In vivo kinetics and DNA-binding properties of the Ah receptor in the golden Syrian hamster

The in vivo long-term cytosolic-nuclear kinetics and DNA-binding properties of the Ah receptor were examined in liver from the golden Syrian hamster. For the kinetic studies, a dose of [3H]2,3,7,8-tetrachlorodibenzo-p-dioxin ([3H]TCDD) that has been previously shown to produce maximal and sustained hepatic enzyme induction without substantial toxicity was used. Following an intraperitoneal dose of 10 micrograms/kg of [3H]TCDD, occupied cytosolic receptor levels reached a peak within 8 h and then decreased rapidly to a level that was approximately 2% of the total receptor. Throughout the 35-day period, unoccupied cytosolic receptor represented from 65 to 80% of the total receptor content. At 8 h following dosing, less than 30% of the total amount of receptor was associated with the nuclear fraction; this percentage declined slowly to less than 5% of the total at Day 35. The half-life for the decline in detectable nuclear receptor levels was 13 days and was similar to the half-life for the decline in [3H]TCDD content of the whole liver, cytosol, and nuclear extract. The Ah receptor contained in hamster hepatic cytosol underwent a ligand-dependent transformation in vitro to two forms having affinity for DNA-Sepharose, one of which was isolated from nuclei of animals treated with [3H]TCDD in vivo. A comparison of the specific binding recovered following various analytical procedures revealed that the binding of [3H]TCDD to the form not found in nuclear extracts was more labile under certain experimental conditions. These studies indicate the heterogeneity of the Ah receptor in hamster hepatic cytosol and suggest that DNA binding in vitro and nuclear uptake in vivo occur through a ligand-dependent transformation process. The maintenance of maximal hepatic enzyme induction is, in part, a consequence of the sustained presence in the nucleus of only a small percentage of the total receptor content. The whole-tissue kinetics of TCDD appears to be a major factor regulating the long-term retention of the TCDD-receptor complex in the nucleus.     

Photoaffinity labeling of the nuclear Ah receptor from mouse Hepa 1c1c7 cells using 2,3,7,8-[3H]tetrachlorodibenzo-p-dioxin

The photoinduced formation of the covalently labeled cytosolic and nuclear aryl hydrocarbon (Ah) receptors was studied using 2,3,7,8-[3H]tetrachlorodibenzo-p-dioxin (TCDD) as the photoaffinity label. Irradiation of TCDD alone at wavelengths of greater than 300 nm resulted in rapid degradation of this compound (t 1/2 = 8 min). In a separate experiment, the unliganded cytosolic Ah receptor was only slowly inactivated (t 1/2 = 48 min) using the greater than 300 nm light source. Preliminary experiments with rat hepatic cytosol did not result in significant formation of specifically bound [3H]TCDD-protein covalent adducts which could be visualized by autoradiography. Irradiation of [3H]TCDD-nuclear Ah receptor complexes isolated from mouse Hepa 1c1c7 cells for 15 min gave approximately a 40% overall yield of the radiolabeled Ah receptor protein adduct. Denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the [3H]TCDD-nuclear Ah receptor photoadduct gave a single major radiolabeled protein with an apparent molecular size of 91 kDa. The chromatographic properties of the control (dark) and photolabeled nuclear Ah receptor complexes were comparable using Sephacryl S-300 and DNA-Sepharose columns. Velocity sedimentation of both the control (dark) and irradiated nuclear Ah receptor complexes gave specifically bound peaks which sedimented at 6.5 S. However, the trichloroacetic acid-precipitable (buffer-reconstituted) [3H]TCDD-nuclear Ah receptor photo-covalent adduct was eluted from the Sephacryl S-300 column in the void volume and did not exhibit a specifically bound peak after velocity sedimentation analysis due to protein aggregate formation. In contrast, the elution profile of the aggregate on a DNA-Sepharose column was similar to that observed for the control (dark) and photolabeled complexes, which were eluted from the column with salt concentrations between 0.24 and 0.28 M. These photolabeling studies show that [3H] TCDD can act as a photoaffinity label for the Ah receptor and can be utilized as photoligand to probe further the structure and function of this protein.     

