The ability to convert the 4 S glucocorticoid receptor to the 7–8 S form is dependent on both RNA and protein factors

The DEAE-cellulose-purified 4 S form of the rat liver glucocorticoid receptor can associate with cytosolic factors, as evidenced by an alteration of the sedimentation value of the 7–8 S form. On the basis of sedimentation profile, this form is indistinguishable from the activated, low-salt 7–8 S form isolated from rat liver cytosol. In addition, both the endogenous and reconstituted 7–8 S receptor can bind DNA as the 7–8 S form. In keeping with our reports that the endogenous form of the 7–8 S receptor is sensitive to RNAase digestion, treatment of the cytosol with RNAase prior to mixing with the 4 S receptor prevents the formation of the 7–8 S material. Moreover, warming the cytosol to 50°C prior to mixing with the 4 S receptor also eliminates the ability to form the heavier material. Since RNA is heat-stable, this suggests that other factors may be involved. Treatment of the cytosol with N-ethylmaleimide, a reagent reported to be specific for sulfhydryl groups, also eliminates 7–8 S generating ability. These observations suggest that a protein may be a component of the 7–8 S generating material. This is substantiated by the observation that trypsin or chymotrypsin treatment of the cytosol mitigates the ability of the cytosol to form the 7–8 S material and results in the appearance of a form of the receptor that sediments at approximately 6 S. Protease treatment of partially purified material eliminates the 7–8 S generating activity entirely. We conclude that the 7–8 S form of the receptor can be reconstituted from the 4 S receptor via association with at least two other cytosolic factors, a protein and an RNA.     

Binding of the rat liver 7-8 S dexamethasone receptor to deoxyribonucleic acid

The 7-8 S form of the [3H]dexamethasone (9 alpha-fluoro-11 beta,17,21-trihydroxy-16 alpha-methylpregna-1,4-diene-3, 20-dione) receptor from rat liver cytosol can be converted to the 3-4 S form by RNase treatment or high salt, suggesting a salt-sensitive association between the receptor protein and RNA. In DNA-cellulose column assays, the gradient-purified 3-4 S form bound DNA more efficiently than the 7-8 S form, though the 7-8 S form was also capable of binding to DNA-cellulose to a significant extent. Activated 7-8 S dexamethasone receptor could be released from its association with soluble DNA by treatment with DNase I. Sucrose gradient analysis showed that the released receptor sedimented as the 7-8 S form and was sensitive to RNase treatment, which induced a conversion to the 3-4 S form. Activated RNase-generated 3-4 S receptor again displayed a higher degree of binding to soluble DNA and was recovered in the 3-4 S form following DNase extraction. The fact that the 3-4 S form bound immobilized or soluble DNA more efficiently suggests that the associated RNA of the 7-8 S form interferes directly or indirectly with the receptor association with DNA. The observation that the receptor binds to DNA in its 7-8 S form suggests that the receptor complex is capable of binding RNA and DNA concurrently.     

Studies with chymotrypsin and RNAase showing a heterooligomeric structure of the glucocorticoid receptor complex from rat liver which is stabilized by a low molecular weight factor

The glucocorticoid receptor from rat liver displays a differential sensitivity toward digestion by chymotrypsin and RNAase A that is dependent on its activation state. Unactivated (9-10 S) receptor is not digested by these enzymes, while activated 7-8 S receptor is. Chymotrypsin treatment yields an approx. 3 S form, while RNAase treatment yields a 4.9 S form that is distinct from the high-salt 4 S form. To firmly establish that the results are due to specific hydrolytic activities of the particular enzymes, we show that the chymotrypsin effect is inhibited by diisopropylfluorophosphate and not RNAasin, while the reverse is true for RNAase A. We further show that the differential sensitivity toward chymotrypsin is due to the association of a proteinase-resistant, heat-stable low molecular weight factor with the unactivated glucocorticoid receptor. When this factor is removed by warming, dialysis or molecular sieving of the receptor complex, the complex becomes sensitive to chymotrypsin. We also show that moderate chymotrypsin treatment yields a 6-7 S form of the receptor which is composed of, at least, RNA and the 4 S receptor. On the basis of these results, we propose that the 9-10 S receptor is composed of a low molecular weight stabilizing factor whose presence apparently alters the conformation of the complex such that the RNA and the RNA-binding site of the receptor are protected, a chymotrypsin-sensitive factor, RNA and the 4 S receptor itself.     

