Androgen-binding Capacity Research Articles

Substitution of Aspartic Acid-686 by Histidine or Asparagine in the Human Androgen Receptor Leads to a Functionally Inactive Protein with Altered Hormone-Binding Characteristics

We have identified two different single nucleotide alterations in codon 686 (GAC; aspartic acid) in exon 4 of the human androgen receptor gene in three unrelated families with the complete form of androgen insensitivity. One mutation (G----C) results in an aspartic acid----histidine substitution (with 15-20% of wild-type androgen-binding capacity), whereas the other mutation (G----A) leads to an aspartic acid----asparagine substitution (with normal androgen-binding capacity, but a rapidly dissociating ligand-receptor complex). The mutations eliminate a Hinfl restriction site. Screening for the loss of the Hinfl site in both families with the Asp----Asn mutation resulted in the recognition of heterozygous carriers in successive generations of each. Both mutant androgen receptors were generated in vitro and transiently expressed in COS and HeLa cells. The receptor proteins produced had the same altered binding characteristics as those measured in fibroblasts from the affected subjects. R1881-activated transcription of a GRE-tk-CAT reporter gene construct was strongly diminished by both mutant receptors and was only partially restored using a 100-fold higher concentration of ligand compared with wild-type receptor. Thus, aspartic acid-686 appears essential for normal androgen receptor function. Substitution of this amino acid residue, by either histidine or asparagine, results in androgen insensitivity and lack of androgen-dependent male sexual differentiation.

A single base mutation in the androgen receptor gene causes androgen insensitivity in the testicular feminized rat.

The complete form of androgen insensitivity is an inherited X-linked syndrome in which genetic males fail to undergo masculinization in utero due to defective functioning of the androgen receptor (AR). The molecular basis of androgen insensitivity was investigated in the testicular feminized (Tfm) rat with this syndrome. AR mRNA size and amount, as well as nuclear AR protein revealed by immunocytochemistry, suggested normal expression of the AR gene in the Tfm rat. Sequence analysis of the AR coding region from Tfm and wild-type littermate male rats revealed a single transition mutation, guanine to adenine, within exon E, changing arginine 734 to glutamine within the steroid-binding domain of the AR. This arginine is highly conserved among the family of nuclear receptors and may be part of a phosphorylation recognition site. A recreated mutant AR (Arg734----Gln) expressed in COS cells had only 10-15% of the androgen-binding capacity of wild-type AR; the reduced androgen-binding capacity was similar to that of AR in tissue extracts of the Tfm rat. Stimulation of transcriptional activity by the recreated mutant AR was reduced relative to wild-type AR in cotransfection assays in CV1 cells using as reporter plasmid the mouse mammary tumor virus promoter linked to the chloramphenicol acetyltransferase gene. Thus, arginine 734 appears essential for normal AR function both in androgen binding and transcriptional activation. Absence of these functions results in androgen insensitivity and lack of male sexual development.

Association of androgen binding sites with the endoplasmic reticulum of rat ventral prostate

Microsomes from ventral prostate of 24-h castrated rats contain a single set of tissue-specific high-affinity, low-capacity androgen binding sites. These sites are indigenous to the endoplasmic reticulum, as shown by purification procedures associated with marker enzymes and electron microscopic analyses. When prostatic microsomal membranes are separated from plasma membranes using the nuclear or the mitochondrial pellets as the source of fractionation in sucrose gradients, the androgen binding activity is selectively associated with fractions rich in rough endoplasmic reticulum and ribosomes. Eighty-four percent of the total content of Na+/K+ adenosine triphosphatase (ATPase) and only 27% of the total binding capacity were concentrated in fractions rich in smooth-surfaced vesicular membranes, when nuclear suspensions constituted the membrane source. In contrast, the region of the same gradient when enriched in rough endoplasmic reticulum and deficient in plasma membrane content contained 73% of the androgen-binding capacity and only 14% of the ATPase. For fractions collected using mitochondrial suspensions as starting material, the ratio (total glucose-6-phosphatase/total binding capacity) was closer to 1.0 than similar ratios of ATPase/binding capacity, indicating co-sedimentation of binding sites with microsomal membranes and not with plasma membranes. Na+/K+ ATPase, but not 5' nucleotidase, is a valid plasma membrane marker for ventral prostate. Microsomal androgen receptors may constitute a new level of regulation of androgen action in target cells.

