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Biomedical subjects

L Pinsky

Publications and source records attributed to L Pinsky.

At least 73 records · Page 4Linked to original sources
PubMed1993

Kinetic evidence for a unique testosterone-receptor complex in 5 alpha-reductase sufficient genital skin fibroblasts and the effects of 5 alpha-reductase deficiency on its formation.

When 5 alpha-reductase-sufficient genital skin fibroblast (GSF) monolayers are incubated with testosterone (T), they first form androgen (A)-receptor (R) complexes that dissociate at a fast rate [k(37 degrees C = 0.024 min-1]. As T is converted to 5 alpha-dihydrotestosterone (DHT), this population of T-R complexes is eventually replaced by one that dissociates much more slowly [k(37 degrees C) = 0.006 min-1], at a rate typical of DHT-R complexes. During the course of T to DHT conversion, one may observe a population of A-R complexes that has a linear (monophasic) intermediate dissociation rate constant [k(37 degrees C) = 0.012 min-1]; this population cannot simply reflect a mixture of T- and DHT-R complexes. The rate at which the complexes are processed from one dissociative form to the next varies with the incubation temperature and the presence or absence of serum in the medium; it also varies within and among GSF strains under apparently constant conditions. To explain these facts, we propose a model that enables the 5 alpha-reductase enzyme to influence the processive dissociative behaviour of T-R complexes by engaging in some sort of coupling with the AR. The proposal is strengthened by a set of observations in cells with constitutive, mendelian or inhibitor-induced 5 alpha-reductase deficiency that preclude a simple quantitative relation between A-R complex processing and the extent of T to DHT conversion.

3-Oxo-5-alpha-Steroid 4-Dehydrogenase↗
PubMed1993

Reduced transcriptional regulatory competence of the androgen receptor in X-linked spinal and bulbar muscular atrophy.

Expansion of the long (CAG; glutamine)n repeat in the first exon of the X-linked human androgen receptor gene (hAR) causes spinal and bulbar muscular atrophy, frequently in association with mild androgen insensitivity. The relevant normal motor neurons are preferentially stimulated by androgen, however no motor neuron disorder occurs with any other known AR mutation, including those that cause complete androgen insensitivity. We have found that a polyglutamine (Gln) expanded AR transactivates an androgen-responsive reporter gene subnormally. Other groups have reported that a poly Gln-deleted AR transactivates normally. A parsimonious interpretation of all these facts is that poly Gln expansion causes the AR to lose a function that is necessary for full androgen sensitivity and to gain a function that is selectively motor neuronotoxic.

Androgens↗
PubMed1993

Substitution of valine-865 by methionine or leucine in the human androgen receptor causes complete or partial androgen insensitivity, respectively with distinct androgen receptor phenotypes.

We have identified two different single nucleotide missense substitutions at valine-865 in exon 7 of the human androgen receptor (AR) gene in two families with androgen resistance. Val-->methionine is associated with the complete syndrome; Val-->leucine is associated with the partial form. In genital skin fibroblasts, both alterations yield a normal maximum binding capacity, but an increased apparent equilibrium dissociation constant for all androgens tested. In genital skin fibroblasts, Val865-Met A-R complexes have increased rate constants of dissociation with 5 alpha-dihydrotestosterone, and the nonmetabolized ligands methyltrienolone or mibolerone (MB); their Val865-Leu counterparts have increased rates with methyltrienolone and MB, but not with 5 alpha-dihydrotestosterone. In transiently transfected COS-1 or PC-3 cells, Met865 AR is more severely impaired than Leu865 AR in transactivating two different androgen-responsive reporter constructs, thereby correlating with clinical phenotype. In COS-1 cells exposed to MB for 74 h, this relative impairment correlates with the relative instability of the MB-binding activity of each mutant AR, suggesting that their respective intrinsic transcriptional regulatory competence is normal. Notably, these mutant ARs lose significantly more MB-binding activity than immunoreactivity, suggesting that prolonged MB exposure induces them to adopt a nonbinding state. The position homologous to Val865 in the AR is occupied by Leu or Met in the three steroid receptors closely related to the AR. This indicates the structural subtlety that underlies the steroid-binding activity of different steroid receptors.

Amino Acid Sequence↗
PubMed1992

The human androgen receptor: structure/function relationship in normal and pathological situations.