Structure and function of the Ah receptor: sulfhydryl groups required for binding of 2,3,7,8-tetrachlorodibenzo-p-dioxin to cytosolic receptor from rodent livers

Cytosol from rodent liver was exposed to a variety of sulfhydryl-modifying reagents to determine if the cytosolic Ah receptor contained reactive sulfhydryl groups that were essential for preservation of the receptor's ligand binding function. At a 2 mM concentration in rat liver cytosol, all sulfhydryl-modifying reagents tested (except iodoacetamide) both blocked binding of [3H]2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) to unoccupied receptor and caused release of [3H]TCDD from receptor sites that had been labeled with [3H]TCDD before exposure to the sulfhydryl-modifying reagent. Exposure of cytosol to iodoacetamide before labeling with [3H]TCDD prevented subsequent specific binding of [3H]TCDD, but iodoacetamide was not effective at displacing previously bound [3H]TCDD from the Ah receptor. The mercurial reagents, mersalyl, mercuric chloride, and p-hydroxymercuribenzoate, were more effective at releasing bound [3H]TCDD from previously labeled sites than were alkylating agents (iodoacetamide, N-ethylmaleimide) or the disulfide compound 5,5'-dithiobis(2-nitrobenzoate). Presence of bound [3H]TCDD substantially protected the Ah receptor against loss of ligand binding function when the cytosol was exposed to sulfhydryl-modifying reagents. This may indicate that the critical sulfhydryl groups lie in or near the ligand binding site on the receptor. Subtle differences exist between the Ah receptor and the receptors for steroid hormones in response to a spectrum of sulfhydryl-modifying reagents, but the Ah receptor clearly contains a sulfhydryl group (or groups) essential for maintaining the receptor in a state in which it can bind ligands specifically and with high affinity.     

Defective binding of 3-methylcholanthrene to the Ah receptor within C3H/1OT1/2 clone 8 mouse fibroblasts in culture

C3H/1OT1/2 clone 8 mouse fibroblasts (C3H/1OT1/2 cells) exhibit induction of aryl hydrocarbon hydroxylase (cytochrome P1-450) when exposed in culture to benzo(a)pyrene, benz(a)anthracene or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), but do not display the induction response when treated with 3-methylcholanthrene (MCA), the classical inducer of cytochrome P1-450. Induction of cytochrome P1-450 is regulated by the Ah receptor which initially binds inducing chemicals in the cytoplasm, after which the inducer x receptor complex translocates into the nucleus. Cytosolic and nuclear forms of the Ah receptor can be detected in C3H/1OT1/2 cells using [3H]TCDD as the radioligand in culture, but specific Ah receptor binding is not detectable within C3H/1OT1/2 cells incubated with [3H]MCA. In contrast, in Hepa-1c1 cells, which exhibit cytochrome P1-450 induction when treated with MCA, cytosolic and nuclear Ah receptor can be detected by incubation of the cells either with [3H]MCA or with [3H]TCDD. Nonradioactive MCA is able to compete with [3H]TCDD for Ah receptor sites in C3H/1OT1/2 cells, but the relative potency of MCA as a competitor is lower within C3H/1OT1/2 cells than in C3H/1OT1/2 cytosol during extracellular incubation. Specific binding of [3H]MCA to Ah receptor can be detected by incubation of [3H]MCA with C3H/1OT1/2 cytosol outside the cell. The selective loss of response to MCA as a cytochrome P1-450 inducer (while retaining response to other inducers) appears to be due to defective interaction of MCA with the Ah receptor within the intracellular environment. The specific molecular alteration which makes the MCA x receptor complex ineffective within C3H/1OT1/2 cells is unknown. Some fibroblast lines other than C3H/1OT1/2 also selectively fail to respond to MCA; thus, this variation in Ah receptor function may not be due to a mutational change in the Ah regulatory gene which codes for the Ah receptor.     

