Androgen Receptor Phosphorylation, Turnover, Nuclear Transport, and Transcriptional Activation SPECIFICITY FOR STEROIDS AND ANTIHORMONES*

Nuclear transport, phosphorylation, ligand binding, and degradation rate of the recombinant androgen receptor (AR) were analyzed in transfected COS cells in the presence of various steroids and antiandrogens. Transcriptional activition was assessed in CV1 cells by cotransfection with an androgen-responsive chloramphenicol acetyltransferase (CAT) reporter vector. Hormone binding specificity of recombinant AR was essen-tially identical to endogenous AR. AR localized in the nucleus in the presence of methyltrienolone (R1881, a synthetic androgen), dihydrotestosterone, testosterone, hydroxyflutamide, cyproterone acetate, estradiol, progesterone, and RU486. In the absence of hormone or with the antiandrogen, flutamide, AR remained largely in the cytoplasm with a perinuclear distribution. AR was degraded rapidly (tllz = 1 h) except in the presence of androgen (tllz = 6 h) which accounted for an apparent 2-4-fold androgen-induced increase in AR phosphorylation, indicating that AR phosphorylation was not enhanced by androgen. CAT activity was stimulated by R1881, dihydrotestosterone, testosterone, cyproterone acetate, estradiol,

Nuclear transport, phosphorylation, ligand binding, and degradation rate of the recombinant androgen receptor (AR) were analyzed in transfected COS cells in the presence of various steroids and antiandrogens. Transcriptional activition was assessed in CV1 cells by cotransfection with an androgen-responsive chloramphenicol acetyltransferase (CAT) reporter vector. Hormone binding specificity of recombinant AR was essentially identical to endogenous AR. AR localized in the nucleus in the presence of methyltrienolone (R1881, a synthetic androgen), dihydrotestosterone, testosterone, hydroxyflutamide, cyproterone acetate, estradiol, progesterone, and RU486. In the absence of hormone or with the antiandrogen, flutamide, AR remained largely in the cytoplasm with a perinuclear distribution. AR was degraded rapidly (tllz = 1 h) except in the presence of androgen (tllz = 6 h) which accounted for an apparent 2-4-fold androgen-induced increase in AR phosphorylation, indicating that AR phosphorylation was not enhanced by androgen. CAT activity was stimulated by R1881, dihydrotestosterone, testosterone, cyproterone acetate, estradiol, progesterone, and RU486 in a dose-dependent manner. The antiandrogens, flutamide and hydroxyflutamide, lacked agonist activity and inhibited R1881-induced activation of CAT and androgen stabilization of AR. Steroids and antiandrogens with moderate to low affinity for AR promoted both nuclear transport and transcriptional activation but only at high hormone concentrations. Hydroxyflutamide acted as a true antiandrogen since it lacked agonist activity and was an inhibitor of androgen-induced transcriptional activation.
The biological effects of androgens can be inhibited by androgen antagonists known as antiandrogens. On the basis of structure, antiandrogens are subdivided into two groups, steroidal and nonsteroidal, both of which can inhibit agonist binding to the androgen receptor (AR)' (1). Flutamide (LY,LY,(Ytrifluoro-2-methyl-4'-nitro-m-propionotoluidide) is considered a pure antiandrogen because it lacks agonist activity and is specific for the AR. Administered to animals, flutamide has potent antiandrogenic effects on male reproductive tissues, decreasing organ size (2,3) and blocking androgen effects in castrated rats. It was postulated that since flutamide shows little binding affinity for AR, its effects in vivo are mediated through a hydroxylated metabolite, hydroxyflutamide ((Y,LY,LYtrifluoro-2-methyl-4'-nitro-rn-lactotoluidide) (4)(5)(6)(7). Subsequent studies demonstrated that hydroxyflutamide administered in vivo is an effective antiandrogen (8). More recently flutamide itself was shown to inhibit receptor binding of labeled androgen but only at very high concentrations (Ki 10 W ) (9).
RU486 is a synthetic progestin and glucocorticoid antagonist that binds with high affinity to glucocorticoid and progesterone receptors (10,11) and causes efficient nuclear localization of these receptors (12)(13)(14). RU486 promotes specific binding of progesterone (15)(16)(17) and glucocorticoid receptors (18) to their hormone response elements, does not induce a transcriptional response (15), and blocks the constitutive transcriptional function of a truncated mutant form of the progesterone receptor (19).