Discrete functions have been attributed to precise regions of the human androgen receptor (hAR) by expression of deletion mutants in COS and HeLa cells. A large C-terminal domain constitutes the hormone-binding region and a central basis, cysteine-rich domain is responsible for DNA binding. In addition, separate domains responsible for transactivation and nuclear translocation have been identified. In LNCaP cells (a prostate tumor cell line) the hAR is a heterogeneous protein which is synthesized as a single 110 kDa protein, but becomes rapidly phosphorylated to a 112 kDa protein. Metabolic labeling experiments using radioactive orthophosphate also indicated that the hAR is a phosphoprotein. Structural analysis of the AR gene in LNCaP cells and in 46, XY-individuals displaying androgen insensitivity (AIS) has revealed several different point mutations. In LNCaP cells the mutation affects both binding specificity and transactivation by different steroids. In a person with complete AIS a point mutation was identified in the splice donor site of intron 4, which prevents normal splicing and activates a cryptic splice donor site in exon 4. The consequence is a functionally inactive AR protein due to an in-frame deletion in the steroid-binding domain. In two unrelated individuals with complete AIS, two different single nucleotide alterations in codon 686 (Asp) were found. Both mutations resulted in functionally inactive ARs due to rapidly dissociating hormone-AR complexes. It is concluded that the hAR is a heterogeneous phosphoprotein in which functional errors have a dramatic impact on phenotype and fertility of 46, XY-individuals.

Amino Acid Sequence↗
PubMed1992

Androgen resistance due to mutation of the androgen receptor.

The androgen receptor (AR) is a 'one-stop' signal transduction system that is the core of the intracellular androgen-response apparatus. It is an androgen-regulated, DNA-binding protein that regulates the expression of certain target genes, primarily at the transcriptional level. Mutations at the X-linked AR locus cause deficient or defective AR activity and, thereby, an extraordinarily wide spectrum of clinical androgen resistance. At one extreme, the affected 46,XY person is an infertile phenotypic female; at the other, he is a phenotypic male who may even be fertile, yet have gynecomastia or other focal signs of postpubertal subvirilization. We have identified 32 proven or putatively pathogenic alterations in the AR gene of 38 androgen-resistant families. This permits heterozygote detection and prenatal diagnosis whenever relevant. Most of the mutations affect the AR's androgen-binding domain, partly because our search has been targetted on those whose genital skin fibroblasts have impaired androgen-binding activities. The AR is a prototypic member of a subfamily that includes the receptors for progesterone, glucocorticoid, and mineralocorticoid. Observations that correlate AR genotype with clinical and receptor phenotypes of androgen resistance will help to generate a fine structure-function map of the AR and its close relatives. Constitutional variation in androgen sensitivity, that may be restricted to an organ (or organ system), could contribute to the pathogenesis of certain diseases whose sex ratio departs significantly from one.

Amino Acid Sequence↗
PubMed1992

Replacement of arginine 773 by cysteine or histidine in the human androgen receptor causes complete androgen insensitivity with different receptor phenotypes.

We have discovered two different point mutations in a single codon of the X-linked androgen-receptor (AR) gene in two pairs of unrelated families who have complete androgen insensitivity (resistance) associated with different AR phenotypes in their genital skin fibroblasts. One mutation is a C-to-T transition at a CpG sequence near the 5' terminus of exon 6; it changes the sense of codon 773 from arginine to cysteine, ablates specific androgen-binding activity at 37 degrees C, and eliminates a unique KpnI site at the intron-exon boundary. The other mutation is a G-to-A transition that changes amino acid 773 to histidine and eliminates an SphI site. This mutant AR has a normal androgen-binding capacity at 37 degrees C but has a reduced affinity for androgens and is thermolabile in their presence. Transient transfection of COS cells with cDNA expression vectors yielded little androgen-binding activity at 37 degrees C from Arg773Cys and abundant activity with abnormal properties from Arg773His, thereby providing the pathogenicity of both sequence alterations. This conclusion coincides with the following facts about evolutionary preservation of the position homologous to Arg773 in the AR: it is occupied by Arg or lysine in the progesterone, glucocorticoid, and mineralocorticoid receptors, and it is within a 14-amino-acid region of their steroid-binding domains that share approximately 85% amino acid identity.

Adolescent↗
PubMed1991

Amber mutation creates a diagnostic MaeI site in the androgen receptor gene of a family with complete androgen insensitivity.