Increased Androgen Binding Capacity in Sebaceous Glands in Scalp of Male-pattern Baldness

Sebaceous glands were isolated by manual dissection under a microscope from surgical specimens of scalp skin with male pattern baldness and skin specimens of hairy and bald scalp obtained at autopsy. The 800 X g pellet (nuclear fraction) and the 164,000 X g supernatant fraction (cytosol) of homogenates of the sebaceous glands were used for measurements of androgen binding characteristics, using dextran-coated charcoal and sucrose gradient methods. Scatchard plots showed high affinity binding for [3H]dihydrotestosterone (DHT) and [3H]methyltrienolone (R1881). Nuclei prepared from bald scalp contained greater total androgen binding capacity than nuclei of hairy scalp, although Kd values of type I binding were similar (0.68 vs 0.56 nM, respectively). On sucrose gradient, the binding protein from cytosol was found in the 7 to 8S density range. Androgen binding by cytosol of sebaceous glands of hairy scalp had Kd of 1.89 +/- .79 and 2.05 +/- .56 nM for DHT and R1881, respectively, and Bmax of 18.7 +/- 4.4 and 20.0 +/- 4.6 fmol/mg protein for DHT and R1881, respectively. Cytosol from sebaceous glands of bald scalp had Kd values approximately half those of hairy scalp, and Bmax values 50%-100% higher. The bound 3H labeled DHT and R1881 could be partially displaced by testosterone (40-50%), moxestrol (28-32%), promegestone (19-26%), and delta 4-androstenedione (6-12%), but not by dehydroepiandrosterone. These data demonstrate the presence of specific androgen binding protein in sebaceous glands, and that sebaceous glands of bald scalp have greater binding affinity and capacity for androgens than those in hairy scalp. This difference may explain the greater androgenic response in androgenic alopecia.

Androgen receptor in sexual differentiation

Androgens play an essential role in sexual differentiation and their action is mediated by the androgen receptor (AR). The normal AR is a soluble protein, highly thermolabile, with a mol. wt of 90 kDa and a pI of 5.2 as determined by 2 dimensional (2D) gel electrophoresis. It is regulated by androgens in culture conditions but the physiological relevance of this regulation remains controversial. The presence of a functional AR is an absolute requirement for male sexual differentiation and its absence results in complete insensitivity. However, androgen insensitivity (complete or partial) can develop in the presence of a normal androgen binding capacity and there is no correlation between the clinical and the biochemical findlings. A number of qualitative abnormalities have been described to explain the failure of androgen action in these cases: they all emphasize the extreme instability of the abnormal AR. It is difficult at the present time to determine whether these abnormalities result from structural mutations of the AR gene, transcriptional or post-transcriptional abnormalities. Further elucidation of these defects awaits for an antibody and/or a cDNA probe for the AR.

Multiple binding components for methyltrienolone in canine prostatic epithelial cells

Using a whole cell assay system, the androgen binding capacity of canine prostatic epithelial cells was evaluated in relation to their function. Radiolabeled Methyltrienolone (R1881) was used as the ligand in the presence of an excess of Triamcinolone acetonide and the amount of [ 3H]R1881 bound to the cells at equilibrium was determined by either displacement or saturation studies. With immature cells in culture (3 days of attachment), displacement analysis revealed the presence of high affinity binding sites which were also present in cells cultured for 10 days. With freshly dispersed prostatic cells (mostly secretory epithelial cells) as well as with older cells in culture (17 and 24 days), only less specific binding sites were observed with both unlabeled R1881 and/or dihydrotestosterone (DHT). In contrast, only the high affinity androgen receptor (AR) was present in cytosolic extracts prepared from normal glands. Displacement studies performed with cultured cells at different stages of growth also showed that the basal level as well as the degree of low affinity binding increased during the maturation of non-proliferating cells. The presence of multiple binding components was demonstrated by saturation studies performed with either cultured or freshly dispersed cells. The first component, that was saturated at 5 nM of [ 3H]R1881, was due to AR while the other two binding components, showing positive-cooperativity (Hill coefficients of 1.90 and 5.07, respectively), were saturated at concentrations of 15 and 30 nM of [ 3H]R1881. In contrast, the Hill coefficient for the AR was 0.88 indicating the presence of an independent component. It was calculated that only 11.4% of the total uptake of R1881 was attributed to AR binding, suggesting that the remainder may represent an intracellular pool of androgens. Thus, a whole cell binding assay represents a dynamic system for the detection, by saturation studies, of binding components that are not revealed using the conventional displacement studies or cell-free systems. It is proposed that these acceptor sites may play a role in differentiated prostatic function rather than in cell proliferation.

