Androgen-receptor Complex Research Articles

Differential modulation of androgen receptor action by deoxyribonucleic acid response elements.

In addition to the steroid response elements (SREs), which are recognized by several steroid receptors, a second class of DNA elements exhibiting selectivity for the androgen receptor (AR) and named androgen response elements (AREs) has been identified. Here we provide evidence for the differential role of these element classes in modulating AR function. AR complexes attached to response elements representative of each class were purified. Limited protease digests of ARE- or SRE-bound AR complexes led to the generation of different patterns, in line with differential accessibilities. In transactivation assays, mutations in the AR dimerization interface of the DNA-binding domain had various effects, depending on the response elements tested. The R598D mutant displayed much enhanced activity on SREs, whereas far less effect was seen on the selective AREs. The A596T mutant had reduced activity on AREs but not on SREs. Ectopic expression of the coactivators transcriptional intermediary factor 2 (TIF2) and ARA55 stimulated AR activity to different extents, depending on the response element. When using cysteine-rich secretory protein 1 (CRISP-1) SRE as reference, the most significant difference was observed with Pem ARE-2. A differential response of each element class was furthermore observed in the presence of two enzymes involved in the sumoylation pathway. Ubiquitin-conjugating enzyme 9 (Ubc9) overexpression enhanced AR action conveyed by SREs, whereas little effect was seen on Pem ARE-1 and repression on Pem ARE-2. Protein inhibitor of activated STAT (PIAS)xalpha overexpression had little influence on SRE-mediated AR activity but was repressive when using AREs. Altogether, these results demonstrate that DNA response elements play an important modulatory role in transmitting AR action and may be determinative for specificity of gene expression in cell or tissue types.

Interaction of organophosphate pesticides and related compounds with the androgen receptor.

Identification of several environmental chemicals capable of binding to the androgen receptor (AR) and interfering with its normal function has heightened concern about adverse effects across a broad spectrum of environmental chemicals. We previously demonstrated AR antagonist activity of the organophosphate (OP) pesticide fenitrothion. In this study, we characterized AR activity of analogues of fenitrothion to probe the structural requirements for AR activity among related chemicals. AR activity was measured using HepG2 human hepatoma cells transfected with human AR plus an androgen-responsive luciferase reporter gene, MMTV-luc. AR antagonist activity decreased as alkyl chain length of the phosphoester increased, whereas electron-donating properties of phenyl substituents of the tested compounds did not influence AR activity. Oxon derivatives of fenitrothion, which are more likely to undergo hydrolytic degradation, had no detectable AR antagonist activity. Molecular modeling results suggest that hydrogen-bond energies and the maximum achievable interatomic distance between two terminal H-bond capable sites may influence both the potential to interact with the AR and the nature of the interaction (agonist vs. antagonist) within this series of chemicals. This hypothesis is supported by the results of recent AR homology modeling and crystallographic studies relative to agonist- and antagonist-bound AR complexes. The present results are placed in the context of structure-activity knowledge derived from previous modeling studies as well as studies aimed toward designing nonsteroidal antiandrogen pharmaceuticals. Present results extend understanding of the structural requirements for AR activity to a new class of nonsteroidal, environmental, OP-related chemicals.

Effect of fatty acids on estradiol and testosterone binding to whole DU-145 prostate cells

Cancer of the prostate is one of the leading causes of cancer related deaths in men. An important role in the development of prostate cancer is played by androgens and androgen ablation is therefore currently used in cancer treatment. In the past, estrogens were widely used in treatment of prostate cancer, but there are indications that estrogens could also be involved in carcinogenesis. Lately, much research has been done on the modulation of the binding of steroid hormones to their receptors by polyunsaturated fatty acids (PUFAs), which could interfere with the steroid hormone's message. Therefore, the aim of this study was to determine in whole DU-145 human prostate cells the effect of EFAs and their metabolites on the binding and affinity of the estrogen receptor (ER) and androgen receptor (AR) to estradiol (E2) and testosterone (T), respectively. Fatty acids were dissolved in ethanol and added to the cell culture in a final ethanol concentration of 0.2% on the fourth day of incubation. The results showed that the PUFAs under investigation inhibited the AR's capacity, in contrast to the ER's capacity which was stimulated. However, the dissociation constants (Kd) of the AR and ER complexes in the presence of the PUFAs, were as follows. Except for eicosapentaenoic acid (EPA) which decreased the AR dissociation constant and EPA and α -linolenic acid (ALA) which increased the ER dissociation constant, the remaining FAs had no significant effect on the Kd values of both the AR and ER complexes. According to these priliminary results it is postulated that men should benefit with a diet rich in certain essential polyunsaturated fatty acids although its function remains to be clarified.

