J. Steroid Biochem. Molec. Biol. Vol. 44. No. 3, pp. 277-285, 1993 09604)760/93 $6.00 + 0.00 Printed in Great Britain Pergamon Press Ltd

C H A R A C T E R I Z A T I O N O F R A T U T E R I N E E S T R O G E N

R E C E P T O R S I N V I V O

EMANUEL LEVIN,* ANDREA M. Ac'ns and SILVIA LOPEZ Departamento de Bioquimica, Facultad de Medicina, Universidad de Buenos Aires, Paraguay 2155 (1121),

Buenos Aires, Argentina

(Received 2 March 1992; accepted 13 November 1992)

Summary--In vivo binding of [3H]estradiol ([3H]E2) in the rat uterus was performed by an intraluminal perfusion of the ligand for different time periods. In this way the binding takes place in the intact organ before processing the tissue. In 10 min, with 10 nM [3H]E 2 apparent saturation or steady state incorporation of the [3I-I]E2 was achieved with a similar distribution of the label between cytosol and nuclear fractions. In vitro, the subcellular localization of the estrogen receptor (ER) is influenced by the extent of tissue damage. With the intact organ the ER subcellular distribution approaches that of the/n vivo perfusion. With increasing [3H]E2 in the perfusate it was possible to obtain a "saturation" curve and to derive the kinetic parameters. For cytosol: Kd 16 nM; Bm~ 235 fmol/mg prot. For nucleus: Kd 2.7 nM; Bm~ 103 fmol/mg prot. To follow the time course of the ER movement/n vivo, "pulse and wait" experiments were designed. Both uterine horns were peffused for 1 min. One of the horns was immediately processed (0 time) and the other was left in place after the peffusion for different periods. At 0 time 90% of the bound label appeared in the cytosol. At 5, 15 and 30 min, the label in the cytosol decreased and that of the nucleus increased approx, to 50%. Thus, translocation of the bound label from cytosol to nucleus was apparent. The role of the cytoplasm-nucleus ER traffic in the regulation of gene transcription by estrogens is discussed.

INTRODUCTION

The regulation and functional modalities of estrogen receptors (ER) in vivo are not suffi- ciently known, partly because most of their dynamic parameters like dissociation constants, capacity, isoform conversions, translocation, and interaction with other macromolecules, were determined in in vitro binding to their corresponding ligand and analogues. The extra- polation of these data to a receptor func- tioning in a tissue of a living animal has a

limited meaning because the regulatory mech- anisms operating in vivo may impose import- ant variations and modify qualitatively and quantitatively the receptor behaviour.

Following the precursor studies of Jensen and Jacobson [1] in vivo incorporation of labelled estradiol (E2) was performed by parenteral administration [2, 3]. Few studies tried to main- tain or to control a steady humoral concen- tration [4], which is mandatory for equilibrium

*To whom correspondence should be addressed.

or dynamic studies between supply and uptake compartments.

We have developed a perfusion method for studying the/n vivo binding of F_q to the uterine ER in the rat, based on the intraluminal supply of [3I-I]E2 to both uterine horns in anesthesized animals. Thus, the humoral and nervous regu- lations are preserved when the binding is taking place and the ligand concentration is kept con- stant in the perfusate. Moreover, the contact of the ligand is confined to the organ under study without distribution and dilution in the general circulation. The results reflect the pro- portion of receptors bound to the ligand ([3H]F_q-ER complex) under regulatory mech- anisms that maintain unoccupied part of the whole receptor population. It is not necessary to apply exchange, equilibrium or "protection" techniques common for in vitro incubations (protease inhibitors, hypotonicity, different tem- peratures, etc). The subcellular distribution of the receptors is another process under regulat- ory mechanisms operating in the whole organ- ism and it is quite different from results obtained in vitro and in cell cultures.

