THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1988 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 263, No. 27, Issue of September 25, pp. 13802-13806 1988 Printed in L?S.A.

Association of the Ah Receptor with the 90-kDa Heat Shock Protein* (Received for publication, May 9, 1988)

Gary H. Perdew From the Department of Foods and Nutrition, Purdue University, West Lafayette, Indiana 47907

Partially purified Ah receptor preparations were used to produce a monoclonal antibody, designated as 8D3, that is capable of immunoprecipitating the Ah receptor. Hepa l c l c 7 cytosol was photoaffinity- labeled with [ ‘261]-2-azido-3-iodo-7,8-dibromodi- benzo-p-dioxin followed by immunoprecipitation, and the resulting precipitate was applied to a sodium dodecyl sulfate-polyacrylamide electrophoretic gel. These gels were stained with Coomassie Blue and re- vealed the presence of a major immunoprecipitated 90- kDa protein, and after autoradiography a radiolabeled 95-kDa protein (Ah receptor) was detected. The 90- kDa protein was determined to be the 90-kDa heat shock protein (HSP9O) by western blot analysis using an antibody (AC88) previously shown to be specific for HSP9O. An increase in the sedimentation of the Ah receptor on sucrose density gradients was seen upon addition of monoclonal antibody 8D3 to Hepa l c l c 7 cytosol. Monoclonal antibody 8D3 immunoprecipitates the Ah receptor from Hepa 1 cells (murine), HeLa cells (human), and rat liver cytosolic extracts, indicating that the Ah receptor is complexed with HSP9O in sev- eral mammalian species tested. These results illustrate another physicochemical property that the supergene family of soluble steroid receptors and the Ah receptor have in common.

Halogenated aromatic hydrocarbons are ubiquitous in the environment (1) and produce a wide range of species- and tissue-specific toxic effects (2 ) . These effects are thought to be mediated by the Ah’ receptor, a soluble protein capable of binding halogenated aromatic hydrocarbons with high affin- ity. 2,3,7,8-Tetrachlorodibenzo-p-dioxin has been shown to have the highest affinity for the receptor, and thus is a potent inducer of aryl hydrocarbon hydroxylase activity. After bind- ing 2,3,7,8-tetrachlorodibenzo-p-dioxin the Ah receptor is be- lieved to enhance transcription of PI-450 (aryl hydrocarbon hydroxylase) by binding to dioxin-responsive genomic ele- ments located upstream of the PI-450 promoter site in mouse hepatoma cells (3). Little is known about the biochemical

* This work was supported in part by National Institute of Envi- ronmental Health Science Grant ES-01884, National Cancer Institute Core Grant 07175, National Cancer Institute Postdoctoral Training Grant T32-CA09020, American Cancer Society Institutional Grant IN-17, and an Indiana Elks Institutional Grant. This is technical paper No. 11,683, Indiana Agricultural Experiment Station. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduer- tisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ’ The abbreviations used are: Ah, aryl hydrocarbon; HSP90, 90- kDa heat shock protein; MOPS, 3-(N-morpholino)propanesulfonic acid PBS, phosphate-buffered saline; Hepa 1, hepatoma cell line lclc7; LDS, lithium dodecyl sulfate; mAb, monoclonal antibody; SDS, sodium dodecyl sulfate; GAM, goat anti-mouse IgG+IgM antibodies.

properties of the Ah receptor (eg. phosphorylation, protein composition) and the sequence of events from ligand binding to subsequent binding to DNA. The physicochemical proper- ties of the rat hepatic Ah receptor and the glucocorticoid receptor are similar (4). Much more information about the biochemical properties of the glucocorticoid receptor has been obtained. It is useful to examine this data and compare it to possible properties of the Ah receptor. It has been established that a dimeric form of the 90-kDa heat shock protein (HSPSO) is associated with the glucocorticoid receptor (5). HSPSO has been found associated with steroid receptors (6, 7), several oncogenic tyrosine protein kinases (6), and actin (8). The specific function(s) of HSPSO has not been established. It has been proposed that HSPSO caps the DNA binding region of the glucocorticoid receptor, thus maintaining the receptor in an inactive form (9). It may also be possible that HSPSO has a stabilizing effect on the proteins that it associates with.

