Mode of action and dose-response framework analysis for receptor-mediated toxicity: The aryl hydrocarbon receptor as a case study.

Dioxins and dioxin-like compounds are tumor promoters that cause liver cancer in rats and mice. The aryl hydrocarbon receptor (AHR) has been implicated as a key component in this tumor promotion response. Despite extensive knowledge of the toxicology of dioxins, no mode of action (MOA) hypothesis for their tumorigenicity has been formally documented using the Human Relevance MOA framework developed by the International Programme on Chemical Safety (IPCS). To address this information gap, an expert panel was convened as part of a workshop on receptor-mediated liver tumorigenicity. Liver tumors induced by ligands of the AHR were assessed using data for dioxins and related chemicals as a case study. The panel proposed a MOA beginning with sustained AHR activation, eventually leading to liver tumors via a number of other processes, including increased cell proliferation of previously initiated altered hepatic foci, inhibition of intrafocal apoptosis and proliferation of oval cells. These processes have been identified and grouped as three key events within the hepatocarcinogenic MOA: (1) sustained AHR activation, (2) alterations in cellular growth and homeostasis and (3) pre-neoplastic tissue changes. These key events were identified through application of the Bradford-Hill considerations in terms of both their necessity for the apical event/adverse outcome and their human relevance. The panel identified data supporting the identification and dose-response behavior of key events, alteration of the dose-response by numerous modulating factors and data gaps that potentially impact the MOA. The current effort of applying the systematic frameworks for identifying key events and assessing human relevance to the AHR activation in the tumorigenicity of dioxins and related chemicals is novel at this time. The results should help direct future regulatory efforts and research activities aimed at better understanding the potential human cancer risks associated with dioxin exposure.

Okadaic acid increases ARNT homodimer transactivation potential.

The human aryl hydrocarbon nuclear translocator (hARNT) protein belongs to the family of basic helix-loop-helix (bHLH) PAS transcription factors and regulates a range of cellular processes by either homodimerizing or heterodimerizing with other bHLH-PAS proteins. hARNT has been shown to be almost exclusively phosphorylated on serine residues. However, regulation of hARNT with respect to phosphorylation remains poorly understood. The phosphatase inhibitor okadaic acid was used to explore whether a change in hARNT phosphorylation status could influence hARNT homodimer activity. The hARNT homodimer has been shown to bind to E boxes and E-box binding factors are believed to be important in the regulation of cell differentiation and proliferation. Okadaic acid significantly increased hARNT-mediated class B, E-box-driven reporter activity in COS-1 cells, transiently expressing hARNT without affecting hARNT protein levels. This alteration in hARNT-mediated class B, E-box-driven reporter activity correlates with an observed increase in [32P]orthophosphate incorporation into hARNT. Treatment with okadaic acid resulted in a 12-fold increase in [32P]orthophosphate incorporation into hARNT that was transiently expressed in COS-1 cells; an increase in the number of tryptic phosphopeptides generated from hARNT digests on two-dimensional phosphopeptide maps was also observed. Despite the significant increase in [32P]orthophosphate incorporation into hARNT, serine remained the predominantly phosphorylated residue. Clearly, increased serine phosphorylation does not appear to negatively regulate hARNT homodimerization or transactivation potential. These results demonstrate that increased hARNT homodimer signaling in COS-1 cells may result from a direct change in hARNT phosphorylation status.

Monitoring nuclear import with GFP-variant fusion proteins in digitonin-permeabilized cells.

A convenient assay for monitoring nuclear localization signal-mediated nuclear import of green fluorescent protein (GFP)-variant fusion proteins has been developed. This modified assay relies upon indirect immunofluorescence microscopy for visualization of transported substrates. The use of GFP-variant fusion proteins allows for the rapid assessment of optimal digitonin concentration and permits nuclear import to be monitored with minimal sample preparation in real time.

The Q-rich subdomain of the human Ah receptor transactivation domain is required for dioxin-mediated transcriptional activity.