Extracellular correction of the androgen-receptor transformation defect in two families with complete androgen resistance

We have characterized the cellular and extracellur phenotype of the mutant androgen receptor (AR) from two families who have complete androgen resistance despite a normal androgen-binding capacity ( B m ) in their genital skin fibroblasts (GSF). The cellular receptors fail to up-regulate their basal AR activity in response to prolonged incubation with 5α-dihydrotestosterone (DHT), or with two synthetic androgens, methyltrienolone (MT) and mibolerone (MB), and form A-R complexes with increased equilibrium ( K d ) and non-equilibrium (k) dissociation constants. In addition, they are thermolabile when recently dissociated, but not in their native state. A-R complexes made in normal or mutant cytosol at 4°C elute from DEAE-Sephacel at ~0.25 M KCl (untransformed), with or without prior passage through Sephadex G-25; when made in cells at 37°C, extracted with 0.4 M KCl in a buffer containing 10 mM Na 2MoO 4, and desalted by G-25, they elute at ⩽0.1 M KCl. Normal KCl-extracted DHT- and MB-R complexes dissociate (37°C) at the same slow, linear rate as their in-cell counterparts (transformed); the mutant ones dissociate more slowly than their rapidly-dissociating in-cell counterparts and, to a variable extent, nonlinearly—an early faster phase, a later slower (transformed). Thus, as judged by two conventional criteria of steroid-R complex transformation, the mutant A-R complexes can transform, possibly in two steps, under certain cell-free conditions. This behavior differentiates a class of structural AR mutations whose molecular definition awaits application of recombinant DNA techniques to the X-linked AR locus.

Suppression of testosterone-estradiol binding globulin by medroxyprogesterone acetate in polycystic ovary syndrome

Serum androgen binding capacity is decreased in hirsute women and normal women receiving medroxyprogesterone acetate. A sensitive radioimmunoassay was used to measure the main androgen binding protein, testosterone-estradiol-binding globulin, in 13 patients with hirsutism and the polycystic ovary syndrome. Testosterone-estradiol-binding globulin concentrations were determined before, during, and after treatment with medroxyprogesterone acetate. Testosterone-estradiol-binding globulin was lower in untreated polycystic ovary syndrome patients than in normal controls (21.9 +/- 3.7 versus 64 +/- 4 nmol/L, P less than .01). Administration of medroxyprogesterone acetate (400 mg intramuscularly every 15 days for nine months) to polycystic ovary syndrome patients caused a decrease of serum testosterone and further lowering of testosterone-estradiol-binding globulin (7.6 +/- 1.9 nmol/L, P less than .01). In two patients the concentration of testosterone-estradiol-binding globulin reached values as low as 0.9 and 0.8 nmol/L, approximately 1/60 and 1/70 of the normal level. Testosterone-estradiol-binding globulin returned to basal levels (15.1 +/- 1.3 nmol/L) between one and two years after discontinuation of medroxyprogesterone acetate. Corticosteroid binding globulin levels were normal in polycystic ovary syndrome and did not change with medroxyprogesterone acetate. It was concluded that: medroxyprogesterone acetate causes marked lowering of plasma testosterone-estradiol-binding globulin; the effect is not mediated by sex hormone levels; and the decrease in testosterone-estradiol-binding globulin offsets, at least partially, the beneficial action of medroxyprogesterone acetate.

Thyroidal modulation of androgenic expression in mice submandibular gland.