277

278 EMA~nJEL LEVIN et al.

MATERIAI.~ AND METHODS

Perfusion procedure

Young adult female Wistar rats (60-80 days old) were used throughout. Eight to 15 days after oophorectomy the animals were anes- thetized and each uterine horn was cannulated with an appropriate polyethylene cannula at the ovary extreme for entrance of the perfusion fluid and with another cannula at the cervix end for exit of the perfusate. Each uterine horn acted as a separate entity of the same tissue for the binding studies. Each horn was simultaneously or successively perfused with a continuous pump at 0.15 ml/min, using a PBS buffer (NaCI, 0.8%; KC1, 0.02%; Na2HPO4, 0.12%; 0.5% bovine serum albumin; pH 7.4) as vehicle. [3H]E 2 at a given concentration was added for total binding in one uterine horn and the same labelled ligand concentration plus 200-fold excess of nonradioactive ~ for the nonspecific binding in the other horn. The specific binding was calculated by subtracting the nonspecific binding of one horn from the total binding of the other one. Diethylstilbestrol was used in some experiments in substitution of E2 as dis- placer for the nonspecific binding and the results did not differ significantly from those with the natural hormone. In several experiments both uteri were utilized for the total binding.

Processing of the tissue

After perfusion with the labelled ligand, each uterus was rinsed with PBS for 30 s through the cannula at the same rate; then it was excised and washed through three vials containing cold saline solution. All remaining manipulations were performed on crushed ice. The uteri were cleaned from tissue debris, weighed, minced with scissors and homogenized in a Potter type homogenizer with a hypotonic molybdate buffer: Tris 10 raM; EDTA 1.5 mM; Na molyb- date 10mM; glycerol 10% (v/v); 2-mercap- toethanol 2 raM; pH 7.4. Subcellular fractions were obtained by differential centrifugation. The nuclear pellet obtained after 10rain cen- trifugation at 900 g was washed three times with the molybdate buffer and further resuspended for radioactivity measurements. The cytosolic bound E2 in the supernatant of the 30min 100,000g was separated by treatment with a carbon--dextran (C-D) (1-0.1%) suspension. The radioactivity was measured in an aliquot of the supernatant (triplicate). Proteins were deter- mined in each of the subcellular fractions by

the Lowry method. The resuspended particles of the nuclear fraction did not interfere with the spectrophotometric measurement due to the small volume used (10 and 20/~I in a final 1.4 ml volume). In the initial experiments, the mito- chondrial-microsome pellet from the 100,000g centrifugation was processed in the same way as the nuclear fraction.

TLC chromatography

Subcellular fractions from uterine homogen- ates obtained after 10 min perfusion of the rat uteri with 10nM [3H]E 2, were submitted to chloroform-methanol (2: l, v/v) extraction for chromatography in a 0.25 mm silica gel plate (Merck) to study the E2 metabolic products. The perfusate was collected along the peffusion and was also extracted with chloroform-methanol. All extracts were evaporated at 37°C under N current and dried residues were redissolved in chloroform and applied to the plates. F~, estriol and estrone standards were simul- taneously processed. The running solvent was chloroform-ethyl acetate (80:20, v/v) and the location of the spots was accomplished with I2 vapours. Each lane was cut in 1 cm sections for radioactivity counting.

Reagents

[2,4,6,73H]E2 was from NEN. Nonlabelled E2 and the rest of the drugs were analytical grade. (Sigma, Baker, Merck). Radioactivity was measured in a PPO-POPOP toluene scintillation fluid with 52-59% efficiency.

RESULTS

TLC chromatography

After 10min uterine perfusion with 10nM [all]E2, samples from the cytosol (without C-D treatment) and from the nuclear fraction were processed for the chromatography run. 85% of the radioactivity was recovered as E2; 12 to 15% corresponded to estrone. After C-D treat- ment of the cytosol and washing of the nuclear fractions, chromatography of the correspond- ing samples showed that all the radioactivity comigrated with E2 in both fractions. In the perfusate collected during the perfusion, no significant radioactivity was associated to estrone and estriol. Serum (2 ml) obtained from blood collected by heart puncture after the pcrfusion period, extracted with chloro- form-methanol, evaporated and redissolved

Rat uterine estrogen receptors /n vwo

Table i. In v ~ Ez binding in the rat uterus

Cytosol Nucleus

Exper. Total Nonsp. Specific Total Nonsp. Specific (fmol FoJm s protein)