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A R Phosphorylation and
Functional Activity 969 induction of the chloramphenicol acetyltransferase gene linked to an androgen-responsive promoter (26).

EXPERIMENTAL PROCEDURES
Materials-The following reagents were purchased monkey kidney COS-7 and CV1 cells from the American Type Culture Collection; Dulbecco's modified essential medium with high glucose and Eagle's modified essential medium without phosphate from GIBCO; bovine serum from HyClone Laboratories, Inc.; ~-threo- [ Cell Transfection-Nuclear transport, receptor turnover, and phosphorylation were determined in monkey kidney COS-7 cells transfected with 10 pg of the full-length human AR expression vector pCMVhAR (p5HBhAR-A) (20, 26) using DEAE-dextran (27). COS cells were maintained in 10% fetal calf serum in Dulbecco's minimal essential medium containing high glucose and antibiotics and plated a t 1.0 X lo6 cells/lO-cm dish. Immediately after transfection cells were placed in 5% serum containing medium, and 24 h later, into serum-free, phenol red-free medium in the presence or absence of hormone. For immunocytochemical studies, COS cells were transfected on two chamber glass slides as previously described (20). Twenty-four h prior to fixation, cells were placed in serum-free, phenol red-free medium in the presence or absence of steroid. Medium with or without hormone was replaced with fresh medium 2 h prior to fixation. Transfection efficiency ranged from 4-8% as reflected by AR immunostaining of transiently transfected COS cells. The level of AR expression in COS cells was 340 +-22 fmol/mg protein, determined in a whole cell binding assay described below.
AR functional activity was assessed in monkey kidney CVI cells by transient transfection as previously described (20). CV1 cells were maintained in 10% fetal calf serum in medium as described above.
Cells were plated at 1.3 X lo6 cells/lO-cm dish so that approximately 80-90% confluence was achieved at the time of transfection 24 h later. Cells were transfected with 1-3 pg of AR expression vector pCMVhAR and 1-3 pg of the reporter vector using the calcium phosphate DNA precipitation method (27). The CAT reporter vector pMTV29VTM contains two glucocorticoid response elements separated by 29 base pairs and positioned 5' of the CAT gene (28). AR expression in CV1 cells was 30 +-10 fmol/mg protein determined in the whole cell binding assay and was below the detection level by AR immunocytochemistry. CAT assays representative of at least six than 100-fold. independent experiments are shown with maximal induction greater Binding Specificity-Steroid and nonsteroidal antiandrogen binding specificities were determined in COS cells transiently expressing recombinant AR using a whole cell binding assay previously described (27). COS cells in 24-well tissue culture plates containing lo5 cells/ well were transfected with 1 pg of pCMVhAR DNA/well using DEAEdextran (27). Cells were maintained for 24 h in Dulbecco's modified Eagle's medium containing 5% fetal calf serum until 24 h prior to labeling when they were placed in serum-free, phenol red-free media. Cells were labeled for 2 h with 5 nM [3H]methyltrienolone (R1881) in the presence or absence of increasing concentrations of unlabeled hormones. Nonspecific binding was accounted for by parallel incubations in the presence of a 100-fold excess unlabeled R1881. Labeling medium was removed and the cells washed twice in phosphatebuffered saline and harvested in 2% SDS, 10% glycerol, and 10 mM Tris, pH 6.8, and radioactivity determined by scintillation counting.
