Studies on estrogen entry into uterine cells and on estradiol-receptor complex attachment to the nucleus — Is the entry of estrogen into uterine cells a protein-mediated process?

320 (1973) 267-283 ~) Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands

Blochlmica et Biophysica Acta,

BBA 27219

STUDIES O N E S T R O G E N E N T R Y INTO U T E R I N E CELLS A N D ON ESTRAD I O L - R E C E P T O R C O M P L E X A T T A C H M E N T TO T H E N U C L E U S -- IS T H E E N T R Y OF E S T R O G E N INTO U T E R I N E CELLS A P R O T E I N - M E D I A T E D PROCESS.'?

EDWIN MILGROM, MICHEL ATGER and ETIENNE-EMILE BAULIEU Unitd de Recherches sur ie Mdtabolism¢ Moldculaire et la Physio-Pathoiogie des St#roides de r lnstitut National de la Santd et de la Recherche Mddicale, Ddpartement de Biochimie, Facultd de Mddicine Paris-Sud, 78 Avenue du Gdndrai Leclerc - 94270 - Bic#tre (France)

(Received March 12th, 1973)

SUMMARY I. The entry of estrogens into immature rat uterus target cells has been studied under experimental conditions where it could be distinguished from the subsequent binding to the receptor and also from the adsorption to the tissue outside the cells. Several lines o f evidence suggest that the entry of estradiol cannot be ascribed to simple diffusion. Various SH-group blocking reagents inhibit the entry of estradiol (and estrone and estriol) into uterine cells. Correlative experiments indicate that they inhibit to a lesser degree (or even not at all) the binding of the steroid by the receptor. The entry process becomes saturated in the range of physiological hormonal concentrations. It shows estrogen specificity since the incorporation of estradiol is not in competition with an excess of cortisol, corticosterone, progesterone and testosterone. However, this selectivity differs from that of the intracellular receptor since diethylstilbestrol exhibits less affinity than estradiol for the entry mechanism, whereas both estrogens are bound to the receptor with the same affinity. It seems very probable then that there is a protein-mediated step on the way of estrogens to the receptor, which may be involved in their passage through the uterine target cell membrane. 2. The estrogen uterus cytosol receptor, when it has bound estradiol, is able to bind itself to a nuclear acceptor structure. 0t-Iodoacetamide inhibits the nuclear accumulation of estradiol-receptor complexes. This inhibition can be reproduced in a reconstituted acellular system (cytosol+nuclei)under conditions where there is no noticeable modification of the estrogen binding by the receptor. These experimental results differentiating between ligand binding and binding of the hormone-receptor complex to the nuclear acceptor, suggest that they are operative through two distinct sites of the receptor protein.

* A preliminary account of these experiments has been published in (1972) 274, 2771.

C. R. Acad. Sci. Paris

268

E. MILGROM et ah

INTRODUCTION It is usually admitted that steroids, like other lipophilic compounds, diffuse freely through cell membranes. Therefore, the first specific event in target cells would be the binding to the intracellular cytosol receptor. However, in most studies, the incorporation of radioactivity during in vitro incubations of a tissue with a radioactive steroid has been considered as a direct estimate of the hormone entering the cells. With this approach two experimental pitfalls are not taken into consideration. First, an important amount of the incubated steroid is adsorbed to the tissue; it does not enter the cells, but is taken into account when considering the incorporation of radioactivity. Williams and Gorski I have described an experimental procedure (see Material and Methods) which solves this difficulty in the case of estradiol incubations with rat uteri. Secondly, modifications in receptor content of tissue lead to modifications in hormone incorporation. It is thus necessary when studying such an incorporation to distinguish between the variations due to the entry itself and the variations due to the subsequent binding to the receptor. These considerations and the necessity to use an organ containing a stable and well characterised receptor system, led to the use of the rat uterus for the study of the penetration of steroid hormones into responsive tissues. In experimental conditions where the receptor binding capacity was carefully controlled and distinguished from the entry process, the characteristics which differentiate passive diffusion from protein-mediated entry (saturability, specificity and inhibition by amino acid reagents) were examined. During these experiments, the cytosol --, nucleus transfer of estradiol-receptor complexes was also studied. It has been shown previously that the binding of estradiol gives the receptor the capacity to bind to a nuclear acceptor component (see reviews in ref. 2). Thus the receptor has operationally at least two distinct sites, one for hormone binding and the second for interacting with a nuclear structure. The results presented here indicate that they can be experimentally differentiated. MATERIALS AND METHODS 1. Buffer

A 0.01 M Tris-HCl, pH 7.4, 0.0015 M EDTA buffer (Tris-EDTA buffer) was used in most experiments. 2. Steroids

[6,7-3H2]Estradiol (N.E.N., specific activity 50Ci/mmole), [2,4,6,7-3H4] estradiol (N.E.N., 110 Ci/mmole), [6,7-3H2]estriol (N.E.N., 42 Ci/mmole), [6,7-3H2] estrone (C.E.A. France, 48 Ci/mmole) were used. For comparison, the results were normalised to a specific activity of 50 Ci/mmole. The purity of radioactive steroids was checked by thin-layer chromatography on silica gel using a benzene--ethyl acetate (3:2, v/v) system for estrone and estradiol and a 72 % ethyl acetate-13.5 % water saturated in hexane-4,5 % absolute ethanol-10 % acetic acid system for estriol. Non radioactive, chromatographically pure steroids were gifts of RousselUCLAF.