The aryl hydrocarbon receptor (AhR), a basic helix-loop-helix/Per-Arnt-Sim transcription factor, mediates many of the toxic and biological effects of the environmental contaminant, 2,3,7,8-tetrachlorodibenzo-p-dioxin, which include the transcriptional activation of dioxin-responsive genes such as CYP1A1. Many aspects of this process are known; however, the mechanism of transcriptional activation and the proteins that are key to this process remain to be determined. The hAhR has a complex transactivation domain, composed of three potentially distinct subdomains. Deletional analysis of the hAhR transactivation domain indicates that removal of the P/S/T-rich subdomain enhances transcriptional activity, whereas the Q-rich subdomain is critical for hAhR transactivation potential, and the acidic subdomain by itself fails to activate a dioxin response element-driven reporter gene. Deletional analysis of the Q-rich subdomain identified a critical stretch of 23 amino acids between residues 666 and 688 of the hAhR, which are required for transactivation potential. Alanine scanning mutagenesis of this region identified a leucine residue (Leu-678), which is required for hAhR activity. Functional analysis of this point mutant revealed that it is capable of binding ligand, heterodimerization, and subsequent binding to dioxin response elements. Further, when hAhR/L678A and hAhR containing only the acidic subdomain were overexpressed they acted as dominant negative receptors and repressed wild-type hAhR activity. In addition, the hAhR/L678A failed to activate CYP1A1 gene transcription in transfected BP-8 cells and exhibited reduced binding to RIP140 in vitro. Thus, Leu-678 appears to be critical for efficient transactivation activity of the hAhR and appears to disrupt recruitment of co-regulators.

Aryl hydrocarbon receptor (AhR)/AhR nuclear translocator (ARNT) activity is unaltered by phosphorylation of a periodicity/ARNT/single-minded (PAS)-region serine residue.

The aryl hydrocarbon nuclear translocator (ARNT) protein belongs to the family of basic helix-loop-helix (HLH)-periodicity/ARNT/single-minded [Per/ARNT/Sim (PAS)] transcription factors and regulates a range of cellular processes by either homodimerizing or heterodimerizing with other basic HLH-PAS proteins. To date, it has been shown that both the HLH and PAS domains are required for aryl hydrocarbon receptor (AhR) ARNT heterodimerization and that phosphorylation of ARNT is also required for this heterodimerization. Presently, regulation of ARNT with respect to phosphorylation is poorly understood. In an earlier study, murine ARNT was shown to be a phosphoprotein, to display charge heterogeneity, and to have a shift in its predominant isoforms after heterodimerization with the AhR. It was hypothesized that this shift may represent a change in ARNT phosphorylation status. Metabolic [(32)P]orthophosphate labeling of human ARNT-transfected COS-1 cells, in conjunction with phosphoamino acid analysis, Edman degradation, and phosphopeptide mapping, demonstrated that ARNT is predominantly phosphorylated on serine residues and that serine 348 (S348) in the PAS domain is phosphorylated. Alanine and glutamic acid substitutions were used to demonstrate that loss of phosphorylation at this site did not influence AhR-mediated xenobiotic response elements-driven or ARNT-mediated class B E-box-driven signaling. Additionally, the phosphorylation pattern of ARNT was unaltered after AhR heterodimerization. Although phosphorylation of S348 did not modulate AhR-ARNT or ARNT-ARNT signaling, phosphorylation of this PAS-region serine residue may be important in other ARNT-mediated gene expression systems.

A tetratricopeptide repeat half-site in the aryl hydrocarbon receptor is important for DNA binding and trans-activation potential.

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 to trans-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 of trans-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.

Aryl hydrocarbon (Ah) receptor levels are selectively modulated by hsp90-associated immunophilin homolog XAP2.