Receptor Turnover and Phosphorylation-Phosphorylation and turnover studies were performed using methodology similar to that previously described (32). Forty-eight h after COS cell transfection with pCMVhAR, serum-free, phenol red-free medium was aspirated and the cells washed with 3 ml of either phosphate-free Eagle's minimal essential medium containing 20 mM HEPES, pH 7.2, or methionine-free Eagle's minimal essential medium containing 20 mM HEPES, pH 7.2. Four ml of fresh phosphate-free or methionine-free media was added, and the cells were incubated at 37 "C in a 5% Con incubator for 15 min. Steroids or nonsteroidal antiandrogens and 200 pCi of [32P]orthophosphate or 200 pCi of T r a n~~~S -l a b e l were added and incubated for the indicated times at 37 'C. Cells were washed twice with Dulbecco's modified Eagle's media and harvested by scraping into 1 ml of RIPA buffer (1% Triton X-100, 1% deoxycholate, 0.1% sodium dodecyl sulfate, 0.15 M NaCl, 0.5 mM sodium vanadate, 5 mM EDTA, and 50 mM Tris, pH 7.4). DNA was sheared by repeated passage through a Pasteur pipette and the samples rotated for 20 min at 4 "C. Supernatants of a 15-min centrifugation at 13,000 X g a t 4 "C were transferred to new microfuge tubes and 60 pg of AR52 AR antipeptide IgG (26,29) added and incubated overnight at 4 "C. Pansorbin Staphylococcus aureus cells (Calbiochem Corp., La Jolla, CA) were prepared fresh by washing with six volumes of 10% SDS followed by three washes with RIPA with 1-min centrifugations to pellet the cells. The final Pansorbin pellet was resuspended in an equal volume of RIPA and 20 pl added to each sample followed by incubation for 2 h at 4 "C. After a 1-min centrifugation, a series of 0.3-ml washes of the pellets with 30 s centrifugations included twice RIPA; 0.5 M NaC1, RIPA; 1 mg/ml bovine serum albumin, RIPA; 0.5 M NaCl, RIPA; 1 mg/ml bovine serum albumin, RIPA; twice RIPA. The final pellets were resuspended in 75 pl of 2.5% SDS, 12.5% glycerol, 12.5 mM Tris, pH 6.8, and incubated at room temperature for 10 min. Following a 2-min centrifugation, the supernatants were transferred to new tubes and made 4% 2-mercaptoethanol and 0.05% bromphenol blue. Samples were heated at 95 "C for 5 min, cooled to room temperature, and loaded onto 8% SDS-polyacrylamide gels prepared as described (27,33). The extent of radiolabel incorporation was determined by computer analysis of the transferred membranes on an Ambis radioscanner. The data shown are representative of at least three independent experiments.

RESULTS
Recombinant AR Steroid Binding Specificity-In a whole cell competitive binding assay, transiently expressed AR had highest apparent binding affinity for R1881, followed by di- Binding data not shown included testosterone and progesterone inhibition curves which were similar to dihydrotestosterone and estradiol, respectively. AR Phosphorylation and Functional Activity was similar to dihydrotestosterone. Cyproterone acetate, estradiol (Fig. l), and progesterone (not shown) compete for ['H]R1881 bindingwith 6040% inhibition at a 100-fold molar excess of unlabeled hormone (Fig. 1). The antiandrogen flutamide failed to inhibit binding of [3H]R1881 in this concentration range as previously reported for the endogenous receptor (9)) and hydroxyflutamide inhibited binding by about 50% at a 100-fold molar excess concentration (Fig. 1). RU486, a glucocorticoid and progesterone receptor antagonist (10, l l ) , inhibited binding by about 70% at a 100-fold molar excess of labeled hormone (Fig. 1). Thus, the binding specificity of recombinant AR expressed in COS cells was similar to that of the endogenous receptor (34)(35)(36) in its limited specificity a t elevated steroid concentrations. Both endogenous and transiently expressed AR bind a variety of steroid hormones and antagonists with apparent moderate affinities (lo-' K J . How this binding effects receptor function was examined by analysis of AR nuclear localization, degradation rate, phosphorylation, and transcriptional activition. Hormone Specificity of AR Nuclear Transport-Androgendependent transport of AR to the nucleus was previously demonstrated in transiently transfected COS cells (20) and is shown in Fig. 2, A-C. In the absence of hormone, AR is localized in the perinuclear region of the cytoplasm (Fig. 2 A ) . Nuclear localization of AR was observed following incubations AR phosphorylation were investigated by incubating transfected COS cells with [R2P]orthophosphate and immunoprecipitating receptor using anti-peptide AR antibody AR52 described previously (29). Transfection with the parent expression vector (P5) which lacks AR coding sequence produced no major phosphorylated products (Fig. 3A, lane 1 ). In the absence of