Redox-dependent DNA binding of the purified androgen receptor: evidence for disulfide-linked androgen receptor dimers.

Full-length histidine-tagged, dihydrotestosterone-bound human androgen receptor (AR) was purified to homogeneity by affinity and gel-filtration chromatography for antibody production and analysis of AR dimerization and DNA binding properties. A monoclonal antibody was raised that recognized human and rat AR epitope (360)ArgAspTyrTyrAsnPheProLeuAla(368) in the NH(2)-terminal domain and slowed migration of AR-DNA complexes in mobility shift assays. AR binding to androgen response element DNA had a K(d) of 2.0 nM and a Hill coefficient of 2.1, indicating high-affinity, cooperative binding. AR solution dimerization was detected only at >/=0.2 microM AR, and DNA binding increased dimerization up to 30-fold. Slow- and fast-migrating AR-DNA complexes were detected under different reducing conditions that differed 5-fold in their dissociation rates from DNA. Treatment with the sulfhydryl oxidizing reagent diamide formed the faster migrating, slower dissociating complex, indicating it represents disulfide-linked AR dimers bound to DNA. The results indicate that high concentrations of purified AR are required for solution dimerization and that cooperative DNA binding stabilizes two dimer forms that differ in redox state.     

Zinc potentiation of androgen receptor binding to nuclei in vitro

Zn2+ potentiates binding of the 4.5S [3H]dihydrotestosterone-receptor complex to isolated rat prostate Dunning tumor nuclei in vitro when assayed in the presence of 300 microM ZnCl2, 3 mM MgCl2, 0.25 M sucrose, 5 mM mercaptoethanol, 0.15 M KCl, and 50 mM tris(hydroxymethyl)aminomethane, pH 7.5. In the presence of 5 mM mercaptoethanol, the concentration of 50 microM total Zn2+ required to promote half-maximal receptor binding to nuclei corresponds to a free Zn2+ concentration of 50 nM. The receptor-nuclear interaction appears to be selective for Zn2+; other divalent cations when added at a concentration of 1 mM to a buffer containing 5 mM mercaptoethanol are less effective (Ni2+) or have essentially no effect (Ca2+, Mg2+, Mn2+, Co2+, Cu2+, and Cd2+). Zn2+ does not alter the sedimentation rate of the 4.5S [3H]dihydrotestosterone receptor in the presence of mercaptoethanol; however, in the absence of mercaptoethanol, Zn2+ causes the receptor to aggregate. Zn2+-dependent nuclear binding of the 4.5S [3H]dihydrotestosterone receptor is saturable at 1.4 X 10(-13) mol of receptor sites/mg of DNA, corresponding to approximately 1150 sites/nucleus. In the presence of excess nuclei, up to 60% of added receptor is nuclear bound. An apparent binding constant for the receptor-nuclear interaction of 10(13) M-1 was approximated. Pyridoxal 5'-phosphate (less than or equal to 10 mM), but not 0.4 M KCl, inhibits Zn2+-dependent nuclear binding of the [3H]dihydrotestosterone receptor. Up to 66% of nuclear-bound receptor can be extracted in buffer containing 3 mM ethylenediaminetetraacetic acid plus either 0.4 M KCl or 10 mM pyridoxal 5'-phosphate.(ABSTRACT TRUNCATED AT 250 WORDS)     

DNA and ribonucleotide binding characteristics of two forms of the androgen receptor from rat prostates

Androgen receptors (sedimentation value approximately 4S and Stokes radius 2.8 nm) present in the cytoplasmic fraction obtained from prostates of castrated rats bind to DNA-Sepharose and double stranded DNA. A receptor fragment (sedimentation value approximately 3S and Stokes radius 2.3 nm) obtained from rat prostates in the course of a purification procedure showed greatly diminished binding affinity for both DNA-Sepharose and soluble DNA. In contrast, both the 4S cytosol receptor and the 3S receptor form interacted with equal affinity with prostate RNA or poly(UG). These observations provide evidence that for DNA binding a different or additional part of the receptor molecule is required than for RNA and polyribonucleotide binding.     