Ribonuclease-induced transformation of progesterone receptor from rabbit uterus

The effect of RNase on the transformation of progesterone receptor from rabbit uterus was studied by density-gradient centrifugation and DNA-cellulose binding assay. The 7S form of the receptor in crude cytosol was RNase sensitive, and converted to the 4S form after RNase treatment. This reaction was prevented by an RNase inhibitor and reversed by the addition of ribosomal RNA. RNase treatment also caused a two-fold increase in the DNA binding of cytosolic receptor, and reduced the time required for heat-induced transformation. However, sucrose-gradient-purified progesterone receptor (7S) did not undergo transformation by warming unless exogenous RNase was added, thereby suggesting that a cytosolic factor, which might be endogenous RNase, is necessary for the heat-induced transformation of progesterone receptor. Furthermore, degradation of the receptors which occurred after prolonged warming at 25 degrees C in the presence of RNase could be prevented by the addition of DNA-cellulose to the reaction mixture. These results indicate that RNA is associated with the 7S form of progesterone receptor, and that its hydrolysis by RNase might be involved in the transformation of this receptor.     

Physical measurements of the liver glucocorticoid receptor.

Physical measurements were made on the cytosolic form of the liver [3H]dexamethasone receptor. These include a Stokes radius of 3.5 nm, determined by gel filtration, and sedimentation coefficients of 5.1 and 7-8S, by sucrose-density-gradient centrifugation. From these measurements, the following physical properties were calculated: apparent mol. wt. 78000 (the 5.1 S form); D app. 6.1 X 10(-7) cm2-S-1; f/fo 1.25; axial ratio 4.7; these indicate a globular protein. Measurements of sedimentation coefficient of cytosol steroid-receptor complexes previously subjected to various activating conditions gave different values and lead to the conclusion that the mechanism of activation in vitro enabling the steroid-receptor complex to bind to DNA is more complex than simple disaggregation to a uniform size.     

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 of the 8-9 S molybdate-stabilized estrogen receptor from low-affinity to high-affinity state without dissociation into subunits

The rate of dissociation of labeled estradiol from [3H] estradiol-8-9 S receptor complexes ([3H]E2-8-9 S ER) molybdate-stabilized was determined in the presence of either an excess of unlabeled hormone ("chase") or of charcoal/dextran suspension ("stripping"). Biphasic dissociation of the hormone was observed in both cases, but the fraction of the fast-dissociating component was dramatically reduced (5% instead of 60%) when stripping was used. As the dissociation patterns were independent of the degree of saturation of the receptor, the results do not favor the possibility of cooperative effects between binding sites in the 8-9 S ER. After pretreatment of cytosol by charcoal at 28 degrees C for 15 min, the dissociation studied by chase displayed only the slowly dissociating component (t1/2 approximately 65 min). This effect was dependent on temperature and influenced by the ligand bound to 8-9 S ER, being pronounced with estradiol (E2) and absent with [3H]4-hydroxytamoxifen. The slow-dissociating component obtained after charcoal treatment was reconverted to fast-dissociating state by adding dithiothreitol or by incubation with cytosol at 20 degrees C. The charcoal treatment did not change the sedimentation coefficient (approximately 9 S) and the Stokes radius (approximately 7 nm) of the [3H]E2-8-9 S ER, and the slow-dissociating form obtained did not bind to DNA-cellulose either in the presence or absence of molybdate ions. Thus there are likely small but functionally significant changes of structure in the 8-9 S ER which remain in a non-DNA-binding form, whereas the rate of estradiol dissociation is modified.     

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.     

Tyrosine phosphorylation of the receptor for insulin-like growth factor II is inhibited in plasma membranes from insulin-treated rat adipocytes.