We investigated the influence of testes and thyroid gland on the maintenance of biochemical parameters and of [3H]R1881 binding sites of adult mice submandibular gland (SMG). Castration (Cx) performed at beginning of puberty prevented sex-dependent SMG development without interfering with maximal androgen binding capacity. Thyroidectomy (Tx) had strong effects on SMG, mainly by lowering the number of androgen binding sites. All alterations could be fully reverted after treatment with testosterone (5 mg/animal, single dose) or with thyroxine (T4, 250 micrograms/animal per day during 5 days). The effects of Cx on SMG could be reverted by therapy with testosterone, T4, or with both hormones (testosterone + T4) in a non-synergistic fashion. It is shown the importance of thyroidal activity on the physiological maintenance of androgen receptors in the murine SMG; the role played by thyroid gland seems to be essential for the full expression of the androgen-dependent SMG activity in adult mice.

Seminiferous tubule androgen receptors in experimental cryptorchidism.

As an initial approach to the study of seminiferous tubule androgen receptors in disordered spermatogenesis, cytosol androgen receptors were studied in rats with experimental cryptorchidism. Two weeks after the testis had been repositioned in the abdomen of 6-week-old rats, the animals were hypophysectomized to deplete the testis of androgen, and 1 week later they were killed. Androgen receptor binding was studied in seminiferous tubule cytosol using [3H]methyltrienolone as the radiolabelled probe. The androgen-binding capacity of cryptorchid testis, when expressed as fmol bound/testis, was reduced to 50% of control, in parallel with the decline in testis weight. No change in binding affinity was found. Sucrose density gradient centrifugation using a vertical tube rotor revealed a 9S molybdate-stabilized receptor under low-salt conditions in both cryptorchid and scrotal seminiferous tubule cytosol. Receptor-complex stability studies, analysis by gel filtration and DEAE-cellulose chromatography produced similar results in cryptorchid and scrotal tubules. The mechanism for the reduction in testicular receptor content of an abdominal testis remains to be clarified. The demonstration that testicular androgen receptors can be reduced by cryptorchidism suggests that further studies may indicate the role of receptor binding in testicular function.

Ontogenesis of androgen receptors in the mouse submandibular gland: correlation with the developmental profiles of circulating thyroid and testicular hormones.

Specific binding of the synthetic androgen, [17 alpha-methyl-3H]methyltrienolone, to the cytosol fraction of the submandibular salivary gland (SMG) of male mice was studied in relation to the developmental profiles of testosterone and thyroid hormones in blood. The peak rise of serum triiodothyronine (T3) at prepubertal age was closely related to both the increase of maximal androgen-binding capacity in SMG and the conspicuous surge of proliferative activity as indicated by increased rate of glandular DNA content. Also, 2-month thyroidectomized mice had an age-related, strong reduction in the number of androgen-binding sites. On the other hand, the development of the secretory functions of the gland could be better related to the rise of circulating testosterone by days 25-30 of age. The results suggest that thyroid hormones play a very important role in the early induction and further maintenance of androgen receptors in the murine SMG.

Suppression of testosterone-estradiol binding globulin by medroxyprogesterone acetate in polycystic ovary syndrome.

Serum androgen binding capacity is decreased in hirsute women and normal women receiving medroxyprogesterone acetate. A sensitive radioimmunoassay was used to measure the main androgen binding protein, testosterone-estradiol-binding globulin, in 13 patients with hirsutism and the polycystic ovary syndrome. Testosterone-estradiol-binding globulin concentrations were determined before, during, and after treatment with medroxyprogesterone acetate. Testosterone-estradiol-binding globulin was lower in untreated polycystic ovary syndrome patients than in normal controls (21.9 +/- 3.7 versus 64 +/- 4 nmol/L, P less than .01). Administration of medroxyprogesterone acetate (400 mg intramuscularly every 15 days for nine months) to polycystic ovary syndrome patients caused a decrease of serum testosterone and further lowering of testosterone-estradiol-binding globulin (7.6 +/- 1.9 nmol/L, P less than .01). In two patients the concentration of testosterone-estradiol-binding globulin reached values as low as 0.9 and 0.8 nmol/L, approximately 1/60 and 1/70 of the normal level. Testosterone-estradiol-binding globulin returned to basal levels (15.1 +/- 1.3 nmol/L) between one and two years after discontinuation of medroxyprogesterone acetate. Corticosteroid binding globulin levels were normal in polycystic ovary syndrome and did not change with medroxyprogesterone acetate. It was concluded that: medroxyprogesterone acetate causes marked lowering of plasma testosterone-estradiol-binding globulin; the effect is not mediated by sex hormone levels; and the decrease in testosterone-estradiol-binding globulin offsets, at least partially, the beneficial action of medroxyprogesterone acetate.