Monoclonal antibodies are needed to more effectively study the biochemical properties of the Ah receptor. In the data presented here we report the production of a monoclonal antibody to the murine HSPSO that is able to co-precipitate the Ah receptor. There has been no previous direct evidence for the Ah receptor-HSP9O complex.

EXPERIMENTAL PROCEDURES

Materiuls-Enzyme grade ammonium sulfate was obtained from Schwarz/Mann. Cell culture media and serum were purchased from Gibco Laboratories, unless otherwise noted. 2-Azid0-3-[’*~1]iodo-7,8- dibromodibenzo-p-dioxin was synthesized as described (10). Nitro- cellulose membrane BA83 was purchased from Schleicher & Schuell. “Control” ascites TEPC 183 IgM was obtained from Sigma. Acryl- amide, N,N”methylenebisacrylamide, and ammonium persulfate were purchased from Bio-Rad. All other chemicals were obtained from Sigma unless otherwise noted.

Animak-C57BL/6J, SJ/L, and BALB/c mice were obtained from Jackson Laboratory, Bar Harbor, ME and bred in the laboratory of Dr. Alan Poland. Outbred Sprague-Dawley rats were obtained from Harlen Sprague-Dawley Inc., Indianapolis, IN.

Preparation of Cytosolic Extracts and the Ammonium Sulfate Frac- tion-Mouse liver cytosol and the 40-55% ammonium sulfate fraction were prepared from the livers of C57BL/6J mice as described (10). Sprague-Dawley liver cytosolic fraction was prepared as described (11). Confluent Hepa 1 or HeLa cells were harvested in trypsin/ EDTA and washed three times in Dulbecco’s phospbate-buffered saline. The cells were suspended in 25 mM MOPS, 1 mM EDTA, 0.02% NaN3, pH 7.5 (at 0 “C) + 10% glycerol (MEN + l0XG) at 7.6 X IO6 cells/ml and homogenized in a Dura-Grind Dounce tissue grinder (Wheaton Instruments, Millville, NJ) with 15 strokes by hand. Sodium molybdate (20 mM) was included in the buffer during preparation of rat liver and HeLa cell extracts. Cytosolic extracts were stored at -80 “C until ready for use.

Zmmunization-The Ah receptor was purified 20,000-fold as pre- viously described (12). Briefly, Ah receptor was purified using ion- exchange chromatography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and C-4 reverse-phase chromatography under dena- turing conditions. Ah receptor (95 kDa) preparations after high- performance liquid chromatography were dialyzed against 0.1% tri- fluoroacetic acid, followed by the addition of Nonidet P-40 on an equal (w/w) basis. Samples were then lyophilized and resolubilized in

13802

A h Receptor Is Associated with 90-kDa Heat Shock Protein 13803

a minimal volume of 0.1 N NaOH, followed by neutralization with 0.5 N HC1 at 4 "C. Female SJL/J mice, 8 weeks old, were immunized with 95-kDa receptor preparations. The first injection containing 40 pg of protein mixed with Freund's complete adjuvant was given intraperitoneally. Mice were once again injected intraperitoneally after 4 weeks, with 40 pg of receptor material in Freund's incomplete adjuvant. After a 2-week rest, a final booster injection of 20 pg of receptor preparation in saline was given by intrasplenic injection (13).

Cells and Media-The mouse myeloma cell line SP2/O-Ag 14 was obtained from Dr. Bill Sugden (McArdle Laboratory for Cancer Research, University of Wisconsin-Madison) and used as a fusion partner. This cell line was grown in complete medium, consisting of RPMI 1640 (Gibco Laboratories), supplemented with 20% (v/v) calf serum (Hyclone, Logan, UT), 2 mM glutamine, 1 mM pyruvic acid, 0.5 mM oxaloacetic acid, and 0.2 units/ml bovine insulin. After fusion the cell mixture was cultured in the above medium with the addition of 0.1 mM hypoxanthine, 0.2 p~ aminopterin, and 1.6 mM thymidine (HAT medium). Normal human fibroblasts SL-68, provided by Dr. Norman Drinkwater, and HeLa cells, provided by Dr. Jeffrey Ross, both from McArdle Laboratory, were cultured in Dulbecco's high glucose medium supplemented with 10% fetal calf serum. Hepa lclc7 cells were obtained from Dr. James P. Whitlock (Department of Pharmacology, Stanford, CA) and grown in a-minimum essential medium (Sigma) containing 8% fetal calf serum at 37 "C in 94% air,

