Changes in the sedimentation properties of cytosolic androgen receptors associated with activation in vitro and in vivo

In cell-free systems androgen receptor (AR) labeled with (3H)DHT at 0 degrees C in the presence of 50mM molybdate remains unactivated (less than 3% binding to nuclei) and untransformed (7-8S on sucrose density gradients containing 0.4M KCl and 50mM molybdate). In the absence of molybdate, however, these complexes undergo activation and transformation even at 0 degrees C, albeit, very slowly. Incubation of unactivated, untransformed AR complexes at 18 degrees C, or at 0 degrees C in the presence of 0.4M KCl, greatly accelerated both activation and transformation. Activation and transformation are also associated with formation of high affinity (3H)DHT-receptor complexes as indicated by decreased rates of (3H)DHT dissociation from the receptor. Cytosolic AR complexes labeled with (3H)DHT in tissue slices at 37 degrees C, or in vivo, undergo rapid activation, transformation and nuclear translocation. The data suggest that activation and transformation of cytosolic AR in cell-free systems is associated with changes in the physicochemical properties of AR similar to those occurring upon hormone binding in intact cells and in vivo.     

Modulation of steroid affinity for the androgen receptor by sucrose

The effects of sucrose on androgen binding to its receptor were investigated. Sucrose decreased the rate of thermal inactivation of unoccupied and occupied androgen receptor (AR) and the rates of [3H]5 alpha-dihydrotestosterone [( 3H]DHT) dissociation from both activated and nonactivated AR complexes. Binding of [3H]DHT to AR in vivo, or in intact cells at 37 degrees C, caused reduction of [3H]DHT dissociation from cytosolic and nuclear complexes, as compared to in vitro labeled receptor complexes. Further, exposure of these complexes to sucrose at 0 degrees C caused an additional reduction of dissociation rates. Thus, the decrease of [3H]DHT dissociation induced by sucrose is independent of the reaction that reduces DHT dissociation from activated and transformed AR. Sucrose also reduced the ability of mersalyl acid to inactivate AR complexes. This effect of sucrose was markedly diminished in the presence of 2M urea. Sucrose did not significantly affect the association rate, sedimentation properties, or nuclear binding ability of AR complexes, but it did decrease the equilibrium dissociation constant. Other monosaccharides and disaccharides also stabilized AR. These data suggest that sucrose induces conformational changes in the steroid binding domain of androgen receptor, thereby reducing the rates of inactivation, steroid dissociation, and the accessibility of sulfhydryl groups to mersalyl.     

Characterization of nontransformed and transformed androgen receptor from rat submandibular gland

Rat submandibular gland cytosol contained androgen receptor which had a single class of specific binding and an apparent dissociation constant of (1.1-1.2) X 10(-9) M. The process of transformation was investigated by a slightly modified minicolumn method in which the transformed receptor complexes were separated from the nontransformed receptor and meroreceptor. 10 mM ATP or pyrophosphate at 0 degrees C induced transformation of androgen receptor as did heat or salt treatment. 20 mM of sodium molybdate completely inhibited transformation that resulted from ATP, heat or salt treatment. The nontransformed androgen receptor complexes sedimented at 8 S and eluted at 250-260 mM KCl from DEAE-Sephacel, and its molecular weight was found to be 220 000 on Sephacryl S300 gel chromatography. On the other hand, the transformed androgen receptor complexes sedimented at 4.1-4.3 S (ATP or KCl treatment) or 3.5-3.8 S (heat treatment) and eluted at 60-80 mM KCl from DEAE-Sephacel. The molecular weight of the transformed androgen receptor complexes was 80 000-85 000 (ATP or KCl treatment) or 70 000-80 000 (heat treatment). These results suggest that the transformation of androgen-receptor complexes from rat submandibular gland was induced by the subunit dissociation and that salt bridges may be involved in the subunit interaction.     

Dialysis-induced transformation of mouse submandibular androgen-receptor

Dialysis induced transformation of cytosol androgen receptor from mouse submandibular gland. On DEAE-Sephacel chromatography, this dialyzed [3H]methyltrienolone receptor complex was eluted at 0.10 M KCl which was lower than 0.25 M KCl required to elute the nontransformed androgen receptor complex, but higher than 0.05 M KCl required to elute the heat transformed receptor. On glycerol density gradient centrifugation, the dialysis transformed receptor complex shifted its sedimentation coefficient to 6 S from 8 S of the nontransformed condition, whereas the heat transformed receptor was sedimented at 4 S. Molybdate inhibited the dialysis-induced transformation on DEAE-Sephacel chromatography. The charge and the molecular size of dialysis-induced transformed receptor complex were different from those of heat-induced transformed receptor complex.     

