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Glucocorticoid receptors: Relations between steroid binding and biological effects
J. Mol. Biol. (1972) 67, 99-115
Glucocorticoid
Receptors : Relations between Steroid Binding and Biological Effects
GUY G. ROUSSEAU,JOHN D. BAXTER AND GORDON M. TOMKINS Department of Biochemistry and Biophysics University of California, San &an&co, Calif. 94122, U.S.A. (Received 1 October 1971) Steroid binding has been studied in cytoplasmic extracts of cultured rat hepatoma cells to investigate the mechanism of enzyme induction by glucocorticoids. The affinity of inducer steroids for the specific receptors contained in the extracts is directly related to the potency of these steroids as inducers of tyrosine aminotransferase. The ability of anti-inducer steroids to compete with inducers for binding is similar to their ability to inhibit induction. Furthermore, the affinity of an anti-inducer for the receptors can be predicted from its ability to inhibit inducer binding. These and other correlations allow distinction between the specific cytoplasmic receptors and a number of other molecules, includingplasma transcortin, which also bind glucocorticoid hormones. Further experiments were carried out to determine whether an allosteric model, proposed earlier, could explain the differential effects of inducer, suboptimal inducer and anti-inducer steroids. According to the model, steroids interact with either, or both, of two conformational states of the receptors; the uncomplexed receptors are predominantly in one (inactive) form and binding by inducers, but not by anti-inducers, increases the concentration of the other (active) conformation. Consistent with this idea, we find that receptors are less stable when they are free, or complexed by an anti-inducer, than when they are bound by an inducer. Furthermore, kinetic studies are in accordance with the proposal that binding by inducers, but not anti-inducers, is associated with conformational changes in the receptor molecules. Specific glucocorticoid receptors were also characterized in other tissues and found to be similar to those in hepatoma tissue culture cells.
1. Introduction Induction of tyrosine aminotransferase by glucocorticoids in rat hepatoma tissue culture cells is being studied to elucidate the mechanisms of steroid regulation of gene expression in eukaryotes. The earliest step known in the interaction of steroid hormones with target cells is the binding of these compounds to “specific cytoplasmic receptors” (for review, see Jensen, Numata, Brecher & DeSombre, 1971). In rat hepatoma cells we have found glucocorticoid receptors which appear to be the direct mediators of enzyme induction (Baxter & Tomkins, 1970; Baxter & Tomkins? 1971a,b). We have now studied the steroid-receptor interaction in greater detail. The results further support the idea that these receptors are involved in induction and suggest that the biological activity of steroid hormones may depend on their actions as allosteric effeotors. 99
100
G. G. ROUSSEAU,
J.
D.
BAXTER
AND
G. M. TOMKINR
2. Materials and Methods (a) Mutevials The source and purity of [1,2,4-3H]dexamethasone (9 to 12 Ci/m-mole) and of nonradioactive steroids were previously described (Samuels & Tomkins, 1970; Baxter & Tomkins, 1971~~). [1,2-3H]progesterone (33.5 Ci/m-mole), [1,2-3H]cortisol (42 Ci/m-mole) and [1,2-3H]corticosterone (44 Ci/m-mole) were obtained from New England Nuclear. Phosphate-buffered saline consisted of 0.1 M-NaCl and 0.026 M-potassium phosphate, pH 7.6. Homogenization buffer was composed of 0.02 M-N-Tris-(hydroxymethyl) methylglycine (Tricine), 0.002 M-C&I,, 0.001 M-MgCl,, pH 7.4. Activated charcoal (Norit-A, Fisher) was prepared as described (Baxter & Tomkins, 1971a). Growth medium, pH 7.6 to 7.8, was Swim’s 77 (Grand Island Biological Company, New York) supplemented with NaHC03 (0.5 g/l.), 0.05 M-Tricine, 0.002 rvr-glutamine and 10% of either calf serum or a mixture (1: 1) of fetal calf and calf serum. (b) Cell culture,
enzyme induction
and preparation
of tissue extracts
HTCt cells were grown in suspension at 37°C (Hershko & Tomkins, 1971) and harvested while in the log or early stationary phase of growth, at a density of 2 to 15 x lo5 cells/ml. Induction of tyrosine aminotransferase was performed in serum-free medium as described previously (Samuels & Tomkins, 1970). HTC cell cytoplasmic extracts were prepared according to Baxter & Tomkins (1971a). Briefly, cells were washed with ice-cold phosphate-buffered saline, disrupted in one volume of homogenization buffer using a Teflon-glass tissue grinder, and centrifuged at 100,000 g for 1 hr. The supernatant fraction (cytoplasmic extract or cytosol), kept at 0°C and used within 48 hr, was not further fractionated to avoid alterations of receptor properties observed during purification (Rousseau, Baxter & Ton&ins, unpublished observations). (c) Measurement
of steroid
binding
Many techniques employed to measure steroid binding (e.g. density-gradient centrifugation, gel filtration and equilibrium dialysis) are either time consuming or not sufficiently accurate for detailed quantitative studies of glucocorticoid-receptor interactions. The charcoal assay (Baxter & Tomkins, 1971a) is devoid of these drawbacks and, when performed as described below, can be used even if bound steroid dissociates rather rapidly. This method depends on the fact that charcoal adsorbs free, but not macromolecule-bound, steroid (Murphy, 1967; Heyns, Van Baelen & De Moor, 1967; Korenman, Perrin & McCallum, 1969). Following reaction of 3H-labeled steroid with a cell extract, the unbound steroid can be adsorbed on the charcoal which is then discarded. Providing the charcoal-containing reaction mixture is vigorously, but briefly, agitated and the charcoal immediately centrifuged, a single determination of the radioactivity remaining in the supernatant medium is sufficient for an accurate measurement of bound steroid. This procedure is more convenient than other types of charcoal assays (cf. Murphy, 1967; Heyns et al., 1967; Milgrom & Baulieu, 1969). Specific binding$ of dexamethasone was measured as follows. Portions (0.4 ml.) of cytoplasmic extract containing radioactive dexamethasone (up to 10m7 m) were incubated at 0°C. Samples containing the same concentration of radioactive dexamethasone plus a 500- to lO,OOO-fold excess of non-radioactive dexamethasone as a competitor were run in parallel for “background” (Baxter & Ton&ins, 1971a) determination. After 90 min, 50 ~1. of a suspension of activated charcoal (100 mg/ml.) was added to the samples which were immediately vigorously agitated (Vortex mixer) for 3 set and centrifuged at 600 g for 1 min. The supernatant medium was centrifuged at 10,000 g for 5 min and portions of the second supernatant fraction were counted (charcoal-resistant radioactivity). The amount of steroid specifically bound by the cytoplasmic extract was calculated by subtracting the samples from the charcoal-resistant charcoal-resistant radioactivity of the “background” radioactivity of the corresponding competitor-free sample. Free steroid concentration was t Abbreviation used; HTC, rat hepatoma tissue culture. $ By “speciflo binding” we refer to high affinity ‘binding of steroids to a limited number components without implying that the binding reaction per 8s ha a biological significance.
of
GLUCOCORTICOID
RECEPTORS
101
cslculated by subtracting the tot81 concentration of steroid bound in the 8bsence of competitor from the steroid concentration initially present in the same incubstion mixture. The following experiments establish the validity of this assay. First, charcoal is not saturated with steroid at the concentrations of steroid employed. When buffer solutions contttining 8 const8nt amount of [3H]dex8methasone and increasing concentrations of nonradioactive dexamethasone (up to 10m4 M) 8re treated with charcoal, the amount of radioactivity removed by charcoal remains constant. Also, the measured itmount of steroid specific8lly bound by the cgtoplasmic extract does not change when the concentration of added charcoal is increased from 100 to 300 mg/ml. (Fig. l(8)).
Charcoal added (mg/ml.) (0)
Time of ogitotion bet) (b)
FIQ. 1. Effect of the 8mount of charcoal and of the time of agitation with charcoal on the measurement of steroid binding. HTC cell cytoplssmic extracts (protein concentretion 1.65 mg/ml.) were incubated at 0°C with 2 x10e8 M-[3H]dex8meth8sone (12 Ci/m-mole) in the absence (-O-O-) or presence (-a----a-) of lOwE M-non-radioactive dexctmethasone. Following 90 min of incubation, 50 ~1. of a suspension of activated charcoal were agitated with 0.4 ml. of incubation mixture, and charcoal-resistant radioactivity was determined as described in Materials and Methods. Specific binding corresponds to the difference between the upper and lower curves in both parts of the Fig. (a) Effect of the amount of charcoal. The 50.~1. charcoal suspension contained varying amounts of activated charcoal; (b) effect of the time of agitation. Following the addition of 50 pl. of the charcoal suspension (100 mg/ml.), the samples were agitated for varying time-periods before centrifugation. Second, appreciable dissociation of bound steroid does not occur under the assay conditions. It c8n be seen from Fig. l(b) that measured specific binding of dexamethasone rectches, after 8 very short period, 8 level which remains relatively constant even over 60 set of agitation. This might be expected, since the dissociation of dexamethasone from the receptors 8t 0°C is quite slow (Baxter & Tomkins, 1971a). Third, the amount of specifically bound steroid is linearly related to the concentr8tion of cytoplasmic extrect. Fig. 2 shows that this holds true when the reaction mixture contains 1 to 30 mg protein/ml. Fourth, it is unlikely that charcoal destroys or binds a significant proportion of the receptors, since the use of higher concentrations of chrtrcoal (Fig. l(8)) or more prolonged 8gitation in the presence of charcoal (Fig. l(b)) do not decrease the 8mount of specificslly bound steroid. The fact that the relationship shown in Fig. 2 is linear also supports this inference. Finally, t,he amount of specifically bound dexamethasone measured by the assay is comparable to
102
G. G. ROUSSEAU,
J. D.
Concentration
BAXTER
of cytoplosmic
AND
extract
G. M. TOMKTNS
(mg protein/ml.)
Fm. 2. Relation between cytoplasmic extract concentration and specific binding. The amount. of specifically bound [oH]dexamethasone at 2 x 10e8 M was determined by the charcoal assay in various dilutions of a HTC cell cytoplasmic extract.
TABLE 1 Comparison
of charcoal assay and gel f&ration for dexamethasoneT Specific binding Charcoal assay Gel filtration
measurement
(cts/min/mg
protein)
6360 53804
(6330, 6390) (4500-6120)
of speci$c binding
oj
t HTC cell cytoplasmic extracts were incubated for 90 min at 0°C in the presence of 2 x 10-r M-[3H]dexamethasone (12 Ci/m-mole) with or without competing 10e5 M-non-radioactive dexamethasone. Two 0.4-ml. portions were assayed by the charcoal method and 3 other portions (0.4 to 1 ml.) were filtered at 4°C through a G25 Sephadex column (1 cm x 7.2 cm, flow rate 0.9 ml./min) which was eluted with homogenization buffer. $ Difference in the specific radioactivity of excluded volume between competitor-free and competitor-containing extracts (mean and range of 3 experiments).
that estimated by gel filtration (Table 1). With the charcoal assay, the results are more reproducible and denaturation of the receptors as seen upon gel filtration (Baxter & Tomkins, 19716) can be avoided. The assay can be adapted to measurement of specific binding of other steroids by HTC cell receptors. Experiments similar to those described in Figs 1 and 2 have established that progesterone binding can be determined accurately even though it dissociates from HTC cell receptors much faster than dexamethasone (see below). The charcoal assay was also used for studying steroid binding by rat liver cytosol and plasma transcortm. In the latter case, plasma must be diluted 20- to 200-fold. Otherwise, most of the steroid may be bound at the concentration ordinarily used (about lo-* M), rendering difficult an accurate determination of the free steroid concentration. (d) Other methods Tyrosine aminotransferase activity and protein concentration were assayed as described previously (Samuels L Tomkins, 1970). For measurement of radioactivity, aqueous samples were dissolved in toluene containing 4 g Omnifluor (New England Nuclear)/l. and 10% Biosolve (formula BBS-3, Beckman Instrument Company) and counted in a Beckman scintillation spectrometer with an efficiency of 44%.