The aryl hydrocarbon receptor (AhR) is a ligand-inducible transcription factor that mediates biological responses to halogenated aromatic hydrocarbons. The unliganded AhR is a cytoplasmic, tetrameric complex consisting of the AhR ligand-binding subunit, a dimer of hsp90, and the hepatitis B virus X-associated protein 2 (XAP2). The role of XAP2 as a member of the AhR core complex is poorly understood. XAP2 shares significant homology with the immunophilins FKBP12 and FKBP52, including a highly conserved, C-terminal, tetratricopeptide repeat (TPR) domain. XAP2 forms a complex with hsp90 and the AhR but can also bind to both independently. This binding is mediated by the conserved TPR domain. Single-point mutations in this region are sufficient to disrupt the association of XAP2 with both the AhR and hsp90 in cells. Cotransfection of the AhR and XAP2 in COS-1 cells results in increased AhR levels compared with cells transfected with the AhR alone. In contrast, coexpression of the AhR with the TPR containing proteins FKBP52, protein phosphatase 5 (PP5), or XAP2 TPR-mutants deficient in binding to the AhR and hsp90 does not affect AhR levels and coexpression of the AhR with the TPR domain of PP5 results in AhR down-regulation. These results demonstrate that XAP2 is apparently unique among hsp90-binding proteins in its ability to enhance AhR levels. A yellow fluorescent protein (YFP)-XAP2-FLAG was constructed and biochemically characterized, and no loss of function was detected. YFP-XAP2-FLAG was transiently transfected into NIH 3T3 and was found to localize in both the nucleus and the cytoplasm when visualized by fluorescence microscopy. Treatment of Hepa-1 cells with the hsp90-binding benzoquinone ansamycin, geldanamycin, and the macrocyclic antifungal compound radicicol resulted in AhR but not XAP2 or FKBP52 turnover. Taken together, these results suggest that XAP2/hsp90 and FKBP52/hsp90 complexes are similar yet exhibit unique functional specificity.

Subcellular localization of the aryl hydrocarbon receptor is modulated by the immunophilin homolog hepatitis B virus X-associated protein 2.

The hepatitis B virus X-associated protein 2 (XAP2) is an immunophilin homolog and core component of the aryl hydrocarbon receptor (AhR). Immunophilins are components of many steroid receptor complexes, serving a largely unknown function. Transiently expressed AhR.YFP (yellow fluorescent protein) localized to the nuclei of COS-1 and NIH-3T3 cells. Co-expression of AhR.YFP with XAP2 restored cytoplasmic localization, which was reversed by 2,3,7, 8-tetrachlorodibenzo-p-dioxin treatment (TCDD). The effect of XAP2 on AhR localization was specific involving a nuclear localization signal-mediated pathway. Examination of the ratio of AhR to XAP2 in the AhR complex revealed that approximately 25% of transiently expressed AhR was associated with XAP2, in contrast with approximately 100% when the AhR and XAP2 were co-expressed. Strikingly, TCDD did not influence these ratios, suggesting that ligand binding initiates nuclear translocation prior to complex dissociation. Analysis of endogenous AhR in Hepa-1 cells revealed that approximately 40% of the AhR complex was associated with XAP2, predicting observed AhR localization to cytoplasm and nuclei. This study reveals a novel functional role for the immunophilin-like component of a soluble receptor complex and provides new insight into the mechanism of AhR-mediated signal transduction, demonstrating the existence of two structurally distinct and possibly functionally unique forms of the AhR.

Lack of an absolute requirement for the native aryl hydrocarbon receptor (AhR) and AhR nuclear translocator transactivation domains in protein kinase C-mediated modulation of the AhR pathway.

Protein kinase C (PKC)-mediated modulation of the aryl hydrocarbon receptor (AhR) pathway was examined in CHOK1-derived L10.I cells stably transfected with the pGUDLUC6.1 reporter; pGUDLUC6.1 is solely controlled by four dioxin-responsive enhancer elements. Co treatment of L10.I cells with 10 nM 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD) and 81 nM phorbol 12-myristate 13-acetate (PMA), an activator of sn-1,2-diacylglyerol binding PKCs, enhanced transactivation of the reporter construct several-fold relative to cells treated with a saturating 10 nM TCDD dose alone; this effect was dubbed the "PMA effect." A domain swapping and deletional analysis of the native AhR and AhR nuclear translocator (ARNT) protein transactivation domains (TADs) was performed to determine if these domains are absolutely required for the AhR x ARNT dimer-mediated PMA effect in the L10.I model system; controls demonstrate the suitability of the L10.I model for these analyses and that endogenous AhR and ARNT levels are extremely low in this model. Transient coexpression of the AhR and ARNT-474-FLAG, an ARNT protein lacking the native ARNT TAD, in L10.I cells reveals the native ARNT TAD is not absolutely required for the AhR x ARNT-474-FLAG dimer to mediate the PMA effect. Transient coexpression of AhRDeltaCVP, a chimeric AhR protein in which the native AhR TAD has been replaced with the VP16 (herpes simplex virus protein 16) TAD (which control experiments demonstrate is unaffected by PMA), and ARNT in L10.I cells indicates that the native AhR TAD is not absolutely required for this AhRDeltaCVP x ARNT dimer to mediate the PMA effect. These observations strongly suggest that PKC-mediated modulation of the AhR pathway is not absolutely dependent on coactivators recruited to the AhR. ARNT dimer by the native TADs of the AhR and its heterodimerization partner ARNT.