Properties of free and occupied androgen receptor in rat skeletal muscle cytosol: effect of testosterone

Our aim was to investigate the effect of a single testosterone (T) injection on the androgen receptor (AR) in rat skeletal muscle (SM) cytosol. The properties of AR were studied in order to establish the protocol for differential determination of free and hormone-occupied AR in SM cytosols from non-hormone-deficient animals. Using the developed ligand-exchange protocol, we demonstrated that injection of T (1 mg/kg) caused alternating changes of the total AR binding. The binding minimum (23% of the control) was measured 1 h after the injection. It was followed by pronounced and lasting elevation of the AR binding. In the control cytosols, AR complexes constituted ∼25% of the total receptor content. Changes of their relative content immediately after T administration were consistent with rapid nuclear translocation of the AR. Inhibition of protein synthesis by cycloheximide (CHI) injection demonstrated that delayed and lasting increase of the AR binding after T injection partially depended on the stimulated protein synthesis. Altogether, the obtained evidence supports the assumption that the AR mediates elevation of its own gene expression in SM upon administration of T.

Specific Androgen Receptor Activation by an Artificial Coactivator

Transcription activation of steroid receptors, such as the androgen receptor (AR), is mediated by coactivators, which bridge the receptor to the preinitiation complex. To develop a tool for studying the role of the AR in normal development and disease, we constructed artificial coactivators consisting of the transcription activation domains of VP16 or p65/RelA and the AR hinge and ligand-binding domain (ARLBD), which has been shown to interact with the AR N-terminal domain. The artificial VP16-ARLBD and ARLBD-p65 coactivators interacted with the AR N terminus and wild-type AR in an androgen-dependent and androgen-specific manner. VP16-ARLBD and ARLBD-p65 enhanced the AR transactivity up to 4- and 13-fold, respectively, without affecting the expression of the AR protein. The coactivators did not enhance the transcription activity of the progesterone receptor (PR) or the glucocorticoid receptor (GR), showing their specificity for the AR. In addition, to construct PR- and GR-specific coactivators, the VP16 activation domain was fused to the PR and GR hinge/ligand-binding domain. Although VP16-PRLBD and VP16-GRLBD interacted with the C-terminal portion of steroid receptor coactivator-1, they did not enhance the transcription activity of their receptor. The presented strategy of directing activation domains or other protein activities into the DNA-bound AR complex provides a novel means of manipulating AR function in vitro and in vivo.

Shortage of glutamine (CAG) homopolymeric repeats suppresses the expression of the androgen receptor in familial cases with complete androgen insensitivity syndrome

The androgen receptor gene of two familial cases with complete androgen insensitivity syndrome was analyzed. The shortage of glutamine homopolymeric repeats (13 repeats) in the N-terminal domain of the androgen receptor of the patients was identified by DNA sequence analysis. In vitro transfection experiments with the patients' androgen receptor gene indicated that the expression of the androgen receptor in transfected COS-7 cells was decreased by 10% as compared to that of the wild type androgen receptor gene. The thermal stability of the 5α-dihydrotestosterone—androgen receptor complex was also partially impaired. The capacity of the androgen receptor to activate target gene transcription was partially disturbed in a luciferase assay. The shortened glutamine homopolymeric repeats might therefore be related to the pathogenesis of complete androgen insensitivity syndrome.