I 99. i 7.4 9 i.7 87.2 ! 2.2 75.0 2 129.0 3.9 125.1 113.5 6.4 107.1 3 1 i 1.9 5.4 106.6 158.2 8.9 149.3 4 143.0 3.2 140.0 188.3 8.6 179.9 5 160.5 3.0 157.5 161.2 17.0 144.2

128.7 4.6 124.2 141.7 10.6 131.1 +SD +22 +1.6 -t-23 +36 +3.7 ±36

10 min perfusion of one uterine horn with 10 nM [3HIE2 (total binding) and of the other horn with 10 nM [3HIE 2 plus 2/~M F-, 2 (nonspecific binding). Specific binding is the difference between total and nonspecific values.

279

in ethanol did not show significant radioac- tivity. It can be concluded that after 10 rain uterine perfusion, the bound radioactivity (not adsorbed by C-D) in the cytosol and after washing in the nuclear fraction) corresponds to E2. The passage of radioactivity to blood during the uterine perfusion was negligible.

Uterine binding

In the initial experiments, total E2 binding in one uterine horn was compared with that of the other horn after 10rain perfusion with 10 nM [3H]E 2. Values for each horn did not differ more than 13 + 0.9% of the mean (n -- 6). The time for a given [3HIE 2 concentration to attain a steady state/11 vivo, was explored with a 10 nM [3HIE2 for different perfusion periods. One horn was perfused for I0 rain as a reference time and the other horn for longer periods up to 40 rain. The E2 binding in each of both uteri was not significantly different even for the longest period tested (data not shown). Consequently, 10 min was adopted as the time necessary to reach a [3H]E2-ER steady state in the uterus when per- fused with 10 nM [3H]E2. The values obtained in 5 experiments for total, nonspecific and specific binding in the cytosol and in the nuclear frac- tion showed that the specific binding was 96 and 93% of the total binding in the cytosolic and nuclear fractions, respectively (Table 1). The data for total and specific binding are not significantly different from each other. Thus, when rat uterine horns are perfused with 10 nM [3HIE 2 in vivo, total binding values can be considered as representative of the E2 specific binding to the uterine ER. The bound and free [3HIE 2 in the cytosol and in the nucleus can be calculated from the following radio- activity measurements: total homogenate- untreated cytosol=total nucleus; untreated cytosol - C-D treated cytosol = free cytosolic [3HJ~; and, total nucleus- washed nucleus = free [3I-I]E2 in the nucleus.

In the cytosol, bound [3HIE 2 corresponded to 85% of the total cytosolic radioactivity and in the nucleus it was 58% of the total nuclear radioactivity (average of three measurements).

The subcellular distribution of the E2-ER complex found in our experiments shows a cytoplasmic and a nuclear location of the com- plex. For comparison with that of the in vitro binding, we designed two types of incubations preserving the cell integrity to avoid the possible liberation of the unoccupied nuclear ER by the mechanical disruption of the tissue before bind- ing. The in vitro incubation was performed: (a) opening longitudinally the uterus after its excision and incubating the "open" organ with the ligand in a PBS-albumin solution and (b) an isolated organ preparation, perfusing the excised uterus with the same solution as for in vivo assays in a Krebs-Ringer bath gassed with O2--CO 2 (95-5%). In both conditions the incubation time was 10 rain and the temperature 37°C (Fig. 1). Under minimal damage in vitro (conditions a and b) the radioactivity is higher in the nuclear fraction than in the cytosol. When the binding was performed in the cytosolic and nuclear fractions obtained by homogenization of the uterus (destruction of the tissue), 3% of

IOO

80

60 %

4o

2o

o In vitro inc. In vitro Perf. In vivo I~rf,

E ~ cytoso~ r[TT] Nucleus

Fig. 1. F_~ binding under conditions o f minimal damage of the tissue. In vitro inc: the uterus was slit longitudinally and incubated in a PBS-albumin buffer. In vitro perf'. The excised uterus was perfused in a Krebs-Ringer ba th with a PBS-albumin buffer. In v/to perf." see Materials and Methods. Experiments were performed for 10 min at

37°C.