An androgen-dependent pilosebaceous tumor spontaneously developed in the japanese house musk shrew Suncus murinus

Following administration of [ 3H]testosterone to castrated male Japanese house musk shrews ( Suncus murinus), radioactive metabolites were detected in sidegland nuclei and the major one of them was dihydrotestosterone (DHT). The androgen binding capacity of the cytoplasmic fraction of sideglands was measured in vitro by the use of [ 3H]R1881 as a ligand. The binding showed a high affinity for R1881 ( K d = 6.2 × 10 −10 M) and a low capacity ( B max = 22 fmol/mg protein). p]Sucrose density gradient centrifugation brought about a peak of [ 3H]R1881 in the 7S region in low ionic strength buffer. Their characteristics as described above are consistent with those of other androgen target organs. A cutaneous pilosebaceous tumor, which spontaneously developed on the sidegland of old male S. murinus, was transplanted to nude athymic mice. It grew in males only and failed to grow in females and castrated males. A specific androgen binding was found in this tumor ( K d = 7.8 × 10 −10M, B max = 100 fmol/mg protein). Therefore, this transplantable pilosebaceous tumor is androgen-dependent and can be utilized as a new suitable model in the study of the mechanism of androgen on tumor development.

Androgen binding capacity and 5 alpha-reductase activity in pubic skin fibroblasts from hirsute patients.

We have measured the total (cytosolic plus nuclear) androgen binding capacity of pubic skin fibroblasts from nine patients with hirsutism of various origin. Confluent intact cell monolayers were incubated with increasing concentrations (0.05-2 nM) of [3H]dihydrotestosterone ([3H]DHT) with or without a 200-fold excess of unlabeled DHT. The androgen binding capacities (mean +/- SD) were similar in normal men (411 +/- 171 fmol/mg DNA), women (310 +/- 103 fmol/mg DNA), and hirsute patients (313 +/- 141 fmol/mg DNA) regardless of the plasma androgen levels. In contrast, the 5 alpha-reductase level in pubic skin fibroblasts (mean +/- SD) was, as previously described, higher in hirsute women (3.3 +/- 2.6 fmol/micrograms DNA . h) than in normal women (1.1 +/- 0.6 fmol/microgram DNA . h; P less than 0.05). We conclude from these data that: 1) increased androgen binding capacity cannot be held responsible for hypersensitivity to androgens in hirsutism; 2) the androgen receptor is not regulated by androgens in human skin, as similar levels are observed in men, women, and hirsute patients; 3) this contrasts with 5 alpha-reductase activity and emphasizes the importance of this enzyme as an amplifier of androgen action in areas where it is stimulated by androgens, such as pubic skin.

Characterization of the cytosol androgen receptor of the human prostate.

Direct measurement of the binding of endogenous androgens to the androgen receptor of human tissues has not been possible because of contamination of tissue with traces of plasma proteins, such as testosterone-binding globulin (TeBG), that contain more androgen-binding capacity than does the receptor itself. Molybdate is known to stabilize the 8-9S forms of other steroid hormone receptors. We took advantage of this phenomenon to characterize the androgen receptor of hyperplastic prostates removed at surgery, using sucrose density gradient centrifugation in a vertical rotor. In 10 mM sodium molybdate, the androgen receptor sediments as a distinct 9.2 +/- 0.5S moiety, easily separable from TeBG. Unlike TeBG, the 9S receptor is not removed by absorption with Concanavalin A. [3H]Dihydrotestosterone (3H-labeled 17 beta-hydroxy-5-alpha-androstan-3-one) binding to the 9S receptor is not removed by absorption with Concanavalin A. [3H]Dihydrotestosterone (3H-labeled 17 beta-hydroxy-5 alpha-androstan-3-one) binding to the 9S receptor is not competed for by excess triamcinolone acetonide (9 alpha-fluoro-11 beta, 16 alpha, 17 alpha, 21-tetrahydroxypregna-1,4-diene-3,20-dione cyclic 16,17-acetonide) or promegestone (17,21-dimethyl-19-non-pregna-4,9-diene-3,-20-dione), which are known to bind to the progestin receptor. In contrast, [3H]methyltrienolone (3H-labeled 17 beta-hydroxy-17 alpha-methyl-estra-4,9,11-trien-3-one) binds to both androgen and progestin receptors, and consequently, the binding of this ligand to the androgen receptor was assessed in the presence of a 500-fold excess of triamcinolone acetonide. The amounts of 9S binding (7.8 and 5.8 fmol/mg protein) are similar for dihydrotestosterone and methyltrienolone. The amount of 9S binding of testosterone to the receptor was also similar to that of dihydrotestosterone, but the affinity of testosterone for the 9S receptor was only a fifth or less of that for dihydrotestosterone. The observation that testosterone binds less avidly than dihydrotestosterone to the receptor may explain the role of dihydrotestosterone formation in androgen physiology.