Androgen hormone binding to adipose tissue in rats

Nuclear binding of androgen was examined, using R 1881, a synthetic androgen. The amount of androgen-receptor complexes bound to isolated nuclei was determined in isolated adipocytes from the epididymal (Epi), retroperitoneal (Ret), inguinal (Ing) and mesenteric (Mes) adipose tissues from intact and castrated rats. The binding was specific and saturable with a K d in the nanomolar range. Binding was examined after 2 days and after 1 and 2 weeks after castration, showing a higher binding in the Mes tissue in comparison with Ing at all time-points ( P < 0.05). Mes adipocytes showed a trend (0.05 < P < 0.1) to up-regulate their binding capacity 2 days after castration, and a significant ( P < 0.05) downregulation 2 weeks after castration. Two days after castration, R 1881 binding, expressed per mg triacylglycerol (TG), was generally higher in the Mes region ( P < 0.05). This was not fully significant in comparison with Epi tissue in intact rats. When expressed per cell the differences were somewhat diminished, due to differences in cell sizes. Androgen binding showed a negative correlation with T(i-uptake in vivo ( r = 0.85, P < 0.01), suggesting that a higher density of androgen receptors leads to a more inhibited lipid uptake. In conclusion, a specific androgen receptor was demonstrated in adipose tissue in rat, showing regional differences and a negative correlation with the lipid accumulation of the tissue.

Localization and hormonal stimulation of phosphorylation sites in the LNCaP-cell androgen receptor.

Phosphorylation of the androgen receptor in human prostate tumour cells (LNCaP) is increased by addition of androgens to intact cells. Double-label studies, using [35S]methionine incorporation into receptor protein, and [32P]P(i) to label metabolically receptor phosphorylation sites, have enabled us to determine the phosphate content, relative to receptor protein, of both nontransformed and transformed and androgen receptors generated in intact LNCaP cells. No net change in the phosphorylation of the intact 110 kDa steroid-binding component of the androgen-receptor complex was found upon transformation to the tight nuclear binding form in the intact cell. Partial proteolysis of androgen receptor protein metabolically labelled with [32P]P(i) and photolabelled with [3H]R1881 (methyltrienolone) revealed that phosphorylation occurs mainly in the N-terminal trans-activation domain, whereas no phosphorylation was detected in the steroid- and DNA-binding domains. The location of most (> 90%) of the hormonally regulated phosphorylation sites in the N-terminal trans-activation domain suggests a role of phosphorylation of the androgen receptor in transcription regulation.

Molecular genetics of human androgen insensitivity.

Androgen insensitivity syndromes represent one cause of human male pseudohermaphroditism related to defects in the androgen receptor. The formation of a biologically active androgen receptor complex with testosterone and 5 alpha-dihydrotestosterone is required for normal androgen action during fetal development and differentiation of the internal accessory sex glands and external genitalia. Cloning of the human androgen receptor complementary DNA and genetic screening of human subjects with the clinical and biochemical features of androgen insensitivity using the polymerase chain reaction, denaturing gradient gel electrophoresis and nucleotide sequencing techniques have led to the identification of molecular defects in the androgen receptor. The complexity of phenotypic presentation by affected subjects with the complete or partial forms of androgen insensitivity is represented by the heterogeneity of androgen receptor gene mutations which include deletions and point mutations, with the latter causing inappropriate splicing of RNA, premature termination of transcription and amino acid substitutions. The naturally occurring mutations in the androgen receptor of subjects with androgen insensitivity represent a base upon which we can increase our understanding of the structure and function of the androgen receptor in normal physiology and disease.

FUNDAMENTAL AND CLINICAL STUDIES ON A NEW NONSTEROIDAL 5α-REDUCTASE INHIBITOR AND A NEW STEROIDAL ANDROGEN-RECEPTOR COMPLEX FORMATION INHIBITOR

