Nuclear binding of glucocorticoid receptors: Relations between cytosol binding, activation and the biological response

NUCLEAR BINDING OF GLUCOCORTICOID RECEPTORS: RELATIONS BETWEEN CYTOSOL BINDING, ACTIVATION AND THE BIOLOGICAL RESPONSE ERNEST BUXIM, DANIELT. MATULICH.NANCYC. LAN, STEPHENJ. HIGGINS,* STONEYS. StMoNst and JOHN D. BAXTER From the Howard Hughes Medical Institute Laboratories, the Departments of Medicine and Biochemistry and Biophysics and the Metabolic Research Unit, University of California, San Francisco, California, U.S.A.

SUMMARY To better understand the early steps in glucocorticoid hormone action from cytoplasmic receptor-steroid binding to nuclear binding of the resulting complexes. we have in the current studies examined in cultured hcpatoma (HTC) cells the kinetics of the nuclear binding of glucocorticoid receptors, the activation of the reaptor-steroid complex required for this binding and the nature of the nuclear binding sites. The quantitative relationships of these events have also been compared to the glucocorticoid response. When intact cells are incubated with dexamethasonc at 0°C. cytosol binding of the steroid occurs readily but nuclear binding proceeds very slowly. After heating the cells at 37°C for 40 s and then chilling them, the kinetics of the nuclear binding at 0°C are markedly increased. Thus, in the intact cell at 0°C. like ceil-free systems, the heat-sensitive activation and not the nuclear binding itself appears to occur and be rate-limiting for nuclear binding of the complexes. Nuclear binding measured at 37°C was similar at 304Omin. and I6 h, by which time induction of tyrosine aminotransferase is maximal. Thus, unlike the case with most other classes of hormones, in this system glucocorticoids do not affect the concentration of their receptors. Nuclear binding was linearly related to the content of cytoplasmic receptor-glucocorticoid complexes and a Scatchard plot (nuclear-bound over cytosol-bound vs nuclear-bound dexamethasone) was parallel to the abscissa. These observations suggest that in the cell the nuclear acceptors are far from saturated with receptors. It has been proposed that there are multiple orders of affinity for binding receptors in the nucleus. Salt sensitivity of bound receptors has been used on one criterion. Indeed, whereas most of the nuclearbound receptors can be extracted with 0.5 M KCI, about 20% resist this extraction. In the current studies, the proportion of salt-extractable and salt-resistant nuclear-bound receptors was found to be constant with time (to 16 h) and cytosol receptor-steroid complex concentration. Thus, the thermodynamics of receptor association with the salt-labile and salt-resistant nuclear-bound receptors appear similar and the evidence does not support the idea that these receptors have fundamental differences; the incomplete extraction of the receptors with salt may refkct some property of the solubilization process rather than two types of nuclear acceptors. The content of nuclear-bound receptors was found to be linearly related to the induction of tyrosine aminotransferase; this suggests that the number of glucocorticoid receptors, and not some distal event, is the limiting factor in the ghmocorticoib hormone response and that Yspare receptors” are not present. These data also do not support the ides that in these cells. a physiological “acceptor” that binds receptors with an affinity much higher than the observed nuclear-binding (“high-affinity operators”) mediates glucocorticoid action; these hypothetical sites would have been more saturated at the receptor concentrations achieved. Instead, the data are consistent with the possibility that a nuclear acceptor population with an affinity that does not vary greatly from the observed nuclear binding is responsibk for the glucocorticoid response. The data are discussed in terms of a model in which receptors bind similarly to acceptors present in excess, many of which are located at sites where a physiological response is not elicited. By this model, the probability of obtaining a glucocorticoid response is proportional to the chance association of receptors with those acceptors located at important loci.

INTRODUCTION

Glucocorticoid hormones bind to specific receptor proteins inside the cells of responsive tissues [l-4]. Active steroids appear to influence the receptor in such a way that the receptor-steroid complex can l Current address: Department of Biochemistry, University of Leeds, Leeds LS2 9LS. England. i Current address: National- Institute of Arthritis, Metabolism and Digestive Diseases, Laboratory of Chemistry, NIH. Bethesda, Maryland 20014, U.S.A.

undergo an “activation” step which then allows it to bind to chromatin [l, S-93. This latter interaction in some way influences the transcription of specific mRNAs [lO-133. A number of aspects of the steps in these proccsscs are poorly understood and are the subject of the current studies; in these we have attempted to gain information from an analysis in cultured hepatoma (HTC) cells of the kinetics and the dose-response relations between formation of the cytosol receptor-steroid complex, its activation. 175

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nuclear binding of the complexes and a glucocorticoid hormone response. For the studies, steroids were incubated with intact cells so that binding of hormone by the receptor occurred under conditions similar to those in which the biological response was elicited. The activation

