Male pseudohermaphroditism.
Explore the source record for details and available documents.
Adrenal Hyperplasia, Congenital↗
Biomedical subjects
F S French
Publications and source records attributed to F S French.
An androgen-inducible expression system for Saccharomyces cerevisiae.
A novel controllable expression system for Saccharomyces cerevisiae has been developed. Expression of the gene encoding the human androgen receptor, from a strong yeast promoter, results in transactivation of a hybrid promoter carrying androgen-responsive sequences such that a target gene may be expressed in an androgen-dependent manner. By selection of an appropriate combination of androgen receptor level, target-gene copy number and concentration of the androgenic ligand, dihydrotestosterone, the expression level can be set within a 1400-fold range with no detectable effect on normal cell growth.
Androgens↗
Transcriptional activation and nuclear targeting signals of the human androgen receptor.
The androgen receptor (AR) is a signal-transducing protein required for sexual differentiation, development, and expression of the male phenotype. A series of human AR deletion mutants were created either by site-directed mutagenesis using restriction enzyme digestion, the polymerase chain reaction, or, for a series of unidirectional NH2-terminal deletions, exonuclease III digestion. Receptor mutants were expressed in monkey kidney COS cells as truncated AR proteins between 20 and 107 kDa as revealed on immunoblots, where wild type AR was a doublet of 114 and 108 kDa. Subcellular localization by immunocytochemical staining demonstrated androgen-dependent nuclear uptake of AR from a perinuclear region of the cytoplasm. A nuclear targeting signal similar in sequence and position to the glucocorticoid receptor and homologous to the SV40 large T antigen was required for androgen-induced nuclear uptake of wild type AR. AR mutants lacking the NH2-terminal and/or steroid binding domains were constitutively nuclear with reduced transcriptional activity. Transcriptional activation by wild type AR was androgen-dependent in cotransfection studies of CV1 cells using the chloramphenicol acetyltransferase reporter gene linked to the mouse mammary tumor virus promoter. Deletion mutagenesis revealed within the NH2-terminal region a domain required for full transcriptional activity and within the steroid binding domain, an inhibitory function, deletion of which yielded a constitutively active receptor. Inhibition of wild type AR by coexpression with an inactive NH2-terminal fragment suggested competition for nuclear factors required for transcriptional regulation. These studies demonstrate a concerted interplay among the domains of the AR protein in regulating gene transcription.
Amino Acid Sequence↗
Molecular analysis of the androgen receptor.
Explore the source record for details and available documents.
Amino Acid Sequence↗
A frameshift mutation destabilizes androgen receptor messenger RNA in the Tfm mouse.
A composite mouse androgen receptor DNA sequence was obtained by amplifying genomic DNA or cDNA using the polymerase chain reaction. The open reading frame was 2,697 basepairs, encoding a polypeptide of 899 amino acids (98,204 mol wt). Amino acid sequence comparisons indicated that the mouse androgen receptor (AR) is 97% homologous with rat AR and 83% with human AR. The amino acid sequences of the three receptors are identical within the DNA- and steroid-binding domains. Northern blot analysis revealed the predominant mouse AR mRNA to be 10 kilobases (kb). A 1.7-kb mRNA species was detected in mouse kidney using a cDNA probe containing only 5' untranslated AR sequence. Lack of hybridization with AR-coding sequence probes suggested that the 1.7-kb mRNA was not a truncated form of AR mRNA. Sequencing of genomic DNA isolated from testicular feminized (Tfm) mice revealed a single base deletion in the N-terminal domain, resulting in a frameshift mutation. Cycloheximide treatment caused a dramatic increase in AR mRNA in kidneys of Tfm mice, but not wild-type mice, suggesting that the Tfm mutation results in an unstable AR mRNA.
Amino Acid Sequence↗
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.
Amino Acid Sequence↗
Expression and localization of androgen receptor in the R-3327 Dunning rat prostatic adenocarcinoma.
The Dunning R-3327 rat prostatic adenocarcinoma and its sublines have been developed as a model system to study prostate tumor progression. We have used this system to study the changes in androgen receptor (AR) and AR mRNA expression which occur during tumor progression from androgen dependent to androgen independent growth. Dorsal prostate and all tumor sublines contained a 10-kilobase AR mRNA on Northern blot analysis. The levels of AR mRNA in each subline compared to dorsal prostate (100%) were: H (75%) greater than G (48%) greater than HI (25%) greater than HI-F = AT-1 = AT-3 = MAT-Lu = MAT-Ly-Lu = less than 5%. Immunocytochemistry showed AR predominantly in acinar epithelial cells of dorsal prostate and the androgen sensitive H subline. In the H subline, both acinar epithelial cells and locally invasive adenocarcinoma cells within the stroma showed positive immunostaining. The androgen responsive, anaplastic G subline also showed strong positive immunostaining. The androgen resistant AT-1 and MAT-Lu sublines lacked immunostaining for the AR. Steroid autoradiography revealed a similar cellular distribution of AR. These data suggest that in the Dunning system the loss of androgen binding and responsiveness is primarily due to selective changes in gene expression and not to gene rearrangements or posttranscriptional or translational modification of the AR mRNA or protein.