In the case of benign prostatic hypertrophy (BPH), operative treatment presents many problems and drug therapy is still important. We have examined the effects of a new steroidal antiandrogen (TZP4238, 17α-acetoxy-6-chloro-2-oxapregna-4, 6-diene-3, 20-dione) and a nonsteroidal 5α-reductase inhibitor (ONO3805, sodium- (-) -4- [2- {2, 3-dimethyl-4- [1- (4-isobutylphenyl) ethoxy] benzoylamino} phenoxy] butyrate), which are effective in reducing prostate weight, using Wistar rats. We also examined the clinical effects of these compounds in patients with BPH. Under endogenous androgen conditions, we assessed the effects of some antiandrogens on reduced accessory sex organ weight. As a result, it was suggested that progestin drugs (TZP4238 etc.) produced this effect at low doses and that the effect lasted for more than six weeks. Under exogenous androgen conditions, that is, without central nervous system action on the prostate, the effect of ONO3805 was almost equal to that of chlormadinone acetate (CMA). Furthermore, we examined TZP4238 and other drugs in terms of on the action of androgen-receptor complex formation in the cytosol and nuclear extracts. TZP4238 showed a competitive inhibitory action, in contrast to ONO3805. On the other hand, only ONO3805 had a 5α-reductase inhibitory action. Among tested compounds neither TZP4238 nor ONO3805 significantly changed blood testosterone concentrations in the rat. Clinical results are as follows : progestin drugs (TZP4238 and CMA) were more effective than others tested in reducing prostate volume and improving voiding disturbance and residual urine volume but produced frequent loss of libido. On the other hand, the 5α-reductase inhibitor was associated with no loss of libido. To maintain the patient's quality of life, it is hoped that a drug based on 5α-reductase inhibition, which has few side effects, will be developed.

Modulation of androgen-responsive gene expression by estrogen

Studies on hormonal action frequently focus on a single hormone. In intact animals, however, genes may respond to the balance of multiple hormones. Therefore, we have studied the mutual influence of sex steroids on eight genes previously known to be testosterone-responsive in kidneys of mice. A variety of responses to estrogen were recorded. Effects occurred primarily at the transcriptional level although in several cases there was also evidence of decreased mRNA stability. Estrogen did not affect the nuclear location of the androgen receptor. Apparently each gene interacts with both androgen-receptor complex and estrogen-receptor complex, and the ultimate outcome depends on each gene's detailed regulatory structure.

Purification of Androgen Receptors in Human Sebocytes and Hair

Human sebaceous glands (SG) and hair follicles (HF) are target structures in the skin for androgen action. They contain steroid enzymes, capable of transforming weak androgens into the target-tissue-active androgens testosterone (T) and dihydrotestosterone (DHT), which bind to the androgen receptor (AR) to regulate cellular transcription. The AR from HF and SG from human scalp tissue has been purified greater than 86,000 times by phenyl-sepharose, DEAE-sephacel, gel filtration chromatography, and ultrafiltration. Sucrose density gradient analysis and non-denaturing gradient polyacrylamide gel electrophoresis (PAGE) and sodium dodecyl sulfate (SDS)-PAGE revealed two molecular species of AR, an active form called monomer, capable of binding DHT with great specificity (4S, m = 62,000 Da, Kd = 0.6 nM, Bmax 8260 fmol/micrograms protein), and the other, an inactive form of the monomer called tetramer (10.8S, m = 252,000 Da, Kd = 2.9 nM). The two species are interconvertible, and after purification each appeared as a single band on SDS-PAGE. The conversion of the monomer to the tetramer AR form is influenced by reduced and oxidized glutathione, and possibly by an endogenous disulfide converting factor (DCF). Furthermore, biochemical events in the androgenic signal transduction sequence were shown to be stimulated by androgens via the AR. These include the total nuclear AR content, chromatin binding of AR complexes, and stimulation of RNA polymerase II, thus influencing gene expression, which is important in understanding regulation of HF growth and SG proliferation.

A protein in rat prostatic chromatin interacting with androgen regulated gene

2M NaCl-insoluble fraction of rat ventral prostate chromatin (residual proteins) contain proteins able to interact specifically with androgen-receptor complex and is, therefore, a part of the acceptor complex. Among residual proteins, a 97 KDa protein has been found which binds significantly to a genomic fiagment containing an androgen-regulated gene coding for a 22 KDa protein. The biological significance of this binding in androgen action need to be further studied. A mini-plasmid clone containing 22 KDa protein coding sequence was cloned into Charon 4A genomic library from which a 5.7 Kb genomic fragment was isolated, identified by hybridization with a 5′ and a 3′ cDNA probes, and shown to contain the 5′ flanking sequence. Restriction enzyme treatment of this fragment yielded a 4.7 Kb restriction fragment representing the 5′ upstream region and a 1.0 Kb containing part of the coding sequence. Deletion studies indicated that the 97 KDa protein bound only to a subclone of about 300 bp segment. Furthermore, gel shifting experiment supported its DNA-protein binding.