ESTROGEN ENTRY INTO UTERINE CELLS

269

3. lnhibitors 2,4-Dinitrophenol B grade, iodoacetic acid sodium salt (iodine free) and ctiodoacetamide were obtained from Calbiochem (Los Angeles, Calif.), oligomycin (15 % oligomycin A and 85 % oligomycin B), p-chloromercurophenylsulfonic acid monosodium salt and 5,5'-dithiobis(2-nitrobenzoic acid) from Sigma (Saint-Louis, Mo.), Ouabain was donated by the Nativelle Laboratories (Paris). 4. In vitro measurement o f 3H-labeled steroid entry into the rat uterus It has recently been shown 1 that after in vitro incubation of uteri with [3H]estradiol, some of the radioactive steroid is trapped in the tissue, outside the cells, and cannot be washed out. When the preparation is subsequently homogenized, this hormone is bound secondarily to the receptor proteins. To prevent this artifact, Williams and Gorski I suggested the addition of an excess of non-radioactive estradiol prior to homogenization. This isotopic dilution prevents further binding of radioactive steroid while the previously bound radioactive estradiol does not dissociate because of the very slow rate of dissociation of the hormone-receptor complex at low temperature. The rate of association of estradiol to the receptor is not a limiting step since it has been shown to be very fast 3. Under these conditions, the measurement of [aH]estradiol bound to high-affinity receptor protein can be an estimate of the steroid entering into the target cells (provided there is no alteration of the binding). In each incubation, six uterine horns of 19-22 day Wistar rats were used. They were incubated in 4 ml Dulbecco modified Eagle medium containing the alllabeled steroid and eventually the inhibitor, under air and with agitation. In some cases they were preincubated in the absence of the all-labeled steroid. The length and the temperature of incubation varied in different experiments and are indicated in the corresponding sections. All subsequent steps were performed at 0 °C. The vials were cooled for 5 min and the uteri washed twice for 10 min with Tris-EDTA buffer containing 1/~M of unlabeled estradiol. The uterine horns were homogenized in 1 ml Tris-EDTA buffer containing 1/~M unlabeled estradiol. The homogenate was centrifuged at 800 × 10 g • min and the supernatant was further centrifuged at 105 000 × 60 g . min. The final supernatant was called the cytosol. The pellet of the initial centrifugation was washed twice in 3 ml Tris-EDTA and extracted 1 h with 1 ml of the same buffer containing 0.4 M KCI. After a 105 000 × 60 g • rain centrifugation, the supernatant was called the nuclear extract. In the cytosol and in the nuclear extract the receptor-bound radioactivity was measured (see Section 6). 5. Measurement o f the uterus receptor content after incubation Uterine horns were incubated under the same conditions as in 4, except that no radioactive or unlabeled steroid was added at the incubation or the homogenization step. The cytosol was identically prepared and incubated for 90 min at 0 °C with 5 nM [aH]estradiol, a saturating concentration for the receptor a. The receptor-bound radioactivity was measured (see Section 6). 6. Measurement o f receptor-bound all-labeled steroid at 0 °C 0.3 ml of the soluble preparations (cytosol or nuclear extract after tissular or cellular labeling with hormone) were incubated in duplicate for 1 h with agitation,

270

E. MILGROM et al.

with 0.3 ml of a suspension of dextran-coated charcoal (0.05 % dextran, 0.5 % charcoal in Tris-EDTA buffer) at 0 °C. During this period the "non specific" (low affinity) complexes dissociate, whereas the specific (high affinity receptor) complexes practically do not (see the"differential dissociation" technique in (4)). The suspension was then centrifuged at 800× 10 g . rain and 0.2 ml of the supernatant counted in duplicate. 7. Measurement o f receptor-bound 3H-labeled steroid at 37 ° C

Taking into account the fast dissociation of the complexes and the inactivation of the receptor at 37 °C, the adsorption technique was modified as follows. Receptor solutions were prepared in buffer containing 15 % sucrose. To 0.3 ml of the various incubations was added, as quickly as possible and avoiding agitation, 0.3 ml of the dextran-coated charcoal suspension previously heated to 37 °C. All tubes were centrifuged together for 2 rain at 3000 × g at 37 °C. The supernatant was immediately separated from the pellet and aliquots were counted for radioactivity. Parallel incubations containing the same amount of radioactive steroid in buffer were identically processed, thus giving blank values which were then substracted from the values obtained with receptor solutions. Under these experimental conditions, the contact between the charcoal and the binding solution occurs only for a very short time during the centrifugation step, before which the two phases are separated due to differences in density. The time of contact is also strictly identical for all the incubations of the same experiment. With this approach reproducible measurements of receptor-estradiol binding at 37 °C could be obtained. 8. Radioactivity

Radioactivity was measured in Packard glass vials with a mixture of 3 ml ethanol and 10 ml of a Omnifluor (N.E.N.)-toluene solution (4 g/l). RESULTS Estrogen entry into prepuberal rat uteri Saturability o f the process o f estradiol entry into uterine cells. Protein-mediated

transport is saturable whereas simple diffusion is not. In order to study the characteristics of estradiol entry into rat uteri without being limited by the saturation of the receptor, very short incubations (5 rain) at 37 °C in presence of various amounts (0.1-50 riM) of [3H]estradiol were performed. The entry process was linear until the 15th rain (see Fig. 5) and initial velocities of entry could thus be measured. The entry rate was expressed as the amount of hormone entering one uterine horn per rain (i in Fig. 1) and it was measured under the experimental conditions detailed in Materials and Methods, Section 4. As the range of steroid concentrations in the incubation medium T was very wide a Lineweaver-Burk 5 type plot could not be used and the results are given with a Eadie 6 or Scatchard ~ type plot where the ratio i/T is plotted vs i. In such a plot a non-saturable interaction will give an horizontal line since i will be proportional to T, whereas a saturable interaction will give a straight line of negative slope. The entry of estrogen into the uterus (Fig. 1) seems to depend on a saturable process at low steroid concentrations and a non-saturable process at higher concentrations. When the experimental points were studied with a IBM 360 computer using a program previously described a, the best fit was obtained assuming the exis-

ESTROGEN ENTRY INTO UTERINE CELLS

271

tence of both a saturable and a non-saturable process. For the former the dissociation constant was KD=3.21 nM and the maximum entry rate was ira,,----4.0 fmoles/min per uterine horn. The theoretical line computed with these values is given in Fig. 1 and can be compared with the experimental points. Actually, these quantitative data are only approximate due to the experimental impossibility of handling a very large number of simultaneous incubations. But the important point in the results of this experiment appeared to be the mere fact that the entry process was saturable. However, it was important to see that this saturation could not be simply ascribed to the characteristic hormone-receptor interaction, and thus, that the proportion of unbound hormone present in the uteri at the end of the 5-min incubations was very low. The concentration of unbound ligand could be calculated using the relationship 9. (Binding sites). (Unbound ligand) (Bound ligand) = ~ t i - ~ n e q ~ c ~ ) +

(Unbound ligand)

The concentration of bound ligand being experimentally (Fig. 1) measured, the concentration of binding sites and the dissociation constant at 37 °C had to be measured in order to calculate the concentration of unbound ligand. These binding parameters were measured in separate experiments performed with a soluble system, the uterine cytosol. 0,2 (T~ ~

0.1.

lOS

\

0.05.