GLUCOCORTICOID
RECEPTORS
103
3. Results (a) Interaction of inducer steroids with the receptors From earlier work (Baxter & Ton&ins, 1971a) we concluded that the interaction between dexamethasone and the receptors conforms to a reversible equilibrium reaction with a single class of receptor sites. Indeed, a plot (Scatchard, 1949) of the ratio of bound to free dexamethasone as a function of bound dexamethasone is linear. Also, the kinetics of binding are second order (proportional to the concentration of steroid and receptors) and the kinetics of dissociation are first-order (proportional to the concentration of the complex). This interaction appears to be involved in the hormonal induction of enzymes. Consistent with this view, the dexamethasone concentrations required for binding, and for induction of tyrosine aminotransferase or cell adhesiveness are very similar, and the ability of other steroids to compete with dexamethasone for binding is related to their effects on induction (Baxter & Tomkins, 1971a; Tomkins et al., 1970). In the present study we examined the binding characteristics of other steroids which, like dexamethasone, induce tyrosine aminotransferase to a high level (“optimal inducers”, Samuels & Tomkins, 1970). Maximal induction is attained at steroid concentrations which differ for various optimal inducers. The biological potencies of four of these, dexamethesone, corticosterone, cortisol and eldosterone, were compared by examining the steroid concentration required for half-maximal induction (Table 21). Binding of the same steroids by the receptors was also measured. The Scatchard plots of the binding data (see Fig. 3 and Rousseau, Baxter, Funder, Edelman & Tomkins, 1972) are linear, suggesting that each optimal inducer tested TABLE 2
Comparison of specijc binding properties of optimal inducers with their ability to induce tyrosine aminotransferase
Steroid
Dexamethasone Corticosterone Cortisol Aldosterone
Steroid concentration (an) required for halfmaximal inductiont
1.5 x10-s (1-2*5x 10-Q 1~3x10-‘(10-s-2x10-7)~ 1.9x10-7 (1.3-3x10-78 4x10-‘/[
Apparent equilibrium (dissociation) constant
at O”’ (“)s
3.1 x10-e 4.3x10-9 1.1 x10-8 2.5x10-s
Cytoplasmic receptor-site concentration (pmoles/mg protein)1 0456 0436 063 0.67
t HTC cells were incubated at 37“C in serum-free medium in the presence of various ooncentrations of steroid and were assayed for tyrosine aminotransferase activity as described in Materials and Methods. $ HTC cell cytoplasmic eatracts were incubated with various concentrations of 3H-labeled steroid at 0°C. Specific binding was determined by the charcoal assay and a Scatchard plot of the data (see Fig. 3) was linear in each case. The apparent equilibrium (dissociation) constant was calouleted from the slope of the Scatchard plot and the concentration of receptor sites was estimated from the intercept with the abscissa. $ Range from 3 or 4 experiments. jl Taken from Samuels I% Tomkins, 1970. t The present finding that corticosterone observations (Samuels & Tomkins, 1970).
is more potent than cortisol is in contrast with previous
104
G. G. ROUSSEAU,
J. D.
BAXTER
Bound steroid
AND
G. M.
TOMKINS
i M x IO9 i
Fro. 3. Specific cytoplasmic binding of cortisol and corticosterone. Specific binding of various or [sH]cortioosterone (-+---•--) by HTC cell concentrations of [sH]cortisol (- O----O--) cytoplasmic extracts (3.4 mg protein/ml.) was measured by the charcoal assay after 90 min of incubation. The data are plotted by the Scatchard technique. That 90 min of incubation allowed for equilibrium to be established w&s determined in separate experiments. interacts with a single class of receptor sites. Since the concentration of sites is the same for each steroid (Table 2) and since all these steroids compete with each other for binding (Table 4, Baxter & Tomkins, 1971a and unpublished observations), the same sites are probably involved in the binding reactions. Comparison of the equilibrium (dissociation) constants of the specific binding reactions for the four optimal inducers (Table 2) shows that their biological potency is directly related to their affinity for the receptors. These correlations strongly support the view that HTC cell cytosol contains a receptor molecule which is involved in the hormonal action.
(b) Interaction of anti-inducer steroids with the receptors A number of steroids have been classified as anti-inducers since at appropriate concentrations they inhibit *he induction of tyrosine aminotransferase by inducers (Samuels & Tomkins, 1970). Anti-inducers also inhibit specific cytoplasmic binding of dexamethasone (Baxter & Tomkins, 1971a). The data in Figure 4 show that the antiinducer progesteronet (see Table 3) behaves as a competitive inhibitor of dexamethasone binding. Assuming that progesterone + receptor + progesterone-receptor complex, we calculated from the competition data the apparent equilibrium (dissociation) constant to be 2.9 f O-8(standard error of the mean) x lo- * M at 0°C. Furthermore, t Progesterone and 1’7 ar-hydroxyprogesterone were olassified (Samuels & Tomkins, 1970) as sub-optimal inducers since they were found to induce tyrosine aminotransferase by about 5% as observed in our compared to cortisol (see Table 4). However, this effect was not consistently studies. Consequently, we have considered these steroids as anti-inducers because they strongly inhibit induction by inducers.
GLUCOCORTICOID TABLE
Inhibition
105
RECEPTORS
3
by anti-inducers of speci$c binding or enzyme induction by dexamethaaone Induction of tyrosine aminotransfer8%3 by 10 -s na-dexamethasone in the presence of anti-inducer ( oJ of control)t
Anti-inducer
None Progesterone 0 lo-’ M lo-’ M lo-‘M
lo-srd
[sH]Dexamethasone (10-s M) bound in the presence of anti-inducer (Oh of control)$
100
100
113 93 44 12
100 70 9 2
97 100 44 18
105 103 64 22
17 m-methyltestosterone lo-‘M lo-’ lo-’
M
M
10-s M
t Inductions were performed as described in Materials and Methods, using the steroid concentrations indicated. 100% induction was 130 to 178 m-units of enzyme/mg protein depending on the experiment. $ HTC cell cytoplasmio extracts were incubated for 90 min at 0°C with 10-s M-[sH]dexa.methasone (12 Ci/m-mole) plus various concentrations of non-radioactive steroids, and the amount of specific dexamethasone binding was determined. The 100% value was 13,500 to 36,700 ots/min/ ml. incubation depending on the concentration of cytoplasmic extraot. 5 Mean from 2 or 3 experiments.
when interaction of [3H]progesterone with HTC cell extracts is examined directly, speckfit binding can be demonstrated (Fig. 5), and a Scatchard plot of the data is linear (inset of Fig. 5), suggesting that the assay detects binding to a single class of receptor sites. The apparent equilibrium (dissociation) constant for the reaction was found to be 2.2 to 2.5 x 10-s M (3 experiments), in good agreement with the value calculated
FIG. 4. Competitive inhibition of specific dexamethasone binding by progesterone. Specific binding of [3H]dexamethasone by HTC cell oytoplasmic extracts was determined (protein concentration: 5.9 mg/ml.) at various concentrations of [3H]dexamethasone and competing nonradioactive progesterone. The co-ordinates refer to the reciprocal of the molar concentration of bound (l/B) or free (l/P) dexamethasone.
106
G. G.
ROUSSEAU,
J. D.
BAXTER
AND
Free progesterone
(M x IO’)
G. M.
TOMKINS
FIQ. 5. Progesterone binding by HTC cell receptors. Specific binding of various concentrations of [3H]progesterone by HTC cell cytoplasmic extracts (protein concentration: 5.4 mg/ml.) was determined by the charcoal assay. The time of incubation was 90 min. The So&chard analysis of the data is shown in the inset.
from the competition studies. Also the concentration of progesterone receptor sites (indicated by the intercept of the Scatchard plot on the abscissa) differed by less than 20% from the value obtained with dexamethasone. In other experiments, dexamethasone prevented specific binding of [3H]progesterone to the extent predicted from the relative aflinities of the two steroids for the specific receptors. We conclude that dexamethasone and progesterone bind to the same receptor site and that competitive inhibition of inducer binding by anti-inducers accounts for the biological effects of the
I
I
I
I
I
I
I
20
40
60
90
120
180
Time(min)
FIG. 6. Kinetics of dexamethasone and progesterone association with the cytoplasmic receptors. HTC cell cytoplasmic extracts were incubated (6.0 mg protein/ml.) at 0°C with [3H]dexamethaaone or [3H]progesterone at the concentrations indicated in the Fig. (with or without low5 M competing non-radioactive steroid, see Materials and Methods). At the time-intervals indicated, 0.4-ml. portions were agitated with 50 ~1. of charcoal and the amount of speifioally bound steroid was determined.
GLUCOCORTICOID
107
RECEPTORS
latter. In support of this idea, the anti-inducers progesterone and 17 or-methyltesto.. sterone inhibit induction to a degree which closely parallels their ability to compete with dexamethasone for binding (Table 3). Similar correlations were found when the effect of these anti-inducers on induction or binding by cortisol or corticosterone was examined. (c) Different interactions of inducers and anti-inducers with the receptors The effect of steroids on the synthesis of tyrosine aminotransferase has been explained by proposing that these hormones interact with an allosteric receptor system (Samuels & Tomkins, 1970). According to this model, uncomplexed receptors exist predominantly in an inactive conformation. Inducer binding promotes a shift toward an active conformation, while anti-inducers bind to the inactive form and do not cause an increase in the concentration of active receptors. It is consistent with the model that certain properties of the dexamethasone-bound receptors (presumably in the active conformation) differ from those of uncomplexed receptors (Baxter & Tomkins, 1971a). Furthermore, the association of dexamethasone with the specific receptors is slow despite the high affinity of the interaction, suggesting that conformational changes might be associated with the binding (Baxter & Tomkins, 1971a). If this interpretation is correct, the anti-inducer progesterone might bind faster than dexamethasone in spite of its lower affinity. This would arise because, in the absence of steroid, the receptor form which binds progesterone (i.e. the inactive form) would exist at a higher concentration than the active form bound by dexamethasone. This prediction was verified (Fig. 6). At the receptor-site concentration A
A
2.0
J
bU
YV
120
150
180
Time( min)
FIQ. 7. Kinetics of dexamethasone and progesterone dissociation from the cytoplasmic receptors. HTC cell cytoplasmic extracts were incubated at 0°C (protein concentration: 5 mg/ml.) in the presence of 10-s M-[3H]dexamethssone (with or without 10-r competing non-radioactive dexamethasone for baokground determination, see Materials and Methods). At equilibrium, i.e. after 90 min of incubation (see Fig. 6) non-radioactive dexamethasone to a final concentration of 10e5 M was added to half of each incubation mixture (zero-time on the Fig.). At the time intervals indicated, charcoal was agitated with 0.4-ml. portions and the amount of specifically bound steroid was determined. The same procedure was simultaneously performed using radioactive (4 x 10-s M) and non-radioactive ( 10m5 M) progesterone. The amount of specifically bound dexamethasone (--A----A--) and progesterone (-A---A-) without added competitor or following the addition of the competitor (--O----e-for dexamethasone and -O-O-for progesterone) is shown.
