Ribonucleic acid association with androgen receptor from rat submandibular gland

The transformed androgen receptor from rat submandibular gland converts to a faster sedimenting form (6-8S) on a glycerol gradient centrifugation after withdrawal of a transformation-inducing reagent (KCl or ATP). In this report, the association of cytosolic RNA with the transformed androgen receptor was investigated as a possible mechanism of molecular conversion of the androgen receptor. When the transformed and converted androgen receptors were treated with RNase A, these receptors sedimented at 4.5S in a low-salt glycerol gradient. Addition of RNA from rat submandibular gland to the RNase-Sepharose-treated transformed receptor caused a shift of receptor peak from 4.5S to 5.8S. RNA from rat submandibular gland, yeast RNA and E. coli rRNA inhibited DNA-cellulose binding of a RNase-treated transformed receptor in the absence of molybdate. These observations suggest that conversion from the transformed 4S androgen receptor to a 6-8S form resulted from the association of RNA(s) with the transformed receptor.     

Occurrence of a 6S intermediate form of the progesterone receptor that is sensitive to ribonuclease

Steroid receptors exist in cytosol as 9S, non-DNA-binding species and as 4S (transformed) species that bind to DNA or nuclei. Labeling the progesterone receptor from rabbit uterine cytosol with [³H]progesterone in the presence of 10 mM sodium molybdate revealed a 9S species on sucrose gradient centrifugation. Without molybdate, the receptor sedimented as an intermediate species of 6S, which converted to 4S in 0.3 M NaCl. The 6S species could also be generated from the 4S species by dialysis. Dilution of the same 4S species gave only partial re-aggregation with 50% of the receptor remaining as 4S. Dialysis appeared to retain the association of a macromolecular aggregation factor present in cytosol. Serum did not seem to be the source of the aggregation factor, as perfusion of the uterine vasculature before excision did not affect the S value of the receptor. We tested whether RNA was involved by treating receptor with RNase A (100 µg/400 µl cytosol). While the molybdate-stabilized cytosol receptor (9S) was unaffected, RNase A partially (50%) converted the 6S form of receptor to 4S. RNase A also partially converted the re-aggregated form back to 4S. Protease inhibitors had no effect on this action of RNase. Formation of receptor-ribonucleotide protein particles may play a role in steroid action in the cell.     

Separation and characterization of 7 and 9 S forms of rat liver aryl hydrocarbon receptor

Two forms of rat liver aryl hydrocarbon receptor were separated by chromatography on DEAE-cellulose in the presence of molybdate. After labeling for 2 h at 0 degrees C, the receptor separated on the DEAE column into a flow-through peak (peak I) and a peak eluting at 80 mM KCl (peak II). It had been reported previously that exposure to high salt in the presence of molybdate caused the appearance of both 9 and 5-6 S receptor forms. After confirming this, I examined the relationship of the peak I and peak II receptors to these receptor forms. In high salt buffer containing molybdate, the peak I receptor sedimented in the 5-6 S region and the peak II receptor at 9 S. High salt buffer lacking molybdate converted both peak I and peak II receptors to forms sedimenting in the 5-6 S region. In low salt buffer containing molybdate, the peak I receptor sedimented at slightly more than 7 S and the peak II receptor at 9-10 S. Thus, the peak II receptor could be stabilized by molybdate as a 9 S form, and the peak I receptor was converted by high salt from a 7 to a 5-6 S form, despite the presence of molybdate. Most of the peak I receptor bound to a DNA-cellulose column and was eluted by high salt. The peak II receptor showed very little DNA binding.     

Sodium molybdate converts the RNA-associated transformed, oligomeric form of the glucocorticoid receptor into the transformed, monomeric form

The glucocorticoid receptor from rat liver cytosol prepared in 2 ml buffer/g tissue sedimented at approximately 10 S in low salt density gradient centrifugation without molybdate. When the receptor was heated at 25 degrees C, both approximately 10 S and approximately 7 S forms were seen in low salt gradient. The approximately 10 S form was not capable of binding to DNA-cellulose and was stabilized by sodium molybdate, namely it corresponded to untransformed receptor. The approximately 7 S form was capable of binding to DNA-cellulose and regarded as transformed receptor. On the other hand, partially-purified transformed receptor labeled with [3H]dexamethasone-21-mesylate sedimented at approximately 5 S, which migrated as a approximately 94 kDa species in SDS-polyacrylamide gel electrophoresis. The reconstitution analysis of this partially-purified approximately 5 S receptor and liver cytosol, showed the shift to approximately 7 S form. RNase A or T1 converted approximately 7 S transformed form into approximately 5 S but it did not affect approximately 10 S untransformed form. 5-20 mM sodium molybdate also shifted approximately 7 S to approximately 5 S. These results indicate that the approximately 7 S transformed form of the glucocorticoid receptor observed in low salt conditions might be an oligomer, probably including both approximately 5 S steroid-binding component and RNA/ribonucleoprotein, and that molybdate dissociates these interactions in a specific manner.     