We have identified a factor from rat liver cytosol that enhances the DNA-cellulose-binding ability of the glucocorticoid receptor and lowers the sedimentation value from 9-10 S to 4-5 S. Cytosol is prepared in the presence of molybdate, and unactivated receptor is isolated by chromatography on DEAE-cellulose in the presence of molybdate. This receptor sediments at 9-10 S and has little affinity for DNA. If the molybdate is removed and the receptor is incubated at 25 degrees C with the low-salt wash of the DEAE-cellulose column, DNA binding is enhanced by 50-600% relative to controls incubated with buffer only. In addition, the factor present in the low-salt wash converts the 9-10 S receptor into a mixture of 5 S and 4 S forms. The factor must be present during the incubation in order to exert its maximal effect. Factor added after the incubation has only marginal effects on the DNA-binding ability of the receptor, indicating that the factor does not increase the DNA-binding ability of activated receptor. Moreover, the factor is significantly less effective on receptor that has been activated before incubation with the factor. These results suggest that the factor acts as an activation enhancer. Preliminary characterization indicates that the activation enhancer is a trypsin-sensitive protein of approx. 70,000 Da, whose activation-enhancing properties are inhibited by ATP. RNAase A, which has effects similar to those described above on the 7-8 S receptor, does not mimic the effects of the activation enhancer on the 9-10 S receptor.     

DNA and ribonucleotide binding characteristics of two forms of the androgen receptor from rat prostates

Androgen receptors (sedimentation value approximately 4S and Stokes radius 2.8 nm) present in the cytoplasmic fraction obtained from prostates of castrated rats bind to DNA-Sepharose and double stranded DNA. A receptor fragment (sedimentation value approximately 3S and Stokes radius 2.3 nm) obtained from rat prostates in the course of a purification procedure showed greatly diminished binding affinity for both DNA-Sepharose and soluble DNA. In contrast, both the 4S cytosol receptor and the 3S receptor form interacted with equal affinity with prostate RNA or poly(UG). These observations provide evidence that for DNA binding a different or additional part of the receptor molecule is required than for RNA and polyribonucleotide binding.     

Intracellular inhibition of chromatin binding and transformation of androgen receptor by 3'-deoxyadenosine

Incubation of minced rat ventral prostate with 3'-deoxyadenosine (3'-dA) prior to labeling with the androgen, tritiated 7 alpha, 17 alpha-dimethyl-19-nortestosterone, reduced the level of androgen receptor bound to chromatin and increased the level of cytosolic androgen receptor and the fraction of cytosolic androgen receptor that did not bind to DNA. This effect was specific for 3'-dA and not mimicked by adenosine, 2'-deoxy-adenosine, cytidine, guanosine, or uridine. Adenosine was a competitive inhibitor of the 3'-dA effect. Labeled cytosolic androgen receptor from 3'-dA-treated prostate had properties that were similar to those exhibited by untransformed androgen receptor from prostate cytosol prepared in the presence of Na2MoO4, an inhibitor of receptor transformation in cell-free systems. Both androgen receptors had sedimentation coefficients of 8-9 S in low-salt gradients, did not bind to DNA tightly, and had a high affinity for DEAE-cellulose. The 3'-dA effect on these properties was not observed if androgen receptor from 3'-dA-treated prostate was isolated on high-salt gradients. These findings show that androgen receptor transformation does take place in intact prostate cells and suggest that 3'-dA inhibits chromatin binding of androgen receptor by interfering with androgen receptor transformation. The transformation process appears to involve removal of components from androgen receptor. Since 3'-dA is a potent inhibitor of the synthesis, polyadenylation, and nucleocytoplasmic transport of RNA, the 3'-dA effect may indicate a role for RNA in the mechanism of receptor transformation in intact target cells.     

