ARA9 Modifies Agonist Signaling through an Increase in Cytosolic Aryl Hydrocarbon Receptor
2000; Elsevier BV; Volume: 275; Issue: 9 Linguagem: Inglês
10.1074/jbc.275.9.6153
ISSN1083-351X
AutoresJohn J. LaPres, Edward Glover, Elizabeth E. Dunham, Maureen K. Bunger, Christopher A. Bradfield,
Tópico(s)Mass Spectrometry Techniques and Applications
ResumoThe aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that mediates the effects of agonists like 2,3,7,8-tetrachlorodibenzo-p-dioxin. In the current model for AHR signaling, the unliganded receptor is found in the cytosol as part of a complex with a dimer of the 90-kDa heat shock protein and an immunophilin-like molecule, ARA9. In yeast, expression of ARA9 results in an increase in the maximal agonist response and a leftward shift in the AHR dose-response curve. To better understand the mechanism by which ARA9 modifies AHR signal transduction, we performed a series of coexpression experiments in yeast and mammalian cells. Our results demonstrate that ARA9's influence on AHR signaling is not due to inhibition of a membrane pump or modification of the receptor's transactivation properties. Using receptor photoaffinity labeling experiments, we were able to show that ARA9 enhances AHR signal transduction by increasing the available AHR binding sites within the cytosolic compartment of the cell. Our evidence suggests that ARA9's effects are related to its role as a cellular chaperone;i.e. we observed that expression of ARA9 increases the fraction of AHR in the cytosol and also stabilized the receptor under heat stress. The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that mediates the effects of agonists like 2,3,7,8-tetrachlorodibenzo-p-dioxin. In the current model for AHR signaling, the unliganded receptor is found in the cytosol as part of a complex with a dimer of the 90-kDa heat shock protein and an immunophilin-like molecule, ARA9. In yeast, expression of ARA9 results in an increase in the maximal agonist response and a leftward shift in the AHR dose-response curve. To better understand the mechanism by which ARA9 modifies AHR signal transduction, we performed a series of coexpression experiments in yeast and mammalian cells. Our results demonstrate that ARA9's influence on AHR signaling is not due to inhibition of a membrane pump or modification of the receptor's transactivation properties. Using receptor photoaffinity labeling experiments, we were able to show that ARA9 enhances AHR signal transduction by increasing the available AHR binding sites within the cytosolic compartment of the cell. Our evidence suggests that ARA9's effects are related to its role as a cellular chaperone;i.e. we observed that expression of ARA9 increases the fraction of AHR in the cytosol and also stabilized the receptor under heat stress. aryl hydrocarbon receptor FK506-binding protein heat shock protein of 90 kDa upstream activating sequence polyacrylamide gel electrophoresis phosphate-buffered saline β-napthaflavone dexamethasone transactivation domain glucocorticoid receptor tetratricopeptide repeat synthetic drop-out 4-morpholinepropanesulfonic acid The AHR1 is a ligand-activated transcription factor that mediates the biological effects of halogenated dioxins and related compounds (1.Schmidt J.V. Bradfield C.A. Annu. Rev. Cell Dev. Biol. 1996; 12: 55-89Crossref PubMed Scopus (796) Google Scholar). In a widely held model of dioxin signal transduction, the AHR is found in the cytosol, in a complex with Hsp90 and a newly discovered protein known as ARA9 (2.Dolwick K.M. Swanson H.I. Bradfield C.A. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8566-8570Crossref PubMed Scopus (206) Google Scholar, 3.Whitelaw M.L. Gottlicher M. Gustafsson J.A. Poellinger L. EMBO J. 1993; 12: 4169-4179Crossref PubMed Scopus (124) Google Scholar, 4.Fukunaga B.N. Probst M.R. Reisz-Porszasz S. Hankinson O. J. Biol. Chem. 1995; 270: 29270-29278Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar). 2ARA9 was identified and named by three separate groups. It is also known as XAP2, for hepatitis B virus X-activating protein 2, and AIP, for AHR-interacting protein. 