Point mutation in the steroid-binding domain of the androgen receptor gene in a family with complete androgen insensitivity syndrome (CAIS)

An exonic single nucleotide substitution in the human androgen receptor gene (hAR) could be detected in an Italian family with two children affected by complete androgen insensitivity syndrome (CAIS), also called testicular feminization. This mutation leads to a guanine to adenine transition in exon 5, changing the sense of the codon from methionine (ATG) to valine (GTG). As this mutation abolishes a NcoI restriction site, a rapid test for the mutation can be performed by digestion of the polymerase chain reaction products with this enzyme. Previous results of indirect gene diagnosis in this family could be confirmed by this method.     

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.     

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.     

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.     

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     

A mutation in the ligand binding domain of the androgen receptor of human LNCaP cells affects steroid binding characteristics and response to anti-androgens

LNCaP prostate tumor cells contain an abnormal androgen receptor system. Progestagens, estradiol and anti-androgens can compete with androgens for binding to the androgen receptor and can stimulate both cell growth and excretion of prostate specific acid phosphatase. We have discovered in the LNCaP androgen receptor a single point mutation changing the sense of codon 868 (Thr to Ala) in the ligand binding domain. Expression vectors containing the normal or mutated androgen receptor sequence were transfected into COS or Hela cells. Androgens, progestagens, estrogens and anti-androgens bind the mutated androgen receptor protein and activate the expression of an androgen-regulated reporter gene construct (GRE-tk-CAT). The mutation therefore influences both binding and the induction of gene expression by different steroids and antisteroids.     

Plasticity of the ecdysone receptor DNA binding domain

Ecdysteroids coordinate molting and metamorphosis in insects via a heterodimer of two nuclear receptors, the ecdysone receptor (EcR) and the ultraspiracle (Usp) protein. Here we show how the DNA-recognition alpha-helix and the T box region of the EcR DNA-binding domain (EcRDBD) contribute to the specific interaction with the natural response element and to the stabilization of the EcRDBD molecule. The data indicate a remarkable mutational tolerance with respect to the DNA-binding function of the EcRDBD. This is particularly manifested in the heterocomplexes formed between the EcRDBD mutants and the wild-type Usp DNA-binding domain (UspDBD). Circular dichroism (CD) spectra and protein unfolding experiments indicate that, in contrast to the UspDBD, the EcRDBD is characterized by a lower alpha-helix content and a lower stability. As such, the EcRDBD appears to be an intrinsically unstructured protein-like molecule with a high degree of intramolecular plasticity. Because recently published crystal structures indicate that the ligand binding domain of the EcR is also characterized by the extreme adaptability, we suggest that plasticity of the EcR domains may be a key factor that allows a single EcR molecule to mediate diverse biological effects.     

Unspecific DNA binding of the DNA binding domain of the glucocorticoid receptor studied with flow linear dichroism

The unspecific interaction between the DNA-binding domain of the human glucocorticoid receptor and DNA was studied using linear dichroism (LD) and circular dichroism (CD) spectroscopy. The amplitude of the LD signal was found to increase upon addition of protein at ionic strengths less than 60 nM Na+, indicating an increased persistence length of the complex compared to uncomplexed DNA. Analysis of the LD spectrum suggests that the binding does not involve intercalation of tyrosine residues. Evidence of saturation is found at a binding stoichiometry of approximately 5 DNA base pairs per protein monomer.     

The androgen receptor in LNCaP cells contains a mutation in the ligand binding domain which affects steroid binding characteristics and response to antiandrogens

The human prostate tumor cell line LNCaP containd an abnormal androgen receptor system with broad steroid binding specificity. Progestagens, estradiol and several antiandrogens compete with androgens for binding to the androgen receptor in the cells to a higher extent than in other androgen sensitive systems. Optimal growth of LNCaP cells is observed after addition of the synthetic androgen R1881 (0.1 nM). In addition, estrogens, progestagens and several antiandrogens do not inhibit androgen responsive growth, but have striking growth stimulatory effects and increase EGF receptor level and acid phosphatase secretion. We have found that the androgen receptor in the LNCaP cells contains a single point mutation changing the sense of codon 868 (Thr to Ala) in the ligand binding domain. Expression vectors containing the normal or mutated androgen receptor sequence were transfected into COS or HeLa cells. Androgens, progestagens, estrogens and several antiandrogens bind the mutated androgen receptor proteon and activate the expression of an androgen-regulated reporter gene (GRE-tk-CAT), indicating that the mutation directly affects both binding specificity and the induction of gene expression. Interestingly, the antiandrogen casodex showed antiandrogenic properties in growth studies of LNCaP cells and did not induced reporter gene activity in Hela cells transfected with the mutant receptor. The mutated androgen receptor of LNCaP cells is therefore a useful tool in the elucidation of different levels of action of steroids and antisteroids.     

The binding of androgen receptor to DNA and RNA

The androgen receptor from mouse kidney cytosol has been studied for its nucleic acid binding properties by DNA-cellulose centrifugation assay. The receptor appears to bind to RNA (mRNA, tRNA, rRNA) as well as to DNA. Salt and heat activation of the androgen receptor enhances both DNA and RNA binding. The receptor binds slightly better to denatured DNA than to native DNA. The androgen receptor binds about 2-fold tighter to poly(dG-dC) than to poly (dA-dT). The interaction of the receptor with DNA is not greatly affected by the BrdUrd substitution. The observation that androgen receptor shows a significant affinity to RNA may imply that androgen receptor-RNA interaction could play a role in gene regulation.     

Molecular mechanism of angelman syndrome in two large families involves an imprinting mutation.

Patients with Angelman syndrome (AS) and Prader-Willi syndrome with mutations in the imprinting process have biparental inheritance but uniparental DNA methylation and gene expression throughout band 15q11-q13. In several of these patients, microdeletions upstream of the SNRPN gene have been identified, defining an imprinting center (IC) that has been hypothesized to control the imprint switch process in the female and male germlines. We have now identified two large families (AS-O and AS-F) segregating an AS imprinting mutation, including one family originally described in the first genetic linkage of AS to 15q11-q13. This demonstrates that this original linkage is for the 15q11-q13 IC. Affected patients in the AS families have either a 5.5- or a 15-kb microdeletion, one of which narrowed the shortest region of deletion overlap to 1.15 kb in all eight cases. This small region defines a component of the IC involved in AS (ie., the paternal-to-maternal switch element). The presence of an inherited imprinting mutation in multiple unaffected members of these two families, who are at risk for transmitting the mutation to affected children or children of their daughters, raises important genetic counseling issues.