0021-972x/92/7506-1474$03.00/0 Journal of Clinical Endocrinology and Metabolism Copyright 0 1992 by The Endocrine Society
Immunoreactive Subjects with CAROL SONIA
Vol. 75, No. 6 Printed in U.S.A.
Androgen Receptor Androgen Resistance*
M. WILSON, JAMES ZOPPI, AND MICHAEL
E. GRIFFIN, J. McPHAUL
JEAN
D. WILSON,
Expression MARCO
in
MARCELLI,
Departments of Internal Medicine (C.M. W., J.E.G., J. D. W., M. M., S.Z., M. J.M.) and Pharmacology (C.M. W.), University of Texas Southwestern Medical Center, Dallas, Texas 75235-8857 ABSTRACT
els of immunoreactive AR correlated closely with androgen-binding capacity. In 15 androgen-resistant subjects with qualitatively abnormal AR, immunoreactive AR levels tended to be higher than predicted from the ligand-binding capacity. Discordance between immunoreactivity and androgen binding also occurred in fibroblasts from 3 other subjects. One carries a stop codon in the AR gene and produces a truncated AR that is immunoreactive but does not bind androgen. Two carry single point mutations in the hormone-binding domain and produce immunoreactive AR that is normal in size but does not bind androgen. (J Clin Endocrinol Metab 75: 1474-1478, 1992)
Individuals with androgen resistance encompass a spectrum of phenotypic abnormalities ranging from complete testicular feminization to undervirilized men. Such subjects have been classified according to the hormone-binding characteristics in genital skin fibroblasts and on the basis of the mutation in the androgen receptor (AR) gene. Antibodies to the amino-terminal region of the human AR were used to develop an immunoblot assay for the comparison of androgen binding with the amount of AR expressed in genital skin fibroblasts. In controls and 4 androgen-resistant subjects with DNA-binding domain mutations, lev-
T
HE HUMAN androgen receptor (AR) is expressed in fibroblasts cultured from genital skin, and demonstration of impaired ligand binding in fibroblasts is the most reliable means of diagnosing androgen resistance (1). Using such assays,androgen resistance has been characterized as associated with absent, reduced, abnormal, or no demonstrable defect in ligand binding (2). The cloning of the AR cDNA and the sequencingof many AR mutant geneshave provided additional insight into androgen resistance(reviewed in Ref. 3). In most instances, ligand binding can be predicted from the sequencing data. For example, androgen resistancewith no demonstrable defect in ligand binding is commonly due to mutation in the DNA-binding domain of AR, a defect that would not be expected to affect ligand binding or AR levels (4, 5). Likewise, complete or partial gene deletions (6-9), premature termination codons (lo-13), and aberrant processing of mRNA (14) cause the loss of ligand binding and are expected to cause a parallel loss of full-length AR. Furthermore, single amino acid substitutions that impair androgen binding (15) might result in the selective loss of ligand binding despite normal amounts of immunoreactive AR. In the caseof mutations that causequalitatively abnormal AR, the relationship of ligand binding to the amount of AR is difficult to predict. Some receptors in this category have Received December 13, 199 1. Address all correspondence and requests for reprints to: Carol M. Wilson, Ph.D., Department of Internal Medicine, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, Texas 75235-8857. * This work was supported by Grant DK-03892 from the NIH, the March of Dimes (FY92-0788), the Robert A. Welch Foundation (I-1090), and a grant from the Perot Family Foundation. Portions of this work were presented at the 73rd Annual Meeting of The Endocrine Society, Washington, D.C., June 19-22, 1991.
relatively subtle androgen binding defects, and one would expect ligand binding to correlate with AR (16). Other such subjects form inherently unstable receptor-steroid complexes, in which ligand-binding assaysmight underestimate AR levels (17). We have developed an immunoblot assay to assessthe level of AR in cultured skin fibroblasts and have compared this assaywith ligand binding in fibroblasts from 13 control subjects, 15 subjects with androgen resistance associated with qualitatively abnormal AR, and 7 subjects with other categories of androgen resistance. Materials Cell culture
and Methods
and sample preparation
Fibroblast strains were established from genital skin biopsies from control and androgen-resistant individuals, as previously described (18). For monolayer binding studies, fibroblasts were grown to confluence in 6-cm well plates in standard medium Eagle’s Minimum Essential Medium with Earle’s salts (Gibco, Grand Island, NY) containing 10% fetal calf serum and changed to standard medium containing 5 mg/mL BSA 1 day before assay. Fibroblasts for immunoblot assays were grown to confluence in standard medium containing 10% fetal calf serum in 15cm plates, rinsed three times with Tris-saline (50 rnM Tris-HCI and 150 mM NaCl, pH 7.4), and scraped into Tris-saline. Cells from lo-40 plates were centrifuged at 1000 x g and rinsed three times by resuspension in Tris-saline. The washed cell pellet (1 vol) was resuspended in 1.5 vol water and 2.5 vol l-fold concentrated sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer (200 mM dithiothreitol, 4% SDS, 20% glycerol, 0.004% bromophenol blue, and 160 rnM Tris, pH 6.9), homogenized on ice in a Dounce homogenizer (Blaessig Glass Specialties, Rochester, NY) with 10 strokes of the loose pestle and 20 strokes of the tight pestle, and centrifuged for 30 min at 200,000 x g. Aliquots of the supernatant, termed fibroblast extract, were stored in liquid nitrogen until used for immunoblot assays. Recovery was investigated in control strain 704 using repeated cycles of boiling and sonication to solubilize the unfractionated washed cell pellet and the 200,000
1474
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 12 November 2015. at 16:45 For personal use only. No other uses without permission. . All rights reserved.
IMMUNOREACTIVE
ANDROGEN
X g pellet. No immunoreactive AR was detected in the 200,000 X g pellet, and no difference was apparent between immunoblots of unfractionated cells and the 200,000 X g supernatant, indicating that essentially all of the AR is recovered in the fibroblast extract. The number of cells per plate was estimated by counting aliquots of dispersed cells from duplicate plates.