1

2

i moln x*1014

Fig. 1. Saturability o f [3H]cstradiol entry into rat uteri. 5-min incubation at 37 °C in the presence of various concentrations (0.1-50 riM) o f [3Hlestradiol. i, moles of steroid incorporated/min per uterine horn; 7", total concentration o f stroid in the incubation medium. The theoretical computerized representation of the entry process (broken line) has been obtained on the basis of a program 8 for a double system with one high affinity saturable component and another component of lower affnnity and non saturable. The binding parameters for the saturable component arc: dissociation constant (KD) 3.21 riM, maximal entry rate (i,,,) 4.0 fmoles/min per uterine horn. For the non saturable component 1 / K v • i s , , is 3.56 10-3/min per uterine horn.

Since the estrogen receptor is very unstable at 37 °C, it was necessary to find a technique allowing a fast measurement of the steroid-receptor interaction. Using such a technique (see Materials and Methods, Section 7) it was possible to show (Fig. 2) that the association between estradiol and the uterine receptor was highest after 5 min followed by a decay probably due to receptor inactivation. Therefore, incubating uterine cytosol with various concentrations (0.2 riM-10 nM) of [3H]estradiol at 37 °C for 5 min enabled the measurement (Fig. 3) of the dissociation con-

E. MILGROM et al.

272

stant (KD=0.35 nM) and of the binding site concentrations (0.3 nM in the incubation corresponding to a concentration in the uteri of 20 pmoles/g wet weight or 20 nM if the density of uteri is assumed to be 1) of the receptor. (B)

(B)/(U)

nM

0.1 \* O"

'\

0.05

"""'°'~°~ . . . . . . . . . 8-Q. 0

1~

1'5

2'0rain

0

0~,

i(B,nM

Fig. 2. [3H]estradiol-uterine receptor association at 37 °C. Rat uterine cytosol (protein concentration 0.7 mg/ml) was prepared in Tris-EDTA buffer with 15 ~ sucrose. Aliquots of this cytosol were incubated 5 min at 0 °C with 0.2 nM [~H]estradiol and then for various periods of time at 37 °C, and bound hormone was measured (see Materials and Methods, Section 7). Fig. 3. Binding of [3Hlestradiol at various concentrations (0.2-10 riM) to uterine receptor at 37 °C. 20 uterine horns (wet wt 8.75 rag/horn)were homogenized in Tris-EDTA buffer with 15 ~ sucrose. 12 ml of cytosol were obtained, incubated 5 rain at 0 °C and 5 rain at 37 °C with l~H]estradioi. The bound hormone was measured. The theoretical computerized binding representation (broken line) has been obtained on the basis of a programs for a double system with one high affinity saturable component and another component of low affinity and non saturable. The binding parameters for the saturable binding are: dissociation constant (Kn) 0.35 nM, binding sites concentration (N) 0.3 riM. For the non-saturable binding I/Ko" N is 0.07. These values were calculated using a program for an IBM 360 computer 8. The fit of the experimental and of the theoretical points is shown in Fig. 3. The concentration of binding sites calculated at 37 °C was identical to that measured at 0 °C. Using these values, the relation previously given, and the concentration of bound hormone measured in these 5-min incubations it was possible to calculate the proportion of unbound hormone in the tissue. Since the amount of bound steroid present per uterine horn after the 5-min incubations varied between 0.79 fmoles (0.1 nM incubation) and 105 fmoles (50 nM incubation) representing a concentration o f bound hormone of 0.09 and 12 nM, it was calculated that the proportion of unbound hormone would vary between 1.7 and 4.2 ~o. This shows that due to the high concentration of receptor in the uterine tissue and to its high aftinity for estradiol, the amount of hormone entering the tissue in these short incubations could not saturate the receptor. Thus, the observed saturation can be interpreted in terms of an entry process into the uteri and not of receptor-hormone interaction. Effect o f SH-blocking agents on estradiol entry into target cells. In the presence o f SH-blocking agents the entry at 37 °C of radioactive estradiol into uterine cells was markedly impaired (Table I). Under these conditions, control measurements showed that the uterine content in receptor binding sites was also altered. However, in all cases, there was a significant difference between the extent of inhibition of both processes, the entry being more inhibited than the receptor capacity. In some experiments the steroid having entered the tissue exceeded the receptor capacity at the end

ESTROGEN ENTRY INTO UTERINE CELLS

273

TABLE I EFFECT OF SH-BLOCKING AGENTS ON THE ENTRY OF [SH]ESTRADIOL INTO THE RAT UTERUS AND ON ESTROGEN RECEPTOR CONTENT Preincubation (10 rain) and incubation (30 rain) were performed at 37 °C. The incorporation o f [3H ]estradiol (10 aM ) in seven different triplicate incubations in the absence o f inhibitors corresponds to 11 500± 1 100 dpm ( ± S.E.) per uterine horn. The receptor content after the same incubation in absence of hormone was equivalent to 4 200 ± 350 dpm per uterine horn. Results are given as the mean of three experiments d= S.E.

Inhibitor

Entry*

Receptor content per* after identical incubation

No 2 mM 0t-iodoacetamide 10 mM ct-iodoacetamide 5 mM iodoacetate 30 mM iodoacetate 3 mM 5,5"-dithiobis(2-nitrobenzoic acid) lO mM 5,5'-dithiobis(2-nitrobenzoic acid)

100 4.7±0.1 3.3 ± 1.3 25.9 4-0.7 7.3 ± 1.0 74.6±2.3 62.0-/-3.5

I00 21.6--/-1.5 23.5±3.0 31.5± 1.9 33.6-/-1.5 99.9±9.5 89.7±0.8

< < < < < <

0.001 0.01 0.I O.OOl 0.1 O.Ol

* Percent of the total (cytosol+nuclear) entry of [3H]estradiol in the absence of inhibitor. ** Statistical significance (Student's t test) of the difference between the effects on entry and receptor content.

of the incubation. This was probably due to protection of the receptor by the steroid in the studies on the entry process. ct-lodoacetamide and iodoacetate exerted the greatest inhibition. Dithiobisnitrobenzoic acid had only a very small effect on the receptor content and a moderate effect on the entry. p-Chioromercurophenylsulfonate is reputed not to enter cells. Its effect was studied at various concentrations with the hope that the entry of the steroid could be inhibited without any effect on the receptor content. In fact, p-chloromercurophenylsulfonate was found to be very similar to other SH-blocking reagents, and thus probably does enter uterine cells to some extent. Again, at all the studied concentrations, the inhibition ofestradiol entry was higher than the decrease in receptor content (Fig. 4). %i

•

.