Ah receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin in rat liver: lack of sensitivity to alkaline phosphatase when compared with that of glucocorticoid receptor

Rat hepatic cytosol was treated with alkaline phosphatase in order to determine if dephosphorylation altered the ability of Ah receptor to bind 2,3,7,8-[3H]tetrachlorodibenzo-p-dioxin (TCDD). Glucocorticoid receptor was studied for comparison. As previously had been shown in other laboratories, treatment of cytosol with purified alkaline phosphatase dramatically reduced the subsequent ability of glucocorticoid receptor to bind hormone. However, alkaline phosphatase had no effect on the ability of Ah receptor to bind [3H]TCDD. If either glucocorticoid receptor or Ah receptor was occupied by its ligand prior to exposure to alkaline phosphatase there was no loss in ligand binding capacity. Crude alkaline phosphatase (containing some protease activity) substantially reduced the ability of glucocorticoid receptor to bind hormone and shifted the sedimentation position of the glucocorticoid receptor from approximately 8 S to approximately 2 S. Crude alkaline phosphatase did not reduce the ability of Ah receptor to bind [3H]TCDD and did not alter sedimentation of the 9 S [3H]TCDD. Ah receptor complex. Although the Ah receptor appears to be a member of the steroid receptor superfamily, the lack of effect of alkaline phosphatase on Ah receptor (compared to the sensitivity of glucocorticoid receptor) highlights another significant difference in molecular characteristics between the Ah receptor and the receptors for steroid hormones.     

Characterization of the Ah receptor mediating aryl hydrocarbon hydroxylase induction in the human liver cell line Hep G2

The Ah receptor, a soluble cytoplasmic receptor that regulates induction of cytochrome P450IA1 and mediates toxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), was detected and characterized in the continuous human liver cell line Hep G2. The mean concentration of specific binding sites for TCDD was 112 +/- 26 (SEM) fmol/mg cytosol protein as determined in eight separate cytosol preparations in the presence of sodium molybdate. This is equivalent to 14,000 binding sites per cell, approximately 40% of the sites per cell found in the mouse hepatoma line Hepa-1. The cytosolic Ah receptor from Hep G2 cells sedimented at 9 S and was specific for those halogenated and nonhalogenated aromatic compounds known to be agonists for the Ah receptor in rodent tissues and cells. Specific binding in the 9 S region was detected with both [3H]TCDD and 3-[3H]methylcholanthrene. 3-[3H]Methylcholanthrene did not bind to any component besides that at approximately 9 S. Phenobarbital, dexamethasone, and estradiol did not compete with [3H]TCDD for binding to the Hep G2 Ah receptor. Specific binding of [3H]triamcinolone acetonide to glucocorticoid receptor could also be demonstrated in Hep G2 cytosol. The apparent equilibrium dissociation constant (Kd) for binding of [3H]TCDD to Hep G2 Ah receptor was 9 nM by Woolf plot analysis, about an order of magnitude weaker than the affinity of [3H]TCDD for the mouse Hepa-1 Ah receptor or for the C57BL/6 murine hepatic Ah receptor. [3H]TCDD.Ah receptor complex, which was extracted from nuclei of Hep G2 cells incubated with [3H]TCDD at 37 degrees C in culture, sedimented at approximately 6 S under conditions of high ionic strength. Aryl hydrocarbon hydroxylase (AHH) activity was significantly induced after 24 h of incubation with polycyclic aromatic hydrocarbons: the EC50 for AHH induction was 5.3 microM for benz(a)anthracene and 1.3 microM for 3-methylcholanthrene. Modification of the preparative technique for cell cytosol, especially inclusion of 20 mM sodium molybdate in homogenizing and other buffers, was necessary to detect cytosolic Hep G2 Ah receptor. Hep G2 cells appear to conserve drug-metabolizing activity associated with cytochrome P450IA1 as well as the receptor mechanism which regulates its induction.     

Characterization of an inducible aryl hydrocarbon receptor-like protein in rat liver.

The individual pretreatment of Sprague-Dawley rats with either 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or 2,2',4,4',5,5'-hexachlorobiphenyl (HCB) has been previously shown to result in the "induction" of [3H]TCDD specific binding activity in hepatic tissue. In the present work, the coadministration of TCDD and HCB increased the concentration of hepatic proteins capable of binding [3H]TCDD specifically by at least 2-3-fold. This increase was shown not to be the result of activation, by HCB, of a form of the receptor having low affinity toward [3H]TCDD into a form with high affinity. Kinetic analysis of the time course of binding of [3H]TCDD to induced cytosol was consistent with the presence of an "inducible" binding protein in addition to the "constitutive" aryl hydrocarbon (Ah) receptor present in cytosol from untreated animals. The liganded ([3H]TCDD) form of the inducible binding component lost its ligand much faster than the liganded form of the constitutive Ah receptor at 37 degrees C; apparent first order rate constants for loss of [3H]TCDD were 0.55 min-1 and less than 0.0024 min-1, respectively. Conversely, the unliganded form of the induced binding component was slightly more stable (approximately 2-fold) toward thermal inactivation than the unbound constitutive Ah receptor. The [3H]TCDD-bound protein(s) in uninduced and induced cytosols behaved identically in a sucrose gradient; 8.7-8.9 S in the absence of salt, shifted to 5.5 S by 0.4 M KCl. They were also indistinguishable by gel permeation chromatography, and by photoaffinity labeling their TCDD-binding subunits, approximate molecular weights 105,000. These results show the hepatic TCDD-binding protein(s) induced upon pretreatment of Sprague-Dawley rats with TCDD/HCB to be kinetically distinct from the Ah receptor, but structurally very similar.     