Cell Fusion-Three days after final immunization, the spleen was removed from the SJ/L mouse. The previous day 8 X lo3 irradiated (4000 rads) human fibroblasts, SL-68, were plated out in 96-well plates a t 8 X lo3 cells/well in 100 p1 of complete myeloma medium. The spleen cells were isolated and added to S P 2/0 myeloma cells maintaining a ratio of 5:l. The cell mixture was fused by the addition of 2 ml of 38% polyethylene glycol 1450 (Kodak, Rochester, NY), 5% dimethyl sulfoxide in RPMI 1640. The cells were gently mixed and then centrifuged for a total of 6 min. The polyethylene glycol solution was aspirated off and the cells were gently resuspended in 6 ml of serum-free RPMI 1640, and then centrifuged for 7 min. The resultant cell pellet was resuspended in complete media and 100 pl (2.5 X lo5 spleen cells/well) was added to each well of the 96-well plates seeded with fibroblasts. After incubation for 5 h, 50 pl of complete medium containing 5 X HAT medium was added to each well. After 14 days, hybridomas were screened by the solid-phase immunoassay described below.

Screening Assay-One-ml polypropylene tubes (Flow labs, 45 X 8.8 mm, PPN tubes) were incubated with 500 pl of 5 pg/ml affinity- purified goat anti-mouse IgG and IgM (Jackson Immunoresearch Lab, West Grove, PA) in PBS at 4 "C. The following day the tubes were incubated with 500 pl of 1% bovine serum albumin in PBS for 1 h; this solution was removed and replaced with PBS until the tubes were used. From each well positive for hybridoma growth, 100 pl of supernatant was removed and placed in a 1-ml assay tube and brought up to a total volume of 500 p1 with PBS. Assay tubes were allowed to stand for 1 h, followed by two washes in PBS. To each tube, 500 pl of [''61]2-iodo-7,8-dibromodibenzo-p-dioxin-labeled 40-55% ammo- nium sulfate fraction (procedure for binding outlined below) was added and allowed to sit for 1 h at 4 "C. Finally, the tubes were washed twice with ice-cold PBS and counted in a gamma counter.

Preparation of Radioligand-Receptor Complexes for Screening As- say-A 40-55% ammonium sulfate fraction was solubilized in ice- cold MEN + 10%G to a final concentration of 9 mg of protein/ml. Ammonium sulfate precipitation only causes partial dissociation of the murine Ah receptor-HSP9O complex. In a separate tube was added 9.5 X lo6 cpm (specific activity 2,176 Ci/mmol) of ['251]-2-iodo- 7,8-dibromodibenzo-p-dioxin in 5 pl of dimethyl sulfoxide followed by the addition of 250 pl of the ammonium sulfate fraction; tubes were then incubated for 30 min at room temperature. The details of the radiosynthesis of this ligand will be described elsewhere.' The radioligand-protein mixture was then added to 10 ml of charcoal/ dextran (final concentration 1%/0.1%) in MEN + 10%G and stirred for 5 min. The charcoal was removed by centrifugation at 10,000 X g X 10 min at 4 "C, and the supernatant was stored on ice.

Cloning and Ascites Production-Wells determined to be positive by solid-phase immunoassay were cloned twice by limiting dilution using 96-well microtiter plates and 8 X lo3 fibroblasts/well as a feeder

primed SJL/J X BALB/c ByJ (F,) mice with 1 X lo7 hybridoma cells. layer. Large amounts of antibody were obtained by injecting pristane-

' Bradfield, C. A., Kende, A. s., and Poland, A. (1988) Mol. Phar-

6% COZ.

macol., in press.

Antibody subtyping was performed using a subtyping kit from Boeh- ringer Mannheim Biochemicals.

