Androgen-receptor Complex Research Articles

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.

Incomplete regression of Müllerian ducts in the androgen insensitivity syndrome

The biochemical and histological features of two related patients with the complete form of the androgen insensitivity syndrome (AIS) coexisting with incomplete regression of the Müllerian ducts are described. Both patients presented unilateral Müllerian derivatives (fallopian tube) identified by microscopic examination of surgically excised internal genital tissue. Biochemical studies performed in genital skin-derived fibroblasts from one of the affected subjects showed the existence of a specific and saturable 8.2 to 8.4 S cytosolic and 3.4 S nuclear androgen receptor exhibiting a Kd of 1.32 nmol/L. These mutant cells, however, clearly presented a significantly low maximal nuclear [3H]-5 alpha-dihydrotestosterone uptake (71.0 fmol/mg of deoxyribonucleic acid [DNA]; control strain, 284 fmol/mg DNA). Thus, an impaired uptake of the androgen receptor complex at the nuclear level was probably the cause of the complete absence of phenotypic expression of androgen action in this family. The overall findings are on line with the well-demonstrated genetic and molecular heterogeneity of the AIS.

The 56-59-kilodalton protein identified in untransformed steroid receptor complexes is a unique protein that exists in cytosol in a complex with both the 70- and 90-kilodalton heat shock proteins

It has previously been shown that 9S, untransformed progestin, estrogen, androgen, and glucocorticoid receptor complexes in rabbit uterine and liver cytosols contain a 59-kDa protein [Tai, P. K., Maeda, Y., Nakao, K., Wakim, N. G., Duhring, J. L., & Faber, L. E. (1986) Biochemistry 25, 5269-5275]. In this work we show that the monoclonal antibody KN 382/EC1 raised against the rabbit 59-kDa protein reacts with 9S, untransformed glucocorticoid receptor complexes in cytosol prepared from human IM-9 lymphocytes but not with 4S salt-transformed receptors. The human protein recognized by the EC1 antibody is a 56-kDa protein (p56) of moderate abundance located predominantly in the cytoplasm by indirect immunofluorescence. There are at least six isomorphs of p56 by two-dimensional gel analysis. N-Terminal sequencing (20 amino acids) shows that p56 is a unique human protein. When p56 is immunoadsorbed from IM-9 cell cytosol, both the 70- and 90-kDa heat shock proteins are coadsorbed in an immune-specific manner. Neither heat shock protein reacts directly with the EC1 antibody. We conclude that p56 exists in cytosol in a higher order complex containing hsp70 and hsp90, both of which in turn have been found to be associated with untransformed steroid receptors.

The binding of 3H-labelled androgen-receptor complexes to hypothalamic chromatin of neonatal mice: Effect of sex and androgenization

The binding of 3H-labelled androgen-receptor complexes, prepared by (NH 4) 2SO 4 precipitation from the 105,000 g supernatant of hypothalamic cytosol, to hypothalamic chromatin of neonatal mice covalently coupled to cellulose was measured in vitro. Saturation binding was also determined after extraction of histones and the masking of acidic proteins with high molarities of guanidine hydrochloride. This investigation showed the presence of high-affinity, low-capacity acceptor sites for [ 3H]-testosterone-receptor complexes in male hypothalamic chromatin ( K d value = 0.39 × 10 −10 M and binding sites of 41 fmol per mg of DNA). Acceptor activity seems to be associated with the acidic protein fraction of chromatin. No specific acceptor sites of similar nature were found in chromatin taken from the hypothalami of female mice. On the basis of these results, it is suggested that the androgen-unresponsiveness of female mice is related to the absence of acceptors for the androgen-receptor in female mice hypothalami.

Characterization and partial purification of the 8-9S androgen receptor from benign prostatic hyperplasia tissues.

The present study reports a 3,800-fold purification of the 8-9S androgen-receptor complex from benign prostate hyperplasia (BPH) tissues using differential chromatography. In addition, the BPH androgen receptor complexes have been characterized using sucrose density gradient (SDG) ultracentrifugation, gel permeation, and anion exchange high performance liquid chromatography (HPLC). Results indicate that a) under nontransforming conditions, BPH cytosols contained both 8-9S (40-78%) and 4S (22-60%) androgen-receptor forms, b) apparent molecular weights of these androgen-receptor apparent molecular weights of these androgen-receptor complexes, as analyzed by gel permeation HPLC, were estimated to correspond at 270 kDa, and 90 kDa respectively, c) 8-9S androgen-receptor complexes were retained on an anion exchange HPLC column and could be eluted at 0.22 M KCl at a linear gradient, whereas 4S complexes were not retained on anion exchange columns under identical experimental conditions, d) 10X dilution of BPH cytosols containing only the 4S (0.6 M KCl) form and subsequent chromatography on anion exchange HPLC system was indicative of fragmentation (these fragments were retained on anion exchange columns and could be eluted by 0.33 M KCl on a linear gradient HPLC), and e) increased temperature (22 C) was permissive of proteolytic fragmentation (fragments were estimated to correspond at 30, 15, and 5 kDa). The results are discussed in relationship with the composition of the nontransformed androgen-receptor molecules.

