Clinical and endocrinological characterization of two subjects with Reifenstein syndrome associated with qualitative abnormalities of the androgen receptor

The androgen receptor in fibroblasts cultured from a biopsy of scrotal skin from 1 subject with Reifenstein syndrome has been found to be normal in amount and to bind dihydrotestosterone with normal affinity but to be qualitatively abnormal as evident by thermolability and instability upon ultracentrifugation. The family study of this subject and endocrine studies document androgen resistance in the index patient and his affected uncle. These findings provide evidence for X-linkage of this disorder, and suggest that the mutations that give rise to this phenotype are probably allelic to the mutations of the androgen receptor that cause testicular feminization.     

Proper receptor signalling in a mutant catfish gonadotropin-releasing hormone receptor lacking the highly conserved Asp(90) residue

The negatively charged side chain of an Asp residue in transmembrane domain 2 is likely to play an important role in receptor signalling since it is highly conserved in the whole family of G protein-coupled receptors, except in mammalian gonadotropin-releasing hormone (GnRH) receptors. In this paper we show that the conserved Asp(90) of the catfish GnRH receptor can be substituted by a neutral Asn(90) without abolishing receptor signalling if another negatively charged Glu(93) is introduced in a proximal region of the receptor interior, thereby mimicking the Glu(90)-Lys(121) salt bridge of mammalian GnRH receptors.     

Molecular analysis of the androgen receptor gene in 52 patients with complete or partial androgen insensitivity syndrome: a collaborative study.

In patients with androgen insensitivity syndrome (AIS), RFLP study of the androgen receptor gene made it possible to analyze whether deletions or mutations could be responsible for abnormalities in androgen responsiveness. We studied RFLPs of DNA from 25 46,XY patients with partial AIS (PAIS), defined as a concentration of androgen receptor in genital-skin fibroblasts less than 340 fmol/mg DNA, and DNA from 27 46,XY patients with complete AIS (CAIS) with no detectable androgen receptor site. DNA samples were digested with BamHI, EcoRI, HindIII and TaqI restriction enzymes and hybridized with three cDNA probes covering the three domains of the androgen receptor. When we had the maternal and an unaffected brother's DNA, we analyzed the two androgen receptor gene polymorphisms described, the HindIII and the exon 1 CAG repeat polymorphisms, in order to distinguish the two maternal X chromosomes, and to detect carriers of AIS. We did not find any large deletion among the 52 patients. We observed a heterozygous mother in 3 of 14 families studied with the HindIII polymorphism, and in 12 of 25 families using the exon 1 CAG repeat polymorphism. This study suggests that in AIS, abnormalities in androgen receptor response could be related to point mutations or microdeletions rather than to gross structural alterations of the androgen receptor gene. Furthermore, unless the point mutation has been described, exon 1 and HindIII polymorphism studies would enable the identification of carriers in 50% of families, and the prenatal diagnosis of AIS.     

Expression of a mutant androgen receptor in cloned fibroblasts derived from a heterozygous carrier for the syndrome of testicular feminization

Thermolability of androgen binding was compared in fibroblasts cloned from normal female skin, skin from a subject with testicular feminization whose mutation is known to be associated with a thermolabile androgen receptor, and from the mother of the subject with testicular feminization. Seven of 28 clones studied from the mother exhibited thermolability of binding, indicating that the mutant gene that causes thermolability of binding, like the gene responsible for the normal androgen receptor, is X-linked.     

Substitution of conserved glycine residue by alanine in natural and synthetic neuropeptide ligands causes partial agonism at the stomoxytachykinin receptor

A few naturally occurring insect tachykinin-related peptides, such as stomoxytachykinin (Stc-TK), contain an Ala-residue instead of the highly conserved Gly-residue that is present in most other members of this peptide family. Stc-TK is a potent, partial agonist of the stable fly (Stomoxys calcitrans) tachykinin receptor, STKR. By means of synthetic analogues, the Gly/Ala exchange, representing the addition of a single methyl group in the active core region of these peptides, was shown to be fully responsible for the generation of this partial agonism, which was also accompanied by an increase in agonistic potency. Surprisingly, this Ala-dependent reduction in maximal response levels was only observed for the agonist-induced cellular calcium rise. Stomoxytachykinin, Stc-TK, did not display partial agonism for the STKR-mediated cyclic AMP response. A possible explanation for this differential partial agonism is that the Gly-containing and Ala-replaced peptides recognize and stabilize active receptor conformations that differ in their functional coupling efficacies towards these response pathways. Drosotachykinins, Drm-TK, tachykinin-like peptides encoded in the fruit fly genome, were shown to be STKR-agonists. Interestingly, one of these peptides, which contains an Ala-residue instead of the conserved Gly-residue, also proved to be a potent, partial agonist for STKR.     