Sucrose Gradient Analysis-Hepa 1 cytosol (1 mg/ml) was incu- bated with 100,000 cpm of [''51]-2-iodo-7,8-dibromodibenzo-p-dioxin (specific activity, 2,176 Ci/mmol) for 30 min at room temperature. Excess ligand was removed by addition of the protein/ligand mixture to a charcoal/dextran pellet that is resuspended (final concentration, l .O%/O.l%) for 5 min. The charcoal was then removed by centrifu- gation (3,000 X g) for 10 min at 4 "C. Aliquots (300 pl) of Hepa 1 cytosolic samples were layered on 5.1 ml of 10-30% sucrose gradients prepared in MEN + 10%G buffer. The tubes were centrifuged for 105 min at a 390,196 X gmaX in a Beckman VTi80 vertical rotor. After centrifugation, 3-drop fractions were collected and counted in a TmAnalytic 1191 gamma counter (TmAnalytic, Inc., Elk Grove Vil- lage, IL).

Photoaffinity Labeling-2-Azido-3-["5I]-2-iodo-7,8-dibromodiben- zo-p-dioxin was added to the Hepa 1 cytos01(400 pg/ml) at a final concentration of 240 fmol/ml (specific activity, 2,176 Ci/mmol, 9.2 X lo5 cpm/ml), and the solution was incubated for 30 min at 20 'C. The protein/ligand solution was then placed on ice for 5 min, followed by transfer of the mixture to a tube containing pelleted charcoal/dextran (final concentration, 1%/0.1%) and incubated on ice for 20 min. The charcoal was pelleted and the solution was irradiated with two 15- watt lamps (UV Products, >302 nm) at a distance of 8 cm for 4 min. The Hepa 1 cytosol was carefully removed from the charcoal pellet, and 8-mercaptoethanol was added to a final concentration of 20 mM.

Immunoprecipitation-Affinity-purified goat anti-mouse IgG+IgM antibodies (GAM) were coupled to cyanogen bromide-activated Seph- arose CL-4B at 1 mg/ml gel. The coupling efficiency was >98%. To 100 p1 of GAM-Sepharose was added 200 pl of ascites and allowed to stand for 2 h, followed by three washes with phosphate-buffered saline. To this gel was added 400 p1 of photoaffinity-labeled Hepa 1, HeLa, or rat liver cytosolic protein (1 mg/ml) and incubated for 90 min at 4 "C. The labeling conditions used were as described above. This mixture was subsequently washed three times with MEN + 10%G at 4 "C (HeLa and rat liver samples were washed with MEN + 10%G + 20 mM sodium molybdate), followed by solubilization in 4% LDS, 24% glycerol, 100 mM Tris-HC1, 2 mM EDTA, 20 mM dithiothreitol, pH 6.8 (2 X LDS sample buffer).

Gel Electrophoresis and Autoradiography-Protein samples were subjected to sodium dodecyl sulfate discontinous polyacrylamide slab gel electrophoresis, gels were stained with Coomassie Brilliant Blue R, dried, and subjected to autoradiography as described (10).

Immunoblotting and Immunochemical Staining of the 90-kDa Heat Shock Protein-After polyacrylamide gel electrophoresis the proteins were transferred to nitrocellulose membranes essentially as described by Gibson (14). The electrotransfer was performed in a Genie elec- troblot unit (Idea Scientific Co., Corvallis, OR) for 90 min at a constant voltage of 12 V. Following the transfer the blot was stored in 10 mM sodium phosphate, pH 7.4, 150 mM NaC1, 0.5% Tween 20 (v/v), and 0.1% (w/v) bovine serum albumin (blotting buffer) for 16 h at 4 "C. The blots were incubated with 10 pg/ml monoclonal antibody AC88 in blotting buffer for 1 h (the remaining steps were performed at room temperature), followed by three 10-min washes in blotting buffer. The blot was then incubated in 10 pg/ml anti-mouse polyvalent immunoglobins (Sigma) for 1 h. After washing, the blot was incubated in 10 pg/ml mouse peroxidase-anti-peroxidase (Jack- son Immunoresearch, West Grove, PA) for 45 min and washed as stated above. Finally, the antigenic bands were stained in 50 mM Tris-HC1, pH 7.4, 3,3-diaminobenzidine (0.3 mg/ml), and 0.005% hydrogen peroxide. Control blots were subjected to the same proce- dures except without addition of the primary antibody.

