In vitro binding of progesterone to receptors in the human endometrium and the myometrium

Biochimiea et Biophysica Acta, 317 (1973) 4o3-419 © Elsevier Scientific Publishing Comapny, Amsterdam - Printed in The Netherlands

BBA 36473 I N V I T R O BINDING OF P R O G E S T E R O N E TO RECEPTORS IN T H E

HUMAN ENDOMETRIUM AND T H E MYOMETRIUM

URMILA VERMA AND KESHO R. LAUMAS

Department of Reproductive Biology, All India Institute of Medical Sciences, New Delhi-i6, (India) (Received February 22nd, 1973)

SUMMARY

High affinity, low capacity progesterone binding receptors have been identified in the cytosol fractions of the human endometrium and the myometrium. The endometrial and the myometrial progesterone binding proteins had sedimentation coefficients of 4.5 S and 4.1 S, respectively. Analysis of the bound steroids revealed that, along with progesterone, small amounts of its metabolites (2oa-hydroxy-4-pregnene3-one, 5a-pregnane-3,2o-dione, 5a-pregnane-2oa-ol-3-one) were also bound to the receptor proteins. Among the steroids studied for ligand specificity, 5a-pregnane3,2o-dione showed the highest competition for progesterone binding sites. Progestational steroids, like chlormadinone acetate and norgestrel, did not compete for the progesterone receptors. The endometrial and the myometrial progesterone binding receptors were thermolabile and protein in nature. The molecular weight of the endometrial progestrone binding protein was about 60 000-67 ooo with a molecular (Stokes) radius of 33 A and the frictional ratio of 1.26. The myometrial progesterone binding protein had a molecular weight of 56 000-58 ooo with a molecular (Stokes) radius of 31 A and a frictional ratio of 1.23. The binding of corticosterone to the myometrial cytosol was only 2 2 - 3 4 % , whereas with progesterone it was 70-95 %. A study of the immunoabsorption of the plasma proteins from the endometrial and the myometrial cytosol suggested the presence of specific progesterone binding receptors in the cytosol that were different to plasma proteins. The association constant of progesterone for the endometrial progesterone receptor was 1. 9- lO9 M-1 and for the myometrial progesterone receptor it was 1.4. lO9 M-I, values that are higher than the association constant of progesterone for corticosteroid binding globulin, which is 7" lO8 M-1. The evidence suggested that the human endometrial and the myometrial progesterone binding proteins are different to the corticosteroid-binding globulin.

INTRODUCTION

Current research on the mechanism of action of steroid hormones has revealed

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u. VERMA, K. R. LAUMAS

that one of the primary steps in the hormone action is the binding of the steroid with the cytoplaslnic receptor proteins1, 2. It was demonstrated as early as 1962 that oestradiol is selectively taken up and retained in the uterus a, however, similar evidence for the retention of progesterone in the uterus was not forthcoming. It was shown for the first time by Laumas and Farooq< 5 that an injection of I1,2-aH2 progesterone to ovariectomized oestrogen-primed rats resulted in an initial rapid uptake of the steroid and of its metabolites, an initial rapid disappearance, fl}llowed by a slow disappearance, indicating some retention of the steroid. The levels of progesterone and/or its metabolites in the uterus were found to be higher than those in blood and skeletal muscle. Specific retention of progesterone in the rat uterus was also seen by constant infusion of 1,2-aH2]progesterone to rats 6. Later, similar observations on the selective uptake of progesterone in the uterus wer rep{)rted by us s and other workers 9 12. Preliminary evidence for the binding of progesterone to a supernatant fraction of the rat uterus was reportedL Various characteristics of the progesterone binding proteins in the cytosol fractions of rat, rabbit and guinea pig uterus have now been studied~°, la-21. Wiest and Rao 2~ and H a u k k a m a a el al. 22 have studied the binding of progesterone to the human endometrium. However, the specificity and the characteristics of the progesterone binding proteins in the human endometrium and the myometrium remain to be investigated. The present study is a detailed investigation of the progesterone binding proteins in the human endometrial and the myometrial cytosol fractions. The electrophoretic pattern of the binding proteins and the effect of hydrolytic enzymes on the binding of progesterone to the cytosol proteins and their other physical characteristics have been analysed. Steroid specificity of the binding proteins and competitive displacement of progesterone from the binding sites by natural and synthetic steroids have been studied. MATERIALS AND METHODS