Monolayer
binding
and sucrose density centrifugation
assays
Methods for estimation of specific androgen-binding capacity, temperature sensitivitv of binding, and the dissociation rate of ligand binding have been previously described (19). In brief, fibroblast monolayers were incubated with 0.25-3.0 nM [‘Hldihydrotestosterone for 60 min at 37 C, harvested with trvpsin, and disrupted by sonication. Aliquots were assayed for radioactivity and protein (20). The thermostabilityof monolayer binding was investigated by incubation with [3H]dihydrotestosterone at 41 or 37 C in the same experiment. Samples were classified as thermolabile if 60% or less dihydrotestosterone was bound at 41 C compared to 37 C. The dissociation rate of ligand-receptor complexes was investigated by incubating prelabeled monolayers with a 500-fold excess of nonradioactive dihydrotestosterone for various times before harvesting. Dissociation rates were classified as abnormal if the percentage of baseline specific binding after 5.5 h was less than half that obtained with the control strain (704) in the same experiment. AR from one strain (450) was classified as qualitatively abnormal because the ligand-receptor complex was not stable in the density gradient centrifugation assay (21). SDS-PAGE
and immunoblot
assays
Fibroblast extracts were thawed, diluted if necessary with SDS-PAGE loading buffer, boiled 3 min, sonicated, subjected to SDS-PAGE, and transferred to nitrocellulose membrane filters, as previously described (22), except that the transfer buffer contained 0.1% SDS. For quantitative assays, electrophoresis was performed in standard running gels (7.5% acrylamide and 0.2% N,N’-methylene-bis-acrylamide) until the dye front reached the bottom of the gel. Under these conditions control AR appeared as a poorly resolved doublet [apparent mol wt (Mr), -110 kilodaltons (kDa)]. In experiments designed to enhance the resolution of bands in the lOO-kDa range, low bis running gels (7.5% acrylamide and 0.05% N,N’-methylene-bis-acrylamide) were used, and electrophoresis was continued until a 69-kDa prestained standard Mr marker (Bio-Rad Laboratories, Richmond, CA) was 0.5 cm from the bottom of the gel. Under these conditions, control AR appeared as two distinct bands (apparent Mr, -116 and -122 kDa). Immunoreactive bands were visualized by incubating the nitrocellulose filter sequentially with affinitypurified antibodies from a rabbit (U402) immunized with a synthetic peptide containing the amino-terminal 21-amino acid sequence of human AR and with iz51-labeled antirabbit immunoglobulin G F(ab’h, followed by autoradiography (22). Mr values were estimated from a standard curve obtained with Y-labeled Mr markers (Rainbow Markers, Amersham Corp., Arlington Heights, IL) run in the same gel. A comuutine densitometer (model 300A, Molecular Dvnamics, Sunnvvale, CA) Las uled to estimate-the amount of immunoreactive AR in bands of the appropriate Mr. Relative immunoreactivity per fibroblast cell was calculated by comparison with a standard curve obtained with samples of a control strain (704) run in the same gel. Characterization
of
RECEPTOR
1475 Results
Correlation
of
immunoreactive
AR with cell number
To determine the sensitivity of the immunoblot assay, we assayed various amounts of extract from the control strain 704 (Fig. 1). AR was detected in immunoblots as a doublet of bands with an apparent Mr of about 110 kDa, consistent with previous reports of AR size (22, 24), and the amount of AR correlated with the number of cells between 6 X lo4 and 1 X lo6 fibroblasts (13-205 pg protein). The findings were independent of the total amount of protein applied, as similar results were obtained when the samples were diluted with SDS-PAGE loading buffer or when the amount of protein applied was kept constant by diluting with extracts from fibroblasts that contain no immunoreactive AR (data not shown). The average amount of dihydrotestosterone bound by strain 704 was 36 + 5 fmol/mg protein (*SD; n = 25; range, 26-44). These data indicate that immunoreactive AR should be detected in lo6 fibroblasts that encompassthe detectable range of ligand binding (4-70 fmol/mg protein). Level of immunoreactive AR is concordant androgen-binding sites in controls
with the number
of
Genital skin fibroblasts from 13 control subjects were assayedfor specific dihydrotestosterone binding and immunoreactive AR (Fig. 2). In strains in which the dihydrotestosterone-binding capacity varied from lo-47 fmol/mg protein, levels of immunoreactive AR correlated positively with ligand-binding capacity (1:= 0.81). As anticipated, levels of immunoreactivity and hormone-binding capacity were also normal in four androgen-resistant subjects known to carry point mutations in the DNA-binding domain of AR (4, 5) iFig. 2).
r= 0.99
AR mutations
The AR genes of subjects N717, N69, and N909 were amplified by polymerase chain reaction, using genomic DNA derived from cultured genital skin fibroblasts, as previously described (10). The amplified fragments were subcloned into the sequencing vector Ml3 and sequenced. A single base substitution in exon 7 was found at position 2714 (C + G) for N717. This point mutation results in the conversion of the triplet encoding serine 851 (TCA) to a premature termination codon (TGA). Single base substitutions were found in exon 5 at position 2377 (T + C) for N69 (23) and in exon 8 at position 2657 (A -+ G) for N909, resulting in single amino acid substitutions within the AR hormone-binding domain: Trp739 + Arg for N69 and Tyrs3* + Cys for N909.
Cells
(x 105)
FIG. 1. Correlation of normal immunoreactive AR protein and cell number in extracts from genital skin fibroblasts. Samples containing normal AR (strain 704) extracted from varying numbers of fibroblasts were fractionated by electrophoresis on 7.5% SDS-polyacrylamide gels under reducing conditions and transferred to nitrocellulose filters. Normal llO-kDa AR bands recognized by antibodies to the aminoterminal region of the human AR were labeled with [iz51]antirabbit immunoglobulin G F(ab’h!, visualized by autoradiography, and quantified by densitometry, as described in the text. The graph presents data obtained from the immunoblot shown in the inset.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 12 November 2015. at 16:45 For personal use only. No other uses without permission. . All rights reserved.
WILSON
1476
JCE & M. 1992 Vol75.No6
ET AL.
1.5
NO’
.
4
v
10
’
0
00
I
20 30 Dihydrotestosterone (DHT) Binding (fmollmg protein)
_I
40 Capacity
50
FIG. 2. Relation between immunoreactive AR protein and androgenbinding capacity in fibroblasts from control subjects. The level of immunoreactive AR per cell for each strain is expressed relative to values obtained in the same immunoblot with a control stain (704), as described in Fig. 1, and is plotted as a function of the androgen-binding capacity, as described in the text. The average androgen-binding capacity of 704 fibroblast extracts (star) and the range of values (bracketed line) observed with this strain in 25 experiments is indicated. Twelve other controls are represented on the graph by open circles. Data from these controls were used to determine the regression line (dashed line) and coefficient by least squares. Open squares represent four androgen-resistant subjects with mutations in the DNA-binding domain of the AR. The inset contains an immunoblot illustrating the relationship between varying amounts of 704 and fibroblasts from controls with relatively low and high levels of androgen-binding capacity.