RESULTS AND DISCUSSION

Partially purified Ah receptor preparations (20,000-fold enriched relative to liver cytosol) were used in an attempt to produce monoclonal antibodies to the Ah receptor. After screening hybridomas from several fusions, two positive clones were isolated and subtyped, and both 8D3 and 8H1 were found to be IgM antibodies. Initial observations indi- cated that the antibodies had an apparent low affinity for the Ah receptor. Each antibody was tested for the ability to immunoprecipitate the radiolabeled Ah receptor, mAb 8D3 was able to precipitate greater than 50% of the Ah receptor, compared with 5% using mAb 8H1 (data not shown). mAb

13804 A h Receptor Is Associated with 90-kDa Heat Shock Protein

8D3 immobilized on GAM-Sepharose was incubated with photoaffinity-labeled Hepa 1 cytosol, followed by SDS-poly- acrylamide gel electrophoresis and stained with Coomassie Blue (Fig. lA, lane 2) . The mAb 8D3 was able to precipitate a -90-kDa protein, no immunoprecipitated protein was ob- served with control antibody (Fig. lA, lune 3). The autoradi- ogram shown in Fig. 1B reveals that 70% of the photoaffinity- labeled Ah receptor, which migrates at 95 kDa, is co-immu- noprecipitated by mAb 8D3 (lane 2). The nonspecific receptor adsorption seen in Fig. 1B, lane 3, is probably due to both the "sticky" nature of the Ah receptor and the low salt washing buffer used.

It has been suggested that the Ah receptor is part of a supergene family containing the soluble steroid receptors. The glucocorticoid receptor has been shown to be associated with the murine HSPSO (15). In addition, recent evidence strongly suggests that the glucocorticoid receptor is associated with HSPSO in uiuo (16). Thus, it would be logical to determine whether the mAb 8D3-immunoprecipitated -90-kDa protein is the murine HSPSO. This can be tested by immunostaining a Western blot of mAb 8D3-immunoprecipitated Hepa 1 cytosol with an mAb to HSPSO (Fig. 2 ) . Monoclonal antibody AC88 has previously been shown to bind with high specificity to the murine HSPSO on Western blots (15). The results in Fig. 2 indicate that the 90-kDa protein immunoprecipitated by mAb 8D3 is the murine HSPSO. The apparently low affinity of mAb 8D3 for the coprecipitation of the Ah receptor is due to a >1000-fold molar excess of free heat shock protein in liver cytosol. Highly purified and denatured preparations of receptor used for antibody production evidently contained HSPSO as a contaminating protein.

The addition of heparin to mouse hepatic cytosol has pre- viously been shown to cause an altered sedimentation value from 8s to 4s on sucrose density gradients (17). From this result, one would expect that heparin would cause dissociation

A B 1 2 3 4 5 1 2 3 4

- 200 -

- 116 - - 92 -"-

" - -"(L- 66 -

""L - - 4 5 - -

FIG. 1. Immunoprecipitation of the Ah receptor. Hepa 1 cy- tosol was photoaffinity-labeled and 400 pl (1 mg/ml) of this prepa- ration was incubated with 100 pl of GAM-Sepharose previously saturated with a monoclonal antibody. The GAM-Sepharose was washed, solubilized in 2 X LDS sample buffer, and applied to an SDS- polyacrylamide gel. The gel was then Coomassie Blue-stained. A , lane I , a 100-pg aliquot of photoaffinity-labeled cytosolic protein; lane 2, immunoprecipitate of Hepa 1 cytosol with mAb 8D3; lane 3, immu- noprecipitate of 400 pg of Hepa 1 cytosol with control antibody TEPC 183 IgM; lane 4, immunoprecipitate of Hepa 1 cytosol (400 pg) incubated for 2 h with 500 pg of heparin prior to incubation with mAb 8D3-GAM-Sepharose; lane 5, mAb 8D3-GAM-Sepharose not incubated with Hepa 1 cytosol. B, autoradiography of the gel in Fig. 1A; lanes 1-4 are as in A .

1

200-

11 6- 92- - 66-

45-

2 3

" FIG. 2. Immunochemical staining of the mAb 8D3-immu-

noprecipitated 90-kDa heat shock protein on a Western blot. Lanes 1-3 correspond to Fig. 1A, lanes 1-3. After transfer to nitro- cellulose membrane the blot was incubated with mAb AC88 and the bound antibody was visualized as described in experimental proce- dures.

0 10 15 20 25

I?otlom TOP

1 r-action No.