Physicochemical characterization of the nuclear form of Ah receptor from mouse hepatoma cells exposed in culture to 2,3,7,8-tetrachlorodibenzo-p-dioxin

Molecular properties of nuclear aromatic hydrocarbon (Ah) receptor from Hepa-1c1c9 (Hepa-1) cells were assessed by velocity sedimentation on sucrose gradients and by gel permeation chromatography on Sephacryl S-300. Nuclear Ah receptor was obtained by exposing intact cells to [3H]-2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) for 1 h at 37 degrees C in culture followed by extraction of receptor from nuclei with buffers containing 0.5 M KCl. The nuclear Ah receptor was compared to the cytosolic Ah receptor from the same cells. Under conditions of low ionic strength, the Ah receptor from Hepa-1 cytosol sedimented as a single 9.4 +/- 0.63 S binding peak that had a Stokes radius of 7.1 +/- 0.12 nm and an apparent relative molecular mass of 271,000 +/- 16,000. After prolonged (24 h) exposure to high ionic strength (0.5 M KCl), cytosol labeled with [3H]TCDD exhibited two specific binding peaks. The large form of cytosolic Ah receptor seen under high ionic strength conditions sedimented at 9.4 +/- 0.46 S, had a Stokes radius of 6.9 +/- 0.19 nm, and an apparent Mr 267,000 +/- 15,000. The smaller ligand-binding subunit generated by exposing cytosol to 0.5 M KCl sedimented at 4.9 +/- 0.62 S, had a Stokes radius of 5.0 +/- 0.14 nm, and an apparent Mr 104,000 +/- 12,000. Nuclear Ah receptor, analyzed under high ionic strength conditions, sedimented at 6.2 +/- 0.20 S, had a Stokes radius of 6.8 +/- 0.19 nm, and an apparent Mr 176,000 +/- 7000. Nuclear Ah receptor from rat H4IIE hepatoma cells was analyzed and found to have physicochemical characteristics identical to those of nuclear Ah receptor from the mouse Hepa-1 cells. The molecular mass of Hepa-1 nuclear Ah receptor was found to be statistically different from both the Mr approximately 267,000 cytosolic Ah receptor and the Mr approximately 104,000 subunit which were present in cytosol under high ionic strength conditions. Hepa-1 nuclear Ah receptor could not be converted to a smaller ligand-binding subunit by treatment with alkaline phosphatase, ribonuclease, or sulfhydryl-modifying reagents or prolonged exposure to 1.0 M KCl. Cytosolic Ah receptor from Hepa-1 cells was "transformed" by heating at 25 degrees C in vitro into a form with high affinity for DNA-cellulose. The transformed cytosolic Ah receptor, when analyzed under conditions of high ionic strength, sedimented at approximately 6 S, had a Stokes radius of approximately 6.7 nm, and an apparent Mr approximately 167,000.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

Isolation and physical study of the 13S fragment of 16S RNA and its complex with ribosomal protein S4

A fragment of E. coli 16S RNA has been obtained by its hydrolysis with pancreatic RNAase A coupled to Sepharose 4B. This fragment has a molecular weight of 170 000 and a sedimentation coefficient of 13S. It does not aggregate in solution and binds with the ribosomal protein S4. The 13S fragment and it complex with the protein S4 have been studied by different physical methods in the first place, by neutron scattering. It has been shown that this fragment is compact in solution. The radii of gyration of the fragment (50 +/- 3 A) and of the protein S4 within the complex (17 +/- 3 A) coincide, within limits of experimental error, with the radii of gyration for the free RNA fragment (47 +/- 2 A) and the free ribosomal protein S4 in solution (18 +/- 2 A). Hence, the conclusion is made that the compactness of the 13S fragment of the 16S RNA and the ribosomal protein S4 does not change at the complex formation. The compact 13S fragment of the 16S RNA is shown to be contrast matched in the H2O/D2O mixture containing 70% D2O which corresponds to its partial specific volume v equal to 0.537 cm3/g.     