Adenocarcinoma↗
Molecular basis of androgen insensitivity.
Explore the source record for details and available documents.
Amino Acid Sequence↗
Immunohistochemical localization of the androgen receptor in rat and human tissues.
Immunohistochemical localization of the androgen receptor (AR) was performed in reproductive tissues, submaxillary gland, pituitary, and brain of the rat and in human prostate. AR was visualized using either of two polyclonal antibodies raised against peptides with sequences derived from rat and human AR. Tissue sections of 6-8 microns, frozen in isopentane and fixed in paraformaldehyde, were stained using immunoglobulin G fractions of immune, preimmune, and peptide-adsorbed immune sera in the avidin-biotin peroxidase procedure. AR was prominent in nuclei of acinar epithelial cells of epididymis, ventral prostate, seminal vesicle, and ductus deferens from the intact rat. Androgen withdrawal, 3 days after castration, resulted in the loss of receptor immunostaining, which was restored within 15 min of androgen administration. Stromal cell staining was absent or weak in the ventral prostate of intact rats, but was more evident in the epididymis. AR was confined to nuclei of cells within and bordering the interstitial compartment of the testis, including Sertoli cells, peritubular myoid cells, and interstitial cells, and was undetectable in germ cells. Submaxillary gland epithelial cells and a population of rat anterior pituitary cells showed strong nuclear staining of AR. In rat brain, AR was present in the medial preoptic, arcurate, and ventromedial nuclei of the hypothalamus, the medial nucleus of the amygdala, the CA-1 hippocampus, and the cortex. AR was prominent in acinar epithelial cells in human benign prostatic hyperplasia and was also present in stroma of fibromuscular benign hyperplasia. Heterogeneous staining was observed in stromal and epithelial cells of prostatic adenocarcinoma. The results of these studies indicate that AR can be detected immunohistochemically in a variety of tissues and cell types using antipeptide polyclonal antibodies. The presence of AR in tissues correlated with their known androgen responsiveness.
Animals↗
Autologous down-regulation of androgen receptor messenger ribonucleic acid.
Autoregulation of androgen receptor (AR) mRNA was investigated using Northern blot analysis with AR cDNA fragments as probes. The amount of AR mRNA increased 2- to 10-fold with androgen withdrawal and decreased below control levels after androgen stimulation in rat ventral prostate, coagulating gland, epididymis, seminal vesicle, kidney, and brain, and in a human prostate cancer cell line, LNCaP. In rat ventral prostate, AR mRNA increased 2- to 3-fold within 24 h after castration and remained elevated for 4 days. Treatment with testosterone propionate beginning 24 h after castration reduced ventral prostate AR mRNA 4-fold within 8 h of androgen replacement. Administration of estradiol 24 h after castration had no significant effect on prostatic AR mRNA. Androgens, including testosterone and the synthetic androgen methyltrienolone (R1881), or the antiandrogen cyproterone acetate down-regulated AR mRNA in vitro in LNCaP cells, whereas estradiol was without effect. Administration of testosterone propionate to rats with androgen insensitivity did not decrease AR mRNA. Down-regulation of AR mRNA by androgen is therefore a receptor-mediated process which occurs in vivo in rat tissues that differ in androgen responsiveness and in cultured human prostate cells.
Animals↗
Functional characterization of naturally occurring mutant androgen receptors from subjects with complete androgen insensitivity.
