Androgen receptor defects: historical, clinical, and molecular perspectives.
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Amino Acid Sequence↗
Biomedical subjects
F S French
Publications and source records attributed to F S French.
Specificity of ligand-dependent androgen receptor stabilization: receptor domain interactions influence ligand dissociation and receptor stability.
The molecular basis for the different physiological effects of testosterone (T) and dihydrotestosterone (DHT) was investigated using recombinantly expressed wild-type and mutant androgen receptor (AR). Rates of androgen dissociation from nuclear and cytoplasmic AR were compared with hormone- and concentration-dependent receptor degradation rates. T dissociates from AR 3 times faster than DHT or methyltrienolone (R1881) and is less effective in stabilizing the receptor. Analysis of AR deletion mutants and AR/glucocorticoid receptor chimeras indicates that the AR NH2-terminal domain has a specific role in stabilizing the receptor by slowing the rate of ligand dissociation and AR degradation. Amino acid mutations that abolish receptor dimerization, nuclear localization, or DNA-binding activity have no significant effect on androgen dissociation or AR degradation. A naturally occurring steroid-binding domain mutation (Val889 to Met) that causes androgen insensitivity, but does not alter equilibrium androgen binding affinity, lowered the androgen-binding capacity as a result of increased rates of androgen dissociation and AR degradation. Thus, AR stabilization and function require prolonged receptor occupancy with androgen, with a similar extent of stabilization observed at higher concentrations of faster dissociating androgens and lower concentrations of slower dissociating androgens. Retention of receptor-bound androgen is enhanced by an interaction between the AR NH2-terminal and steroid-binding domains. The ligand specificity and concentration dependence of receptor stabilization provide an explanation for physiological differences in the actions of T and DHT.
Animals↗
Androgen and glucocorticoid receptors interact with insulin degrading enzyme.
We recently identified a protein, designated receptor accessory factor (RAF), that interacts directly with and enhances the DNA binding of androgen (AR) and glucocorticoid (GR) receptor peptide fragments. To determine its identity, RAF was purified from HeLa cell extracts by anion-exchange and DNA affinity chromatography. RAF activity co-purified with a 110-kDa protein, the partial amino acid sequence of which shares 97.5% identity with insulin degrading enzyme (IDE), a metalloendoprotease implicated in the intracellular degradation of insulin. The identity of RAF was confirmed in gel shift assays by demonstrating that AR.RAF and GR.RAF DNA complexes shifted to a slower mobility in the presence of an IDE antibody. Purified preparations of RAF had insulin degrading activity, and this activity was inhibited by AR. In addition, the interaction of AR or GR with RAF was competed by insulin and bacitracin, a competitive inhibitor of IDE. The interactions of AR and GR with IDE may have important implications for both insulin- and steroid-mediated signaling.
Base Sequence↗
Regulatory mechanism of human factor IX gene: protein binding at the Leyden-specific region.
Hemophilia B-Leyden is characterized by the gradual amelioration of bleeding after the onset of puberty. All Leyden phenotype mutations found to date lie within the Leyden-specific region, which spans roughly nt-40 to +20 in the 5' end of the human factor IX gene. With HepG2 cell nuclear extracts, the Leyden-specific region and its immediate neighboring region of the normal factor IX gene showed five DNase I footprints: FP-I (nt +4 to +19), FP-II (nt -16 to -3), FP-III (nt -27 to -19), FP-IV (nt -67 to -49), and FP-V (nt -99 to -77). Protein binding affinities of short oligonucleotides containing sequences of FP-I, FP-II, or FP-III were substantially reduced in the presence of Leyden phenotype mutations in these areas, correlating well with the negative effects of these mutations on factor IX gene expression. A Leyden phenotype mutation at nt -20 (T to A) caused a loss of both footprints FP-III and FP-II but generated a new footprint, FP-III' (nt -34 to -23), partially overlapping with FP-III, indicating mutation-dependent competitive protein binding at these sites. Although the FP-III' area contains an androgen responsive element-like sequence, the nuclear protein that binds at FP-III' is not androgen receptor. The protein was not recognized by anti-androgen receptor antibody and, furthermore, was present not only in liver but also in both androgen receptor-positive and androgen receptor-negative cells in electrophoretic mobility shift assays. The nuclear concentration of this protein increased significantly upon treatment of the HepG2 cells with testosterone. Its binding affinity to an oligonucleotide (-32sub) containing the FP-III' sequence was greatly reduced in the presence of exogenous androgen receptor, suggesting a possible interaction of this protein with androgen receptor. The affinities of both this protein and a protein which binds to FP-III (presumably HNF-4) to -32sub with a mutation at nt -26 were grossly lowered. These findings suggest that the amelioration of hemophilia B-Leyden with a mutation at nt -20 after puberty involves binding of a specific non-androgen receptor nuclear protein at FP-III' and it is able to substitute for the function of a protein bound at FP-III in the normal gene optimally through its elevated interaction with androgen receptor upon a surge of testosterone.(ABSTRACT TRUNCATED AT 400 WORDS)
Animals↗
Characterization of mutant androgen receptors causing partial androgen insensitivity syndrome.