step has mostly been studied

in cell

free systems where only the activated form of the glucocorticoid-receptor complex binds to nuclei or DNA [l. S-93. Activation is enhanced by increased temperatures and salt [l, 5-93, and probably involves a conformational change Cl. 5-9, 143 that may be associated with removal of pyridoxal phosphate from the receptor [7]. In intact cells at 0°C little nuclear binding occurs after up to 6 h, even though cytosol receptor-steroid complex formation occurs readily [IS]. It is conceivable that the salt concentrations inside the call are sufficient for activation to occur and that the slow kinetics are due to a slow rate at 0°C of nuclear’ binding itself. Revious studies have not differentiated whether the slow rate of observed nuclear binding at O’C is rate limited in this way or by the activation step. In the current study we have separated the two processes by incubating intact cells at 0°C. interrupting this incubation by a brief heating 37°C. and then following the kinetics of nuclear binding. An attempt to estimate the number of nuclear sites (acceptors)* that bind the receptors in the intact HTC cells has not been made. However, Higgins et a/.[161 found that complexes bound in the intact cell do not inhibit subsequent cell-free nuclear binding of complexes, suggesting that the nuclear sites are not fully saturated in the intact cell even though there is saturation of the receptor by the steroid. Estimates of the nuclear capacity for binding receptors in intact cells in other systems have been attempted. Williams and GorskiC17-j found. in the uterus. that as the fractional saturation of the estrogen receptors with estradiol progressed the ratio of nuclear and cytosol complexes remained constant. From these data, they suggested that it is likely that the nuclear acceptor capacity exceeds by several-fold the cytosol receptor content. Similar estimates have been ma& using dexamethasone binding by rat kidney slices; it was found that there was a linear relation between the amount of nuclear binding and the concentration of cytosol receptor-dexamethasone complexes [lS]. The idea that the quantity of nuclear acceptors exceeds that of the cytosolic receptors is also derived from cell-free experiments performed in a number of laboratories [I. 6. 163. In the current studies, the quantity of nuclear acceptors for glucocorticoid receptors in intact hepatoma cells was investigated by comparing the amount of nuclear binding as a function of the cytosolic receptor-steroid complexes. * For these studies, the term acceptor refers to any site in the nucleus that binds receptor-steroid complexes without implying whether or not these are the sites for receptor action.

It has been fashionable to think that the receptorsteroid complexes are binding to nuclei anywhere there is “open” DNA [6, IO. 19,201. Such observed nuclear binding could be nonspecific in analogy with the bacterial systems studied in greater detail. In these, regulatory molecules can be bound to a few “operators” with high affinity and to a much larger number of nonspecific sites on the DNA. Thus, with steroid-responsive systems, it is proposed that the true “operators” mediating steroid hormone action bind the receptors much tighter than do the nonspecific sites that are present in excess and account for the observed binding. Consequently. this “operator” binding would not be observed. Indeed, the glucocorticoid receptor appears to be capable of nonspecific DNA binding-especially at low ionic strength-and this binding is agonist steroid- and activationdependent [I, 211. Other data. however, suggest that factors other than DNA contribute to and enhance nuclear binding [I]. First, whereas nuclear binding of glucocorticoid receptors occurs readily at more physiological ionic strength (0.1-0.15 M salt) [I, 5. 6, 12, l&24]. very little DNA binding is observed [I, 19.25261. Secondly, nuclear-bound complexes dissociate much more slowly than DNA-bound complexes [ 1.5, 16,211. Thirdly, there is evidence in systems responsive to other classes of steroids that proteins isolated from chromatin stimulate chromatin- or DNA-binding of receptors [27-291. Such an acceptor protein has not been identified in systems responsive to glucocorticoids. but these could explain the observed differences between nuclear and DNA binding of receptors and they could dictate the quantity and location of “acceptor” sites. In the current studies we have attempted to obtain an indication of the relationship of the observed nuclear binding to the biological response by relating its magnitude to the relative induction of tyrosine aminotransferase by glucocorticoids. If nuclear “operators” exist that bind receptors with a much higher affinity than do those sites responsible for most of the observed nuclear binding, then these sites should approach saturation with receptors at a lower concentration of receptors than does the observed binding. In this case, a linear relationship between the observed nuclear binding and the biological response might not occur. To obtain some indication of the possibility of heterogeneity of the nuclear acceptors for steroid receptors, investigators have studied the release from nuclei of receptors with salt [l, 16.30-321. It has been demonstrated that with increasing salt concentrations to about 0.5 M, about 80?.$of the bound receptors can be released from isolated nuclei; it has been suggested that the population that resists the 0.5 M KCl digestion may be hound with a higher affinity to the nucleus and could be more biologically important. For instance. Clark and coworkers [30] found with estrogen receptors and the uterus that estradiol remained

Nuclear binding of glucocorticoids bound to the salt-resistant sites longer than to the salt-sensitive sites. However, from other studies with the uterus. it was suggested that the salt-sensitive receptors did not differ from those that were saltresistant; the latter may merely be trapped in the chromatin during the salt extraction [32]. In the current studies we have examined this in the case of glucocorticoid receptors in cultured hepatoma cells by investigating the quantity of 0.5 M NaCl sensitive and resistant nuclear-bound receptors at different times and concentrations of cytosol receptor-steroid complexes. If the receptor-steroid complexes bound saltresistant sites associate with these with higher affinity, then the relative proportion of these in the nucleus should be higher at lower concentrations of cytosol complexes. Binding studies are commonly performed after a brief incubation of cells with the steroid. Since biological responses are usually measured at some later time, it is crucial to know whether there are changes in any of the parameters mentioned above over the time course in which the biological response is measured. Thus, in the current studies, the binding early after steady-state levels have been achieved is compared to that measured at the time the biological response is maximal. These data also give information about the possibility of whether glucocorticoids can regulate the cellular concentrations of their receptors.

MATERIALS

AND

METHODS

Materials

Phosphate-buffered saline (PBS) contained 0.1 M NaCl and 0.025 M potassium phosphate, pH 7.6. Homogenization buffer (buffer A) contained 0.02 M N-Tris (hydroxymethyl) methyl gfycine (Tricine), 2 mM CaCi, and 1 mM Mg&, pH 7.4. Induction medium was a defined mixture of nutrients designed for tissue culture use, and growth medium was induction medium plus 5-10% calf serum [ 151. [3HJ-Dexamethasone was from Amersham-Searle, SA 22-35 Ci/ mmol; nonradioactive dexamethasone was from Sigma. Its purity was determined as previously described [15,22]. Cell binding experiments Cultured hepatoma cells were grown in spinner cultures in growth medium as described previously [!5]. The experiments were performed with cells in the logarithmic phase of growth at a concentration of 300.000 to 1$00,000 per ml. Cells were harvested by centrifugation at 3000g. The supematant medium was discarded and the cells were taken up (in approx. l/S of the original volume) in fresh growth medium (to which 0.1% sodium bicarbonate had been added) containing varying concentrations of radioactive dexamethasone with or without 10pM nonradioactive dexamethasone for determination of “background” binding [ 153. The cells were then incu-