We have discovered in the X-linked androgen receptor gene a single nucleotide substitution that is the putative cause of complete androgen insensitivity (resistance) in a family with affected individuals in 2 generations. Earlier studies on the family indicated co-segregation of mutant phenotype and the RFLPs at the loci DXS1 and DXYS1. The mutation is an adenine-to-thymine transversion in exon 8 that changes the sense of codon 882 from lysine to an amber (UAG) translation termination signal. The substitution creates a recognition sequence for the restriction endonuclease MaeI: this permits ready recognition of hemizygotes and heterozygotes after amplification of genomic exon 8 by the polymerase chain reaction. The mutation predicts the synthesis of a truncated receptor that lacks 36 amino acids at the carboxy terminus of its 252-amino acid androgen-binding domain. The cultured genital skin fibroblasts of the one affected patient examined have normal levels of androgen receptor mRNA, but negligible androgen-receptor binding activity. These results accord with a variety of data from spontaneous and artificial mutations indicating that all portions of the steroid binding domain contribute to normal steroid binding by a steroid receptor.

Amino Acid Sequence↗
PubMed1991

The 56 kDa androgen binding protein is an aldehyde dehydrogenase.

We have described a 56 kDa protein from genital skin fibroblasts that specifically binds androgen and that is generally not expressed in genital skin fibroblasts from patients with androgen insensitivity due to genetic defects of the androgen receptor. We have isolated a partial cDNA clone for the 56 kDa protein from an expression library of genital skin fibroblasts. In vitro translation of message selected with this clone faithfully produces the 56 kDa protein which can be immuneprecipitated with an anti-56 kDa antiserum. Northern blots probed with this clone show a 2.2 kb message, which parallels the expression of the 56 kDa protein. The sequence of this 998bp clone is identical to human liver aldehyde dehydrogenase 1, the cytoplasmic isoenzyme. On activity gels of genital skin fibroblast cytosol covalently labelled with androgen, aldehyde dehydrogenase activity comigrates with the single band labelled specifically with androgen. Thus, the 56 kDa androgen binding protein is an aldehyde dehydrogenase, which is prominently expressed in normal genital skin fibroblasts, but not in non-genital skin fibroblasts.

Alcohol Dehydrogenase↗
PubMed1991

The 56/58 kDa androgen-binding protein in male genital skin fibroblasts with a deleted androgen receptor gene.

Human genital skin fibroblasts (GSF) make a relatively abundant 56/58 kDa protein that binds androgens. The protein shares many properties with the approximately 100 kDa androgen receptor that is encoded by a locus in the q12 region of the X chromosome. It does not appear to be androgen-induced, yet is absent in GSF of most patients with complete androgen insensitivity (CAI). A precursor-product relation with the androgen receptor (AR) protein has been largely excluded; that it may be an unorthodox product of the AR gene has not. The 56/58 kDa protein is made by the GSF of a mentally retarded subject who has CAI because of a complete deletion of the coding portion of the AR gene. Hence, the strong constitutional and statistical correlations that have been demonstrated between the two proteins cannot arise because they share the same gene. The subject's genomic DNA hybridizes normally with 11 single-copy probes from Xq11-Xq13. Therefore, we cannot attribute her mental retardation to a contiguous gene syndrome.

Androgen-Binding Protein↗
PubMed1991

Androgen receptor abnormalities.

The human androgen receptor is a member of the superfamily of steroid hormone receptors. Proper functioning of this protein is a prerequisite for normal male sexual differentiation and development. The cloning of the human androgen receptor cDNA and the elucidation of the genomic organization of the corresponding gene has enabled us to study androgen receptors in subjects with the clinical manifestation of androgen insensitivity and in a human prostate carcinoma cell line (LNCaP). Using PCR amplification, subcloning and sequencing of exons 2-8, we identified a G----T mutation in the androgen receptor gene of a subject with the complete form of androgen insensitivity, which inactivates the splice donor site at the exon 4/intron 4 boundary. This mutation causes the activation of a cryptic splice donor site in exon 4, which results in the deletion of 41 amino acids from the steroid binding domain. In two other independently arising cases we identified two different nucleotide alterations in codon 686 (GAC; aspartic acid) located in exon 4. One mutation (G----C) results in an aspartic acid----histidine substitution (with negligible androgen binding), whereas the other mutation (G----A) leads to an aspartic acid----asparagine substitution (normal androgen binding, but a rapidly dissociating androgen receptor complex). Sequence analysis of the androgen receptor in human LNCaP-cells (lymph node carcinoma of the prostate) revealed a point mutation (A----G) in codon 868 in exon 8 resulting in the substitution of threonine by alanine. This mutation is the cause of the altered steroid binding specificity of the LNCaP-cell androgen receptor. The functional consequences of the observed mutations with respect to protein expression, specific ligand binding and transcriptional activation, were established after transient expression of the mutant receptors in COS and HeLa cells. These findings illustrate that functional errors in the human androgen receptor have an enormous impact on phenotype and fertility.