13 Increased androgen binding capacity in experimental prostatic carcinomas treated with estrogen

13 Increased androgen binding capacity in experimental prostatic carcinomas treated with estrogen

Androgen receptors in human skin cytosol: physiological and pathological variations.

The androgen binding capacity has been measured in thé cytosol of genital and pubic skin of normal men and women and patients with disorders of androgen sensitivity. The binding capacity was highest in genital skin, but no differences were observed with age or sex. This, and the presence of a normal binding capacity in some patients with patent clinical insensitivity, suggests that the androgen receptor in human skin cytosol is not regulated by androgens.

Nuclear androgen receptors in prostates from diabetic rats.

It is shown that the binding of the synthetic androgen [3H] methyltrienolone ([3H] R1881) to the nuclear fraction of prostates of streptozotocin-diabetic rats is 38% reduced in comparison to controls. Although the exogenous replacement therapy of diabetic animals wih insulin was capable of restoring to normality several body and tissue parameters, the androgen binding capacity of prostate nuclei is not recovered. Normal binding values were observed in diabetic animals which have been treated with exogenous testosterone. It is postulated that the decrease in the nuclear androgen receptor content in the prostatic gland of streptozotocin-diabetic rats is due to the lower circulating testosterone levels in these animals.

Androgens in the fetal guinea-pig after maternal infusion of radioactive testosterone.

Testosterone was measured in plasma pools from male and female fetal guinea-pigs between the ages of 30 and 55 days of pregnancy. Between days 33 and 36 the testosterone concentration in the plasma of males (1.4 ng/ml) was several times higher than that found at other ages or that measured in female fetuses. After infusion of tritiated testosterone for 2 h into pregnant guinea-pigs at day 36 of pregnancy, high levels of testosterone and androstenedione were found in maternal plasma. Nevertheless, tritiated testosterone and androstenedione could hardly be shown in the fetuses. Similar large differences in plasma progesterone levels appeared to exist between the maternal and the fetal circulation. Therefore, only a very small fraction of these steroids can penetrate from the maternal circulation into that of the fetus. This finding might be explained by the large difference in androgen-binding capacity between maternal and fetal plasma, as was shown by equilibrium dialysis.

Preliminary characterization of a new androgen-binding protein in human seminal plasma.

In order to obtain comparative analysis between the steroid binding proteins from blood serum and seminal plasma, fresh samples were incubated and assayed in parallel with the following tritiated steroids: dihydrotestosterone, testosterone, cortisol, progesterone and estradiol. Thereafter, and using techniques such as (1) polyacrilamide disk gel electrophoresis with elution of proteins from the gel slices, (2) ammonium sulphate precipitation, (3) enzymatic hydrolysis, and (4) gel filtration on Sephadex G-100, there was a resemblance in the electrophoretic patterns confirming the presence in both fluids of proteins like TeBG, CBG, and Albumin, previously described in blood serum. In seminal fluid, but not in blood serum, another small protein with androgen binding capacity was found and its preliminary characterization demonstrated: disappearance with enzymatic treatment, low molecular weight, negative charge, and precipitation range with ammonium sulphate from 30 to 35%. Its elution profile on Sephadex G-100 was contaminated with albumin.