Replacement of arginine 773 by cysteine or histidine in the human androgen receptor causes complete androgen insensitivity with different receptor phenotypes

We have discovered two different point mutations in a single codon of the X-linked androgen-receptor (AR) gene in two pairs of unrelated families who have complete androgen insensitivity (resistance) associated with different AR phenotypes in their genital skin fibroblasts. One mutation is a C-to-T transition at a CpG sequence near the 5' terminus of exon 6; it changes the sense of codon 773 from arginine to cysteine, ablates specific androgen-binding activity at 37 degrees C, and eliminates a unique KpnI site at the intron-exon boundary. The other mutation is a G-to-A transition that changes amino acid 773 to histidine and eliminates an SphI site. This mutant AR has a normal androgen-binding capacity at 37 degrees C but has a reduced affinity for androgens and is thermolabile in their presence. Transient transfection of COS cells with cDNA expression vectors yielded little androgen-binding activity at 37 degrees C from Arg773Cys and abundant activity with abnormal properties from Arg773His, thereby providing the pathogenicity of both sequence alterations. This conclusion coincides with the following facts about evolutionary preservation of the position homologous to Arg773 in the AR: it is occupied by Arg or lysine in the progesterone, glucocorticoid, and mineralocorticoid receptors, and it is within a 14-amino-acid region of their steroid-binding domains that share approximately 85% amino acid identity.     

Characteristics of cytosol androgen receptor in rat thymus

Male and female rat thymic cytosol contained specific androgen receptor. The apparent dissociation constants (Kd) were 2.4 nM in males and 2.5 nM in females, and the number of binding sites (NBS) were 23.7 fmol/mg protein in males and 34.2 fmol/mg protein in females. Transformation of receptor to the DNA binding state was achieved by heat or KCl treatment of [3H]R1881-receptor complex, and the characteristics of transformed and nontransformed receptors were investigated. The nontransformed androgen-receptor complex eluted at 0.20-0.25 M KCl from DEAE-Sephacel and sedimented at 9.1 S and its molecular weight was 255,000 on agarose gel chromatography, while the transformed receptor complex eluted at 0.03-0.15 M KCl with a broad peak and sedimented at 4.5 S and its molecular weight was 80,000-85,000. The minicolumn binding assay revealed that approximately 57% of the total receptor complexes bound to DNA-cellulose following heat treatment (20 degrees C, 1 h). Castration exerted no effect on the physicochemical properties of cytosol androgen receptor, but it increased the number of binding site to the female level.     

The state transitions of normal and mutant androgen-receptor complexes in human genital skin fibroblasts

We have incubated cells from controls and subjects with receptor-defective androgen resistance with 3H-labelled testosterone (T), methyltrienolone (MT), dihydrotestosterone (DHT) or mibolerone (MB) and studied the temperature dependence of the dissociation rate constants of these various androgen-receptor (A-R) complexes both within cells and after they were extracted from them. In control cells, Arrhenius plots for T-, MT-, DHT- and MB-R complexes were linear and formed a hierarchy of dissociation states with energies of state IV greater than III greater than II, greater than I, respectively. Relative to this hierarchy, the dissociation states of the MB-, DHT- and MT-R complexes in mutant cells were displaced to higher, androgen-inappropriate energies in a mutant-distinctive pattern. When extracted from cells control or mutant T- or MT-R complexes, and mutant (but not control) DHT- or MB-R complexes lowered their respective dissociation rates by undergoing state transitions in conformity with the hierarchy. Hence we propose that different A-R complexes reach different dissociative states by undergoing sequential transitions along a common pathway, and that these transitions are co-regulated both by the chemical characteristics of the bound androgen and by other cellular non-receptor factors.     