2ARA9 was identified and named by three separate groups. It is also known as XAP2, for hepatitis B virus X-activating protein 2, and AIP, for AHR-interacting protein. In the presence of agonist, the AHR translocates to the nucleus, where it binds to its nuclear partner, ARNT. This AHR·ARNT heterodimer is capable of binding DNA and promotes the transcription of a battery of responsive genes including those encoding a number of xenobiotic-metabolizing enzymes (5.Whitlock Jr., J.P. Chichester C.H. Bedgood R.M. Okino S.T. Ko H.P. Ma Q. Dong L. Li H. Clarke-Katzenberg R. Drug Metab. Rev. 1997; 29: 1107-1127Crossref PubMed Scopus (65) Google Scholar). Hsp90 has been shown to play a role in maintaining AHR in a conformation that can bind ligand with high affinity (6.Pongratz I. Mason G.G. Poellinger L. J. Biol. Chem. 1992; 267: 13728-13734Abstract Full Text PDF PubMed Google Scholar, 7.Carver L.A. Jackiw V. Bradfield C.A. J. Biol. Chem. 1994; 269: 30109-30112Abstract Full Text PDF PubMed Google Scholar, 8.Perdew G.H. J. Biol. Chem. 1988; 263: 13802-13805Abstract Full Text PDF PubMed Google Scholar). Although ARA9 has been shown to increase AHR function in yeast and mammalian cells, its role in AHR signaling is not understood (7.Carver L.A. Jackiw V. Bradfield C.A. J. Biol. Chem. 1994; 269: 30109-30112Abstract Full Text PDF PubMed Google Scholar, 8.Perdew G.H. J. Biol. Chem. 1988; 263: 13802-13805Abstract Full Text PDF PubMed Google Scholar, 9.Carver L.A. Bradfield C.A. J. Biol. Chem. 1997; 272: 11452-11456Abstract Full Text Full Text PDF PubMed Scopus (346) Google Scholar, 10.Kuzhandaivelu N. Cong Y.-S. Inouye C. Yang W.-M. Seto E. Nucleic Acids Res. 1996; 24: 4741-4750Crossref PubMed Scopus (94) Google Scholar).ARA9 was initially identified in yeast two-hybrid screens, in which the AHR or the hepatitis B virus protein X were used as bait (9.Carver L.A. Bradfield C.A. J. Biol. Chem. 1997; 272: 11452-11456Abstract Full Text Full Text PDF PubMed Scopus (346) Google Scholar, 10.Kuzhandaivelu N. Cong Y.-S. Inouye C. Yang W.-M. Seto E. Nucleic Acids Res. 1996; 24: 4741-4750Crossref PubMed Scopus (94) Google Scholar, 11.Ma Q. Whitlock Jr., J.P. J. Biol. Chem. 1997; 272: 8878-8884Abstract Full Text Full Text PDF PubMed Scopus (361) Google Scholar). Later, it was purified from monkey cells in a complex with the AHR (12.Meyer B.K. Pray-Grant M.G. Vanden Heuvel J.P. Perdew G.H. Mol. Cell. Biol. 1998; 18: 978-988Crossref PubMed Scopus (305) Google Scholar). ARA9 contains two notable structural motifs. In its amino terminus, ARA9 contains an FKBP homology domain. This domain shares 28% amino acid sequence identity to FKBP12 (9.Carver L.A. Bradfield C.A. J. Biol. Chem. 1997; 272: 11452-11456Abstract Full Text Full Text PDF PubMed Scopus (346) Google Scholar, 11.Ma Q. Whitlock Jr., J.P. J. Biol. Chem. 1997; 272: 8878-8884Abstract Full Text Full Text PDF PubMed Scopus (361) Google Scholar, 12.Meyer B.K. Pray-Grant M.G. Vanden Heuvel J.P. Perdew G.H. Mol. Cell. Biol. 1998; 18: 978-988Crossref PubMed Scopus (305) Google Scholar). However, ARA9 is unable to bind FK506 and does not appear to have peptidyl prolyl isomerase activity 3L. A. Carver and C. A. Bradfield, unpublished results. 3L. A. Carver and C. A. Bradfield, unpublished results. (13.Carver L.A. LaPres J.J. Jain S. Dunham E.E. Bradfield C.A. J. Biol. Chem. 1998; 273: 33580-33587Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar). In its carboxyl terminus, ARA9 contains three TPRs. TPRs are defined by strings of 34 amino acids that have been shown to play roles in protein-protein interaction (14.Lamb J.R. Tugendreich S. Hieter P. Trends Biochem. Sci. 1995; 20: 257-259Abstract Full Text PDF PubMed Scopus (547) Google Scholar). This domain structure is similar to that found in the GR-associated immunophilin, FKBP52, which contains two FKBP domains in its amino terminus and three TPRs in its carboxyl terminus (15.Tai P.K. Albers M.W. Chang H. Faber L.E. Schreiber S.L. Science. 