Hepatic Ah receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin. Partial stabilization by molybdate

In structure and general mode of action, the Ah receptor is very similar to the receptors for steroid hormones. Molybdate previously has been shown to be highly effective at preserving ligand-binding function in steroid receptors during their exposure to elevated temperature or high ionic strength and at stabilizing steroid receptors as high molecular weight oligomeric complexes. Since such stabilization by molybdate can be very useful during characterization and purification of receptors, we tested the effects of molybdate on the Ah receptor to determine if the Ah receptor, like the receptors for steroid hormones, might be stabilized. In hepatic cytosols from C57BL/6N mice and Sprague-Dawley rats, molybdate concentrations up to 30 mM in homogenizing and analysis buffers did not alter the concentration of specific Ah receptor sites detected by binding of [3H]2,3,7,8-tetrachlorodibenzo-p-dioxin. However, inclusion of 20 mM molybdate in the homogenizing buffer did significantly protect unliganded Ah receptor from thermal inactivation at 20 degrees C and from KCl-induced loss of ligand-binding ability. In accord with previous reports, 20 mM molybdate in homogenizing and analysis buffers greatly increased the concentration of detectable glucocorticoid receptor in rat hepatic cytosol and estrogen receptor in rat uterine cytosol. Exposure to 0.4 M KC1 caused the glucocorticoid receptor from rat liver to shift sedimentation from approximately equal to 8 S to approximately equal to 4 S and caused a severe loss of specific glucocorticoid binding. Presence of 20 mM molybdate stabilized the glucocorticoid receptor as a single discrete peak sedimenting at approximately equal to 8 S. In contrast, the Ah receptor from rat liver exposed to 0.4 M KC1 in the presence of molybdate sedimented as biphasic peaks; one peak (approximately equal to 9.5 S) corresponded to the form of Ah receptor observed at low ionic strength, while the other peak (approximately equal to 5.5 S) corresponded to the form of Ah receptor seen in cytosol treated with 0.4 M KC1 in the absence of molybdate. Addition of heparin to hepatic cytosols from mice or rats shifted sedimentation of Ah receptor from approximately equal to 9.5 S to approximately equal to 5.5 S. Molybdate, again, provided stabilization in the approximately equal to 9.5 S form, but only for about one-half the total Ah receptor content in both rat and mouse hepatic cytosols. In sum, molybdate is far less effective at stabilizing rodent Ah receptors than it is at stabilizing steroid receptors in the same species.     

Characterization of steroid hormone receptors with ion-exchange fast protein liquid chromatography

Androgen, estrogen and progesterone receptors have been characterized with anion exchange Fast Protein Liquid Chromatography (FPLC) on a Mono Q column (Pharmacia). In the presence of sodium molybdate androgen receptors in cytosols from rat prostate, rat epididymis and calf uterus eluted as a single sharp peak at 0.32 M NaCl with recoveries of approx 90%. The molybdate-stabilized form of the androgen receptor from rat prostate was purified about 75-fold. The receptor containing FPLC-peak fractions sedimented in high salt (0.4 M KCl) linear sucrose gradients at 3.6 S (prostate) and at 4.6 S (epididymis and calf uterus) respectively. Multiple forms of the androgen receptor were present in cytosols from rat prostate prepared in the absence of sodium molybdate, probably due to proteolytic breakdown of the native form. Calf uterine estradiol and progesterone receptors prepared in the presence of sodium molybdate (20 mM) eluted from the Mono Q column at 0.32 M NaCl. The molybdate-stabilized forms of the oestradiol and progesterone receptors were purified approx 70-fold and 30-fold respectively. In the absence of molybdate the estradiol receptor dissociated into two major forms eluting at 0.23 M NaCl and 0.37 M NaCl. After heat induced transformation (30 min at 25 degrees C) of the estradiol receptor one major peak was eluted at 0.42 M NaCl, indicating a change in the surface charge of the estradiol receptor as a result of the 4 S to 5 S transformation. It is concluded that the FPLC anion exchange system is a powerful, fast tool for characterization and partial purification of steroid receptors. In addition this technique could be applied as a rapid procedure for the quantitative estimation of steroid receptors in small biological samples.     