Amino Acid Sequence↗
PubMed1990

The state transitions of normal and mutant androgen-receptor complexes in human genital skin fibroblasts.

We have incubated cells from controls and subjects with receptor-defective androgen resistance with 3H-labelled testosterone (T), methyltrienolone (MT), dihydrotestosterone (DHT) or mibolerone (MB) and studied the temperature dependence of the dissociation rate constants of these various androgen-receptor (A-R) complexes both within cells and after they were extracted from them. In control cells, Arrhenius plots for T-, MT-, DHT- and MB-R complexes were linear and formed a hierarchy of dissociation states with energies of state IV greater than III greater than II, greater than I, respectively. Relative to this hierarchy, the dissociation states of the MB-, DHT- and MT-R complexes in mutant cells were displaced to higher, androgen-inappropriate energies in a mutant-distinctive pattern. When extracted from cells control or mutant T- or MT-R complexes, and mutant (but not control) DHT- or MB-R complexes lowered their respective dissociation rates by undergoing state transitions in conformity with the hierarchy. Hence we propose that different A-R complexes reach different dissociative states by undergoing sequential transitions along a common pathway, and that these transitions are co-regulated both by the chemical characteristics of the bound androgen and by other cellular non-receptor factors.

Cells, Cultured↗
PubMed1990

The 56 kDa androgen-binding protein in human genital skin fibroblasts: its relation to the human androgen receptor.

We have recently described in genital skin fibroblasts (GSF) a relatively abundant 56 kDa protein with androgen-binding activity. This protein is missing in GSF of most patients with complete androgen insensitivity syndrome (CAI). The protein has many characteristics compatible with the androgen receptor; it has in fact been tentatively considered as a precursor or degradation form of the prototypic (approximately 100 kDa) human androgen receptor. We have prepared an antiserum to this protein, which allowed us to detect it as a direct product by in vitro translation of mRNA from GSF. It is thus very unlikely to be a degradation product of a larger precursor. Furthermore, covalent photolytic labeling of this protein with the androgen analogue [3H]mibolerone revealed a much lower affinity for this protein than is known for the androgen receptor. Finally, the GSF of two exceptional patients with complete androgen insensitivity syndrome due to negligible androgen receptor-binding activity express this protein normally, as determined on two-dimensional gels by Western blot analysis with the antiserum and by photolytic covalent labeling with androgen analogues. These data indicate that the protein is not a precursor or a degradation product of the receptor; nor is it androgen-induced. They are more compatible with the idea that the protein is another member of the steroid/thyroid/retinoic acid receptor supergene family, perhaps as an unorthodox product of the human androgen receptor gene.

Androgen-Binding Protein↗
PubMed1990

Androgen receptor defects in patients with minimal and partial androgen resistance classified according to a model of androgen-receptor complex energy states.

We have characterized intracellularly the androgen-receptor (A-R) complexes formed by genital skin fibroblasts from 2 unrelated males with qualitative defects of the androgen receptor: one has a small nonhypospadic penis as part of a syndrome of mild androgen resistance; the other was born with ambiguous external genitalia. The dissociation rate constants of testosterone, methyltrienolone (MT), dihydrotestosterone (DHT) and mibolerone (MB) from normal androgen receptors were determined at various temperatures: when plotted by the method of Arrhenius, they yielded a linear hierarchy of dissociation states with energies of state IV greater than III greater than II greater than I, respectively. Relative to this hierarchy, patient A-R complexes were displaced to higher, androgen-inappropriate energies in a mutant-distinctive pattern. MB- or MT-R complexes of both patients were thermolabile; however, both up-regulated normally in response to prolonged incubation with either hormone. Apparent equilibrium affinity constants (Kd) of the DHT- and MB-R complexes formed by both patients were normal; however, the binding capacity (Bmax) for MB in 1 case was subnormal. The distinctive biochemical phenotypes of A-R complexes in these 2 patients with androgen resistance will facilitate the definition of structure-function relations in the androgen receptor, a classical DNA-binding, transcription-regulating protein.

Adolescent↗
PubMed1990

An exonic point mutation of the androgen receptor gene in a family with complete androgen insensitivity.

