Characterization Of Steroid Receptors Research Articles

Characterization of Steroid Receptors in Human Ovarian Cancer

Steroid receptors for estrogen, progestogen and androgen in human ovarian cancer were characterized. 159,800xg supernatant was used as cytosol. In the cytosol, sucrose density gradients showed 5S and 8S binding peaks for each receptor. Steroid specificity studies showed that estrogen for [3H]-estradiol-receptor binding, progestogen for [3H]-R5020-receptor binding and androgen for [3H]-DHT-receptor binding were specific inhibitors. Equilibrium studies showed that the dissociation constant(Kd) was (15.3±3.1)x10-10M(n=8) for [3H]-estradiol receptor binding, (2.3, 4.6)x10-10M(n=2) for [3H]-R5020 receptor binding and (6.6, 26.0)x10-10M(n=2) for [3H]-DHT receptor binding.

Steroid receptors and their associated proteins.

Those investigators involved in the purification and characterization of steroid receptors have been confronted with a number of difficulties, one being the expanding list of cellular proteins that can bind and copurify with these receptors. Research over the past 8-10 yr has established a high degree of acceptance for the concept that most inactive steroid receptors are sequestered in a complex with the 90-kilodalton (kDa) heat shock protein, hsp90 (see refs. 1, 2 for review). Initial observations on the copurification of this 90-kDa protein were confusing, and in some cases it was thought to be a contaminant or to actually be the receptor. Clarification of a receptor:hsp90 complex was provided by several approaches which mainly involved the use of antibodies to rapidly and gently isolate receptor complexes. That such a complex exists in the cell is supported primarily by three types of experiments The metabolic labeling studies of Howard and Distelhorst (3) argued that hsp90 binding was not simply occurring after cell lysis. Yeast mutants having an abnormally low level of hsp90 have been shown to be inefficient for the expression of functional steroid receptors (4). Most recently, Gehring and coworkers (5) have used chemical cross-linking in intact cells to show an association of glucocorticoid receptor with hsp90 and a second protein, p59. While a role of hsp90 in receptor structure is strongly supported, the topic of receptor-associated proteins is still quite confusing and uncertain. Most more recent members of this list (see Table 1) suffer from a lack of information regarding their identity and their presence or absence in a variety of receptor systems. Whether they truly represent intracellular receptor-associated proteins of significance to hormone action is unknown, and this represents a key topic for future research in this area. In this brief review, we will attempt to consolidate studies from a variety of systems, clarify some of the confusing terminology about receptor-associated proteins, and suggest some rationale for their participation

Characterization of steroid receptors in the gut and kidney of the frog ( Rana catesbeiana) and in the gut of the turtle ( Chrysemys picta)

Para glucocorticoid- and paramineralocorticoid-binding cytosolic receptors ( pGR, pMR)‡ were demonstrated in the intestine and kidney of the frog, Rana catesbeiana and in the intestine of the turtle, Chrysemys picta, in the presence of sodium molybdate. These receptors were of high affinity and low capacity with the following binding parameters: pGR: K d: frog intestine (FI), triamcinolone acetonide (TA): 3.3 nM, corticosterone (B): 3.4 nM; frog kidney (FK), TA: 4.3 nM, B: 9.3 nM; turtle intestine (TI), TA: 4.8 nM; N max: FI, TA: 357, B: 371; FK, TA: 301, B: 157; TI, TA: 350 fmol/mg protein. pMR: K d. FI, aldosterone: 0.9 and 90 nM (biphasic curves); FK, aldosterone: 0.6 and 36 nM (biphasic curves); N max: FI, 13 and 147 fmol/mg protein; FK, 78 and 109 fmol/mg protein. The receptor had the following ligand affinities: pGR: FI and FK: triamcinolone acetonide ⪢ DOC ⪢ 11β -hydroxyprogesterone ⪢ progesterone ⪢ corticosterone ⪢ cortisol ⪢ aldosterone ⪢ 11-dehydrocorticosterone ⪢ 17α-hydroxyprogesterone ⪢ cortisone; TI: triamcinolone acetonide ⪢ corticosterone ⪢ progesterone ⪢ DOC ⪢ cortisol ⪢ aldosterone; pMR: FI and FK: corticosterone ⪢ 11β-hydroxyprogesterone ⪢ aldosterone ⪢ triamcinolone acetonide = cortisol ⪢ DOC ⪢ 11-dehydrocorticosterone ⪢ progesterone ⪢ 17α-hydroxyprogesterone ⪢ cortisone. Androgens, estrogens or 18-hydroxycorticosterone did not compete for binding in either tissue. The heat activated frog receptors did not bind to naked DNA, though the turtle receptor did. It was possible to show that cytosol receptor-ligand complexes from all tissues were bound by nuclear acceptor sites. On linear sucrose gradients, the FI TA-receptor complex sediments with a single peak (7.5S), the FK TA-receptor complex gave two peaks (8.0 and 4.4S) and the TI TA-receptor complex showed a single peak (9.0S). The hydrodynamic parameters of the pGR's were determined by gel exclusion on Sephacel S-300. The following results were obtained: Mr: FI, 265, 80, 40 kDa (multiple proteins); FK, 280, 60, 20 kDa (multiple proteins); TI, 366 kDa; R s: FI, 6.9, 3.9 nm; FK, 6.9, 2.9 nm; TI, 7.6 nm; f/f 0: FI, 1.6; FK, 1.6; TI, 1.6. It is suggested on the basis of the binding and hydrodynamic parameters that non-mammalian epithelial corticosterone receptors have undergone biochemical evolution from one class of vertebrates to another.

