Chick oviduct progesterone receptor: structure, immunology, function

Molecular and Cellular Endocrinology, 37 (1984) Elsevier Scientific Publishers Ireland. Ltd.

1- 13

MCE 01188

Review

Chick oviduct progesterone

receptor:

Jack-Michel INSERM

activation;

antibodies;

oligomeric

immunology,

function

Renoir and Jan Mester

U 33, Laboratoire des Hormones, 94270 Bictitre (France) (Received

Keywork

structure,

structure;

3 May 1984)

subcellular

localization.

Summary

Analysis of the purified chick oviduct progesterone receptor using biochemical and immunological approaches indicates that while the ‘activated’ receptor (‘4s’) is a mixture of two progestin-binding polypeptides, ‘A’ (M, - 79 kDa) and ‘B’ (M, - 110 kDa), the non-activated receptor (‘8s’) is a population of complexes containing a hormone-binding polypeptide (A or B, but probably not both) bound to a non-hormone-binding protein (M, - 90 kDa). Two molecules of the 90 kDa protein appear to be present in each ‘8s’ receptor molecule. The 90 kDa protein is also associated with the non-activated forms of receptors of other steroid hormones in the chick. Molybdate stabilizes the non-activated receptors, probably by forming weak coordination bonds with radicals provided by the subunits of the ‘8s’ structure. Activation implies separation of the subunits, without a change in their primary structure, and does not require intervention of any protein other than those present in the ‘8s receptor form. The presence of ligand at the binding site accelerates the activation process but, in vitro, is not necessary for it to occur. Unlike the non-activated form, activated receptors bind to theecell nuclei. However, histological studies with anti-progesterone receptor antibodies indicate that in the non-hormone-exposed tissue the (nonactivated) receptors could be localized in the nuclei.

It is generally believed that biological actions of steroid hormones in target tissues are mediated by specific intracellular binding proteins (receptors) (reviews in Jensen and Jacobson, 1962; Baulieu et al., 1975). In the absence of exposure to the respective hormones, the receptors are found predominantly in the soluble fraction of tissue homogenates (‘cytosol’) and display properties of what has been termed the ‘native’ form. Irrespective of hormone, animal or tissue, the ‘native’ receptors are characterized by their large size (7-10s in density gradients) and low affinity of binding to isolated nuclei and to certain polyanions (review in Milgrom, 1981). Hormone treatment leads to a change in the apparent subcellular distribution of 0303-7207/84/$03.00

Q 1984 Elsevier Scientific

Publishers

Ireland,

the receptor so that a considerable portion of its total cellular content is recovered in the nuclear fraction of tissue homogenate from which it can be extracted by high salt buffers. Changes in physico-chemical properties (‘activation’) of the receptor protein appear to precede this apparent ‘translocation’. It is not certain that a real shift of receptor molecules from the cytoplasm to the nucleus occurs in the intact target cell. For a more detailed analysis our discussion will focus on the chick oviduct progesterone receptor (PR). Chick oviduct has been studied extensively as a target tissue for steroid hormones because of their large-scale specific effects in this tissue, permitting investigation of the mechanism of Ltd.

2

hormonal regulation at a molecular level. Upon estradiol stimulation, the chick oviduct increases in size from 20 mg (in immature birds) to several grams, and in the laying hen, as a result of the interplay of estradiol, progesterone and androgen, it reaches a weight of 20-40 g. In addition, estradiol (E) and progesterone (P) induce synthesis of the major proteins of the egg white (ovalbumin, conalbumin, ovomucoid lysozyme) in a manner suggesting independent control of gene expression by the two hormones (Palmiter, 1972). Study of the structure and function of the progesterone receptor (PR) from oviduct tissue is therefore important for understanding how it can mediate the observed effects of progesterone.

