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DNA-binding of androgen and estrogen receptors from mouse brain: Behavior of residual androgen receptor from Tfm mutant
Brain Research, 140 (1978) 159-164 (g~Elsevier/North-Holland Biomedical Press
159
DNA-binding of androgen and estrogen receptors from mouse brain: behavior of residual androgen receptor from Tfm mutant
S. J. WIELAND, T. O. FOX and C. SAVAKIS Department of Neuropathology, Harvard Medical School and Department of Neuroscience, Chihlren's Hospital Medical Center, Boston, Mass. 02115 ( U.S. A.)
(Accepted August 24th, 1977)
The mediation of androgen and estrogen effects on the developmental and adult functions of the brain may require several macromolecules. An estradiol binding protein, presumed to be a receptor, has been demonstrated in the hypothalamus and limbic regions of the mouse 1,a-5 and rat TM, and activity that catalyzes the aromatization of androgen to estrogen has been reported in specific brain regions 14. These two activities alone could theoretically account for the different actions of estrogens and androgens. However, an androgen binding protein which has both low capacity and high affinity has also been detected in hypothalamus and other brain regions~-, 3,%11, raising the possibility that there may be a direct androgen receptor system. Neither proposed androgen-response mechanism, aromatization nor direct androgen receptors precludes the existence of the other: one or both may be necessary for normal androgen-dependent development and neural response. The vastly lowered level of the androgen receptor in mice affected by the testicular feminization mutation, Tfm a, is consistent with the suggestion that this protein is functioning as a receptor, since male mice affected by this X-linked recessive mutation exhibit phenotypic androgen resistance. However, androgen receptors in T f m / Y , while greatly lowered, are detectable in hypothalamus and other tissues 1,3,v,9. The properties of these residual receptors are of interest in characterizing both normal and mutant androgen receptors and in probing their possible functions. The estrogen receptor, although present in very low levels in mouse brain s , has been detected by the use of DNA-cellulose chromatography, and we have achieved its partial purification using this affinity method 4,s. Androgen binding in mouse hypothalamus a and cerebellum 7 is also very low; therefore, a method for easier detection and selective enrichment of these putative androgen receptors would be very useful. In this report, we have compared the DNA-binding properties of putative androgen and estrogen receptors from normal and T f m / Y mouse hypothalamus-preoptic area (HPOA). In initial experiments (Fig. 1) with [all]steroid-labeled extracts from normal mice, the estradiol receptor and the androgen receptor from HPOA both adhere to DNA-cellulose when applied in EB50 ('extract buffer' with a NaC1 concentration of
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Fig. 1. Adherence of androgen receptor and estrogen receptor to DN A-cellulose and cellulose. Sephadex void volumes from hypothalamus-preoptic area (HPOA) extracts (10 animals each), labeled with either [all]testosterone (a) or [ZH]estradiol (b) were divided into equal aliquots and chromatographed on parallel DNA-cellulose (open squares) and cellulose (closed circles) columns. To prepare these extracts, hybrid (C57BI/6J × C3H/HeJ)F1 mice were taken in their fourth week of age, killed by cervical dislocation and the HPOA was removed from each, with an average weight of 25 rag. All subsequent steps were performed at 2-4 °C. Samples were homogenized by hand in ground-glass Duall hcmogenizers with a final extract volume adjusted to 15 tissue blocks/ml using a buffer which contained 10 m M Tris • HCI (pH 8.1 at 21 °C), 1 m M mercaptoethanol, l m M Na3EDTA, 10% glycerol, and 50 m M NaCI. Buffer designations indicate the NaC1 concentration (raM) of the buffer; for example, EB50 indicates that this extract buffer contains 50 m M NaCI. The homogenate was centrifuged at 140,1300 × g for 60 min, after which the supernatant was labeled with 10 n M of either [l,2,6,7-SH(N)]testostero ne (85 Ci/mmole) or [2,4,6,7-aH(N)]estradiol (91.3 Ci/mmole) from New England Nuclear. After 30 min of incubation, each was passed through a 6.5 ml Sephadex G-25 column to remove unbound hormone, and the macromolecular peak was then applied to a I ml DNA-cellulose or cellulose column equilibrated with EBS0, also containing 0.2 mg/ml bovine serum albumin, and then chromatographed 46-8. The columns were eluted with the indicated steps. For the purposes of this report, it should ~ce emphasized that by 'receptor' we mean high affinity, low capacity binding to available sites in high speed supernatants prepared under these conditions.