Differential recruitment of coactivator RIP140 by Ah and estrogen receptors. Absence of a role for LXXLL motifs.

The Ah receptor (AhR), a soluble cytosolic protein, mediates most of the toxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related environmental contaminants. The mechanism of ligand-mediated AhR activation has been, in part, elucidated. The sequence of events following the binding of the AhR/AhR nuclear translocator protein (ARNT) heterodimer to dioxin response elements has yet to be completely understood. The role of coactivator, RIP140, in the modulation of transcriptional activity of AhR/ARNT heterodimer was examined. RIP140 enhanced TCDD-mediated, dioxin response element-driven reporter gene activity in three cell lines. Co-immunoprecipitation and co-localization assays revealed that RIP140 interacted with AhR, but not with ARNT, both in vitro and in cells. Mapping of the interaction sites revealed that RIP140 was recruited by the AhR transactivation domain via the Q-rich subdomain. The RIP140 domain that interacts with the AhR was mapped to a location between amino acid residues 154 and 350, which is distinct from those involved in estrogen receptor binding. The signature motif, LXXLL, which is responsible for binding of several coactivators to nuclear receptors, is not required for RIP140 binding to AhR. These results demonstrate that the AhR recruits coactivators that are capable of enhancing transcription and, thus, the AhR may compete with steroid receptors for a common coactivator pool. In addition, the data suggest that there are distinct motif(s) for the recruitment of RIP140 to AhR and possibly other non-steroid receptors/transcription factors.

Characterization of the AhR-hsp90-XAP2 core complex and the role of the immunophilin-related protein XAP2 in AhR stabilization.

The unliganded aryl hydrocarbon receptor (AhR) exists in the cytoplasm in a tetrameric 9S core complex, consisting of the AhR ligand-binding subunit, a dimer of hsp90, and the hepatitis B virus X-associated protein 2 (XAP2), an immunophilin-related protein sharing homologous regions with FKBP12 and FKBP52. Interactions between the recently identified XAP2 subunit and other members of the unliganded AhR complex and its precise role in the AhR signal transduction pathway are presently unknown. Mapping studies indicate that XAP2 requires the PAS, hsp90, and ligand binding domain(s) of the AhR for binding, and that both proteins directly interact in the absence of hsp90. XAP2 is also able to interact with hsp90 complexes in the absence of the AhR, and C-terminal sequences of XAP2 are required for this interaction. XAP2 binds to the C-terminal end of hsp90, which contains a tetratricopeptide repeat domain acceptor site, whereas the AhR binds to a domain in the middle of hsp90. XAP2 was not found to be associated with the AhR-Arnt heterocomplex either in vitro or in nuclear extracts isolated from Hepa 1 cells treated with TCDD. Transient expression of XAP2 in COS-1 cells resulted in enhanced cytosolic AhR levels, suggesting a role for XAP2 in regulating the rate of AhR turnover.

Protein kinase C modulates aryl hydrocarbon receptor nuclear translocator protein-mediated transactivation potential in a dimer context.

Protein kinase C (PKC)- and protein kinase A (PKA)-mediated modulation of the transactivation potential of human aryl hydrocarbon receptor nuclear translocator (hARNT), a basic helix-loop-helix (bHLH)-PAS transcription factor, and the bHLH-ZIP transcription factors USF-1 (for upstream regulatory factor 1) and c-Myc were examined. An 81 nM dose of the PKC activator phorbol-12-myristate-13-acetate (PMA), shown here to specifically activate PKC in COS-1 cells, or a 1 nM dose of the PKA activator 8-bromoadenosine-3',5'-cyclic monophosphate (8-Br-cAMP) results in 2. 6- and 1.9-fold enhancements, respectively, in hARNT-mediated transactivation of the class B, E-box-driven reporter pMyc3E1bLuc relative to identically transfected, carrier solvent-treated COS-1 cells. In contrast, 81 nM PMA and 1 nM 8-Br-cAMP did not enhance transactivation of pMyc3E1bLuc-driven by USF-1 and c-Myc expression relative to identically transfected, carrier-treated COS-1 cells. Co-transfection of pcDNA3/ARNT-474-Flag, expressing a hARNT carboxyl-terminal transactivation domain deletion, and pMyc3E1bLuc does not result in induction of reporter activity, suggesting PMA's effects do not involve formation of unknown hARNT-protein heterodimers. Additionally, PMA had no effect on hARNT expression relative to Me2SO-treated cells. Metabolic 32P labeling of hARNT in cells treated with carrier solvent or 81 nM PMA demonstrates that PMA does not increase the overall phosphorylation level of hARNT. These results demonstrate, for the first time, that the transactivation potential of ARNT in a dimer context can be specifically modulated by PKC or PKA stimulation and that the bHLH-PAS and bHLH-ZIP transcription factors are differentially regulated by these pathways in COS-1 cells.