Androgen-binding capacity and immunoreactive AR protein in fibroblasts from patients with qualitatively abnormal receptors
Androgen-binding capacity and immunoreactive AR levels were also compared in 15 subjects from 12 families with qualitatively abnormal AR. Receptor binding in fibroblasts from these subjects ranged from 7-46 fmol/mg protein and was thermolabile and/or exhibited an increased rate of ligand dissociation. The correlation of immunoreactivity with ligand binding in these sampleswas also positive, but not as strong as that in normal samples(r = 0.75; Fig. 3). Several of these individuals had more immunoreactive AR than predicted from ligand-binding capacity (Fig. 2). Immunoreactive ligand binding
AR in androgen-resistant
subjects with absent
In the course of these studies immunoblotting was performed with samples from several androgen-resistant subjects with no detectable ligand binding (the receptor-negative category; Table 1). Fibroblasts from subjects N69 and N909 contained reduced levels of immunoreactive llO-kDa AR [72% and 34% compared to control (704) fibroblasts]. Single amino acid substitutions that impair androgen binding would be expected to cause such a selective loss of ligand binding. As predicted, nucleotide analysis of the AR genes carried by these subjects revealed single point mutations located at position 2377 (T-K) for N69 and position 2657 (A-+G) for N909, resulting in amino acid substitutions (Trp739---, Arg
p/i
,
4
10
I
20 Dihydrotestosterone (fmol/mg
I
I
I
30 Binding protein)
40
50
Capacity
FIG. 3. Relation between immunoreactive AR and androgen-binding capacity in fibroblasts from subjects with qualitatively abnormal AR. Androgen-binding capacity and relative levels of immunoreactive AR were determined as described in Fig. 2. The dashed line is the control regression line from Fig. 2. Three subjects (H) had the phenotype of incomplete testicular feminization. Twelve subjects (0) had the Reifenstein syndrome or were undervirilized fertile males.
TABLE subjects AR
1. Immunoreactive AR levels in fibroblasts from three with androgen resistance, absent ligand binding, and mutant
Patient
ID no.
N717 N69 N909
Immunoreactive (% of normal control)
11 77 38
AR Mutation
CyP --) stop TrpTa9 -+ Arg TyP + cys
Immunoreactivity was detected in immunoblots, as described in the text, quantified by densitometry, and expressed relative to the immunoreactivity of a control sample (704) representing the same number of cells run in the same electrophoresis gel. Mutations in AR were deduced from the results of nucleotide analysis of the AR gene, as described in the text.
and TyrB3*+ Cys, respectively) in the AR hormone-binding domain. In contrast, N717 fibroblasts contained lower levels (11% of control value) of immunoreactive AR, which appeared to migrate more rapidly than normal in standard 7.5% SDSpolyacrylamide gels, suggesting truncation at the carboxylterminus (data not shown). Under conditions designed to improve the separation of proteins in the lOO-kDa range, the difference in mobility was enhanced (Fig. 4), and two major AR bands were evident with each strain (apparent Mr, -116 and -122 kDa for 704, -110 kDa and -114 kDa for N717). In keeping with these results, N717 was found to carry a premature termination codon (TCA + TGA) in exon 7 of the AR gene at the position normally encoding amino acid 851. Expression of this mutant gene would result in a truncated AR protein approximately 93% the size of normal AR, as was observed. The presencein both 704 and N717 fibroblasts of doublet immunoreactive bands with the predicted differencesin Mr suggeststhat both normal and truncated protein may undergo the same posttranslational modification. Both
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 12 November 2015. at 16:45 For personal use only. No other uses without permission. . All rights reserved.
IMMUNOREACTIVE
ANDROGEN
kDa
- 69 FIG. 4. The truncated AR protein for subject N717 is detected by the immunoblot assay. Fibroblast extracts representing 2.5 X 10’cellsfrom a control subject(704) or 9 x lo6 cells from an androgen-resistant subject(N717)carrying a stop codonat the positionencodingamino acid 851wereseparatedby SDS-PAGE,using7.5%acrylamideand 0.05%N,N’-methylene-bis-acrylamide in the running gel. Immunoblotswerevisualizedasdescribedin the text. Numberson the right indicatethe positionof standardMr markers(MW) run in the center lane.
samplesalsoproduced a second set of immunoreactive bands about 35-40 kDa smaller. The origin of these minor bands is not known, but the facts that the N717 bands also move faster than normal and are recognized by anti-N-terminal antibodies suggestthat they may represent proteolytic cleavage fragments.
RECEPTOR
1477
in the DNA-binding domain, the quantity of AR correlated with the amount of specific dihydrotestosterone binding. In subjects with androgen resistanceassociatedwith qualitatively abnormal AR, levels of dihydrotestosterone binding also correlated positively with immunoreactive AR, but in several instances the amount of immunoreactive AR was greater than predicted from binding assays.This phenomenon is probably due to mutations in which the stability of hormone binding is impaired more severely than the level of immunoreactive AR expression. In one androgen-resistant subject (N717), truncated AR protein was shown to be the consequence of a premature termination codon. In two subjects (N69 and N909), single point mutations decreased hormone binding and immunoreactive AR without affecting AR size, suggestingthat mutant AR mRNA or protein produced by these subjectsis lessstable than normal. In summary, we employed an immunoblot assay to quantitate the level of immunoreactive AR in unfractionated extracts of cultured skin fibroblasts. These data were used to compare AR levels with ligand-binding capacity in individual patients and to identify a subset of patients in whom hormone binding and AR levels are discordant. This technique will facilitate the characterization of selected AR mutants and allow direct assessmentof the effects that mutations have on the level of AR expression in mutant cell strains. Acknowledgments Experttechnicalassistance wasprovidedby DianeAllmanandElsa Yang.
References
2.
JE, Wilson JD, 1989 The androgenresistance syndromes: 5ol-reductase deficiency,testicularfeminization,and relatedsyndromes.In: Striver CR, BeaudetAL, Sly WS, Valle D, eds.The metabolicbasisof inheriteddisease, 6th ed. New York: McGrawHill; pp 1919-1944. Wilson JD. 1992 Syndromes of androgenresistance. Biol Reprod.
3.
McPhaul MJ, Marcelli
1.
Griffin
46:168-173.