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.

Tissue‐specific decline in cytosolic casein kinase II of the ventral prostate in aging rats

An age-associated decline in the activity of cytosolic casein kinase II in the rat ventral prostate was observed. The decrease in specific and total activity of casein kinase II of prostatic cytosol obtained from 12-month old rats compared with that from 3-month old rats was 50% and 70%, respectively. This decrease was tissue specific since no such alteration in enzymic activity was found in liver, lung, or heart. The decline in activity was not due to an increase in the concentration of a casein kinase inhibitor or to altered androgenic status with aging. Rather, the reduction in the activity of the enzyme was commensurate with the decrease in the total concentration of the enzyme protein as determined by an ELISA using anti-casein kinase II antibodies. The activity of casein kinase II in the nuclear fraction of the rat prostate was not altered in aging animals. It may be concluded that the cytosolic form of casein kinase II in the prostate of older animals is regulated at the transcriptional level (but not via the androgen-receptor complex mechanism). An alternative explanation for this observation may relate to altered stability of the enzyme in the prostate of older animals.

Progressive induction of mRNA synthesis for androgen-responsive genes in mouse kidney

A number of mRNAs present m kidney are selectively induced by the administration of androgen to mice. Using a pulse-labelling method to measure in vivo rates of mRNA synthesis, seven androgen-responsive mRNAs were tested. The time courses of induction following testosterone treatment indicated that androgen-responsive mRNA synthesis increases progressively. Depending on the mRNA examined, it took 2–10 days after the start of hormone administration for synthesis rates to reach a maximum. Even the fastest of these inductions is slow compared to response times in other steroid-responsive systems, and is very slow compared to the time required for androgen-receptor complex to accumulate in the nucleus. We conclude that gene activation in response to androgen is a prolonged and incremental process rather than a single event. Two alternative models are proposed: (1) these genes are actually responding to intermediate transcription factors that accumulate progressively in response to androgen; (2) the androgen-responsive genes contain multiple binding sites that have a cumulative effect on transcription as the number of receptor complexes bound increases.

Characteristics of the rat prostate androgen receptors analyzed by sucrose density gradient and high-performance liquid chromatofocusing

Rat prostate cytosolic androgen-receptor complexes were analyzed by sucrose density gradient (SDG) centrifugation and by high-performance liquid chromatofocusing (HPCF). Without protecting agents, these complexes were resolved by HPCF at basic (8.25–7.1), intermediary (7.0–5.0) and acidic (4.6–4.2) pH. Sodium molybdate stabilized labeled complexes which migrated in the 8–9S and 3.5–6S areas on SDG. These were further stabilized by the presence of sodium molybdate and four protease inhibitors: complexes then sedimented mainly in the 8–9S area with a shoulder at 6–7S. Forms eluting at acidic pH on HPCF were favored by the presence of sodium molybdate and further enhanced by the addition of inhibitors, to the detriment of basic ones. Furthermore, when chromatographed on phosphocellulose (P-c), unretained complexes sedimented as a symmetrical peak on SDG centrifugation in the 8–9S area, but were eluted from HPCF columns as two entities at pH 4.1 and 4.6. The P-c retained complexes subsequently detached by 0.6 M KC1, were resolved into three entities by HPCF with a major component at pH 8.2, which sedimented in the 4S areas. These results demonstrate that the gradual decrease in the negative net charge of androgen receptor correlates with the gradual reduction in mass of the androgen-receptor complex. Moreover, this can be interpreted as further evidence for a heterogeneity of androgen receptor population in rat prostate, suggesting the involvement of a multistep mechanism preceding the induction of specific gene transcription by the hormone.

Studies on the characterization of rat prostate androgen receptors.