FIG. 3. Binding of mAb 8D3 to the murine Ah receptor during sucrose gradient centrifugation. Hepa 1 cytosol (1 mg/ ml) was incubated with 26 fmol/ml ["sI]-2-iodo-7,8-dibromodibenzo- p-dioxin in the absence or presence of a 200-fold molar excess of 2,3,7,8-tetrachlorodibenzofuran (TCDBF), followed by velocity sedi- mentation on 10-30% sucrose gradients. After incubation with [1251] -2-iodo-7,8-dibromodibenzo-p-dioxin, samples (300 p l ) were also in- cubated for 30 min a t 4 "C with 5 pl of TEPC 183 ascites (control antibody) or 5 pl of 8D3 ascites and layered on parallel sucrose density gradients. After centrifugation, 3-drop fractions were col- lected from the bottom of the tube and counted in a gamma counter. The marker protein used was catalase (11s).

of the Ah receptor from HSPSO; this result can be seen in Fig. 1, lane 4, in both A and B. Upon addition of heparin, HSPSO remains bound to mAb 8D3, but 90% of the specifically bound radiolabeled Ah receptor is no longer found associated with the immunoprecipitate. Sucrose density gradients were per- formed to provide additional evidence of the Ah receptor complex-mAb 8D3 interaction. Addition of 8D3 ascites to the Hepa 1 cytosol resulted in an increased sedimentation of the

Ah Receptor Is Associated with 90-kDa Heat Shock Protein 13805

1 2 3 4 5 . 5 = , 7 J . ,., # 9 :

200-

116- 9 2 - e

66-

45- 4, - - -

FIG. 4. Immunoprecipitation of the Ah receptor from Hepa 1, HeLa, and rat liver cytosolic fractions. Each cytosolic fraction was photoaffinity-labeled as described under “Experimental Proce- dures.” To 100 pl of GAM-Sepharose, previously incubated with a given monoclonal antibody, was added 400 pl (1 mg/ml) of each preparation in separate tubes. After 90 min the GAM-Sepharose was washed, solubilized in LDS buffer, and applied to an SDS-polyacryl- amide gel. The gel was fixed, dried, and subjected to autoradiography. The resulting autoradiograph is shown. Lane I , a 150-pg aliquot of photoaffinity-labeled Hepa 1 cytosolic protein; lane 2, immunoprecip- itate of 400 pl of Hepa 1 (1 mg/ml) cytosol with mAb 8D3; lane 3, immunoprecipitate of 400 pl of Hepa 1 cytosol with antibody TEPC 183; lane 4, a 150-pg aliquot of photoaffinity-labeled rat liver cytosolic protein; lane 5, immunoprecipitate of 400 p1 of rat liver cytosol (1 mg/ml) with mAb 8D3; lune 6, immunoprecipitate of 400 pl of rat liver cytosol with antibody TEPC 183; lane 7, a 150-pg aliquot of photoaffinity-labeled HeLa cytosolic protein; lane 8, immunoprecip- itate of 400 pl of HeLa (1 mg/ml) cytosol with mAb 8D3; lane 9, immunoprecipitate of 400 pl of HeLa cytosol with antibody TEPC 183.

Ah receptor compared to control ascites (Fig. 3). It was of interest to investigate the ability of mAb 8D3 to

immunoprecipitate HSPSO in other species to determine its usefulness, and in addition, whether the A h receptor is also co-precipitated. Fig. 4 indicates that mAb 8D3 is able to immunoprecipitate 70% of the Ah receptor-HSP9O complex from Hepa 1 (murine), 50% from rat liver, and 55% from HeLa (human) cytosol. The apparent decreased level of Ah receptor co-immunoprecipitation seen in rat and human cell cytosols may be due to instability of the Ah receptor-HSPSO complex in these species compared to Hepa 1 cytosol (murine). The Ah receptor-HSPSO complex is more resistant in vitro to high salt exposure in C57BL/6J mouse liver cytosol compared to the receptor complex in rat hepatic cytosol (4, 18). The high degree of molecular weight polymorphism in the Ah receptor between species seen in Fig. 4 has been previously described (19). Thus, association of the HSPSO protein with the Ah receptor occurs in vitro across the mammalian species tested.

mAb 8D3 is also capable of immunoprecipitating both mu- rine glucocorticoid and estrogen receptors? This is the only mAb presently available that binds to soluble receptor-HSPSO

‘ D. Toft, personal communication.

complexes in mammals: mAb 8D3 was unable to immuno- precipitate the 88-kDa protein from water mold Achlya am- bisexualis extracts (data not shown); this protein is apparently the equivalent of the vertebrate HSPSO (20). Attempts to use mAb 8D3 on protein blots to bind to the denatured form of HSPSO were unsuccessful.