Dual role of FoxA1 in androgen receptor binding to chromatin, androgen signalling and prostate cancer

High androgen receptor (AR) level in primary tumour predicts increased prostate cancer-specific mortality. However, the mechanisms that regulate AR function in prostate cancer are poorly known. We report here a new paradigm for the forkhead protein FoxA1 action in androgen signalling. Besides pioneering the AR pathway, FoxA1 depletion elicited extensive redistribution of AR-binding sites (ARBs) on LNCaP-1F5 cell chromatin that was commensurate with changes in androgen-dependent gene expression signature. We identified three distinct classes of ARBs and androgen-responsive genes: (i) independent of FoxA1, (ii) pioneered by FoxA1 and (iii) masked by FoxA1 and functional upon FoxA1 depletion. FoxA1 depletion also reprogrammed AR binding in VCaP cells, and glucocorticoid receptor binding and glucocorticoid-dependent signalling in LNCaP-1F5 cells. Importantly, FoxA1 protein level in primary prostate tumour had significant association to disease outcome; high FoxA1 level was associated with poor prognosis, whereas low FoxA1 level, even in the presence of high AR expression, predicted good prognosis. The role of FoxA1 in androgen signalling and prostate cancer is distinctly different from that in oestrogen signalling and breast cancer.     

Synthesis and androgen receptor binding of dihydrotestosterone hemisuccinate homologs

Dihydrotestosterone-succinyl-agarose is the most common form of androgen affinity column. To investigate the effect of variation in the number of methylene groups between the ester and amide functions, a homologous series, varying the number of methylenes between the functional groups, has been synthesized and evaluated. In addition, since structure studies show 17 alpha-methyldihydrotestosterone binds with high affinity, a 17 alpha-carboxymethyl ligand (3) was studied. Relative binding affinities of the dicarboxylates (assayed as the n-butyl amides) range from 0.003 to 0.044 (dihydrotestosterone = 1.00), while there was no detectable binding for 3. Only the suberate binds better than the much-used succinate, and it would be a likely candidate for an affinity ligand.     

Specificity of sigma-dependent binding of RNA polymerase to DNA

Summary Although a large number of E. coli RNA polymerase molecules can bind to phage T3 DNA, not more than three remain bound per DNA template after addition of poly inosinic acid (poly I) which has a high affinity for the enzyme. These stable complexes are able to initiate RNA chains without lag as the enzyme is resistant against rifampicin if substrate is added simultaneously with the drug. Poly I resistant complexes decay very rapidly in the cold (Fig. 2) and are not formed in the absence of the polymerase factor (Table 2). The data provide additional support for the idea that the factor effects the binding of the enzymes to specific sites on the DNA template.     

Domains of the human androgen receptor and glucocorticoid receptor involved in binding to the nuclear matrix

Steroid receptors have been reported to bind to the nuclear matrix. The nuclear matrix is operationally defined as the residual nuclear structure that remains after extraction of most of the chromatin and all soluble and loosely bound componnets. To obtain insight in the molecular mechanism of the interaction of steroid receptors with the nuclear matrix, we studied the binding of several deletion mutants of the human androgen receptor (hAR) and the human glucocorticoid receptor (hGR) to the nuclear matrix. Receptor binding was tested for two different nuclear matrix preparations: complete matrices, in which most matrix proteins are retained during the isolation procedure, and depleted matrices, which consist of only a subset of these proteins. The results show that the C-terminal domain of the hAR binds tightly to both depleted and complete matrices. In addition, at least one other domain of the hAR binds to complete matrices but not to depleted matrices. In contrast to the hAR, the hGR binds only to complete matrices. For this interaction both the DNA-binding domain and the C-terminal domain of the hGR are required, whereas the N-terminal domain is not. We conclude that specific protein domains of the hAR and the hGR are involved in binding to the nuclear matrix. In addition, our results indicate that the hAR and the hGR are attached to the nuclear matrix through different molecular interactions.     

Estrogen and androgen receptor binding affinity of 10 beta-chloro-estrenen derivatives

10 beta-Chloroestradien-3-one and its derivatives with chlorine substitution in ring A have been prepared. Efficient synthetic methods for 2-chloro- and 4-chloroestradiol are described. The binding affinity of these chlorinated estrogens to the uterine estrogen receptor was measured by a competitive binding assay using [3H]estradiol as ligand. 4-Chloroestradiol showed high binding affinity for the receptor (110% of that of estradiol). 2-Chloroestradiol, 10 beta-chloroestradien-3-one and 4,10 beta-dichloroestradien-3-one had moderate binding affinity. The structures of 10 beta-chloroestradien-3-one and androst-1,4-dien-3-one are very similar and can almost be superimposed. However, their binding affinities to the estrogen and androgen receptor were different. Androst-1,4-dien-3-one displayed no measurable affinity for the estrogen receptor and measurable affinity for the androgen receptor whereas 10 beta-chloroestradien-3-one had very low affinity for the androgen receptor.