Nuclear receptor coactivator SRC-1 interacts with the Q-rich subdomain of the AhR and modulates its transactivation potential.

The aryl hydrocarbon receptor (AhR), a soluble cytosolic protein, mediates many of the toxic effects of TCDD and related chemicals. The toxic effects are largely cell, tissue, and promoter context dependent. Although many details of the overall dioxin signal transduction have been elucidated, the transcriptional regulation of dioxin-induced genes like cyp1A1 is not yet completely understood. Previously, we have shown that the co-regulator RIP140 is a potential AhR coactivator. In this report, the role of coactivator, SRC-1, in AhR-mediated transcriptional regulation was examined. SRC-1 increased AhR-mediated, TCDD-dependent reporter gene activity three-fold in Hepa-1 and COS-1 cells. In in vitro interaction assays, SRC-1 was found to interact with AhR but not with ARNT. SRC-1 interacted weakly with AhR in the absence of TCDD and the addition of ligand further increased SRC-1 binding to AhR. Deletional mapping studies of the AhR revealed that SRC-1 binds to the AhR transactivation domain. Finer mapping of the SRC-1-interacting subdomains in the AhR transactivation domain suggested that the Q-rich subdomain was necessary and sufficient for interaction, similar to that seen with RIP140. Using GFP-tagged constructs, SRC-1 was shown to interact with AhR in cells. Unlike RIP140, LXXLL motifs in SRC-1 were necessary for interaction with AhR in vitro and for coactivation in Hepa-1 cells. The recruitment of certain coactivators by a variety of receptors suggests possible common coactivator pools and competition among receptors for limiting coactivators. Examination of the role of SRC-1 in AhR/ARNT transactivation in ARNT-deficient mutant Hepa-1 c4 cells demonstrates that the AhR transactivation domain is sufficient for enhanced coactivation mediated by SRC-1 in the presence of a transactivation domain deleted ARNT protein.

AH receptor, ARNT, glucocorticoid receptor, EGF receptor, EGF, TGF alpha, TGF beta 1, TGF beta 2, and TGF beta 3 expression in human embryonic palate, and effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD).

Protein and mRNA for epidermal growth factor (EGF), transforming growth factor-alpha (TGF alpha), EGF receptor, transforming growth factor-beta 1 (TGF beta 1), TGF beta 2, TGF beta 3, glucocorticoid receptor (GR), the aryl hydrocarbon receptor (AhR), and the Ah receptor nuclear translocator (ARNT) were localized in gestational days (GD) 49-59 human embryonic secondary palates. The response to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was determined for expression of these genes following palatal organ culture. Craniofacial tissues were shipped in medium from the Human Embryology Laboratory, University of Washington, Seattle, WA. Half of each specimen was cultured in control medium and half in medium containing TCDD at either 1 x 10(-8) or 1 x 10(-10) M. After fixation and paraffin-embedding, sections were examined either immunohistochemically or by in situ hybridization. Expression patterns were determined for each gene for the major stages of palatogenesis and in response to TCDD and compared to previously determined patterns of expression in the same developmental stages of palatogenesis for the mouse (GD49-59 in human palatogenesis corresponds to GD12-16 in the mouse). Human and mouse palates were dissimilar in particular spatiotemporal patterns of expression of these genes. Relative to patterns in mouse palatal development, human tissues demonstrated expression of EGF at early palatal stages, expression of EGF receptor and TGF alpha throughout fusion events, and uniform expression of TGF beta 3 in all epithelial regions without specifically higher levels in the medial cells. The responses to TCDD also differed in patterns of gene expression as well as in concentration required to induce hyperplasia of the medial epithelium. In summary, human palates expressed all of these regulatory genes, responses to TCDD were detected, and comparison between mouse and human palates revealed interspecies variation that may be a factor in each species' response to TCDD, as well as other teratogenic exposures.