Activation of the dioxin and glucocorticoid receptors to a DNA binding state under cell-free conditions

The activation in vitro of dioxin and glucocorticoid receptors from a non-DNA binding to a DNA binding state was characterized. Ligand-free dioxin and glucocorticoid receptors were partially co-purified from rat liver cytosol, and both receptors sedimented at 9 S following labeling with the respective ligand. The 9 S forms of the dioxin and glucocorticoid receptors have previously been shown to represent heteromeric complexes containing the Mr approximately equal to 90,000 heat shock protein. The 9 S ligand-free or ligand-bound glucocorticoid receptor was converted to the monomeric 4-5 S form upon exposure to 0.4 M NaCl even in the presence of the stabilizing agent molybdate. Under identical conditions, the 9 S ligand-free and ligand-bound dioxin receptor forms remained essentially intact. However, in the absence of molybdate, the dioxin receptor could be converted to a 4-5 S form upon exposure to high concentrations of salt. These results indicate that the glucocorticoid receptor readily dissociates from the 9 S to the 4-5 S form even in the absence of hormone, whereas both the ligand-free and ligand-occupied 9 S dioxin receptor forms represent more stable species. Gel mobility shift experiments revealed that the 4-5 S glucocorticoid receptor interacted with a glucocorticoid response element both in the absence and presence of ligand. On the other hand, occupation of the dioxin receptor by ligand greatly enhanced the ability of the receptor to be activated to a form that binds to its target enhancer element. Once dissociated, the monomeric form of the dioxin receptor was also able to interact with its DNA target sequences even in the absence of ligand. Thus, ligand binding efficiently facilitates subunit dissociation of the dioxin receptor but is not a prerequisite for DNA binding per se. Given the apparent stability of its non-DNA binding 9 S form, the dioxin receptor system might be a useful model for the investigation of the mechanism of activation of soluble receptor proteins.     

Salt-induced transformation of the glucocorticoid-receptor complex in the presence of molybdate

The glucocorticoid hormone-receptor complex has been shown to exist in several forms. The transformation status of various forms of the complex isolated from rat thymus cytosol in the presence of molybdate was determined. The non-transformed receptor had a higher affinity for DEAE-cellulose than the transformed receptor. The rate at which the non-transformed complex was transformed to a smaller form with a low affinity for DEAE-cellulose by exposure to salt was greater in the absence of molybdate than in its presence. We conclude that salt-induced transformation of the complex is retarded but not prevented by molybdate and is associated with subunit dissociation.     

Effects of antiestrogen versus antiprogestin on transformed and nontransformed steroid receptors

In order to determine if different physicochemical properties exist among antihormone-receptor complexes, we have compared the interaction of the antiprogestin RU486 with progesterone receptor (PR) versus the triphenylethylene antiestrogen H1285 (4-(N,N-diethyl-aminoethoxy)-4'-methoxy-alpha-(p-hydroxyphenyl-alp ha'- ethylstilbene] with estrogen receptor (ER) from rabbit uterine tissue. Contrary to other reports, we observed no difference in the sedimentation properties of transformed PR (4S) when bound by the antagonist RU486 versus the progesterone agonist R5020 in either cytosol or DEAE partially-purified receptor preparations analyzed on sucrose gradients containing 0.3 M KCl. In addition, we found no difference in the sedimentation properties of these receptor preparations in the presence of 10 mM sodium molybdate: the nontransformed RU486-PR and nontransformed R5020-PR both sedimented as a 6S species. These same results were obtained when the receptor preparation and gradient analysis were performed in the absence of monothioglycerol. Likewise, there was no change in the sedimentation properties of the transformed PR when the receptor, partially purified in the absence of molybdate, was analyzed on sucrose gradients containing 10 mM sodium molybdate to prevent receptor alteration during centrifugation. From DNA-cellulose assays performed with partially purified PR in the absence of molybdate we determined that the 4S form of R5020-PR and RU486-PR is transformed receptor; whereas in the presence of molybdate, the 6S species is nontransformed. In contrast, we found a different pattern of sedimentation when comparing transformed antiestrogen-receptor complexes with transformed estrogen-receptor complexes. In this case, transformed H1285-ER sedimented as 6S and estradiol-ER sedimented as 4S. We conclude from these experiments that these two antihormones, RU486 and H1285, may have different mechanisms of action in their antagonism of steroid hormone action. Antiestrogen stabilizes the salt-transformed ER as a dimer while antiprogestin appears to permit dissociation of the oligomeric form of the receptor to the monomeric form.     