iI.0

Pa

1. ‘Native’ progesterone receptor and ‘activation’ In the oviduct homogenate of immature chicks not-treated with progesterone, close to 100% of the PR is found in the high-speed supernatant (cytosol) fraction. The cellular content of PR is under estradiol control; its level increases from about 3 X lo3 molecules/celI without estradiol treatment to 5-7 x lo4 molecules/cell after 2 weeks of estrogen priming. The PR level falls after 4 weeks of withdrawal of E to - 1.5 X lo4 molecules/cell (Mester and Baulieu, 1977) (assuming one binding site per molecule of PR; see further). Under these conditions, the PR behaves as a ‘native’ species; it sediments at 7.9s in sucrose gradients, its Stokes radius (R’,) is - 7.7 nm and it does not bind to phosphocellulose (Wolfson et al., 1980; Yang et al., 1982a; Renoir et al., 1982a). These characteristics have been obtained when studying the PR in the presence of molybdate, which stabilizes the PR and receptors for other steroid hormones in their non-activated state (Nielsen et al., 1977; Toft and Nishigori, 1979; Wolfson et al., 1980, 1981). In the absence of molybdate the [3H]P-PR complexes are transformed to an ‘activated’ state, in a process that occurs slowly at 0’ C but is quite rapid at 25 “C (1 h) (Fig. 1 (a)). The activated PR can also be obtained by incubation of the cytosolic receptor-hormone complexes at high ionic strength (> 1 h in 0.3 M KC1 at O°C; Buller et al., 1975). Yet other ways to ‘activate’ the chick oviduct PR are incubation with heparin (Yang et al., 1982a) or with ATP (Moudgil and Toft, 1977) at 0°C and in

10 fractions

20

Fig. l(a). Sucrose gradient ultra~ntrifugation of i3H]P-PR. 0, molybdate-stabilized complexes; 0, complexes treated with 0.3 M KC1 for 1 h at 0 o C. Internal markers, glucose oxidase (g.0. 7.9s) and peroxidase (p.o. 3.6s). are shown.

low ionic strength medium. The mechanism by which these two compounds acts on the PR molecules is not known. The activated PR is smaller (- 4s in density gradients) and binds well to nuclei, to DNA and to phosphocellulose (Grody et al., 1982). The additional 4S -+ 5s transformation which has been observed with estrogen receptor (Yamamoto and Alberts, 1972; Notides and Nielsen, 1974) apparently does not occur with the PR. Physico-chemical properties of the ‘activated’ receptors have been shown to be similar or identical to those of nuclear receptors (Mester et al., 1981). An additional parameter which characterizes ‘activation’ is the increased stability of the [3H]P-PR complexes as seen by studying the dissociation kinetics (Fig. l(b)). Both ‘native’ and ‘activated’ complexes dissociate according to firstorder kinetics, but the activated receptor has a half-life about 3 times greater than the nonactivated form. Thermodynamically, this increased stability results from greater enthalpy change upon

i

,

i

23

48

72

Ttme

(h)

Fig. l(b). Dissociation kinetics of activated, non-activated and molybdate-stabilized receptor-hormone complexes. The [ 3HIP-PR complexes in chick oviduct cytosol in buffer without molybdate were formed by incubation at 25 ‘C for 1 h (0); an ahquot prepared in the same way was supplemented with molybdate (50 mM) after the 25 o C incubation (0). Another aliquot of cytosoi was labelled by incubation at O°C for 3 h (a); to a portion of the incubate, molybdate (50 mM) was added (A). The last aliquot of the cytosol was supplemented with molybdate (50 mM) prior to incubation with [3H]P (1 h, 25 OC) (0). Dissociation kinetics was studied after adding lOOO-fold excess of unlabelled progesterone; at different times of incubation at 0 o C, portions of the solutions were treated with charcoal-dextran suspension to remove the unbound hormone (for details see Wolfson et al., 1980).