Radioligand-dependent differences in the molecular properties of the mouse and rat hepatic aryl hydrocarbon receptor complexes

A comparison of the molecular properties of the male Long-Evans rat and male C57BL/6 mouse hepatic cytosolic aryl hydrocarbon (Ah) receptor complex was determined using 2,3,7,8-[3H]tetrachlorodibenzo-p-dioxin (TCDD) and 2,3,7,8-[3H]tetrachlorodibenzofuran (TCDF) as radioligands. In low salt buffer, the sedimentation coefficients, Stokes radii, relative molecular masses, frictional ratios, axial ratios and gel permeation chromatographic properties of the rat receptor complexes were ligand independent. In contrast, there were several ligand-dependent differences in the mouse Ah receptor complexes formed after incubation in low salt buffer and these include: sucrose density gradient analysis of the 2,3,7,8-[3H]TCDF receptor complex gave a 9.5 S specifically bound peak and a 2.6 S nonspecifically bound peak whereas the corresponding 2,3,7,8-[3H]TCDD receptor complex gave a single 9.6 S specifically bound peak; sucrose density gradient analysis of the two major peaks eluted from a Sephacryl S-300 column chromatographic separation of the 2,3,7,8-[3H]TCDF receptor complex gave two specifically bound peaks at 9.2 and 5.1 S. The molecular properties of the rat hepatic cytosolic receptor complexes incubated in high salt (0.4 M KCl) buffer were ligand independent with one exception, namely the significant difference in the sedimentation coefficient of the specifically bound disaggregated 2,3,7,8-[3H]TCDD receptor complex (6.8 S) and the corresponding 2,3,7,8-[3H]TCDF receptor complex (5.0 S). The major ligand-dependent differences in the mouse receptor complexes incubated in high salt (0.4 M KCl) were associated with the sedimentation coefficients of the complexes derived after direct incubation and after gel permeation chromatography. For example, both ligands gave two specifically bound complexes after chromatography on Sephacryl S-300 column and centrifugation of these fractions gave both the approximately 9 and approximately 5 S peaks; this suggested that there was some equilibration between the aggregated and disaggregated receptor complexes. The behavior of the 2,3,7,8-[3H]TCDF mouse receptor complex was similar after incubation in low or high salt buffer except that sucrose density gradient analysis of the gel permeation chromatographic fractions gave an additional specifically bound peak which sedimented at 7.2 S. These studies demonstrate that the molecular properties of the Ah receptor were dependent on the source of the cytosolic receptor preparation, the ionic strength of the incubation media, and the structure of the radioligand.     

Interaction of hexachlorobenzene with the receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin in vitro and in vivo. Evidence that hexachlorobenzene is a weak Ah receptor agonist

Hexachlorobenzene (HCB) produces hepatic porphyria and induces the hepatic cytochrome P450 isozymes P450c (P450IA1) and P450d (P450IA2) in rodents. These and other effects of HCB resemble those of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), which acts via its binding to the aromatic hydrocarbon (Ah) receptor. We therefore examined the ability of HCB to interact with this receptor in vitro and in vivo. HCB, at concentrations of 1 microM or higher, inhibited the specific binding of [3H]TCDD (0.3 nM) to the Ah receptor in vitro, whereas the solubility of [3H]TCDD was affected only at 100 microM HCB. The inhibition was competitive, with a KI of approximately 2.1 microM. In rats fed a diet containing 3000 ppm HCB for varying times (4 h to 7 days), the specific binding of [3H]TCDD in hepatic cytosol was reduced by up to 40%, as observed previously for known Ah receptor agonists. The decrease in [3H]TCDD specific binding in cytosol of HCB-treated rats was due principally to a decrease in the number of binding sites for [3H]TCDD rather than competition from residual HCB. As shown by immunoblotting and radioimmunoassay, HCB induced the cytochrome P450 isozymes P450c and P450d, which are regulated by the Ah receptor, as well as the phenobarbital-inducible isozymes P450b and P450e. Together these results indicate that HCB is a weak agonist for the Ah receptor, and suggest that some of its effects may be mediated by its interaction with this gene-regulatory protein.     