,

,

,,

b .... °,~

o:s I;5

3

5

16 mU

Fig. 4. Effect of p-chloromercuriphenylsulfonate on estrogen entry into rat uteri (b) and on their receptor content (a). Preincubation 10 rain and incubation 30 rain at 37 °C. The entry of [3Hlestradiol (1 nM) in two different triplicate incubations in the absence of inhibitor was 2 781 ±104 dpm (4S.E.) per uterine horn. The receptor content was as in Table I.

274

E. MILGROM et al.

The effects of other SH reagents (N-ethylmaleimide, parahydroxy- or parachloromercuribenzoate) could not be studied since they prevent the binding of estrogen to the receptor t°' 11. The time course of u-iodoacetamide effects on estradiol entry and receptor stability was studied at two different temperatures. At 37 °C (Fig. 5) the decrease of the receptor (Fig. 5B) during incubation was very fast, even in the absence of a-iodoacetamide (e); when the latter compound was added (f), a relatively small difference was observed initially, and later became more significant after 20 and 30 rain. In contrast, the rate of entry of [3H]estradiol into the tissue (Fig. 5A) was greatly reduced by ~-iodoacetamide (a v s b) even at the start of the incubation. Moreover, in experiments without u-iodoacetamide, it was observed that a 5-min preincubation (c) did not change the estradiol entry during the first

dpm/horn 15000. , •

500(; g

A, ~ w •

1500C

C

A

1000(

'

?

500( fo

dpfn/horn 50000t

2o

~

40mh, I0

20

dpm/horn

B

4OOO(]

30

40

5~

~m

B

4OOO( 3000( ~

~

20OO0

¢

1000( 10

2'0

3'0

40m~

0

~

2'0

3o

,io

5o

eom

Fig. 5. Comparison of the time course of the effects at 37 °C of --iodoacetamide on the entry of radioactive estradiol (10 riM) into rat uteri (A) and on their receptor content (B). a, entry of [3H]estradiol in the absence o f ~-iodoacetamidc, without preincubation; b, entry o f [aH]estradioi in the presence of 2 m M ~-iodoacetamide, without preincubation; c, entry of [3H]estradioi in the absence of ~-iodoacetamide after preincubation of 5 rain; d, entry of [3H]estradiol in the presence of 2 mM cc-iodoacetamide, after preincubation of 5 rain; e, receptor content in the absence of cc-iodoacetamide; f) receptor content in the presence of 2 mM ~-iodoacetamide. Fig. 6. Comparison of the time course on the effects at 25 °C of ~-iodoacetamide on the entry of radioactive estradiol (10 nM) into rat uteri (A) and on their receptor content (B). 10 rain preincubation, a, entry of [3H]estradiol in the absence o f ~.iodoacetamide; b, entry of [3H]estradiol in the presence of 2 m M ~-iodoacetamide; c) receptor content in the absence of ~-iodoacetamide; d, receptor content in the presence of 2 mM ~-iodoacetEunide.

E S T R O G E N E N T R Y INTO U T E R I N E CELLS

275

minutes, although during the s a m e p e r i o d there was a marked decrease of the receptor content, thus showing that the latter was not a limiting factor. The different time course of a-iodoacetamide effects on estradiol entry and on receptor content at 37 °C was also observed at 25 °C (Fig. 6), where the diminution of the receptor content (Fig. 6B) was less than in experiments at 37 °C. A possible interpretation of these experimental data is that two different sets of SH groups are present in a protein regulating the entry process and in the receptor. More reactivity of the former compared to the latter would explain why it is possible to obtain a time course and quantitative differences between the effects of SH-blocking agents on entry into the cells and binding of the hormone to the receptor, respectively. Hormone specificity of the estrogen entry process into uterine cells. (a). Since estrone and estriol can bind to the estrogen receptor and also slowly dissociate from the formed complex t2, the entry of radioactive estrone and estriol in the presence or absence of 0c-iodoacetamide was also measured. The inhibitor impaired the entry of both estrogens in a similar manner to that observed with estradioi (Table II). TABLE II EFFECT OF INHIBITORS ON T H E E N T R Y O F ~H-LABELED ESTROGENS INTO THE RAT UTERUS Total binding, cytosol binding and nuclear binding in the absence of inhibitors were, respectively, 4 179, 831, 3 358 dpm per uterine horn when estrone was incubated and 8 861,848, 8 013 dpm per uterine horn when estriol was incubated. Experimental conditions as in Table I. ~H.labeled estrogen

Inhibitor

Entry*

Estrone Estriol Estradiol Estradiol Estradiol Estradiol

~,-Iodoacetamide (2 mM) "~-Iodoacetamide (2 mM) Cortisol (1 p M ) Corticosterone (1 p M ) Testosterone (1 # M ) Progesterone (1 # M )

7.7 2.4 95.8 96.0 102.0 106.1

* Percent of the entry of the same [3H]estrogen (10 nM) in the absence of inhibitor.

Whether or not the three estrogens have one transport system in common cannot be established from these experiments. (b). The entry of non estrogenic steroids could not be tested directly since they are not bound by the estrogen receptor and there are no tests available for measuring their entry into the uterine cells. However, it was possible to study the eventual inhibition of entry of radioactive estradiol. Nonradioactive steroids, cortisol, corticosterone, testosterone or progesterone, at a concentration of 1 gM, were preincubated with uteri for 10 rain at 37 °C. The incubation at 37 °C for 30 min was performed in presence of 10 nM [3H]estradiol and 1 #M of the unlabeled competing steroid. As seen in Table II none of the tested non-estrogenic steroids inhibited the entry of radioactive estradiol. (c). Finally, experiments were designed to see if there were competition between different estrogens for the entry process and if the respective affinities for this process and for receptor binding were different. Here again short incubations (5 rain) and relatively low steroid concentrations were used in order to prevent as much as possible receptor saturation.

276

E. M I L G R O M

et al.