SBMB 44/3---F

280 ~ I . £ ~ et aL

the label was in the nucleus and the rest in the cytosol (not shown in the figure). When compar- ing the in vitro with the in vivo perfusions, the former showed the specific binding more localized in the nuclear fraction (cytosol: 32%, nucleus: 68%), while in vivo the label was more equitatively distributed between the nuclei and the cytosol. These results indicate that the sub- cellular distribution of the specific binding of E2 is different /n vitro and in rico. When the experimental conditions are closer to the in vivo situation, the contribution of the cytosol gradu- ally increases while that of the nuclear fraction decreases (Fig. 1). Assuming that the more physiological situation is that of the /n vivo perfusion, we can conclude that in the living tissue the subcellular localization of the ER is under the control of regulatory mechanisms for maintaining part of the ER in the cytoplasm to bind the E2 entering the cell. Alternatively, two kinds of ER molecules or different aggregation states of the same receptor molecule could be found in the cytoplasm and in the nucleus, the first one for the incoming E 2 and that of the nucleus for activating the genome. Any alter- ation of this equilibrium state leads to changes in the localization of the receptor molecules.

To follow the time course of the ER subcellu- lar distribution, "pulse and wait" experiments were designed where both uterine horns were peffused for 1 rain and subsequently rinsed /n situ. One horn was immediately processed and the other was kept in place in the anesthetized animal for different time periods before pro- cessing the tissue. Assuming that the cytosol is expression of the cytoplasmic extract, a

1°° I~- . .~osol } _ . oo! l

O' 5' 15' SO' Time

Total ~ Cytosol I T ~ Nucleus

Fig. 2. Translocation of the E2-ER complex in the rat uterus in vivo. Pulse and wait experiments: after I min [3H]E2 perfusion, one horn was immediately processed (0 time). The other horn was kept in place and processed at the indicated time. Bars represent E2 binding. Insert: Lines were drawn according to the mean values of the cytosol and nuclear bars. Total binding (cytosol + nucleus) shows a

progressive decline with time.

Table 2. Perfudon ~ vwo and further incubation /n vitro of the perfmed rat uterus

Perfusion Incubation (P/I).IO0 n

Cytosol 370.0 + 65.7 552.3 + 73.2 67% 7 Nuclei 329.6 + 89.9 286.9 + 35.1 I 15% 6

Specific binding in fmol Ez/mg protein. Perfmion time 10 rain: [3HIE 2 concentration in the perfmate: 10riM for total binding in one uterine horn and with the addition of 2 #M DES for nonspzcific binding in the other horn. Incubation time: 20 h at 4°C; 10 nM [3HIE 2 for total binding and 10nM [~H]E2 plus 2/~M DES for nonspccific binding in the corrcslmading perfmcd horn.

cytoplasm-nucleus translocation of the label can be appreciated (Fig. 2).

The in vivo occupation of the receptors by the ligand should also be under regulatory mechanisms that cannot be evidenced/n vitro. To approach this aspect, uteri were perfused in vivo for 10 min with [3I-I]E2 when a steady state is achieved and then utilized for an in vitro incubation. One horn was perfused with 10 nM [~H]E2 for total binding and the other with 10riM [~I-I]~ plus 2#M diethylstilbestrol (DES) for nonspecific binding. After excision, the uteri were homogenized in TEGMo buffer and cytosol and nuclear fractions were obtained by differential centrifugation. Part of these frac- tions were processed as described to measure the [~H]Fq-ER binding/11 vivo. Other al/quots of the corresponding uteri were incubated with 10 nM [3HIE2 for 20h at 4°C without and with 2#M DES for measurement of the total and nonspe- cific binding, respectively. Thus, unoccupied

200- -

100

5O

o

(t)~ 100

50

o

I I

o

~o

0 0

o o

o o

I I 25 50

[SH]E 2 (nM)

Fig. 3. "Saturation" curves for the E,-ER binding in the rat uterus in vivo. Pcrfusion time: 10 min. Upper graph: cytosol. Lower graph: nucleus. Specific binding in fmol

E~Jmg protein.