177

bated at 37°C for 30 min to allow maximal binding in a gyrotory shaker bath at 100 rev./min. Following incubation. the cell suspensions were transferred to conical tubes and were centrifuged at 3000g for Zmin. The supernatant medium was assayed for radioactivity from which the free dexamethasone concentration was determined. The pellet was immediately washed with 30ml of ice-cold PBS and the supernatant medium was discarded. All remaining procedures were performed at WC. The cells were then resuspended in 2 ml of PBS, transferred to 12 x 75 mm tubes and pelleted by centrifugation for 5 min at 600g. The supematant medium was discarded and the pellet was frozen in liquid nitrogen. Then 0.7 ml of homogenization medium was added. The pellet was thawed, resuspended with a glass rod, and the mixture was centrifuged at 600g for 5 min. The volume of the supernatant medium was recorded and 0.5 ml of it was filtered over a Sephadex G-25 (5 x 70mm) column, and eluted in buffer A. Radioactivity in the excluded volume (macromolecular-bound steroid) was assayed. The pellet from the 600 g centrifugation of the cell homogenate was resuspended in 2 ml buffer A and was transferred to small homogenizer tubes. The tubes were then washed with 2 ml of buffer A and these washes were added to the homogenization tube. These suspensions were homogenized (six strokes at 2000 rev./min) and then centrifuged 2500 g for 5 min. The pellet was washed twice with 1Oml buffer A. These “partially purified nuclei” were finally resuspended in 0.6 ml of water. Portions were taken for determination of radioactivity as described above and for measurement of DNA [33] and protein [34]. The specifically-bound radioactivity in the nuclei was measured by subtracting from the total bound at a given concentration, the amount of radioactivity bound in the presence of excess dexamethasone (nonspecific binding). Enzyme induction studies

Induction of tyrosine aminotransferase measured with the Diamondstone assay [35].

was

Whole-cell actiuution experiments

Hepatoma ceils were harvested in the log phase of growth, washed three times in induction media and suspended in induction media at 0°C and 5,000,000 cells/ml. At time Oh, 1OnM [3H]dexamethasone was added to the cells. A parallel experiment with 10 nM dexamethasone plus 10 PM of the nonradioactive steroid was run to determine nonspecific binding. At 1.75 h, 1 ml portion of the mixture was transferred into each of nine test tubes. At time 2 h, four tubes were heated at 37°C for 45-60 s. Thereafter, 2, 2.25, 2.5, 3 and 4 h, cells were washed three times in PBS, frozen 10 min on dry ice, thawed at 4°C homogenized in 3 ml of buffer A and centrifuged 5 min at 1200 g. The pellet was washed three times in induction medium and the supernatant medium was run over a

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ERNESTBLOOMet al.

5 x 70 mm Sephadex G-25 column. Both the nuclear pellet and the G-25 exclusion volume were assayed for radioactivity as previously described (22-J. Salt extraction

of nuclear

bound

receptor experiments

The procedure was as in the 30 min cell binding experiments except as follows. Cells were incubated up to 16 h in induction medium. Nuclear pellets were divided into two aliquots after their resuspension; one was extracted for 30 min in buffer A and the other in buffer A plus 0.5 M KCl. RESULTS Steroid nucleur

concentration

dependency

on cytosol

and

binding

Nuclear and cytosol binding of dexamethasone at different concentrations of the hormone incubated with intact cells at 37°C is shown in Fig. 1. Both fractions become relatively saturated with steroid at similar concentrations. In this experiment. about 60X, of the binding is nuclear and 40% is cytoplasmic; the maximal nuclear-bound steroid varied in different experiments from 40% to 60% of the total. A Scatchard analysis of these data is shown in Fig. 2. As indicated, the slopes are similar. suggesting that the extent of relative saturation of cytosol and nuclear binding is similar over a wide range of steroid concentrations. From these data and from other experiments we estimate that the “apparent” equilibrium dissociation constants for cytosol nuclear or total ceil binding of dexamethasone are approximately the same, ail around 10 nM. C.rtosol receptor-dexamethasone on nuclear

complex

dependency

binding

The relationship between the nuclear binding, and the cytosol receptor-steroid compiex concentration, is shown in Fig. 3. As might be anticipated.

since slopes

3 a

BOUND

(pmolewsample)

Fig. 2. Scatchard plot of the data shown in Fig. 1. The nuercept on the abscissa corresponds to 4.0 pmol/mg DNA for nuclear binding and 0.3 pmol/mg protein for cytosol binding. In four experiments, the extrapolated maximal nuclear binding varied from 2.3 to 4.9 (mean = 4.0) pmol/mg DNA and from 0.2 to 0.4 (mean, 0.3) pmol/mg protein.

of the cytosol and nuclear binding are identical, this relationship is linear. Nuclear binding shows no tendency to plateau; these data suggest that at the concentrations of cytosol receptor-dexamethasone complexes maximally achievable in these cells (i.e. when the receptors are near saturated with steroid), the nuclear acceptors are not saturated with receptordexamethasone complexes. A Scatchard analysis of the nuclear binding of receptor-dexamethasone complexes, as related to the concentration of cytosol receptor-dexamethasone complexes (data pooled from four separate experiments), is shown in Fig. 4. The proportion of receptors in the nuclei and cytosol varied somewhat in different experiments. Thus, the data were normalized to mean the ratio of nuclear to cytoplasmic binding for all the determinations in a given experiment A

i!i#icb NUCLEI

P

CYlOsoL 2

0

20

40

60

FREE DEXAMETHASONE

80

100

bMl

Fig. 1. Dexamethasone concentration dependency of total cellular. nuclear and cytosol binding in intact HTC cells at 37C measured as described in the Methods. Each 2Oml incubation contained 8 x LO’ cells. Maximal total binding corresponds to about 20.000 binding sites per cell.