'50 mM'). When these extracts are applied to parallel columns of cellulose, there is virtually no detectable retention of the tracers. Therefore, the binding to DNAcellulose is DNA-dependent (Fig. 1). The androgen-labeled material which adheres to DNA-cellulose can be eluted by raising the ionic strength with EB130, which in one step elutes approximately 85-90%; the remainder elutes with EB210. In contrast, approximately 90% of the estradiol-labeted material elutes with EB210 under these conditions. The small remaining fraction of estradiol-labeled material eluting with EB400 corresponds to a form of the estradiol receptor with a higher sedimentation rate than the EB210 fraction (5S vs. 4S, unpublished data). The 5S form appears to be derived from the 4S form by forming a complex with a second protein 17. The heat and time dependence for quantitative conversion of estradiol receptor from mouse HPOA has been reported~,6. We examined more closely the DNA-cellulose elution profiles of androgen and estradiol receptors to determine how sharply their salt dependence could be defined (Fig. 2). We also compared the DNA-cellulose adherence of receptor with either testosterone or dihydrotestosterone as the ligand. Elution with small steps of salt indicates clear separation of androgen receptor elution (EB110 and EB140) from the
161 ELUTION
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DNA-CELLULOSE
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110 140 170 210 400
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Fig. 2. Elution patterns of sex steroid complexes on DNA-cellulose. Equal portions (10 animals each) of HPOA extract were labeled with [3H]estradiol, [3H]testosterone or [3H]dihydrotestosterone and applied to identical DNA-cellulose columns as in the legend to Fig. 1, with the addition that [1,2,4,5,6, 7-3H(N)]5a-dihydrotestosterone (80 Ci/mmole) also was used. The elution steps with increments of NaCI concentration were 4 column volumes each. Total cpm for each column were: estradiol, 3840; testosterone, 1700; DHT, 2270.
region of estradiol receptor elution (above EBI70). The similarity of the elution profiles for labeling with testosterone and dihydrotestosterone is in agreement with our other evidence 3 that these hormones bind to the same receptor. To confirm that the androgen-labeled, DNA-adhering material is the androgen receptor, the Tfm mutant was examined. The total androgen receptor from the HPOA of Tfm/Y is reduced to 10-15% of normal levels when examined by Sephadex chromatography and sucrose density gradient sedimentation3,16. All of this residual androgen receptor adheres to DNA-cellulose in EB50 and can be eluted with EB210 (Fig. 3 and ref. 16). Therefore, DNA-cellulose chromatography provides an independent criterion for determining the level of androgen receptor in TritelY. In this experiment (Fig. 3), the amount of androgen receptor for Tfm/Y is 15 % of the control. In contrast, estradiol receptor is present at normal amounts and adheres to DNA in the normal proportion (Fig. 3). While the reduction in Tfm/Y of androgen receptor serves as a control for the detection of normal androgen receptor, the existence of detectable residual receptor in Tfm/Y is of intrinsic interest. The behavior of this residual receptor was examined in more detail (Fig. 4a). Again (see Fig. 1), normal androgen receptor elutes primarily with EB130, while a small fraction elutes with EB210. However, the residual receptor
162 ADHERENCE
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Fig. 3. Adherence to DNA-cellulose of androgen receptor and estrogen receptor from TJm/Y and cob trol HPOA. Extracts of HPOA from Tfm/Y and sibling mice (Ta/Y and Ta/Ta) were divided into two equal portions and labeled with Jail]testosterone or [3H]estradiol. They were then applied to D N A cellulose columns. Material adhering to the columns was eluted in a single step with EB210. Each bar represents 4 HPOAs.
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Fig. 4. Elution profiles of androgen receptor and estrogen receptor from Tfm/Y and control HPOA on DNA-cellulose. Extracts of H P O A from Tfm/Y (squares) and sibling mice (circles) were labeled with [3H]dihydrotestosterone (a) or [aH]estradiol (b) and applied to DNA-ceUulose. The material adhering to the columns was eluted first with EB130, then with EB21& (For this experiment, we used [aH]dihydrotestosterone as for Fig. 2, but with a specific activity of 123 Ci/mmole.)
