Functional analysis of six androgen receptor mutations identified in patients with partial androgen insensitivity syndrome
Functional analysis of six androgen receptor mutations identified in patients with partial androgen insensitivity syndromeCharlotte L. Bevan*, Betty B. Brown, Helen R. Davies, Bronwen A. J. Evans1, Ieuan A. Hughes and Mark N. Patterson
University Department of Paediatrics, University of Cambridge, Cambridge CB2 2QQ, UK and 1University Department of Child Health, University of Wales College of Medicine, Cardiff CF4 4XN, UK
Received September 28, 1995;Revised and Accepted November 17, 1995
Partial androgen insensitivity syndrome (PAIS) is caused by defects in the androgen receptor gene and presents with a wide range of undervirilization phenotypes. We studied the consequences of six androgen receptor ligand-binding domain mutations on receptor function in transfected cells. The mutations, Met742Ile, Met780Ile, Gln798Glu, Arg840Cys, Arg855His and Ile869Met, were identified in PAIS patients with phenotypes representing the full spectrum seen in this condition. In all cases the androgen receptor was found to be defective, suggesting that the mutation is the cause of the clinical phenotype. The Gln798Glu mutation is exceptional in that it did not cause an androgen-binding defect in our system, although the mutant receptor was defective in transactivation assays. This mutation may affect an aspect of binding not tested, or may be part of a functional subdomain of the ligand-binding domain involved in transactivation. Overall we found milder mutations to be associated with milder clinical phenotypes. There is also clear evidence that phenotype is not solely dependent on androgen receptor function. Some of the mutant receptors were able to respond to high doses of androgen in vitro, suggesting that patients carrying these mutations may be the best candidates for androgen therapy. One such mutation is Ile869Met. A patient carrying this mutation has virilized spontaneously at puberty, so in vivo evidence agrees with the experimental result. Thus a more complete understanding of the functional consequences of androgen receptor mutations may provide a more rational basis for gender assignment in PAIS.
Testosterone and its derivative 5[alpha]-dihydrotestosterone (DHT) are the two major androgens that mediate male sexual differentiation. They are responsible for masculinization of the urogenital tract of the 46XY embryo and play the major role in development of secondary sexual characteristics at puberty (1 ). While the roles of testosterone and DHT are quite distinct in that some target cells respond to testosterone and others to DHT, the action of both is mediated by one androgen receptor.
Androgen insensitivity syndrome (AIS) is a disorder of male sexual differentiation caused by an absent or dysfunctional androgen receptor (2 -4 ). Individuals with AIS have a 46XY karyotype with testes and normal or elevated testosterone levels. Vestigial or incomplete Müllerian or Wolffian duct structures are present in some cases. The syndrome is classified into two forms: complete androgen insensitivity syndrome (CAIS) characterized by a normal female external appearance apart from a usual lack of pubic and axillary hair; and partial androgen insensitivity syndrome (PAIS) which presents at birth with a broad spectrum of incompletely virilized genital phenotypes ranging from primarily female with some virilization such as clitoromegaly or labial fusion to primarily male with undervirilization such as hypospadias or micropenis. PAIS patients are usually, but not invariably, infertile and there is some dispute whether PAIS can also present in a very mild form as infertility in otherwise normal males (5 -8 ).
The androgen receptor (AR) is a protein of 919 amino acids encoded by a gene of about 90 kilobases located at Xq11-12 (9 -11 ). The androgen receptor belongs to the nuclear receptor superfamily (12 ) and has three main domains encoded by eight exons (13 -16 ). The large N-terminal fragment encoded by exon 1 is the least conserved region and is apparently involved in transcriptional activation of target genes by the AR (17 ). The central section, encoded by exons 2 and 3, comprises two `zinc finger'-like structures (18 ). This is the most highly conserved domain in the nuclear receptor superfamily and is responsible for binding to DNA at the androgen response element (ARE) in the promoter region of target genes. The DNA-binding domain also appears to contain a dimerization function (19 ). A nuclear translocation signal is located in the hinge region between the DNA-binding and ligand-binding domains (20 ,21 ). The C-terminal 250 amino acids encoded by exons 4-8 are involved in high-affinity ligand binding. There is also evidence, directly and by analogy with other steroid hormone receptors, that the ligand-binding domain contains subdomains involved in dimerization and transcriptional activation (22 -31 ).