CYTOSOL COMPtEXES

(pmolot/mglJNAl

Fig 3. kuclear binding as a function of cytosol binding. Data are taken from three experiments including the one shown in Fig I.

Nuclear binding of glucocorticoids 160

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SATURATION

OF THE NUCLEAR

BINDING SITES

Fig. 4. Scatchard plot of receptor-steroid complex binding to the nucleus. The ratio of nuclear to cytoplasmic binding is plotted as a function of the maximal binding. Data points are taken from four separate experiments. The binding capacity is taken from a Scatchard plot of the wholecell binding data in each experiment. The mean ratios of the nuclear-bound/cytoplasmic-bound receptor-steroid complexes obtained in each experiment were normalized to one. and the individual points are plotted relative to this ratio.

ratio is plotted as a function of fractional saturation of the receptor by the steroid. As shown, although there is considerable scatter, at all levels of fractional saturation (to nearly 90”/,), the ratio remains constant and does not appear to approach the abscissa. These data therefore do not provide any evidence that the nuclear acceptor sites are approaching saturation with receptor-dexamethasone complexes as the receptors become saturated with steroid. If the line extrapolates to the abscissa, it does so at a distance considerably farther out on it than indicated on the figure. These data further imply that the nuclear acceptor capacity exceeds, by at least several-fold the maximal nuclear binding. These observations combined with knowledge that about half of the total receptors are nuclear-bound, suggest that the acceptor capacity exceeds by at least severalfold the content of the cytosol receptors. (taken as 1) and this

Fig. 5. Nuclear bound receptor-hormone complex after “activation” in the intact cell. Cells were incubated at 0°C with lo-* M dexamethasone and nuclear binding was measured at various times (0). After 2 h of incubation at 0°C. half of the samples were heated 45-60 s at 37°C and then brought back to 0°C and nuclear binding was measured at various times thereafter (0). Cytosol binding was 1700 + 8OOc.~.m./5 x IO’ cells prior to heating. After heating and chill& it was 1105 +‘645. 1315 f 785. and 1660 f 1020c.p.m./5 x lo6 cells at respectively 30. 60 and 12Omm. Binding in parallel tubes that were not heated corresponding to the same time periods was 1210 & 930. 1565 f 680 and 2645 i 1600 c.p.m./5 x IO6 cells. The data rdlect the mean values from three experiments.

Salt extractible

receptors

The kinetics and other properties 6f the extraction of receptor-steroid complexes have been reported previously [16,22]. Figure 6 shows that about 80% of the receptor-dexamethasone complexes can be soiubilized by 0.5 M KCl. This value is invariant of the steroid concentration in the 5-17 nM range, a span covering more than a three-fold range of bound receptors. Thus, the data do not provide evidence for the idea that the salt-resistant receptors are bound at sites that associate with the receptor with higher affinity than those that are salt-sensitive. Longer

term kinetics

We have previously reported the kinetics of nuclear and cytosol binding by cultured hepatoma cells [ 1.4, 15,36,373. In the current studies the binding at

Acfiwtiou in the cell

Figure 5 shows that nuclear binding proceeds slowly when cells are incubated with the steroid at O’C. However. when the ells are briefly exposed to an increase in temperature (which minimally increased the total or cytosolic bound steroid. see the legend to Fig. 5). the rate of nuclear binding increases rapidly; nuclear binding then plateaus within an hour. Since much of the nuclear binding occurred at 0°C. the temperature shift must have promoted some alteration that affects the ability of the receptor-steroid complex to bind to nuclei. It is likely that this alteration is the activation

of the receptor-steroid

complex.

L

I

5

I

IO

I

I5

DEXAMETHASONE (nm 1 Fig. 6. Salt-labile nuclear-bound receptor-steroid complexes as a function of dexamethasone concentration. Cells were incubated a1 37°C for 16 h with dexamethasone. The data are taken from eight experiments done in singlicate (see Methods, Fig. 7).

180

ERNESTBL~CIMer (11. TOTAL

I

CYTOSOL

NUCLEI

-7

Fig.

7. Distribution of receptor-steroid complexes at 40 min compared to 16 h. Ceils (300,0001.000.000ml) were incubated at 37°C with 10nM dexamethasone. After harvesting and separation of cytosol and nuclei (Methods), the nuclei were washed three times and resuspended in 0.5 ml of buffer A. The nuclear suspension was then divided and centrifuged at 12009 for 2 min. One portion was incubated with agitation (vortex) for 30min in buffer A and the other in buffer A with 0.5 M KCI. The mixtures were then centrifuged and the pellets washed three times in buffer A and finally assayed for specifically-bound radioactivity. Shown are mean values (f SEM) for singlicate incubations in I2 experiments. The average total binding was about 13,000 molecules of dexamethasone specifically bound per

cell. a relatively early time (4Omin) was compared with that at a later time. Also, the relative cytosol and nuclear-bound steroid and the salt-sensitive and saltresistant nuclear-bound receptors were quantified. As Fig. 7 shows. the relative binding in the various fractions was invariant over this time period. These data therefore indicate that there is no preferential accumulation of receptors in any fraction over this time period and that the homologous hormone does not regulate the cellular concentrations of glucocorticoid receptors.