The glucocorticoid antagonist 17 alpha-methyltestosterone binds to the 10 S glucocorticoid receptor and blocks agonist-mediated dissociation of the 10 S oligomer to the 4 S deoxyribonucleic acid-binding subunit

The glucocorticoid antagonist 17 alpha-methyltestosterone inhibits binding of the agonist [3H]triamcinolone acetonide ot the glocucorticoid receptor in cytosol prepared from rat pituitary tumor GH1 cells. Competitive binding studies indicate that the dissociation constant for 17 alpha-methyltestosterone is about 1 microM. After incubation of intact GH1 cells with 10 nM [3H]triamcinolone acetonide at 37 C and subsequent cell fractionation at 4 C, three glucocorticoid receptor forms are observed: cytosolic 10 S receptor, cytosolic 4 S receptor, and nuclear receptor. Concurrent incubation with 17 alpha-methyltestosterone reduces the amount of [3H]triamcinolone acetonide bound to each of these receptor forms. Ligand-exchange assays performed at 0 C in intact cells using [3H]triamcinolone acetonide show that the exchangeable antagonist is associated predominantly with cytosolic 10 S receptor. Immunochemical analysis using monoclonal antibody BuGR2 indicates that 17 alpha-methyltestosterone does not cause substantial accumulation of glucocorticoid receptors in GH1 cell nuclei and, when present together with agonist, reduces nuclear accumulation of receptor seen with agonist alone. Results from dense amino acid labeling studies show that unlike [3H]triamcinolone acetonide, 17 alpha-methyltestosterone does not reduce the total amount of cellular glucocorticoid receptor and does not reduce receptor half-life. These results are consistent with a model for glucocorticoid receptor transformation in which binding of agonist promotes the dissociation of an oligomeric 10 S cytosolic receptor protein to its DNA-binding 4 S subunit. The antagonist 17 alpha-methyltestosterone competes with agonist for binding to the 10 S cytosolic receptor but does not appear to promote dissociation of the oligomer, thus inhibiting agonist-mediated nuclear actions of the glucocorticoid receptor.     

Characterization of the Ah receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin: use of chemical crosslinking and a monoclonal antibody directed against a 59-kDa protein associated with steroid receptors

The Ah receptor regulates induction of cytochrome P450IA1 (aryl hydrocarbon hydroxylase) by "3-methylcholanthrene-type" compounds and mediates the toxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin and related halogenated aromatic hydrocarbons. Hepatic Ah receptor from untreated rodents is localized in the cytosol and has an apparent molecular mass of 250 to 300 kDa. This large form can be dissociated into a smaller ligand-binding subunit upon exposure to high ionic strength. The Ah receptor displays many structural similarities to the receptors for steroid hormones. Two non-ligand-binding proteins have been identified to be associated with the cytosolic forms of the steroid hormone receptors. The first is a 90-kDa heat shock protein (hsp 90); the second is a 59-kDa protein (p59) of unknown function. The cytosolic Ah receptor ligand-binding subunit previously has been shown to be associated with hsp 90. In the present study, we used a monoclonal antibody, KN 382/EC1, generated against the 59-kDa protein which is associated with rabbit steroid receptors to determine if p59 also is a component of the large cytosolic Ah receptor complex. Cytosolic forms of rabbit progesterone receptor, glucocorticoid receptor, and Ah receptor were analyzed by velocity sedimentation on sucrose gradients under low-ionic-strength conditions and in the presence of molybdate. Progesterone receptor from rabbit uterine cytosol and glucocorticoid receptor from rabbit liver each had a sedimentation coefficient of approximately 9 S. In the presence of KN 382/EC1 antibody the progesterone receptor and the glucocorticoid receptor both underwent a shift in sedimentation to a value of approximately 11 S. The increase in sedimentation velocity is an indication that the receptor-protein complexes are interacting with the antibody. Under low-ionic-strength conditions the Ah receptors from rabbit uterine cytosol and liver cytosol had a sedimentation coefficient of approximately 9 S. However, in contrast to the steroid receptors, the Ah receptor showed no change in its sedimentation properties in either tissue in the presence of KN 382/EC1, indicating that the antibody is not interacting with the Ah receptor. Multimeric Ah receptor complexes that were chemically crosslinked still did not show any interaction with KN 382/EC1. These data indicate that the 59-kDa protein either is not associated with the Ah receptor or is present in an altered form which the antibody cannot recognize.     

