Subcellular Localization of the Aryl Hydrocarbon Receptor Is Modulated by the Immunophilin Homolog Hepatitis B Virus X-associated Protein 2

The dioxin receptor is a ligand-dependent transcription factor that mediates the biological effects of dioxin and related environmental pollutants. In the absence of ligand the receptor is present in the cytoplasmic compartment of the cell associated with the hsp90-dependent chaperone complex. This complex regulates several functions of the receptor such as ligand binding and nuclear import. Furthermore, intracellular localization of the receptor is modulated by multiple factors such as the export protein CRM-1 and the hsp90-associated immunophilin XAP-2. We have identified the mechanism of XAP-2-induced cytoplasmic localization of the receptor and studied the potential cross-talk between CRM-1 and XAP-2. We show that XAP-2 anchors the ligand-free receptor to cytoskeletal structures. This effect is blocked upon treatment with the actin inhibitor cytochalasin B, whereas the tubulin inhibitor colchicine had no effect on receptor localization. In addition, we show that the receptor interacts with CRM-1 both in the presence and absence of ligand. CRM-1-mediated nuclear export occurs independently of XAP-2. Our data provide evidence that CRM-1 and XAP-2 act in parallel through different mechanisms and target different interfaces of the receptor. These results suggest that two pathways cooperate to localize the non-activated receptor in the cytoplasmic compartment of the cell.In mammalian cells, multiple systems such as ligand binding, dimerization, and post-translational modifications have evolved to regulate the function of transcription factors. Recently, intracellular compartmentalization has emerged as a major pathway to modulate transcription factor activity. For instance, the dioxin receptor is a ligand-dependent transcription factor which, in the absence of ligand, is found in the cytoplasmic compartment of the cell or, depending on cell type, evenly distributed between the cytoplasm and the nucleus (1, 2). This non-activated form of the receptor interacts with the hsp90 1 -dependent molecular chaperone complex (3) and its associated proteins, such as the hsp90-associated factor p23 (4, 5) and the dioxin receptor specific immunophilin XAP-2 (6 -8).The interaction between the dioxin receptor and hsp90 is mediated by two distinct structural determinants, the DNA binding bHLH domain (9), and the ligand binding region within the PAS-B subdomain of the receptor (10). In the presence of ligand the receptor accumulates in the nucleus (1, 11) where it interacts with the general dimerization partner factor ARNT (12). This event induces release of the hsp90 complex (4, 13). Nuclear accumulation of the receptor is regulated by a nuclear localization signal present in the bHLH domain (14) and by the hsp90 complex which regulates the interaction between the import machinery and the activated form of the receptor (1). In addition, we have recently shown that the non-activated form of the receptor is shuttling between the nucleus and the cytoplasm (2). In this respect, nuclear export of the dioxin receptor is likely to be me...

Section: Discussionmentioning

confidence: 99%

Two Parallel Pathways Mediate Cytoplasmic Localization of the Dioxin (Aryl Hydrocarbon) Receptor

Berg

Pongratz

2002

“…XAP2 associates with the hepatitis B-virus protein X, the AhR (28,47), and the Epstein-Barr virus nuclear protein (EBNA-3) (48), which is a nuclear antigen that is necessary for B-cell transformation. Interestingly, XAP2 enhances AhR levels (35,46) and plays a role in the cytoplasmic localization of the dioxin receptor (49). XAP2, also known as ARA-9 and AIP, is highly expressed in spleen and thymus and minimally expressed in liver, kidney, and lung (28,50).…”

Section: Fkbp52 P50mentioning

confidence: 99%

Evidence That Peroxisome Proliferator-activated Receptor α Is Complexed with the 90-kDa Heat Shock Protein and the Hepatitis Virus B X-associated Protein 2