The androgen insensitivity syndrome (AIS) is an X-linked disorder caused by mutations of the androgen receptor (AR) gene resulting in a spectrum of sex phenotypes that ranges from complete female (complete AIS) to nearly complete male (partial AIS). Using the polymerase chain reaction and denaturing gradient gel electrophoresis, we have analyzed the AR gene in three 46,XY individuals with partial AIS. In one subject whose androgen insensitivity was manifest at birth by clitoromegaly, posterior labial fusion, and a urogenital sinus, androgen-binding affinity in genital skin fibroblasts was similar to that of the control. In this subject, a mutation was identified in exon C encoding the second zinc finger of the androgen receptor. The mutation converted a leucine residue at position 616 to arginine, causing greatly reduced binding of receptor to an androgen-response element DNA sequence. However, the mutant AR retained a low level of transcriptional activity at physiological androgen concentrations in keeping with the subject's phenotype of partial AIS. In the second subject, who also had an ambiguous external genital phenotype, a single base mutation was identified in exon G, converting arginine at position 840 to histidine. Androgen-binding affinity in genital skin fibroblasts of this subject was 7-fold lower than control, and the mutant receptor had reduced transcriptional activity. In the third subject, who had a female phenotype with normal pubic hair reflecting a low degree of androgen responsiveness, the valine residue at position 889 was replaced by methionine. This mutant receptor had apparent normal androgen-binding affinity but reduced androgen-binding capacity when examined by expression of the recreated mutant AR in COS 7 cells. These results demonstrate the clinical, functional, and molecular heterogeneity in the syndrome of partial androgen insensitivity.
Adolescent↗
Androgen insensitivity syndromes.
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Adolescent↗
Androgen receptor immunohistochemistry on paraffin-embedded tissue.
Androgen deprivation remains the primary therapy for patients with metastatic prostate cancer. Response to hormonal manipulation, however, has been quite variable. Androgen receptor (AR) expression has been used to predict clinical response to antiandrogenic treatment. However, methods of detection of AR expression have been limited to receptor biochemical assays in cytosolic or nuclear fractions of frozen tissue homogenates with obvious contamination problems by nonmalignant epithelial and stromal cells. As a result, studies correlating AR expression with response to hormonal therapy have been limited and controversial. More recently, immunohistochemical methods of analysis have become available, but only on frozen tissue. We describe a simple method to evaluate ARs on formalin-fixed, paraffin-embedded tissue sections using antigen retrieval methods. Primary, as well as metastatic, prostate carcinomas showed nuclear staining for ARs. Secretory cells stained uniformly in hyperplastic and normal prostatic glands. The majority of stromal cells had strong nuclear positivity. All other cancers tested (colon, breast, lung, skin, kidney) were negative. This immunohistochemical technique allows evaluation of prostate cancer AR status in routinely processed tissues. Thus, application of this technology to archival material should permit assessment of whether AR expression is predictive of response to endocrine therapy in advanced prostate cancer.
Adenocarcinoma↗
A complex response element in intron 1 of the androgen-regulated 20-kDa protein gene displays cell type-dependent androgen receptor specificity.
The androgen-regulated 20-kDa protein gene consists of four exons that code for a major secretory protein of rat ventral prostate. Analysis of its potential cis-acting transcriptional regulatory elements revealed that a large intron 1 region (In-1) had stronger androgen response element (ARE) activity than did the 5'-flanking DNA. In cotransfected CV1 cells, In-1 and its most active subfragment In-1c functioned as AREs but not glucocorticoid response elements (GRE). Nevertheless several ARE/GRE-like partial palindromic sequences are present in In-1c, and it bound both androgen receptors and glucocorticoid receptors in mobility shift assays. A cluster of three ARE/GRE-like sequences contained within a 39-base pair sequence of In-1c had both ARE and GRE activities when analyzed as an isolated oligonucleotide, suggesting that other elements within In-1c determined its ARE specificity. In addition to ARE/GRE-like sequences, In-1c contains putative response elements for the transcription factors AP1, CREB, AP2, OCT-1, C/EBP, and a number of inverted and direct repeats. The ARE specificity of In-1c observed in CV1 cells was diminished in PC3 and HeLa cells transiently cotransfected with an androgen receptor or glucocorticoid receptor expression vector together with an In-1c reporter vector; however, the ARE activity of In-1c was greater than its GRE activity in these cell lines. Interestingly, a 131-base pair subfragment of In-1c retained ARE specificity in all three cell lines.