dissociation of the ‘activated’ compared to the ‘native’ receptor-ligand complex (Yang et al., 1983). Slower dissociation of the activated complexes therefore suggests a conformational change leading to a better fit between the binding site and the ligand. The possible physiolo~c~ significance of such change is also suggested by the observation that in the estrogen receptor system a similar increase in the stability of the receptor-ligand complex occurs upon the 4s + 5s transformation (Notides and Nielsen, 1974). Surprisingly, PR activation does not require the presence of the ligand at the binding site (Yang et al., 1982a), although it does proceed faster with PR-P complex than with unliganded PR (Fig. l(c)). This observation does not follow the prevailing view that unoccupied receptors fail to undergo activation (Milgrom, 1981). Within the cell, however, the presence of the ligand appears to be a prerequisite to ‘receptor translocation’ to the nucleus; incubation in vitro at 37OC of tissue explants in the absence of P leads only to a rapid loss of PR binding in the cytosol without the appearance of P-binding sites in the nuclear fraction (Wolfson et al., 1981).

time’(hrs

)

Fig. l(c). Effects of hgand on the 85 -+ 45 transformation by KCI. Chick oviduct cytosol was incubated at 0 o C in 0.3 M KC1 for periods of time as shown, either after labelling with [‘HIP (A) or prior to labelling (A). Molybdate was added in this case before [ 3H]P, in order to block any further transformation. The 8s and 4S complexes were resolved by density gradient ultracentrifugation; the percentage of total PR in the 4S form is shown.

Although the mechanism of activation is not completely clear, it seems reasonable to propose that it consists of separation of PR subunits forming the ‘native’ 8s molecule. The separation of subunits may be facilitated by a conformational change that occurs upon binding of hormone or ATP (Moudgil and Toft, 1977). It can also be triggered either by interruption of ionic bonds involved in subunit interaction (high salt, heparin), or by an increase in temperature. No enzyme activity seems to be involved as activation can be obtained with purified PR (Yang et al., 1982b; Put-i et al., 1983). Moreover, the subunits of the ‘native’ PR are unchanged by ‘activation’, as judged by their electrophoretic and immunological characteristics (see further). In the intact cell there must also exist a mechanism which rn~nt~ns the receptor in its ‘native’ form in the absence of ligand at the physiological temperature. Whether there is resemblance between the ‘physiological native form stabilizer’ and molybdate remains to be elucidated.

4

II. Structure of the chick progesterone receptor A. Multiple forms and proteolysis fragments of the chick oviduct PR Initial reports (Sherman et al., 1970, 1974, 1976) on the chick oviduct cytosol PR had recognized the presence of several forms which could be resolved by gel filtration. These comprise: large form (form I) eluting in the V, of a Biogel A 0.5 M column; two smaller forms (II and III) which eluted very close to each other with R, - 6.3 nm and 4.9 nm respectively, and two small ones (IV and V) with R, - 3.5 nm and 2.1 nm respectively (Sherman et al., 1974). Further studies (Birnbaumer et al., 1983~; Vedeckis et al., 1980a, b) showed that while forms IV and V were proteolytic fragments, forms II and III correspond to intact or authentic P-binding proteins. They can be easily separated by ion-exchange chromatography of ‘activated’ PR; form III, which elutes at < 0.15 M KC1 from DEAE-cellulose, was named ‘subunit A’, while form II, eluted with 0.25 M KCl, is now known as ‘subunit B’ (Schrader et al., 1975, 1977). Moderate proteolysis of either the A or the B subunit yields similar or identical hormone-binding fragments, ‘form IV’ (M, - 43 kDa) and ‘form The latter still possesses v’ (M, - 23 kDa). steroid-binding characteristics similar to those of the intact PR, and has been called ‘mero-receptor’ (Sherman et al., 1976, 1978; Vedeckis et al., 1980a, b). A smaller fragment (M, - 9.5 kDa) containing the hormone-binding site was obtained by Staphylococcus aureus V8 protease digestion of affinity-labelled receptor (Birnbaumer et al., 1983b; Gronmeyer et al., 1983). All these fragments include the NH, terminus of the protein. The steroid-binding site is therefore situated close to the NH, terminus of both the A and B proteins. Two lines of evidence support the notion that the A and B subunits are not related to each other by a precursor-product relationship. Firstly, all attempts to convert one form into the other have failed. Secondly, when the two proteins were radioiodinated and subjected to peptide mapping, they were found to yield some common peptides, but also peptides specific for either the A or the B protein (Birnbaumer et al., 1983b). This indicates that the A and B subunits possess a similar or