163 from the Tfm/Y appears to be mainly in the EB210 peak, while the EB130 peak is greatly reduced. This figure also illustrates that the residual androgen receptor binds dihydrotestosterone as well as binding testosterone (see Fig. 3). As a control, the elution profile of the Tfm/Y estrogen receptor does not differ from normal (Fig. 4b). Our report is the first demonstration of both DNA-dependence and ionic strength requirements for the adherence of androgen receptor to DNA-cellulose. Adherence to DNA-cellulose of putative androgen receptors in salt-free cytosol from peripheral target tissue had been reported 1~. The adherence to DNA-cellulose of androgen receptor and estradiol receptor from HPOA differs in two ways. All of the androgen receptor can adhere to DNA-cellulose (unpublished data), while only half of the estradiol receptor adheres e,s. Secondly, the androgen receptor adheres at lower ionic strength, while the adhering portion of the estradiol receptor elutes only at higher ionic strengths (Figs. I and 2). The difference in elution conditions between androgen receptor and estrogen receptor facilitates efficient physical separation of the two species. We can now pursue the study of their independent behavior, and their possible interactions with each other. The small amount of androgen receptor which we detect in cytosols of HPOA from the Tfm/Y mouse agrees with some previous studies which have revealed residual receptor-like binding in Tfm/Y kidney and brain 1,a,9,16. The residual androgen receptor of mouse Tfm/Y has not lost its ability to adhere to DNA-cellulose (Figs. 3 and 4, and ref. 16). However, the elution profile of the residual receptor differs from the normal pattern (Fig. 4) with either dihydrotestosterone or testosterone (not shown) as ligand. Not only has residual androgen receptor been demonstrated in mouse Tfm/Y, but a partial androgen induction of protease 'A' and nerve growth factor activity in the TJm/Y submandibular gland has been reported 15. Using dihydrotestosterone, we have also noted androgen-induced behavioral, metabolic and enzyme-induction effects in the Tfm/Y (unpublished data). For these reasons, we have accepted the description of Tfm as 'androgen resistant q~, since these mutants are not totally 'androgen-insensitive'. This may not be unlike the phenotype of the rat Tfm mutant, for which the level of androgen receptor is approximately 15 % of normal and partial responses to androgen are reported ~a. In summary, androgen and estradiol receptors from mouse hypothalamuspreoptic area (HPOA) were chromatographed on DNA-cellulose, their adherence was shown to be DNA-dependent, and the ionic strength requirements for the adherence of these receptors differed, allowing separation of the androgen receptor from the estradiol receptor. For the T/m mutant which had a reduced level of androgen receptor in HPOA, the elution profile of the residual androgen receptor on DNA-cellulose differed from the normal pattern. This research was supported by a National Research Service Award in Cell and Developmental Biology, on Grant 5 T32 GM 07226 02, awarded by General Medical Sciences, DHEW (S.J.W.) and a Basil O'Connor Starter Research Grant from The National Foundation-March of Dimes (T.O.F.).
t64 1 Attardi, B., Geller, L. N. and Ohno, S., Androgen and estrogen receptors in brain cytosol from male, female and testicular feminized (Tfm/Y) mice, Endocrinology, 98 (1976) 864-874. 2 Barley, J., Ginsberg, M., Greenstein, B. D., MacLusky, N. J. and Thomas, P. J., An androgen receptor in rat brain and pituitary, Brain Research, 100 (1975) 383-393. 3 Fox, T. O., Androgen- and estrogen-binding macromolecules in developing mouse brain: biochemical and genetic evidence, Proc. nat. Acad. Sci. (Wash.), 72 (1975) 4303-4307. 4 Fox, T. O., Oestradiol receptor in neonatal mouse brain, Nature (Lurid.), 258 (1975) 441-444. 5 Fox, T. O., Nuclear estradiol receptor formation : serum complexing activity (5S-CA) utilized to compare mouse hypothalamus and uterus. In Proceedings of the Second Annual (1976) Maine Biomedical Science Symposium, University of Maine Press, Orono, 1977, pp. 544-572. 6 Fox, T. O.,Conversionofthehypothalamicestradiolreceptor to the'nuclear'form, Brain Research, 120 (1977) 580-583. 7 Fox, T. O., Estradiol and testosterone binding in normal and mutant mouse cerebellum: biochemical and cellular specificity, Brain Research, 128 (1977) 263-273. 