Cytosolic Receptor Complex Research Articles

Possible heterogeneity of glucocorticoid receptors: Studies on liver and heart cytosols

After incubation of liver and heart cytosol preparations with 4 × 10 −8 M [ 3H]-TA (triamcinolone acetonide), passing the incubate through a phosphocellulose (PC) column, thermal activation, and chromatography on a second PC-column, three glucocorticoid-binding components were found, which eluted at about 0.32 M, 0.43 M and 0.51 M KCl. Fractions detected are believed to be specific glucocorticoid-binding proteins, since their labeling by [ 3H]-TA is eliminated by heating the cytosol for 30 min at 37°C or the addition of excess non-labeled TA. The first glucocorticoid-binding component is readily absorbed during passage of thermally activated cytosol through DEAE-cellulose while the two latereluting components are more weakly retained by this synthetic polycation. These components interact with DNA-cellulose and can be eluted in the narrow range of elute KCl concentrations. The values of [ 3H]-TA-binding to the liver glucocorticoid-binding fractions are several times higher than for heart cytosol. Nevertheless, three peaks of the activated form of heart cytosol receptor complexes can be detected, more or less evidently, at the same elution positions on the PC column at various endocrine states of animals, in normal female and male subjects, in experimental thyreotoxicosis, and in glucocorticoid deficiency caused by adrenalectomy. In the latter case specific [ 3H]-TA binding was increased more than 2-fold in each of the three glucocorticoid-binding fractions. Incorporation of [ 3H]-TA into the three glucocorticoid-binding fractions of heart cytosol is severely depressed by a 100-fold excess of non-labeled deoxycorticosterone (DOC), while in liver cytosol preparations diluted as much as 5-fold and containing amounts of [ 3H]-TA receptor complexes comparable to that of heart cytosol, this cannot be achieved. Estimation of specific [ 3H]-aldosterone binding to heart cytosol of both intact and adrenalectomized rats showed that none of the glucocorticoid-binding fractions described can be attributed to a “pure” mineralocorticoid receptor.

Inactivation of rat liver glucocorticoid receptor by molybdate. Effects on both non-activated and activated receptor complexes.

When freshly prepared glucocorticoid-receptor complex from rat liver cytosol was incubated at 23 degrees C in the presence of sodium molybdate, its subsequent binding to isolated nuclei, DNA-cellulose and ATP-Sepharose was blocked. In addition, binding to these acceptors by cytosol receptor complex fractionated with (NH4)2SO4 was also blocked by incubation of the complexes with 50 mM-sodium molybdate. However, molybdate had no effect on the binding of activated receptor complexes to ATP-Sepharose. Molybdate was also effective in extracting the nuclear- and DNA-cellulose-bound glucocorticoid-receptor complexes in a dose-dependent manner. Molybdate appears to exert its effects directly on the receptor by interacting with both non-activated and activated receptor forms.

Interaction of a high affinity anti-estrogen (alpha-[4-pyrrolidinoethoxy]phenyl-4-hydroxy-alpha'-nitrostilbene, CI628M) with uterine estrogen receptors.

The polar metabolite of the anti-estrogen alpha-[4-pyrrolidinoethoxy]phenyl-4-methoxy-alpha'-nitrostilbene (CI628), that is selectively accumulated by the nuclear estrogen receptor in vivo, is identified as the corresponding free phenol (alpha-[4-pyrrolidinoethoxyl]phenyl-4-hydroxy-alpha'-nitrostilbene (CI628M) and has been prepared in high specific activity tritium-labeled form to study its interaction with the rat uterine estrogen receptor. CI628M has an affinity for the cytosol receptor that is comparable to that of estradiol (Kd (CI628M) = 0.16 nM; Kd (estradiol) = 0.28 nM), and the binding of these two compounds is mutually competitive. Dissociation of the CI628M complex with the estrogen receptor is somewhat slower than that of the estradiol complex, and prewarming the complexes at 28 degrees C causes a characteristic decrease in the rate of dissociation. On low salt sucrose gradients, the CI628M and estradiol cytoplasmic receptor complexes both sediment as 8S species; their sedimentation profiles on high salt gradients are somewhat different, however. While the estradiol receptor complex shows mainly a 4.6S species that shifts to a 5.8S species upon warming, the unwarmed CI628M complex is predominantly 5.8S and becomes somewhat more disperse upon warming. Under high salt conditions, there is a progressive shift of the 4.6S to the 5.8S species as cytosol concentration increases; the CI628M complex shows a greater tendency to form the more rapidly sedimenting form at all cytosol concentrations. After in vivo injection of [3H]CI628M, the uterine nuclear receptor sediments as a characteristic 5S species. Cytosol receptor complexes with estradiol and CI628M, maintained at 0 degrees C, show very little binding to ATP-Sepharose, while after warming, 50-60% of both complexes are bound by the resin. Both complexes also show similar elution profiles from DNA-cellulose columns; however, the extent of binding of the [3H]CI628M receptor complex to the column is consistently only 40-50% that of the estradiol receptor complex. These results indicate that the properties of the uterine estrogen receptor complexed with estradiol and with the high affinity anti-estrogen CI628M are very similar. The differences observed in the behavior of the CI628M cytosol receptor complex on high salt sucrose gradients and on DNA-cellulose columns, though quantitative rather than qualitative, may be indicative of differences in receptor properties that are important to the antagonist activity of this compound.