Functional characterisation of mutations in the ligand-binding domain of the androgen receptor gene in patients with androgen insensitivity syndrome

Five mutations in the ligand-binding domain of the androgen receptor gene were identified in patients with complete (A765T, C784Y, R831X and M895T) or partial (R840G) androgen insensitivity. A765T and R831X have been reported previously whereas the other three mutations are novel. Receptors carrying these mutations were transiently expressed in COS-1 cells, and androgen binding and capacity to transactivate an androgen-responsive reporter gene were assayed. C784Y led to abolished androgen binding and transactivating capacity, R840G and M895T showed reduced specific binding and partial transactivation. The in vitro functions of the R840G and M895T mutants were improved with supraphysiological concentrations of steroid. Received: 10 June 1998 / Accepted: 10 September 1998     

Association of the Hind III polymorphism with the androgen receptor gene in partial androgen insensitivity syndrome.

Partial androgen insensitivity syndrome (PAIS) is an X-linked disorder resulting from defects in the intracellular androgen receptor (AR). The cloning of the AR cDNA has provided the molecular tools to identify gene abnormalities. Gene deletions being the exception in PAIS, prenatal diagnosis of PAIS resulting from a single base mutation in high risk families is not practical unless the mutation is already known. Brown et al. (1989) reported that 10% of normal X chromosomes present a Hind III 6.7/3.5 kb polymorphism. In this study, we report the association of the Hind II polymorphism in a woman whose son has a PAIS associated with a very low androgen receptor concentration: we differentiated the two maternal X chromosomes and characterized the affected allele. These data demonstrate that the presence of Hind III polymorphic fragments could be used in prenatal diagnosis of androgen insensitivity syndrome in high risk families.     

Anabolic-androgenic steroid interaction with rat androgen receptor in vivo and in vitro: a comparative study

Anabolic steroids are synthetic derivatives of testosterone and are characterized by their ability to cause nitrogen retention and positive protein metabolism, thereby leading to increased protein synthesis and muscle mass. There are disagreements in the literature in regards to the interaction of anabolic steroids with the androgen receptor (AR) as revealed by competitive ligand binding assays in vitro using cytosolic preparations from prostate and skeletal muscle. By use of tissue extracts, it has been shown that some anabolic steroids have binding affinities for the AR that are higher than that of the natural androgen testosterone, while others such as stanozolol and methanedienone have significantly lower affinities as compared with testosterone. In this study we show that stanozolol and methanedienone are low affinity ligands of the rat recombinant AR as revealed by a ligand binding assay in vitro, however, based on a cell-based AR-dependent transactivation assay, they are potent activators of the AR. We also show that a single injection of stanozolol and methanedienone causes a rapid cytosolic depletion of AR in rat skeletal muscle. Based on these results, we conclude that anabolic steroids with low affinity to AR in vitro, can in fact in vivo act on the AR to cause biological responses.     

Multiple roles of the conserved key residue arginine 209 in neuronal nicotinic receptors.

We have examined the role of a highly conserved arginine (R209), which flanks the M1 transmembrane segment of nAChRs, in the biogenesis and function of neuronal nAChRs. Point mutations revealed that, in alphaBgtx-sensitive neuronal alpha7 nAChRs, the conserved arginine is required for the transport of assembled receptors to the cell surface. By contrast, R209 does not play any role in the transport of assembled alpha-Bgtx-insensitive neuronal alpha3beta4 nAChRs to the cell surface. However, a basic residue at this position of alpha3 and beta4 subunits is necessary for either synthesis, folding, or assembly of alpha3beta4 receptors. Moreover, electrophysiological experiments revealed that in alpha3beta4 receptors the conserved arginine of the alpha3 subunit is involved in either coupling agonist binding to the channel or regulating single channel kinetics.     

Definition of the human androgen receptor gene structure permits the identification of mutations that cause androgen resistance: premature termination of the receptor protein at amino acid residue 588 causes complete androgen resistance

We have isolated and characterized the gene encoding the human androgen receptor. The coding sequence is divided into eight coding exons and spans a minimum of 54 kilobases. The positions of the exon boundaries are highly conserved when compared to the location of the exon boundaries of the chicken progesterone and human estrogen receptor genes. Definition of the intron/exon boundaries has permitted the synthesis of specific oligonucleotides for use in the amplification of segments of the androgen receptor gene from samples of total genomic DNA. This technique allows the analysis of all segments of the androgen receptor gene except a small region of exon 1 that encodes the glycine homopolymeric segment. Using these methods we have analyzed samples of DNA prepared from a patient with complete androgen resistance and have detected a single nucleotide substitution at nucleotide 1924 in exon 3 of the androgen receptor gene that results in the conversion of a lysine codon into a premature termination codon at amino acid position 588. The introduction of a termination codon into the sequence of the normal androgen receptor cDNA at this position leads to a decrease in the amount of mRNA encoding the human androgen receptor and the synthesis of a truncated receptor protein that is unable to bind ligand and is unable to activate the long terminal repeat of the mouse mammary tumor virus in cotransfection assays.     