[3H]Estradiol entry (Fig. 7) was strongly inhibited by unlabeled estradiol, confirming the saturability of the process. Diethylstilbestrol exerted a lower inhibition at all the concentrations studied, and the inhibition due to estriol was even smaller. In contrast, when [3H]estradiol binding to uterine receptor was studied at 37 °C, the inhibition due to unlabeled estradiol and diethylstilbestrol was essentially identical (Fig. 8). %l 100

(B) . M c

~

80

7"~

lb(cI.M

o

. . . .

b

~;

l~)(c)n"

Fig. 7. Inhibition by estradiol (a), diethylstilbestrol (b) and estriol (c) of [3H]estradiol entry (i) into rat uteri at 37 °C. laH]Estradiol (I riM) and various concentrations (C) of competing steroids were incubated 5 rain at 37 °C with prepuberal rat uteri. Results are given as percent of entry of [3H]estradiol (I nM) in the absence of competing steroids (1.34 • 10-t5 moles/rain per uterine horn) + S.E. (triplicate incubations). Fig. 8. Inhibition by unlabeled estradiol and diethylstilbestrol of 13H]estradiol binding to rat uterus receptor at 37 °C. Uterine cytosol was prepared in Tris-EDTA buffer with 15 % sucrose (protein concentration 1.1 mg/ml). 0.4-ml aliquots were incubated in duplicate 5 rain at 0 °C and 5 rain at 37 °C with [aH]estradiol (0.5 riM) in the presence or in the absence of unlabeled estradiol (a) or diethylstilbestrol (b) (I-10 nM). Receptor bound radioactivity (B) was measured at 37 °C. C, concentration of competing steroid. These experiments suggest that a single saturable system controls the entry of estradiol and diethylstilbestrol into uterine cells and that it has an affinity for the two estrogens different to that of the uterine receptor. It is not possible to decide whether the relatively small inhibition exerted by estriol was due to competition for the entry system or for the receptor.

Effect of energy depletion or metabolic inhibitors on the entry of [aH]estradiol into the uterine cells. Metabolic inhibitors did not inhibit the entry of estradiol into uterine cells (Table III). Oligomycin and ouabain had no effect but seemed in some way to protect the receptor. K C N and 2,4-dinitrophenol had nearly identical effects on the entry process and on receptor content suggesting that their initial effect might have been on the latter. Absence of energy sources in the preincubation and incubation media had no effect on the entry or on the receptor content. Temperature effect on estradioi entry into the rat uterus. The rate of entry of radioactive estradiol was measured at 37 °C, 25 °C, 10 °C and 0 °C (Fig. 9). The initial linear part of each curve was used to determine the entry rate of radioactive estradiol. The observed temperature dependence cannot be explained in terms of limitation by the association between steroid and receptor since the association rate constant is very high even at 0 °C (1.5 • 10s-10 • 105 M -1 • sec-1) 3 and in these experiments the steroid concentration in the medium (10 nM) and the receptor concentration in the tissue (about 20 nM) were quite high (see Fig. 3).

ESTROGEN ENTRY INTO UTERINE CELLS

277

TABLE III EFFECT OF ENERGY DEPLETION ON THE ENTRY OF [3H]ESTRADIOL (10 nM) INTO THE RAT UTERUS AND ON ESTROGEN RECEPTOR CONTENT Experimental conditions as in Table I. In this experiment the Eagle--Dulbecco medium was replaced by a Krebs-Ringer phosphate solution with or without 1 m8 glucose/ml. Entry and receptor content in the absence of glucose were compared to the same parameters as those in the presence of glucose. Results are given as the mean of three experiments 4- S.E.

Inhibitor

Entry

Receptor content after identical incubation

No Dinitrophenol (5 mM) KCN (30 raM) Oligomycin (0.14 raM) Ouabain (1 raM) Absence of energy so urces

100 54.7t2.9 24.4+ 1.4 102.2=£6.6 101.8+2.0

100 74.0+ 10.4 33.1 +3.3 115.94-5.6 134.7+!3.2

103.5 -t-6.4

91.7 4-7.6

i moles x l 0 ls

270

29o

me T

Fig. 9. Temperature effect on estradiol entry into rat uteri. [3H]Estradiol (10 nM) was incubated for various times and various temperatures with rat uteri. The initial linear part of each curve was used to measure the entry rate (/=moles incorporated/rain per uterine horn). 7", absolute temperature.

Attachment o f the estradiol-receptor complex to the nucleus Tissue incubations. (a). In the experiments a l r e a d y r e p o r t e d in T a b l e I a n d Fig. I, the effect o f various inhibitors o n the subccllular d i s t r i b u t i o n o f 3H-labeled estrogens in the uterus was also studied. T a b l e IV shows t h a t in the absence o f inhibitors, 8 0 - 9 0 % o f the b o u n d estradiol was f o u n d in the nuclear fraction after 30 min incub a t i o n at 37 °C. O f all the inhibitors tested, g - i o d o a c e t a m i d e , i o d o a c e t a t e , dithiobisn i t r o b e n z o i c acid a n d p - c h l o r o m e r c u r i p h e n y l s u l f o n a t e were the o n l y ones significantly lowering this p r o p o r t i o n . These results indicate t h a t S H g r o u p s are p r o b a b l y involved in the n u c l e a r b i n d i n g o f the s t e r o i d - r e c e p t o r c o m p l e x a n d t h a t the affinity for the nucleus can be dissociated f r o m the l i g a n d b i n d i n g c a p a c i t y o f the receptor. Similar results were o b s e r v e d with estrone a n d estriol. (b). T h e dissociation between the t w o p h e n o m e n a was again o b s e r v e d when the effgct o f a - i o d o a c e t a m i d e o n the time course o f the b i n d i n g in the cytosol a n d in the nuclei was studied. A t 37 ° C (Fig. 10), in the a b s e n c e o f inhibitor, the c o n c e n t r a t i o n o f b o u n d steroid in the ¢ytosol r e m a i n e d

278

E. MILGROM et ai.

TABLE IV EFFECT OF VARIOUS INHIBITORS ON ESTROGEN DISTRIBUTION BETWEEN UTERINE CYTOSOL AND NUCLEI Estrogen

Inhibitor

Nuclear binding*

Estradiol (10 nM)

No ~,-Iodoacetamide (2 mM) ~-Iodoacetamide (10 mM) Iodoacetate (5 raM) Iodoacetate (30 mM) 5,5'-Dithiobis(2-nitrobenzoic acid) (3 raM) 5,5'-Dithiobis(2-nitrobenzoic acid) (10 mM) Dinitrophenol (5 raM) KCN 30 (raM) Oligomycin (0.14 mM) Ouabain (1 raM) p-Chloromercuriphenylsulfonate(0.5 raM)

84.7~0.4 27.5+5.4 19.5~4.6 67.7±2.0 29.9 ±4.1 78.1-4-2.0 70.0±2.5 79.1 +3.1 81.9-/-1.4 86.9=/=0.4 85.4-/-0.2 75.6 + 1.1

Estradiol (1 nM)

No p-Chloromercuriphenylsulfonate (0.5 raM) p-Chloromercuriphenylsulfonate (1.5 mM) p-Chloromercuriphenylsulfonate (3 raM) p-Chloromercuriphenylsulfonate (10 raM)

83.2-/-2.1 84.2±1.1 76.9±0.4 22.8 ± 1.8 21.9±0.2

Estrone (10 nM)

No --lodoacetamide (2 raM)

80.4 15.9

Estriol (10 nM)

No e-Iodoacetamid¢ (2 raM)

90.4 14.0

* Percent of total (cytosol+nuclear) binding ± S.E. (3 experiments).