Relation

between nuclear

binding

und

the glucocorri-

coid response

We found a close correlation between the observed nuclear binding and the biological response of induction of tryrosine aminotransferase (TAT) (Fig. 8). Both processes were half maximal at about I5 nM dexamethasone. The insert emphasizes the linear relationship between these processes. No evidence is obtained for a tendency of the biological response to plateau with increasing nuclear binding. Thus, there is no tendency of the physiological nuclear acceptors mediating giucocorticoid hormone action to be saturated in the cell even when all the receptors are bound by an active steroid. DISCUSSION

Fig. 8. Relutron between relative nuclear binding and tyrosine aminotr;msferase induction. The nuclear binding data 10). pooled from four separate experiments. are plotted as a per cent of the maximal nuclear binding (taken from the abscissa intercept of a Scatchard plot analogous to the one shown in Fig. 3). The induction data (0) represent the mean (ISEM) of three separate experiments performed with duplicate incubations in each. The maximal induction was taken from the plateau values from inductions at I-IOpM dexamethasone in each experiment. This varied from 65-110 miliiunit of enzyme/mg total cell protein depending on the experiment. The insert shows the induction in relation to nuclear binding.

The present studies using intact cultured hepatoma cells have focused on the quantitative relations between formation of cytosol receptor-dexamethasone complexes. their binding to the nucleus and the glucocorticoid response of induction of tyrosine : aminotransferase. In the ceil the apparent equilibrium dissociation constant (K,) for receptor binding by dexamethasone is around 10nM: the constant is the same whether cytosol. nuclear or whole cell binding is measured. A plot of the nuclear-bound as a function of the concentration of cytosol receptor-dexamethasone complexes was linear. These data suggest that the nuclear capacity for binding receptor-steroid complexes is not approaching saturation in the cell. even though the nuclei bind receptor-dexamethasone complexes rather tightly (they are retained after cell fractionation and multiple washes of nuclei) [I. 5.6, 16.22.361. Further, a Scatchard plot of the ratio of nuclear-bound to cytosol complexes as a function of the nuclear-bound complexes appears to parallel the abscissa (Fig. 4). Therefore, the point of

Nuclear binding of glucocorticoids intercept of this line on the abscissa (an indication of the total acceptor capacity). if it occurs. is several-fold greater than the maximal nuclear binding observed. Since almost half of the receptors are nuclear-bound, it appears that there are several-fold more acceptors for re~ptor-gluc~orticoid complexes than there are cytoplasmic receptors; there are more than 100,000 acceptors per cell. The current studies support the notion that activation of the receptor-glucocorticoid complexes can occur in the intact cell-as has been more rigorously shown in cell-free incubations. The slow kinetics of nuclear binding that occur at 0°C are not due to an inherently slow nuclear receptor interaction; instead they are slow because some temperature-dependent intracellular event is required. Nuclear binding proceeds very rapidly at 0°C if the cells are briefly (45-6Os.t exposed to 37°C. AIthough other explanations are possible. it is likely that the intracellular event is activation. These results are also consistent with those of Wira and Munck[38] who incubated cells at 3°C. raised the temperature to 37°C for a brief period and then chilled them. In contrast to our finding they observed an expiate increase in nuclear binding that was maximal at the end of the warming and there was an increase in the total steroid-bound; thus. it was not possible in their studies to differentiate between an effect on nuclear binding per se combined with an increase. in total binding and a sole e&t on activation. Since activation readily proceeds at 37°C. the temperature at which biological responses are measured, it might be asked to what extent is it a limiting factor in the cell? Is this step merely a transition, important at lower temperatures, but not limiting at 37”C? We have sought to answer this question by isolating the cytosol receptor-steroid compiexes after in~bation of cells at 37°C with [3H]-dexamethasone and asking what proportion of these are in the activated form by incubating them with a large excess of nuclei under conditions described previously [U). Under these conditions, about 507: of the receptors are in the activated form. However, in other experiments, we (and others 3941) have found that the gel-filtration of the cytosol used prior to the nuclear binding assay can itself cause activation, so that the value above may be an overestimate. Finally, in yet other studies (unpublished), we have found that after their extraction from nuclei, only about half of the r~ptor~exame~asone complexes are in the activated form. Since all of these receptors had been activated (as they were nuclearbound) this experiment suggests that some of the receptors can be converted to the inactivated form by the extraction techniques. AI1 of these observations. taken together. suggest that it is not possibIe to know from the current methodology what percentage of the receptors in the cytosol were. in the cefls, actually in a form that can bind to the nucleus. Munck and Foley[S] have more recently obtained further evidence that activation can occur in the cell.

Iill

They measured the kinetics of accumulation of activated and nonactivated forms (following incubation of rat thymus cells with [jH]-steroid at 37’C) by fractionating the cells and separating the forms on DEAE-celluIose columns. After five minutes. most of the receptors appear to be in the activated state. Thus in spite of the experimental problems discussed above, their data suggest that in the cell. conversion to the activated state appears to be complete enough that this step does not significantly limit the amount of nuclear binding that can occur. As discussed in the Introduction. there has been interest in the possibility that nuclear-bound receptor-glucocorticoid complexes that resist extraction with 0.5 M KCl may be associated with sites that bind the receptors with higher affinity than those sites that bind 0.5 M KCI extractible receptors. In the current studies, we found that the proportion of salt-sensitive and salt-resistant nuclear-bound receptor-glucocorticoid complexes was invariant over a range of cytosol receptor-steroid complex concentrations (generated by incubating the cells with varying concentrations of steroid that resulted in 25-801, saturation of the receptors). Ttrese data suggest that the salt-resistant receptors are not binding to acceptor sites with a higher affinity for the receptors than acceptors associating with the salt-sensitive receptors, as there should be a greater proportion of the salt-resistant receptors formed at lower concentrations of cytosot receptor-steroid complex. It is more likely that the salt studies do not indicate multiple types of binding. but instead, the salt-resistant receptor-steroid complexes reflect some characteristic of the extraction process itself such as trapping in the chromatin. Such a conclusion was reached by Traish et ul.[32] who studied estrogen receptor binding in the uterus. Thus, studies with salt digestion of nuclei do not provide evidence for multiple orders of acceptor affinity for receptors. In other experiments, we found that the proportion of cytosol and nuclear-bound complexes were similar after 4Omin and 16 h of incubation of cells with dexamethasone. The proportion of salt-sensitive and saltresistant sites was also constant. Thus. quantitative studies of nuclear binding of receptors at earlier times do reflect the situation at later times where the biological responses were measured. These data also suggest that in these cells the glucocorticoid does not regulate the cellular con~ntration of its homologous receptor. This contrasts with the case with most classes of hormones studied (for review see [42]) whereby the homologous receptor regulates the cellular content of its receptors. These observations are consistent with a general theme that physiological regulation of the cellular sensitivity to giucocorticoids occurs mostly through influences other than.on the cellular concentration of receptors (for review. see C431). When the steroid concentrations required for a glucocorticoid response in the cultured hepatoma cells of