Mutations in the androgen receptor (AR) are thought to cause complete androgen insensitivity (CAIS) in 46,XY human subjects who have a female phenotype despite normal adult male concentrations of plasma testosterone. Assays of AR binding in cultured skin fibroblasts from subjects with CAIS show either an apparent absence of AR (AR-) or normal levels of AR (AR+) binding. In several subjects with CAIS, AR-, no gross AR mutation was detected by Southern blot analyses of genomic DNA and normal sized 10 kilobase mRNA was present on Northern blots of poly(A+) RNA from cultured genital skin fibroblasts. We have used the polymerase chain reaction to amplify individual exons within the human AR gene of subjects with CAIS and have identified point mutations in three subjects. In one AR- subject (R774C), amino acid 774 was changed from arginine (CGC) to cysteine (TGC), in another AR- subject (R831Q), arginine (CGA) was changed to glutamine (CAA) at position 831, and in an AR+ subject (V866M) a methionine (ATG) was substituted for valine (GTG) at position 866. Transfection of wild type and mutant AR cDNA clones into COS cells results in detection of AR protein by immunoblotting. AR ligand binding activity is absent in cells transfected with AR mutants R774C and R831Q, but present with AR mutant V866M. Androgen binding in cells transfected with AR mutant V866M has a 6-fold lower apparent binding affinity than that of wild-type AR. Transcriptional activation of the MMTV-CAT reporter gene was androgen dependent and specific and nearly maximal at physiological concentrations (10(-10) M) of androgen when wild-type AR was transfected into cells, whereas neither AR mutants R774C nor R831Q were able to stimulate CAT activity even at 10(-8) M androgen. AR mutant V866M was able to stimulate CAT activity but the androgen dose dependency was shifted toward pharmacological concentrations of steroid that exceed in vivo levels. The molecular basis of CAIS in humans exhibits genetic heterogeneity. Our study shows that some cases of CAIS are explained by an inability to form a functional AR-steroid complex and hence, the AR is unable to activate transcription of genes essential for male sex differentiation during fetal development.
Amino Acid Sequence↗
Follicle-stimulating hormone regulation of androgen-binding protein messenger RNA in sertoli cell cultures.
FSH plays an important role in testicular Sertoli cell differentiation and function in spermatogenesis. Previous studies using rat androgen-binding protein (ABP) as a marker of FSH action on Sertoli cells have demonstrated in vivo and in vitro regulation of ABP. We now have extended these studies to examine FSH regulation of ABP mRNA using Northern blot hybridization. In the immature rat testicular ABP mRNA [1.7- and 2.3-kilobase (kb) species] increased with age and reached a maximum 20 days postpartum, coincident with an increased plasma FSH concentration. To determine the direct effect of FSH on Sertoli cells, we examined ABP mRNA in vitro. In Sertoli cell-enriched cultures FSH was found to maintain the major 1.7-kb ABP RNA transcript level over 5 days of treatment in a dose-dependent manner, whereas in the absence of FSH, ABP mRNA declined with time in culture. The ABP mRNA maintenance by FSH was accompanied by higher concentrations of secreted immunoreactive ABP, which declined in the absence of FSH. This FSH effect on ABP mRNA and secreted ABP was mimicked by the cAMP analog (Bu)2cAMP. After the decline of ABP mRNA during culture, administration of FSH did not result in a detectable increase in the 1.7-kb ABP mRNA within 3 days, whereas cAMP and c-fos mRNA were rapidly induced within 15 min. On the contrary, the level of the minor hybridizing ABP mRNA (2.3 kb) was altered by FSH, indicating differential regulation of the 1.7- and 2.3-kb hybridizing species. Also, after FSH deprivation, tissue plasminogen activator and inhibin alpha mRNA were substantially increased within 6 h of FSH treatment.(ABSTRACT TRUNCATED AT 250 WORDS)
Androgen-Binding Protein↗
Expression of recombinant androgen receptor in cultured mammalian cells.
Full-length rat and human androgen receptor (AR) cDNA clones were expressed in COS-7 and CV1 monkey kidney cells to analyze the AR protein using immunological and cotransfection techniques. The studies were aided by the development of two rabbit polyclonal antibodies, designated AR32 and AR52, directed against epitopes within the N-terminal region of AR. Each antibody recognizes native AR by sucrose gradient analysis and detects a 114-kilodalton protein in COS cells transfected with human or rat AR cDNA. Covalent binding of the synthetic androgen [3H]methyltrienolone (R1881) to the 114-kDa protein was saturable. The endogenous native AR was similarly 114 kDa on immunoblots of a human prostate adenocarcinoma cell line, LNCaP, and rat sex accessory gland extracts. AR was localized in nuclei of transfected COS cells and in LNCaP cells by immunocytochemical staining. Androgen induction of CAT activity was dose dependent in CV1 cells cotransfected with the AR expression vector and a reporter plasmid containing the mouse mammary tumor virus promoter linked to the chloramphenicol acetyltransferase gene. It is concluded that antipeptide antibodies are useful reagents in characterizing both native and denatured forms of the AR protein. The 114-kDa protein expressed transiently in cultured cells represents the full-length AR protein, has a molecular size equivalent to that of endogenous AR, and mediates androgen-dependent transcriptional activation in CV1 cells.
Amino Acid Sequence↗
Primary structure and androgen regulation of a 20-kilodalton protein specific to rat ventral prostate.