The purpose of the work described here was to investigate the functional consequences of AR mutations identified in PAIS patients, with a view to understanding the relationship between the functional deficit in the receptor and the clinical phenotype in the patient. In particular, we have studied six AR mutations previously reported in PAIS patients (32 ) (B. Evans and L. Gregory, in preparation). Genital skin fibroblast (GSF) cell lines from five of these patients showed decreased androgen-binding ability and the mutations identified all lie in the androgen-binding domain of the receptor. We aimed to introduce these mutations individually into a human androgen receptor expression vector and assay the effect they have on receptor function in vitro. In this way, we were able to compare the results of assays using in vitro engineered mutant constructs with those in the patient cell line and demonstrate whether the single identified mutation indeed causes the phenotype. We went on to compare the degree and nature of receptor dysfunction with the severity of undervirilization in the patients. Finally, we investigated whether increasing doses of androgen could overcome the defect of the androgen receptor in vitro, in order to identify patients who are more likely to respond to androgen therapy in vivo. This paper presents evidence that the mutation is the cause of the clinical phenotype in all six cases and demonstrates the effect each has on various aspects of receptor function.
The transactivation assay measures the ability of the receptor to stimulate transcription from an androgen response element. This assay is therefore a more complete test of androgen receptor function. Each mutant AR construct was cotransfected into HeLa cells with the reporter vector pG29GtkCAT (35 ) which carries the chloramphenicol acetyl transferase (CAT) gene under the control of an androgen-responsive promoter (two progesterone/glucocorticoid response elements upstream of the thymidine kinase promoter). HeLa cells rather than COS cells were used as the latter exhibited high background levels of CAT activity when no androgen was added, or when transfected with the reporter vector alone (unpublished data). A wild-type construct was assayed in parallel with all mutant constructs. The activation of the CAT gene by wild-type receptor at the highest MB concentration (10 nM) was defined as 100% activation for that experiment and the activity at other concentrations and of the parallel mutant construct was expressed relative to this value. As the wild-type assay was highly reproducible the mean values (n = 6) are shown for comparison with each mutant activation pattern (Fig. 2 ).
All the mutant receptors reconstructed showed impaired function and so are likely to be the mutation causing the clinical phenotype in each of the six patients studied. Met742Ile, Met780Ile, Arg840Cys, Arg855His and Ile869Met each showed qualitatively abnormal androgen binding; all have increased apparent Kd and all but Met742Ile are thermolabile. The Bmax values obtained for Met742Ile, Met780Ile, Arg840Cys and Ile869Met suggest that receptor levels in cells transfected with these mutant constructs are lower than in cells transfected with wild-type construct; however, these quantitative differences are not so striking as the qualitative differences observed. The abnormal binding is not surprising as the mutations all lie in the region identified as the hormone-binding domain of the receptor. An exception is the Gln798Glu mutation. GSF cells from the patient in this case showed apparently normal binding. Similarly, the mutant receptor expressed in COS cells had values for Kd, thermolability and Bmax within the normal range.
The binding data obtained in GSF cells from the six patients are also shown in Table 2 for comparison with the results of COS cell assays. Comparing the two different systems the order of severity of binding defect, as represented by the fold increase in Kd, is slightly different. The mutations Arg840Cys and Arg855His confer a relatively more severe defect in COS cells, where these receptors have the lowest binding affinities of all the receptors studied. A variable factor in GSF assays is the genetic background of the patient, which could mask or exacerbate the effect of a receptor defect. This variation is absent in the COS cell assay. Although the internal milieu of COS cells is different to those in which the AR functions in vivo, the COS cell background provides a constant environment in which to compare the functional consequences of different mutations.
The effect each mutation has on the overall function of the receptor was studied further in a transactivation assay using an androgen-responsive reporter vector. This assay showed that each mutation causes receptor dysfunction as exhibited by a decreased ability to stimulate transcription of the reporter compared with the wild-type construct. Met742Ile and Met780Ile showed severely impaired transcriptional activation at all concentrations of MB with only a very slight increase in activity at the highest concentration. Arg840Cys, Arg855His and Ile869Met all showed some increase in activity with increasing hormone levels. Interestingly, the transactivation assay showed that Gln798Glu does cause impairment of receptor function, despite its normal affinity for androgen. It is possible that the substitution affects some aspect of binding not tested, for instance it may alter the stability of the hormone-receptor complex at 37oC (which may in turn increase the rate of degradation of the AR) (36 ). Further experiments will measure other binding parameters of this receptor. Another explanation could be that Gln798 affects an AR function other than binding, e.g. transactivation, and lies in a functional subdomain within the ligand-binding domain (LBD).