Amino Acid Sequence↗
Receptor accessory factor enhances specific DNA binding of androgen and glucocorticoid receptors.
Protein-protein interactions are common among transcriptional activators and may have important consequences for gene regulation. Using the mobility shift assay, we have identified a factor that enhances specific DNA binding of truncated rat androgen (AR) and glucocorticoid (GR) receptors by 25- and 6-fold, respectively, through the formation of heteromeric complexes. This factor, designated receptor accessory factor, or RAF, also potentiates DNA binding of full-length human GR. RAF is temperature and trypsin sensitive and is present in a variety of cultured mammalian cells. By gel filtration RAF has a predicted molecular mass of 130 kDa. RAF enhancement of AR-DNA binding is optimal with androgen response element DNA. RAF appears to interact directly with AR because 1) deoxycholate, which interferes with protein-protein but not protein-DNA interactions, prevents RAF.AR.DNA complex formation, 2) RAF activity is recovered from an androgen response element DNA affinity column only in the presence of AR, and 3) RAF increases the size of an AR.DNA complex by gel filtration. Mutagenesis of truncated AR fragments indicates that a region in the NH2-terminal domain is required for RAF to enhance AR-DNA binding. The interaction of RAF with AR and GR suggests that RAF might influence the ability of these nuclear receptors to activate transcription.
Animals↗
Transcriptional repression of the glycoprotein hormone alpha subunit gene by androgen may involve direct binding of androgen receptor to the proximal promoter.
Testicular androgens suppress the synthesis and secretion of the pituitary gonadotropins, in particular, luteinizing hormone. This suppressive effect includes transcription of both the common alpha subunit gene and the unique beta subunit genes. Herein, we demonstrate that 1500 base pairs (bp) of proximal 5'-flanking region derived from the human alpha subunit gene and a shorter 315-bp segment of the bovine alpha subunit gene confer negative regulation by androgen to the gene encoding bacterial chloramphenicol acetyltransferase in transgenic mice. Cotransfection assays with human androgen receptor indicated that the 1500-bp promoter region of the human alpha subunit gene also confers androgen regulation (transcriptional suppression) to reporter genes in both pituitary and placental cell lines. This raises the possibility of a role for DNA binding in suppression of alpha subunit transcription by activated androgen receptor. Consistent with this possibility, we have used a gel-mobility shift assay to detect several high affinity binding sites for androgen receptor located in the proximal promoter of the human alpha subunit gene. The strongest androgen receptor binding site is located at approximately -101 in the proximal 5'-flanking region. This steroid receptor binding site overlaps another binding site that defines one of several contiguous cis-acting regulatory elements required for basal transcriptional activity. Thus, binding of activated androgen receptor to this region may block the binding of a requisite trans-acting factor and lead to an attenuation in transcription. We conclude that this interaction, which occurs directly at the level of the pituitary, represents one of several physiological avenues through which androgens regulate gonadotropin gene expression.
Androgens↗
Characterization of the DNA-binding domain of the mouse uterine estrogen receptor using site-specific polyclonal antibodies.
The DNA-binding domain of the mouse uterine estrogen receptor (ER) was characterized using site-specific polyclonal antibodies. The peptides used as antigens have sequences corresponding to amino acids 185-199 and 227-245, the two zinc finger regions of the DNA-binding domain of the human ER, and produced anti-sera designated A-1542 and A-1554, respectively. Mouse uterine nuclear ER and salt-activated 4 S cytosol receptor, as well as 8 S untransformed cytosol receptor, were observed to react with the antisera by Western blot and sucrose density gradient centrifugation analyses indicating that the DNA-binding domain of the 8 S cytosol receptor is not completely masked by heat shock protein 90 or other proteins. Only A-1554 detected a nuclear-specific doublet form of the ER on Western blot analysis. In a gel shift assay, neither antisera altered the pattern of the nuclear ER interaction with the vitellogenin A2 estrogen response element (VRE). In contrast, antiserum A-1554 partially shifted the 8 S cytosol receptor-VRE complex. This concurs with mutational analysis and x-ray crystallography studies with the human ER that have shown that the second finger is not in contact with the DNA. The results of the gel shift assay were confirmed by sucrose density gradient analysis using the same buffer conditions. The nuclear receptor-VRE complex did not react with either antisera, suggesting that when the dimeric nuclear receptor form binds the VRE, the specific receptor epitopes involved with the DNA binding may be blocked and unable to bind the antisera. The cytosol receptor-VRE complex reacted only partially with the second finger antisera A-1554, suggesting that on receptor monomers the second finger epitope is not completely blocked by DNA binding or dimer formation.