Discussion The syndromes of androgen resistance were initially categorized basedon qualitative and quantitative assessmentsof androgen binding in cultured genital skin fibroblasts (1). The development of methods for the structural analysis of the AR gene has permitted the characterization of mutations responsible for defective AR function (reviewed in Ref. 3). Most studies have focused on mutations causing profound androgen resistance and assessmentof mutant AR function using assays of hormone binding or transcriptional activation While such in vitro studies are informative, the results cannot be related directly to the level of endogenous AR expressedin cells. We have developed an immunoblot assay to quantitate immunoreactive AR and have examined the relation between androgen-binding capacity and AR levels in fibroblasts. The technique is sufficiently sensitive to allow estimation of immunoreactive AR in skin fibroblasts over a wide range. In normal subjects and subjects with mutations
M, Tilley WD, Griffin JE, Wilson JD. 1991
Androgen resistance caused gene. FASEB J. 5:2910-2915. 4.
complete androgen
receptor
testicular feminization in a patient resistance. J Clin Invest. 87:1123-
Zoppi S, Marcelli M, Deslypere J-P, Griffin JE, Wilson JD, McPhaul MJ. 1992 Amino acid substitutions in the DNA-binding domain of the human androgen receptor receptor-positive androgen resistance. Mol
6.
in the androgen
Marcelli M, Zoppi S, Grino PB, Griffin JE, Wilson JD, McPhaul MJ. 1991 A mutation in the DNA-binding domain of the androgen receptor gene causes with receptor-positive 1126.
5.
by mutations
are a frequent cause of Endocrinol. 6:409-415.
Brown TR, Lubahn DB, Wilson EM, Joseph DR, French FS, Migeon CJ. 1988 Deletion of the steroid-binding domain of the human androgen receptor in one family with complete androgen insensitivity s&drom& evidence for further genetic heterogeneity in this svndrome. Proc Nat1 Acad Sci USA. 85:8157-8155.
7.
McClean HE, Warne GL, French FS, Lubahn DB, Wilson EM, Zajac JD. Identification of a deletion in the androgen receptor gene in two Annual
siblings with androgen Meet of the Endocrine
insensitivity syndrome. Proc of the Sot of Australia. 1990; 33:l. M, Gottlieb 8, Pinsky L, et al. 1991 The 56/58 kDa 8. Trifiro androgen-binding protein in male genital skin fibroblasts with a
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 12 November 2015. at 16:45 For personal use only. No other uses without permission. . All rights reserved.
1478
WILSON
deleted androgen receptor gene. Mol Cell Endocrinol. 75:37-47. 9. Quigley CA, Friedman KJ, Johnson A, et al. 1992 Complete deletion of the androgen receptor gene: definition of the null phenotype of the androgen insensitivity syndrome and determination of carrier status. J Clin Endocrinol Metab. 74:927-933. 10. Marcelli M, Tilley WD, Wilson CM, Griffin JE, Wilson JD, McPhaul MJ. 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. 11. Marcelli M, Tilley WD, Wilson CM, Wilson JD, Griffin JE, McPhaul MJ. 1990 A single nucleotide substitution introduces a premature termination codon into the androgen receptor gene of a patient with receptor-negative androgen resistance. J Clin Invest. 85:1522-1528. 12. Sai T, Seino S, Chang C, et al. 1990 An exonic point mutation of the androgen receptor gene in a family with complete androgen insensitivity. Am J Hum Genet. 46:1095-1100. 13. Trifiro M, Prior RL, Sabbaghian N, et al. 1991 Amber mutation creates a diagnostic Mae1 site in the androgen receptor gene of a family with complete androgen insensitivity. Am J Med Genet. 40:493-499. 14. Ris-Stalpers C, Kuiper GGJM, Faber PW, et al. 1990 Aberrant splicing of androgen receptor mRNA results in the synthesis of a nonfunctional receptor protein in a patient with androgen insensitivity. Proc Nat1 Acad Sci USA. 87:7866-7870. 15. Brown TR, Lubahn DB, Wilson EM, French FS, Migeon CJ, Corden JL. 1990 Functional characterization of naturally occurring mutant androgen receptors from patients with complete androgen insensitivity. Mol Endocrinol. 4:1759-1772. 16. Guerami A, Griffin JE, Kovacs WJ, Grino PB, MacDonald PC, Wilson JD. 1990 Estrogen and androgen production rates in two
ET
17.
18.
19.
20.
21.
22.
23.
24.
.-
JCE & M. 1992 Vol75.No6
brothers with Reifenstein syndrome. J Clin Endocrinol Metab. 71:247-251. Marcelli M, Tilley WD, Zoppi S, Griffin JE, Wilson JD, McPhaul MJ. 1991 Androgen resistance associated with a mutation of the androgen receptor at amino acid 772 (Arg-+Cys) results from a combination of decreased messenger ribonucleic acid levels and impairment of receptor function. J Clin Endocrinol Metab. 73:318325. Griffin JE, Punyashthiti K, Wilson JD. 1976 Dihydrotestosterone binding by cultured human fibroblasts: comparison of cells from control subjects with hereditary male pseudohermaphroditism due to androgen resistance. J Clin Invest. 57:1342-1351. Grino PB, Isidro-Gutierrez RF, Griffin JE, Wilson JD. 1989 Androgen resistance associated with a qualitative abnormality of the androgen receptor and responsive to high dose androgen therapy. J Clin Endocrinol Metab. 68:578-585. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. 1951 Protein measurement with the Folin phenol reagent. J Biol Chem. 193:265275. Griffin JE, Durrant JL. 1982 Qualitative receptor defects in families with androgen resistance: failure of stabilization of the fibroblast cytosol androgen receptor. J Clin Endocrinol Metab. 55:465-474. Husmann DA, Wilson CM, McPhaul MJ, Tilley WD, Wilson JD. 1990 Antipeptide antibodies to two distinct regions of the androgen receptor localize the receptor protein to the nuclei of target cells in the rat and human prostate. Endocrinology. 126:2359-2368. Marcelli M, Zoppi S, Wilson CM, Griffin JE, Wilson JD, McPhaul MJ. Amino acid substitution in a small segment of exon 5 of the human androgen receptor (AR) gene cause complete testicular feminization (CTF) by different mechanisms. Proc of the 74th Annual Meet of The Endocrine Sot. 1992;107. van Laar JH, Bolt-deVries J, Voorhorst-Ogink MM, Brinkmann AO. 1989 The human androgen receptor is a 110 Kd protein. Mol Cell Endocrinol. 63:39-44.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 12 November 2015. at 16:45 For personal use only. No other uses without permission. . All rights reserved.