Polyanionic-binding properties of the receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin. A comparison with the glucocorticoid receptor

The interaction of the rat hepatic receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin (dioxin) with immobilized heparin (heparin-Sepharose) or DNA (DNA-cellulose) has been compared to the polyanionic-binding properties of the rat hepatic glucocorticoid receptor. Both the nonoccupied and in vitro occupied forms of the receptors interacted with heparin-Sepharose but with varying strength, as determined by ligand binding assays or an enzyme-linked immunosorbent assay based on a monoclonal antibody against the steroid- and DNA-binding Mr approximately 94,000 glucocorticoid receptor protein. In the absence of ligand, both the dioxin and glucocorticoid receptors eluted from heparin-Sepharose at 0.1-0.2 M KCl, in contrast to the in vitro occupied receptor forms which eluted at 0.3-0.4 M KCl. Following elution of the in vitro occupied dioxin receptor from heparin-Sepharose, it was efficiently retained on DNA-cellulose and eluted at an ionic strength of approximately 0.2 M KCl. In the presence of 20 mM sodium molybdate which is known to inhibit the activation of steroid hormone receptors to a DNA-binding form, both the dioxin and glucocorticoid receptors eluted at 0.1-0.2 M KCl from heparin-Sepharose. In analogy to what has previously been shown for the glucocorticoid receptor, sodium molybdate stabilized a large dioxin-receptor complex with a sedimentation coefficient, S20,w, of 9-10 S, a Stokes radius of approximately 7.5 nm, and a calculated Mr of 290,000-310,000. Limited proteolysis of both the dioxin and glucocorticoid receptors with trypsin which is known to eliminate the DNA-binding property of both receptor forms also resulted in a decreased strength in the interaction of both in vitro occupied receptors with heparin-Sepharose (elution at 0.1-0.2 M KCl). In line with these data, calf thymus DNA in solution competed for receptor binding to heparin-Sepharose. In conclusion, the chromatographic properties of the dioxin receptor on heparin-Sepharose are indistinguishable from those of the glucocorticoid receptor, and both receptors appear to be structurally and functionally closely related proteins.     

Effects of molybdate on steroid receptors in intact GH1 cells. Evidence for dissociation of an intracellular 10 S receptor oligomer prior to nuclear accumulation

Treatment of intact GH1 cells with sodium molybdate inhibits the subsequent rate of nuclear accumulation of hormone-occupied glucocorticoid and estrogen receptors. Cells were incubated at 23 degrees C for 1 h with 30 mM molybdate and then for up to 30 min with [3H]triamcinolone acetonide or [3H]estradiol in the continued presence of molybdate. Although molybdate did not affect the rate of receptor occupancy with either steroid, cells treated with molybdate had more occupied cytosolic and fewer occupied nuclear receptors than control cells. For the glucocorticoid receptor, cells treated with molybdate had more 10 S and fewer 4 S cytosolic receptors than control cells. In low salt cytosol molybdate inhibits the temperature-mediated subunit dissociation of occupied 10 S glucocorticoid receptor. These results suggest that a hormone-mediated dissociation of an intracellular 10 S oligomeric glucocorticoid receptor form to its 4 S subunits is required prior to accumulation of occupied receptors in the nuclear fraction. In cells incubated at 37 degrees C for 1 h or longer with [3H]triamcinolone acetonide, molybdate shifts the steady state intracellular distribution of receptor toward the 10 S cytosolic receptor form, consistent with the interpretation that molybdate affects the rapidly exchanging subunit equilibrium between the 10 S and 4 S cytosolic forms by slowing the rate of 10 S receptor dissociation. Molybdate prevents loss of glucocorticoid-occupied 10 S but not 4 S receptors in heated cytosol by stabilizing the relatively protease-resistant 10 S receptor. Since molybdate stabilizes 10 S oligomeric steroid receptors in vitro, the effects of molybdate on nuclear accumulation of occupied receptors in intact cells support the intracellular existence and physiological relevance of 10 S glucocorticoid and estrogen receptors. These results support a general model for steroid receptor activation in which binding of hormone promotes dissociation of intracellular 8-10 S oligomeric receptors to their DNA-binding subunits.     