Rat uterine estrogen

Table 3. Parameter5 derived from the "saturation" data in vtvo

Parameter Cytmol Nuclei

Maximal binding 235 + 13 103 :[: 10 (fmol/mg protein)

/~ (riM) 16 + 2 2.7 ± 0.8

Michaelis--Menten equation.

sites after the in vivo perfusion should be satu- rated by the subsequent in vitro incubation. Separation of the free ligand in the cytosol was accomplished by C-D treatment and in the nuclear fraction by two washings in cold buffer. The in vivo occupied binding sites in the cytosol represent 67% of the maximal binding obtained in vitro. In the nuclear fraction the binding values for in vivo and in vitro are of the same magnitude (Table 2).

By using increasing concentrations of [3I-I]E2 in a 10 rain perfusion period it was possible to draw a "saturation" curve in vivo from where apparent dissociation constant and maximal binding values can be derived. These parameters correspond to an in vivo condition where a real saturation cannot be attained. However they are valid for our experimental design and represent a measure of the affinity and capacity, respect- ively of the uterine E2-ER binding in the living animal. In these experiments, for every concen- tration point total and nonspecific binding were determined in the same animal, perfusing one uterine horn with [3I-I]E2 alone and the other horn with the same [3HIE 2 concentration plus 200-fold of the unlabelled ligand. Specific binding was the difference between both values (Fig. 3). Data were analysed by Michaelis- Menten and by Hill equations according to a computer program for nonlinear equations. Hill coefficient (nil) was not significantly different from 1, thus, a positive cooperativity between multiple sites model can be discarded. The data fit better to the Michaelis-Menten model for a single binding site in both subcellular fractions (Table 3). The Kd values for the E2 binding in the cytosol and in the nucleus are significantly distinct (P <0.01), as an expression of the different functionality of each binding protein.

DISCUSSION

Most of E2-ER binding studies correspond to in vitro experimental conditions which cannot always be extrapolated to the tissue in the living animal. In our in vivo model the ligand con- centration is kept constant in the perfusate

receptors /n v/vo 281

and at the established speed it practically does not change along the run. Values for maximal binding (Table 3) are different from those of Table I for cytosol and nuclei. Probably, using a 10 nM [3I-1]E2 concentration, "saturation" was not reached in the 10 to 40m in perfusion periods tried; thus, the results in Table 1 are valid for the [~H]E2 concentration used.

The subeellular ER distribution is a topic under discussion. The initial two steps con- ception [5], with transformation and transloca- tion of the cytoplasmic receptor to the nucleus under the ligand entrance and binding, was substituted by the one step model, where the receptor protein was exclusively in the nucleus and was activated by the ligand binding. With respect to the way the hormone travelled from the cell membrane to the nucleus, there are two nonexcluding alternatives: one, where the hor- mone due to its liposolubility would cross the cytoplasm to find the receptor in the nuclear compartment [6]. The other alternative is the existence of a transport protein or receptor favouring the cytoplasmic passage of the lig- and [7]. The translocation model postulated that the same molecule under the ligand binding followed a series of transformations (oligomer to monomer and dimer) or conformational changes (unfolding or exposure of radicals) and the complex formed travelled to the nucleus. The transport model distinguishes one binding protein in the cytoplasm different from that in the nucleus, the "activable" receptor molecule. We detected two receptor species, the cytosoli¢ (cytoplasmic) with a Ka of 16 nM and the nuclear, with a Ka of 2.7 riM. Conformational changes could be responsible for the different Ka found for the cytosol and for the nuclear fraction receptors. Further experiments to characterize both molecular entities are necess- ary. The existence of more than one ER species has already been demonstrated [8-10].

As for other members of the steroid recep- tor superfamily the higher affinity found for the ER nuclear receptor should be related to its regulatory transcriptional function Ill] The lower affinity of the cytosolic receptor is more in accordance with a translocation function or with the liberation of the hormone at the nucleus vicinity. In any case, the cytoplasmic and the nuclear E2-ER complexes are clearly differentiable both by their affinity values and by the time sequence of the binding events (first the cytoplasmic and then the nuclear) and should not be enclosed as one entity.