Amino Acid Sequence↗
Immunohistochemistry of the androgen receptor in human benign and malignant prostate tissue.
The role of the androgen receptor in the development and progression of prostatic carcinoma has not been defined. The development of androgen receptor antibodies has provided new opportunities for direct immunohistochemical analysis. We compared the androgen receptor staining characteristics of fresh human prostatic carcinoma with benign prostatic hyperplasia (BPH) using an avidin-biotin complex method. Cancer and BPH obtained from the same radical retropubic prostatectomy specimen in 10 prostate cancer patients (68.5 +/- 7.3 years old standard deviation) and BPH from 10 noncancer patients (71.5 +/- 7.7 years old) were incubated with AR52, a rabbit polyclonal antibody against a synthetic androgen receptor peptide. Nuclei within each section were graded for intensity of androgen receptor staining (0-absent, 1-weak, 2-moderate or 3-strong) and the percentage (0 to 100%) of nuclei sampled staining at each of these intensity levels was determined. A total intensity score (0 to 300) was the summation of the products of each intensity score (0 to 3) and their corresponding percentages. Cancer sections (166 +/- 69) stained less intensely and more heterogeneously than BPH in cancer patients (246 +/- 41, Student's t test p < 0.05) and noncancer patients (225 +/- 39, p < 0.05). The decreased intensity and greater heterogeneity of androgen receptor staining in cancer tissue may implicate a quantitative or functional difference in androgen receptor between prostatic carcinoma and BPH.
Aged↗
Molecular cloning of rat prostate transglutaminase complementary DNA. The major androgen-regulated protein DP1 of rat dorsal prostate and coagulating gland.
Complementary DNA (cDNA) that codes for a major androgen-dependent secretory protein of rat coagulating gland and dorsal prostate, dorsal protein 1 (DP1), was isolated by molecular cloning. Recombinant DP1 cDNA clones were identified from a bacteriophage lambda gt11 rat coagulating gland expression library using an affinity purified polyclonal antibody. Amino acid sequence deduced from DNA contained sequences identical with several DP1 cyanogen bromide cleavage fragments. Northern blot hybridization of poly(A) RNA isolated from intact rat dorsal prostate and coagulating gland revealed a predominant messenger RNA (mRNA) species of approximately 3200 nucleotides. Tissue-specific expression of DP1 mRNA was indicated by the absence of DP1 mRNA in ventral prostate and other tissues of the rat. Expression of DP1 mRNA was androgen-dependent, decreasing approximately 80% 7 days after castration and increasing rapidly following androgen replacement. Southern blot analysis of restriction enzyme-digested rat DNA indicated that DP1 is encoded by a single gene and that no major genomic rearrangements accounted for its lack of expression in the dorsal prostate-derived rat Dunning tumor. Sequence comparisons revealed that rat prostate DP1 shares sequence identity with Factor XIIIa and tissue transglutaminase, including the active center, GQCWVF, indicating that DP1 is a member of the transglutaminase gene family.
Amino Acid Sequence↗
Response elements of the androgen-regulated C3 gene.
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Androgens↗
Response elements of the androgen-regulated C3 gene.
Intron and 5'-flanking regions of the androgen-regulated C3 subunit gene contain potential cis-acting transcription control sequences including several 15-base pair (bp) partial palindromes resembling response elements for glucocorticoid (GRE) and progesterone (PRE) receptors. Specific DNA binding of the androgen receptor (AR) and androgen-dependent activation of transcription indicate that some of these GRE/PRE-like sequences are capable of functioning as androgen response elements (ARE). A 0.3-kilobase pair (kbp) 5'-flanking fragment including the promoter region contains one such sequence (element A) and a 0.5-kbp region of the first intron contains two sequences (elements B and C). Androgen-dependent enhancement of transcription was assayed by cotransfection of CV1 cells with a rat AR expression vector, pCMVrAR, and C3 genomic fragments or synthetic elements cloned into the reporter vector ptkCAT. Enhancement of chloramphenicol acetyltransferase activity with the 0.5-kbp first intron fragment was 16 +/- 4-fold, while with the 0.3-kbp 5'-flanking fragment no response was detected and element C alone was greater than B or A. Binding of AR in the mobility shift assay correlated with androgen-dependent enhancement of chloramphenicol acetyltransferase activity. The intensity of transcriptional enhancement with the 0.5-kbp intron fragment suggested that other regulatory sequences within this intron region potentiated the ARE activities of elements B and C. ARE activity of the strongest C3 gene response element (C) was similar to that of a potent GRE (element M) of the mouse mammary tumor virus gene.