Mechanism of action of 2,3,7,8-tetrachlorodibenzo-p-dioxin antagonists: characterization of 6-[125I]methyl-8-iodo-1,3-dichlorodibenzofuran-Ah receptor complexes.

6-Methyl-8-iodo-1,3,-dichlorodibenzofuran (I-MCDF) and its radiolabeled analog [125I]MCDF have been synthesized and used to investigate the mechanism of action of 1,3,6,8-substituted dibenzofurans as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) antagonists. Like 6-methyl-1,3,8-trichlorodibenzofuran (MCDF), I-MCDF partially antagonized the induction by TCDD of microsomal aryl hydrocarbon hydroxylase (AHH) and ethoxyresorufin O-deethylase (EROD) activities in rat hepatoma H-4-II E cells and male Long-Evans rat liver. Incubation of rat liver cytosol with [125I]MCDF followed by velocity sedimentation analysis on sucrose gradients gave a specifically bound peak which sedimented at 9.6 S. This radioactive peak was displaced by coincubation with a 200-fold excess of unlabeled I-MCDF, 6-methyl-1,3,8-trichlorodibenzofuran (MCDF), 2,3,7,8-tetrachlorodibenzofuran (TCDF), and benzo [a]pyrene. Based on the velocity sedimentation results and the elution profile from a Sephacryl S-300 gel permeation column, the Stokes radius and apparent molecular weights of the cytosolic [125I]MCDF-Ah receptor complex were 6.5 nm and 259,200, respectively. In addition, the nuclear [125I]MCDF-receptor complex eluted at a salt concentration of 0.29 M KCl from a DNA-Sepharose column. Velocity sediment analysis of the nuclear [125I]MCDF-Ah receptor complex from rat hepatoma H-4-II E cells gave a specifically bound peak at 5.6 +/- 0.8 S. All of these properties were similar to those observed using [3H]TCDD as the radioligand. In addition, there were several ligand-dependent differences observed in the properties of the I-MCDF and TCDD receptor complexes; for example, the [125I]MCDF rat cytosolic receptor complex was unstable in high salt buffer and was poorly transformed into a form with increased binding affinity on DNA-Sepharose columns; Scatchard plot analysis of the saturation binding of [3H]TCDD and [125I]MCDF with rat hepatic cytosol gave KD values of 1.07 and 0.13 nM and Bmax values of 137 and 2.05 fmol/mg protein, respectively. The nuclear extract from rat hepatoma H-4-II E cells treated with I-MCDF or TCDD interacted with a dioxin-responsive element in a gel retardation assay. These results suggest that the mechanism of antagonism may be associated with competition of the antagonist receptor complex for nuclear binding sites.     

Ah receptor in primate liver: binding of 2,3,7,8-tetrachlorodibenzo-p-dioxin and carcinogenic aromatic hydrocarbons

Ah receptor in hepatic cytosols from adult cynomolgus monkeys (Macaca fasicularis) was identified and quantitated by its binding of the highly toxic chemical 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and the carcinogens 3-methylcholanthrene, benzo[a]pyrene, and dibenz[a,h]anthracene. The concentration of Ah receptor in cynomolgus hepatic cytosols (approximately 10 fmol/mg cytosol protein) was about one-quarter of that typically detected in rodent hepatic cytosols. Receptor concentrations were equal in male and female cynomolgus. [3H]TCDD bound to cytosolic receptor with high affinity (Kd approximately 3 nM). In rodents, Ah receptor is known to play a central role in toxicity caused by halogenated aromatic compounds and in carcinogenesis caused by polycyclic aromatic hydrocarbons. Existence of Ah receptor in monkeys indicates that the receptor also may mediate such responses in primates.     