Immunoreactive Subjects with CAROL SONIA
Vol. 75, No. 6 Printed in U.S.A.
Androgen Receptor Androgen Resistance*
M. WILSON, JAMES ZOPPI, AND MICHAEL
E. GRIFFIN, J. McPHAUL
JEAN
D. WILSON,
Expression MARCO
in
MARCELLI,
Departments of Internal Medicine (C.M. W., J.E.G., J. D. W., M. M., S.Z., M. J.M.) and Pharmacology (C.M. W.), University of Texas Southwestern Medical Center, Dallas, Texas 75235-8857 ABSTRACT
els of immunoreactive AR correlated closely with androgen-binding capacity. In 15 androgen-resistant subjects with qualitatively abnormal AR, immunoreactive AR levels tended to be higher than predicted from the ligand-binding capacity. Discordance between immunoreactivity and androgen binding also occurred in fibroblasts from 3 other subjects. One carries a stop codon in the AR gene and produces a truncated AR that is immunoreactive but does not bind androgen. Two carry single point mutations in the hormone-binding domain and produce immunoreactive AR that is normal in size but does not bind androgen. (J Clin Endocrinol Metab 75: 1474-1478, 1992)
Individuals with androgen resistance encompass a spectrum of phenotypic abnormalities ranging from complete testicular feminization to undervirilized men. Such subjects have been classified according to the hormone-binding characteristics in genital skin fibroblasts and on the basis of the mutation in the androgen receptor (AR) gene. Antibodies to the amino-terminal region of the human AR were used to develop an immunoblot assay for the comparison of androgen binding with the amount of AR expressed in genital skin fibroblasts. In controls and 4 androgen-resistant subjects with DNA-binding domain mutations, lev-
T
HE HUMAN androgen receptor (AR) is expressed in fibroblasts cultured from genital skin, and demonstration of impaired ligand binding in fibroblasts is the most reliable means of diagnosing androgen resistance (1). Using such assays,androgen resistance has been characterized as associated with absent, reduced, abnormal, or no demonstrable defect in ligand binding (2). The cloning of the AR cDNA and the sequencingof many AR mutant geneshave provided additional insight into androgen resistance(reviewed in Ref. 3). In most instances, ligand binding can be predicted from the sequencing data. For example, androgen resistancewith no demonstrable defect in ligand binding is commonly due to mutation in the DNA-binding domain of AR, a defect that would not be expected to affect ligand binding or AR levels (4, 5). Likewise, complete or partial gene deletions (6-9), premature termination codons (lo-13), and aberrant processing of mRNA (14) cause the loss of ligand binding and are expected to cause a parallel loss of full-length AR. Furthermore, single amino acid substitutions that impair androgen binding (15) might result in the selective loss of ligand binding despite normal amounts of immunoreactive AR. In the caseof mutations that causequalitatively abnormal AR, the relationship of ligand binding to the amount of AR is difficult to predict. Some receptors in this category have Received December 13, 199 1. Address all correspondence and requests for reprints to: Carol M. Wilson, Ph.D., Department of Internal Medicine, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, Texas 75235-8857. * This work was supported by Grant DK-03892 from the NIH, the March of Dimes (FY92-0788), the Robert A. Welch Foundation (I-1090), and a grant from the Perot Family Foundation. Portions of this work were presented at the 73rd Annual Meeting of The Endocrine Society, Washington, D.C., June 19-22, 1991.
relatively subtle androgen binding defects, and one would expect ligand binding to correlate with AR (16). Other such subjects form inherently unstable receptor-steroid complexes, in which ligand-binding assaysmight underestimate AR levels (17). We have developed an immunoblot assay to assessthe level of AR in cultured skin fibroblasts and have compared this assaywith ligand binding in fibroblasts from 13 control subjects, 15 subjects with androgen resistance associated with qualitatively abnormal AR, and 7 subjects with other categories of androgen resistance. Materials Cell culture
and Methods
and sample preparation
Fibroblast strains were established from genital skin biopsies from control and androgen-resistant individuals, as previously described (18). For monolayer binding studies, fibroblasts were grown to confluence in 6-cm well plates in standard medium Eagle’s Minimum Essential Medium with Earle’s salts (Gibco, Grand Island, NY) containing 10% fetal calf serum and changed to standard medium containing 5 mg/mL BSA 1 day before assay. Fibroblasts for immunoblot assays were grown to confluence in standard medium containing 10% fetal calf serum in 15cm plates, rinsed three times with Tris-saline (50 rnM Tris-HCI and 150 mM NaCl, pH 7.4), and scraped into Tris-saline. Cells from lo-40 plates were centrifuged at 1000 x g and rinsed three times by resuspension in Tris-saline. The washed cell pellet (1 vol) was resuspended in 1.5 vol water and 2.5 vol l-fold concentrated sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer (200 mM dithiothreitol, 4% SDS, 20% glycerol, 0.004% bromophenol blue, and 160 rnM Tris, pH 6.9), homogenized on ice in a Dounce homogenizer (Blaessig Glass Specialties, Rochester, NY) with 10 strokes of the loose pestle and 20 strokes of the tight pestle, and centrifuged for 30 min at 200,000 x g. Aliquots of the supernatant, termed fibroblast extract, were stored in liquid nitrogen until used for immunoblot assays. Recovery was investigated in control strain 704 using repeated cycles of boiling and sonication to solubilize the unfractionated washed cell pellet and the 200,000
1474
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 12 November 2015. at 16:45 For personal use only. No other uses without permission. . All rights reserved.
IMMUNOREACTIVE
ANDROGEN
X g pellet. No immunoreactive AR was detected in the 200,000 X g pellet, and no difference was apparent between immunoblots of unfractionated cells and the 200,000 X g supernatant, indicating that essentially all of the AR is recovered in the fibroblast extract. The number of cells per plate was estimated by counting aliquots of dispersed cells from duplicate plates.