hormone, AR was detected as a 114-kDa phosphoprotein (Fig. 3A, lune 2). Addition of R1881, dihydrotestosterone, or testosterone at 5-50 nM appeared to stimulate the incorporation of radiolabeled phosphate 2-4-fold following a 3-h incubation (lanes 3-5). AR phosphorylation was not significantly enhanced by treatment with 100 nM estradiol, progesterone, or the antiandrogens, cyproterone acetate, flutamide, or hydroxyflutamide (lunes [6][7][8][9][10]. However, 100 nM RU486 caused an apparent increase in AR phosphorylation (lune 11 ) similar to that observed with androgens. None of the hormones altered basal receptor phosphorylation observed in the absence of androgen. Treatment with 100 nM R1881 20 min prior to harvest showed a 1.5-fold increase in apparent phosphorylation (lane 13), but no increase was observed when R1881 was added to the harvest buffer (lane 14). Thus the steroid requirements for hormone-induced phosphorylation appeared to be androgen specific with the exception of RU486. We investigated whether the apparent increase in AR phosphorylation described above resulted from androgen-enhanced AR immunoreactivity or receptor stabilization. Parallel incubations were performed with "S-labeled methionine and cysteine for 3-or 24-h labeling periods in the presence and absence of 10 nM androgens or 100 nM nonandrogenic steroids or antiandrogens. Androgens specifically increased 3-5-fold the amount of "S-labeled receptor (Fig. 3B), which was slightly greater than the apparent increase in receptor phosphorylation observed in Fig. 3A. Steroids and antiandrogens which did not enhance phosphorylation also did not increase "S-labeled AR. A 20-min exposure to androgen or androgen addition to the harvest medium (Fig. 3B, lanes 13  and 14) yielded a pattern similar to the phosphorylation  results. Thus, the apparent androgen-stimulated increase in AR phosphorylation appeared to be due to a parallel increase in AR protein. Sodium dodecyl sulfate-gel electrophoresis and immunoblot analysis of AR in whole cell extracts not immunoprecipitated (not shown) indicated that preferential immunoprecipitation of the androgen-bound receptor did not occur.
The hormone dependence of receptor turnover was examined in a pulse-chase experiment. Following a 30-min incubation with 200 pCi of [35S]methionine, cells in monolayer culture were incubated in the presence or absence of 100 nM R1881, harvested at increasing time intervals, and subjected to immunoprecipitation and SDS-gel electrophoresis. As shown in Fig. 4, a major decrease in the rate of receptor degradation occurred in the presence of androgen. AR degraded intracellularly at 37 "C with a half-time of approximately 1 h in the absence of androgen and 6 h in the presence of androgen (Fig. 4, A and B ) . Nonandrogenic steroids included in this study had little or no effect on the degradation rate observed in the absence of androgen (not shown). The antiandrogens, hydroxyflutamide and cyproterone acetate, inhibited androgen-induced stabilization when included a t a 100-fold molar excess. It is concluded that the &fold enhancement of receptor stability by androgen binding could account for the apparent androgen-stimulated increase in AR phosphorylation, and therefore, androgen does not promote receptor phosphorylation in this assay system.
TranscriptionalActiuation-Transient cotransfection of the AR expression plasmid with a CAT reporter vector in CV1 cells was performed to determine whether other steroids and antiandrogens that bind the receptor stimulate AR-mediated gene transcription. R1881, dihydrotestosterone, and testosterone each induced CAT activity in a dose-dependent manner between 0.001-1 nM (Fig. 5 ) . Low basal activity was observed with the parent pCMV expression vector (P5) with or without 10 nM R1881 or with the AR expression vector in the absence of hormone (Figs. 5 and 6). Estradiol and progesterone stimulated CAT activity nearly equal to that of androgen but only at 1000-fold higher hormone Concentrations (Fig.  5 ) . Estradiol (1 nM) and progesterone (10 nM) produced a transcriptional response similar to that of 0.001 nM R1881 (Fig. 5 ) but were inactive at 0.001 nM. At 10 nM estradiol or 100 nM progesterone, CAT activity was equivalent to that induced by 0.01 nM R1881 (Fig. 5).
CAT activity induced by 100 nM cyproterone acetate or 100 nM RU486 was approximately 50 and 15-20%, respectively, of that observed with 0.01 nM R1881 (Fig. 6). In a control study not shown, RU486 failed to induce CAT activity when the full-length glucocorticoid receptor was coexpressed in CV1 cells. No significant AR-induced CAT activity was detected following incubations with flutamide or hydroxyflutamide at concentrations up to 100 nM (Fig. 6).