Amino Acid Sequence↗
Complete deletion of the androgen receptor gene: definition of the null phenotype of the androgen insensitivity syndrome and determination of carrier status.
The molecular basis of androgen insensitivity was investigated in a family with the complete form of the syndrome. Polymerase chain reaction amplification and Southern blot analysis of genomic DNA revealed a deletion of the entire androgen receptor (AR) gene in affected individuals. The carrier status of female members of this family was examined using a HindIII restriction fragment length polymorphism associated with the AR gene. Obligate carriers were hemizygous for one of the two alleles at this locus, while heterozygosity for the polymorphic alleles, implying the presence of two copies of the AR gene, indicated noncarrier status. This conclusion was supported by gene dosage studies using comparative densitometric analysis of Southern blots hybridized simultaneously with an AR cDNA probe and a control cDNA probe from an unrelated gene. Finally, the pattern of inheritance of another X-linked DNA polymorphism allowed us to conclude that the original mutation had occurred in the germ line of the maternal great-grandfather of the index patient. Although rare, complete deletion of the AR gene is of particular importance in terms of correlation between molecular defect and phenotype, as it represents the quintessential form of complete androgen insensitivity, the null phenotype.
Alleles↗
Single base mutations in the human androgen receptor gene causing complete androgen insensitivity: rapid detection by a modified denaturing gradient gel electrophoresis technique.
Mutations of the human androgen receptor (AR) gene impair normal sexual differentiation and development in karyotypic males, resulting in a spectrum of external genital phenotypes ranging from complete female to nearly complete male. Identification and characterization of these mutations can provide valuable information regarding the functional importance of specific amino acids of the AR. To screen for point mutations in the AR gene underlying the phenotypic abnormalities in the androgen insensitivity syndrome (AIS), the eight exons of the AR gene were amplified from genomic DNA using the polymerase chain reaction (PCR) and analyzed by denaturing gradient gel electrophoresis. A computer program, MELTMAP, was used to identify optimum sequences for denaturing gradient gel electrophoresis, and mutation detection sensitivity was enhanced by forming heteroduplexes between control and subject PCR products to create base mismatches. In seven families with complete AIS, single base mutations were found in the region of the AR gene encoding the steroid-binding domain of the receptor. The mutations that converted amino acid 774 from Arg to His and amino acid 864 from Asp to Gly were recreated using site-directed mutagenesis and the mutant ARs expressed in COS 7 and CV1 cells. In both cases, abnormalities of androgen binding and transcriptional activation were consistent with the observed sex phenotype. These results together with others reported previously demonstrate that single amino acid changes within the region encoded by exons D to H of the AR gene can alter androgen binding and are a common cause of complete androgen resistance. The strategy used herein, employing denaturing gradient gel analysis of heteroduplex PCR products, provides a valuable aid to rapid detection of single base mutations in AIS.
Adolescent↗
Complete androgen insensitivity due to deletion of exon C of the androgen receptor gene highlights the functional importance of the second zinc finger of the androgen receptor in vivo.
Androgen-dependent gene transcription is mediated by the androgen receptor (AR) through interaction of its central zinc finger region with specific DNA sequences on target genes. Failure of this receptor-mediated gene transcription results in end organ resistance to androgens-the androgen insensitivity syndromes. In a pair of siblings with complete androgen insensitivity who had supranormal levels of androgen binding in genital skin fibroblasts, polymerase chain reaction and Southern blot analysis of the androgen receptor gene confirmed by polymerase chain reaction and sequence analysis of AR cDNA, revealed an in-frame deletion of exon C encoding the second zinc finger of the receptor. The mutant receptor in cultured genital skin fibroblasts had normal androgen binding affinity and was localized in the nucleus but had markedly reduced DNA-binding affinity. When recreated in vitro and tested in a cotransfection assay system the mutant receptor failed to activate transcription of an androgen-responsive reporter gene. This naturally occurring mutation highlights the functional dependence of the AR upon its second zinc finger in vivo and explains the complete insensitivity to androgen manifest by the affected individuals despite increased androgen binding. The elevated AR levels in the subjects' genital skin fibroblasts further suggests a possible role for the second zinc finger in autoregulation of receptor levels in vivo.
Androgen-Insensitivity Syndrome↗