1992; 256: 1315-1318Crossref PubMed Scopus (267) Google Scholar, 16.Renoir J.M. Radanyi C. Faber L.E. Baulieu E.E. J. Biol. Chem. 1990; 265: 10740-10745Abstract Full Text PDF PubMed Google Scholar). In addition to their structural similarities, ARA9 and FKBP52 are found associated to the cytosolic complexes of AHR and the GR or progesterone receptor, respectively (9.Carver L.A. Bradfield C.A. J. Biol. Chem. 1997; 272: 11452-11456Abstract Full Text Full Text PDF PubMed Scopus (346) Google Scholar, 11.Ma Q. Whitlock Jr., J.P. J. Biol. Chem. 1997; 272: 8878-8884Abstract Full Text Full Text PDF PubMed Scopus (361) Google Scholar, 12.Meyer B.K. Pray-Grant M.G. Vanden Heuvel J.P. Perdew G.H. Mol. Cell. Biol. 1998; 18: 978-988Crossref PubMed Scopus (305) Google Scholar, 17.Smith R.H. Zhao Y. O'Callaghan D.J. Virology. 1994; 202: 760-770Crossref PubMed Scopus (49) Google Scholar, 18.Pratt W.B. J. Biol. Chem. 1993; 268: 21455-21458Abstract Full Text PDF PubMed Google Scholar).In both mammalian cells and yeast, the presence of ARA9 enhances AHR signaling (11.Ma Q. Whitlock Jr., J.P. J. Biol. Chem. 1997; 272: 8878-8884Abstract Full Text Full Text PDF PubMed Scopus (361) Google Scholar, 12.Meyer B.K. Pray-Grant M.G. Vanden Heuvel J.P. Perdew G.H. Mol. Cell. Biol. 1998; 18: 978-988Crossref PubMed Scopus (305) Google Scholar, 13.Carver L.A. LaPres J.J. Jain S. Dunham E.E. Bradfield C.A. J. Biol. Chem. 1998; 273: 33580-33587Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar). Although ARA9 is capable of directly interacting with the AHR·Hsp90 complex, the mechanism by which it influences receptor signaling is unknown. Our experiments in yeast have shown that heterologous expression of ARA9 increases the maximum response and shifts the dose response of the AHR agonist, βNF, to the left (13.Carver L.A. LaPres J.J. Jain S. Dunham E.E. Bradfield C.A. J. Biol. Chem. 1998; 273: 33580-33587Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar). In our laboratory, dose-response curves are typically performed using a LexA or GAL4 DNA binding domain fused to the AHR. The chimeras have been shown to have similar pharmacology to that observed for the full-length AHR·ARNT system3 (7.Carver L.A. Jackiw V. Bradfield C.A. J. Biol. Chem. 1994; 269: 30109-30112Abstract Full Text PDF PubMed Google Scholar). Given that such chimeras homodimerize to drive transcription, they also do not require ARNT for signal transduction. This simplifies protocols and allows us to focus on factors that influence the AHR without considering effects on ARNT. In this report, we employ a Gal4-AHRNΔ166 chimera that allowed us to assess the functional consequences of ARA9 expression on AHR signaling in mammalian cells.RESULTSGiven the role that the transporter, Pdr5p, has been shown to play in GR signaling and given the similarities between the AHR and GR signaling pathways, we first set out to determine if Pdr5p plays a role in our yeast AHR signaling system. L40 yeast were transformed with pBTMNΔ166AHR in the presence of pYXARA9 or the corresponding vector control. The resulting plates were replicated onto SD media with or without 10 nm βNF and/or 10 μm FK506. Assessment of β-galactosidase activity indicated that ligand-independent signaling of AHR was stimulated 10-fold in the presence of ARA9 (Fig. 1). The addition of βNF led to an additional 9-fold increase above controls (total 90-fold increase; Fig. 1). Treatment with FK506 had no effect on reporter activity in the absence of βNF and slightly inhibited the response to βNF when ARA9 was present. This inhibition was not significant at p ≤ 0.05. As a positive control for FK506 activity as an inhibitor of Pdr5p, the L40 strain was transformed with the GR expression plasmid pY2NLxC along with the FKBP52 expression plasmid pYXFKP52 or the control, pYX242. These experiments were also carried out in the presence or absence of the GR ligand, DEX. We observed that the DEX-induced GR signaling was dependent on the presence of FK506. In the presence of FK506, GR ligand inducibility was 4-fold, and this increase was independent of FKBP52 (Fig. 1).To analyze the role of ARA9 in AHR signaling in mammalian cells, COS-1 cells were cotransfected with the GAL4-NΔ166 AHR chimera (pSGAHRNΔ166), an ARA9 expression vector (pTarget-ARA9), or control plasmid. The transfected cells were then treated with various concentrations of βNF. We observed that the overexpression of ARA9 increased the βNF-driven luciferase expression greater than 4-fold at every βNF concentration tested (Fig.2). Ligand-independent activation of pSGAHRNΔ166 was also increased approximately 4-fold in the presence of ARA9.Figure 2COS-1 cell transfection with pSGAHRNΔ166. Top, schematic diagram of GAL4-AHRNΔ166 chimera (pSGAHRNΔ166), ARA9 expression plasmid (pTarget-ARA9), and GAL4-UAS reporter (pG5luc).Bottom, COS-1 cells were transfected with the GAL4-AHRNΔ166 chimera in the presence of ARA9 (open circles) or an empty expression vector (solid circles). The cells were treated with the indicated amount of βNF for 16 h and harvested by cell lysis. The resulting lysates were assayed for luciferase and β-galactosidase activity. Each value is normalized to its β-galactosidase value to control for transfection efficiency, and all values were normalized within the experiment by setting the control sample in the absence of ligand to a value of 1. Error bars represent the S.E. from triplicate determinants.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The ability of ARA9 to influence transactivation was assessed by cotransfecting various GAL4-TAD chimeras (pSGAHRΔ409, pSGARNT, pSG424, and pSGVP16TAD) in the presence and absence of pTarget-ARA9. Transfections were normalized to β-galactosidase expression, and the ratio of transactivation activity in the presence of ARA9 to vector control was determined. ARA9 was capable of increasing the activity of pSGAHRNΔ166 greater than 4-fold. ARA9 did not significantly influence the activity of any other construct including the AHR TAD construct, pSGAHRNΔ409 (Fig. 3).Figure 3Cotransfection of ARA9 and various TAD constructs. Left, schematic diagrams of GAL4-TAD constructs used in the cotransfections. Right, various GAL4-TAD constructs were cotransfected with ARA9 or an empty expression vector into COS-1 cells. Cells were incubated for 16 h and assayed for luciferase and β-galactosidase activity. Each sample was normalized to β-galactosidase and then expressed as value of activity in the presence of ARA9 over the activity in the presence of empty vector. NΔ166AHR, pSGAHRNΔ166; ARNT, pSGARNT;VP16, pSG-VP16 TAD; GAL, pSG424;NΔ409AHR, pSG AHR NΔ409, which contains the TAD of AHR.Error bars represent the S.E. from triplicate determinants. bHLH, basic helix-loop-helix.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The specificity of the effect of ARA9 on AHR was tested by cotransfection with the ARA9 paralogue, FKBP52. We cotransfected pSGAHRNΔ166 with pTarget-ARA9, pTarget-FKBP52, or the vector control. As shown in Fig. 4 A, we observed that FKBP52 was unable to influence pSGAHRNΔ166 signaling in the presence or absence of βNF. In contrast, ARA9 increased activity in both a ligand-dependent and -independent fashion. Expression of ARA9 and FKBP52 in transfected cells was confirmed by Western blot analysis (Fig. 4 B).Figure 4Cotransfection of pSGAHRNΔ166 and ARA9 or FKBP52. A (top), schematic representation of constructs used in transfection. A (bottom), COS-1 cells were transfected with pSGAHRNΔ166 (the GAL4-AHRNΔ166 chimera) in the presence of an empty expression vector (Ctrl) or ARA9 or FKBP52 expression vectors. Cells were incubated in the absence (hatched bars) or presence of 3.0 × 10−8m βNF (solid bars). Cells were assayed for luciferase and β-galactosidase activity. Each value is normalized to its β-galactosidase value to control for transfection efficiency, and all values were normalized within the experiment by setting the control sample in the absence of ligand to a value of 1. Error bars represent the S.E. from triplicate determinants.B, Western blot analysis was performed on cytosolic extracts from identical transfections described in A. Extracts were prepared as described under “Materials and Methods” and separated on SDS-polyacrylamide gel. Following transfer to nitrocellulose, the blots were probed with antibodies specific for ARA9 (lanes 1 and 2) or FKBP52 (lanes 