108
G. G. ROUSSEAU,
J. D.
BAXTER
AND
G. M.
TOMKINS
ordinarily employed in the binding experiments (2 x lows M), the time for half maximum binding is 4 minutes for 10e8 M-progesterone and 22 minutes for lo-* M-dexamethasone. The rate of specific binding of cortisol, another inducer whose afinity is slightly higher than that of progesterone (see Table 2), was also measured. As expected, this rate was slower than that of progesterone (12 min for half-maximal binding of 4 x IO-* M-cortisol as compared with 2 min for 4 x lo-* M-progesterone). Consistent with its rapid rate of association and lower affinity, progesterone dissociates from the receptor more rapidly than dexamethasone (Fig. 7). The rate of progesterone dissociation is first-order since a @ot of the log of bound steroid as a function of time is linear. This is in accord with the proposed reaction : progesterone + receptor +Gprogesterone-receptor complex. The dissociation rate constant (determined from the time of half-dissociation) is 3.4 (range 2.7 to 5.8) x 10W2min-l as compared to 3.0 x 10m3 min-l for dexamethasone (see Baxter & Tomkins, 1971a). Uncomplexed receptors are more thermolabile than dexamethasone-bound receptors (Baxter & Ton&ins, 1971a). If the receptors which bind progesterone are in the same (inactive) conformation as unbound receptors, the thermostability of progesterone-receptor complexes might be different from that of inducer-bound receptors. In fact, receptors bound by dexamethasone (Fig. 8) or cortisol were found to be more thermostable than those which are bound by progesterone or are uncomplexed.
Dexamethasone,
I 30
I 60
I 90 Time (min)
I 120
I 150
20°C
I 180
FIQ. 8. Kinetics of heat denaturation of dexamethasone-bound and progesterone-bound cytoplasmic receptors. An HTC cell cytoplasmic extract was incubated (final protein concentration: 5 mg/ml.) in the presence of lo-* m-[3H]dexamethasone for 90 min at 0°C. Then (zero-time on the Fig.) half of the incubation mixture was transferred into a water-bath at 20°C. At the time periods indicated, samples were removed from both incubations, chilled to 0°C for 10 min, and then specific binding was determined. An identical experiment was simultaneously performed using the same cytoplasmic extract and, instead of dexamethasone, 4 x 10 -* ar-[3H]progesterone.
(d) Relation between steroid structure, qveci$c binding and biological activity The consequence of variations in steroid structure on the biological effect of glucocorticoids has been discussed previously (Samuels & Tomkins, 1970). We have now examined the influence of such modifications on steroid-receptor binding (Table 4). Addition of an hydroxyl group in the a-configuration on carbon 17 of the steroid molecule decreases both the affinity and the biological potency of the steroid, but the resulting compound retains the properties of the original steroid as an optimal inducer,
ll-deoxycortisol 1 l-deoxycortioosterone Progesterone 11 -deoxyoortisol Progesterone
5 B-H
keto
j3-OH
or-OH
A 4-5
None
Xone
NO%3
17 a-methyltestosterone
1 l-deoxycortisol Progesterone
Cortisol
B
30
11 B-OH progesterone Epicortisol 11 a-OH progesterone 11 a-OH, 17 01methyltestosterone
78
Corticosterone
Ob
30
48
48
30
48
47
47
74
68 30
A
0
2
0
74
68
47
17
Ob
43
17
31
47 14
B
Inhibition of dexam&hasone bindingC
11 -keto progesterone Cortisol
Cortisol 17 or-OH progesterone 2 1-deoxycortisol 6 or-dihydrocortisol 5 ,$dihydrocortisol Cortisone
Steroids
to cytoplasmic
4
0
O-6
24
o-6
44
24
&6
24
100
100
33
100 O-6
A
0
0
0
0
33
100
100
-
-
34
71
100 5
B
Tyrosine aminotransferase inductiond
0
0
0
0
0
41
-
52
-
10-s -M-cort,isnl:
-
Inactive
Anti
at a higher
Inactive
Suboptimal Inactive
Optimal
Optimal
Anti
Suboptimal Suboptimal -
Optimal Anti
B
Suboptimal Anti
Suboptimal Suboptimal Anti
Suboptimal Anti
Optimal
Suboptimal Optimal
31
Optimal Anti 0 84
they are anti-inducers
88
85
55
86
21
55
85
55
0
0
41
0 85
A
Classification of the steroid (type of inducer’)
actions
Inhibition of tyrosine amino transferase inductione A B
receptors and on biological
a Induction data are taken from Samuels I% Tomkins, 1970. b Although lo-’ M-cortisone or 17 a-methyltestosterone do not inhibit binding of 10-s M-[sH]dexamethasone concentration (Baxter & Tomkins, 1971a). 0 Amount (%) by which competing steroids ( low7 M) inhibit the specific binding of 10-s M-[3H]dexamethasone. 6 Induction (net steady-state enzyme activity) by 10m5 M-steroid as percentage of the induction obtained with e Amount (94) by which competing steroids (10m5 M) inhibit induction by lo-? nr-oortisol. I According to Samuels & Tomkins (1970). See also Footnote on page 104.
c-11
5 LX-H
A 4-5
11 B-OH Progesterone Cortisol
Corticosterone Progesterone
A
c-4,5
ar-OH
Substituted (‘3
None
Unsubstituted (4
Substitution
TABLE
in the steroid molecule on binding
c-17
Position on steroid molecule
Effect of substitutions
110
G. G. ROUSSEAU,
J. D.
BAXTER
AND
G. M.
TOMKINS
a sub-optimal inducer or an anti-inducer. In terms of the allosteric model, this substitution diminishes to a comparable extent the affinity of the steroid for both the inactive and the active conformations of the receptors. Addition of an hydrogen atom in the 01configuration on carbon 5, or addit,ion of a keto group on carbon 11 yields a steroid with less affinity but more anti-inducer activity; according to the model, this might be due to a preferential decrease in the affinity of the steroid for the active form of the receptor. Conversely, addition of an hydroxyl group in the /3-configuration on carbon 11 increases the inducing effect of the steroid while its affinity for the receptor can be increased or decreased. This change appears, t.herefore, to enhance the affinity of the steroid for the active conformation and to decrease its affinity for t,he inactive form. Finally, upon addition of an hydroxyl group in the a-configuration on carbon I 1, both the binding affinity and the ability to influence induction are lost. Thus, the biological potency of steroids as well as their qualitative effects as inducers, antiinducers and sub-optimal inducers may be explained in terms of their relative affinities for the two conformations of the receptor.
(e) Difference between the specrific glucocorticoid receptors and rat transcortin Mammalian plasma contains steroid-binding proteins and the question of whet’her these proteins play an obligatory role in steroid action has been considered (for review, 1966). Corticosteroid-binding see Sandberg, Rosenthal, Schneider & Slaunwhite, globulin (transcortin or CBG) is the only plasma protein which binds cortisol, corticosterone and progesterone with high affinity (for review, see Westphal, 1969). These steroids are also bound by the HTC cell specific receptors. On the other hand, transcortin, presumably of liver origin, might be present in HTC cells. The binding properties of the receptors were therefore compared with those of rat transcortin. As discussed above, specific binding of cortisol by rat plasma can be determined by the charcoal assay (Fig. 9). The value found for the apparent equilibrium (dissociation) constant (2.3 x 10m8 M at 0°C) and the concentration of binding sites (5 x low7 M) are similar to those reported for rat transcortin by other investigators using different techniques (Westphal, 1969). The specific cortisol-binding activity of rat plasma measured by the charcoal assay was abolished upon heating for 30 minutes at 60°C but
Free cortisol
( M x IO*)
FIG. 9. Specific binding of cortisol by rat transcortin. Incubations containing diluted (1: 80) plasma from male adrenalectomized Sprague-Dawley rats and various concentrations of [3H]cortisol (with or without 10m5 M competing non-radioactive cortisol) were performed. After 90 min at 0°C specific binding was determined by the charcoal assay.
GLUCOCORTICOID
111
RECEPTORS
TABLE 5
Binding of dexamethasone and2wrtisol by HTC cell specific receptors and by rat transwrtin Amount
of specifically
bound 3H-labeled loll)
steroid
(M X
Rat plasma HTC cell oytoplasmic
Cortisol 581 86
extract
Dexamethasone 0 96
Endogenous steroids were removed from a mixture of female and male Buffalo rat plasma using the method described by Heyns et al. (1967). Specific binding of 10-s M-[3B]cortisol and lo-* M-[3H]dexamethasone by plasma (diluted 1: 7) or by HTC cell cytoplasmic extracts (4 mg protein/ml.) was measured after incubation at 0°C.
TABLE 6
Effect of dexamethasone and cortisol on specific cortisol binding by transcortin Concentration of nonradioactive steroid (M)
None 2x10-s lo-’ 10-s 10-s
Amount
of specifically bound [sH]oortisol (%) in presence of: Cortisol Dexamethasone 100 94 84 40 6
100 102 101 94 89
Rat plasma was prepared as described in the legend of Table 5 and specific binding of 10 -* M-[sH]oortisol in the presence of non-radioactive dexamethasone or oortisol was determined.
not at 45”C, as expected from the known properties of transcortin (Westphal, 1969). The specific HTC cell receptors are clearly different from rat transcortin since binding of dexamethasone by the latter was not detectable (Table 5). This conclusion is fortified by competition studies (Table 6). Whereas a lOOO-fold excess of non-radioactive cortisol completely prevents binding of lo-* M-[3H]cortisol by transcortin, the same excess of non-radioactive dexamethasone decreases [3H]cortisol binding by only 11 y’. These results make it very unlikely that transcortin plays an obligatory role in enzyme induction. (f) Glucocorticoid receptors in liver Contrary to rat plasma, rat liver cytoplasmic extracts a0 contdn specific receptors for dexamethasone (Fig. 10). A Scatchard plot of the binding data is linear (inset of Fig. 10) suggesting a single class of specific sites for this steroid. The apparent equilibrium (dissociation) constant, 7.4 x 10-O M at O”C, suggests that the afEnity of the rat liver receptors for dexamethasone is slightly lower than that of the HTC cell receptors. Yet, the ability of various steroids to compete with dexamethasone for specific binding is similar in both HTC cell and liver extracts (Table 7). Therefore, the specific HTC cd and liver clexamethasone receptors appear to be very much alike.
