phorbol 12-myristate 13-acetate, a potent PKC activator, were examined on the ligand-induced transcriptional activation of the CYP1A1 gene and cellular function of the AhR in human HepG2 101L cells. The 101L cells carry a stable transgene consisting of 1800 bases of 5'-flanking DNA and the promoter of the human CYP1A1 gene linked to the firefly luciferase structural gene. Pretreatment of cells with 12-myristate 13-acetate enhances ligand-induced CYP1A1 gene expression two- to three-fold. Inhibition of PKC activity blocks directly the transcriptional activation and the transactivation of the CYP1A1 gene, indicating a role for PKC in the AhR-mediated transcriptional activation process. However, the DNA binding activities of the invitro activated and the induced nuclear AhR as measured by electrophoretic mobility shift analysisare not affected when CYP1A1 transcription is inhibited, indicating the actions of PKC to be anuclear event that works in concert with or precedes AhR binding to the gene. These results illustratethat PKC is absolutely essential for the cellular and molecular events that control induction of CYP1A1gene transcription (Chen, 1996).

Evolutionary homologs continued: part 3/3 | back to part 1/3


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