Androgenic expression in the submandibular gland of zinc-deficient mice.

The effects of zinc deficiency were studied in mice submandibular salivary glands (SMG). Zn-restricted mice (Zn-) were maintained from weaning until adult age (60 days) with a powdered diet containing 3 mg Zn2+/kg. Pair-fed animals (30 mg Zn2+/kg powdered diet) and control animals fed a regular pelleted diet were also used. Total protein content and proteolytic activity of SMG did not differ among the groups, but morphometric evaluations revealed significant alterations in the nucleus/cytoplasm size ratios, most likely due to an absolute reduction in nuclear volume (control = 122.5 +/- 6.4; Zn- = 91.6 +/- 10.5; pair-fed = 125.1 +/- 6.8 microns 3) paralleled by an increase of the height of the duct epithelium (control = 70.5 +/- 3.0; Zn- = 90.5 +/- 4.2; pair-fed = 81.7 +/- 3.0 microns). The altered food consistency could be responsible for these morphological changes. In order to assess the subcellular distribution of SMG androgen receptors in conditions of chronic Zn deficiency, Zn- animals were mated and the F1 generation was fed as their dams until the age of 45 days. Cytosolic (in 105,000 g supernatants) and nuclear (KCl-extracted) SMG receptors were determined with [3H]R1881. The Zn- animals had reduced nuclear/cytosolic ratios of androgen receptors (control = 0.62; Zn- = 0.14), as an indication that chronically deficient Zn intake determines a sort of destabilization of the interactions of androgen-receptor complexes with target cell nucleus.

Rapid homologous up-regulation of binding capacity of androgen receptors in intact cells

Incubation of MCF 7 cells with 5 alpha-dihydrotestosterone (DHT) at 37 degrees C led to a 70% increase in the Bmax of androgen receptor, as compared to the values measured at 2 degrees C, without detectable changes in equilibrium dissociation constants. When MCF 7 cells were incubated with hormone at 2 degrees C, to reach steady-state levels of androgen-receptor complex, a subsequent temperature shift to 37 degrees C induced a rapid (t 1/2 = 3 min) cycloheximide-insensitive increase in DHT binding to androgen receptor. MCF 7 cell treatments at 37 degrees C either before or after incubation with DHT at 2 degrees C showed that up-regulation of binding capacity of androgen receptor could be observed only if hormone is present during incubation at physiological temperature.

Androgen receptor abnormalities

The human androgen receptor is a member of the superfamily of steroid hormone receptors. Proper functioning of this protein is a prerequisite for normal male sexual differentiation and development. The cloning of the human androgen receptor cDNA and the elucidation of the genomic organization of the corresponding gene has enabled us to study androgen receptors in subjects with the clinical manifestation of androgen insensitivity and in a human prostate carcinoma cell line (LNCaP). Using PCR amplification, subcloning and sequencing of exons 2–8, we identified a G → T mutation in the androgen receptor gene of a subject with the complete form of androgen insensitivity, which inactivates the splice donor site at the exon 4/intron 4 boundary. This mutation causes the inactivation of a cryptic splice donor site in exon 4, which results in the deletion of 41 amino acids from the steroid binding domain. In two other independently arising cases we identified two different nucleotide alterations in codon 686 (GAC; aspartic acid) located in exon 4. One mutation (G → C) results in an aspartic acid → histidine substitution (with negligible androgen binding), whereas the other mutation (G → A) leads to an aspartic acid → asparagine substitution (normal androgen binding, but a rapidly dissociating androgen receptor complex). Sequence analysis of the androgen receptor in human LNCaP-cells (lymph node carcinoma of the prostate) revealed a point mutation (A → G) in codon 868 in exon 8 resulting in the substitution of threonine by alanine. This mutation is the cause of the altered steroid binding specificity of the LNCaP-cell androgen receptor. The functional consequences of the observed mutations with respect to protein expression, specific ligand binding and transcriptional activation, were established after transient expression of the mutant receptors in COS and HeLa cells. These findings illustrate that functional errors in the human androgen receptor have an enormous impact on phenotype and fertility.