In the presence of sodium molybdate and protease inhibitors, two forms of androgen-receptor complexes were observed which sedimented in the areas of 8-9S and 5-7S by SDG centrifugation. The intermediary 5-7S form was better seen when complexes were incubated at low KCl concentrations. The sedimentation coefficient of this form fluctuated between 5 and 7S depending on the KCl concentration. At high ionic strength (0.6M KCl) in all media, one form only was observed having a sedimentation coefficient value of 4.3S. By gel exclusion chromatography, we also observed two specific entities at 75A and 68A; in the presence of 0.6M KCl, however, two entities were found at 68A and 43A. The constant presence of protease inhibitors in all buffers was necessary to separate the intermediary 68A form. We calculated molecular weights of about 270 kDa, 190 kDa, and 80 kDa respectively for these three forms. [3H]R1881-receptor complexes bound to DEAE-cellulose and were eluted in the absence of glycerol at 0.1M and 0.2M KCl. Material found at 0.1M KCl sedimented in the areas of 5-7S and 8-9S in nearly equal proportion, and that found at 0.2M KCl sedimented in the 8-9S area only. When the cytosol was chromatographed at a fast flow rate (4 ml/min), untransformed 8-9S receptors did not bind to phosphocellulose, but transformed complexes were retained, could be eluted with 0.4M KCl and sedimented in the 4S area on KCl free SDG centrifugation. When the excluded untransformed 8-9S complexes were re-chromatographed at a slow flow rate (1 ml/min), they were retained on phosphocellulose, and could be eluted with 0.3M KCl.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding of androgen-receptor complexes to α 2u-globulin genes and to the long terminal repeat of mouse mammary tumor virus

The binding of androgen-receptor complexes to fragments derived from two α 2u-globulin genes (RAP 01 and RAO 01) was studied using a DNA-cellulose competition assay. Rat prostate cytosol labelled with [ 3H]mibolerone was used as a source of the androgen receptor. Two controls were included in these studies: the long terminal repeat (LTR) of mouse mammary tumor virus which has previously been shown to act as an androgen response element and a fragment of the C3 gene of prostatic binding protein which has been demonstrated to bind androgen-receptor complexes. Our experiments indicate that androgen-receptor complexes bind specifically and with comparable affinity to the C3 gene fragment, the LTR and a fragment of RAP 01 located in the 5'-upstream region (bp −642 up to −584). No specific interaction was observed with fragments derived from RAO 01. The region of RAP 01 which binds androgen-receptor complexes has previously been shown to interact with glucocorticoid receptors and contains a 17 bp sequence homologous with the consensus sequence for glucocorticoid-receptor binding. A mutation in this sequence in RAO 01 may be responsible for the loss of glucocorticoid and androgen-receptor binding. It is concluded that at least one member of the α 2u-globulin gene family interacts directly with androgen-receptor complexes with an affinity comparable to that observed for other androgen-dependent genes. The binding is observed in a region displaying also affinity for the glucocorticoid receptor.

Partial purification and characterisation of the human skin fibroblast androgen receptor: Detection of abnormal receptor complexes in cells from patients with androgen insensitivity syndromes

After incubation of hGSF with [ 3H]α-dihydrotestosterone, 17β-hydroxy-7α,17α-dimethyl-4-estrene-3-one, or 17β-hydroxy-17α-methyl-4,9,11-estrien-3-one, androgen-receptor complexes were extracted with 0.5 M KCl and precipitated by 35% ammonium sulphate. Receptor complexes from control hGSF sedimented at approximately 4S on linear 5–20% sucrose gradients. The 4S peak was diminished or absent in cells from androgen insensitive patients exhibiting absent, deficient or unstable binding of androgens in intact hGSF. This procedure may be a useful means of distinguishing quantitative and qualitative defects in androgen binding to receptor, since one cell line found to have normal levels of androgen receptor complexes in whole cell assays had a profile resembling that of receptor negative cells on sucrose gradients. The complexes from one patient with complete androgen insensitivity having normal androgen binding in intact hGSF were indistinguishable from control complexes after sucrose gradient analysis and ADP-Sepharose chromatography. Receptor complexes were eluted from the ADP-Sepharose between 0.5–1.0 M KCl. HPLC-gel filtration of androgen receptor complexes at 22°C revealed two peaks, the larger had a M r of 60–65K, Stokes radius of 3.16 nm and a frictional ratio between 1.21 and 1.43. The second peak, M 4 of 15K, was believed to represent a fragment of the receptor containing the steroid binding domain. On gel filtration at 22°C the complexes from a patient with partial androgen insensitivity, who showed a diminished 4S receptor peak on sucrose gradients, revealed only the small “meroreceptor” fragment, suggesting that the mutation in this individual might render the androgen receptor more susceptible to proteolysis in vitro.