Monolayer
binding
and sucrose density centrifugation
assays
Methods for estimation of specific androgen-binding capacity, temperature sensitivitv of binding, and the dissociation rate of ligand binding have been previously described (19). In brief, fibroblast monolayers were incubated with 0.25-3.0 nM [‘Hldihydrotestosterone for 60 min at 37 C, harvested with trvpsin, and disrupted by sonication. Aliquots were assayed for radioactivity and protein (20). The thermostabilityof monolayer binding was investigated by incubation with [3H]dihydrotestosterone at 41 or 37 C in the same experiment. Samples were classified as thermolabile if 60% or less dihydrotestosterone was bound at 41 C compared to 37 C. The dissociation rate of ligand-receptor complexes was investigated by incubating prelabeled monolayers with a 500-fold excess of nonradioactive dihydrotestosterone for various times before harvesting. Dissociation rates were classified as abnormal if the percentage of baseline specific binding after 5.5 h was less than half that obtained with the control strain (704) in the same experiment. AR from one strain (450) was classified as qualitatively abnormal because the ligand-receptor complex was not stable in the density gradient centrifugation assay (21). SDS-PAGE
and immunoblot
assays
Fibroblast extracts were thawed, diluted if necessary with SDS-PAGE loading buffer, boiled 3 min, sonicated, subjected to SDS-PAGE, and transferred to nitrocellulose membrane filters, as previously described (22), except that the transfer buffer contained 0.1% SDS. For quantitative assays, electrophoresis was performed in standard running gels (7.5% acrylamide and 0.2% N,N’-methylene-bis-acrylamide) until the dye front reached the bottom of the gel. Under these conditions control AR appeared as a poorly resolved doublet [apparent mol wt (Mr), -110 kilodaltons (kDa)]. In experiments designed to enhance the resolution of bands in the lOO-kDa range, low bis running gels (7.5% acrylamide and 0.05% N,N’-methylene-bis-acrylamide) were used, and electrophoresis was continued until a 69-kDa prestained standard Mr marker (Bio-Rad Laboratories, Richmond, CA) was 0.5 cm from the bottom of the gel. Under these conditions, control AR appeared as two distinct bands (apparent Mr, -116 and -122 kDa). Immunoreactive bands were visualized by incubating the nitrocellulose filter sequentially with affinitypurified antibodies from a rabbit (U402) immunized with a synthetic peptide containing the amino-terminal 21-amino acid sequence of human AR and with iz51-labeled antirabbit immunoglobulin G F(ab’h, followed by autoradiography (22). Mr values were estimated from a standard curve obtained with Y-labeled Mr markers (Rainbow Markers, Amersham Corp., Arlington Heights, IL) run in the same gel. A comuutine densitometer (model 300A, Molecular Dvnamics, Sunnvvale, CA) Las uled to estimate-the amount of immunoreactive AR in bands of the appropriate Mr. Relative immunoreactivity per fibroblast cell was calculated by comparison with a standard curve obtained with samples of a control strain (704) run in the same gel. Characterization
of
RECEPTOR
1475 Results
Correlation
of
immunoreactive
AR with cell number
To determine the sensitivity of the immunoblot assay, we assayed various amounts of extract from the control strain 704 (Fig. 1). AR was detected in immunoblots as a doublet of bands with an apparent Mr of about 110 kDa, consistent with previous reports of AR size (22, 24), and the amount of AR correlated with the number of cells between 6 X lo4 and 1 X lo6 fibroblasts (13-205 pg protein). The findings were independent of the total amount of protein applied, as similar results were obtained when the samples were diluted with SDS-PAGE loading buffer or when the amount of protein applied was kept constant by diluting with extracts from fibroblasts that contain no immunoreactive AR (data not shown). The average amount of dihydrotestosterone bound by strain 704 was 36 + 5 fmol/mg protein (*SD; n = 25; range, 26-44). These data indicate that immunoreactive AR should be detected in lo6 fibroblasts that encompassthe detectable range of ligand binding (4-70 fmol/mg protein). Level of immunoreactive AR is concordant androgen-binding sites in controls
with the number
of
Genital skin fibroblasts from 13 control subjects were assayedfor specific dihydrotestosterone binding and immunoreactive AR (Fig. 2). In strains in which the dihydrotestosterone-binding capacity varied from lo-47 fmol/mg protein, levels of immunoreactive AR correlated positively with ligand-binding capacity (1:= 0.81). As anticipated, levels of immunoreactivity and hormone-binding capacity were also normal in four androgen-resistant subjects known to carry point mutations in the DNA-binding domain of AR (4, 5) iFig. 2).
r= 0.99
AR mutations
The AR genes of subjects N717, N69, and N909 were amplified by polymerase chain reaction, using genomic DNA derived from cultured genital skin fibroblasts, as previously described (10). The amplified fragments were subcloned into the sequencing vector Ml3 and sequenced. A single base substitution in exon 7 was found at position 2714 (C + G) for N717. This point mutation results in the conversion of the triplet encoding serine 851 (TCA) to a premature termination codon (TGA). Single base substitutions were found in exon 5 at position 2377 (T + C) for N69 (23) and in exon 8 at position 2657 (A -+ G) for N909, resulting in single amino acid substitutions within the AR hormone-binding domain: Trp739 + Arg for N69 and Tyrs3* + Cys for N909.
Cells
(x 105)
FIG. 1. Correlation of normal immunoreactive AR protein and cell number in extracts from genital skin fibroblasts. Samples containing normal AR (strain 704) extracted from varying numbers of fibroblasts were fractionated by electrophoresis on 7.5% SDS-polyacrylamide gels under reducing conditions and transferred to nitrocellulose filters. Normal llO-kDa AR bands recognized by antibodies to the aminoterminal region of the human AR were labeled with [iz51]antirabbit immunoglobulin G F(ab’h!, visualized by autoradiography, and quantified by densitometry, as described in the text. The graph presents data obtained from the immunoblot shown in the inset.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 12 November 2015. at 16:45 For personal use only. No other uses without permission. . All rights reserved.
WILSON
1476
JCE & M. 1992 Vol75.No6
ET AL.
1.5
NO’
.
4
v
10
’
0
00
I
20 30 Dihydrotestosterone (DHT) Binding (fmollmg protein)
_I
40 Capacity
50
FIG. 2. Relation between immunoreactive AR protein and androgenbinding capacity in fibroblasts from control subjects. The level of immunoreactive AR per cell for each strain is expressed relative to values obtained in the same immunoblot with a control stain (704), as described in Fig. 1, and is plotted as a function of the androgen-binding capacity, as described in the text. The average androgen-binding capacity of 704 fibroblast extracts (star) and the range of values (bracketed line) observed with this strain in 25 experiments is indicated. Twelve other controls are represented on the graph by open circles. Data from these controls were used to determine the regression line (dashed line) and coefficient by least squares. Open squares represent four androgen-resistant subjects with mutations in the DNA-binding domain of the AR. The inset contains an immunoblot illustrating the relationship between varying amounts of 704 and fibroblasts from controls with relatively low and high levels of androgen-binding capacity.