Figure 3 shows a comparison of part of the androgen-binding domain of the AR and the homologous regions of other members of the nuclear receptor superfamily. The receptors for progesterone (PR), mineralocorticoid (MR) and glucocorticoid (GR) are the most closely related to AR and within this subgroup there is a high degree of amino acid conservation. The crystal structure of the ligand-binding domain of human retinoid X receptor (RXR) has recently been elucidated (30 ) and, due to the high degree of conservation of various structurally important residues, is believed to be a prototype for all members of the steroid receptor superfamily. Conservation of functional subdomains has also been found between various members of the superfamily, so comparing AR to other receptors may provide further information about the functional consequences of the mutations discussed here.
The mutant receptors are identified using the numbering convention of Lubahn et al. (42 ), to comply with the AR gene mutations database (43 ). Five of the subjects, with mutations Met742Ile, Met780Ile, Gln798Glu, Arg855His and Ile869Met, have been described in a previous paper (32 ). The mutation Arg840Cys was found in two siblings, one raised as a male and the other as a female. Clinical phenotypes of the subjects and androgen-binding activity of GSF cell lines are summarized in Table 1 .
All the mutations were introduced into the human androgen receptor expression construct pSVARo (15 ), driven by the SV40 early promoter. The Escherichia coli strain DH5[alpha] (44 ) was used for all cloning. All restriction enzymes used were from Stratagene.
For mutations at residues 840, 855 and 869 (see Fig. 4 a), cDNA of the C-terminal part of the androgen receptor was synthesized using MMLV reverse transcriptase (Gibco-BRL) from total RNA prepared from the patients' GSF cell lines (45 ). A 494 bp EcoRI fragment encompassing exons 7 and 8 was amplified from the cDNA by PCR using Promega Taq polymerase. The corresponding EcoRI fragment was removed from pSVARo which was then treated with shrimp alkaline phosphatase (USB) to prevent recircularization. The mutant EcoRI fragment was ligated into pSVARo using T4 DNA ligase (Stratagene).
Androgen-binding ability of the mutant receptors was measured in COS-1 cells using a whole cell binding assay (47 ). COS-1 cells were grown in Dulbecco's modified essential medium (DMEM) supplemented with 2 mM glutamine, 50 IU/ml penicillin, 50 µg/ml streptomycin (all ICN-Flow) and 10% fetal calf serum (FCS) (Gibco), at 37oC in 5% CO2.
Briefly, COS-1 cells at 70-80% confluence were trypsinised and plated out into three 10 cm plates at 106 cells per plate. Twenty-four hours later these were transferred to serum-free medium and transfected with 15 µg pSVARo and 3 µg pSV-[beta]-galactosidase control vector (Promega) using the DEAE-dextran method (48 ). The plates were incubated with transfection mix for 4 h at 37oC in 5% CO2 before being subjected to a DMSO shock. The cells were then allowed to recover for 48 h in DMEM with 10% FCS. The medium was changed to serum-free DMEM for a further 24 h before the cells were trypsinized and pooled for the androgen-binding assay. Cells were incubated at 37oC for 1 h with the following concentrations of 3H-labelled MB (Dupont NEN): 0.125, 0.25, 0.5, 1, 2, 4 nM, in each case with and without a 200-fold excess of unlabelled MB. 2×105 cells were used for each assay point and each point was assayed in duplicate. The highest concentration point was also duplicated at 40oC to measure thermolability of binding. Cells were collected on Whatman GF/A glass microfibre filters and washed using a vacuum harvesting device; the filters were then immersed in Packard Ultima Gold MV for counting on a 2500 TR Packard liquid scintillation counter. Scatchard analysis was performed using the Combicept 2000 Steroid Receptor Assay software (Packard). This gave the binding site concentration Bmax and dissociation constant Kd of binding. Protein was determined using the Bradford assay (48 ) and [beta]-galactosidase measured using a colorimetric assay (49 ) to determine transfection efficiency. The counts were normalised for transfection efficiency so that they could be plotted on the same axes and to determine Bmax.