Inherited Impairment of Nuclear Androgen Uptake as a Cause of Familial Androgen Insensitivity

ULLOA-AGUIRRE, ALFREDO; CHAVEZ, BERTHA; MENDEZ, JUAN PABLO; SAAVEDRA, DOLORES; PEREZ-PALACIOS, GREGORIO Author Information

Modulation of steroid affinity for the androgen receptor by sucrose

The effects of sucrose on androgen binding to its receptor were investigated. Sucrose decreased the rate of thermal inactivation of unoccupied and occupied androgen receptor (AR) and the rates of [ 3H] 5α-dihydrotestosterone ([ 3H]DHT) dissociation from both activated and nonactivated AR complexes. Binding of [ 3H] DHT to AR in vivo , or in intact cells at 37°C, caused reduction of [ 3H] DHT dissociation from cytosolic and nuclear complexes, as compared to in vitro labeled receptor complexes. Further, exposure of these complexes to sucrose at 0°C caused an additional reduction of dissociation rates. Thus, the decrease of [ 3H] DHT dissociation induced by sucrose is independent of the reaction that reduces DHT dissociation from activated and transformed AR. Sucrose also reduced the ability of mersalyl acid to inactivate AR complexes. This effect of sucrose was markedly diminished in the presence of 2M urea. Sucrose did not significantly affect the association rate, sedimentation properties, or nuclear binding ability of AR complexes, but it did decrease the equilibrium dissociation constant. Other monosaccharides and disaccharides also stabilized AR. These data suggest that sucrose induces conformational changes in the steroid binding domain of androgen receptor, thereby reducing the rates of inactivation, steroid dissociation, and the accessibility of sulfhydryl groups to mersalyl.

Intranuclear androgen-receptor complex binding sites of mouse submandibular gland.

Nuclear androgen-receptor binding sites in chromatin regions of mouse submandibular gland were studied. Cytosol androgen receptors prelabeled with [3H]androgen interacted with the crude nuclei in mouse submandibular gland and activation of the receptor was a prerequisite for this interaction. After in vivo administration of [3H]androgen to female mice, radioactivities were found in the nuclei purified from submandibular gland tissues and the [3H]androgen-labeled purified nuclei were further digested with micrococcal nuclease. The androgen receptor was found in solubilized, active chromatin fractions which contained mono- and dinucleosomes. By an in vitro exchange assay, endogenous androgen-receptor complexes associated with chromatin binding sites in intact males were found in the solubilized fraction after micrococcal nuclear digestion, whereas such complexes were not found in females. These results suggest that the androgen receptors translocated to the nuclei and became associated with chromatin and that this association occurred in transcriptionally active chromatin regions that were preferentially sensitive to micrococcal nuclease.

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 5α-dihydro testosterone, 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.

Characterization of the nontransformed and transformed androgen receptor and heat shock protein 90 with high-performance hydrophobic-interaction chromatography

The hydrophobicity of the nontransformed and transformed androgen receptor from rat submandibular gland and heat shock protein 90 (hsp90) from rat submandibular gland and liver was characterized by using high-performance hydrophobic-interaction chromatography on TSK gel Ether-5PW. In the absence of molybdate, cytosol [ 3H]R1881 -androgen receptor complexes were mainly eluted in the 1.3 M region (Peak 1) with a small peak in the 0.8 M region (Peak 2) of a descending salt gradient (2 to 0 M) of ammonium sulfate. In the presence of molybdate, Peak 2 was predominant. When labeled-cytosol was applied after being heated at 25°C for 30 min, a third peak (Peak 3) at around 0.64 M ammonium sulfate was newly observed. Peaks 2 and 3 were observed, while Peak 1 completely disappeared with the labeled-cytosol precipitated at 40% saturated ammonium sulfate. The Stokes radius of Peak 1 was 7 nm, and of Peak 2 was 8 nm. Both peaks were retained poorly by DNA-cellulose but bound rather well to DEAE-cellulose. These results suggest that these two peaks represent the nontransformed receptor, indicating that there are isoforms of the nontransformed androgen receptor which are distinguished by their hydrophobic properties and Stokes radii. Peak 3 had a Stokes radius of 5 nm and preferentially bound to DNA-cellulose, suggesting that this peak corresponds to the transformed receptor. These results indicated that the transformation of the androgen receptor accompanies the enrichment of the hydrophobicity of the receptor molecule. Hsp90 purified from rat livers and hsp90 in the cytosol both from livers and submandibular glands were eluted from Ether-5PW at 0.8 M ammonium sulfate, at almost the same position as Peak 2. This finding suggests that the enrichment of hydrophobicity on transformation is due to dissociation of hsp90 from the nontransformed androgen receptor.