3and 4). Cells transfected with empty vector were used as control (lanes 1 and 4). Hu ARA9 is the transfected human ARA9, COS-1 ARA9 is endogenous ARA9, and Rb FKBP52 is transfected rabbit FKBP52.View Large Image Figure ViewerDownload Hi-res image Download (PPT)One possible explanation for the increase in maximal response and leftward shift in the dose-response curve (Fig. 1 and Ref. 13.Carver L.A. LaPres J.J. Jain S. Dunham E.E. Bradfield C.A. J. Biol. Chem. 1998; 273: 33580-33587Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar) is that ARA9 is increasing available AHR in these cells. To test this hypothesis, we transfected pSGAHRNΔ166 in the presence and absence of pTarget-ARA9 in COS-1 cells. Soluble protein fractions were analyzed for their ability to bind the AHR photoaffinity ligand, 2-azido-3-[125I]iodo-7,8-dibromodibenzo-p-dioxin. Saturation binding isotherms indicate that the presence of ARA9 increased the number of ligand binding sites by 2.4-fold (Fig.5, A and B). Scatchard analysis of these data confirms the increase in binding sites (B max = 2.6 × 10−11 ± 0.3 × 10−11m for control and 6.5 × 10−11 ± 0.2 × 10−11min the presence of ARA9), with no significant change inK D (K D = 1.8 × 10−10 ± 1.1 × 10−10 for control and 1.5 × 10−10 ± 0.4 × 10−10 in the presence of ARA9) (Fig. 5 C).Figure 5Photoaffinity labeling of the GAL4-AHRNΔ166 chimera. A(top), schematic diagram of GAL4-AHRNΔ166 chimera used in labeling. A (bottom), raw data. COS-1 cells were transfected with pSGAHRNΔ166 in the presence (+ARA9) or absence (Ctrl) of an ARA9 expression vector. Cytosols were prepared as described under “Materials and Methods.” Cytosols were prepared, and photoaffinity labeling was performed in the presence of increasing concentrations of 2-azido-3-[125I]iodo-7,8-dibromodibenzo-p-dioxin. Proteins were separated by SDS-PAGE and visualized by autoradiography.B, saturation binding isotherm. Bands corresponding to labeled AHR were excised, counted (Bound (DPM)) and plotted as a function of total ligand (Probe (M)).Squares, control; circles, with ARA9.C, Scatchard analysis. Scatchard analysis was performed on the data from the saturation binding curves as described under “Materials and Methods” (control: K D = 1.8 × 10−10m and B max = 2.6 × 10−11; with ARA9: K D = 1.5 × 10−10 and B max = 6.5 × 10−11). Squares, control;circles, with ARA9.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To extend these results to the full-length AHR, COS-1 cells were cotransfected with a full-length AHR construct (pSportAHR), and pTarget-ARA9 or an empty expression vector. Soluble protein fractions were prepared from the cells, and the AHR was identified by photoaffinity labeling or by Western blot analysis. Using a saturating concentration of photoaffinity ligand, we observed that the presence of ARA9 increased the amount of full-length AHR labeling approximately 2-fold (Fig. 6, p ≤ 0.05). To determine if ARA9 was increasing the amount of AHR in these soluble extracts, total receptor was estimated by Western blot analysis. Given the qualitative nature of Western blot analysis, multiple independent experiments were performed, and representative results are shown in Fig. 7. These blots show a change in total AHR protein that is consistent with a 2-fold increase. (Fig. 7, A and B). This experiment has been repeated under a variety of conditions with similar results.Figure 6Photoaffinity labeling of full length AHR. Top, schematic diagram of full-length AHR construct (FL-AHR) used in labeling. Bottom, full-length AHR construct was cotransfected with ARA9 (+ARA9) or an empty expression vector (Ctrl). Cytosols were prepared and labeled with 1 nm2-azido-3-[125I]iodo-7,8-dibromodibenzo-p-dioxin. Proteins were separated by SDS-PAGE and visualized by autoradiography. Bands corresponding to labeled AHR were excised, counted, and plotted (*, p ≤ 0.05). Error barsrepresent the S.D. from triplicate determinants. bHLH, basic helix-loop-helix.