Partial purification and characterisation of the human skin fibroblast androgen receptor: detection of abnormal receptor complexes in cells from patients with androgen insensitivity syndromes

After incubation of hGSF with [3H]5 alpha-dihydrotestosterone, 17 beta-hydroxy-7 alpha, 17 alpha-dimethyl-4-estrene-3-one, or 17 beta-hydroxy-17 alpha-methyl-4,9,11-estrien-3-one, androgen-receptor complexes were extracted with 0.5 M KCl and precipitated by 35% ammonium sulphate. Receptor complexes from control hGSF sedimented at approximately 4S on linear 5-20% sucrose gradients. The 4S peak was diminished or absent in cells from androgen insensitive patients exhibiting absent, deficient or unstable binding of androgens in intact hGSF. This procedure may be a useful means of distinguishing quantitative and qualitative defects in androgen binding to receptor, since one cell line found to have normal levels of androgen receptor complexes in whole cell assays had a profile resembling that of receptor negative cells on sucrose gradients. The complexes from one patient with complete androgen insensitivity having normal androgen binding in intact hGSF were indistinguishable from control complexes after sucrose gradient analysis and ADP-Sepharose chromatography. Receptor complexes were eluted from the ADP-Sepharose between 0.5-1.0 M KCl. HPLC-gel filtration of androgen receptor complexes at 22 degrees C revealed two peaks, the larger had a Mr of 60-65K, Stokes radius of 3.16 nm and a frictional ratio between 1.21 and 1.43. The second peak, Mr of 15K, was believed to represent a fragment of the receptor containing the steroid binding domain. On gel filtration at 22 degrees C the complexes from a patient with partial androgen insensitivity, who showed a diminished 4S receptor peak on sucrose gradients, revealed only the small "meroreceptor" fragment, suggesting that the mutation in this individual might render the androgen receptor more susceptible to proteolysis in vitro.     

Studies of surface immunoglobulins on human B lymphocytes. I. Dissociation of cell-bound immunoglobulins with acid pH or at 37 degrees C.

Lymphocyte preparations isolated from the human peripheral blood were exposed to different acid pH or incubated at 37 degrees C and the presence of immunoglobulin (Ig) on the cell surface was examined by immunofluorescence (IF) tests. Subsequently, such treated cells were incubated in the autologous serum or in the purified IgG, IgA or IgM proteins and their ability to bind each class of Ig was examined. The results showed that IgG molecules dissociated from large proportions of IgG-positive cells upon exposure to pH 4 at 1 degrees C for 1 min or upon incubation at 37 degrees C for 20 min. The cells from which IgG had been dissociated could again combine with IgG, whereupon the number of positive cells increased, being restored to the number of equivalent to or higher than those before acid or 37 degrees C treatment. These results indicated that the treatment could elute the cell-bound IgG present on the cell and that the receptor sites were not degraded by the treatment and could combine with IgG. These cell-bound IgG were observed not only on the monocytes, but also on the small lymphocytes. It was also found that certain proportions of mononuclear cells carried the cell-bound IgA that could be dissociated with acid pH or 37 degrees C. No cell-bound IgM was observed on any mononuclear cells. Microscopic observations before and after acid or 37 degrees C treatment revealed that the staining distribution of the cell-bound IgG and IgA on the cell was granular, appearing as a discontinuous fluorescence ring and forming multiple aggregates but no typical polar caps on warming. In contrast, IgG, IgA, and IgM stable to acid or 37 degrees C treatment were found on the lymphocytes but not on the monocytes, and their staining distribution was uniformaly diffuse, appearing as a continuous ring and forming a typical cap on warming. Exposure of the cells to pH 4 or 37 degrees C could also elute the cell-bound IgG passively adsorbed to the human lymphoid cells in a culture, but did not affect the intrinsic S.Ig on the lymphoid cells in a culture or on the lymphoma cells. These results indicate that the exposure of the cells to acid pH or to 37 degrees C may enable us to detect unfailingly S.Ig lymphocytes by removing the cell-bound IgG and IgA present on the monocytes and/or lymphocytes. Thus, an average value of approximately 10% was obtained for the S.Ig lymphocyte in the lymphocyte preparations from 11 healthy individuals. In addition, the results provided the evidence that, even in normal peripheral blood lymphocytes, there may be a population of B lymphocytes which lack the S.Ig but carry the cell-bound Ig.     

Phosphorylation and nuclear processing of the androgen receptor.

Although transformed androgen receptor (AR) complexes derived from cytosol and nuclear AR complexes have been shown to bind with high affinity to nuclei and DNA, we have shown that the binding characteristics of the two receptor populations to rat ventral prostate nuclei are different. To account for these differences, we investigated the possibility that the two receptor populations differed in phosphorylation status. Significantly, an anti-phosphotyrosine antibody immunoprecipitated androgen binding from the nuclear AR preparation but not from the transformed cytosolic receptor preparation. These studies suggest that (i) further processing of the AR complex takes place after it has become transformed, and (ii) phosphorylation of the complex is one modification which occurs during the processing of the nuclear receptor.     