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EaNasT BLOOM et

induction of tyrosine aminotransferase were compared for those required for nuclear binding, identical relationships were observed (Fig. 8). Since identical relations were also observed for cytosol or whole-cell binding, the data indicate that “spare” receptors are not present in this gIuc~orti~oid-re~nsive system. Instead, the receptors appear to be limiting for the extent of the response.. As might be anticipated from the above considerations, when the observed nuclear binding was plotted as a function of the magnitude of the induction of tyrosine ~no~~~er~ by the steroid, a linear relationship was obtained. From such an analysis (Fig. 8), it appears that with more nuclear binding, a greater biological response would be achieved. The data further suggest that those nuclear acceptors that are the actual sites of glucocorticoid hormone action are not saturated or even approaching saturation with receptor-steroid complexes with increasing accumulations of cytosol or nuclear-bound complexes. Otherwise, the dose-response curve should be shifted to the left of the receptor-binding curve which is not the CaSe. The question also arises of the relationship of the observed nuclear binding to that which actually mediates glucocorticoid hormone action. In cultured hepatoma cells, and probably in steroid-responsive systems in general, there is a restricted biological response; only a small subset ( < 1%) of the expressed genes of the cell appear to be affected by the steroid This thinking is mainly based on analyses with the use of two-dimensional gels of the proteins syntha sized by hormone-treated and control cells [44-j. These gels allow a detection of perhaps up to 10% of the genes whose products are synthesized at higher levels in the cell, and it is likely that the analysis will in general give at least a rough index of the total complexity of the response. If the expression of as few as 100 genes per cell (assuming _ lo4 expressed genes per cell) is under glucocorticoid hormone control, then the number of &clear acceptors, estimated in the current studies possibly to exceed 100,000, clearly exceeds this value. Thus, unless (as is possible) many receptors are acting at each locus, it is likely that many of the nuclear-bound receptors are not associated with sites where biological responses are elicited. If many receptors are bound at sites where biological responses are not elicited, then is that the lowafhnity binding anticipated from studies of bacterial systems? As said earlier, the observed nuckear binding may be tighter than the nonspecific DNA binding studied earlier. In addition, the linear correlation between the observed nuclear binding and the observed biological response is striking, and from the analyses presented above, it is unlikely that the accep tors that are the sites for receptor action are ever near saturation with receptors in the cell even when all of the receptors are bound by the steroid. Thus, there is no evidence that there exist “operators” that have a higher affinity for the receptors than do the observed

ul.

acceptors; a biological response curve shifted to the left of the nuclear-binding curve would be required in support of such a conclusion. Instead the data are consistent with the idea that acceptor sites which bind receptors with an affinity similar to that of the observed nuclear binding are responsible for the glucocorticoid hormone response. In other studies [lo, 451, we have found that the binding of receptor-glucocorticoid complexes to nuclei is followed by an alteration in chromatin structure detected by an increase in the ability of chromatin to bind bacterial RNA polymerase. Analogous results have been found in systems responsive to other classes of steroids [46,47]. The glucocorticoidmediated changes in chromatin involve an increase (or decrease in certain systems where the glucocorticoid response is inhibitory [iO, 481) by about ~,~/~ell in the number of polymerase binding sites. This number exceeds the quantity of cellular receptors and also the number of gene products affected. The effect on chromatin requires receptor properties other than nuclear binding per se, as it is not observed in certain lymphoma cell lines with mutant receptors that nonetheless bind to nuclei in response to the steroid [48]. It is possible, therefore, that the extensive nuclear binding observed in the cell also extensively affects chromatin. How then is the selectivity of the biological response generated? Of course, this question has not been answered. Nevertheless, we feel that the folbwing working hypothesis based on the current studies and previous work should be considered. FolIowing binding of the glucocorticoid by the receptor and activation of the receptor-steroid complex, the complexes associate with nuclear acceptors that are in excess of the receptors. These may contain both DNA and protein(s). In fact, it is possible that the latter could have the specificity of binding DNA that directs the recep tors to specific loci, but this is not known. In any event, the acceptors, in a few cases, are located at sites where receptor binding to them can result in effects on gene expression. However, in most instances, receptor binding to these acceptors may not affect soecific gene exoression. &cause of the extensive influences on chromatin discussed above, it is possible that much of this acceptor binding by receptors results in a modification in chromatin structure, but that many of these modifications do not affect gene expression. The latter possibility is analogous to the case with cyclic AMP that modifies (phosphorylates) a number of proteins wherever the correct contiguration is present allowing such modifications; many of these phosphorylations in all likelihood do not result in changes in cellular function (for discussion, see [49]). Thus, the receptors would be binding with roughly similar (aIthou~ not necessarily identical) affinities to a number of sites that are in excess. The probability that a given receptor-steroid complex could affect gene expression could then depend on the chance of its associating with certain of the acceptors