Steroids [ ] i , 2 - a H ~ ] P r o g e s t e r o n e ( i . o Ci/6.o mg) and [i,2-aH2]corticosterone (i.o Ci/7.o rag) were obtained from New England Nuclear Corp., Boston, Mass., U.S.A. and purified by thin-layer chromatography. Steroids used were: progesterone, corticosterone, oestradiol, testosterone (Sigma Chemical Co.), norgestrel (Wyeth Labs), megestrol acetate (B.D.H.), chlormadinone acetate (E. Merck), ethinyl oestradiol-3-methyl ether (Searle and Co.), norethisterone acetate (Schering AG, Berlin), 5a-pregnane3,2o-dione (Ikapharm), 2oa-hydroxy-4-pregnene-3-one (Upjotm Co.) and 3fi-hydroxy5a-pregnane-2o-one (Sigma Chemical Co.).

U t e r i n e tissue

H u m a n uterine tissue was collected from the operating theatre, imnlediately after hysterectomy of the patients who were in the secretory phase of the menstrual cycle. The tissue was brought to the laboratory on ice, and the endometrium was scraped from the myometrium under ice-cold conditions. All experiments were carried out at 0-4 °C unless otherwise stated.

PROGESTERONE RECEPTORS

405

Preparation of uterine cytosol fraction for the binding study Sliced myometrial tissue (i.o g) was washed in cold o.oi M Tris-HC1 buffer (pH 7.4) (Buffer A). Slices were minced immediately and homogenized in a Polytron homogenizer with Buffer A containing 0.25 M sucrose. During homogenization, between every IO s of homogenization, the tissue was cooled for 2 min. Cytosol (Supernatant) was prepared by centrifuging the homogenate at lO5 ooo × g for I h. Endometrial and human skeletal muscle cytosols were prepared similarly.

Sephadex column chromatography The endometrial and the myometrial cytosols were incubated with I.O #ci (6 ng) [I,2-SH~lprogesterone for 30 min at o °C. The cytosol-progesterone complex was layered on the top of the Sephadex G-2oo (Pharmacia Uppsala, Sweden) column (2.5 cm × 5o cm) and eluted with Buffer A. Fractions of 4 ml were collected and protein was measured at 280 nm. Aliquots from the fractions were taken for radioactivity counting.

Sucrose density gradient A 4.8-ml linear 5-2o% sucrose gradient in Buffer A was prepared in cellulose nitrate tubes using a Beckman density gradient former and left undisturbed overnight at 4 °C. 0.2 ml of the cytosol incubated with 0.5 #Ci of EI,2-aH~]progesterone for 30 min at o °C was layered on the sucrose gradient and it was centrifuged at 48 ooo rpm for 15 h in a S.W.-5 o rotor in a Beckman Ultracentrifuge Model L. At the end of the centrifugation, the tubes were pierced at the bottom and fractions (30-35) were collected. Tube nmnber was normalized to 30. Radioactivity was determined in each fraction. Bovine serum albumin was run as a marker and the sedimentation coefficient s°20,w of the endometrial progesterone binding protein and the myometrial progesterone binding protein was calculated according to Martin and Ames 23.

Polyacrylamide gel electrophoresis Disc electrophoresis was carried out in a Shandon disc electrophoresis apparatus. 7.5% acrylamide was used with a running pH of 9.5. Both gels and buffers contained [SH~progesterone according to Milgrom and Baulieu la (0.05 /~Ci/gel and 0.5 #Ci/5oo ml buffer). A current of 1.25 mA/gel column was applied for I h to remove the ammonium persulphate ions in the gel. Cytosol (20o/~1) was incubated with 0.5 #Ci of [I,2-3H23progesterone and IO #1 was applied on the polymerised gel. The steroid protein complex was electrophoresed at 4 °C, first for 3 ° rain at 1.25 mA/gel, then 2.5 mA/gel was applied until the free bromophenol blue was 4-5 mm above the bottom of the gel. As a control, Ei,2-SHz]progesterone without cytosol was also electrophoresed simultaneously. After electrophoresis, the gel was removed, frozen at --20 °C for 2o h and then cut into 1.5-mm slices. The slices were dissolved in 30% H20~ at 8o °C for 18 h and counted by adding diotol scintillation fluid. Both experimental and control gels were treated similarly. From the experimental gels, the counts from control gels were subtracted to get the actual experimental counts.