identical portion situated at the NH,-terminal part of the polypeptide chains. The existence of such a highly conserved segment in the A and B molecules is also supported by the observation that antibodies raised against the B subunit cross-react with the A subunit as well as with the ‘mero’-fragment of the PR (see section III; Fig. 2) (Tuohimaa et al., 1984). Precise identification of the progestin-binding proteins had to ’ await finer techniques, namely affinity labelling (D&e et al., 1980), purification to near homogeneity (Kuhn et al., 1975; Coty et al., 1979; Renoir et al., 1984) and antibody studies of purified (Renoir et al., 1984). Analysis ‘activated’ receptor forms A and B has shown their M, to be 79 kDa and 108 kDa, respectively. This is in agreement with the data obtained by fluorography of [3H]R5020 covalently labelled receptor in crude or partially purified preparations (Birnbaumer et al., 1983a) (Table 1). Immunoblotting experiments with crude cytosol and with receptor purified by steroid affinity chromatography yield

Molecular mass kDa

1 2

3

4

5

6

Fig. 2(a). SDS-polyacrylamide gel (SDS-PAGE) electrophoresis of PR. Purification of the molybdate-stabilized (lanes 1 and 4) and of the ‘activated’ (lanes 2, 5 and 6) PR. Lanes 1 and 2, eluates after affinity chromatography; lane 3, ‘mock’ purification (cytosol in molybdate containing buffer which had been preincubated with an excess of unlabelled progesterone (2 CM) was loaded on to the affinity gel and processed in the same way as that in lanes 1 and 2); lane 4, molybdate-stabilized PR purified on DEAE-Sephacel after elution from the affinity chromatography column; lanes 5 and 6, A and B subunit of PR, respectively, isolated by DEAE-Sephacel chromatography of the affinity gel &ate shown in lane 2.

5 TABLE

1

CHARACTERISTICS

OF THE ‘NATIVE’

AND ‘ACTIVATED

Native PR

FORMS

OF THE CHICK

Subunit B

Subunit A

7.1 nm (7.7 nm) a 1.55 245 kDa (265 kDa) a 270 kDa b

Frictional ratio M, calculated from s2+ and R 5 M, calculated from SDS-PAGE data Elution from DEAESephacel (M KCI) Binding to polyanions: ’ Phosphocellulose DNA-cellulose Heparin-agarose ATP-sepharose M, of polypeptides detected in SDS-PAGE (1) by antibodies: Polyclonal IgG-G3

0.1 (form I) 0.16 (form II) _ -

Polyclonal IgG-RB Monoclonal BF4 (2) by affinity Iabelling ([3H]R5020)

3.6s

4.2s

4.6 nm

6.1 nm

1.5 12 kDa

1.77 111 kDa

19 kDa

110 kDa

0.08

0.2

+

+ + + +

+ _

110 79 110 90 110

PR

Activated PR

1.9s

Sedimentation coefficient (~*c.~) Stokes radius (R,)

OVIDUCT

kDa; 90 kDa; kDa kDa; 79 kDa kDa kDa; 79 kDa

f + +

79 kDa

110 kDa

79 kDa none 19 kDa

110 kDa none 110 kDa

. a Found with PR in crude cytosol. b PR cross-linked with glutaraldehyde. ’ + , strong binding; + , weak binding; - , very weak or no binding. Sources: Renoir et al. (1982a, 1984) Yang et al. (1982a). Baulieu et al. (1984).

1

23

4

678

9

205Kt ‘##Z

-

-

-

45K+ 29K+

similar results (Fig. 2) (Baulieu et al., 1984; Renoir et al., in preparation). It is important to note that the same results were obtained when ‘activation’ of PR had been prevented by inclusion of molybdate in the homogenization buffer. This indicates that the primary structure of PR is not altered upon activation. The size of the activated [3H]P-PR complexes as estimated by use of the formula of Siegel and Monty (1966) suggests that _