112
G. G. ROUSSEAU,
J. D.
BAXTER
AND
Free dexameihosone
G. M.
TOMKINS
(M x 10’)
FIG. 10. Specific binding of dexamethasone by rat liver. An adrenalectomized male SpragueDawley rat was anesthetized with ether and the liver was flooded with ice-cold phosphate-buffered saline by injection through the aorta. The liver was removed and cut into small pieces which were resuspended in one volume of homogenization buffer. The cytoplasmic extract was then prepared as for HTC cells. The liver extract was incubated (4.1 mg protein/ml.) for 90 min at 0°C with various concentrations of [3H]dexamethasone and the amount of specific binding measured by the charcoal assay. A Scatchard analysis of the data is shown in the inset. Separate experiments verified that the 90 min period of incubation allowed for equilibrium to be reached.
TABLE
Effect of various
7
steroids on speci$c dexamethasone binding cytoplusmic extracts
Competing
steroid
(10e5 M)
None Androstenedione Epicortisol Testosterone 17 cu-Hydroxyprogesterone Dexamethasone
by rat liver
or HTC
cell
Amount (%) of specifically bound [3H]dexamethasone in the presence of competing steroid HTC cell Liver 100 92 84 56 22 0
100 19 99 27 2 0
Specific binding of 10-s M-[aH]dexamethasone (12 Ci/m-mole) by Buffalo rat liver cytoplasmic extracts (13.6 mg protein/ml.) was measured. The extracts were prepared as described in the legend of Fig. 10 except that the rats used were not adrenalectomized. The 100% value was 7290 cts/min/ml. Duplicate determinations differed by less than 5 %. Data for HTC cells were baken from a previous report (Baxter & Tomkins, 1971a).
4. Discussion In the present study a number of correlations were established between the specific interaction of steroids with HTC cell cytoplasmic receptors and the biological effect of these hormones. Such findings, along with those already presented (Baxter & Tomkins, 1970;1971a,b), strengthen our impression that the receptors are involved in the hormonal induction of tyrosine aminotransferase. Optimal inducers bind to the receptors with an a%nity which is directly related to their biological potency (Table 2).
GLUCOCORTICOID
RECEPTORS
113
The concentrations of cortisol, corticosterone and aldosterone necessary for halfmaximal binding are 15 to 30 times lower than those which produce half-maximal induction. This may be due to the fact that, in contrast to dexamethesone, these inducers are metabolized by the cells (Baxter & Tomkins, 1970) but not by cell-free extracts. Furthermore, the cell-free birding reactions are performed at 0°C while the induction studies &re conducted in whole cells at 37°C. Therefore, a close correlation between steroid binding and induction (Baxter t Tomkins, 1971a; Tomkins et al., 1970) may be best established in the case of dexamethasone. Our data also indicate that anti-inducers bind to the receptors with an afEnity predicted from their capacity to compete with optimal inducers for binding. This effect is, in turn, related to the ability of anti-inducers to inhibit tyrosine aminotransferase induction. As previously shown (Baxter & Ton&ins, 1970;1971a) and described here in greater detail, suboptimal inducers compete with inducers for binding, whereas inactive steroids have very little capacity to do so. Although the kinetics of clexamethasone binding and dissociation at 0°C are very slow (Baxter & Tomkins, 1971a) they are rapid enough at 37°C (Baxter 6 Tomkins, 1970) to account for the kinetics of induction ancl deinduction (Tomkins et al., 1970). Another indication that the glucocorticoicl receptors mediate the hormonal effect is provided by studies on lymphoid tissue (Hollander $ Chiu, 1966; Baxter, Harris, Tomkins & Cohn, 1971), fibroblasts (Hackney, Gross, Aronow & Pratt, 1970) and other HTC cell lines (Levisohn & Thompson, 1972) which showed that an absent or decreased sensitivity to steroids is accompanied by a diminished concentration of specific glucocorticoicl receptor sites. Dexamethasone-binding molecules very similar to the HTC cell cytoplasmic receptors are probably present in most, if not all, glucocorticoicl-responsive tissues. They have been detected in rat liver (see above) and kidney (Rousseau et al., 1972), and in mouse lymphoma cells (Baxter et al., 1971; Rosenau et al., 1972). The specific glucocorticoid receptors differ from a number of other steroid binding components which do not seem to be directly involved in steroid action. The protein termed P2, present in HTC cells (Gardner & Ton&ins, 1969), can be distinguished from the receptors since it has a greater affinity for cortisol than for clexamethasone, it strongly binds the “inactive” steroid androsteneclione and it is not s&nded at physiological concentrations of inducer steroids. Rat plasma transcortin (Westphal, 1969) which is also present in the liver, and another liver cytoplasmic protein (binder A) described by Be&o, Schmicl, BrLenclle, Biesewig & Sekeris (1971) bind cortisol and corticosterone with high affinity. In contrast with the binding component we have characterized in the liver, both transcortin and binder A (Be&o, M. & Koblinski, M., personal communication) have a very low affinity for the potent glucocorticoid clexamethasone. Distinction can also be made, on the basis of the steroid structural requirements for binding, between the specific glucocorticoicl receptors and the steroid-binding fractions I, III and IV isolated from rat liver cytosol by Litwack, Morey & Ketterer (1972). On the other hand, fraction II studied by these investigators shares several binding properties with the HTC cell receptors ancl may be identical to them. Glucocorticoid binding by histones has been reported (Sluyser, 1966,1969; Sunaga & Koicle, 1967). Yet, this interaction is of questionable biological significance since it involves a covalent linkage between the arginine or lysine residues of the histone and the biologically inactive 21dehydro derivative of the steroids (Moncler & Walker, 1970). We have also found, like many investigators, that steroids bind to subcellular components other than the specific receptors. These are termed “non-specific” binding fractions 8
114
G. G. ROUSSEAU,
J. D. BAXTER
AND
G. M. TOMKINS
because they are not saturated with steroid at concentrations well above those which elicit the maximal biological response. A further suggestion that these compounds are not involved in the induction of tyrosine aminotransferase in HTC cells is that they do not bind steroids in relation to their biological potency. We have interpreted the data presented in terms of the proposed model (Samuels & Tomkins, 1970) that steroid hormones intluence the equilibrium between active and inactive conformations of receptor molecules. The model predicts that molecules in the inactive conformation predominate in the absence of steroid, and that anti-inducers bind to this form. It also provides an explanation for the effects of sub-optimal inducers by assuming that they bind to both conformations of the receptor (Samuels & Tomkins, 1970; Rubin & Changeux, 1966). Consistent with the model are the observations that uncomplexed receptors and receptors bound to anti-inducers are more thermolabile than inducer-bound receptors, and that anti-inducers bind steroids much more rapidly than inducers. Even if this model is valid, the available data do not, allow distinction between induced conformational changes (Koshland, 1963) or stabilization of pre-existing states (Monod, Wyman & Changeux, 1965). In any case, we find no evidence of co-operativity either for inducer or anti-inducer binding, or for competition of anti-inducers (progesterone or 17 or-methyl testosterone) with dexamethasone for binding. The allosteric model is further supported by studies on intact cells where dexamethasone-bound but not progesterone-bound receptors accumulate in the cell nucleus (Baxter, Rousseau t Tomkins, 1971). The ability of the receptor to localize in the nucleus could then be a property of the active but not of the inactive conformational state. Although the uncomplexed or anti-inducer-bound receptor has been termed inactive, it may still play a physiological role, for example, as a negative control element in the cytoplasm. It is noteworthy that estrogens influence the properties of crystalline bovine live1 glutamate dehydrogenase in vitro. Detailed physical, kinetic and immunological studies have shown that this is due to alterations in conformation, catalytic activity
and substrate specificity of this enzyme (Tomkins & Yielding, 1961; Tomkins, Yielding, Talal & Curran, 1963). Such observations and the similarities between glucocorticoid
receptors
and receptors
for
other
steroid
hormones
(Jensen,
Numata,
Brecher & DeSombre, 1971) reinforce our impression that steroid hormones act as allosteric effecters in carrying out their biological function. A preliminary account of some of these results has been presented (Baxter et al., 1971). We thank Mrs Barbara Levinson for growing the HTC cells, Mrs Diane Marver and Dr Isidore Edelman for providing adrenalectomized rats and Miss Christina Benson for help. This work was supported by grant no. GM 17239 from the National Institute of General Medical Sciences of the National Institutes of Health. One of us (G. R) is Charge de Recherches du Fonds National de la Recherche Soientifique (Belgium) and recipient of a Public Health Service International Postdoctoral fellowship (no. lF05-TW-1725). Ono of us (J. B.) is a Dernham Senior Fellow of the American Cancer Society, California Division (no. D-177).
Baxter, Baxter, Baxter,
REFERENCES J. D., Harris, A. W., Tomkins, G. M. & Cohn, M. (1971). Science, 171, 189. J. D., Rousseau, G. G. & Tomkins, G. M. (1971). Fed. PTOC. 30, 1048. J. D. & Tomkins, G. M. (1970). PTOC.Nat. Acad. Sci., Wash. 65, 709.