Protein kinase C activity is required for aryl hydrocarbon receptor pathway-mediated signal transduction.

The role of protein kinase C (PKC) in the human aryl hydrocarbon receptor (hAhR) signal transduction pathway was examined in cell lines stably transfected with pGUDLUC6.1, in which luc+ is solely controlled by four dioxin-responsive elements (DREs). These cell lines, P5A11 and HG40/6, were derived from HeLa and HepG2 cells respectively. Simultaneous treatment of these cells with 2,3,7,8, -tetrachlorodibenzo-p-dioxin (TCDD) and phorbol-12-myristate-13-acetate (PMA) enhanced trans-activation of the reporter construct several-fold relative to cells treated with TCDD alone. PKC inhibitors block the PMA effect and hAhR-mediated signal transduction, demonstrating these processes require PKC activity. Examination of other independently generated, HeLa-derived cell lines stably transfected with pGUDLUC6.1 demonstrates the PMA effect in P5A11 cells is not a clonal artifact. Transient transfections indicate the PMA effect is not due to a luciferase message/gene product stabilization mechanism or stimulation of the basal transcription machinery. Examination of cytosolic preparations demonstrates PKC stimulation or inhibition does not alter hAhR and hAhR nuclear translocator protein levels or TCDD-induced down-regulation of hAhR levels. Similarly, examination of nuclear extracts indicated PKC stimulation or inhibition does not alter nuclear AhR levels or hAhR/hAhR nuclear translocator protein heterodimer DRE-binding activity as assessed by electrophoretic mobility shift assay. These results demonstrate a PKC-mediated event is required for the hAhR to form a functional transcriptional complex that leads to trans-activation and that the DRE is the minimal DNA element required for PMA to enhance AhR-mediated trans-activation.

Hsp90-containing multiprotein complexes in the eukaryotic microbe Achlya.

In the oomycete fungus Achlya ambisexualis, hyphae of the male strain undergo sexual differentiation in the presence of the steroid hormone antheridiol. Earlier studies demonstrated that antheridiol binds with high affinity to a 9S multiprotein complex from A. ambisexualis cytosols. Although these complexes were found to contain the heat shock protein Hsp90, the other components were not known. It was of interest to determine if any of the other protein components in the Achlya Hsp90-heterocomplexes would be homologous to those found in the steroid receptor-Hsp90-heterocomplexes of vertebrates. Cytosolic proteins of 110 kDa, 74 kDa, 64 kDa, 61 kDa, 56 kDa, 47 kDa, 27 kDa and 23 kDa, were found in repeated trials, to co-immunoprecipitate with Achlya Hsp90. The 74 kDa protein was identified as the heat shock protein Hsp70, the 23 kDa protein was found to be related to the vertebrate protein p23 and the 56 kDa protein was found to be related to immunophilin FKBP51. All three of these proteins are components of the vertebrate receptor heterocomplexes. The 110 kDa, 61 kDa and 27 kDa proteins appeared to be unique to the Achlya complexes. Unlike the seven other proteins co-immunoprecipitating with Hsp90, the 61 kDa protein was observed only in the co-immunoprecipitates produced from in vitro translates of RNA isolated from antheridiol-treated mycelia.

Hepatitis B virus X-associated protein 2 is a subunit of the unliganded aryl hydrocarbon receptor core complex and exhibits transcriptional enhancer activity.