Identification of the Ah receptor in selected mammalian species and induction of aryl hydrocarbon hydroxylase

The Ah receptor protein, important in the mechanism of induction of aryl hydrocarbon hydroxylase activity, has been identified and partially characterized in hepatic cytosolic preparations from rat, BALB/c mouse, gerbil, hamster, rabbit, ferret and guinea-pig by means of sucrose density centrifugation analysis and hydroxyapatite binding assays. Using 2,3,7,8-tetrachloro[3H]dibenzo-p-dioxin (TCDD) as the ligand, total specific binding capacities ranged over 74-691 fmol [3H]TCDD/mg cytosolic protein and apparent dissociation constants ranged over 0.30-7.8 nM. There was no quantitative correlation between the concentration of cytosolic Ah receptors and the 3-methylcholanthrene-mediated induction of aryl hydrocarbon hydroxylase activity in the species studied. Competitive binding studies with a series of monohydroxylated benzo[a]pyrene derivatives suggested the importance of electronic character in their ability to bind to the Ah receptor and to compete with TCDD for specific binding sites on the receptor.     

Unoccupied and in vitro and in vivo occupied 1,25-dihydroxyvitamin D3 intestinal receptors. Multiple biochemical forms and evidence for transformation

The data presented herein indicate that the chick intestinal 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) receptor which is localized in the chromatin fraction of a low salt homogenate (Walters, M. R., Hunziker, W., and Norman, A. W. (1980) J. Biol. Chem. 255, 6799-6805) can exist in three distinct biochemical forms. The three 1,25(OH)2D3 receptor forms depended on the absence or presence of ligand and additionally whether the ligand was acquired in vitro (4 degrees C incubation for 3-4 h) or in vivo (13 nmol of 1,25(OH)2D3 administered intramuscularly 2 h prior to sacrifice). The receptor forms were distinguished by their relative KCl extractabilities from the target tissue chromatin preparation and from a reconstituted nontarget tissue (liver) chromatin preparation, as well as their relative elution from DNA-cellulose columns when applied as a mixture. In all cases the rank order "affinity" of the receptor for chromatin or DNA was: unoccupied 1,25(OH)2D3 receptors less than in vivo occupied receptors less than in vitro occupied receptors. These changes in DNA-binding "affinity" occurred without a major change in overall surface charge of the receptor molecules as evaluated by co-elution of all three receptor forms from DEAE-Sepharose columns. Similarly, these changes in DNA-binding characteristics were not accompanied by changes in the apparent molecular weights of these receptor species (91,900 +/- 3300; 99,700 +/- 9400; 93,100 +/- 5600, respectively) as assessed by Sephacryl S-200 gel filtration chromatography in the presence of the protease inhibitor phenylmethylsulfonyl fluoride, included throughout these experiments to protect from proteolytic damage. These results represent the first demonstration of biochemical heterogeneity in the 1,25(OH)2D3 receptor system and suggest the existence of a two-step transformation process for the 1,25(OH)2D3 receptor.     

Protamine sulfate precipitation: a new assay for the Ah receptor

The ability of protamine sulfate to effect the quantitative precipitation of 2,3,7,8-[3H]tetrachlorodibenzo-p-dioxin (TCDD):Ah receptor complexes from rat liver cytosol has been developed into a new assay for the identification, quantitation, and characterization of the Ah receptor. The method is reliable, uncomplicated, and rapid, and can be applied to large numbers of samples. The major advantage of the assay is that protamine sulfate appears to selectively precipitate the Ah receptor protein and does not precipitate a number of other proteins that bind [3H]TCDD nonspecifically.     