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 7Western blot analysis of transfected AHRNΔ166 and full-length AHR in the presence or absence of ARA9. Top, schematic diagram of GAL4-AHRNΔ166 chimera and full-length AHR constructs used in transfection. Bottom, A, pSGAHRNΔ166 was transfected in the presence of ARA9 (+ARA9) or control vector (Ctrl). Twenty-five micrograms of cytosol was analyzed by Western blot using an antibody specific for the AHR.Bottom, B, full-length AHR was transfected in the presence of ARA9 (+ARA9) or control vector (Ctrl). Twenty-five micrograms of cytosol was analyzed by Western blot using an antibody specific for the AHR. bHLH, basic helix-loop-helix.View Large Image Figure ViewerDownload Hi-res image Download (PPT)One possible explanation for the observed increase in total and functional AHR in our soluble fractions was that ARA9 is capable of altering the cellular localization of the AHR in the cell. To test this idea, subcellular localization of the AHR was determined by immunohistochemistry in COS-1 cells that were transfected with pSportAHR in the presence or absence of ARA9 (28.Jain S. Bradfield C.A. Mech. Dev. 1998; 73: 117-123Crossref PubMed Scopus (290) Google Scholar). As seen in Fig.8 A, in COS cells, the transiently expressed AHR is found predominantly in the nuclear compartment, with only limited cytoplasmic staining. This expression pattern is shifted to almost completely nuclear in the presence of ligand, demonstrating that the cytosolic fraction of the AHR is functional (Fig. 8 A). In the presence of cotransfected ARA9, transiently expressed AHR is almost completely cytosolic. To demonstrate that this receptor is functional, we showed that the addition of agonist leads to translocation of the AHR to the nuclear compartment when ARA9 is expressed.Figure 8Immunocytochemistry of AHR and heat denaturation of GAL4-AHRNΔ166 chimera. A, COS-1 cells were transfected in the presence or absence of ARA9 and stained with antibodies specific for the AHR as described in the methods. B, pSGAHRNΔ166 was transfected in the presence of ARA9 (circles, solid line) or control vector (squares, dashed line). Soluble protein fractions were prepared and subjected to heat stress at 45 °C for various times. Receptor integrity was analyzed by binding assays with 1 nm2-azido-3-[125I]iodo-7,8-dibromodibenzo-p-dioxin. Samples were counted and plotted as a described under “Materials and Methods.” Error bars represent the S.E. from three separate experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT)A second explanation for the increased functional AHR was that ARA9 stabilized the protein against degradation. To test this hypothesis, we transfected pSGAHRNΔ166 in the presence and absence of ARA9 in COS-1 cells. Soluble protein fractions were prepared and heat-stressed at 45 °C for various times. Samples were analyzed by reversible binding, and heat denaturation curves were determined. The coexpression of ARA9 increased the half-life of AHR by almost 2-fold (from 7.9 to 12.8 min) (Fig. 8 B).DISCUSSIONSeveral laboratories have shown that ARA9 is a component of the AHR·Hsp90 complex (9.Carver L.A. Bradfield C.A. J. Biol. Chem. 1997; 272: 11452-11456Abstract Full Text Full Text PDF PubMed Scopus (346) Google Scholar, 11.Ma Q. Whitlock Jr., J.P. J. Biol. Chem. 1997; 272: 8878-8884Abstract Full Text Full Text PDF PubMed Scopus (361) Google Scholar, 12.Meyer B.K. Pray-Grant M.G. Vanden Heuvel J.P. Perdew G.H. Mol. Cell. Biol. 1998; 18: 978-988Crossref PubMed Scopus (305) Google Scholar). Our previous work in yeast has shown that ARA9 is capable of modifying AHR signaling by shifting the βNF dose-response curve of AHR to the left and increasing the maximal response (13.Carver L.A. LaPres J.J. Jain S. Dunham E.E. Bradfield C.A. J. Biol. Chem. 1998; 273: 33580-33587Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar). Two other laboratories have shown that at a fixed dose of ligand, ARA9 can increase AHR signaling in mammalian cells (11.Ma Q. Whitlock Jr., J.P. J. Biol. Chem. 1997; 272: 8878-8884Abstract Full Text Full Text PDF PubMed Scopus (361) Google Scholar, 12.Meyer B.K. Pray-Grant M.G. Vanden Heuvel J.P. Perdew G.H. Mol. Cell. Biol. 1998; 18: 978-988Crossref PubMed Scopus (305) Google Scholar). We undertook