GLUCOCORTICOID
RECEPTORS
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Baxter, J. D. & Tomkins, G. M. (1971a). Proc. Nat. Acud. Sci., Waeh. 68, 932. Baxter, J. D. & Tomkins, G. M. (1971b). Advances in Biosciences, 7, Schering Workshop on. Steroid Hormone “Receptors”. Ed. by G. Rasp& p. 331. Oxford: Pergamon Press. Beato, M., S&mid, W., Brliendle, W., Biesewig, D. & Sekeris, C. E. (1971). Advances in Biosciences, 7, Schering Workshop on Steroid Hormone “Receptors”, Ed. by G. Rasp& p, 349. Oxford: Pergamon Press. Gardner, R. S. & Tomkins, G. M. (1969). J. Biol. Chem. 244, 4761. Hackney, J. F., Gross, S. R., Aronow, L. & Pratt, W. B. (1970). Mol. Pharmacol. 6, 500. Hershko, A. & Tomkins, G. M. (1971). J. Biol. Chem. 246, 710. Heyns, W., Van Baelen, H. & De Moor, P. (1967). Cl&. Chim. Acta, 18, 361. Hollander, N. & Chiu, Y. W. (1966). Biochem. Biophys. Res. Comm. 25, 291. Jensen, E. V., Numata, M., Brecher, P. I. & DeSombre, E. R. (1971). The Biochemistry oj Steroid Hormone Action. Biochemistry Society Symposium no. 32, Ed. by R. M. S.. Smellie, p. 133. London: Academic Press. Korenman, S. G., Perrin, L. E. & McCallum, T. P. (1969). J. C&in. Endocr. 29, 879. Koshland, D. E. (1963). Cold Sz)r. Harb. Symp. Quant. BioZ. 28, 473. Levisohn, S. R. & Thompson, E. B. (1972). Nature New BioZ. 235, 102. Litwack, G., Morey, K. S. & Ketterer, B. (1972). In Efsects of Drugs on Cellular ControE Mechanisms. Ed. by B. Rabin & R. Freeman, London: Macmillan. Milgrom, E. & Baulieu, E. E. (1969). Biochim. biophys. Acta, 194, 602. Monder, C. & Walker, M. C. (1970). Bioctimistry, 9, 2489. Monod, J., Wyman, J. & Changeux, J. P. (1966). J. Mol. BioZ. 12, 88. Murphy, B. E. P. (1967). J. CZin. Endocr. 27, 973. Rosen&u, W., Baxter, J. D., Rousseau, G. G. & Tomkins, G. M. (1972). Nature New BioZ. in the press. Rousseau, G. G., Baxter, J. D., Funder, J., Edelman, I. S. & Tomkins, G. M. (1972). J. Steroid Biochemistry, in the press. Rubin, M. M. & Changeux, J. P. (1966). J. Mol. BioZ. 21, 265. Samuels, H. H. & Ton&ins, G. M. (1970). J. Mol. BioZ. 52, 57. Sandberg, A. A., Rosenthal, H., Schneider, S. L. & Slaunwhite, W. R., Jr. (1966). Steroid Dynamics, Ed. by G. Pincus, T. Nakao & J. F. Tait. p. 1. New York: Academic Press.. Scatchard, G. (1949). Ann. N. Y. Acud. Sci. 51, 660. Sluyser, M. (1966). J. Mol. BioZ. 19, 591. Sluyser, M. (1969). Biochim. biophys. Acta, 182, 235. Sunaga, K. & Koide, S. (1967). Biochem. Biophys. Res. Comm. 26, 342. Tomkins, G. M., Martin, D. W., Jr., Stellwagen, R. H., Baxter, J. D., Mamont, P. & Levinson, B. B. (1970). Cold Spr. Harb. Symp. Quant. BioZ. 35, 635. Tomkins, G. M. & Yielding, K. L. (1961). CoZd Spr. Harb. Symp. Quunt. Biol. 26, 331. Tomkins, G. M., Yielding, K. L., Talal, N. &z Curran, J. F. (1963). Cold Spr. Harb. Symp. Quant. BioZ. 28, 461. Westphal, U. (1969). Methods in. Enzymology, 15, 761.
Glucocorticoid
Receptors : Relations between Steroid Binding and Biological Effects
GUY G. ROUSSEAU,JOHN D. BAXTER AND GORDON M. TOMKINS Department of Biochemistry and Biophysics University of California, San &an&co, Calif. 94122, U.S.A. (Received 1 October 1971) Steroid binding has been studied in cytoplasmic extracts of cultured rat hepatoma cells to investigate the mechanism of enzyme induction by glucocorticoids. The affinity of inducer steroids for the specific receptors contained in the extracts is directly related to the potency of these steroids as inducers of tyrosine aminotransferase. The ability of anti-inducer steroids to compete with inducers for binding is similar to their ability to inhibit induction. Furthermore, the affinity of an anti-inducer for the receptors can be predicted from its ability to inhibit inducer binding. These and other correlations allow distinction between the specific cytoplasmic receptors and a number of other molecules, includingplasma transcortin, which also bind glucocorticoid hormones. Further experiments were carried out to determine whether an allosteric model, proposed earlier, could explain the differential effects of inducer, suboptimal inducer and anti-inducer steroids. According to the model, steroids interact with either, or both, of two conformational states of the receptors; the uncomplexed receptors are predominantly in one (inactive) form and binding by inducers, but not by anti-inducers, increases the concentration of the other (active) conformation. Consistent with this idea, we find that receptors are less stable when they are free, or complexed by an anti-inducer, than when they are bound by an inducer. Furthermore, kinetic studies are in accordance with the proposal that binding by inducers, but not anti-inducers, is associated with conformational changes in the receptor molecules. Specific glucocorticoid receptors were also characterized in other tissues and found to be similar to those in hepatoma tissue culture cells.
1. Introduction Induction of tyrosine aminotransferase by glucocorticoids in rat hepatoma tissue culture cells is being studied to elucidate the mechanisms of steroid regulation of gene expression in eukaryotes. The earliest step known in the interaction of steroid hormones with target cells is the binding of these compounds to “specific cytoplasmic receptors” (for review, see Jensen, Numata, Brecher & DeSombre, 1971). In rat hepatoma cells we have found glucocorticoid receptors which appear to be the direct mediators of enzyme induction (Baxter & Tomkins, 1970; Baxter & Tomkins? 1971a,b). We have now studied the steroid-receptor interaction in greater detail. The results further support the idea that these receptors are involved in induction and suggest that the biological activity of steroid hormones may depend on their actions as allosteric effeotors. 99
100
G. G. ROUSSEAU,
J.
D.
BAXTER
AND
G. M. TOMKINR
2. Materials and Methods (a) Mutevials The source and purity of [1,2,4-3H]dexamethasone (9 to 12 Ci/m-mole) and of nonradioactive steroids were previously described (Samuels & Tomkins, 1970; Baxter & Tomkins, 1971~~). [1,2-3H]progesterone (33.5 Ci/m-mole), [1,2-3H]cortisol (42 Ci/m-mole) and [1,2-3H]corticosterone (44 Ci/m-mole) were obtained from New England Nuclear. Phosphate-buffered saline consisted of 0.1 M-NaCl and 0.026 M-potassium phosphate, pH 7.6. Homogenization buffer was composed of 0.02 M-N-Tris-(hydroxymethyl) methylglycine (Tricine), 0.002 M-C&I,, 0.001 M-MgCl,, pH 7.4. Activated charcoal (Norit-A, Fisher) was prepared as described (Baxter & Tomkins, 1971a). Growth medium, pH 7.6 to 7.8, was Swim’s 77 (Grand Island Biological Company, New York) supplemented with NaHC03 (0.5 g/l.), 0.05 M-Tricine, 0.002 rvr-glutamine and 10% of either calf serum or a mixture (1: 1) of fetal calf and calf serum. (b) Cell culture,
enzyme induction
and preparation
of tissue extracts
HTCt cells were grown in suspension at 37°C (Hershko & Tomkins, 1971) and harvested while in the log or early stationary phase of growth, at a density of 2 to 15 x lo5 cells/ml. Induction of tyrosine aminotransferase was performed in serum-free medium as described previously (Samuels & Tomkins, 1970). HTC cell cytoplasmic extracts were prepared according to Baxter & Tomkins (1971a). Briefly, cells were washed with ice-cold phosphate-buffered saline, disrupted in one volume of homogenization buffer using a Teflon-glass tissue grinder, and centrifuged at 100,000 g for 1 hr. The supernatant fraction (cytoplasmic extract or cytosol), kept at 0°C and used within 48 hr, was not further fractionated to avoid alterations of receptor properties observed during purification (Rousseau, Baxter & Ton&ins, unpublished observations). (c) Measurement
of steroid
binding
Many techniques employed to measure steroid binding (e.g. density-gradient centrifugation, gel filtration and equilibrium dialysis) are either time consuming or not sufficiently accurate for detailed quantitative studies of glucocorticoid-receptor interactions. The charcoal assay (Baxter & Tomkins, 1971a) is devoid of these drawbacks and, when performed as described below, can be used even if bound steroid dissociates rather rapidly. This method depends on the fact that charcoal adsorbs free, but not macromolecule-bound, steroid (Murphy, 1967; Heyns, Van Baelen & De Moor, 1967; Korenman, Perrin & McCallum, 1969). Following reaction of 3H-labeled steroid with a cell extract, the unbound steroid can be adsorbed on the charcoal which is then discarded. Providing the charcoal-containing reaction mixture is vigorously, but briefly, agitated and the charcoal immediately centrifuged, a single determination of the radioactivity remaining in the supernatant medium is sufficient for an accurate measurement of bound steroid. This procedure is more convenient than other types of charcoal assays (cf. Murphy, 1967; Heyns et al., 1967; Milgrom & Baulieu, 1969). Specific binding$ of dexamethasone was measured as follows. Portions (0.4 ml.) of cytoplasmic extract containing radioactive dexamethasone (up to 10m7 m) were incubated at 0°C. Samples containing the same concentration of radioactive dexamethasone plus a 500- to lO,OOO-fold excess of non-radioactive dexamethasone as a competitor were run in parallel for “background” (Baxter & Ton&ins, 1971a) determination. After 90 min, 50 ~1. of a suspension of activated charcoal (100 mg/ml.) was added to the samples which were immediately vigorously agitated (Vortex mixer) for 3 set and centrifuged at 600 g for 1 min. The supernatant medium was centrifuged at 10,000 g for 5 min and portions of the second supernatant fraction were counted (charcoal-resistant radioactivity). The amount of steroid specifically bound by the cytoplasmic extract was calculated by subtracting the samples from the charcoal-resistant charcoal-resistant radioactivity of the “background” radioactivity of the corresponding competitor-free sample. Free steroid concentration was t Abbreviation used; HTC, rat hepatoma tissue culture. $ By “speciflo binding” we refer to high affinity ‘binding of steroids to a limited number components without implying that the binding reaction per 8s ha a biological significance.
of
GLUCOCORTICOID
RECEPTORS
101
cslculated by subtracting the tot81 concentration of steroid bound in the 8bsence of competitor from the steroid concentration initially present in the same incubstion mixture. The following experiments establish the validity of this assay. First, charcoal is not saturated with steroid at the concentrations of steroid employed. When buffer solutions contttining 8 const8nt amount of [3H]dex8methasone and increasing concentrations of nonradioactive dexamethasone (up to 10m4 M) 8re treated with charcoal, the amount of radioactivity removed by charcoal remains constant. Also, the measured itmount of steroid specific8lly bound by the cgtoplasmic extract does not change when the concentration of added charcoal is increased from 100 to 300 mg/ml. (Fig. l(8)).
Charcoal added (mg/ml.) (0)
Time of ogitotion bet) (b)
FIQ. 1. Effect of the 8mount of charcoal and of the time of agitation with charcoal on the measurement of steroid binding. HTC cell cytoplssmic extracts (protein concentretion 1.65 mg/ml.) were incubated at 0°C with 2 x10e8 M-[3H]dex8meth8sone (12 Ci/m-mole) in the absence (-O-O-) or presence (-a----a-) of lOwE M-non-radioactive dexctmethasone. Following 90 min of incubation, 50 ~1. of a suspension of activated charcoal were agitated with 0.4 ml. of incubation mixture, and charcoal-resistant radioactivity was determined as described in Materials and Methods. Specific binding corresponds to the difference between the upper and lower curves in both parts of the Fig. (a) Effect of the amount of charcoal. The 50.~1. charcoal suspension contained varying amounts of activated charcoal; (b) effect of the time of agitation. Following the addition of 50 pl. of the charcoal suspension (100 mg/ml.), the samples were agitated for varying time-periods before centrifugation. Second, appreciable dissociation of bound steroid does not occur under the assay conditions. It c8n be seen from Fig. l(b) that measured specific binding of dexamethasone rectches, after 8 very short period, 8 level which remains relatively constant even over 60 set of agitation. This might be expected, since the dissociation of dexamethasone from the receptors 8t 0°C is quite slow (Baxter & Tomkins, 1971a). Third, the amount of specifically bound steroid is linearly related to the concentr8tion of cytoplasmic extrect. Fig. 2 shows that this holds true when the reaction mixture contains 1 to 30 mg protein/ml. Fourth, it is unlikely that charcoal destroys or binds a significant proportion of the receptors, since the use of higher concentrations of chrtrcoal (Fig. l(8)) or more prolonged 8gitation in the presence of charcoal (Fig. l(b)) do not decrease the 8mount of specificslly bound steroid. The fact that the relationship shown in Fig. 2 is linear also supports this inference. Finally, t,he amount of specifically bound dexamethasone measured by the assay is comparable to
102
G. G. ROUSSEAU,
J. D.
Concentration
BAXTER
of cytoplosmic
AND
extract
G. M. TOMKTNS
(mg protein/ml.)