Fig. 2(b). Detection of PR by immunoblotting with IgG-G3 (lanes l-4), BF4 (lanes 5-8) and IgG-RB (lane 9). Samples fractionated by the SDS-PAGE were: total oviduct cytosol (lanes 1 and 5); purified A subunit (lanes 2 and 6); purified B subunit (lanes 3 and 7); purified molybdate-stabilized (‘8s’) PR

(lanes 4, 8 and 9). The IgG-G3 antibody was raised in a goat against the 8S PR; it recognizes all receptor subunits. The antibody IgG-RB was obtained from a rabbit immunized with the pure B subunit and it reacts with the A and B subunits but not with the 90 kDa protein. The monoclonal antibody BF4, in contrast, recognizes only the 90 kDa protein. For further information see section HI.

6

Molecular masskDa

Molecular 1

2

3

216K,

identified. They bind also to polyanions such as phosphocellulose and DNA-cellulose. These properties were used for their purification (Kuhn et al., 1975; Coty et al., 1979).

130K _ 97.4K,

,i ”

45K,

29K

_

Fig. 2(c). Identification of hormone-binding components of the IS-PR by affinity labelling. Cytosol in molybdate-containing buffer was loaded on to an affinity gel column. [ ‘H]R5020 was used to elute biospecifically the 8S-PR. A portion of the eluate was further purified by DEAE-Sephacel chromatography where elution was carried out by buffer without molybdate; under these conditions, the PR becomes activated and the A and B subunits are eluted separately with 0.1 M KC1 (A) and with 0.25 M KC1 (B). An aliquot of the 0.25 M KC] fraction was incubated with 2 pM unlabelled P for 1 h at 25 o C (lane 7). All PR-containing fractions were irradiated by UV lamps and analysed by SDS-PAGE, 5515% polyacrylamide gradient (lanes 1-3) and 10% polyacrylamide (lanes 4 and 7). The gels were stained by silver nitrate (lanes 1 and 3) and fluorographed (lanes 4 and 7). Lanes 1 and 4, affinity gel eluted 8s PR; lanes 2 and 5, DEAE-Sephacel fraction eluted with 0.1 M KCI; lanes 3, 6 and 7, DEAE-Sephacel fraction eluted with 0.25 M KCl. Absence of labelled band in lane 7 confirms the lack of non-specific protein labelling with [3H]R5020 during UV irradiation.

A and B subunits are present as independent molecules in the activated PR preparations. Beside the hormone-binding site, the A subunit also carries a DNA-binding site. This site is situated between 23 kDa and 31 kDa (counted from the NH, terminus) on the A subunit (Birnbaumer et al., 1983~). Since, according to Birnbaumer et al. (1983b), the A and B subunits share the a common domain of - 60 kDa containing NH, terminus, the B subunit should also possess the DNA-binding domain. Why the B subunit binds poorly to DNA remains unclear. Both A and B subunits bind strongly to ATP (Moudgil, 1983). The domain of the polypeptide chain necessary for this binding has not yet been

B. Protein composition of the 8S PR The I form of PR (Sherman et al., 1976) has been suggested to represent the ‘native’ complexes consisting of AB dimers or possibly A,B, tetramers (Schrader et al., 1975; Sherman et al., 1984). However, purification of the molybdate-stabilized, non-transformed PR has shown that it contained, in addition to the A and B subunits, another protein of IV, - 90 kDa (Baulieu et al., 1984; Renoir et al., in preparation). This protein does not bind progesteron and its role is unknown. The complexes between the progestin-binding A and B subunits and the 90 kDa protein may exist in the living cell, or may be formed after homogenization of the tissue. They show, however, properties which make the latter possibility less likely. First, they exist also in cytosol prepared by homogenization in 0.15 M KC1 containing buffer, and molybdate is not required for their formation (Mester and Renoir, unpublished). Second, when stabilized by molybdate, they are stable under conditions which largely prevent non-specific protein-protein interactions (high ionic strength, Yang et al., 1982a; urea < 2.5 M, Buchou et al., 1983). Third, once dissociated, the 8s PR complexes cannot be reformed (Yang et al., 1982a). Finally, association of the 90 kDa protein and the PR subunits appears to be specific: the 90 kDa protein co-purifies with the A and B subunits under conditions applied for affinity chromatography which includes washing at high ionic strength and with 2.5 M urea (Renoir et al., 1984), but not with other proteins under similar conditions (Fig. 2(a); Baulieu et al., 1984; Renoir et al., in preparation). The 90 kDa protein may be necessary for maintaining the PR molecules in their ‘non-activated’ state in the intact cell in the absence of hormone. It is also present in other steroid hormone receptors of the chick in their non-activated forms (Joab et al., 1984) and in all tissues of the chick. Since there is no known tissue from which all steroid hormone receptors are absent, it is not clear whether the 90 kDa protein might have other roles in the cell housekeeping than its interaction with