Precipitation of the myometrial progesterone binding protein with (NH4)~SO 4 In order to differentiate uterine progesterone binding protein from that of corticosteroid binding globulin, progesterone binding protein in the myometrial

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cytosol was salted out at different concentrations of (NH4)2S Q according to Mctiuirc and Bariso 15. i ml of the (NH4)2SO 4 (IO 8o%) solution was added to tubes c(mtaining Ioo /A of myometrial cytosol, previously incubated with equal amounts of !:~H progesterone or [3Hlcorticosterone. The tubes were stirred for 15 rain at 4 C and the precipitate was spun at 12 ooo × g for 2o min. The supernatant was decanted and the radioactive steroid was determined in the precipitate and expressed as per cent bound steroid to receptor protein.

lmmunoabsorption of plasma proteins from the endometrial and the ,Lvometrial cylosol Rabbit anti-normal h u m a n serum was polymerized using gluteraldehyde "~. Plasma proteins, if any, in the cytosol were absorbed by mixing the cytosol with the polymerized anti-normal h u m a n serum. The binding capacity of the cytosol was then studied b y equilibrium dialysis.

Equilibrium dialysis Radioactive and non-labelled steroids used for the binding study were dried in a counting vial and redissolved in IO ml of Buffer A. I ml of the endoinetrial oi the myometrial cytosol in dialysis tubing (Union Carbide Corp.) was dialysed against the buffer at 4 °C with constant stirring for 4 ° 11. After 4o h of dialysis, tile radioactivity was analysed in duplicate aliquots from inside and outside the bag. The percent binding of progesterone to the protein was calculated as: Percent bound =

cpm inside dialysis sac -- cpm outside dialysis sac .
Competition of other steroids with progesterone binding to myometrial cytosol Competition of various progestational and non-progestational steroids with progesterone binding sites in io-fold diluted myometrial cytosol (1.4 mg protein/ml) was studied by equilibrium dialysis and the competition efficiency was measured aGas : Competition efficiency =

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Treatment of the cytosol with sulphydml group protecting agent To s t u d y the effect of 2-mercaptoethanol on the binding ot 1,2-~H2]progester one to the myometrial cytosol, the myometrial tissue was homogenized in Buffer A containing 0.25 M sucrose and 5 mM 2-mercaptoethanol. The homogenized tissue was fractionated and the cytosol tlms prepared (14 nag protein/ml) was incubated with [i,2-aH2]progesterone (2 ttCi). The cytosol was loaded on a Sephadex G-2oo column, previously equilibrated with Buffer A containing 5 mM 2-mercaptoethanol, and was eluted with the same buffer. The binding ability of the 2-mercaptoethanoltreated cytosol was compared with the control cytosol, prepared simultaneously from the same tissue without 2-mercaptoethanol. The control cytosol was loaded on a Sephadex G-200 column and eluted with Buffer A without 2-mercaptoethanol.

Analysis of the bound steroid To analyse the protein-bound steroid, endometrial and the myometrial cytosols

PROGESTERONE RECEPTORS

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were incubated at 37 °C for i h with i #Ci of [I,2-3H21progesterone. This cytosol was then chromatographed on Sephadex G-2oo column as described earlier. The proteinbound steroid, eluted through Sephadex G-2oo, was extracted with solvent diethyl ether, dried under N 2 and chromatographed on silica gel G plates using hexaneethyl acetate (5:2, v/v) solvent system. In this system, 2oa-hydroxy-4-pregnene-3one, and 5a-pregnane-3,2o-dione resolved well. 5a-Pregnane-2oa-ol-3-one moved along with progesterone. After the 2oa-hydroxy-4-pregnene-3-one and 5a-pregnane3,2o-dione regions were scraped out, the remaining silica gel of the lane was pooled and radioactivity was extracted with chloroform-methanol (I:I, v/v), dried and rechromatographed on silica gel G in chloroform: acetone (9 :i, v/v) solvent system. This system resolved pregnanediol, 5a-pregnane-2oa-ol-3-one and progesterone. Silica gel corresponding to the authentic standards was scraped off, radioactivity was extracted with chloroform-methanol (I :I, v/v), dried in vials and counted.

Determination of the molecular weight, molecular (Stokes) radius and frictional ratio (fifo) of the binding proteins Molecular weight of the binding proteins by gel filtration was calculated according to Andrews 25, using 2 mg of non-enzymic protein markers (Mann Res. Lab.). Dextran blue 2000 was used to measure the void volume of the column. Molecular weights of the binding proteins from the sucrose gradient sedimentation values were calculated in relation to bovine serum albumin according to Martin and Ames ~a. To calculate the molecular (Stokes) radii of the binding proteins, the distribution coefficients (Ko) of the standard proteins were plotted against their Stokes radii ~6-2s and the Stokes radii of the endometrial and the myometrial progesterone binding proteins were derived by interpolation of their respective Ko values. The frictional ratio f/fo of the binding proteins was calculated from the equation~, ~9 a H0

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where a is Stokes radius; ~, partial specific volume; M, molecular weight; N, Avogadro's number.