The Ah receptor is apparently associated, at least in vitro, with the 90-kDa murine heat shock protein. This co-precipi- tation by mAb 8D3 may be useful in immunoaffinity purifi- cation of the Ah receptor-HSPSO complex. The Ah receptor could then be eluted under gentle conditions from the HSPSO yielding highly enriched functional receptor preparations for further characterization of the Ah receptor or as the initial step in a purification scheme.

Acknowledgments-I would like to thank Dr. Alan Poland for providing financial support and guidance during the monoclonal antibody production phase of this work and Dr. David Toft for kindly providingantibody AC88 and water mold Achlya ambisexualis extracts and for critically reviewing this manuscript. Finally, I wish to thank Marcia Perdew for her assistance in the preparation of the figures.

REFERENCES 1. Young, A. L., Kang, H. K., and Shepard, B. M. (1983) Enuiron.

2. Poland, A., and Knutson, J. (1982) Annu. Rev. Phnrmacol. Toxi-

3. Durrin, L. K., Jones, P. B. C., Fisher, J. M., Galeazzi, D. R., and Whitlock, J. P., Jr. (1987) J. Cell Biochem. 35, 153-160

4. Cuthill, S., Poellinger, L., and Gustafsson, J.-A (1987) J. Biol. Chem. 262,3477-3481

5. Denis, M., Wikstrom, AX. , and Gustafsson, J.-A. (1987) J. Biol. Chem. 262,11803-11806

6. Ziemiecki, A., Catelli, M-G., Joab, I., and Monocharmont, B. (1986) Biochem. Biophys. Res. Commun. 138,1298-1307

7. Sanchez, E. R., Toft, D. O., Schlesinger, M. J., and Pratt, W. B. (1985) J. Biol. Chem. 260,12398-12401

8. Koyasu, S., Nishida, E., Kadowaki, F., Matsuzaki, F., Iida, K.,

Natl. Acad. Sci. U. S. A. 83,8054-8058 Harada, F., Kasuga, M., Sakai, H., and Yahara, I. (1986) Proc.

Sci. Technol. 17,530A-540A

C O ~ . 22,517-554

9. Baulieu, E.-E. (1987) J. Cell. Biochem. 35, 161-174 10. Poland, A., Glover, E., Ebetino, F. H., and Kende, A. S. (1986) J.

11. Poland. A.. and Glover. E. (1987) Biochem. BioDhvs. Res. Com- Biol. Chem. 261,6352-6365

mun.’146, 1439-1449 . . _ -

12. Perdew. G. H.. and Poland. A. (1988) J. Biol. Chem. 263.9848- , . . 9852 ’

munol. Methods 76,332-337 13. Gearing, A., Thorpe, R., Spitz, L., and Spitz, M. (1985) J. Zm-

14. Gibson, W. (1981) Anal. Biochem. 118,l-3 15. Sanchez, E. R., Meshinchi, S., Tienrungroj, W., Schlesinger, M.

J., Toft, D. O., and Pratt, W. B. (1987) J. Biol. Chem. 262,

16. Howard, K. J., and Distelhorst, C. W. (1988) J. Biol. Chem. 263,

17. Denison, M. S., Vella, L. M., and Okey, A. B. (1986) J. Biol. Chem. 261,10189-10195

18. Denison, M. S., Vella, L. M., and Okey, A. B. (1986) J. Biol. Chem. 261,3987-3995

19. Poland, A., and Glover, E. (1987) Biochem. Biophys. Res. Com- mun. 146,1439-1449

20. Riehl, R. M., Sullivan, W. P., Vroman, B. T., Bauer, V. J.,

6591 Pearson, G. R., and Toft, D. 0. (1985) Biochemistry 24,6586-

6986-6991

3474-3481

‘ mAb 8D3 can be obtained by writing to the author.