The Effects of Anti-Androgen TZP4238 (17 α-acetoxy-6-chloro-2-oxapregna-4, 6-diene-3, 20-dione) and Its Related Compounds on the &lt;I&gt;In Vitro&lt;/I&gt; Formation of Androgen-Receptor Complex

It was reported that a new steroidal anti-androgen, TZP4238 (17 alpha-acetoxy-6-chloro-2-oxapregna-4,6-diene-3,20-dione), was 10 times stronger than chlormadinone acetate (CMA) as the in vivo anti-androgenic potential. To confirm this result, the effect of TZP4238 and CMA on ventral prostates of intact rats, or castrated ones treated by testosterone, was investigated. Our experiments demonstrated that TZP4238 was 3-5 times stronger than CMA. The effects of this compound on the androgen-receptor complex formation in the human and rat prostates were investigated and compared to the other anti-androgen and related compounds. An aliquot of cytosol or KCl extract from rat or human prostate was incubated with [3H]R1881 in the presence of various tested compounds. By means of dextran coated charcoal assay and sucrose density gradient centrifugation analysis, TZP4238 and TZP4239 showed a stronger inhibitory effect than other compounds except dihydrotestosterone. Judging from all these results, it was estimated that TZP4238 was 2-3 times stronger than CMA. The Scatchard plot analysis revealed that the inhibitory effect of TZP4238 was a competitive type. Between CMA and TZP4238, the difference of in vivo anti-androgenic potential was not identical with that of the inhibitory effect on the androgen-receptor complex formation.

Specific binding of androgen and androgen-receptor complex by microsomes from rat ventral prostate

Microsomes from rat ventral prostate show the presence of a high affinity-low capacity population of androgen-binding sites with affinity for ionic exchange resin similar to that of cytosol androgen receptor (AR), as manifested by similar results obtained with hydroxylapatite. The affinity for mibolerone was similar for both forms ( K a =0.5–2.9 × 10 10M −1). The membrane-bound form can be extracted in hypotonic buffer, with retention of binding properties. Isotonic sucrose allowed higher degree of extractability of the microsomal AR than 10% (v/v) glycerol. The presence of hormone lends stability to the microsomal AR, while high salt or nonionic detergents have a deleterious effect on their longevity. The microsomal receptor form is not sensitive to serine-proteases as opposed to the cytosol AR. After exhaustive extraction of binding sites, microsomes are capable of accepting cytosol mibolerone-receptor complexes to a level corresponding to the concentration of depleted binding sites; microsomes from non-target tissue do not manifest such capability. Microsomal AR complexes do not bind DNA and they are not activated after heat treatment. Mixed preparations of extracted microsomal complexes with cytosol complexes showed heat-induced increased ability to bind DNA to the same level of diluted cytosol complex alone, indicating the absence of a microsomal inhibitor of DNA binding. The results indicate the co-existence of a non-DNA binding form of the AR in the microsomal membranes with the classical DNA binding form of the AR present in the cytosol of ventral prostate homogenates.

Sequence-specific binding of androgen-receptor complexes to prostatic binding protein genes

Prostatic binding protein is a complex glycoprotein comprising three components, C1, C2 and C3, organized into two different heterodimers (C1-C3 and C2-C3). The rat ventral prostate genes encoding all three constituent polypeptides are expressed under androgenic control. Analysis of genomic fragments containing the genes and flanking sequences revealed in each case one androgen receptor-binding region upstream of or within the promoter and another in the first intron. The effect of androgens on the expression of these genes may, therefore, be mediated by these direct receptor-DNA interactions. The genomic fragments which contain androgen receptor-binding regions all contain nucleotide sequences reminiscent of glucocorticoid response elements (GRE). Mutations in these sequences in restriction fragments and in synthetic oligonucleotides significantly decreased their affinity for androgen-receptor complexes and their introduction into nonspecific sequences conferred affinity for androgen-receptor complexes. Based on these data, a consensus sequence for putative androgen response elements (ARE) is proposed. However, despite the specific recognition of these sequences by the androgen receptor in vitro, only the C3(l) intronic fragment could confer significant androgen responsiveness on a heterologous promoter. While this could be due to the fact that the GRE-like sequences present in the other fragments are not strong AREs, alternative hypotheses are being investigated currently. Not least of these is that the similar localization of the binding sites in each gene might underlie a more complex androgen regulation mechanism.

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.