Sumanasekera

Tien²,

Turpey³

et al. 2003

Self Cite

101

The peroxisome proliferator-activated receptor ␣ (PPAR␣) is a ligand-inducible transcription factor, which belongs to the nuclear receptor superfamily. PPAR␣ mediates the carcinogenic effects of peroxisome proliferators in rodents. In humans, PPAR␣ plays a fundamental role in regulating energy homeostasis via control of lipid metabolism. To study the possible role of chaperone proteins in the regulation of PPAR␣ activity, a monoclonal antibody (mAb) was made against PPAR␣ and designated as 3B6/PPAR. The specificity of mAb 3B6/PPAR in recognizing PPAR␣ was tested in immunoprecipitations using in vitro translated PPAR subtypes. The mAb 3B6/PPAR recognized PPAR␣, failed to bind to PPAR␤ or PPAR␥, and is efficient in both immunoprecipitating and visualizing the receptor on protein blots. The immunoprecipitation of PPAR␣ in mouse liver cytosol using mAb 3B6/PPAR has resulted in the detection of two co-immunoprecipitated proteins, which are heat shock protein 90 (hsp90) and the hepatitis B virus X-associated protein 2 (XAP2). The concomitant depletion of PPAR␣ in hsp90-depleted mouse liver cytosol was also detected. Complex formation between XAP2 and PPAR␣/FLAG was also demonstrated in an in vitro translation binding assay. hsp90 interacts with PPAR␣ in a mammalian two-hybrid assay and binds to the E/F domain. Transient expression of XAP2 co-expressed with PPAR␣ resulted in down-regulation of a peroxisome proliferator response element-driven reporter gene activity. Taken together, these results indicate that PPAR␣ is in a complex with hsp90 and XAP2, and XAP2 appears to function as a repressor. This is the first demonstration that PPAR␣ is stably associated with other proteins in tissue extracts and the first nuclear receptor shown to functionally interact with XAP2.The ability of peroxisome proliferators to activate a receptor in the steroid receptor superfamily was first discovered in 1990, and the cognate protein was designated as PPAR 1 (1). ThePPARs are soluble transcription factors that are activated by a diverse class of lipophilic compounds (2). With the activation of PPAR, a concomitant induction of a number of genes that code for peroxisomal fatty acid metabolizing enzymes was observed in mouse liver. Among these, the peroxisomal enzyme AOx is the most broadly used indicator of peroxisome proliferator action. Transcription of the AOx gene is increased by exposure to the hypolipidemic peroxisome proliferator WY-14,643, and this effect is mediated by a PPRE located 570 base pairs upstream of the transcriptional start site (3). This PPRE contains a direct repeat of the sequence motifs TGACCT and TGTCCT, which is separated by a single nucleotide. A heterodimer of PPAR and RXR binds to PPREs located in upstream regulatory regions of various target genes and the RXR ligand, 9-cis-retinoic acid, increases PPAR/RXR transcriptional activity (4). There are three PPAR subtypes, designated as PPAR␣ (NR1C1), -␤ (NR1C2), and -␥ (NR1C3); and each subtype is capable of binding to DNA after heterodimerizing with RXR (NR2B1) ...

“…In contrast, FKBP52 was unable to modulate AhR levels (13). Interestingly, transient expression of XAP2 also leads to sequestration of the mAhR in the cytoplasm (12,14,15). Regulation of intracellular movement of the AhR both in the presence and absence of ligand is a potentially important aspect of Ah receptor function that warrants further investigation.…”

mentioning

confidence: 99%

The hsp90 Co-chaperone XAP2 Alters Importin β Recognition of the Bipartite Nuclear Localization Signal of the Ah Receptor and Represses Transcriptional Activity

Petrulis

Kusnadi

Ramadoss

et al. 2003

Self Cite

The mouse aryl hydrocarbon receptor (mAhR) is a ligand-activated transcription factor that exists in a tetrameric, core complex with a dimer of the 90-kDa heat shock protein, and the hepatitis B virus X-associated protein 2 (XAP2). Transiently expressed mAhR-YFP (yellow fluorescent protein fused with the mAhR) localizes throughout cells, with a majority occupying nuclei. Coexpression of XAP2 with mAhR-YFP results in a distinct redistribution to the cytoplasm. We have utilized several approaches to attempt to identify the mechanism by which XAP2 modulates the sub-cellular localization of the mAhR. The nuclear export inhibitor, leptomycin B, was used to demonstrate that XAP2 inhibits ligand-independent nucleocytoplasmic shuttling of the receptor. Results from cytoskeletal disruption and the addition of an alternate nuclear localization sequence (NLS) to mAhR-YFP suggest that XAP2 does not physically tether the complex in the cytoplasm. The use of a rabbit polyclonal antibody raised against a portion of the bipartite NLS of the mAhR revealed that XAP2 does not appear to block access to the NLS. However, XAP2 hinders importin ␤ binding to the mAhR complex, suggesting that XAP2 alters the conformation of the bipartite NLS of mAhR. XAP2 also represses the transactivation potential of the AhR, in contrast to previously published reports, perhaps by stabilizing the receptor complex and/or blocking nucleocytoplasmic shuttling of the AhR complex.