Interaction of the antiestrogen [3H]H1285 with the two forms of the molybdate-stabilized calf uterine estrogen receptor

The high affinity antiestrogen [3H]H1285 bound to the cytosol calf uterine estrogen receptor dissociated very slowly (t 1/2 approx 30 h at 20 degrees C) and did not demonstrate a change in dissociation rate in the presence of molybdate, which is characteristic of [3H]estradiol-receptor complexes. [3H]H1285-Receptor complexes sediment at approx 6S on 5-20% sucrose density gradients containing 0.3M KCl with or without 10 mM molybdate. This is in contrast to [3H]estradiol-receptor complexes which sedimented at approx 4.5S without molybdate and at approx 6S with molybdate. These results suggest a physicochemical difference in the estrogen receptor when occupied by antiestrogens versus estrogens. We recently reported that the cytoplasmic uterine estrogen receptor, when bound by estradiol and prepared in 10 mM molybdate, eluted from DEAE-Sephadex columns as Peak I (0.21 M KCl) & Peak II (0.25 M KCl). However, [3H]H1285 bound to the estrogen receptor eluted only as one peak at 0.21 M KCl, also suggesting that the initial interaction of antiestrogens with the estrogen receptor is different. We have extended these studies and report that H1285 can compete with [3H]estradiol for binding to both forms of the estrogen receptor and [3H]H1285 can bind to both forms if the unoccupied receptor is first separated by DEAE-Sephadex chromatography. However, if the receptor is first bound by unlabeled H1285, eluted from the column and post-labeled by exchange with [3H]estradiol, only one peak is measured. Thus, it appears that H1285 binding alters the properties of the receptor such that all receptor components seem to elute as one form. These partially purified [3H]H1285-receptor complexes obtained from DEAE-Sephadex columns sedimented as 5.5S in sucrose density gradients in contrast to the sedimentation values for the [3H]estradiol-receptor components eluting as Peak I (4.5S) and Peak II (6.3S). These differences in the physicochemical characteristics of the estrogen receptor when bound by estrogen versus antiestrogens may be related to some of the biological response differences induced by these ligands.     

Kinetic evidence for a unique testosterone-receptor complex in 5α-reductase sufficient genital skin fibroblasts and the effects of 5α-reductase deficiency on its formation

When 5α-reductase-sufficient genital skin fibroblast (GSF) monolayers are incubated with testosterone (T), they first form androgen (A)-receptor (R) complexes that dissociate at a fast rate [k(37°C = 0.024 min⁻¹]. As T is converted to 5α-dihydrotestosterone (DHT), this population of T-R complexes is eventually replaced by one that dissociates much more slowly [k(37°C) = 0.006 min⁻¹], at a rate typical of DHT-R complexes. During the course of T to DHT conversion, one may observe a population of A-R complexes that has a linear (monophasic) intermediate dissociation rate constant [k(37°C) = 0.012 min⁻¹]; this population cannot simply reflect a mixture of T- and DHT-R complexes. The rate at which the complexes are processed from one dissociative form to the next varies with the incubation temperature and the presence or absence of serum in the medium; it also varies within and among GSF strains under apparently constant conditions. To explain these facts, we propose a model that enables the 5α-reductase enzyme to influence the processive dissociative behaviour of T-R complexes by engaging in some sort of coupling with the AR. The proposal is strengthened by a set of observations in cells with constitutive, mendelian or inhibitor-induced 5α-reductase deficiency that preclude a simple quantitative relation between A-R complex processing and the extent of T to DHT conversion.     

Receptor binding of allylestrenol, a progestagen of the 19-nortestosterone series without androgenic properties

Allylestrenol (17 alpha-allyl-17 beta-hydroxy-4-estren) is an orally active progestagen of the 19-nortestosterone series resembling progesterone since it has no detectable androgenic activity in animal studies and in the human. In the present study, the affinity of its 3-keto metabolite for the transformed progesterone receptor in intact MCF-7 cells was about twice that of progesterone and cyproterone acetate and about 2-3 times less than that of medroxyprogesterone acetate and norethisterone, reflecting the known progestational activity of allylestrenol. The affinity of 3-ketoallylestrenol for the transformed androgen receptor in intact MCF-7 cells was weak (like other progestagens lacking androgenic activity or possessing anti-androgenic activity) and lower than that of weakly androgenic progestagens. On the other hand, the relatively high affinity of 3-keto-allylestrenol for the non-transformed androgen receptor at 4 degrees C in the cytosol fraction did not reflect the known lack of androgenic activity of allylestrenol. Thus competitive studies carried out with transformed receptor complexes in intact cells at 37 degrees C and non-transformed complexes in cytosol distinguish progestagen with weak androgenic activity (e.g. norethisterone) from those displaying no androgenic activity or possessing anti-androgenic activity (e.g. 3-keto-allylestrenol, progesterone, cyproterone acetate and spironolactone).     