Fm. 2. Relation between cytoplasmic extract concentration and specific binding. The amount. of specifically bound [oH]dexamethasone at 2 x 10e8 M was determined by the charcoal assay in various dilutions of a HTC cell cytoplasmic extract.
TABLE 1 Comparison
of charcoal assay and gel f&ration for dexamethasoneT Specific binding Charcoal assay Gel filtration
measurement
(cts/min/mg
protein)
6360 53804
(6330, 6390) (4500-6120)
of speci$c binding
oj
t HTC cell cytoplasmic extracts were incubated for 90 min at 0°C in the presence of 2 x 10-r M-[3H]dexamethasone (12 Ci/m-mole) with or without competing 10e5 M-non-radioactive dexamethasone. Two 0.4-ml. portions were assayed by the charcoal method and 3 other portions (0.4 to 1 ml.) were filtered at 4°C through a G25 Sephadex column (1 cm x 7.2 cm, flow rate 0.9 ml./min) which was eluted with homogenization buffer. $ Difference in the specific radioactivity of excluded volume between competitor-free and competitor-containing extracts (mean and range of 3 experiments).
that estimated by gel filtration (Table 1). With the charcoal assay, the results are more reproducible and denaturation of the receptors as seen upon gel filtration (Baxter & Tomkins, 19716) can be avoided. The assay can be adapted to measurement of specific binding of other steroids by HTC cell receptors. Experiments similar to those described in Figs 1 and 2 have established that progesterone binding can be determined accurately even though it dissociates from HTC cell receptors much faster than dexamethasone (see below). The charcoal assay was also used for studying steroid binding by rat liver cytosol and plasma transcortm. In the latter case, plasma must be diluted 20- to 200-fold. Otherwise, most of the steroid may be bound at the concentration ordinarily used (about lo-* M), rendering difficult an accurate determination of the free steroid concentration. (d) Other methods Tyrosine aminotransferase activity and protein concentration were assayed as described previously (Samuels L Tomkins, 1970). For measurement of radioactivity, aqueous samples were dissolved in toluene containing 4 g Omnifluor (New England Nuclear)/l. and 10% Biosolve (formula BBS-3, Beckman Instrument Company) and counted in a Beckman scintillation spectrometer with an efficiency of 44%.
GLUCOCORTICOID
RECEPTORS
103
3. Results (a) Interaction of inducer steroids with the receptors From earlier work (Baxter & Ton&ins, 1971a) we concluded that the interaction between dexamethasone and the receptors conforms to a reversible equilibrium reaction with a single class of receptor sites. Indeed, a plot (Scatchard, 1949) of the ratio of bound to free dexamethasone as a function of bound dexamethasone is linear. Also, the kinetics of binding are second order (proportional to the concentration of steroid and receptors) and the kinetics of dissociation are first-order (proportional to the concentration of the complex). This interaction appears to be involved in the hormonal induction of enzymes. Consistent with this view, the dexamethasone concentrations required for binding, and for induction of tyrosine aminotransferase or cell adhesiveness are very similar, and the ability of other steroids to compete with dexamethasone for binding is related to their effects on induction (Baxter & Tomkins, 1971a; Tomkins et al., 1970). In the present study we examined the binding characteristics of other steroids which, like dexamethasone, induce tyrosine aminotransferase to a high level (“optimal inducers”, Samuels & Tomkins, 1970). Maximal induction is attained at steroid concentrations which differ for various optimal inducers. The biological potencies of four of these, dexamethesone, corticosterone, cortisol and eldosterone, were compared by examining the steroid concentration required for half-maximal induction (Table 21). Binding of the same steroids by the receptors was also measured. The Scatchard plots of the binding data (see Fig. 3 and Rousseau, Baxter, Funder, Edelman & Tomkins, 1972) are linear, suggesting that each optimal inducer tested TABLE 2
Comparison of specijc binding properties of optimal inducers with their ability to induce tyrosine aminotransferase
Steroid
Dexamethasone Corticosterone Cortisol Aldosterone
Steroid concentration (an) required for halfmaximal inductiont
1.5 x10-s (1-2*5x 10-Q 1~3x10-‘(10-s-2x10-7)~ 1.9x10-7 (1.3-3x10-78 4x10-‘/[
Apparent equilibrium (dissociation) constant
at O”’ (“)s
3.1 x10-e 4.3x10-9 1.1 x10-8 2.5x10-s
Cytoplasmic receptor-site concentration (pmoles/mg protein)1 0456 0436 063 0.67
t HTC cells were incubated at 37“C in serum-free medium in the presence of various ooncentrations of steroid and were assayed for tyrosine aminotransferase activity as described in Materials and Methods. $ HTC cell cytoplasmic eatracts were incubated with various concentrations of 3H-labeled steroid at 0°C. Specific binding was determined by the charcoal assay and a Scatchard plot of the data (see Fig. 3) was linear in each case. The apparent equilibrium (dissociation) constant was calouleted from the slope of the Scatchard plot and the concentration of receptor sites was estimated from the intercept with the abscissa. $ Range from 3 or 4 experiments. jl Taken from Samuels I% Tomkins, 1970. t The present finding that corticosterone observations (Samuels & Tomkins, 1970).
is more potent than cortisol is in contrast with previous
104
G. G. ROUSSEAU,
J. D.
BAXTER
Bound steroid
AND
G. M.
TOMKINS
i M x IO9 i
Fro. 3. Specific cytoplasmic binding of cortisol and corticosterone. Specific binding of various or [sH]cortioosterone (-+---•--) by HTC cell concentrations of [sH]cortisol (- O----O--) cytoplasmic extracts (3.4 mg protein/ml.) was measured by the charcoal assay after 90 min of incubation. The data are plotted by the Scatchard technique. That 90 min of incubation allowed for equilibrium to be established w&s determined in separate experiments. interacts with a single class of receptor sites. Since the concentration of sites is the same for each steroid (Table 2) and since all these steroids compete with each other for binding (Table 4, Baxter & Tomkins, 1971a and unpublished observations), the same sites are probably involved in the binding reactions. Comparison of the equilibrium (dissociation) constants of the specific binding reactions for the four optimal inducers (Table 2) shows that their biological potency is directly related to their affinity for the receptors. These correlations strongly support the view that HTC cell cytosol contains a receptor molecule which is involved in the hormonal action.
(b) Interaction of anti-inducer steroids with the receptors A number of steroids have been classified as anti-inducers since at appropriate concentrations they inhibit *he induction of tyrosine aminotransferase by inducers (Samuels & Tomkins, 1970). Anti-inducers also inhibit specific cytoplasmic binding of dexamethasone (Baxter & Tomkins, 1971a). The data in Figure 4 show that the antiinducer progesteronet (see Table 3) behaves as a competitive inhibitor of dexamethasone binding. Assuming that progesterone + receptor + progesterone-receptor complex, we calculated from the competition data the apparent equilibrium (dissociation) constant to be 2.9 f O-8(standard error of the mean) x lo- * M at 0°C. Furthermore, t Progesterone and 1’7 ar-hydroxyprogesterone were olassified (Samuels & Tomkins, 1970) as sub-optimal inducers since they were found to induce tyrosine aminotransferase by about 5% as observed in our compared to cortisol (see Table 4). However, this effect was not consistently studies. Consequently, we have considered these steroids as anti-inducers because they strongly inhibit induction by inducers.
GLUCOCORTICOID TABLE
Inhibition
105
RECEPTORS
3
by anti-inducers of speci$c binding or enzyme induction by dexamethaaone Induction of tyrosine aminotransfer8%3 by 10 -s na-dexamethasone in the presence of anti-inducer ( oJ of control)t
Anti-inducer
None Progesterone 0 lo-’ M lo-’ M lo-‘M
lo-srd
[sH]Dexamethasone (10-s M) bound in the presence of anti-inducer (Oh of control)$
100
100
113 93 44 12
100 70 9 2
97 100 44 18
105 103 64 22
17 m-methyltestosterone lo-‘M lo-’ lo-’
M
M
10-s M
t Inductions were performed as described in Materials and Methods, using the steroid concentrations indicated. 100% induction was 130 to 178 m-units of enzyme/mg protein depending on the experiment. $ HTC cell cytoplasmio extracts were incubated for 90 min at 0°C with 10-s M-[sH]dexa.methasone (12 Ci/m-mole) plus various concentrations of non-radioactive steroids, and the amount of specific dexamethasone binding was determined. The 100% value was 13,500 to 36,700 ots/min/ ml. incubation depending on the concentration of cytoplasmic extraot. 5 Mean from 2 or 3 experiments.
when interaction of [3H]progesterone with HTC cell extracts is examined directly, speckfit binding can be demonstrated (Fig. 5), and a Scatchard plot of the data is linear (inset of Fig. 5), suggesting that the assay detects binding to a single class of receptor sites. The apparent equilibrium (dissociation) constant for the reaction was found to be 2.2 to 2.5 x 10-s M (3 experiments), in good agreement with the value calculated
FIG. 4. Competitive inhibition of specific dexamethasone binding by progesterone. Specific binding of [3H]dexamethasone by HTC cell oytoplasmic extracts was determined (protein concentration: 5.9 mg/ml.) at various concentrations of [3H]dexamethasone and competing nonradioactive progesterone. The co-ordinates refer to the reciprocal of the molar concentration of bound (l/B) or free (l/P) dexamethasone.
106
G. G.
ROUSSEAU,
J. D.
BAXTER
AND
Free progesterone
(M x IO’)
G. M.
TOMKINS
FIQ. 5. Progesterone binding by HTC cell receptors. Specific binding of various concentrations of [3H]progesterone by HTC cell cytoplasmic extracts (protein concentration: 5.4 mg/ml.) was determined by the charcoal assay. The time of incubation was 90 min. The So&chard analysis of the data is shown in the inset.
from the competition studies. Also the concentration of progesterone receptor sites (indicated by the intercept of the Scatchard plot on the abscissa) differed by less than 20% from the value obtained with dexamethasone. In other experiments, dexamethasone prevented specific binding of [3H]progesterone to the extent predicted from the relative aflinities of the two steroids for the specific receptors. We conclude that dexamethasone and progesterone bind to the same receptor site and that competitive inhibition of inducer binding by anti-inducers accounts for the biological effects of the
I
I
I
I
I
I
I
20
40
60
90
120
180
Time(min)
FIG. 6. Kinetics of dexamethasone and progesterone association with the cytoplasmic receptors. HTC cell cytoplasmic extracts were incubated (6.0 mg protein/ml.) at 0°C with [3H]dexamethaaone or [3H]progesterone at the concentrations indicated in the Fig. (with or without low5 M competing non-radioactive steroid, see Materials and Methods). At the time-intervals indicated, 0.4-ml. portions were agitated with 50 ~1. of charcoal and the amount of speifioally bound steroid was determined.