Correlation of the 4-5 S form and the 8 S form of the cytosolic androgen receptor in murine skeletal muscle

Binding experiments with the cytosolic androgen receptor from murine skeletal muscle yield with testosterone a biphasic saturation curve and a biphasic Scatchard plot. These binding characteristics result from the conversion of 8 S receptor (KD = 1,4 X 10(-10) M) into 4-5 S receptor (KD = 1,2 X 10(-9) M). This conversion is androgen dependent and is facilitated in vitro by either UV-irradiation or by methods known to activate steroid hormone receptor complexes to a nuclear binding form (e.g. high ionic strength or elevated temperature). The measured data show that both receptor forms are in a complex dissociation equilibrium. The reassociation of the 4-5 S receptor to form the 8 S complex is inhibited by RNase.     

Estradiol-17β receptors in the immature rat ovary

Estradiol binding components in the cytosol and nuclear fractions of the ovary from immature rats (22–28 days old) were characterized by $\text{in vitro}$ methods. Several of the biochemical characteristics of the estradiol binding components in the ovarian tissue were compared with the estradiol receptor from the uterus. The results suggest that the ovarian estradiol binding components are similar to the specific high affinity estradiol receptors in the uterus. In the cytosol of intact rat ovary a significant fraction of the total binding sites was found to be occupied, presumably by the endogenous estrogen. Following hypophysectomy there was a significant increase in the available cytosol binding sites. Evidence for translocation of cytosol receptor-estrogen (RE) complex to the nucleus was obtained for the ovary. The sedimentation properties of the RE complex of the ovary and the uterus are similar. The ovarian cytosol RE complex sediments at 7-8S in glycerol gradients at low ionic strength and at 4S in sucrose gradients at high ionic strength. Following extraction with 0.4 M KCl the ovarain nuclear RE complex sediments at 5S in sucrose gradients which is identical to that of the uterine nuclear receptor.     

Interconversion of androgen receptor forms by divalent cations and 8 S androgen receptor-promoting factor. Effects of Zn2+, Cd2+, Ca2+, and Mg2+

The effects of divalent cations (Zn2+, Cd2+, Ca2+, Mg2+) on the cytosol androgen receptor were determined by sedimentation into sucrose gradients. At low ionic strength (25 mM KCl, 50 mM Tris, pH 7.4), Zn2+ (200 microM total, which calculates to 130 nM free Zn2+ in 10 mM mercaptoethanol) causes a shift in the sedimentation coefficient of the rat Dunning prostate tumor (R3327H) cytosol receptor and rat ventral prostate cytosol receptor from 7.5 +/- 0.3 S to 8.6 +/- 0.3 S. Zn2+ stabilizes the 8.6 S receptor form in salt concentrations up to 0.15 M KCl in 50 mM Tris, pH 7.2. In low ionic strength gradients containing Ca2+ (greater than or equal to 200 microM) or Mg2+ (greater than or equal to 1 mM), the receptor sediments as 4.7 +/- 0.3 S. The dissociating effects of Ca2+ and Mg2+ can be fully reversed by sedimentation into gradients containing Zn2+ (200 microM total) or Cd2+ (10 microM total). In the presence of Zn2+ (200 microM total), Ca2+ (10 microM to 3 mM) converts the receptor to an intermediate form with sedimentation coefficient 6.2 +/- 0.2 S, Stokes radius 73 A, and apparent Mr approximately 203,000. The potentiating effect of Zn2+ on formation of the 8.6 S receptor (in the absence of Ca2+) and the 6.2 S receptor (in the presence of Ca2+) requires both the 4.5 S receptor and the 8 S androgen receptor-promoting factor. Sodium molybdate stabilizes the untransformed cytosol receptor but, unlike Zn2+, does not promote reconstitution of the 8.6 S receptor from its partially purified components. These results indicate that divalent cations alter the molecular size of the androgen receptor in vitro and thus may have a role in altering the state of transformation of the receptor.