Androgen-binding capacity and immunoreactive AR protein in fibroblasts from patients with qualitatively abnormal receptors
Androgen-binding capacity and immunoreactive AR levels were also compared in 15 subjects from 12 families with qualitatively abnormal AR. Receptor binding in fibroblasts from these subjects ranged from 7-46 fmol/mg protein and was thermolabile and/or exhibited an increased rate of ligand dissociation. The correlation of immunoreactivity with ligand binding in these sampleswas also positive, but not as strong as that in normal samples(r = 0.75; Fig. 3). Several of these individuals had more immunoreactive AR than predicted from ligand-binding capacity (Fig. 2). Immunoreactive ligand binding
AR in androgen-resistant
subjects with absent
In the course of these studies immunoblotting was performed with samples from several androgen-resistant subjects with no detectable ligand binding (the receptor-negative category; Table 1). Fibroblasts from subjects N69 and N909 contained reduced levels of immunoreactive llO-kDa AR [72% and 34% compared to control (704) fibroblasts]. Single amino acid substitutions that impair androgen binding would be expected to cause such a selective loss of ligand binding. As predicted, nucleotide analysis of the AR genes carried by these subjects revealed single point mutations located at position 2377 (T-K) for N69 and position 2657 (A-+G) for N909, resulting in amino acid substitutions (Trp739---, Arg
p/i
,
4
10
I
20 Dihydrotestosterone (fmol/mg
I
I
I
30 Binding protein)
40
50
Capacity
FIG. 3. Relation between immunoreactive AR and androgen-binding capacity in fibroblasts from subjects with qualitatively abnormal AR. Androgen-binding capacity and relative levels of immunoreactive AR were determined as described in Fig. 2. The dashed line is the control regression line from Fig. 2. Three subjects (H) had the phenotype of incomplete testicular feminization. Twelve subjects (0) had the Reifenstein syndrome or were undervirilized fertile males.
TABLE subjects AR
1. Immunoreactive AR levels in fibroblasts from three with androgen resistance, absent ligand binding, and mutant
Patient
ID no.
N717 N69 N909
Immunoreactive (% of normal control)
11 77 38
AR Mutation
CyP --) stop TrpTa9 -+ Arg TyP + cys
Immunoreactivity was detected in immunoblots, as described in the text, quantified by densitometry, and expressed relative to the immunoreactivity of a control sample (704) representing the same number of cells run in the same electrophoresis gel. Mutations in AR were deduced from the results of nucleotide analysis of the AR gene, as described in the text.
and TyrB3*+ Cys, respectively) in the AR hormone-binding domain. In contrast, N717 fibroblasts contained lower levels (11% of control value) of immunoreactive AR, which appeared to migrate more rapidly than normal in standard 7.5% SDSpolyacrylamide gels, suggesting truncation at the carboxylterminus (data not shown). Under conditions designed to improve the separation of proteins in the lOO-kDa range, the difference in mobility was enhanced (Fig. 4), and two major AR bands were evident with each strain (apparent Mr, -116 and -122 kDa for 704, -110 kDa and -114 kDa for N717). In keeping with these results, N717 was found to carry a premature termination codon (TCA + TGA) in exon 7 of the AR gene at the position normally encoding amino acid 851. Expression of this mutant gene would result in a truncated AR protein approximately 93% the size of normal AR, as was observed. The presencein both 704 and N717 fibroblasts of doublet immunoreactive bands with the predicted differencesin Mr suggeststhat both normal and truncated protein may undergo the same posttranslational modification. Both
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 12 November 2015. at 16:45 For personal use only. No other uses without permission. . All rights reserved.
IMMUNOREACTIVE
ANDROGEN
kDa
- 69 FIG. 4. The truncated AR protein for subject N717 is detected by the immunoblot assay. Fibroblast extracts representing 2.5 X 10’cellsfrom a control subject(704) or 9 x lo6 cells from an androgen-resistant subject(N717)carrying a stop codonat the positionencodingamino acid 851wereseparatedby SDS-PAGE,using7.5%acrylamideand 0.05%N,N’-methylene-bis-acrylamide in the running gel. Immunoblotswerevisualizedasdescribedin the text. Numberson the right indicatethe positionof standardMr markers(MW) run in the center lane.
samplesalsoproduced a second set of immunoreactive bands about 35-40 kDa smaller. The origin of these minor bands is not known, but the facts that the N717 bands also move faster than normal and are recognized by anti-N-terminal antibodies suggestthat they may represent proteolytic cleavage fragments.
RECEPTOR
1477
in the DNA-binding domain, the quantity of AR correlated with the amount of specific dihydrotestosterone binding. In subjects with androgen resistanceassociatedwith qualitatively abnormal AR, levels of dihydrotestosterone binding also correlated positively with immunoreactive AR, but in several instances the amount of immunoreactive AR was greater than predicted from binding assays.This phenomenon is probably due to mutations in which the stability of hormone binding is impaired more severely than the level of immunoreactive AR expression. In one androgen-resistant subject (N717), truncated AR protein was shown to be the consequence of a premature termination codon. In two subjects (N69 and N909), single point mutations decreased hormone binding and immunoreactive AR without affecting AR size, suggestingthat mutant AR mRNA or protein produced by these subjectsis lessstable than normal. In summary, we employed an immunoblot assay to quantitate the level of immunoreactive AR in unfractionated extracts of cultured skin fibroblasts. These data were used to compare AR levels with ligand-binding capacity in individual patients and to identify a subset of patients in whom hormone binding and AR levels are discordant. This technique will facilitate the characterization of selected AR mutants and allow direct assessmentof the effects that mutations have on the level of AR expression in mutant cell strains. Acknowledgments Experttechnicalassistance wasprovidedby DianeAllmanandElsa Yang.
References
2.
JE, Wilson JD, 1989 The androgenresistance syndromes: 5ol-reductase deficiency,testicularfeminization,and relatedsyndromes.In: Striver CR, BeaudetAL, Sly WS, Valle D, eds.The metabolicbasisof inheriteddisease, 6th ed. New York: McGrawHill; pp 1919-1944. Wilson JD. 1992 Syndromes of androgenresistance. Biol Reprod.
3.
McPhaul MJ, Marcelli
1.
Griffin
46:168-173.