282 E ~ m J E L L ~ n N et al.

One important issue is the succession of events followed by the "pulse and wait" exper- iments (Fig. 2). The initial binding is produced almost completely in the cytoplasm if we con- sider that the cytosol contains the cytoplasmic soluble components. There is a small labelling in the nuclear fraction (10%) that can be attributed to the passage already occurring during this first minute. As the time elapses the ligand initially retained by the cytoplasmic receptor-transporter is released or translocated to the nuclear compartment following the kinetics shown in Fig. 2. As to the discrepancy with the one step conception, mainly supported by immunohistochemical findings showing the receptor exclusively located in the nucleus [12], it is possible that the epitope of the nuclear protein is not present in the receptor- transporter localized in the cytoplasm or if it is an oligomeric protein, the epitope could be "protected" or hidden inbetween two units. In the pulse experiments, where the uterus is immediately processed after the perfusion, it cannot be discarded that nuclear receptors not yet reached by the hormone, could be released by homogenization of the tissue and conse-

quendy appear in the cytosol. However, when instead of a pulse, a constant ligand supply is offered during 10 rain, where a steady state is attained, the ligand-reeeptor complex can be detected in both compartments either corre- sponding to one protein suffering confor- mational transformations or to two separate proteic entities. An equilibrium for one or the two ligand-receptor complexes between both compartments would be established for replen- ishment of the activable nuclear complex (Fig. 4). The ER cytoplasm-nucleus equi- librium/exchange is also postulated under different basis by several authors [6, 13, 14].

A comprehensive study by Jakesz, Jassid and Lippmann in 1983 [4] showed the presence of cytosolic and nuclear ER species labelled by previous [3H]E 2 i.p. or s.c. injection in rats, where the [3H]E 2 serum level was checked over a 6 h period. In these experiments the binding was produced in vivo, before any manipulation of the tissue and two ER populations were detected both in the cytosol and in the nucleus, with different physicochemical characteristics for those of each compartment. These results, as well as ours, cannot be explained by the exclu-

. . . . / / Nucleus Cytoplasm 4 ~ . J~/~/

Fig. 4. Model for activation of DNA transcription by E 2. (I) F-z; (2) plasma membrane transfer-receptor; (3) cytoplasmic estrogen transporter-receptor; (4) heat-shock protein; (5) nuclear ER; (6) E2-ER complex;

(7) DNA regulatory (enhancer--silencer) domain; and (8) hormone (estrogen) responsive protein.

Rat uterine estrogen receptors/n vivo 283

sive nuclear location of the ER as it emerged from the immunocytochemical [15] and enucle- ation [16] techniques. It is unlikely that the E2 transit to the nucleus would be driven only by the diffusion and liposolubility properties of the steroid hormone, when membrane pro- teins that selectively bind steroids have been unequivocally shown[17] and even "pho- tographed"[18] at the membrane boundary, The same is valid for cytoplasmic proteins that bind steroids[7], besides their unavoidable presence when synthetized in the ribosomes. [3H]E2 incubations performed with whole cells in culture without disruption of their integrity before binding, show also a cytoplasmic- nuclear translocation process along the exper- imental time with differences between the MCF7 and the T47D cell lines [19]. Any arti- factual movement of the complex can be dis- carded as explanation for the translocation because the cells were submitted to the same procedure at different intervals, but not- withstanding, substantial subeellular location changes occurred.

Previous immunocytochemical procedures have shown cytoplasmic and nuclear location of ER [5, 20]. Although the antigen purification methodology could be questioned, that is, that more than one molecular estrophilin species could be recognized by the antibody, the fact is that such species (one or more) exist in the cytoplasm and in the nucleus. With monoclonal antibodies the presence of a specifically associ- ated cytoplasmic ER protein, not found in the nucleus has also been shown [21].