Determination of the association and the dissociation constants of the binding proteins From the equilibrium dialysis experiments of the endometrial and the myometrial cytosol in the presence of increasing concentrations of unlabelled progesterone, a Scatchard plot of bound vs unbound (B/U) progesterone was plotted on the y axis against the concentrations of bound progesterone (B) on the x axis 8°. An intrinsic association constant (Kass), dissociation constant (Kdiss) and the number of binding sites (n) were calculated.

Radioactivity counting procedure Radioactivity was measured in a liquid scintillation spectrometer (Packard Tricarb Model 3314). The dry samples were counted using IO ml simple scintillation fluid (4 g of 2,5-diphenyloxazole and o.I g of 1, 4 bis-2-(4-methyl-5-phenyloxazolyl)benzene dissolved in I 1 toluene). The aqueous samples were counted using Diotol scintillation fluid (250 ml Dioxan, 15o ml methanol, 250 ml toluene, 52 g naphthalene, 3.25 g 2,5-diphenyloxazole, and 65 mg 1,4-bis-2-(4-methyl-5-phenyloxazolyl)benzene.

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et al. am, u s i n g b o v i n e s e r u m a l b u n f i n as a s t a n d a r d p r o t e i n . RESULTS

Analysis of the endometrial and the myometrial progesterone binding protein by Sephadex G-200 column chromatography T h e e l u t i o n profile o f t h e e n d o m e t r i a l c y t o s o l p r o t e i n - s t e r o i d c o m p l e x , e l u t e d t h r o u g h S e p h a d e x G-200 c o l u m n is s h o w n in Fig. I A . T h e c y t o s o l p r o t e i n r e s o l v e d

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Fig. I. Sephadex G-2oo column chromatographic pattern of the endometrial and the myometrial cytosol [i,2-3H2]progesterone binding protein. A. Endometrial cytosol (2o mg protein) was incubated with I/*Ci of [I,2-3H~]progesterone at o °C. The cytosol-steroid complex was layered on the Sephadex G-2oo column (2.5 cm × 5° cm) and eluted with Tris-HC1 buffer (pH 7-4). B. Sephadex G-2oo column chromatographic pattern of the myometrial cytosol (i8 mg protein) incubated with I/~Ci of [I,2-3H~]progesterone at o °C.

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into three major 28o-nm-absorbing peaks. The A2s0 nm/A2s0 nm ratio showed the presence of proteins in the first two peaks. The A280n m / A 2 e 0 n m absorption ratio of the third peak did not indicate the presence of proteins. A small amount of the radioactivity due to progesterone and/or its metabolites was associated with the first protein peak. Much of the radioactive progesterone and/or its metabolites were eluted with the second protein peak. Control experiments showed that free radioactive progesterone was eluted within the elution volume of the third peak. The Sephadex gel chromatographic pattern of the myometrial cytosol proteinsteroid complex is shown in Fig. IB. Like the endometrial cytosol, the myometrial cytosol also resolved into three 28o-nm-absorbing peaks, much of the radioactivity being associated with the second protein peak. In general, both endometrial and myometrial cytosols gave comparable elution profiles and showed progesterone binding to the uterine proteins. Gel filtration of the muscle cytosol through Sephadex G-2oo did not show any binding of [3Hlprogesterone to the muscle proteins. 4.5 S

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Sucrose gradient sedimentation pattern of progesterone receptors i~l tke e'ndometrial and the myometrial cytosol The sedimentation profile of the endometrial and the mvometrial progesterone receptors is shown in Fig. 2. The calculation of the sedimentation coefficient and the statistical analysis of the values showed that the endometrial progesterone binding protein sedimented slightly earlier than the myometrial progesterone binding protein. The sedimentation coefficient for the endometiial progesterone binding protein was found to be 4.5 ± o.o9 S and that of myometrial progesterone binding protein 4. I }: o.094 S.

Electrophoretie mobility of the endometrial and the myometrial progesterone binding protein On polyacrylamide gel electrophoresis, the endometrial cytosol resolved into a number of proteins, however, the radioactive progesterone was associated with only one protein, which had a relative mobility (RE, relative to the mobility of bromophenol blue) of 0.75 (Fig. 3A). No radioactivity was detected in the albumin region. Gel electrophoresis of the myometrial cytosol (Fig. 3B) showed ttmt the myometrial progesterone binding protein had a mobility different than that of endometrial progesterone binding protein. It had a relative mobility (RE) of o.89. There was another small radioactivity peak having relative mobility of 0.88, similar to that of albumin.