Transformation (4S to 5S) of the nuclear estrogen receptor is reversible but not accompanied by a change in the affinity for DNA

The nuclear estrogen receptor from calf uterus was used to investigate the possible relationship between receptor transformation (4S to 5S) and receptor activation (DNA binding). Receptors extracted from nuclei after exposure of uterine tissue tc [3H]estradiol sedimented at 5.2S, the characteristic value of the transformed receptor. After storage at -20 degrees C the receptor sedimented at 4.0S, indicating conversion of the 5S form into the non-transformed 4S form. Upon reincubation at 28 degrees C the 4S form transformed into the 5S form following second-order kinetics. The rate constant obtained was 4.3 x 10(7) M-1 min-1, a value identical to that reported for the cytosol receptor. These data show that receptor transformation is reversible. Molybdate (10-50 mM) was not able to prevent receptor transformation in the nuclear extract, but was inhibitory in cytosol. This suggests that molybdate does not prevent receptor transformation, but rather inhibits disaggregation of the 8S oligomer into the 4S monomer. In DNA-binding assays (DNA-cellulose or nuclei) the non-transformed (4S) and transformed (5S) states of the nuclear estrogen receptors displayed identical affinities for DNA. The present data show that 4S to 5S transformation of nuclear receptors follows a readily reversible process, but this process is not an essential step for the exposure of the receptors' DNA-binding site. Although the physiological function of the 5S form remains unclear it may be important for the recognition of specific gene regulatory sites.     

Effects of calcium on the chick oviduct progesterone receptor

The chick oviduct cytosol progesterone receptor can be transformed to a small form (Rs = 21A, S20,w:2.9) denoted "mero-receptor" by incubation in the presence of Ca2+ [8]. In the molybdate-free cytosol all the progestin binding components could be completely transformed to mero-form by 1 h treatment with 100 mM Ca2+ at 0 degrees C. If EDTA was secondarily added, the ligand was rapidly released. If molybdate (20 mM) containing cytosol was incubated with Ca2+, no radioactivity was found in the meroposition on the Agarose A 0.5 m column, but the bound steroid sedimented at 2.9 S in sucrose gradients containing Ca2+ (and no molybdate). When 20 nM molybdate was added to cytosol containing receptor activated by 0.3 M KCl, complete mero-transformation by Ca2+ was obtained also by the gel filtration criterion, indicating that molybdate does not inhibit the mero-transforming factor. Ligand-free progesterone receptor could also be completely converted to mero-form by endogenous cytosolic transforming factor and calcium. The transforming factor was completely inactivated, when cytosol was run through Agarose A 0.5 m gel. Mero-transformation was found to be irreversible. The purified progesterone receptor subunit 110 K (B) was partially converted to smaller forms by calcium alone (100 mM, 0 degrees C, 1 h) whereas addition of a small amount of cytosol allowed complete conversion to mero-form.     

Effects of temperature, storage and sodium molybdate on the analysis of estrogen and progesterone receptors in rabbit uterine tissue and gynecologic tumor

The cytoplasmic estrogen receptor (ERc) and progesterone receptor (PRc) in mammary tumors have been recognized as useful biochemical markers for predicting the objective response of patients with advanced breast cancers to endocrine therapy. These proteins are also useful in the prognosis of gynecologic carcinoma. This report presents data showing the effect of sodium molybdate in the stabilization of estrogen and progesterone receptors. In rabbit uterine tissue, molybdate (20 mM) increased the binding of progesterone and estrogen to the receptors in several ways: (a) the apparent loss of detectable receptors during lengthy sucrose gradient analysis and at elevated temperature (30 degrees C) was reduced; (b) the instability of receptors due to storage at -70 degrees C was lessened, and (c) the conversion of the 7S PRc to the 3.5S form was minimized. Similarly, molybdate caused a qualitative and/or a statistically significant quantitative difference in receptor values for some human gynecologic tumors presented herein; the molybdate-associated changes vary with tumor specimen. Of the 8 tumors for which receptor values in the presence of molybdate (M+) and its absence (M-) can be compared, detectable ERc of 6 and PRc of 7 tumors increased with molybdate, and ERc of 2 and PRc of 1 tumor showed no change. In addition to the increase in receptor values, a concomitant shift of the 3-4S molecules to the 7-8S moieties was noted for some tumors (1 of 6 for ERc and 3 of 7 for PRc). In 2 receptor-poor tumor samples, ERc was only detected in M+ cytosols. These results show that molybdate is effective in reducing receptor degradation and stabilizes the 7-8S molecules from converting to 4S moieties. The addition of molybdate may be helpful for better quantitation of steroid receptors in clinical specimens.     