We have discovered in the X-linked androgen receptor gene a single exonic nucleotide substitution that causes complete androgen insensitivity (resistance) in a sibship with three affected individuals. The mutation, a guanine-to-adenine transition, occurs at nucleotide number 2682 and changes the sense of codon 717 from tryptophan to a translation stop signal. Codon 717 is in exon 4, so the mutation predicts the synthesis of a truncated receptor that lacks most of its androgen-binding domain. The substitution abolishes a recognition sequence for the restriction endonuclease HaeIII. Amplification of exon 4 by the polymerase chain reaction followed by double digestion with HinfI and HaeIII permits facile recognition of hemizygotes and heterozygous carriers of the mutation.

Amino Acid Sequence↗
PubMed1989

Impaired spermatogenesis is not an obligate expression of receptor-defective androgen resistance.

We are studying a man who presented at age 21 years with severe extragenital subvirilization despite high-normal to above-normal levels of plasma testosterone for at least 5 years. At puberty, his penis, scrotum, and testes matured normally, and he did not develop gynecomastia; however, his voice, muscularity, and facial, sexual, and body hair remained immature. A 2.5-ml ejaculate yielded normal results for sperm density, morphology, and motility. Because persistent undervirilization was emotionally disabling, he has received pharmacologic doses of testosterone enanthate intramuscularly for 3.5 years. The treatment has improved his virilization and masculine self-image substantially, and his semen analysis has remained well within the normal range. The androgen receptor in his genital skin fibroblasts has a distinctively mutant phenotype: it has a low affinity (increased apparent equilibrium dissociation constant, Kd) for 5 alpha-dihydrotestosterone and two synthetic androgens, mibolerone (MB) and methyltrienolone (MT), and its binding capacity (Bmax) is normal for the other two ligands, but questionably low for MT. In addition, it up-regulates its activity normally in response to prolonged incubation with androgen, and its androgen-receptor complexes are not thermolabile. Our study of this man permits two conclusions: impaired spermatogenesis is not the irreducible expression of receptor-defective androgen resistance in man; and androgen pharmacotherapy may be remedial for those in whom extragenital subvirilization is emotionally costly and subnormal spermatogenesis is not an inevitable side effect of such therapy.

Adult↗
PubMed1989

A single-site allosteric model of intracellular androgen-receptor interaction.

We present a single-site, two-state model for analyzing the effect of time and ligand concentration on the extent and character of 5 alpha-dihydrotestosterone, methyltrienolone or mibolerone binding to the specific androgen receptor within cultured human genital skin fibroblasts. The model has three basic attributes: formation of the initial low-affinity androgen-receptor complex, and its transformation to a higher affinity state are irreversible, first-order processes; and receptors released from complexes in each state not only differ from each other and from their pre-liganded progenitor, but can also reassociate with androgen to yield complexes in their respective parental states. The rate constants of dissociation and apparent equilibrium binding constants of the two affinity states were determined for each of the three androgens within normal cells and those of a transformation-defective mutant. When these values are combined with estimates of the rate constants at which the complexes are formed and transformed, the model accurately simulates time-dependent changes in the slopes and character of experimental Scatchard plots. It can also generate Scatchard plots that are concave, convex or sigmoidal simply by making sequential changes in its formation or transformation constants. Thus, our model can explain complex ligand-receptor binding kinetics that have heretofore been interpreted according to alternate models of transformation and binding-site multiplicity with or without properties of cooperativity, and it supports the notion that receptor recycling involves intermediate receptor states.

Allosteric Regulation↗
PubMed1988

An abundant 56 kD protein with low affinity androgen binding: another member of the steroid/thyroid receptor family?

We have found in genital skin fibroblasts an abundant 56 kD protein which appears related to the androgen receptor. The protein is detected in the soluble fraction by two-dimensional polyacrylamide gel electrophoresis as two spots with isoelectric points of 6.7 and 6.5 respectively. Photoaffinity labelling of GSF with 8 nM or 50 nM [3H]-methyltrienolone selectively labels the two protein spots but with an apparently lower affinity than is known for the androgen receptor. The two protein spots stem from one protein as judged from peptide patterns of partial proteolytic digests and the equal labelling of both spots with methyltrienolone. Cells from subjects with mutant androgen receptors generally lack the 56 kD protein and labelling with methyltrienolone fails, but the protein is not the androgen receptor itself. We propose the hypothesis that the 56 kD protein is synthesized from the same gene as the androgen receptor and that androgen may not be its natural ligand.

Androgens↗