steroid hormone receptors. Its cellular content is 50-lOO-fold greater than would correspond to the amount of steroid hormone receptors (Baulieu et al., 1984). An observation which may be of importance for elucidation of the biological function of the 90 kDa protein is its phosphorylation in vitro and in vivo (Garcia et al., 1983; Dougherty et al., 1982). The 90 kDa protein also co-purifies with a Ca2+-dependent protein phosphorylating activity and may itself in fact be a protein kinase (Garcia et al., 1983). The behaviour of the purified molybdate-stabilized PR in ion-exchange chromatography suggests that the complexes containing both A and B subunits are certainly not common, if at all present in such preparations (Fig. 3). In fact, the DEAE-Sephacel elution profile shows heterogeneity in the subunit composition of the PR in such manner that the complexes eluted at lower KC1 concentrations contain predominantly the A subunit while those eluted at higher ionic strength are rich in B subunit. Similar conclusions can be drawn from immunoblotting data of DEAE-Sephacel elution fractions of total cytosol, in which the 8s PR is stabilized by Na,MoO,. This result confirms that purification does not cause a change in the receptor structure. Toft and co-workers have

Fig. 3. Heterogeneity of 8s PR. PR eluted from affinity gei in molybdate-containing medium was fractionated by DEAESephacel chromatography in the presence of molybdate. Fractions ‘I’ and ‘II’ as indicated were pooled and analysed by SDS-PAGE; detection of the PR components was carried out by silver staining (lanes 1 and 3) and by immunoblotting with IgG-RB (lanes 2 and 4).

also recently achieved the purification of the ‘non-transformed’ molybdate-stabilized chick oviduct PR and characterized two different 8s PR forms, both containing the 90 kDa protein (Dougherty et al., 1982; Puri et al., 1982). The relative quantities of the A and B subunits of chick oviduct in individual cytosol preparations are variable and have been reported to be influenced by season (Boyd and Spelsberg, 1979; Spelsberg and Halberg, 1980). This also argues against the structure of native PR containing the A and B molecules in a 1: 1 ratio. C. Structure of the 8S PR The model we propose (Table 2) postulates that the XS PR is a mixed population of molecules always containing one hormone-binding subunit associated with the 90 kDa protein. This model reposes on the following arguments, in addition to the observed heterogeneity of the 8s PR described in the previous subsection. First, the major part of the 90 kDa protein appears to be present in the cytosol in the form of a dimer. This follows from estimation of the apparent molecular weight of the protein in non-denatured preparations, on the basis of its Stokes radius and sedimentation coefficient (data obtained using the anti-90 kDa monoclonal antibody for detection of the protein in fractions after gel filtration or after sucrose gradient ultracentrifugation (Buchou et al., unpublished data)). The M, calculated using the formula of Siegel and Monty (1966) is 183 kDa. Second, when highly purified preparations of the 8s PR were cross-linked by glutaraldehyde and analysed by SDS-PAGE, a major band of - 270 kDa was detected next to a minor band of - 190 kDa, suggesting that trimeric molecules (2 X 90 kDa + 110 kDa = 290 kDa; 2 X 90 kDa + 79 kDa = 259 kDa, indistinguishable given the heterogeneity of all cross-linked molecules due to varying length of glutaraldehyde polymers) were predominant (Fig. 4) (Baulieu et al., 1984; Renoir et al., in preparation). Molecular weights calculated for the purified 8s PR, - 245 kDa (Renoir et al., 1982a; Puri et al., 1982), and crude 8s PR, - 265 kDa (Yang et al., 1982a; Dougherty et al., 1982), on the basis of their respective sZO,Wand R,, do not differ substantially from data obtained for the glutaraldehyde