Na&ar

binding

in the population. This would be proportional to the observed nuclear binding which in turn, would bc

linearly correlated with the steroid hormone response as we have found This model is of course a hypothesis and therefure needs testing its other ways. Nevertheless, it is hoped. with the acquisition of more sophist&&d biochemical approaches, that this model will generate other experiments that can test it. Acknowledgements-This work was supported by NIH Grant AM 18878 and AM 19997. We thank Susan Bram for assistance and Guy G. Rousseau for advioe.

of glwrti&ds

21. Rousseau G. G., Higgins S. J., Baxter J. D.. Gelfand D. and Tomkins G. M.: J. biol. Chem. 250 (1975) 6015-6021.

22. Higgins S. J., Rousseau G. C.. Baxter J. D. and Tomkins G. M.: J. biol. Chem. 248 (1973) 5866-5872. 23. Ishii D. N, Pratt W. 8. and Aronow L.: Bj~hernis~~ II flQ72) 3896-3904. 24. Cidiowski J. A. and Thanassi J. W.: B~hernj~rrl, IS (1979) 2378-2384. 25. Milgrom E, Atger M. and Bailly A.: Eur. .I. B&hem. 70 (1976) pp. l-6. 26. Bugany N. and Beat0 M.: M&c. Ceil Endocr. 7 (1977) pp. 4966. 27. Kl~jk~S~f~o~

REFERENCES

1. Higgins S. J., Baxter J. D. and Rousseau G. G.: In Glucocorricrrid Hormone Acrion (Edited by J. D, Baxter and G. G. Rousseau). Springer-Verlag (1979) p. 135, 2. Litwack G. S. and Singer S.: In Biochemical Actions of Hormnes(Edited by G. Litwack). Academic Press, Voi II (1972) p. 113. 3. Munck A.: In Receprors and Me&n&m of A&on of Steroid normnes Parr I (Edited bv J. R. Pasoualinci Marcel Dekker, New York (1976) p’. 1. a ~’ 4. Rousseau G. G.: In Glucocorricoid Hormone A&on (Edited by J. D. Baxter and G, E. Rouseau). SpringerVerlag (1979) p. 49. 5. Simons S. S Jr:ibid. p_ 161. fr. Milgrom E. and Atger M.: J. steroid &o&em. 6 (1975) 487492. 7. Cake H. C., DiSorbo D. M. and Litwack G.: J. biol, Chem. 253 (1978) 48864891. 8. Munck A. and Fbley R.: Nature 278 (1979) 752-754. 9. Beat0 M., Kalimi M. and Feigelson P.: Biochemisay I2 (1973) 3372-3379. IO. Johnson L. K, Baxter J. D. and Rousseau G. G.: in Gf~~or~~o~ Hornrona Action (Edited by I. D. Baxter and G. G. Rouseau). Springer-Verlag (1979) p. MS. il. Feigelson F. and Killewich L. A.: ibid p. 243. 12. Varmus H. E., Ringold G. and Yamamoto K. R.: ibid. p. 253. 13. Martial J. A.. Seeburg P. H, Matulich D. T., Goodman H. M. and Baxter J. D.: ibid. p. 279. 14. Sakave Y. and Thompson E B.: Biorlrnn biopkys. Res. Corn-. 77 (197t) 533-541. 15. Rouseau G. 0, Baxter J. a.. Higgins S. J. and Tomkins G. M.: J. Molec. Bioi. 79 (1973) 539-554. 16. Higgins S. Y., Rousseau G. G,, Baxter J. D. and Tomkins G. M,: Proc. nut!. Acad. Sci. U.S.A. 70 (1973) 3415-3418. If. Wiiliams D. and Go&i J.: Proc. nurl. Acad. Sci. U.S,A, 69 (1972) 3464-3468.

18. Funder J. W., Feldman D. and Ed&man I. S: En& crinology 92 (1973) 1005-1013.

19. Yamamoto K. R. and Alberts B. M.: Ann. Rev. Biothem. 45 (1976) 721-146.

20. Beato M.. Kulimi M.. Konstam M. and Feigelbn B&kern&try f2 (f973).

P.:

Mu&. John, that’s beautiful work. I would like to make a comment about your conclusion from the linear correlation between bialo&al activity and the numba of nuclear binding sites, that there may be an excess number of sites. Your data really refers to the affinity of the nuclear sites, not to their number. There might be as few as one site per cell, as long as the affinity is such that they don’t become saturated. Baxter. I agree with that. My point was, whatever are the sit+s of action, these are probably not saturated.

183

28. 29. 30. 31. 32.

L.. Chiu J.-F, T&Y.-H. and Hniiica L. S.: Proc. or. Accrd. Sci. U.S.A. 73 (1976) 1954-1958. Webster R. A., Pikler G. M. and Spelsberg T. C.: Biothem, J. 156 (1976) 409-418. Spelsber8 T. C, Webster R. A. and Pikler G. M.: NoMe 262 (1976) 65-67. Clark J. l-i.. Peck E. 3, Jr, Hwdin 3. W. and Eriksson H.: In Rac~ptors and Hormone Action VoL fl. Acadcmic Press, Inc. (1978) p. 1. Pikler Gy M., Webster R. A. and Spelsberg 1. C.: Biothem. J. 1% (1976) 399-403. Traish A. M., Muller R. E. gnd Wotiz H. H.: J. biol. them

252 (1977) 6823-6830.