,sore

A

,soooJ

I000~

5000

•

/ //"

,

/

'~,/

l

./"~"" ~01~l 5000

1

Fig. 10. Time course of the subcellular distribution of bound [3H]estradiol during organ incubation at 37 °C of rat uteri in the absence (A) or in the presence (B) of 2 nM ~-iodoacetamide. The data in this figure are taken from the same experiments as those reported in Fig. 5. The incubation has been preceded by a 5-min preincubation, a, nuclear binding; b, cytosol binding. practically u n c h a n g e d d u r i n g all the time o f i n c u b a t i o n whereas b o u n d steroid in the nuclear extract a p p e a r e d to rise progressively to values which were 5-8 times higher t h a n in the cytosol. I n the presence o f ~-iodoacetamide, the c o n c e n t r a t i o n o f the b o u n d steroid in the cytosol a n d in the nucleus was practically c o n s t a n t d u r i n g the i n c u b a t i o n ; however, at all times there was m o r e b o u n d steroid in the cytosol t h a n in the nucleus. A t 25 °C (Fig. 11), the nuclear transfer o f the s t e r o i d - r e c e p t o r complexes was smaller t h a n at 37 °C, b u t essentially the same features were observed.

E S T R O G E N E N T R Y INTO U T E R I N E CELLS dpm/hom 751~

279

dpm/hor. A

?SOC

B

SO0

Fig. 11. Time course of the subceUular distribution of bound [aH]estradiol during organ incubation at 25 °C of rat uteri in the absence (A) or in the presence (B) of 2 nM --iodoacetamide. The data in this figure are taken from the same experiments as those reported in Fig. 6. The incubation has been preceded by a 10-rain preincubation, a, nuclear binding; b, cytosol binding. These results suggest that the nuclear binding of the steroid-receptor complex is r i s i n g w i t h t h e t e m p e r a t u r e a n d is i n h i b i t e d b y ~ - i o d o a c e t a m i d e . Studies with an acellular reconstituted system (cytosol+ nuclear fraction). S i n c e u-iodoacetamide alters the transfer to the nucleus of steroid-receptor complexes two c o m p l e m e n t a r y q u e s t i o n s c a n b e r a i s e d . F i r s t , is t h e b l o c k i n g effect d e p e n d e n t o n t h e TABLE V E F F E C T OF ~t-IODOACETAMIDE ON N U C L E A R A'VI'ACHMENT OF ESTRADIOL-CYTOSOL RECEPTOR COMPLEXES IN A C E L L U L A R INCUBATIONS Expt 1.24 uteri were homogenized at 0 °C in 3.6 ml 0.01 M Tris-HCl, pH 7.4, 0.25 M sucrose buffer (Tris-sucrnse buffer). The supernatant of a 700 x 10 g . rain centrifugation at 0 °C, was further centrifuged at 105 000 X 60 g " rain and the cytosol was divided into two parts. One was incubated 60 min at 0 °C with 2 n M [aH]estradiol, the other was incubated with 2 n M [3H]estradiol and 10 mM - iodoacetamide. The pellet of the 700 × 10 g " rain centrifugation (nuclei) was washed twice with 8 ml of the Tris-sucrose buffer and suspended in 2 ml of this buffer. To half of the nuclear suspension was added 1 vol. of buffer; to the other half was added 1 vol. of buffer containing 20 mM --iodoacetamide (final concentration 10 mM). Both were incubated 30 rain at 25 °C. Then three control incubations and three incubations containing the inhibitor were performed for 60 min at 25 °C by mixing 0.5 ml of the cytosol preparation with 0.5 ml of the nuclear preparation. The incubates were cooled in ice for 5 min, centrifuged at 2 000x I0 g • rain. The nuclear pellet was washed twice with 3 ml of buffer and extracted 1 h at 0 °C with 1 ml Tris buffer containing 0.4 M KCI. The receptor-bound radioactivity was measured in the cytosol and in the nuclear extract. Expt 2 was identical to Expt 1 with the exception that the homogenization and incubation buffer did not contain sucrose. Data are expressed as dpm + S.E. (three incubation).

Inh~iwr

Expt 1 Intact nuclei Expt 2 Broken nuclei

Receptor-bound [aH]estradiol at the end of the incubation Cytosol

Nuclei

No --Iodoacetamide (10 m M )

41 090-/- 1410

10 4 6 0 + 140

52 180±520

4 180+80

No u-Iodoacetamide (10 raM)