Mutation of R116C results in highly oligomerized alpha A-crystallin with modified structure and defective chaperone-like function

An autosomal dominant congenital cataract in human is associated with mutation of Arg-116 to Cys (R116C) in alpha A-crystallin. To investigate the molecular basis of cataract formation, rat alpha A-crystallin cDNA was cloned into pET-23d(+), and the site-directed mutants S142C (similar to wild-type human alpha A) and R116C/S142C or R116C (similar to human R116C variant) were generated. These were expressed in E. coli and the recombinant alpha A-crystallins purified by Sephacryl size-exclusion chromatography. The chaperone-like function of mutant R116C determined at 37 degrees C with insulin and alcohol dehydrogenase as target proteins was about 40% lower than those of wild-type and mutant S142C. Based on size-exclusion chromatography data, the oligomeric size of the R116C mutant was about 2000 kDa at 25 degrees C, 1400 kDa at 37 degrees C, and 900 kDa at 45 degrees C. In comparison, alpha A-wild-type and alpha A-S142C ranged from 477 to 581 kDa. Heat stability studies corroborated the effect of temperature on the dynamic quaternary structure of the R116C mutant. Circular dichroism spectra showed secondary and tertiary structural changes, and ANS fluorescence spectra showed loss of surface hydrophobicity in the R116C mutant. These findings suggest that the molecular basis for the congenital cataract with the alpha A-R116C mutation is due to the generation of a highly oligomerized alpha A-crystallin having a modified structure and decreased chaperone-like function.     

Dissecting phenotypic variation among AIS patients

We have created genital skin fibroblast cell lines directly from three patients in a Chinese family affected by androgen insensitivity syndrome (AIS). All patients in the family share an identical AR Arg840Cys mutant but show different disease phenotypes. By using the cell lines, we find that the mutation has not influenced a normal androgen-binding capacity at 37 degrees C but has reduced the affinity for androgens and may cause thermolability of the androgen-receptor complex. The impaired nuclear trafficking of the androgen receptor in the cell lines is highly correlated with the severity of donors' disease phenotype. The transactivity of the mutant is substantially weakened and the extent of the reduced transactivity reflects severity of the donors' disease symptom. Our data reveal that although etiology of AIS is monogenic and the mutant may alter the major biological functions of its wild allele, the function of the mutant AR can also be influenced by the different genetic backgrounds and thus explains the divergent disease phenotypes.     

Characterization of nonactivated and activated glucocorticoid-receptor complexes from intact rat thymus cells

In cells exposed to glucocorticoids at 37 degrees C activated glucocorticoid-receptor complexes (complexes with affinity for nuclei and DNA) are formed after nonactivated complexes. Activation thus appears to be an obligatory physiological process. To investigate this process we have characterized cytoplasmic complexes formed in rat thymocytes at 0 and 37 degrees C. Complexes in cytosols stabilized with molybdate were analyzed by sucrose gradient centrifugation and by chromatography on DNA-cellulose, DEAE-cellulose, and agarose gels. Two major complexes were observed: the nonactivated complex, eluted from DEAE at approximately 200 mM KCl, was formed at 0 and 37 degrees C, gave S20,w = 9.2 S, Stokes radius = 8.3 nm, and calculated Mr = 330,000; the activated complex, eluted from DEAE at approximately 50 mM KCl, appeared only at 37 degrees C, gave S20,w = 4.8 S, Stokes radius = 5.0 nm, and Mr = 100,000. A third, minor complex, probably mero-receptor, which appeared mainly at 37 degrees C, bound to neither DNA nor DEAE, and gave S20,w = 2.9 S, Stokes radius = 2.3 nm, and Mr = 27,000. With three small columns in series (DNA-cellulose, DEAE-cellulose and hydroxylapatite), the three complexes can be separated in 5-10 min. By this method we have examined the stability of complexes under our conditions. We conclude that in intact thymus cells glucocorticoid-receptor complexes occur principally in two forms, nonactivated and activated, and that activation is accompanied by a large reduction in size. The origin of the mero-receptor complex remains uncertain.