33. Giles K. W. and Myers A.: Nature 206 (1965) 93. 72 (1976) 248-254. 34. Bradford M. M.: An&r. B&hem 35. Diamondstone 1. I.: An&y?. BID&m. f6 (1%6f 395-w. 36. Baxter J. D. and Tomkins G. M.: Proc. nor. Acad, Sci. U.S.A. 65 (1970) 709-715. 37. Rousseau G. G.. Baxter J. D. and Tomkins G. M.: J. Molec. Biol. 67 (1972199-l 15. 38. Wira C. R. and M&k A.: J. bid!. Ckem 249 (1974) 5328-5336. 38. Goidt J. A., Cake M. H, Doian L., Parchman L. G. and Litwack G.: Biochenrisrry 16 (t977) 2125-2130. 4p. Sand0 J. J., Nielson C. J. and Pratt W. B.: J. bioL Chem 2% (1977) 7579-7582. 41. Bailly A., SaIlas N. and Milgrom E.: J. biol. Chcm. 252

(1977) 858-863, 42. Catt K. J., Harwood J. P., Aauilcra G. and Dufau M. L.: itbt~e 200 (1979) its-I ia. 43. Harris A, W. and Baxter J. D.: In Gfu~orf~o~ Hormone Action (Edited by J. D. Baxter and G. G* Rousseau). Spring&-Verlagil979) p, 423. 44. Ivarie R. D. and O’Farrell P. II.: Cell 13 11.978141-55. 45. Johnson L. K. and Baxter J. D.: J. bioi. Chbm. 253 (1978) 1991-1997. 46. Mainwaring W. 1. P.: fn The ~ffh~~~ of Action c$ An&ogew. Springer-VerIag, Heidelberg (1977) p. 1. 47. Ghan L. and O‘Malley B. W.: N. Engi. 1. Med. 2!& (1976) 1322-1328, 1372-3382, 143&1437. 48. Johnson L. K., Lan N. and Baxter J. D.: J. biol. Chem. 1979 in press. 49. Baxter J. D. and MacLeod K. M.: in Duncan’s Diseases ofMet&fism (1979) in press.

Mmck Coming to the question of one or a few, can you tell whether your cells are being turned on in an all-ornone manner? Baxt~. We don’t have any data for that in our system. 1 think that the best data that I have ever seen lias been accumulated by Brad Thompson and his colleagues looking at tyrosine aminotransferase. Their data suggest that as induction of tyrosine aminotransferase increased the enzyme cowxntration in each cell increased in a uniform manner. Induction was not heterolpous in this respect.

184

ERNESTBLOOMet

Scbnder. With respect to the receptor being an excess or not. the number of receptor molecules is about equivalent to the number of RNA polymerase 2 molecules in the ceil. I think that one of the problems here is that we tend to forget that we do not know at this time whether the recep tars are acting catalytically or stoichiometrically. Baxter. We know something about the number of binding sites that we observe: we know nothing about the number of binding sites at which the receptor actually acts to effect the expression of specific genes. I hope I did not mislead anyone into thinking that we think that we know that. But what I do think the data imply is that whatever are the sites of action of the receptor, it is likely that these are not saturated by receptors under conditions in which the maximally observed induction occurs in these cells. Further, since there are a lot of activated receptors remaining free in the cytosol. commonly 50% of the total, if the sites of receptor action in the nucleus have an enormously increased affinity for the receptor compared to those sites accounting for the observed binding, then I doubt one would see the close correlation between nuclear binding and the biology that we observe. Although I feel that the point is not established, at least one should consider the possibility that there may not be high affinity operators. M&r. With respect to those comments I’d like to mention the work of Tom Spelsberg in Minnesota (Mayo Clinic). He has done titration experiments with radioactive progesterone and has published (Ann. N.Y. Acad. Sci. 286 (1977) 43-63) figures showing that if one does enough data points on the nuclear uptake versus dose curve. one can see several plateaux in that curve. Receptor uptake is thus not a simple linear function of dose in that case, at least. It may be that. hidden within the non-saturability curve that you showed there may be plateau regions that you just haven’t seen yet. Baxter. We simply do not see that type of pattern for this glucocortcoid-responsive system. Further. even if there were plateaus like Tom has observed, I would have to argue that those plateaus are irrelevant for the biology in our case, because if these become saturated with receptors at some lower concentration of receptor they would not likely be involved in the biology. because it is increasing with further nuclear binding. Thompson. Newby and Bougeoie, I believe, have done a gene dose study using somatic cell hybrids in which they put 1. 2. 3 or 4 doses of the receptor gene into lymphoma

al.

cells. Now maybe John, you remember the data better than I. In fact. I don’t think they saw any greater response with the 2.3 or 4 gene doses of receptors than they did with the single gene. Baxter. No, they did see a difference; the changes in gene dosage (above one) shifted the dose-response. Qark. I would like to make a comment about the relationship between receptor binding and response, only to say what John already knows, that it is a highly complex thing depending upon what time you evaluate the response, what time you evaluate the binding, because one can get all sorts of correlative effects. In the rat uterus when one measures growth you find that very few recep tars are required to maximize. This is under conditions of continuous occupancy so there are many more receptors present than are needed Baxter. Jim, it varies, but my experience with steroidresponsive systems is turning out to be consistent. if you can measure the gene product of interest such as tyrosine aminotransferase. If some end point is measured where there is an accumulation, for example lymphoid cell killing, it is possible to devise the experimental conditions to get the biological dose-response to be shifted to the left of receptor binding. This is due to the fact that a submaximal rate of killing can ultimately result in death of all the cells. However, if in such a system the rate of killing rather than cell killing is examined, then a more linear relationship results. This type of analysis has in fact been performed with lymphoma cells by Alan W. Harris in Melbourne. I would not be surprised to find that a similar type situation exists when you measure uterine growth. I think that these data are telling us that the receptors for steroid hormones unlike the usual case with receptors for polypeptide and catecholamine hormones, are actually limiting in determining the hormone response. Clark. I still believe that the situation is more complex in cells; that respond to a steroid by growing. High doses of hormone may cause a more rapid rate of growth but that response is limited. This same limit is reached by low doses of hormone. The same number of receptors are required to reach this level. Baxter. Nevertheless. I would like to emphasize that with the particular example you mentioned of uterine growth vs. brain RNA polymerase. it is easy to explain why you would see a non-linear relationship in one case and a linear relationship in the other.