Inherited impairment of nuclear androgen uptake as a cause of familial androgen insensitivity

The endocrine and biochemical characteristics of four related 46,XY pseudohermaphrodite patients with the Reifenstein Syndrome are presented. All of them (6 and 9 years old, first generation, and 9 and 12 months old, second generation) exhibited ambiguity of external genitalia and a family pedigree characteristic of an X-linked pattern of inheritance. Serum basal levels of LH, FSH, testosterone (T), androstenedione and 5α-dihydrotestosterone (DHT) were within normal limits. Administration of hCG induced a normal response in terms of serum T in three of the patients, with a concomitant increase in serum DHT. However, an abnormally elevated T : DHT ratio was found in two of these subjects on the day of maximal T response (T : DHT ratio, 24 and 27; normal range, 4–21). Genital skin-derived fibroblasts from all patients were studied for [ 3H]DHT uptake in a whole-cell monolayer assay. Three of the mutant strains exhibited values of [ 3H]DHT uptake at 37 ° C within the lower limits of normality (39.4–47.05 fmol/mg protein/h; normal strains, 36–101 fmol/mg protein/h), whereas fibroblasts from the remaining patient presented a slightly decreased uptake (31.66 fmol/mg protein); when studied at 42 ° C, all mutant strains behaved as the normal controls. The existence of a specific 4.6 S cytosol androgen receptor was clearly seen in the two mutant strains when analysed by sucrose gradient centrifugation. Nevertheless, in one of the mutant strains, a significantly low maximal nuclear [ 3H]DHT uptake was detected (173.6 fmol/mg DNA; control strain, 301.6 fmol/mg DNA). The overall data were interpreted as demonstrating the existence of an impaired uptake of the androgenreceptor complex at the nuclear levels as the cause of the incomplete phenotypic expression of androgen action in this family. In this setting, the presence of low peripheral 5α-reductase activity may be considered as a secondary manifestation of the androgen insensitivity.

Steroid Receptor Recycling and Interaction of Receptor with RNA

Steroid receptors, such as the androgen receptor from the rat ventral prostate, are involved in a recycling process as part of the mechanism by which steroids affect target tissues. Recycling of steroid-receptor complexes may involve interaction of receptors with RNA. Evidence for RNA-receptor complexes in various cells and tissues has been presented, and it has been shown that the ability of RNA to interact with steroid-receptor complexes is dependent on the composition of the RNA. As part of our investigations on the role of RNA in steroid-receptor recycling, we have studied the effects of actinomycin D and cordycepin (3'-deoxyadenosine) on androgen receptors in short-term cultures of rat ventral prostate tissue. Actinomycin D increased nuclear and decreased cytoplasmic levels of androgen-receptor complexes. Cordycepin, in contrast, decreased nuclear and increased cytoplasmic levels of androgen-receptor complexes. Cordycepin markedly decreased the amount of cytosolic androgen-receptor complex that could bind to DNA-cellulose whereas actinomycin D had no effect on DNA-cellulose binding activity. Although both actinomycin D and cordycepin are inhibitors of RNA synthesis, the contrasting effects of cordycepin may be due to inhibition of RNA processing or transport by cordycepin. The effects of these two compounds are consistent with a model of steroid receptor recycling in which RNA facilitates removal of receptors from the nuclear acceptor and the steroid receptor remains associated with the RNA during RNA processing and transport.

ANDROGEN INSENSITIVITY SYNDROME (ATS): CELLULAR RESPONSES TO ANDROGENS

A considerable number of hereditary defects in the mechanism of action of testosterone (T) is known. Most, but not all, can be traced back to either a functionally deficient receptor or a defect in the 5-alpha reductase activity. The current (in vitro) diagnostic approach involves cell culture of genital skin derived fibroblasts followed by studies of whole cell dihydrotestosterone (DHT) or R1881 uptake and the kinetics of T reduction.Several receptor abnormalities can be detected only under special conditions: lability at 42°C; increased dissociation rate of the androgen receptor complex; different behaviour in the absence of molybdate, etc. A more or less complete investigation is therefore extremely time consuming, while in a significant percentage of all patients with the clinical diagnosis AIS, the diagnosis cannot be confirmed or excluded by means of laboratory studies. Therefore, we tried a functional approach: fibroblasts were stimulated for 6 days with T and DHT, followed by 25S-methionine pulse-labeling. Labeled proteins were separated by means of 1- and 2-dimensional electrophoresis and detected by autoradiography. We found no evidence for the induction of new proteins, but the results suggest that under these conditions in normal androgen-responsive cells at least one protein disappears. In AIS patients no such repression was seen. This functional approach is able to corroborate and complement the “classical” receptor studies.