Subcellular distribution of androgen binding sites in cultured Sertoli cells before and after exposure to androgens

When rat Sertoli cells in culture were exposed to [ 3H]-labeled androgen over 20 min at 34°C, the concentration of [ 3H]-androgen-receptor complex in the cytosol fraction rapidly increased and then declined. Over this same interval, total (i.e. occupied plus unoccupied) receptor levels, measured by an exchange assay, also declined. A substantial proportion of the receptor remaining in the cytosol after 20 min was bound to steroid. Concomitant with these changes, the cytosol/nuclear ratio of specifically bound steroid rapidly declined as label accumulated in the paniculate fraction. By 20 min, nuclear bound label often exceeded cytoplasmic receptor depletion. Some cytosol receptor may adhere to particulate matter and be present in the Triton X-100 washes of the crude nuclear pellet. In cells not exposed to androgen, a substantial number of unoccupied, tightly bound nuclear androgen binding sites of high affinity were measured by incubation of washed pellets with labeled steroid at 0°C. Following exposure to labeled steroid at 34°C, the net number of unoccupied nuclear binding sites declined. These data indicate that while some specifically bound steroid in the Sertoli cell nuclear fraction arises from translocation of cytosol receptor complexes, steroid probably also binds to previously unfilled nuclear binding sites.

Studies on cytosol and nuclear binding of estradiol in human breast cancer

Defects in the translocation of the cytosol estradiol receptor (ERC)-estradiol complex to the nucleus could partly explain the failure of endocrine therapy in 40% of breast cancer patients with significant ERC. To examine this possibility, 98 cases of human breast cancer were investigated for cytoplasmic and nuclear (ERN) estradiol receptors. ERC was assayed by a charcoal method following incubation with [ 3H]-estradiol. Nuclei were isolated from the low speed sediment and incubated at 30°C with the ERC-[ 3H]-estradiol complex in the presence and absence of an estrogen competitor. ERN was extracted from the nuclei with 0.4 M KCl and expressed as specifically bound estradiol, fmol/mg DNA. The quantity of ERN ranged between 0–746 fmol/mg DNA. Of 50 cases with significant ERC activity, 11 had no ERN (24%). In the borderline ERC group of 17 tumors, 5 had no ERN (29%), while of the 31 cases with zero or negligible ERC, 27 eases had no ERN (87%). Characteristics of ERC are different from the classical model. Chromatography of ERC on Sephacryl-S-200 columns and sucrose gradient sedimentation values indicate that the original form has an approximate molecular weight of 35,600, which on warming is transformed into an even smaller form (mol. wt — 22,700). The larger form of ERC (mol. wt ∼- 250,000) is produced by aggregation in low ionic strength media. The major form of the ERN complex has a molecular weight of 35,600, the same as that of the cytosolic receptor complex. It appears that the absence of ERN in a significant proportion of ERC positive tumors could contribute to the failure of endocrine therapy in such patients. Results indicate that the determination of both ERC and ERN could improve the selection of patients for endocrine therapy.

5α-Dihydrotestosterone nuclear receptor and rna polymerases activity in the male rat anterior pituitary (in vitro studies)

The presence of specific 5α-DHT receptors in the male rat anterior pituitary has been previously reported. Using a “reonstitutive” cell-free system we demonstrated that the specific 5α-DHT cytosol receptor complex is translocated to the nucleus at 25°C. The complex is 3.6 S after extraction of the nuclei with 1 M NaCl. Experiments to test the role of this 5α-DHT nuclear complex on RNA synthesis in cell free conditions have been performed. No effect on RNA polymerases activity was observed.

Cytosol and nuclear progesterone receptor in cat uterus and oviduct

Binding of progesterone (P) to a specific cytosol receptor and the in vivo translocation of the cytosol receptor complex to the nucleus was studied in the cat uterus and oviduct. Cytosol and nuclear fractions were prepared from the reproductive tracts of ovariectomized animals treated for 14 days with estradiol (E 2) and from those which were subsequently infused with a P solution for 15 min. In vitro cytosol incubation with [ 3H]-P included cortisol to eliminate binding to CBG. The specific binding of [ 3H]-P reached equilibrium within 90 min at 0°C and was stable for at least 4 h in both tissues. Competitive binding studies revealed the following binding affinities for the cytosol receptor; R5020 (17,21-dimethyl-19-nor-4,9-pregnadiene-3,20-dione) = P ⪢ corticosterone = E 2 = testosterone. Scatchard analysis of [ 3H] P binding in cytosols from E 2-treated animals yielded a K D of 3.2 × 10 −8 M in the uterus and 2.5 × 10 −8 M in the oviduct. The P binding protein had a sedimentation coefficient of 6.8 S. The content of nuclear receptor was assessed by an exchange assay using R5020. Equilibrium was reached within 4 h at 22°C and remained unchanged for another 2h. The relative binding affinities for the nuclear receptor were; R5020 > P > corticosterone > testosterone > E 2. The K D was 8.2 × 10 −9 M in uterine nuclei and 6.0 × 10 −9M in oviduct nuclei from E 2 + P treated animals. Following P-infusion the decrease in the quantity of specifically bound [ 3H]-P in the cytosol, and the increase in the quantity of specifically bound [ 3H]-R5020 in the nuclei, were significant in both tissues.