Prior to ligand activation, the unactivated aryl hydrocarbon receptor (AhR) exists in a heterotetrameric 9S core complex consisting of the AhR ligand-binding subunit, a dimer of hsp90, and an unknown subunit. Here we report the purification of an approximately 38-kDa protein (p38) from COS-1 cell cytosol that is a member of this complex by coprecipitation with a FLAG-tagged AhR. Internal amino acid sequence information was obtained, and p38 was identified as the hepatitis B virus X-associated protein 2 (XAP2). The simian ortholog of XAP2 was cloned from a COS-1 cDNA library; it codes for a 330-amino-acid protein containing regions of homology to the immunophilins FKBP12 and FKBP52. A tetratricopeptide repeat (TPR) domain in the carboxy-terminal region of XAP2 was similar to the third and fourth TPR domains of human FKBP52 and the Saccharomyces cerevisiae transcriptional modulator SSN6, respectively. Polyclonal antibodies raised against XAP2 recognized p38 in the unliganded AhR complex in COS-1 and Hepa 1c1c7 cells. It was ubiquitously expressed in murine tissues at the protein and mRNA levels. It was not required for the assembly of an AhR-hsp90 complex in vitro. Additionally, XAP2 did not directly associate with hsp90 upon in vitro translation, but was present in a 9S form when cotranslated in vitro with murine AhR. XAP2 enhanced the ability of endogenous murine and human AhR complexes to activate a dioxin-responsive element-luciferase reporter twofold, following transient expression of XAP2 in Hepa 1c1c7 and HeLa cells.

The Ah receptor is a sensitive target of geldanamycin-induced protein turnover.

Geldanamycin (GA) binds directly to hsp90 and apparently disrupts certain hsp90 heterocomplexes. We have investigated the GA-hsp90 interaction and its effect on other associated proteins. Incubation of 2-[125I]-iodo-3-azido-7,8-dibromo-p-dioxin-labeled Hepa 1c1c7 cytosol with GA-coupled beads revealed a stable association of Ah receptor (AhR)/hsp90 complex with GA. In addition, sucrose gradient sedimentation analysis demonstrated that GA does not disrupt the 9S Ah receptor complex in vitro. HeLa and Hepa 1c1c7 cells were subjected to a dose-response and time-course treatment with GA and the level of the AhR was determined. A 75% depletion in AhR levels was observed within an hour of exposure to 100 nM GA. The relative stability of other proteins that associate with hsp90 was determined with the following rank order of sensitivity to GA exposure: AhR >> c-Raf-1 > glucocorticoid receptor > CDK4 >> p50. A series of hsp90 deletion mutants were used to map the domain that interacts with GA. Deletion of the first 221 amino acids in NH2-terminal domain resulted in loss of binding to solid-phase GA. Epitopes of monoclonal antibodies specific for hsp90 were also determined by direct immunoprecipitation with hsp90 mutants. Results indicated that monoclonal antibodies 8D3 and 3G3 interact with hsp90 via the first 221 amino acids in NH2-terminal region, whereas AC88 requires a COOH-terminal region between amino acids 661-677.

Ah receptor nuclear translocator protein heterogeneity is altered after heterodimerization with the Ah receptor.

The Ah receptor (AhR) and the Ah receptor nuclear translocator (ARNT) are capable of forming a transcriptionally active heterodimeric complex. The biochemical events that are required for dimerization and transactivation are not fully understood. The purpose of this study was to determine whether covalent modifications of ARNT occur between ARNT existing in the monomeric form and after heterodimerization with the AhR and subsequent binding to DNA. Mouse hepatoma cell line 1c1c7 (Hepa 1) cytosol and ARNT immunoprecipitations were subjected to two-dimensional gel electrophoresis. ARNT was visualized with two antibodies, with distinct epitope specificity, and each detected a considerable level of charge heterogeneity. The pI range observed was 5.7-6.4, with the predominant form at a pI of 6.2. The AhR/ARNT heterodimer was immunoprecipitated from high-salt nuclear extract obtained from Hepa 1 cells treated with beta-naphthoflavone using an anti-AhR polyclonal antibody. This immunoprecipitate was subjected to two-dimensional gel electrophoresis, and coimmunoprecipitated ARNT was visualized. The results indicated that ARNT complexed with the AhR in the nucleus has an isoform pattern shifted toward the basic end, with the predominant isoform having a pI of 6.8. Thus, a significant shift in pI occurs during the dimerization and/or after binding to DNA. In vitro transformation of the AhR with 2,3,7,8-tetrachlorodibenzo-p-dioxin in cytosol leads to heterodimerization with ARNT. Two-dimensional gel electrophoresis of ARNT coimmunoprecipitated with the AhR revealed the same isoform pattern as seen in cytosol. This would indicate that each isoform of ARNT is capable of heterodimerizing with the AhRin vitro. ARNT is a phosphoprotein, and the more acidic isoforms appear to have a higher level of phosphorylation.

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.