Kinetic and thermodynamic evidence of a molybdate interaction with glucocorticoid receptor in calf thymus

The effect of molybdate on the kinetic and thermodynamic properties of the dexamethasone-receptor interaction was studied in calf thymus cytosol. In the presence of molybdate both the equilibrium binding studies and the association and dissociation experiments reveal a significantly lower affinity of the receptor for [3]dexamethasone. At 0 degrees C the equilibrium dissociation constant increases from 0.8 nM to 1.8 nM, the association rate constant shifts from 1.5 X 10(8) M-1 h-1 to 0.2 X 10(8) M-1 h-1, whereas the rate of dissociation of the untransformed receptor increases from 0.04 h-1 to 1.1 h-1 in the molybdate-containing buffer. All these effects appear dependent on the concentration of molybdate but the dissociation of the transformed receptor (0.01 h-1) is unaffected. The enthalpy for the association, delta H not equal to, increases at least twofold whereas the entropy, both for the association (delta S not equal to = -25 to +104 J K-1 mol-1) and for the equilibrium (delta S degrees = -100 to +38 J K-1 mol-1), is markedly influenced by the presence of molybdate. Taken all together these data suggest that molybdate interacts with the receptor molecule turning it into a form that displays low affinity for steroid, in addition to the well-documented incapacity to transform itself. This fact leads us to think that both the binding and the transformation are the expression of conformational modifications involving molybdate-sensitive groups.     

Physicochemical analysis of reversible molybdate effects on different molecular forms of glucocorticoid receptor

Several distinct molecular forms of glucocorticoid receptor have been identified in a melanoma model system. We have used velocity sedimentation to monitor molybdate dependent alterations in receptor size and heterogeneity. In the absence of molybdate, native glucocorticoid receptor from dexamethasone-sensitive tumors sediments at 7–8 S and 12–13 S. Under identical conditions, receptor isolated from dexamethasone-resistant tumors sediments at 7–8 S only. However, when molybdate is introduced, either during homogenization or immediately prior to centrifugation, glucocorticoid receptors from both dexamethasone-sensitive and -resistant tumors sediment sharply at 9–10 S. These molybdate induced phenomena are reversible. The activated forms of glucocorticoid receptor isolated from both dexamethasone-sensitive and -resistant tumors by DEAE-cellulose chromatography have similar sedimentation coefficients (4–5 S) which are unaffected by molybdate.     

The receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin in the mouse hepatoma cell line Hepa 1c1c7. A comparison with the glucocorticoid receptor and the mouse and rat hepatic dioxin receptors

The molecular properties of the receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin in the mouse hepatoma cell line Hepa 1c1c7 were investigated. The receptor was found to represent a highly asymmetrical molecule with a sedimentation coefficient, s20,w, of approximately 8 S, a Stokes radius of 7-8 nm, and a calculated Mr approximately equal to 260,000-300,000. In comparison, the Hepa 1c1c7 glucocorticoid receptor in analogy to the glucocorticoid receptor in general as well as the C57BL/6 mouse and rat hepatic dioxin receptors are molecules with an s20,w value of 4-5 S, a Stokes radius of approximately 6 nm, and a calculated Mr approximately equal to 100,000. In the presence of 20 mM sodium molybdate, a large Mr approximately equal to 270,000-310,000 form of the Hepa 1c1c7 glucocorticoid receptor is stabilized which is hydrodynamically indistinguishable from the Mr approximately equal to 260,000-300,000 Hepa 1c1c7 dioxin receptor. Sodium molybdate does not have any effect on the molecular properties of the Hepa 1c1c7 dioxin receptor. In conclusion, the large form of dioxin receptor present in Hepa 1c1c7 mouse hepatoma cells in the absence of sodium molybdate is strikingly similar to molybdate-stabilized steroid hormone receptors as well as the molybdate-stabilized form of the dioxin receptor previously demonstrated in rat hepatic cytosol. Therefore, the Hepa 1c1c7 dioxin receptor might offer an interesting model for studies on the structure and function of Mr approximately equal to 300,000 forms of soluble receptors.     