Selective binding of mouse estradiol.receptor complexes to oligo(dT)-cellulose

As a model for the nonspecific interaction of steroid receptors with DNA, the binding of estradiol.receptor complexes of mouse kidney and uterine cytosols to oligo(dT)-cellulose was studied in detail. A limited concentration range of monovalent cationic salts was required for optimal binding, regardless of prior activation of the receptor complexes or the oligomer ligand. Thermal activation of the receptor complexes did not facilitate binding. The reaction was selective for intracellular steroid hormone.receptor complexes, as extracellular proteins binding estradiol with low affinity (bovine serum albumin) or high affinity (mouse alpha-fetoprotein) were inactive. Both crude and partially purified kidney cytosol receptor complexes bound preferentially to oligo(dT)- and oligo(dC)-celluloses, rather than oligo(dA)-celluloses. These findings suggest that at least part of the nonspecific interaction of estradiol.receptor complexes with native DNA is through a salt-sensitive binding of the complex to pyrimidine-rich surfaces of the DNA.

A new assay method for the determination of oestrogen receptors.

A new assay method was developed to determine the quantity of oestrogen cytoplasmic receptors which are capable of associating with acceptor sites on chromatin. The quantity of oestrogen receptor complexes associated with chromatin is of paramount importance in the mechanism of action of oestrogen. This method is based on the specific binding properties of oestrogen to the receptor and on the binding of the complex to chromatin. Cytosol was pre-incubated with [3H]oestradiol-17 beta ([3H]Oe2) to form a complex, and further incubated with a constant amount of chromatin. After this the quantity of [3H]Oe2 cytosol receptor complexes specifically associated with the chromatin was determined. The binding activity of the chromatin to the [3H]Oe2 receptor complex was stable for at least six months in 0.15 M NaCl at -20 degrees C. This method enables us to determine the quantity of the biologically active oestrogen receptors more specifically than with other methods.

Differences between cytosol receptor complexes with corticosterone and dexamethasone in hippocampal tissue from rat brain

The binding of [ 3H] corticosterone and [ 3H] dexamethasone to soluble macromolecules in cytosol of the hippocampal region of the brain has been studied in adrenalectomized male rats. Unlabeled dexamethasone appears to be a less effective competitor than corticosterone in the binding of [ 3H] corticosterone, while both unlabeled steroids compete equally well for the binding of [ 3H] dexamethasone. Further investigation of macromolecular complexes with [ 3H] dexamethasone and [ 3H] corticosterone revealed that they differ from each other in their behavior during ammonium sulfate precipitation, BioRad A-5M gel permeation chromatography, DE-52 anion exchange chromatography, and DNA-cellulose chromatography. 1. (1) After exposure to a 33% ammonium sulfate solution relatively more [ 3H] dexamethasone complex than [ 3H] corticosterone complex is precipitated. 2. (2) Treatment of the cytosol with 0.3 M KCl gives disaggregation of the supramolecular 3H-labeled corticoid complexes which are seen eluting with the void volume during gel permeation chromatography on Biorad A-5M at low ionic strength. In 0.3 M KCl, the [ 3H] dexamethasone complex has an elution volume somewhat smaller than that of bovine serum albumin, while the [ 3H] corticosterone complex in 0.3 M KCl is to unstable to survive chromatography with A-5M. 3. (3) Chromatography on DE-52 resolved the 3H-labeled corticoid complexes into three binding components. The complex with [ 3H] dexamethasone contains a higher percentage (85%) of a component less firmly attached (i.e. eluted by 0.15 M KCl) to the anion exchange resin than is observed for the complex with [ 3H] corticosterone (49%).

Receptor transformation, the key to estrogen action

The transforrnation of the native (4S) uterine cytosol-receptor complex to an active (5S) form is an estrogen-requiring, temperature-dependent process, the rate of which is affected by the pH and ionic strength of the cytosol. Unlike its 4S precursor, the transformed complex is retained by uterine nuclei and chromatin but by non-target tissue chromatin as well. The specificity of the in vitro nuclear uptake of transformed receptor complex appears to reside in the biochemical response; transformed receptor effects in vitro the same target tissue specific, quantitative stimulation and qualitative change in nuclear RNA synthesis as that observed in the target tissue after administration of the hormone in vivo.