8 Fox, T. O. and Johnston, C., Estradiol receptors from mouse brain and uterus: binding to DNA, Brain Research, 77 (1974) 330-336. 9 Gehring, U. and Tomkins, G. M., Characterization of a hormone receptor defect in the androgeninsensitivity mutant, Cell, 3 (1974) 59-64. 10 Kato, J., Atsumi, Y. and Inaba, M., Estradiol receptors in female rat hypothalamus in the developmental stages and during pubescence, Endocrinology, 94 (1974) 3C9-317. 11 Kato, J. and Onouchi, T., 5a dihydrotestosterone 'receptor' in the rat hypothalamus, L)zdocrinol. Jap., 20 (1973) 429-432. 12 Mainwaring, W. I. P. and Irving, R., The use of deoxyribonucleic acid cellulose chromatography and isoelectric focusing for characterization and partial purification of steroid receptor complexes, Biochem. J., 134 (1973) 113-127. 13 Naess, O., Haug, E., Attramadal, A., Aakvaag, A., Hansson, V. and French, F., Androgen receptors in the anterior pituitary and central nervous system of the androgen 'insensitive' (Tfin) rat: correlation between receptor binding and effects of androgens on gonadotropin secretion, Erdocrinology, 99 (1976) 1295-1303. 14 Naftolin, F., Ryan, K. J., Davies, I. J., Reddy, V. V., Flores, F., Petro, Z., Kuhtl, M., While, R. J., Takaoka, Y. and Wolin, L., The formation of estrogens by central neuroendocrir~e tissues, Rec. Progr. Horm. Res., 31 (1975) 295 319. 15 Schenkein, I., Levy, M., Bueker, E. D. and Wilson, J. D., Immunological and enzynmtic evidence for the absence of an esteroproteolytic enzyme (protease 'D') in the submandibular gland of the Tfm mouse, Endocrinology, 94 (1974) 840-844. 16 Wietand, S. J. and Fox, T. O., D N A affinity separates androgen from estrogen binding activity: study of residual androgen binding macromolecules in Tfm mutant mouse brain, Nearosci. Abstr. ll (1976) 661. 17 Yamamoto, K. R. and Alberts, B, M., In vitro conversion of estradiol receptor protein to its nuclear form : dependence on hormone and DNA, Proc. nat. Acad. Sci. (Wash.), 69 (1972) 2105 2109.
159
DNA-binding of androgen and estrogen receptors from mouse brain: behavior of residual androgen receptor from Tfm mutant
S. J. WIELAND, T. O. FOX and C. SAVAKIS Department of Neuropathology, Harvard Medical School and Department of Neuroscience, Chihlren's Hospital Medical Center, Boston, Mass. 02115 ( U.S. A.)
(Accepted August 24th, 1977)
The mediation of androgen and estrogen effects on the developmental and adult functions of the brain may require several macromolecules. An estradiol binding protein, presumed to be a receptor, has been demonstrated in the hypothalamus and limbic regions of the mouse 1,a-5 and rat TM, and activity that catalyzes the aromatization of androgen to estrogen has been reported in specific brain regions 14. These two activities alone could theoretically account for the different actions of estrogens and androgens. However, an androgen binding protein which has both low capacity and high affinity has also been detected in hypothalamus and other brain regions~-, 3,%11, raising the possibility that there may be a direct androgen receptor system. Neither proposed androgen-response mechanism, aromatization nor direct androgen receptors precludes the existence of the other: one or both may be necessary for normal androgen-dependent development and neural response. The vastly lowered level of the androgen receptor in mice affected by the testicular feminization mutation, Tfm a, is consistent with the suggestion that this protein is functioning as a receptor, since male mice affected by this X-linked recessive mutation exhibit phenotypic androgen resistance. However, androgen receptors in T f m / Y , while greatly lowered, are detectable in hypothalamus and other tissues 1,3,v,9. The properties of these residual receptors are of interest in characterizing both normal and mutant androgen receptors and in probing their possible functions. The estrogen receptor, although present in very low levels in mouse brain s , has been detected by the use of DNA-cellulose chromatography, and we have achieved its partial purification using this affinity method 4,s. Androgen binding in mouse hypothalamus a and cerebellum 7 is also very low; therefore, a method for easier detection and selective enrichment of these putative androgen receptors would be very useful. In this report, we have compared the DNA-binding properties of putative androgen and estrogen receptors from normal and T f m / Y mouse hypothalamus-preoptic area (HPOA). In initial experiments (Fig. 1) with [all]steroid-labeled extracts from normal mice, the estradiol receptor and the androgen receptor from HPOA both adhere to DNA-cellulose when applied in EB50 ('extract buffer' with a NaC1 concentration of