Interaction of vanadyl ribonucleoside complex with the androgen receptor

The addition of vanadyl ribonucleoside complex (VRC), a potent inhibitor of RNase, to the transformed 4.5S androgen receptor from rat submandibular gland caused an increase in the sedimentation coefficient to 7.0S. Moreover, VRC decreased the DNA-cellulose binding of the transformed receptor; 50% inhibition of the DNA-cellulose binding was achieved at 1.8 mM VRC. On the other hand, agents related to VRC and oxoanions of transient metals, such as ribonucleoside, vanadate, molybdate, tungstate and arsenate, exerted no effect on the DNA-cellulose binding ability of the receptor. These findings suggest that VRC binds to the transformed androgen receptor at the DNA-binding site and that both oxovanadium ion and ribonucleoside are indispensable for the binding of VRC to the transformed androgen receptor.     

Resolution of non-activated and activated androgen receptors based on differences in their hydrodynamic properties

This study shows that cytosolic androgen receptor of rat ventral prostate sediments at 10-11 S on conventional low salt sucrose density gradients (SDG), and at 4.6 S on high salt SDG, whether it is activated or not; inclusion of 10 mM Na2MoO4 in all buffers does not alter these sedimentation coefficients. In the presence of 50 mM Na2MoO4 non-activated and activated androgen receptors sediment in high salt SDG at 7-8 S and 4.6 S, respectively. Thus the presence of high concentrations of molybdate during centrifugation inhibits the KCl induced disaggregation of receptor into subunits. Similar effects are observed on Sephacryl-S200 gel filtration; in 50 mM MoO2-4 and 0.4 M KCl non-activated receptor has an estimated Stokes radius of 67 A; this value decreases to 52 A upon activation in the presence of proteolysis inhibitors; omission of molybdate during chromatography yielded 52 A and 27 A entities. Estimated mol. wts are 198,000 Daltons for the non-activated 67 A form and 98,000 Daltons for the activated 52 A receptor. Sodium molybdate (50 mM) prevents temperature (18 degrees C) and high ionic strength (0.4 M KCl) induced receptor activation. This inhibition was overcome by removing molybdate by centrifugal gel filtration, or by increasing the KCl concentration to 0.8 M. The inhibitory effects of molybdate on salt induced receptor disaggregation into activated subunits are no longer observed at pH greater than 7.4 or after chemical modification of sulfhydryl groups. Once androgen receptor has been disaggregated into its activated subunits the activated state is maintained even upon reassociation to 10-11 S aggregates in low salt. The relative concentrations of KCl and molybdate are critical; thus, 10 mM Na2MoO4/0.4 M KCl and 50 mM Na2MoO4/0.8-1.2 M KCl did not differentiate activated from non-activated androgen receptor based on their hydrodynamic properties. In the presence of 0.4 M KCl and 50 mM molybdate, however, the hydrodynamic properties of androgen receptor can be correlated with receptor activation.     

Physicochemical properties of type I corticosteroid receptors from rat brain

[3H]Aldosterone binds with high affinity to Type I corticosteroid receptors in cytosols from adrenalectomized rat forebrains. Physicochemical parameters of these receptors were determined in the presence of molybdate, which stabilized receptors and maintained them in a presumably untransformed state. The Stokes' radius of the molybdate-stabilized receptor was 8.1 nm, as determined by gel filtration on Sephacryl S-300. Its sedimentation coefficient was 9.1S in linear sucrose density gradients. The receptor is asymmetric, with an axial ratio of 8-10 and an apparent mol. wt of 303,000 dalton. The [3H]aldosterone-receptor complex is anionic and elutes from DEAE-Trisacryl in a single peak with a maximum at 160 mM KCl. Exposure to heat or salt in the absence of molybdate, conditions which transform other steroid receptors to smaller DNA-binding forms, causes marked instability of the [3H]aldosterone-receptor complex. The [3H]aldosterone-binding protein of rat forebrain, which displays the binding characteristics of a renal Type I (mineralocorticoid) receptor, is similar in size, shape and charge to the molybdate-stabilized oligomeric forms of other steroid hormone receptors.