Thus, transcriptional activation by AR was androgen specific only at hormone concentrations less than 1 nM. At higher concentrations, induction of CAT activity reflected AR bind-AR Phosphorylation and Functional Activity

ZE), but stimulated only marginal transcriptional activation
at 100 nM (Fig. 6). Hydroxyflutamide acted, therefore, as a pure antiandrogen as proposed previously (9). The antiandrogens, flutamide, hydroxyflutamide, and cyproterone acetate, were tested for their ability to inhibit androgen-induced CAT activity. As shown in Fig. 7, flutamide (500 nM) caused about a 50% inhibition of R1881-(0.05 nM) induced CAT activity. This inhibitory effect may result in part from limited metabolic conversion of flutamide to hydroxyflutamide as previously suggested from in uiuo studies (4-7). Hydroxyflutamide-(500 nM) inhibited R1881 (0.05 nM) induced CAT activity by approximately 90%, while alone, it increased CAT activity only slightly higher than background (Fig. 7). The inhibitory activity of cyproterone acetate at 10 nM was about 50%. Higher concentrations of cyproterone acetate showed strong agonist activity as noted above. Thus all three antiandrogens inhibited androgen-induced CAT activity to different degrees, the most effective being hydroxyflutamide. were placed in 0.2% serum and not further treated (no hormone addition) or treated with 0.05 M R1881 in the presence and absence of 500 nM flutamide or hydroxyflutamide or 10 and 500 nM cyproterone acetate as indicated. Some samples as indicated received only antiandrogen treatment. The acetylated forms of ["CJchloramphenicol were separated by thin layer chromatography and the plate was exposed to x-ray film. The spots were quantitated by elution of the silica plate and radioactive scintillation counting and are shown as fold induction in the bar graph below.

DISCUSSION
The AR has high binding affinity and specificity for the biologically active androgens, but only at low steroid concentrations. Lack of AR steroid binding specificity at elevated steroid concentrations characterizes both the endogenous (34) and transiently expressed recombinant receptor as demonstrated in this report and shown previously for a truncated form of the expressed AR (37). A question addressed in the present study was whether the AR undergoes functional activation when bound to nonandrogenic hormones for which it has low to moderate binding affinity. The studies indicate that the antiandrogens, cyproterone acetate, estrogen, progesterone, and RU486, not only promote nuclear transport, but enhance transcriptional activation by AR. In contrast, only high affinity androgen binding stabilized the AR protein, slowing its rate of degradation and making it appear that androgen induced AR phosphorylation. Of the steroids and antihormones tested in the transient cotransfection assay, hydroxyflutamide had properties closest to a true antiandrogen since it inhibited androgen-induced transcriptional activation and did not significantly enhance AR-mediated transcriptional activation.
AR nuclear transport in transiently transfected COS cells was shown previously to be androgen-dependent (20). In the absence of hormone addition, AR displays a striking punctate perinuclear distribution in the cytoplasm, while androgen addition causes strong nuclear immunostaining. The perinuclear cytoplasmic localization of AR in the absence of androgen was not evident, however, with the endogenous receptor following androgen withdrawal by castration for reasons not fully understood (30). In tissue sections of rat ventral prostate, nuclear staining is strong in the intact animal but castration causes loss of immunoreactivity. It is conceivable that the rapid turnover of the AR protein in the absence of androgen as described in this study, albeit in transfected COS cells, contributes to the loss of immunstaining in ventral prostate tissue sections following castration. Steroids and antihormones that bind AR with moderate affinity and stimulate CAT activity at elevated hormone concentrations also promote AR nuclear transport in transfected COS cells. However, hydroxyflutamide caused strong nuclear immunostaining but failed to induce AR-mediated transactivation. It is clear, therefore, that nuclear localization per se is necessary but not sufficient for transcriptional activation, as suggested earlier in studies with truncated mutant forms of AR. AR deletion mutants lacking the NH2-terminal domain were constitutively nuclear yet lacked gene transcriptional activity due to deletion of sequences critical for AR function (20).
The cell systems (CV1 and COS monkey kidney cells) used in formulating conclusions concerning hormone specificity of nuclear transport, transcriptional activity, phosphorylation, and receptor turnover, could be considered somewhat artificial since they do not express AR endogenously and may be deficient in certain transcription factors required to promote hormone-specific gene activation. However, many of the results of these transient transfection studies parallel earlier observations in uiuo. For example, it was recognized that progestational steroids promote AR-mediated gene activation in uiuo and stimulate growth of the male reproductive tract and virilization of the female fetus (38)(39)(40). Furthermore, progestins potentiate nuclear uptake of AR in uiuo in mouse kidney (41). Cyproterone acetate, a progestational steroid (42), has not been considered a true antiandrogen because it has both agonist and antagonist activities in vivo (40,43).