Phenotypic heterogeneity associated with identical mutations in residue 870 of the androgen receptor

BACKGROUND/AIMS: Mutations in the androgen receptor (AR) gene result in an X-linked recessive form of male pseudohermaphroditism known as the androgen-insensitivity syndrome (AIS). The alterations most frequently observed are missense or nonsense point mutations in exons 4-8 of the AR gene that affect the steroid-binding domain of the receptor in subjects with various degrees of androgen resistance. Despite the increasing number of AR mutations identified, a reliable genotype-phenotype correlation has not been established and individuals with the same molecular defect may exhibit different phenotypes. Here, we studied a patients with an AIS characterized by bilateral gynecomastia, normal male external genitalia, and normal sperm counts. METHODS: Exon-specific polymerase chain reaction, single-stranded conformational polymorphism, and sequencing analysis of the subject's AR gene were performed in addition to hormone-binding assays in skin fibroblasts from the patient. RESULTS: A point mutation at codon 870 of the AR, changing alanine to valine, was detected. CONCLUSION: As AR missense mutations changing alanine 870 to valine have been previously described in 3 unrelated patients showing severe AIS phenotypes, we conclude that phenotypic heterogeneity associated to identical mutations in the AR gene is probably due to individual functional differences in AR coregulator molecules.     

A catalytic function for the structurally conserved residue Phe 100 of ribonuclease T1.

The function of the conserved Phe 100 residue of RNase T1 (EC 3.1.27.3) has been investigated by site-directed mutagenesis and X-ray crystallography. Replacement of Phe 100 by alanine results in a mutant enzyme with kcat reduced 75-fold and a small increase in Km for the dinucleoside phosphate substrate GpC. The Phe 100 Ala substitution has similar effects on the turnover rates of GpC and its minimal analogue GpOMe, in which the leaving cytidine is replaced by methanol. The contribution to catalysis is independent of the nature of the leaving group, indicating that Phe 100 belongs to the primary site. The contribution of Phe 100 to catalysis may result from a direct van der Waals contact between its aromatic ring and the phosphate moiety of the substrate. Phe 100 may also contribute to the positioning of the pentacovalent phosphorus of the transition state, relative to other catalytic residues. If compared to the corresponding wild-type data, the structural implications of the mutation in the present crystal structure of Phe 100 Ala RNase T1 complexed with the specific inhibitor 2'-GMP are restricted to the active site. Repositioning of 2'-GMP, caused by the Phe 100 Ala mutation, generates new or improved contacts of the phosphate moiety with Arg 77 and His 92. In contrast, interactions with the Glu 58 carboxylate appear to be weakened. The effects of the His 92 Gln and Phe 100 Ala mutations on GpC turnover are additive in the corresponding double mutant, indicating that the contribution of Phe 100 to catalysis is independent of the catalytic acid His 92. The present results lead to the conclusion that apolar residues may contribute considerably to catalyze conversions of charged molecules to charged products, involving even more polar transition states.     

A conserved role of a DEAD box helicase in mRNA masking.

Clam p82 is a member of the cytoplasmic polyadenylation element-binding protein (CPEB) family of RNA-binding proteins and serves dual functions in regulating gene expression in early development. In the oocyte, p82/CPEB is a translational repressor, whereas in the activated egg, it acts as a polyadenylation factor. Coimmunoprecipitations were performed with p82 antibodies in clam oocyte and egg lysates to identify stage-regulated accessory factors. p47 coprecipitates with p82 from oocyte lysates in an RNA-dependent manner and is absent from egg lysate p92-bound material. Clam p47 is a member of the RCK/p54 family of DEAD box RNA helicases. Xp54, the Xenopus homolog, with bona fide helicase activity, is an abundant and integral component of stored mRNP in oocytes (Ladomery et al., 1997). In oocytes, clam p47 and p82/CPEB are found in large cytoplasmic mRNP complexes. Whereas the helicase level is constant during embryogenesis, in contrast to CPEB, clam p47 translocates to nuclei at the two-cell stage. To address the role of this class of helicase in masking, Xp54 was tethered via 3' UTR MS2-binding sites to firefly luciferase, following microinjection of fusion protein and nonadenylated reporter mRNAs into Xenopus oocytes. Tethered helicase repressed luciferase translation three- to fivefold and, strikingly, mutations in two helicase motifs (DEAD--> DQAD and HRIGR-->HRIGQ), activated translation three- to fourfold, relative to MS2. These data suggest that this helicase family represses translation of maternal mRNA in early development, and that its activity may be attenuated during meiotic maturation, prior to cytoplasmic polyadenylation.     

A large deletion/insertion-induced frameshift mutation of the androgen receptor gene in a family with a familial complete androgen insensitivity syndrome

Androgen insensitivity syndrome (AIS) is an X-linked recessive genetic disorder with a normal 46, XY karyotype caused by abnormality of the androgen receptor (AR) gene. One Chinese family consisting of the proband and 5 other members with complete androgen insensitivity syndrome (CAIS) was investigated. Mutation analysis by DNA sequencing on all 8 exons and flanking intron regions of the AR gene revealed a unique large deletion/insertion mutation in the family. A 287 bp deletion and 77 bp insertion (c.933_1219delins77) mutation at codon 312 resulted in a frameshift which caused a premature stop (p.Phe312Aspfs*7) of polypeptide formation. The proband's mother and grandmother were heterozygous for the mutant allele. The proband's father, uncle and grandfather have the normal allele. From the pedigree constructed from mutational analysis of the family, it is revealed that the probably pathogenic mutation comes from the maternal side.