Characterization of steroid receptors in human prostate using mibolerone

Accurate quantitation of androgen receptors requires a radioactive ligand which has affinity and specificity for the receptor and which is stable to metabolic enzymes. In this report, we have characterized the properties of 7 alpha,17 alpha-dimethyl-17 beta-hydroxy-4-estren-3-one (mibolerone) in human benign hyperplastic prostate cytosol and compared them to those of 17 beta-hydroxy-17 alpha-methyl-estra-4,9,11-trien-3-one (R1881). Mibolerone was found to have an affinity (Kd = 1.5 nM) greater than R1881. (Kd = 2.3 nM) for the androgen receptor in human prostate tissue. Surprisingly, mibolerone was found to bind with high affinity to the progesterone receptor in both human prostate (Kd = 5.9 nM) and rabbit uterus (Kd = 1.1 nM). However, binding to this receptor in both species could be blocked with a 500-fold excess of triamcinolone acetonide. [3H]Mibolerone binding to the androgen receptor was competed effectively with unlabeled dihydrotestosterone, R1881, and mibolerone but not by progesterone, diethylstilbestrol or R5020, in the presence of triamcinolone acetonide. Interestingly, mibolerone was more resistant to metabolism than R1881 in prostate cytosol when exposed to elevated temperatures (30 degrees C) for extended periods of time. However, when exposed to high-intensity ultraviolet irradiation, both compounds lost 50% of their binding ability in about 30 minutes. Mibolerone was found to have a very low affinity (Ki = 540 nM) for human sex steroid binding protein. These studies demonstrate that mibolerone is a useful ligand for androgen receptor assays. They also emphasize the need for including competitors of progesterone receptor binding in assays utilizing this steroid for androgen receptor measurements.

The use of immobilized estradiol antiserum in the study of receptors and other estradiol-binding proteins

Antiestradiol antibody immobilized on a polymer film is set in competition for tritium-labeled estradiol with estradiol receptor or antiestradiol antibody in solution. Measurement of equilibrium quantity of radiolabel in test solutions in the presence and in the absence of dissolved antibody or receptor gives a data base for evaluating the association constant and the quantity of the estradiol-complexing species in solution. The immobilized antibody method avoids the problems and uncertainties arising in the step of separating the bound from the free hormone which is the pivotal step in all currently used procedures for the determination and characterization of steroid receptors. With the exception of equilibrium dialysis, it is the only procedure presently available to study steroid antibodies and receptors in solution at unperturbed equilibrium. Using the immobilized antibody method at 4°C, 0.86 ± 0.12 × 10 11 m −1 was the association constant found for estradiol receptor in rat uterine cytosol, and 2.6 ± 0.5 × 10 11 m −1 was the association constant found for antiestradiol antibody raised in a rabbit to estradiol linked to bovine serum albumin via a C-6 carboxymethyloxime.

The use of anti-steroid antibodies in the characterization of steroid receptors.

The use of anti-steroid antibodies in the characterization of steroid receptors.