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Fig. 1. Adherence of androgen receptor and estrogen receptor to DN A-cellulose and cellulose. Sephadex void volumes from hypothalamus-preoptic area (HPOA) extracts (10 animals each), labeled with either [all]testosterone (a) or [ZH]estradiol (b) were divided into equal aliquots and chromatographed on parallel DNA-cellulose (open squares) and cellulose (closed circles) columns. To prepare these extracts, hybrid (C57BI/6J × C3H/HeJ)F1 mice were taken in their fourth week of age, killed by cervical dislocation and the HPOA was removed from each, with an average weight of 25 rag. All subsequent steps were performed at 2-4 °C. Samples were homogenized by hand in ground-glass Duall hcmogenizers with a final extract volume adjusted to 15 tissue blocks/ml using a buffer which contained 10 m M Tris • HCI (pH 8.1 at 21 °C), 1 m M mercaptoethanol, l m M Na3EDTA, 10% glycerol, and 50 m M NaCI. Buffer designations indicate the NaC1 concentration (raM) of the buffer; for example, EB50 indicates that this extract buffer contains 50 m M NaCI. The homogenate was centrifuged at 140,1300 × g for 60 min, after which the supernatant was labeled with 10 n M of either [l,2,6,7-SH(N)]testostero ne (85 Ci/mmole) or [2,4,6,7-aH(N)]estradiol (91.3 Ci/mmole) from New England Nuclear. After 30 min of incubation, each was passed through a 6.5 ml Sephadex G-25 column to remove unbound hormone, and the macromolecular peak was then applied to a I ml DNA-cellulose or cellulose column equilibrated with EBS0, also containing 0.2 mg/ml bovine serum albumin, and then chromatographed 46-8. The columns were eluted with the indicated steps. For the purposes of this report, it should ~ce emphasized that by 'receptor' we mean high affinity, low capacity binding to available sites in high speed supernatants prepared under these conditions.
'50 mM'). When these extracts are applied to parallel columns of cellulose, there is virtually no detectable retention of the tracers. Therefore, the binding to DNAcellulose is DNA-dependent (Fig. 1). The androgen-labeled material which adheres to DNA-cellulose can be eluted by raising the ionic strength with EB130, which in one step elutes approximately 85-90%; the remainder elutes with EB210. In contrast, approximately 90% of the estradiol-labeted material elutes with EB210 under these conditions. The small remaining fraction of estradiol-labeled material eluting with EB400 corresponds to a form of the estradiol receptor with a higher sedimentation rate than the EB210 fraction (5S vs. 4S, unpublished data). The 5S form appears to be derived from the 4S form by forming a complex with a second protein 17. The heat and time dependence for quantitative conversion of estradiol receptor from mouse HPOA has been reported~,6. We examined more closely the DNA-cellulose elution profiles of androgen and estradiol receptors to determine how sharply their salt dependence could be defined (Fig. 2). We also compared the DNA-cellulose adherence of receptor with either testosterone or dihydrotestosterone as the ligand. Elution with small steps of salt indicates clear separation of androgen receptor elution (EB110 and EB140) from the
161 ELUTION
FROM
DNA-CELLULOSE
50l 40
TRADIOL
30
50 20
10
[;~Z'////////////////////A 80 110 140 170 210 400
D
W l._J LIJ
40
TOSTERONE
30 20 10
©
I---
80 110 140 170 210 400
b-
©
40
DIHYDROTESTOSTERONE
30 20 10 80
110 140 170 210 400
NaCI (mM)
Fig. 2. Elution patterns of sex steroid complexes on DNA-cellulose. Equal portions (10 animals each) of HPOA extract were labeled with [3H]estradiol, [3H]testosterone or [3H]dihydrotestosterone and applied to identical DNA-cellulose columns as in the legend to Fig. 1, with the addition that [1,2,4,5,6, 7-3H(N)]5a-dihydrotestosterone (80 Ci/mmole) also was used. The elution steps with increments of NaCI concentration were 4 column volumes each. Total cpm for each column were: estradiol, 3840; testosterone, 1700; DHT, 2270.