Transcriptional activation activity of the mutant receptors was measured in HeLa cells. These were cultured in DMEM supplemented with 2 mM glutamine, 50 IU/ml penicillin, 50 µg/ml streptomycin and 10% FCS at 37oC in 5% CO2. Cells at 70-80% confluence were trypsinized and plated out at 2.5 × 105 cells per 3.5 cm plate in DMEM with 10% dextran-coated-charcoal treated (DCC) serum (50 ). Twenty-four hours later they were transfected using calcium phosphate coprecipitiation (48 ). The following quantities of DNA were used per plate: 1.5 µg AR construct; 0.75 µg of the androgen-responsive chloramphenicol acetyl transferase reporter vector pG29GtkCAT (35 ); 0.75 µg pBOS-[beta]-galactosidase (51 ) and 1 µg salmon sperm DNA (Sigma). After incubation with this mix for 4 h the cells were shocked with 15% glycerol then allowed to recover overnight in DMEM with 10% DCC serum. The medium was then supplemented with MB concentrations of 0, 0.01, 0.1, 1 and 10 nM. After a further 48 h the cells were harvested by scraping. Cell extracts were prepared by three freeze-thaw cycles of the resuspended pellet (in 0.25 M Tris-HCl, pH 8), the cell debris was pelleted at 13 000 g and the supernatant used in the assay. CAT assays were performed according to a phase-extraction protocol (52 ). The CAT activity was measured in a 2500 TR Packard liquid scintillation counter. Each MB concentration was assayed in triplicate and each mutant construct was assayed in parallel with wild-type pSVARo for comparison of results. Results were normalized for protein concentration and transfection efficiency by measuring the [beta]-galactosidase activity. The activity of the wild-type receptor at 10 nM MB was taken to be 100% and the other counts expressed relative to this.
The authors thank Dr Albert Brinkmann for the expression vector pSVARo, Dr Rainier Renkawitz for the reporter plasmid pG29GtkCAT and Dr M. Ritzen for samples from the Gln798Glu case. We gratefully acknowledge the contributions of Dr Jennifer Batch and Dr Denise Williams. This work was supported by the Sims Fund (University of Cambridge), the Sir Halley Stewart Trust and the Wellcome Trust.
1 Wilson, J. D., Griffin, J. E., George, F. W. and Leshin, M. (1981) The role of gonadal steroids in sexual differentiation. Rec. Prog. Horm. Res., 37, 1-39.MEDLINE Abstract
2 Patterson, M. N., McPhaul, M. J. and Hughes, I. A. (1994) Androgen insensitivity syndrome. Bailliere's Clin.Endocrinol. Metab., 8, 379-404.
3 Quigley, C., De Bellis, A., Marschke, K., El-Awady, M., Wilson, E. and French, F. (1995) Androgen receptor defects: historical, clinical and molecular perspectives. Endocr. Rev., 16, 271-321.MEDLINE Abstract
4 Pinsky, L., Trifiro, M., Kaufman, M., Beitel, L. K., Mhatre, A., Kazemi-Esfarjani, P., Sabbaghian, N., Lumbroso, R., Alvarado, C., Vasiliou, M. and Gottlieb, B. (1992) Androgen resistance due to mutation of the androgen receptor. Clin. Invest. Med., 15, 456-472.MEDLINE Abstract
5 Tsukada, T., Inoue, M., Tachibana, S., Nakai, Y. and Takebe, H. (1994) An androgen receptor mutation causing androgen resistance in undervirilized male syndrome. J. Clin. Endocrinol. Metab., 79, 1202-1207.MEDLINE Abstract
6 Grino, P. B., Griffin, J. E., Cushard, W. G. and Wilson, J. D. (1988) A mutation of the androgen receptor associated with partial androgen resistance, familial gynaecomastia, and fertility. J. Clin. Endocrinol. Metab., 66, 754-761.MEDLINE Abstract
7 Aiman, J. and Griffin, J. (1982) The frequency of androgen receptor deficiency in infertile men. J. Clin. Endocrinol. Metab., 54, 725-732.MEDLINE Abstract
8 Bouchard, P., Wright, F., Portois, M., Couzinet, B., Schaison, G. and Mowszowicz, I. (1986) Androgen insensitivity in oligospermic men: a reappraisal. J. Clin. Endocrinol. Metab., 63, 1241-1246.