8 TABLE

2

SCHEMATIC

REPRESENTATION

8s forms (non-activated Activation:

receptor):

OF THE CHICK

OVIDUCT

PR STRUCTURE

A(90 kDa),; B(90 kDa), (proposed model) A(90 kDa), it A+ (90 kDa), + A+ + 2(90 kDa) B(90 kDa), Z= B’(90 kDa), + B+ +2(90 kDa) the activated forms of subunits A and B. The postulated intermediaij (A+, B+ designate structures A+ (90 kDa),, B+(90 kDa), have not been identified. It is also unknown whether, after activation, the 90 kDa protein exists as a monomer or a dimer.)

Primary structure of subunits:

NHZks&ed)

B:

coon I

.

1OkDa * (1) (2) The the

Points of high susceptibility to proteolytic enzymes. Highly conserved sequence containing the steroid-binding site. Sequence probably conserved but no DNA-binding site on the B subunit. primary structures of the native (A, B) and activated (A +, Bf ) subunits 90 kDa protein.

cross-linked purified preparations. various forms of PR are summarized

Properties of in Table 1.

b s-7.5 I 1

2345678

+206K

-+-140K *ISOK *9741( -c-701( +661(

Fig. 4. Cross-linking of purified 8s PR. The molybdate-stabilized receptor was purified on the affinity column followed by DEAE-Sephacel chromatography in phosphate buffer. An aliquot was boiled with SDS-mercaptoethanol for SDS-PAGE analysis (part a, lane 2) in 7.5% polyacryiamide. (Lane 1, molecular weight markers.) Portions of the remaining 8s PR were treated with 0.4% ghttaraldehyde for 0, 1, 5, 15, 30 and 60 min at O” (lanes l-6); Iysine (0.15 M) was added to stop the reaction. The samples were analysed by SDS-PAGE in a 5-7.5% polyacrylamide gradient (part b). Lane 7, cross-linked hemocyanin markers (70 kDa, 140 kDa, 280 kDa); lane 8, noncross-linked marker proteins.

are apparently

identical.

They show no similarity

with

D. Role of molybdate in the stabilization of 8S PR How does molybdate stabilize the 8s PR molecules? The initial hypothesis that it would act by inhibiting phosphatase activity is probably incorrect since other phosphatase in~bitors have no effect on 85 + 4s conversion (Wolfson et al., 1980; Seeley and Costas, 1983). Moreover, PR activation does not seem to require an action of a phosphatase since it can be obtained with highly purified preparations. Indirect evidence suggests that molybdate ions form complexes with the 8s receptor molecule (Buchou et al., 1983). Most likely, relatively weak bonds are involved since very high ionic strength (> 1 M KCl) or moderate urea concentrations (> 3 M) transform the receptor to 4s forms even in the presence of molybdate. Moreover, the molybdate effect is readily reversible (Nishigori and Toft, 1980), which implies rapid dissociation of moIybdate-SS PR complexes. A clue about molybdate interaction with the receptor comes from comparison with other similar ions. While vanadate and tungstate have a similar stabilizing effect to molybdate (Nishigori et al., 1980; Murakami et al., 1982) no effect was seen with PO;-, SO:-, CrOi--

9

or NO; ions (Wolfson et al., 1981). The transition metals (which include MO, V and W) are known to form, at neutral pH, weak covalent coordination complexes of the type:

ROOPR ‘Me/R RLMe< R’

0’