42 765-t-500

10 440-4-620

48 085+880

3 650-/-220

E. MILGROM et al.

280

maintenance of the cells structure, or conversely, can it be observed also in acellular conditions? Secondly, is the transfer only dependent on the steroid-receptor complex concentration and the lack of transfer only due to the low level of complexes in the presence of ~-iodoacetamide (due in part to the inhibition of entry of the steroid into the cell)? The cytosol was prepared and first incubated with [3H]estradiol at 0 °C and then incubated with nuclei at 25 °C in the presence or absence of ~-iodoacetamide (Table V). In the absence of ~-iodoacetamide about 20 % of receptor-steroid complexes were transferred into the nuclei as already observed by others 13. ~-Iodoacetamide inhibited the nuclear binding of steroid-receptor complexes, without changing the estradiol binding capacity of the receptor as shown by the increased concentration TABLE VI SITE OF THE ACTION OF Qt-IODOACETAM1DE ON THE N U C L E A R ATTACHMENT OF THE ESTRADIOL CYTOSOL RECEPTOR COMPLEXES Expt 1.40 uteri were homogenized in 6 ml of Tris--sucrose buffer (see Table V). The supernatant of a 700 × 10 O • rain centrifugation was further centrifuged at 105 000 x 60 O • rain and the cytosol was incubated 1 h at 0 °C with 2 nM 13Hlestradiol. The pellet of the 700 × 10 0 • rain centrifugation (nuclei) was washed twice with 13 ml of Tris-sucrose buffer and suspended in 3.3 ml of this buffer. 1.5 ml of the suspension was diluted with 6 ml buffer; another 1.5 ml was diluted with 6 ml buffer containing ~-iodoacetamide (final concentration 10 raM). Both were incubated 1 h at 25 °C and cooled to 0 °C. They were then centrifuged at 2000 × 10 g . rain and the pellets were washed twice with 13 ml of Tris-sucrose buffer. The final pellet was suspended in 3 ml buffer. 0.5-ml aliquots of the nuclear suspension were incubated with 0.5-ml aliquots of the cytosol during 1 h at 25 °C. At the end of the incubation the final cytosol and the nuclear extract were prepared as described in Table II. Expt 2. 6. I ml cytosol was prepared from 40 uteri homogenized in 4.5 ml of Tris-sucrose buffer and centrifuged at 105 000 x 60 g • rain. It was incubated 1 h at 0 °C with 2 nM of [~H]estradiol. To 2.88 ml o f the incubated cytosoi was added 0.32 ml of Tris-sucrose buffer containing ~-iodoacetamide (final concentration 10 mM). The control aliquot was prepared with an identical mixture but contained no ~,-iodoacetamide. Both were incubated 45 rain at 25 °C, cooled 10 rain to 0 °C and dialysed 4 h at 0 °C against 2 1 of Tris-sucrose buffer containing 1 pM [3H]estradiol (this limited the dissociation o f [3H]estradiol-receptor complexes). It was then checked by A2,o ,=, radioactivity and volume measurements that both dialysates differed by less than 5 % for all these criteria. The nuclear pellet was freshly prepared from 40 other uteri homogenized in 6 ml Tris-sucrose buffer and centrifuged 10 rain at 700 × g. The pellet was washed twice by 13 ml of Tris-sucrose buffer and resuspended in 5.2 ml of the same buffer. 0.5-ml aliquots of nuclear suspension were incubated with 0.5-ml aliquots of ~,-iodoacetamide treated or control cytosol 45 rain at 25 °C. After 5 rain cooling at 0 °C the cytosol and nuclei were separated by a 10 rain centrifugation at 2000 × #. The nuclei were washed twice with 3 ml Tris-sucrose buffer and extracted I h at 0 ~C with 1 ml Tris buffer containing 0.4 M KCI. Receptor bound radioactivity was measured in cytosol and nuclear extracts. Data are expressed as d p m + S . E . (five incubations).

Inhibitor

Receptor-bound [3H]estradiol at the end o f the incubation Cytosol

Nuclei

Expt I Nuclei only exposed to inhibitor

No g-Iodoacetamide (I0 mM)

39 540-t-640 40 310 =[=1040

11 810-t-1050 ! ! 650 =i=220

Expt 2 Cytosol only exposed to inhibitor

No ,~-Iodoacetamide (10 raM)

54 380-t-230 58 3505= 530

6 850-k 150 3 180+160

ESTROGEN ENTRY INTO UTERINE CELLS

281

of bound hormone in the cytosol under the effect of ~-iodoacetamide and also by the results of the incubation of [3H]estradiol with the receptor in the presence or absence o f ~-iodoacetamide (Table VI). The inhibition did not depend on the maintenance of the nuclear structure since it was observed when the nuclei were prepared and incubated in isotonic sucrose (Table V, Exp 1) and also if the experiment was performed in hypotonic media where the nuclear membrane was disrupted as confirmed by phase microscopy (Table V, Expt 2). It is therefore probable that u-iodoacetamide inhibition is not related to a hypothetical transport of the steroid-receptor complex through the nuclear membrane. The inhibition by u-iodoacetamide of the nuclear binding could be due to the alkylation of either the nuclear "acceptor" or of the cytosol receptor. Therefore in subsequent experiments, only the nuclei or only the cytosol were previously exposed to the action of u-iodoacetamide. The inhibitor was then eliminated and the cytosol was incubated with the nuclei. In Table VI it can be observed that only the alkylation of the cytosol was effective, and not that of the nuclei. It is known that prolonged dialysis may reverse the effect of ~-iodoacetamide on some enzymes t4, but it is unlikely that the two washes of the nuclei completely suppressed the ~-iodoacetamide effect in this instance. Dialysis was used to remove the ~,-iodoacetamide from the cytosol since addition of cystein or ~-mercaptoethanol at high concentrations to the ~-iodoacetamidetreated receptor solution completely abolished its binding of estradiol. In identical conditions, ~-iodoacetamide itself and cystein or ]Lmercaptoethanol separately had no effect on the binding (Table VII). This suggests that the receptor may contain an TABLE VII EFFECT OF ct-IODOACETAMIDE AND ~-MERCAPTOETHANOL ON ESTROGEN BINDING TO RECEPTOR Cytosol (3 mg/ml protein concentration in 0.25 M Tris--sucrose buffer) was prepared from uteri of prepuberal rats. To aliquots of this cytosoi was added 0.1 of their volume of the same buffer containing either ~-iodoacetamide (final concentration in the incubate 10 mM) or/Lmercaptoethanol (final concentration 100 raM) or both ~,-iodoacetamide and/3-mercaptoethanol. A control without these compounds was also performed. Three incubations were done for 60 min at 25 °C followed by 60 rain at 0 °C with each of these solutions and 5 nM [3H]estradiol. At the end of this incubation receptorbound [3Hlestradiol was measured at 0 oC. Results are given as receptor-bound [3H]estradiol 5: S.E. (three determinations).

Receptor-bound [3]Hestradiol (pM) Control a-lodoacetamide (10 mM) ~-Mercaptoethanol (100 mM) a-Iodoacetamide (10 raM) +/~-mercaptoethanol (100 raM)

758 4-22 712-4-17 7794-17 242 4-41

S-S bridge(s) necessary for hormone binding and which is (are) destroyed by the action of a mixture of ~,-iodoacetamide and eystein or/~-mercaptoethanol; or alternatively, that some c o m p o u n d formed during the reaction of ~-iodoacetamide with cystein or ~-mercaptoethanol destroys the binding.