9 Lubahn, D. B., Joseph, D. R., Sullivan, P. M., Willard, H. F., French, F. S. and Wilson, E. M. (1988) Cloning of human androgen receptor complementary DNA and localization to the X chromosome. Science, 240, 327-330.MEDLINE Abstract
10 Chang, C., Kokontis, J. and Liao, S. (1988) Molecular cloning of human and rat complementary DNA encoding androgen receptors. Science, 240, 324-326.MEDLINE Abstract
11 Trapman, J., Klaasen, P., Kuiper, G. G. J. M., van der Korput, J. A. C. M., Faber, P. W., van Rooij, H. C. J., Geurts van Kessel, A., Voorhorst, M. M., Mulder, E. and Brinkmann, A. O. (1988) Cloning, structure and expression of a cDNA encoding the human androgen receptor. Biochem. Biophys. Res. Commun., 153, 241-248.MEDLINE Abstract
12 Tsai, M-J. and O'Malley, B. (1994) Molecular mechanisms of action of steroid/thyroid receptor superfamily members. Annu. Rev. Biochem, 63, 451-486.MEDLINE Abstract
13 Lubahn, D. B., Brown, T. R., Simental, J. A., Higgs, H. N., Migeon, C. J., Wilson, E. M. and French, F. S. (1989) 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. Proc. Natl Acad. Sci. USA, 86, 9534-9538.MEDLINE Abstract
14 Marcelli, M., Tilley, W. D., Wilson, C. M., Griffin, J. E., Wilson, J. D. and McPhaul, M. J. (1990) 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. Mol. Endocrinol., 4, 1105-1116.MEDLINE Abstract
15 Brinkmann, A. O., Faber, P. W., van Rooij, H. C. J., Kuiper, G. G. J. M., Ris, C., Klaasen, P., van der Korput, J. A. G. M., Voorhorst, M. M., van Laar, J. H., Mulder, E. and Trapman, J. (1989) The human androgen receptor: domain structure, genomic organisation and regulation of expression. J. Steroid Biochem., 34, 307-310.MEDLINE Abstract
16 Brinkmann, A., Jenster, G., Kuiper, G., Ris, C., van Laar, J., van der Korput, J., Degenhart, H., Trifiro, M., Pinsky, L., Romalo, G., Schweikert, H., Veldschote, J., Mulder, E. and Trapman, J. (1992) The human androgen receptor: structure/function relationship in normal and pathological situations. J. Steroid Biochem. Mol. Biol., 41, 361-368.MEDLINE Abstract
17 Jenster, G., van der Korput, H., Trapman, J. and Brinkmann, A. O. (1995) Identification of two transcription activation units in the N-terminal domain of the human androgen receptor. J. Steroid Biochem. Mol. Biol., 53, 443-448.
18 Freedman, L. (1992) Anatomy of the steroid receptor zinc finger region. Endocr. Rev., 13, 129-145.MEDLINE Abstract
19 Wong, C-i., Zhou, Z-x., Sar, M. and Wilson, E. M. (1993) Steroid requirement for androgen receptor dimerization and DNA binding. J. Biol. Chem., 268, 19004-19012.MEDLINE Abstract
20 Zhou, Z-x., Sar, M., Simental, J. A., Lane, M. V. and Wilson, E. M. (1994) A ligand-dependent bipartite nuclear targeting signal in the human androgen receptor. J. Biol. Chem., 269, 13115-13123.MEDLINE Abstract
21 Jenster, G., Trapman, J. and Brinkmann, A. O. (1993) Nuclear import of the human androgen receptor. Biochem. J., 293, 761-768.MEDLINE Abstract
22 Nemoto, T., OharaNemoto, Y., Shimazaki, S. and Ota, M. (1994) Dimerization characteristics of the DNA- and steroid-binding domains of the androgen receptor. J. Steroid Biochem. Mol. Biol., 50, 225-233.MEDLINE Abstract
23 Fawell, S. E., Lees, J. A., White, R. and Parker, M. G. (1990) Characterization and colocalization of steroid binding and dimerization activities in the mouse estrogen receptor. Cell, 60, 953-962.MEDLINE Abstract
24 Danielian, P. S., White, R., Lees, J. A. and Parker, M. G. (1992) Identification of a conserved region required for hormone dependent transcriptional activation by steroid hormone receptors. EMBO J., 11, 1025-1033.MEDLINE Abstract
25 Durand, B., Saunders, M., Gaudon, C., Roy, B., Losson, R. and Chambon, P. (1994) Activation function 2 (AF-2) of retinoic acid receptor and 9-cis retinoic acid receptor: presence of a conserved autonomous constitutive activating domain and influence of the nature of the response element on AF-2 activity. EMBO J., 13, 5370-5382.MEDLINE Abstract