Cytoplasmic and nuclear binding proteins could be different molecular entities or frag- ments from the same core associated with other proteins or forming homo or heteroligomers with a differential functionality reflected by their affinity constants, specificity, aggregation, immunoreactivity, etc. In the model depicted in Fig. 4. the dissociation-binding balance can be switched in such a way as to favour the binding when the hormone enters the cytoplasm and to liberate the ligand when it is close to the nucleus where it is "captured" by and bound to the nuclear receptor with a higher affinity for the ligand. In any case, the existence of a kind of estrogen receptor or transporter precludes the one step model where the ligand is travelling free to the nucleus. The progression of the hormone in the cytoplasm toward the nucleus should be regulated by the dynamic and kinetic parameters of a binding and dissociation pro-

cess between a transporter/receptor protein and its ligand.

From our experiments we cannot ascertain which cell types are involved in the E2 binding. We assume that in the steady state (10rain perfusion) endometrial and myometrial cells are reached by the hormone. ER have been described in these cellular types [22]. The cyto- chemical localization of the [~I-I]E2 complex after the perfusion is under study in our laboratory. The results from measurements of free and bound radioactivity in the cytosol and in the nucleus, indicate that 85% is associated with a macromolecule in the cytosol and the remaining 15% correspond to the free ligand. It could represent the dissociated E 2 in the vicinity of the nuclear membrane, ready to cross to the nucleus and to bind the activable receptor therein. In the nucleus, the free E2 represents 42% of the total hormone in this compartment. This different proportion of the free ligand in the cytosol and in the nucleus could be a reflection of the interrelation between the association-dissocia- tion constants of the E2-ER complex and its functionality in each compartment.

The combined perfusion-incubation exper- iments show that not all the receptor molecules are occupied when the ligand is bound in vivo (Table 3). The value of 67% obtained for the bound ligand in the cytosol by the uterine perfusion, is an approximation in our exper- imental conditions (anesthesized animals, uter- ine horns isolated in situ, intraluminal perfusion, which is not the natural way for the ligand uptake). However, it is the first quanti- tative datum for the maximal capacity of a tissue in vivo.

In the nuclei after in vivo perfusion, the /n vitro incubation values were not significantly different from those of the perfusion (Table 3), that is, the available binding sites apparently were occupied by the in vivo binding. The meaning of this differential occupancy of the cytosolic and nuclear binding sites in the in vivo perfusion is not clear and shall be approached more profoundly in future studies.

The nuclear-cytoplasm protein movement and exchange should be considered as one of the most efficient regulatory mechanisms of gene transcription, where the cytoplasm is a dynamic reservoir for a very rapid provision and subtraction of metabolites (macromol- ecules included) directed to selective DNA do- mains for a given time (from pulses to pro- longed periods) to trigger enhancing or silencing

284 E ~ ~ et ai.

effects [23]. This regulatory traffic is applicable to the steroid receptor molecules through con- formational changes, oligomeric break and formation, association-dissociation and fold- ing-unfolding to other proteic species and other dynamic regulatory changes operating /n vivo. In vitro, such vital adjustments are probably absent and only partially envisaged through the appearance of some configuration of the protagonic molecules.

Summarizing the evidence presented in this article and that of other authors, we postulate a general three step model for the modulation of the genomic transcription by steroid hormones: (I ) hormone binding to (and dissociation from) a plasma membrane receptor for entrance to the cell; (2) hormone binding to (and dissociation from) a receptor/transporter for its passage to the nucleus; and (3) binding to a nuclear recep- tor for its activation, followed by binding of the activated complex to a given genomic DNA regulatory sequence directly or through an association to a hormone responsive protein (Fig. 4).

The identity or homology between nuclear and cytoplasmic receptors cannot be ascertained from our data. Several alternatives are poss- ible, including oligomeric associations and dis- sociations, conformational changes, etc, with different patterns of translocation of these proteins between both compartments accord- ing to the grade of occupancy of the recep- tors, the manipulations of the tissue, etc. In this respect, the extensive use of cytosolic ex- tracts intended to represent the whole receptor population, as well as the exclusive nuclear location of the ER, should be postulated with caution when extended to the in vivo subcellular compartmentation of the estrogen receptors.

Acknowledgements--This study was supported by grants from the Consejo Nacional de Investigaciones Cientificas and from the University of Buenos Aires. The very able technical assistance of Ines Nievas is greatly acknowledged.

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