Discussion The syndromes of androgen resistance were initially categorized basedon qualitative and quantitative assessmentsof androgen binding in cultured genital skin fibroblasts (1). The development of methods for the structural analysis of the AR gene has permitted the characterization of mutations responsible for defective AR function (reviewed in Ref. 3). Most studies have focused on mutations causing profound androgen resistance and assessmentof mutant AR function using assays of hormone binding or transcriptional activation While such in vitro studies are informative, the results cannot be related directly to the level of endogenous AR expressedin cells. We have developed an immunoblot assay to quantitate immunoreactive AR and have examined the relation between androgen-binding capacity and AR levels in fibroblasts. The technique is sufficiently sensitive to allow estimation of immunoreactive AR in skin fibroblasts over a wide range. In normal subjects and subjects with mutations
M, Tilley WD, Griffin JE, Wilson JD. 1991
Androgen resistance caused gene. FASEB J. 5:2910-2915. 4.
complete androgen
receptor
testicular feminization in a patient resistance. J Clin Invest. 87:1123-
Zoppi S, Marcelli M, Deslypere J-P, Griffin JE, Wilson JD, McPhaul MJ. 1992 Amino acid substitutions in the DNA-binding domain of the human androgen receptor receptor-positive androgen resistance. Mol
6.
in the androgen
Marcelli M, Zoppi S, Grino PB, Griffin JE, Wilson JD, McPhaul MJ. 1991 A mutation in the DNA-binding domain of the androgen receptor gene causes with receptor-positive 1126.
5.
by mutations
are a frequent cause of Endocrinol. 6:409-415.
Brown TR, Lubahn DB, Wilson EM, Joseph DR, French FS, Migeon CJ. 1988 Deletion of the steroid-binding domain of the human androgen receptor in one family with complete androgen insensitivity s&drom& evidence for further genetic heterogeneity in this svndrome. Proc Nat1 Acad Sci USA. 85:8157-8155.
7.
McClean HE, Warne GL, French FS, Lubahn DB, Wilson EM, Zajac JD. Identification of a deletion in the androgen receptor gene in two Annual
siblings with androgen Meet of the Endocrine
insensitivity syndrome. Proc of the Sot of Australia. 1990; 33:l. M, Gottlieb 8, Pinsky L, et al. 1991 The 56/58 kDa 8. Trifiro androgen-binding protein in male genital skin fibroblasts with a
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 12 November 2015. at 16:45 For personal use only. No other uses without permission. . All rights reserved.
1478
WILSON
deleted androgen receptor gene. Mol Cell Endocrinol. 75:37-47. 9. Quigley CA, Friedman KJ, Johnson A, et al. 1992 Complete deletion of the androgen receptor gene: definition of the null phenotype of the androgen insensitivity syndrome and determination of carrier status. J Clin Endocrinol Metab. 74:927-933. 10. Marcelli M, Tilley WD, Wilson CM, Griffin JE, Wilson JD, McPhaul MJ. 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. 11. Marcelli M, Tilley WD, Wilson CM, Wilson JD, Griffin JE, McPhaul MJ. 1990 A single nucleotide substitution introduces a premature termination codon into the androgen receptor gene of a patient with receptor-negative androgen resistance. J Clin Invest. 85:1522-1528. 12. Sai T, Seino S, Chang C, et al. 1990 An exonic point mutation of the androgen receptor gene in a family with complete androgen insensitivity. Am J Hum Genet. 46:1095-1100. 13. Trifiro M, Prior RL, Sabbaghian N, et al. 1991 Amber mutation creates a diagnostic Mae1 site in the androgen receptor gene of a family with complete androgen insensitivity. Am J Med Genet. 40:493-499. 14. Ris-Stalpers C, Kuiper GGJM, Faber PW, et al. 1990 Aberrant splicing of androgen receptor mRNA results in the synthesis of a nonfunctional receptor protein in a patient with androgen insensitivity. Proc Nat1 Acad Sci USA. 87:7866-7870. 15. Brown TR, Lubahn DB, Wilson EM, French FS, Migeon CJ, Corden JL. 1990 Functional characterization of naturally occurring mutant androgen receptors from patients with complete androgen insensitivity. Mol Endocrinol. 4:1759-1772. 16. Guerami A, Griffin JE, Kovacs WJ, Grino PB, MacDonald PC, Wilson JD. 1990 Estrogen and androgen production rates in two
ET
17.
18.
19.
20.
21.
22.
23.
24.
.-
JCE & M. 1992 Vol75.No6
brothers with Reifenstein syndrome. J Clin Endocrinol Metab. 71:247-251. Marcelli M, Tilley WD, Zoppi S, Griffin JE, Wilson JD, McPhaul MJ. 1991 Androgen resistance associated with a mutation of the androgen receptor at amino acid 772 (Arg-+Cys) results from a combination of decreased messenger ribonucleic acid levels and impairment of receptor function. J Clin Endocrinol Metab. 73:318325. Griffin JE, Punyashthiti K, Wilson JD. 1976 Dihydrotestosterone binding by cultured human fibroblasts: comparison of cells from control subjects with hereditary male pseudohermaphroditism due to androgen resistance. J Clin Invest. 57:1342-1351. Grino PB, Isidro-Gutierrez RF, Griffin JE, Wilson JD. 1989 Androgen resistance associated with a qualitative abnormality of the androgen receptor and responsive to high dose androgen therapy. J Clin Endocrinol Metab. 68:578-585. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. 1951 Protein measurement with the Folin phenol reagent. J Biol Chem. 193:265275. Griffin JE, Durrant JL. 1982 Qualitative receptor defects in families with androgen resistance: failure of stabilization of the fibroblast cytosol androgen receptor. J Clin Endocrinol Metab. 55:465-474. Husmann DA, Wilson CM, McPhaul MJ, Tilley WD, Wilson JD. 1990 Antipeptide antibodies to two distinct regions of the androgen receptor localize the receptor protein to the nuclei of target cells in the rat and human prostate. Endocrinology. 126:2359-2368. Marcelli M, Zoppi S, Wilson CM, Griffin JE, Wilson JD, McPhaul MJ. Amino acid substitution in a small segment of exon 5 of the human androgen receptor (AR) gene cause complete testicular feminization (CTF) by different mechanisms. Proc of the 74th Annual Meet of The Endocrine Sot. 1992;107. van Laar JH, Bolt-deVries J, Voorhorst-Ogink MM, Brinkmann AO. 1989 The human androgen receptor is a 110 Kd protein. Mol Cell Endocrinol. 63:39-44.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 12 November 2015. at 16:45 For personal use only. No other uses without permission. . All rights reserved.