26 Jenster, G., van der Korput, H. A. G. M., van Vroonhoven, C., van der Kwast, T. H., Trapman, J. and Brinkmann, A. O. (1991) Domains of the human androgen receptor involved in steroid binding, transcriptional activation, and subcellular localization. Mol. Endocrinol., 5, 1396-1404.MEDLINE Abstract
27 Simental, J. A., Sar, M., Lane, M. V., French, F. S. and Wilson, E. M. (1991) Transcriptional activation and nuclear targeting signals of the human androgen receptor. J. Biol. Chem., 266, 510-518.MEDLINE Abstract
28 Tasset, D., Tora, L., Fromental, C., Scheer, E. and Chambon, P. (1990) Distinct classes of transcriptional activating domains function by different mechanisms. Cell, 62, 1177-1187.MEDLINE Abstract
29 Barettino, D., Vivanco Ruiz, M. and Stunnenburg, H. (1994) Characterization of the ligand-dependent transactivation domain of thyroid hormone receptor. EMBO J., 13, 3039-3049.MEDLINE Abstract
30 Bourguet, W., Ruff, M., Chambon, P., Gronemeyer, H. and Moras, D. (1995) Crystal structure of the ligand-binding domain of the human nuclear receptor RXR-[alpha]. Nature, 375, 377-382.MEDLINE Abstract
31 White, R., Fawell, S. E. and Parker, M. G. (1991) Analysis of oestrogen receptor dimerisation using chimeric proteins. J. Steroid Biochem. Mol. Biol., 40, 333-341.MEDLINE Abstract
32 Batch, J. A., Williams, D. M., Davies, H. R., Brown, B., Evans, B. A. J., Hughes, I. A. and Patterson, M. N. (1992) Androgen receptor gene mutations identified by SSCP in fourteen subjects with androgen insensitivity syndrome. Hum. Mol. Genet., 1, 497-503.MEDLINE Abstract
33 Klocker, H., Kaspar, F., Eberle, J., Uberreiter, S., Radmayr, C. and Bartsch, G. (1992) Point mutation in the DNA binding domain of the androgen receptor in two families with Reifenstein syndrome. Am. J. Hum. Genet., 50, 1318-1327.MEDLINE Abstract
34 Hughes, I. A. and Evans, B. A. J. (1987) Androgen insensitivity in forty-nine patients: classification based on clinical and androgen receptor phenotypes. Horm. Res., 28, 25-29.MEDLINE Abstract
35 Shule, R., Muller, M., Kaltschmidt, C. and Renkawitz, R. (1988) Many transcription factors interact synergistically with steroid receptors. Science, 242, 1418-1420.
36 Zhou, Z-x., Lane, M. V., Kemppainen, J. A., French, F. S. and Wilson, E. M. (1995) Specificity of ligand-dependent androgen receptor stabilization: receptor domain interactions influence ligand dissociation and receptor stability. Mol. Endocrinol., 9, 208-218.MEDLINE Abstract
37 Webster, N. J. G., Green, S., Tasset, D., Ponglikitmongkol, M. and Chambon, P. (1988) The transcriptional activation function located in the hormone-binding domain of the human oestrogen receptor is not encoded in a single exon. EMBO J., 8, 1441-1446.
38 Beitel, L. K., Kazemi-Esfarjani, P., Kaufman, M., Lumbroso, R., DiGeorge, A. M., Killinger, D. W., Trifiro, M. A. and Pinsky, L. (1994) Substitution of arginine-839 by cysteine or histidine in the androgen receptor cause different receptor phenotypes in cultured cells and coordinate degrees of clinical androgen resistance. J. Clin. Invest., 94, 546-554.MEDLINE Abstract
39 McPhaul, M. J., Marcelli, M., Zoppi, S., Wilson, C. M., Griffin, J. E. and Wilson, J. D. (1992) Mutations in the ligand binding domain of the androgen receptor gene cluster in two regions of the gene. J. Clin. Invest., 90, 2097-2101.MEDLINE Abstract
40 Marcelli, M., Zoppi, S., Wilson, C., Griffin, J. and McPhaul, M. (1994) Amino acid substitutions in the hormone-binding domain of the human androgen receptor alter the stability of the hormone receptor complex. J. Clin. Invest., 94, 1642-1650.MEDLINE Abstract
41 Chang, Y. T., Migeon, C. J. and Brown, T. R. (1991) Human androgen insensitivity syndrome due to androgen receptor gene point mutations in subjects with normal androgen receptor levels but impaired biological activity. In: Proceedings of the 73rd Annual Meeting of The Endocrine Society, Washington DC. pp. 37, abstract 28.