Binding of progesterone to the endometrial and the myometrial cytosol after immunoabsorption of the plasma proteins from the cytosol To remove plasma proteins, if any, from the cytosol, endometrial and the myometrial cytosols were absorbed with polymerized rabbit anti-normal human serum. The binding of progesterone to this cytosol was not abolished but was decreased from 80 to 56.9% in the endometrium and in the myometrium it was decreased from 7 ° to 51.6%. The inability of the absorption of the binding protein

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Fig. 3. Polyacrylamide gel electrophoretic pattern of the cytosol progesterone binding proteins, A. Endometrial cytosol. The bromophenol blue moved in Fraction 37 and the endometrial binding protein in Fraction 28. (RF 0.75). B. Myometrial cytosol. The bromophenol blue moved in Fraction 36 and the myometrial binding protein in Fraction 25 (RF 0.69) and another radioactivity peak was found in Fraction 32 (RF 0.88).

from the endometrial and the myometrial cytosol confirmed that the endometrial and the myometrial progesterone binding proteins are not of plasma origin and are different to corticosteroid binding globulin.

Comparison of the binding of [I,2-aH~Jprogesterone and [I,2-3HzTcorticosterone to the myometrial progesterone binding protein Binding of ~3H~progesterone and [aHlcorticosterone to the myometrial progesterone binding protein was studied by equilibrium dialysis. The results showed that the binding of [I,2-3H21corticosterone to the myometrial cytosol was only 22 % (Table I), whereas 70% of progesterone was bound under identical conditions. In the presence of increasing concentrations of unlabelled inert corticosterone, a small decrease in the binding appeared, The binding of [~Hlprogesterone to the myometrial cytosol protein was about 3 times more than that of the binding of [3Hlcorticosterone using equilibrium dialysis. This suggested that the protein may have more affinity TABLE I BINDING OF CORTICOSTERONE AND PROGESTERONE TO THE HUMAN MYOMETRIAL CYTOSOL Binding of (a) [I,2-"H2]progesterone to the myometrial cystosol and (b) the binding of [I,2-"Hz]corticosterone to the myometrial cytosol in the presence of lO-4OO ng of unlabelled corticosterone were studied. Steroids

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for progesterone than corticosterone. To further confirm the above results, progesterone binding and corticosteroid binding proteins were fractionated with different concentrations of (NH4)2SO 4. It could be seen from Fig. 4 that the maxinmm binding of [aHicorticosterone to the myometrial cytosol was only 34°o, precipitable with 60% of (NH4)2SO 4 solution, whereas the maximum binding of laHjprogesterone t(~ the myometrial cytosol was 9o--95°i~, precipitable with 5o°/i, (NH4)._,SOa solution. lOO z om F--

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Influence of unlabelled progesterone on the binding of myometrial progesterone binding protein The addition of 2 /~g of unlabelled progesterone to the myometrial cytosol incubated with [aHlprogesterone inhibited the radioactive progesterone binding to the 4.I-S protein (Fig. 5). This indicated that the binding protein in the 4.I-S region 1,500

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has a limited binding capacity, with the increase of progesterone concentration to 5/~g, there was no binding of [SHlprogesterone to 4.I-S protein. However, its binding to a uterine macromolecule which sedimented at the bottom of the gradient was observed.

Competition of various steroids to the progesterone binding sites of the myometriat progesterone binding protein Competition of various progestational and non-progestational steioids to the progesterone binding sites in the myometrial progesterone binding protein was studied by equilibrium dialysis. Competition efficiency was measured as described under Materials and Methods. The compounds tested compete in the following order of decreasing efficiency (Table II) : progesterone > 5a-pregnane-3,2o-dione > corticosterone > 3fl-hydroxy-5a-pregnane-2o-one > 2oa-hydroxy-4-pregnene-3-one > I7fl-oestradiol and testosterone. Contraceptive steroids, like chlormadinone acetate, having progestational activity showed very little competition for the progesterone binding sites. TABLE II COMPETITION OF PROGESTATIONAL AND NON-PROGESTATIONALSTEROIDS TO MYOMETRIAL CYTOSOL PROGESTERONE BINDING PROTEIN Io-fold diluted m y o m e t r i a l cytosol (1. 5 m g protein/ml) was dialysed against Buffer A containing 12 ooo c p m of EI,2-SH,]progesterone and 1.6 . io -~ M of unlabelled steroids. Competition efficiency was m e a s u r e d as described u n d e r Materials and Methods.