GLUCOCORTICOID
107
RECEPTORS
latter. In support of this idea, the anti-inducers progesterone and 17 or-methyltesto.. sterone inhibit induction to a degree which closely parallels their ability to compete with dexamethasone for binding (Table 3). Similar correlations were found when the effect of these anti-inducers on induction or binding by cortisol or corticosterone was examined. (c) Different interactions of inducers and anti-inducers with the receptors The effect of steroids on the synthesis of tyrosine aminotransferase has been explained by proposing that these hormones interact with an allosteric receptor system (Samuels & Tomkins, 1970). According to this model, uncomplexed receptors exist predominantly in an inactive conformation. Inducer binding promotes a shift toward an active conformation, while anti-inducers bind to the inactive form and do not cause an increase in the concentration of active receptors. It is consistent with the model that certain properties of the dexamethasone-bound receptors (presumably in the active conformation) differ from those of uncomplexed receptors (Baxter & Tomkins, 1971a). Furthermore, the association of dexamethasone with the specific receptors is slow despite the high affinity of the interaction, suggesting that conformational changes might be associated with the binding (Baxter & Tomkins, 1971a). If this interpretation is correct, the anti-inducer progesterone might bind faster than dexamethasone in spite of its lower affinity. This would arise because, in the absence of steroid, the receptor form which binds progesterone (i.e. the inactive form) would exist at a higher concentration than the active form bound by dexamethasone. This prediction was verified (Fig. 6). At the receptor-site concentration A
A
2.0
J
bU
YV
120
150
180
Time( min)
FIQ. 7. Kinetics of dexamethasone and progesterone dissociation from the cytoplasmic receptors. HTC cell cytoplasmic extracts were incubated at 0°C (protein concentration: 5 mg/ml.) in the presence of 10-s M-[3H]dexamethssone (with or without 10-r competing non-radioactive dexamethasone for baokground determination, see Materials and Methods). At equilibrium, i.e. after 90 min of incubation (see Fig. 6) non-radioactive dexamethasone to a final concentration of 10e5 M was added to half of each incubation mixture (zero-time on the Fig.). At the time intervals indicated, charcoal was agitated with 0.4-ml. portions and the amount of specifically bound steroid was determined. The same procedure was simultaneously performed using radioactive (4 x 10-s M) and non-radioactive ( 10m5 M) progesterone. The amount of specifically bound dexamethasone (--A----A--) and progesterone (-A---A-) without added competitor or following the addition of the competitor (--O----e-for dexamethasone and -O-O-for progesterone) is shown.
108
G. G. ROUSSEAU,
J. D.
BAXTER
AND
G. M.
TOMKINS
ordinarily employed in the binding experiments (2 x lows M), the time for half maximum binding is 4 minutes for 10e8 M-progesterone and 22 minutes for lo-* M-dexamethasone. The rate of specific binding of cortisol, another inducer whose afinity is slightly higher than that of progesterone (see Table 2), was also measured. As expected, this rate was slower than that of progesterone (12 min for half-maximal binding of 4 x IO-* M-cortisol as compared with 2 min for 4 x lo-* M-progesterone). Consistent with its rapid rate of association and lower affinity, progesterone dissociates from the receptor more rapidly than dexamethasone (Fig. 7). The rate of progesterone dissociation is first-order since a @ot of the log of bound steroid as a function of time is linear. This is in accord with the proposed reaction : progesterone + receptor +Gprogesterone-receptor complex. The dissociation rate constant (determined from the time of half-dissociation) is 3.4 (range 2.7 to 5.8) x 10W2min-l as compared to 3.0 x 10m3 min-l for dexamethasone (see Baxter & Tomkins, 1971a). Uncomplexed receptors are more thermolabile than dexamethasone-bound receptors (Baxter & Ton&ins, 1971a). If the receptors which bind progesterone are in the same (inactive) conformation as unbound receptors, the thermostability of progesterone-receptor complexes might be different from that of inducer-bound receptors. In fact, receptors bound by dexamethasone (Fig. 8) or cortisol were found to be more thermostable than those which are bound by progesterone or are uncomplexed.
Dexamethasone,
I 30
I 60
I 90 Time (min)
I 120
I 150
20°C
I 180
FIQ. 8. Kinetics of heat denaturation of dexamethasone-bound and progesterone-bound cytoplasmic receptors. An HTC cell cytoplasmic extract was incubated (final protein concentration: 5 mg/ml.) in the presence of lo-* m-[3H]dexamethasone for 90 min at 0°C. Then (zero-time on the Fig.) half of the incubation mixture was transferred into a water-bath at 20°C. At the time periods indicated, samples were removed from both incubations, chilled to 0°C for 10 min, and then specific binding was determined. An identical experiment was simultaneously performed using the same cytoplasmic extract and, instead of dexamethasone, 4 x 10 -* ar-[3H]progesterone.
(d) Relation between steroid structure, qveci$c binding and biological activity The consequence of variations in steroid structure on the biological effect of glucocorticoids has been discussed previously (Samuels & Tomkins, 1970). We have now examined the influence of such modifications on steroid-receptor binding (Table 4). Addition of an hydroxyl group in the a-configuration on carbon 17 of the steroid molecule decreases both the affinity and the biological potency of the steroid, but the resulting compound retains the properties of the original steroid as an optimal inducer,
ll-deoxycortisol 1 l-deoxycortioosterone Progesterone 11 -deoxyoortisol Progesterone
5 B-H
keto
j3-OH
or-OH
A 4-5
None
Xone
NO%3
17 a-methyltestosterone
1 l-deoxycortisol Progesterone
Cortisol
B
30
11 B-OH progesterone Epicortisol 11 a-OH progesterone 11 a-OH, 17 01methyltestosterone
78
Corticosterone
Ob
30
48
48
30
48
47
47
74
68 30
A
0
2
0
74
68
47
17
Ob
43
17
31
47 14
B
Inhibition of dexam&hasone bindingC
11 -keto progesterone Cortisol
Cortisol 17 or-OH progesterone 2 1-deoxycortisol 6 or-dihydrocortisol 5 ,$dihydrocortisol Cortisone
Steroids
to cytoplasmic
4
0
O-6
24
o-6
44
24
&6
24
100
100
33
100 O-6
A
0
0
0
0
33
100
100
-
-
34
71
100 5
B
Tyrosine aminotransferase inductiond
0
0
0
0
0
41
-
52
-
10-s -M-cort,isnl:
-
Inactive
Anti
at a higher
Inactive
Suboptimal Inactive
Optimal
Optimal
Anti
Suboptimal Suboptimal -
Optimal Anti
B
Suboptimal Anti
Suboptimal Suboptimal Anti
Suboptimal Anti
Optimal
Suboptimal Optimal
31
Optimal Anti 0 84
they are anti-inducers
88
85
55
86
21
55
85
55
0
0
41
0 85
A
Classification of the steroid (type of inducer’)
actions
Inhibition of tyrosine amino transferase inductione A B
receptors and on biological
a Induction data are taken from Samuels I% Tomkins, 1970. b Although lo-’ M-cortisone or 17 a-methyltestosterone do not inhibit binding of 10-s M-[sH]dexamethasone concentration (Baxter & Tomkins, 1971a). 0 Amount (%) by which competing steroids ( low7 M) inhibit the specific binding of 10-s M-[3H]dexamethasone. 6 Induction (net steady-state enzyme activity) by 10m5 M-steroid as percentage of the induction obtained with e Amount (94) by which competing steroids (10m5 M) inhibit induction by lo-? nr-oortisol. I According to Samuels & Tomkins (1970). See also Footnote on page 104.
c-11
5 LX-H
A 4-5
11 B-OH Progesterone Cortisol
Corticosterone Progesterone
A
c-4,5
ar-OH
Substituted (‘3
None
Unsubstituted (4
Substitution
TABLE
in the steroid molecule on binding
c-17
Position on steroid molecule
Effect of substitutions
110
G. G. ROUSSEAU,
J. D.
BAXTER
AND
G. M.
TOMKINS
a sub-optimal inducer or an anti-inducer. In terms of the allosteric model, this substitution diminishes to a comparable extent the affinity of the steroid for both the inactive and the active conformations of the receptors. Addition of an hydrogen atom in the 01configuration on carbon 5, or addit,ion of a keto group on carbon 11 yields a steroid with less affinity but more anti-inducer activity; according to the model, this might be due to a preferential decrease in the affinity of the steroid for the active form of the receptor. Conversely, addition of an hydroxyl group in the /3-configuration on carbon 11 increases the inducing effect of the steroid while its affinity for the receptor can be increased or decreased. This change appears, t.herefore, to enhance the affinity of the steroid for the active conformation and to decrease its affinity for t,he inactive form. Finally, upon addition of an hydroxyl group in the a-configuration on carbon I 1, both the binding affinity and the ability to influence induction are lost. Thus, the biological potency of steroids as well as their qualitative effects as inducers, antiinducers and sub-optimal inducers may be explained in terms of their relative affinities for the two conformations of the receptor.
(e) Difference between the specrific glucocorticoid receptors and rat transcortin Mammalian plasma contains steroid-binding proteins and the question of whet’her these proteins play an obligatory role in steroid action has been considered (for review, 1966). Corticosteroid-binding see Sandberg, Rosenthal, Schneider & Slaunwhite, globulin (transcortin or CBG) is the only plasma protein which binds cortisol, corticosterone and progesterone with high affinity (for review, see Westphal, 1969). These steroids are also bound by the HTC cell specific receptors. On the other hand, transcortin, presumably of liver origin, might be present in HTC cells. The binding properties of the receptors were therefore compared with those of rat transcortin. As discussed above, specific binding of cortisol by rat plasma can be determined by the charcoal assay (Fig. 9). The value found for the apparent equilibrium (dissociation) constant (2.3 x 10m8 M at 0°C) and the concentration of binding sites (5 x low7 M) are similar to those reported for rat transcortin by other investigators using different techniques (Westphal, 1969). The specific cortisol-binding activity of rat plasma measured by the charcoal assay was abolished upon heating for 30 minutes at 60°C but
Free cortisol
( M x IO*)
FIG. 9. Specific binding of cortisol by rat transcortin. Incubations containing diluted (1: 80) plasma from male adrenalectomized Sprague-Dawley rats and various concentrations of [3H]cortisol (with or without 10m5 M competing non-radioactive cortisol) were performed. After 90 min at 0°C specific binding was determined by the charcoal assay.
GLUCOCORTICOID
111
RECEPTORS
TABLE 5
Binding of dexamethasone and2wrtisol by HTC cell specific receptors and by rat transwrtin Amount
of specifically
bound 3H-labeled loll)
steroid
(M X
Rat plasma HTC cell oytoplasmic
Cortisol 581 86
extract
Dexamethasone 0 96
Endogenous steroids were removed from a mixture of female and male Buffalo rat plasma using the method described by Heyns et al. (1967). Specific binding of 10-s M-[3B]cortisol and lo-* M-[3H]dexamethasone by plasma (diluted 1: 7) or by HTC cell cytoplasmic extracts (4 mg protein/ml.) was measured after incubation at 0°C.