282

E. MILGROMet aL

DISCUSSION A method of studying the entry of steroids into cells must overcome the difficulty due to the adsorption of high amounts of these hydrophobic compounds outside the cells. It is then necessary to devise a technique measuring the amount of steroid which has actually entered the c_ s. Williams and Gorski t have used the binding to intracellular receptor proteins to resolve this problem. When estrogens are bound to their uterine receptor, their dissociation at low temperature is very slow and it is possible in this way to stop the reaction at the desired time by tooling the uteri and adding an excess of unlabeled steroid. Using this approach, we have systematically studied some of the features which allow to distinguish simple diffusion from proteinmediated processes in the entry of various compounds into cells. The entry of estrogens into rat uterus is sensitive to SH-blocking agents in experimental conditions when the estradiol binding ability of the receptor is less (or not at all in the case of dithiobisnitrobenzoic acid or p-chloromercurisulfonate at low concentrations) diminished. However estradiol binding also depends on the integrity of SH-groups but with less reactivity, thus allowing us to find experimentally a different inhibition for entry and binding, The present observations explain the reported contradictory results where estradiol binding by the solubilized receptor was unchanged by ~-iodoacetamidet 5, whereas it was decreased when intact uterine horns were incubated with estradiol t°. This discrepancy might be explained by the newly observed transport step. The entry process is also saturable and its estrogen specificity is different from that of the receptor, since diethylstilbestrol competes markedly less for the entry of radioactive estradiol than estradiol itself whereas both are bound to the same extent by the receptor. The entry of estradiol, estrone or estriol are all inhibited by SH-blocking reagents. However it cannot be said whether or not there is a unique transport system for these three estrogens. Conversely, the lack of competition by a 100-fold excess of non estrogenic steroids favors the concept of estrogen specificity. The most probable explanation of these experimental data is the existence of a protein-mediated process regulating the passage of estrogen through the uterine cell membrane. However for the present time our knowledge of the receptor itself and of the cellular biology of the uterus is very limited. Much of the evidence presented in this paper is based on the different characteristics of the entry process and of the solubilized receptor but it cannot be excluded that the receptor in situ has different properties from those of the solubilized form. The use of a tissue introduces also the problem of the penetration of the steroid through the intercellular space. Isolated uterine cells ~ could represent a better model for such studies although the use of proteolytic enzymes in their preparation may modify the properties of the cell membrane. Other limitations come from the method used to measure the entry of steroid and which is necessarily indirect, passing through an interaction with the receptor. For all these reasons the existence of a protein-mediated transport of estrogen through the uterine cell membranes is very likely but cannot be definitively proven by these experiments. Another protein-mediated transport of steroids has previously been described in fibroblasts t8'~9 but two major differences exist with the transport step described

ESTROGEN ENTRY INTO UTERINE CELLS

283

in this paper since it is a transport of the glucocorticoids out of the cells and since it is an energy dependent process. Alkylation of some structure in the uterine cytosol by 0c-iodoacetamide prevents the cytosol-~ nucleus translocation of estradiol-receptor complexes. It is probably the receptor itself which is alkylated either directly at the site which interacts with the nuclear acceptor or at a distance from this site but then stabilizing a conformation of the receptor which has a lower affinity for the acceptor. Another possibility which cannot be ruled out is that ~-iodoacetamide acts on a hypothetic enzyme necessary for the transformation of the cytosol receptor into a slightly modified molecule able to bind to nuclei tr. The study of this inhibition could perhaps lead to a better understanding of the conformational change which, after the binding of the estrogen, raises the affinity of the cytosolic receptor for the nuclear acceptor. Studies on the effects of various inhibitors on the nuclear binding of the receptor have been performed also by O'Malley, B. W. and Toft, D. O. (personal communication). Many questions are raised in relation to these experiments. Does the proteinmediated entry process exist for hormones other than estrogen; is it restricted only to hormone target organs, and in target organs does it exist only for the steroids which act on them? The answers to these questions would perhaps throw some light on a new point of control in steroid hormone action. ACKNOWLEDGMENTS We thank Professor A. Kepes for his interest. This work has been partially supported by the D616gation G6n6rale ~ la Recherche Scientifique et Technique, the Ford Foundation and Roussel-UCLAF. REFERENCES 1 Williams, D. and Gorski, J. (1971) Biochem. Biophys. Res. Commun. 45, 258-264 2 RaspS, G. (1970) Steroid Hormone Receptors, Advances in the Biosciences 7 Pergamon Press, Vieweg 3 Truong, H. and Baulieu, E.-E. (1971) Biochim. Blophys. Acta 237, 167-172 4 Milgrom, E. and Baulieu, E.-E. (1969) Biochim. Biophys. Acta 194, 602-605 5 Lineweaver, H. and Burk, D. (1934) J. Am. Chem. Soc. 56, 658-666 6 Eadie, G. S. (1942) J. Biol. Chem., 146, 85-93 7 Scatchard, G. (1949) Ann. N.Y. Acad. Sci. 51,600-672 8 Baulieu, E.-E. and Raynaud, J. P. (1970) Eur. J. Biochem. 13, 293-304 9 Bau|ieu, E.-E., Raynaud, L P. and Milgrom, E. (1970) Acta Endocrinol suppl. 147, 64, 104-121 10 Jensen, E. V., Hurst, D. J., DeSombre, E. R. and Jungblut, P. W. (1967) Science 158, 385-387 11 Truong, H. (1970) Thq~sede 3~:meCycle (Sciences), Paris 12 Geynet, C., Millet, C., Truong, H. and Baulieu, E.-E. (1972) Gynecol. Invest. 3, 2-29 13 Rochefort, H. and Baulieu, E.-E. (1972) Biochimie 54, 1303-1317 14 Webb, J. L. (1966) Enzyme and Metabolic Inhibttors, Vol. 2, pp. 635-653, Academic Press, New York and London 15 Puca, G. A. and Bresciani, F. (1970) in Research on Steroids (Finkelstein, M., Klopper, A., Conti, C. and Cassano, C. eds) Vol. 4, pp. 247-255 Pergamon Press, Oxford 16 Jensen, E. V.,~Suzuki, T., Kawashima, T., Stumpf, W. E., Jungblut, P. W. and DeSombre, E. R. (1968) Proc. Natl. Acad. Sct, U.S. 59, 632-638 17 Williams, D. and Gorski, L (1973) Biochemistry 12, 297-306 18 Gross, S. R., Arronow, L. and Pratt, W. B. (1970) J. CellBlol. 44, 103-114 19 Gross, S. R., Arronow, L. and Pratt, W. G. (1968) Btochem. Biophys. Res. Commun. 32, 66-72