Effects of SH-modifying reagents on the rat hepatic Ah receptor: inhibition of ligand binding and transformation, and disruption of the ligand-receptor complex

The importance of sulfhydryl (SH) groups in maintenance of physicochemical properties of the rat hepatic Ah receptor was demonstrated using a variety of sulfhydryl (SH)-modifying reagents. Inhibition of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) specific binding was approximately equivalent by 5,5'-dithiobis(2-nitrobenzoate), mersalyl, N-ethylmaleimide, and p-chloromercuriphenylsulfonate, whose inhibition curves were steep in the concentration range close to that of nonprotein SH groups in cytosol (ED50 values 50-200 microM or 13-48 nmol/mg cytosolic protein). Inhibition by p-hydroxymercuribenzoate (PHMB), although exhibiting a lower ED50, was more gradual over this range; iodoacetamide was an order of magnitude less potent. The ability of dithiothreitol to reverse binding inhibition induced by 150 microM (approximately 60 nmol/mg protein) mersalyl diminished with time; it decreased more rapidly in the simultaneous presence of TCDD and mersalyl than when mersalyl was present alone, consistent with increased accessibility of key SH group(s) due to conformational changes attending TCDD-receptor complex formation. Brief exposure of unoccupied receptor to mersalyl prior to TCDD binding caused slower sedimentation of the complex in 0-KCl sucrose gradients and alterations in its elution profiles on DEAE- and DNA-Sepharose suggestive of some impairment of the transformation process. When reagents were added to the transformed TCDD-receptor complex, loss of binding was observed only at concentrations which were an order of magnitude higher than those inhibiting TCDD binding. Loss of binding by each reagent was biphasic, and except for that caused by mersalyl, was not complete even after 6-8 h. Dithiothreitol was able to reverse the effects of mersalyl or PHMB only partially and only if added during the early phase (10-30 min) of binding loss. Mersalyl was much more potent in disrupting the untransformed than the transformed TCDD receptor complex. Physical alteration of the mersalyl-treated TCDD-receptor complex was evident from gel filtration, sucrose gradients, and DNA- and DEAE-Sepharose chromatography. Our results are in striking contrast to the effects of these reagents on steroid receptors, whose bound steroid hormone ligand is rapidly and reversibly displaced by lower concentrations of reagent.(ABSTRACT TRUNCATED AT 400 WORDS)     

Detection and characterization of the Ah receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin in the human colon adenocarcinoma cell line LS180.

The Ah (aromatic hydrocarbon) receptor mediates induction of aryl hydrocarbon hydroxylase (AHH; an enzyme activity associated with cytochrome P450IA1) by polycyclic aromatic hydrocarbon carcinogens such as 3-methylcholanthrene (MC) and benzo[a]pyrene (BP) and the halogenated toxin 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Until recently the AhR seemed to be present only at very low levels in human cells and tissue. With a modified assay (the presence of sodium molybdate and a reduction in the amount of charcoal used to adsorb "excess" ligand) we found that cytosol from LS180 cells contains a high concentration of AhR (400-500 fmol/mg cytosolic protein) when detected by [3H]TCDD or [3H]MC. Cytosolic receptor also was detected with [3H]BP but at a level that was 35% of that detected with [3H]TCDD or [3H]MC. These levels are similar to those found in mouse Hepa-1 hepatoma cells in which AhR has been extensively characterized. The apparent binding affinity (Kd) of the cytosolic receptor for [3H]TCDD and for [3H]MC was about 5 nM. As with Hepa-1, the human LS180 cytosolic AhR sedimented at about 9 S on sucrose gradients when detected with [3H]TCDD, [3H]BP or [3H]MC. The nuclear-associated ligand.receptor complex recovered from cells incubated in culture with [3H]TCDD sedimented at about 6.2 S. The 9.8 S cytosolic form corresponds to a multimeric protein of a relative molecular mass (Mr) of about 285,000 whereas the 6.2 S nuclear receptor corresponds to a multimeric protein of Mr 175,000. The smallest specific ligand-binding subunit (detected by sodium dodecyl sulfate-polyacrylamide electrophoresis under denaturing conditions of receptor photoaffinity labeled with [3H]TCDD) was about Mr 110,000. AHH activity was induced in cells exposed in culture to TCDD or benz[a]anthracene (BA). The EC50 was 4 x 10(-10) M for TCDD and 1.5 x 10(-5) M for BA. For both inducers the EC50 in LS180 cells was shifted about one log unit to the right as compared to the EC50 for AHH induction in mouse Hepa-1 cells. The lower sensitivity of the LS180 cells to induction of AHH activity by TCDD or BA is consistent with the lower affinity of TCDD and MC for binding to human AhR. The ligand-binding properties, physicochemical properties, and mode of action of the AhR in this human cell line are therefore very similar to those of the extensively characterized AhR in rodent cells and tissues.