TABLE 6
Effect of dexamethasone and cortisol on specific cortisol binding by transcortin Concentration of nonradioactive steroid (M)
None 2x10-s lo-’ 10-s 10-s
Amount
of specifically bound [sH]oortisol (%) in presence of: Cortisol Dexamethasone 100 94 84 40 6
100 102 101 94 89
Rat plasma was prepared as described in the legend of Table 5 and specific binding of 10 -* M-[sH]oortisol in the presence of non-radioactive dexamethasone or oortisol was determined.
not at 45”C, as expected from the known properties of transcortin (Westphal, 1969). The specific HTC cell receptors are clearly different from rat transcortin since binding of dexamethasone by the latter was not detectable (Table 5). This conclusion is fortified by competition studies (Table 6). Whereas a lOOO-fold excess of non-radioactive cortisol completely prevents binding of lo-* M-[3H]cortisol by transcortin, the same excess of non-radioactive dexamethasone decreases [3H]cortisol binding by only 11 y’. These results make it very unlikely that transcortin plays an obligatory role in enzyme induction. (f) Glucocorticoid receptors in liver Contrary to rat plasma, rat liver cytoplasmic extracts a0 contdn specific receptors for dexamethasone (Fig. 10). A Scatchard plot of the binding data is linear (inset of Fig. 10) suggesting a single class of specific sites for this steroid. The apparent equilibrium (dissociation) constant, 7.4 x 10-O M at O”C, suggests that the afEnity of the rat liver receptors for dexamethasone is slightly lower than that of the HTC cell receptors. Yet, the ability of various steroids to compete with dexamethasone for specific binding is similar in both HTC cell and liver extracts (Table 7). Therefore, the specific HTC cd and liver clexamethasone receptors appear to be very much alike.
112
G. G. ROUSSEAU,
J. D.
BAXTER
AND
Free dexameihosone
G. M.
TOMKINS
(M x 10’)
FIG. 10. Specific binding of dexamethasone by rat liver. An adrenalectomized male SpragueDawley rat was anesthetized with ether and the liver was flooded with ice-cold phosphate-buffered saline by injection through the aorta. The liver was removed and cut into small pieces which were resuspended in one volume of homogenization buffer. The cytoplasmic extract was then prepared as for HTC cells. The liver extract was incubated (4.1 mg protein/ml.) for 90 min at 0°C with various concentrations of [3H]dexamethasone and the amount of specific binding measured by the charcoal assay. A Scatchard analysis of the data is shown in the inset. Separate experiments verified that the 90 min period of incubation allowed for equilibrium to be reached.
TABLE
Effect of various
7
steroids on speci$c dexamethasone binding cytoplusmic extracts
Competing
steroid
(10e5 M)
None Androstenedione Epicortisol Testosterone 17 cu-Hydroxyprogesterone Dexamethasone
by rat liver
or HTC
cell
Amount (%) of specifically bound [3H]dexamethasone in the presence of competing steroid HTC cell Liver 100 92 84 56 22 0
100 19 99 27 2 0
Specific binding of 10-s M-[aH]dexamethasone (12 Ci/m-mole) by Buffalo rat liver cytoplasmic extracts (13.6 mg protein/ml.) was measured. The extracts were prepared as described in the legend of Fig. 10 except that the rats used were not adrenalectomized. The 100% value was 7290 cts/min/ml. Duplicate determinations differed by less than 5 %. Data for HTC cells were baken from a previous report (Baxter & Tomkins, 1971a).
4. Discussion In the present study a number of correlations were established between the specific interaction of steroids with HTC cell cytoplasmic receptors and the biological effect of these hormones. Such findings, along with those already presented (Baxter & Tomkins, 1970;1971a,b), strengthen our impression that the receptors are involved in the hormonal induction of tyrosine aminotransferase. Optimal inducers bind to the receptors with an a%nity which is directly related to their biological potency (Table 2).
GLUCOCORTICOID
RECEPTORS
113
The concentrations of cortisol, corticosterone and aldosterone necessary for halfmaximal binding are 15 to 30 times lower than those which produce half-maximal induction. This may be due to the fact that, in contrast to dexamethesone, these inducers are metabolized by the cells (Baxter & Tomkins, 1970) but not by cell-free extracts. Furthermore, the cell-free birding reactions are performed at 0°C while the induction studies &re conducted in whole cells at 37°C. Therefore, a close correlation between steroid binding and induction (Baxter t Tomkins, 1971a; Tomkins et al., 1970) may be best established in the case of dexamethasone. Our data also indicate that anti-inducers bind to the receptors with an afEnity predicted from their capacity to compete with optimal inducers for binding. This effect is, in turn, related to the ability of anti-inducers to inhibit tyrosine aminotransferase induction. As previously shown (Baxter & Ton&ins, 1970;1971a) and described here in greater detail, suboptimal inducers compete with inducers for binding, whereas inactive steroids have very little capacity to do so. Although the kinetics of clexamethasone binding and dissociation at 0°C are very slow (Baxter & Tomkins, 1971a) they are rapid enough at 37°C (Baxter 6 Tomkins, 1970) to account for the kinetics of induction ancl deinduction (Tomkins et al., 1970). Another indication that the glucocorticoicl receptors mediate the hormonal effect is provided by studies on lymphoid tissue (Hollander $ Chiu, 1966; Baxter, Harris, Tomkins & Cohn, 1971), fibroblasts (Hackney, Gross, Aronow & Pratt, 1970) and other HTC cell lines (Levisohn & Thompson, 1972) which showed that an absent or decreased sensitivity to steroids is accompanied by a diminished concentration of specific glucocorticoicl receptor sites. Dexamethasone-binding molecules very similar to the HTC cell cytoplasmic receptors are probably present in most, if not all, glucocorticoicl-responsive tissues. They have been detected in rat liver (see above) and kidney (Rousseau et al., 1972), and in mouse lymphoma cells (Baxter et al., 1971; Rosenau et al., 1972). The specific glucocorticoid receptors differ from a number of other steroid binding components which do not seem to be directly involved in steroid action. The protein termed P2, present in HTC cells (Gardner & Ton&ins, 1969), can be distinguished from the receptors since it has a greater affinity for cortisol than for clexamethasone, it strongly binds the “inactive” steroid androsteneclione and it is not s&nded at physiological concentrations of inducer steroids. Rat plasma transcortin (Westphal, 1969) which is also present in the liver, and another liver cytoplasmic protein (binder A) described by Be&o, Schmicl, BrLenclle, Biesewig & Sekeris (1971) bind cortisol and corticosterone with high affinity. In contrast with the binding component we have characterized in the liver, both transcortin and binder A (Be&o, M. & Koblinski, M., personal communication) have a very low affinity for the potent glucocorticoid clexamethasone. Distinction can also be made, on the basis of the steroid structural requirements for binding, between the specific glucocorticoicl receptors and the steroid-binding fractions I, III and IV isolated from rat liver cytosol by Litwack, Morey & Ketterer (1972). On the other hand, fraction II studied by these investigators shares several binding properties with the HTC cell receptors ancl may be identical to them. Glucocorticoid binding by histones has been reported (Sluyser, 1966,1969; Sunaga & Koicle, 1967). Yet, this interaction is of questionable biological significance since it involves a covalent linkage between the arginine or lysine residues of the histone and the biologically inactive 21dehydro derivative of the steroids (Moncler & Walker, 1970). We have also found, like many investigators, that steroids bind to subcellular components other than the specific receptors. These are termed “non-specific” binding fractions 8
114
G. G. ROUSSEAU,
J. D. BAXTER
AND
G. M. TOMKINS
because they are not saturated with steroid at concentrations well above those which elicit the maximal biological response. A further suggestion that these compounds are not involved in the induction of tyrosine aminotransferase in HTC cells is that they do not bind steroids in relation to their biological potency. We have interpreted the data presented in terms of the proposed model (Samuels & Tomkins, 1970) that steroid hormones intluence the equilibrium between active and inactive conformations of receptor molecules. The model predicts that molecules in the inactive conformation predominate in the absence of steroid, and that anti-inducers bind to this form. It also provides an explanation for the effects of sub-optimal inducers by assuming that they bind to both conformations of the receptor (Samuels & Tomkins, 1970; Rubin & Changeux, 1966). Consistent with the model are the observations that uncomplexed receptors and receptors bound to anti-inducers are more thermolabile than inducer-bound receptors, and that anti-inducers bind steroids much more rapidly than inducers. Even if this model is valid, the available data do not, allow distinction between induced conformational changes (Koshland, 1963) or stabilization of pre-existing states (Monod, Wyman & Changeux, 1965). In any case, we find no evidence of co-operativity either for inducer or anti-inducer binding, or for competition of anti-inducers (progesterone or 17 or-methyl testosterone) with dexamethasone for binding. The allosteric model is further supported by studies on intact cells where dexamethasone-bound but not progesterone-bound receptors accumulate in the cell nucleus (Baxter, Rousseau t Tomkins, 1971). The ability of the receptor to localize in the nucleus could then be a property of the active but not of the inactive conformational state. Although the uncomplexed or anti-inducer-bound receptor has been termed inactive, it may still play a physiological role, for example, as a negative control element in the cytoplasm. It is noteworthy that estrogens influence the properties of crystalline bovine live1 glutamate dehydrogenase in vitro. Detailed physical, kinetic and immunological studies have shown that this is due to alterations in conformation, catalytic activity
and substrate specificity of this enzyme (Tomkins & Yielding, 1961; Tomkins, Yielding, Talal & Curran, 1963). Such observations and the similarities between glucocorticoid
receptors
and receptors
for
other
steroid
hormones
(Jensen,
Numata,
Brecher & DeSombre, 1971) reinforce our impression that steroid hormones act as allosteric effecters in carrying out their biological function. A preliminary account of some of these results has been presented (Baxter et al., 1971). We thank Mrs Barbara Levinson for growing the HTC cells, Mrs Diane Marver and Dr Isidore Edelman for providing adrenalectomized rats and Miss Christina Benson for help. This work was supported by grant no. GM 17239 from the National Institute of General Medical Sciences of the National Institutes of Health. One of us (G. R) is Charge de Recherches du Fonds National de la Recherche Soientifique (Belgium) and recipient of a Public Health Service International Postdoctoral fellowship (no. lF05-TW-1725). Ono of us (J. B.) is a Dernham Senior Fellow of the American Cancer Society, California Division (no. D-177).
Baxter, Baxter, Baxter,
REFERENCES J. D., Harris, A. W., Tomkins, G. M. & Cohn, M. (1971). Science, 171, 189. J. D., Rousseau, G. G. & Tomkins, G. M. (1971). Fed. PTOC. 30, 1048. J. D. & Tomkins, G. M. (1970). PTOC.Nat. Acad. Sci., Wash. 65, 709.
GLUCOCORTICOID
RECEPTORS
115
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