Nuclear and cytosolic forms of a 20-kdalton rat ventral prostate protein were purified and partially sequenced from their N-termini. Isolated nuclei were treated with micrococcal nuclease and extracted in 0.6 M NaCl, and proteins were separated by affinity chromatography on Matrex gel green A, ammonium sulfate fractionation, and fast protein liquid chromatography on Superose 12. The 43 amino acid N-terminal sequence of the nuclear 20-kdalton protein was identical with the cytosolic protein except it lacked 7 N-terminal amino acids present in the cytosolic form. The DNA sequence of a full-length complementary DNA clone isolated from a ventral prostate gt11 library extended the N-terminal sequence of the cytosolic form by an additional nine amino acids from the predicted initiation methionine. The cDNA included the nucleotide sequence for the 43 amino acid N-terminal sequence of the purified 20-kdalton protein and predicted molecular weights of 16,686, 17,521, and 18,650, respectively, for the nuclear, cytoplasmic, and nonprocessed proteins. Northern blot analyses of reproductive tract tissue RNAs using the 20-kdalton protein cDNA as probe revealed a single mRNA species of 0.92 kb detectable only in extracts of rat ventral prostate. Expression of the 0.92-kb mRNA was androgen dependent since the mRNA was undetectable in extracts obtained 4 days after castration and was restored 16 h after restimulation with androgen.
Amino Acid Sequence↗
A form of basic fibroblast growth factor is secreted into the adluminal fluid of the rat coagulating gland.
Rat coagulating gland and its secreted fluid contain a mitogenic substance that, by numerous criteria, appears to be similar to basic fibroblast growth factor (basic FGF). It is a potent competence factor in the BALB/c3T3 mouse embryo fibroblast assay, elutes from heparin-agarose at concentrations of NaCl greater than 1.0 M, and has an isoelectric point (pI) between 8 and 9. In coagulating gland fluid, its molecular weight was 20,000 +/- 5,000, as indicated by gel filtration on Sephadex G-200. Western blot analysis of purified factor from coagulating gland cytosol and fluid, respectively, revealed immunoreactive bands of 16,000 and 17,000 relative molecular weight similar to that of purified bovine basic FGF.
Animals↗
Sequence of the intron/exon junctions of the coding region of the human androgen receptor gene and identification of a point mutation in a family with complete androgen insensitivity.
Androgens act through a receptor protein (AR) to mediate sex differentiation and development of the male phenotype. We have isolated the eight exons in the amino acid coding region of the AR gene from a human X chromosome library. Nucleotide sequences of the AR gene intron/exon boundaries were determined for use in designing synthetic oligonucleotide primers to bracket coding exons for amplification by the polymerase chain reaction. Genomic DNA was amplified from 46,XY phenotypic female siblings with complete androgen insensitivity syndrome. AR binding affinity for dihydrotestosterone in the affected siblings was lower than in normal males, but the binding capacity was normal. Sequence analysis of amplified exons demonstrated within the AR steroid-binding domain (exon G) a single guanine to adenine mutation, resulting in replacement of valine with methionine at amino acid residue 866. As expected, the carrier mother had both normal and mutant AR genes. Thus, a single point mutation in the steroid-binding domain of the AR gene correlated with the expression of an AR protein ineffective in stimulating male sexual development.
Alleles↗
Identification of androgen receptors in normal human osteoblast-like cells.
The sex steroids, androgens and estrogens, are major regulators of bone metabolism. However, whether these hormones act on bone cells through direct or indirect mechanisms has remained unclear. A nuclear binding assay recently used to demonstrate estrogen receptors in bone [Eriksen, E.F., Colvard, D.S., Berg, N.J., Graham, M.L., Mann, K.G., Spelsberg, T.C. & Riggs, B.L. (1988) Science 241, 84-86] was used to identify specific nuclear binding of a tritiated synthetic androgen, [3H]R1881 (methyltrienolone), in 21 of 25 (84%) human osteoblast-like cell strains and a concentration of bound steroid receptors of 821 +/- 140 (mean +/- SEM) molecules per cell nucleus. Binding was saturable and steroid-specific. Androgen receptor gene expression in osteoblasts was confirmed by RNA blot analysis. Relative concentrations of androgen and estrogen receptors were compared by measuring specific nuclear estrogen binding. Nuclear binding of [3H]estradiol was observed in 27 of 30 (90%) cell strains; the concentration of bound estradiol receptor was 1537 +/- 221 molecules per cell nucleus. The concentrations of nuclear binding sites were similar in males and females for both [3H]R1881 and [3H]estradiol. We conclude that both androgens and estrogens act directly on human bone cells through their respective receptor-mediated mechanisms.
Binding, Competitive↗
Structural analysis of the human and rat androgen receptors and expression in male reproductive tract tissues.
Explore the source record for details and available documents.
Amino Acid Sequence↗