42 Lubahn, D. B., Joseph, D. R., Sar, M., Tan, J., Higgs, H. N., Larson, R. E., French, F. S. and Wilson, E. M. (1988) The human androgen receptor: complementary deoxyribonucleic acid cloning, sequence analysis and gene expression in prostate. Mol. Endocrinol., 2, 1265-1275.MEDLINE Abstract
43 Patterson, M., Hughes, I., Gottlieb, B. and Pinsky, L. (1994) The androgen receptor gene mutations database. Nucleic Acids Res., 22, 3560-3562.MEDLINE Abstract
44 Hanahan, D. (1983) Studies on transformation of Escherichia coli with plasmids. J. Mol. Biol., 166, 557-580.MEDLINE Abstract
45 Chomczynski, P. and Sacchi, N. (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem., 162, 156-159.MEDLINE Abstract
46 Higuchi, R. (1990) In Innis, M., Gelfand, D., Sninsky, J. and White, T. (eds), PCR Protocols: a guide to methods and applications. Academic Press Ltd, London. pp. 177-183.
47 Hughes, I. A. and Evans, B. A. J. (1988) The fibroblast as a model for androgen resistant states. Clin. Endocrinol., 28, 565-579.
48 Ausubel, F., Brent, R., Kingston, R., Moore, D., Seidman, J., Smith, J. and Struhl, K., (eds.) (1995) Current protocols on CD-ROM, Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., New York.
49 Sambrook, J., Fritsch, E. F. and Maniatis, T. (1989) Molecular Cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor
50 Ris-Stalpers, C., Trifiro, M. A., Kuiper, G. G. J. M., Jenster, G., Romalo, G., Sai, T., van Rooij, H. C. J., Kaufman, M., Rosenfield, R. L., Liao, S., Schweikert, H-U., Trapman, J., Pinsky, L. and Brinkmann, A. O. (1991) Substitution of aspartic acid-686 by histidine or asparagine in the human androgen receptor leads to a functionally inactive protein with altered hormone-binding characteristics. Mol. Endocrinol., 5, 1562-1569.MEDLINE Abstract
51 Mizushima, S. and Nagata, S. (1990) pEF-BOS, a powerful mammalian expression vector. Nucleic Acids Res., 18, 5322.MEDLINE Abstract
52 Seed, B. and Sheen, J-Y. (1988) A simple phase-extraction assay for chloramphenicol acyltransferase activity. Gene, 67, 271-277.MEDLINE Abstract
53 Kastner, P., Krust, A., Turcotte, B., Stropp, U., Tora, L., Gronemeyer, H. and Chambon, P. (1990) Two distinct estrogen-regulated promoters generate transcript encoding the two functionally different human progesterone receptor forms A and B. EMBO J., 9, 1603-1614.MEDLINE Abstract
54 Arriza, J. L., Weinberger, C., Cerelli, G., Glaser, T. M., Handelin, B. L., Housman, D. E. and Evans, R. M. (1987) Cloning of human mineralocorticoid receptor complementary DNA: structure and functional kinship with the glucocorticoid receptor. Science, 237, 268-275.MEDLINE Abstract
55 Hollenberg, S. M., Weinberger, C., Ong, E. S., Cerelli, G., Oro, A., Lebo, R., Thompson, E. B., Rosenfeld, M. G. and Evans, R. M. (1985) Primary structure and expression of a functional human glucocorticoid receptor cDNA. Nature, 318, 635-641.MEDLINE Abstract
56 Green, S., Walter, P., Kumar, V., Krust, A., Bornert, J. M., Argos, P. and Chambon, P. (1986) Human oestrogen receptor cDNA: sequence, expression and homology to v-erb-A. Nature, 320, 134-139.MEDLINE Abstract
57 Manglesdorf, D. J., Ong, E. S., Dyck, J. A. and Evans, R. M. (1990) Nuclear receptor that identifies a novel retinoic acid response pathway. Nature, 345, 224-229.
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