Steroid

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Effect of sulphydryl group protecting agent To study the effect of 2-mercaptoethanol, human myometrial tissue was homogenised witti 2-mercaptoethanol, fractionated and the cytosol was chromatographed through a Sephadex G-2oo column with Buffer A containing 5 mM 2-mercaptoethanol. The elution profile of this cytosol was compared with the cytosol prepared from the uterine tissue without 2-mercaptoethanol, i.e. the control cytosol. The elution profiles (Fig. 6) showed that the ratio of the bound radioactivity of the cytosol prepared and chromatographed in the presence of 2-mercaptoethanol to the control cytosol prepared and processed in the absence of 2-mercaptoethanol was i :0.48. Such an effect was also observed by equilibrium dialysis, where the cytosol prepared in the presence

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Fig. 6. M y o m e t r i a l lO 5 ooo x g c y t o s o l was p r e p a r e d from t h e m y o m e t r i u m , h o m o g e n i z e d in Buffer A c o n t a i n i n g 0.25 M sucrose a n d 5 mM 2 - m e r c a p t o e t h a n o l . The e yt os ol (:~4 m g p r o t e i n / m l ) was l o a d e d on a S e p h a d e x G-2oo c o l u m n a n d cytosol--steroid m i x t u r e e l u t e d w i t h Buffer A cont a i n i n g 5 mM 2 - m e r c a p t o e t h a n o l . 0 - - 0 , c p m in 2 - m e r c a p t o e t h a n o l t r e a t e d ; ( (), a b s o r b a n c e a t 280 n m in 2 - m e r c a p t o e t h a n o l - t r e a t e d cytosol. I n a control, m y o m e t r i a l c yt os ol w a s p r e p a r e d in Buffer A w i t h o u t 2 - m e r c a p t o e t h a n o l from t h e m y o m e t r i u m of t h e sanIe p a t i e n t . I - - I , c p m in contro l cytosol.

TABLE I I[ ACTION OF ENZ3"MES ON THE PROGESTERONE BINDING COMPONENT OF ENDOMETRIIIM AND MYOMETRIUM IO 5 OOO X g SUPERNATANT Ef f ects of t h e e n z y m e s were s t u d i e d b y e q u i l i b r i u m d i a l y s i s of t h e e n d o m e t r i a l anti tile m y o m e t r i a / c y t o s o l in t h e p r e s e n c e of i o o p g of e n z y m e s / m l of cytosol.

Enzymes

Myometrium (percent bound)

Endometrium (percent bound)

Control Ribonuclease Trypsin

70.4 70.4 27.1

So.0 78.8 20.6

of 2-mercaptoethanol was dialysed in Buffer A containing 5 mM 2-mercaptoethanol, and the cytosol prepared in the absence of 2-mercaptoethanol dialysed in Buffer A only. The ratio of the former to the latter was 1:0.63.

Effect of hydrolytic enzymes Binding of progesterone to the endometrial and the myometrial progesterone binding protein was markedly reduced (Table IIl) on dialysis in the presence of trypsin (Sigma Chemical Co.) only. Ribonuclease (Calbiochem) did not affect the binding of progesterone to the endometrial or the myometriat progesterone binding protein.

Effect of heat treatment Sucrose gradient sedimentation analysis of the endometrial and the myometrial cytosol heated at 60 °C for 20 rain showed (Figs 2A and 2B) that the binding peak was completely abolished in the myometrial cytosol. The binding protein of the endo-

PROGESTERONE RECEPTORS

415

T A B L E IV PROGESTERONE

AND ITS METABOLITES

B O U N D TO T H E E N D O M E T R I A L

AND THE MYOMETRIAL CYTO-

PLASMIC FRACTIONS

Radioactivity in steroid-protein complex was analyzed by silica gel G thin-layer chromatography.

Steroid

Endometrium (%)

Myometrium (%)

Progesterone 2oa-Hydroxy-4-pregnene-3-one 5a-Pregnane-2oa-ol-3-one 5a-Pregnane-3,2o-dione

89.8 7.2 1.5 1.2

72 . 2 4.o lO.5 9.7

metrial cytosol was also denatured by heating; however, a small radioactivity peak was observed in the 4.5-S region of the gradient.

Analysis of the bound steroid The thin-layer chromatography of the steroids bound to the endometrial cytosol showed (Table IV) that 89.3% was present as progesterone, about 7% was present as 2oa-hydroxy-4-pregnene-3-one, 1 . 5 % as 5a-pregnane-2oa-ol-3-one and 1.2% as 5a-pregnane-3,2o-dione. The bound steroids in the myometrial cytosol contained 72.2% progesterone, 4% 2oa-hydroxy-4-pregnene-3-one, lO.5% 5a-pregnane-2oa-ol3-one and 9.7% 5a-pregnane-3,2o-dione.