region of estradiol receptor elution (above EBI70). The similarity of the elution profiles for labeling with testosterone and dihydrotestosterone is in agreement with our other evidence 3 that these hormones bind to the same receptor. To confirm that the androgen-labeled, DNA-adhering material is the androgen receptor, the Tfm mutant was examined. The total androgen receptor from the HPOA of Tfm/Y is reduced to 10-15% of normal levels when examined by Sephadex chromatography and sucrose density gradient sedimentation3,16. All of this residual androgen receptor adheres to DNA-cellulose in EB50 and can be eluted with EB210 (Fig. 3 and ref. 16). Therefore, DNA-cellulose chromatography provides an independent criterion for determining the level of androgen receptor in TritelY. In this experiment (Fig. 3), the amount of androgen receptor for Tfm/Y is 15 % of the control. In contrast, estradiol receptor is present at normal amounts and adheres to DNA in the normal proportion (Fig. 3). While the reduction in Tfm/Y of androgen receptor serves as a control for the detection of normal androgen receptor, the existence of detectable residual receptor in Tfm/Y is of intrinsic interest. The behavior of this residual receptor was examined in more detail (Fig. 4a). Again (see Fig. 1), normal androgen receptor elutes primarily with EB130, while a small fraction elutes with EB210. However, the residual receptor
162 ADHERENCE
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Fig. 3. Adherence to DNA-cellulose of androgen receptor and estrogen receptor from TJm/Y and cob trol HPOA. Extracts of HPOA from Tfm/Y and sibling mice (Ta/Y and Ta/Ta) were divided into two equal portions and labeled with Jail]testosterone or [3H]estradiol. They were then applied to D N A cellulose columns. Material adhering to the columns was eluted in a single step with EB210. Each bar represents 4 HPOAs.
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Fig. 4. Elution profiles of androgen receptor and estrogen receptor from Tfm/Y and control HPOA on DNA-cellulose. Extracts of H P O A from Tfm/Y (squares) and sibling mice (circles) were labeled with [3H]dihydrotestosterone (a) or [aH]estradiol (b) and applied to DNA-ceUulose. The material adhering to the columns was eluted first with EB130, then with EB21& (For this experiment, we used [aH]dihydrotestosterone as for Fig. 2, but with a specific activity of 123 Ci/mmole.)
163 from the Tfm/Y appears to be mainly in the EB210 peak, while the EB130 peak is greatly reduced. This figure also illustrates that the residual androgen receptor binds dihydrotestosterone as well as binding testosterone (see Fig. 3). As a control, the elution profile of the Tfm/Y estrogen receptor does not differ from normal (Fig. 4b). Our report is the first demonstration of both DNA-dependence and ionic strength requirements for the adherence of androgen receptor to DNA-cellulose. Adherence to DNA-cellulose of putative androgen receptors in salt-free cytosol from peripheral target tissue had been reported 1~. The adherence to DNA-cellulose of androgen receptor and estradiol receptor from HPOA differs in two ways. All of the androgen receptor can adhere to DNA-cellulose (unpublished data), while only half of the estradiol receptor adheres e,s. Secondly, the androgen receptor adheres at lower ionic strength, while the adhering portion of the estradiol receptor elutes only at higher ionic strengths (Figs. I and 2). The difference in elution conditions between androgen receptor and estrogen receptor facilitates efficient physical separation of the two species. We can now pursue the study of their independent behavior, and their possible interactions with each other. The small amount of androgen receptor which we detect in cytosols of HPOA from the Tfm/Y mouse agrees with some previous studies which have revealed residual receptor-like binding in Tfm/Y kidney and brain 1,a,9,16. The residual androgen receptor of mouse Tfm/Y has not lost its ability to adhere to DNA-cellulose (Figs. 3 and 4, and ref. 16). However, the elution profile of the residual receptor differs from the normal pattern (Fig. 4) with either dihydrotestosterone or testosterone (not shown) as ligand. Not only has residual androgen receptor been demonstrated in mouse Tfm/Y, but a partial androgen induction of protease 'A' and nerve growth factor activity in the TJm/Y submandibular gland has been reported 15. Using dihydrotestosterone, we have also noted androgen-induced behavioral, metabolic and enzyme-induction effects in the Tfm/Y (unpublished data). For these reasons, we have accepted the description of Tfm as 'androgen resistant q~, since these mutants are not totally 'androgen-insensitive'. This may not be unlike the phenotype of the rat Tfm mutant, for which the level of androgen receptor is approximately 15 % of normal and partial responses to androgen are reported ~a. In summary, androgen and estradiol receptors from mouse hypothalamuspreoptic area (HPOA) were chromatographed on DNA-cellulose, their adherence was shown to be DNA-dependent, and the ionic strength requirements for the adherence of these receptors differed, allowing separation of the androgen receptor from the estradiol receptor. For the T/m mutant which had a reduced level of androgen receptor in HPOA, the elution profile of the residual androgen receptor on DNA-cellulose differed from the normal pattern. This research was supported by a National Research Service Award in Cell and Developmental Biology, on Grant 5 T32 GM 07226 02, awarded by General Medical Sciences, DHEW (S.J.W.) and a Basil O'Connor Starter Research Grant from The National Foundation-March of Dimes (T.O.F.).
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