The present study supports these observations in that cyproterone acetate acts as an androgenic agonist in AR-mediated transcriptional activation as determined by CAT assay at elevated steroid concentrations (100 nM), but as an androgen antagonist at a lower hormone concentration (10 nM). On the other hand, cyproterone acetate at elevated concentrations was antagonistic to androgen-induced AR stabilization.
The parallel between endogenous and transiently expressed AR extends to the activity of the nonsteroidal antiandrogen, hydroxyflutamide. Hydroxyflutamide, unlike cyproterone acetate, lacks agonist activity in vivo yet has antiandrogenic activity nearly equivalent to that of cyproterone acetate (2). Hydroxyflutamide has therefore been considered a pure antiandrogen (2, 3). It maintains these characteristics in the in vitro system described here since it did not enhance CAT activity, yet inhibited androgen induction of AR transcriptional activation. Since hydroxyflutamide binds AR with moderate affinity and induces nuclear transport, its binding may impose an altered receptor conformation not conducive to gene activation as suggested for RU486 binding to the glucocorticoid receptor (18).
It is intriguing to note that the androgen-dependent human prostate cancer cell line, LNCaP, responds to hydroxyflutamide as well as estradiol and progesterone with an increase in cell proliferation (44,45). Estradiol and progesterone binding was inhibited by androgen indicating that their effects were mediated by the AR (44). In the LNCaP cell line, testosterone, R1881, and cyproterone acetate each induced AR mRNA down-regulation. However, estrogen failed to down-regulate AR mRNA both in the LNCaP cell line and in normal rat prostate (46). Thus, while estrogen had agonist activity in the induction of AR gene activation, it did not mimic androgen effects on AR mRNA. The AR gene in LNCaP cells contains a single base mutation that changes amino acid residue 877 from threonine to alanine (47). In cotransfection studies with the reconstructed mutant AR, a striking increase in transcriptional activity was observed with hydroxyflutamide suggesting that an alteration of one amino acid within the steroid-binding domain allowed hydroxyflutamide to acquire agonist activity (48). The LNCaP AR also increased transcriptional activation in response to progesterone and cyproterone acetate (49).
Antihormone binding to steroid receptors can initiate early steps in gene activation, i.e. receptor entry to the nucleus and DNA binding. Progesterone receptor binding of RU486 promotes interaction with response element DNA but fails to stimulate transcription (15). Interestingly, although RU486 is a true antagonist for the glucocorticoid receptor, it had agonist activity when bound to AR. Furthermore, of those tested, RU486 was the only steroid that caused an increase in AR phosphorylation that was not offset by a concomitant increase in receptor protein due to stabilization. It was reported previously that RU486 binds endogenous AR of rat prostate (50) and inhibits androgen-induced prostate growth (51). Antagonistic effects of RU486 mediated through the AR were also suggested when it blocked androgen inhibition of prolactin release in uiuo (52).
Another mechanism proposed for the inability of antihormones to promote receptor-mediated gene transcription involves stabilization of oligomeric receptor forms. It was reported that RU486 bound to the glucocorticoid receptor in uiuo stabilizes the 8 S receptor form which does not enter the nucleus (17) or interact with DNA (53,54). The results of the present study do not support a similar effect on AR.
A question remaining from these studies is the role of AR phosphorylation in transcriptional activation by AR. The ability of AR to induce transcriptional activity in response to steroids or antiandrogens with few exceptions paralleled relative binding specificity and affinity. The apparent effect of androgen on AR phosphorylation was nullified when receptor stability was considered. Androgen increased the amount of AR phosphorylation simply by slowing the rate of degradation of the AR protein. AR stabilization by androgen was observed previously in binding studies on tissue cytosols (34). Moreover, in a ductus deferens smooth muscle tumor cell line, endogenous AR stabilization increased about 2-fold with androgen, from t1/2 3.1 h without androgen to tIl2 6.6 h with R1881 (55). The mechanism of receptor stabilization by androgen is not known but the striking specificity for androgen suggests it may be closely linked with receptor functional activity. Although other groups have reported on steroidinduced phosphorylation of AR (25) and the glucocorticoid and progesterone receptors (21)(22)(23)(24), no androgen-dependent enhancement of recombinant AR phosphorylation was detected in the present study. While the AR is clearly a phosphoprotein, the specific role of phosphorylation in receptor function is unclear. AR sites of phosphorylation are currently being mapped, and it is conceivable that androgen binding may increase phosphorylation of a single site not detectable in our assay system.