Molecular weights, molecular (Stokes) radii and frictional ratios of the endometrial and the myometrial progesterone binding proteins The molecular weights, calculated from Sephadex G-2oo column chromatography and sucrose gradient sedimentation analysis, were found to be 60 ooo and 67 ooo, respectively for the endometrial; 56 ooo and 58 ooo, respectively for the

2O hl LI3

rr LI_ a z z~ 0

\ I0 ",,~ 0 I

I

l

~,

~J

i

i

J

,

i

,

I

2 0 nM

I0 ['BOUND]

Fig. 7. Scatchard plot of the endometrial and the myometrial cytosol progesterone binding proteins. Ei,2-SH~Progesterone was dialysed with endometrial or the myometrial cytosol with increasing concentration of progesterone (3-3 onM). O - - O , endometrial cytosol (1.68 mg protein] ml) ; 0 " - - 0 , myometrial cytosol (1.56 mg protein]ml.).

416

U. VERMA, K. R. I.AUMAS

myometrial progesterone binding proteins. On the basis of the elution w,lumes, tb.c distribution coefficient (Ka) of the standard proteins, as well as that of end~metria/ and myometrial progesterone receptors, was calculated. The K~I~/a for endometnal progesterone binding protein was o.8ol and that for myometrial progesterone binding protein was o.813. The molecular radius of the endometrial progesterone receptor was 33 ~l and that of the myometrial progesterone receptor was 31 i~. Using the values of molecular weights and the molecular radii, the frictional ratios of the endometrial and the myometrial progesterone binding proteins were calculated to be 1.20 and 1.23, respectively.

Association and dissociation constants of the progesterone bindi~¢g proteins Association constants of the endometrial and the myometrial progesterone receptors were calculated after equilibrium dialysis with 3-30 nM of progesterone. The Scatchard plot (Fig. 7) of the data (drawn from the mean of 3 5 observations) showed two types of binding systems. A specific (high affinity) binding with the association constant of 1.9. lO9 M-1 for the endometrial cytosol and 1.4. lO9 M ~ for the myometrial cytosol was obtained. A non-specific binding with low association constant was also observed in this system. The number of binding sites in tile specific receptor, as calculated from the Scatchard plot, was 6. lO -12 moles/mg of myometrial cytosol protein. The dissociation constant for the endometrial progesterone receptor was found to be o.53" lO -9 M and for the myometrial progesterone receptor it was o.71" lO -9 M. DISCUSSION

The results reported here give evidence for the presence of a 4.5-S progesterone binding protein in the human endometrial cytosol and a 4.I-S binding protein in the myometrial cytosol. On Sephadex G-2oo column chromatography, a major part of progesterone both in the endometrium and the myometrium was bound to the lighter macromolecules, the molecular weight being 6o ooo-67 ooo for endometrial and 56 ooo-58 ooo for myometrial progesterone binding proteins. Some binding was also observed to the heavier macromolecules in the endometrium as well as in the myometrium. Characterization of the endometrial and the myometrial progesterone binding proteins showed that the binding macromolecule(s) are of a protein nature. An increase in the binding of progesterone to the uterine protein(s) as an effect of mercaptoethanol treatment suggested the involvement of sulphydryl groups in maintaining the integrity of the receptor molecules. The stabilization of the steroid binding proteins by mercaptoethanol has earlier been reported for oestradio132 and norethynodreP ~. Since the binding of progesterone to the uterine cytosol proteins was observed at o °C (sucrose gradient sedimentation analysis), 4 °C (equilibrium dialysis), as well as at 37 °C (studied for the analysis of the bound steroids), indicated that the formation of the steroid-protein complex m a y not be an activation energy-dependent process 1. The binding of progesterone to the endometrial and the myometrial particulate free cytosol fraction in vitro suggested that the binding process did not depend upon cellular organisation. Binding of progesterone to the uterine proteins