this set of experiments to determine how ARA9 may elicit these effects. Several possible mechanisms were considered: 1) that ARA9 influences the amount of free intracellular ligand (29.Kralli A. Yamamoto K.R. J. Biol. Chem. 1996; 271: 17152-17156Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar); 2) that ARA9 is acting as a general modifier of the receptor's transactivation properties; and 3) that ARA9 acts by increasing the amount of functional AHR in the cytosol.Initially, we hoped to draw mechanistic insights into ARA9 function by considering what was known about the role of FKBP52 in steroid receptor signaling. Interestingly, the FKBP52 literature is quite complicated. Despite strong data for a physical interaction between FKBP52 and the GR·Hsp90 complex, there has been no direct evidence that this interaction has any functional consequence. In fact, it now appears that FK506, in the absence of FKBP52, can influence GR signaling by inhibiting an ATP binding cassette transporter, such as Pdr5p (Ref. 29.Kralli A. Yamamoto K.R. J. Biol. Chem. 1996; 271: 17152-17156Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar; Fig. 1). Thus, it is plausible that FK506 was influencing cellular pumps by interacting with unknown FKBPs. Although the chances for this explanation seemed remote, we set out to test the idea that ARA9 could directly inhibit Pdr5p and that this interaction increased the intracellular concentration of the AHR agonist, βNF, in yeast. In effect, we were testing the idea that ARA9 acted like an unknown FK506·FKBP complex that inhibited Pdr5p. As a positive control, we demonstrated that FK506 led to an increase in GR signal transduction in yeast, presumably due to inhibition of Pdr5p (Fig. 1) (29.Kralli A. Yamamoto K.R. J. Biol. Chem. 1996; 271: 17152-17156Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 30.Kralli A. Bohen S.P. Yamamoto K.R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4701-4705Crossref PubMed Scopus (147) Google Scholar). In contrast, the presence of ARA9 had no influence on DEX-dependent GR signaling, suggesting that ARA9 is incapable of affecting Pdr5p. Also of interest was the observation that FK506 had no influence, or had an inhibitory effect, on βNF signaling through the AHR. These results demonstrate that the pump, Pdr5p, does not significantly influence intracellular concentrations of βNF.In our initial experiments, we confirmed that ARA9 had similar effects on AHR signaling in both yeast and mammalian cells. Similar to what we have previously reported in yeast (11.Ma Q. Whitlock Jr., J.P. J. Biol. Chem. 1997; 272: 8878-8884Abstract Full Text Full Text PDF PubMed Scopus (361) Google Scholar), we observed that the cotransfection of ARA9 and AHR in COS-1 cells resulted in an increase in the maximal response and a leftward shift in the dose-response curve of βNF. Interestingly, in both yeast and mammalian cells, ARA9 also increases the background level of AHR signaling (Fig. 2; Ref. 13.Carver L.A. LaPres J.J. Jain S. Dunham E.E. Bradfield C.A. J. Biol. Chem. 1998; 273: 33580-33587Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar). There are a number of possible explanations for this. For example, ARA9 could be acting as a ligand for the AHR, or it could be enhancing the response of the AHR to a natural ligand present in these systems. A conclusion that is consistent with the other data presented in this report is that ARA9 increases the functional levels of the cytosolic AHR by acting as a cellular chaperone. If we assume that some constant fraction of properly folded AHR will be active, then an increase in properly folded AHR, as the result of ARA9 expression, will result in an increase in background activity. Although our data are consistent with this last possibility, we are unable to distinguish between these possibilities, and further experimentation will be necessary to clarify this issue.To eliminate the possibility that ARA9 was acting as a general modifier of transcriptionally active domains, we also examined its effects on a series of AHR deletion
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