PROGESTERONE

RECEPTORS

417

appeared to be of non-covalent type, since the steroids were completely extractable with organic solvents. Steroids bound to the endometrial progesterone binding protein and the myometrial progesterone binding protein on analysis revealed that the major (70-90%) binding steroid was progesterone with small amounts of 2oa-hydroxy-4-pregnene3-one, 5a-pregnane-2oa-ol-3-one and 5a-pregnane-3,2o-dione (allopregnanedione). Reel et al. 34 have reported that after direct incubation of the rat uterine cytosol, no significant metabolite was found and the maior steroid was progesterone, whereas after whole tissue incubation and in in vivo experiments, the bound steroid is reported to be in the form of progesterone and its metabolite, 3a-hydroxy-5a-pregnane,2o-one. The 5a-reduced metabolites of progesterone have been suggested to play a role in the physiological action of progesterone35. The endometrial and the myometrial progesterone binding proteins had limited binding capacity, the binding was decreased with increasing concentrations of progesterone. In the presence of high concentrations of progesterone, the binding of I3Hlprogesterone to a heavy uterine macromolecule was observed. The formation of a heavy uterine macromolecule in the presence of high concentrations of the steroid suggested a control of conformational alterations of the protein by the steroid with which it folms a specific complex of high affinity3e. Of the steroids tested for the ligand specificity, allopregnanedione was able to compete maximally with the progesterone binding sites in the myometrial progesterone binding protein. Furthermore, along with progesterone, allopregnanedione was bound, to the extent of about 9.7%, to the myometrial progesterone binding protein (Table IV) ; this suggested that allopregnanedione may be playing some role in the mechanism of action of progesterone. Similar competition of allopregnanedione with the progesterone binding sites in the human endometrium and rabbit uterine cytosol has been reported *1. Corticosterone, 3fl-hydroxy-5a-pregnane-2o-one and 2oahydroxy-4-pregnene-3-one also compete to some extent for the progesterone binding sites in the myometrial progesterone binding protein. It was interesting to observe that progesterone, but not progestational contraceptive steroids (viz. norgestrel, megestrol acetate, chlormadinone acetate), competes appreciably for the progesterone binding sites. It thus appears that orally active progestational steroids with high progestational activity may act at the uterine level without interaction with progesterone binding receptors. Chlormadinone acetate, on the other hand, was reported to compete with oestradiol binding sites in the human endometrial and the myometrial estradiol binding proteins (Krishnan, A. R., Hingorani, V and Laumas, K. R., unpublished). The binding of progesterone to the endometrial and the myometrial receptors in the immunoabsorbed cytosol showed that the binding protein was of tissue origin and different to plasma corticosteroid binding globulin. The 20-25 % decrease in the percent binding of progesterone in the anti-normal human serum-absorbed cytosol might be due to the absorption of non-specific plasma progesterone binding proteins in the cytosol. Another possibility of the decrease in binding may be the partial physical denaturation of the receptor protein in the immunoabsorption process. The absence of progesterone binding in the human muscle cytosol also suggested that the uterine binding of progesterone is not due to blood contamination and it may be uterine tissue specific. The 3-fold greater binding affinity of the myometrial pro-

418

u. VENMA, Isi. R. I.A{'MAS

gesterone receptor for progesterone t h a n corticosterone, as seen by equilibrium dialysis a n d (NH4)2SO a fractionation, suggested t h a t it was different t(> c~)rticosteroid b i n d i n g globulin. F u r t h e r m o r e , the association c o n s t a n t s of the uterine protein~ (1. 9. IO° M -1 a n d 1.4" 1o 9 M-: for the endometrial a n d the m y o m e t r i a l t>rogester(me b i n d i n g proteins, respectively) were higher t h a n t h a t of plaslna corticosteroid binding globulin (7.O.lO ~ M-l) 37, i n d i c a t i n g t h a t the endometrial a n d the m y o m e t r i a l progesterone b i n d i n g proteins were different to the corticosteroid b i n d i n g globulin. The present s t u d y on the b i n d i n g of progesterone with the receptor proteins showed the specificity of the i n t e r a c t i o n of the steroid with the receptor proteins in the e n d o m e t r i u m a n d the m y o m e t r i u m . It was clearly d e m o n s t r a t e d that the b i n d i n g protein is organ specific, is not due to blood proteins a n d is different to corticosteroid b i n d i n g globulin. The specific interaction of progesterone with the endometrial and the m y o m e t r i a l cytosol proteins m a y c o n t r i b u t e to the lnechanism ()f action ()t progesterone in the h u m a n uterus. ACKNOWLEDGEMENT This work was supported b y the g r a n t s from the Ford F o u n d a t i o n , New York; World Health Organization, Geneva. One of us (U.V.) acknowledges the fellowship support received from the I n d i a n Council of Medical Research, New Delhi; the Council of Scientific a n d I n d u s t r i a l Research, New Delhi and the Director-General of Health Services, New Delhi.

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