‘R

where R may be a radical such as SH, NH, or OH, provided by protein molecules or by water (Stiefel, 1977). This type of bond could account for the observed stabili~ng effects of transition metal anions on the 8s receptor oligomeric structure (Baulieu et al., 1984). Correct relative positions of the radicals entering into coordination complexes shown above are probably necesary. It would be interesting to test whether other oligomeric proteins are also stabilized by transition metal anions; to our knowledge, such experiments have not been reported. III. Immunological

studies of the chick oviduct PR

A. Antibodies raised against the purified 8s PR Polyclonal antibodies have been obtained in the goat and in the rabbit after injection of highly purified 8S PR preparations (Renoir et al., 1982b). These antibodies recognized the 8S, the 4S A and B subunits, as well as the ‘mero-receptor’ of the chick oviduct PR, as assessed by sucrose gradient or by immunoblotting experiments. Cross-reactivity of the goat IgG-G3 polyclonal antibodies with non-transformed and transformed mammalian PR has been observed by density gradient centrifugation analysis. However, no specific bands could be detected in mammalian tissue extracts by immunoblotting using either goat or rabbit anti-8S PR antibodies. This could be due to the low affinity of these antibodies for the heterologous receptors. Logeat et al. (1981) have shown that polyclonal antibodies, raised in the goat by injecting purified PR from rabbit uterus, did not react with chick oviduct PR. These data indicate relatively poor conservation of the PR, in contrast with the estrogen receptor (Greene et al., 1979). However, immunocytochemical studies performed with IgG-G3 detected the presence of PR

in the rat pituitary cells (Morel et al., 1983). The same localization was found for the PR in this tissue as that of estradiol receptor (Morel et al., 1981). Injection of the 85 PR receptor also gave rise to a monoclonal antibody (BF4; Radanyi et al., 1983). This antibody reacts with the 8S PR but not with the 4S PR forms. After transformation of the 8S PR, the presence of the antigen recognized by the BF4 was still demonstrated in the cytosol and in the purified receptor preparations by the use of 35S-labelled BF4 (Joab et al., 1984). Immunoblots identified this antigen to be the 90 kDa protein detected previously (Fig. 2; Renoir et al., 1984; Joab et al., 1984). In addition, the data obtained using the BF4 monoclonal antibody have indicated that the 90 kDa non-hormone-binding protein is present in four steroid hormone receptors (estradiol, androgen, corticosteroid and progesterone) from the chick oviduct in their non-transformed state (Groyer et al., 1982; Joab et al., 1984). Until now, no cross-reactivity of BF4 with the mammalian receptors has been detected. Also, in immunoblots of mammalian tissue extracts, BF4 failed to reveal any bands, suggesting that if an analogous protein to the 90 kDa exists in mammals, its structure is not highly conserved. B. Antibodies raised against the B subunit Difficulties in raising specific antibodies against the receptor subunits were reported by Schrader et al. (1977) who found that the sheep serum contained spontaneous antibodies against these proteins (Weigel et al., 1982). Recently, monoclonal antibody was obtained against the receptor B subunit (Dicker et al., 1982). This antibody, however, recognized only the denaturated B protein (Edwards et al., 1983). In our laboratory, polyclonal antibodies (IgGRB) were obtained in the rabbit after 2 injections of highly purified B preparation (Tuohimaa et al., 1984). These IgG-RB antibodies recognize the non-transformed SS PR as well as the A and B subunits and the 23 kDa proteoiytic fragment (mero-receptor), in total cytosol and in purified preparations (Fig. 2). It cross-reacts weakly with mammalian (rabbit, human) 4S PR.

10

IV. Subcellular distribution gesterone receptor

of chick oviduct pro-

As already mentioned, virtually all the PR is recovered in the high-speed supernatant fraction of the non-P-treated chick oviduct. It was therefore unexpected that when the same tissue was examined by immunohistological methods using specific rabbit anti-PR antibody (IgG-RB), a strong positive reaction was seen in the nuclei of all oviduct cells. A less specific goat antibody (IgG-G3), which recognizes the A and B subunits as well as the 90 kDa protein (Baulieu et al., 1984; Renoir et al., 1984), gave the same positive reaction in the nuclei. In addition, cytoplasmic staining was dense in epithelial cells, whereas tubular gland cells were stained weakly (Gasc et al., 1984). Treatment with progesterone leads to two kinds of effect on the subcellular distribution of PR as observed by hormone-binding assay after tissue fractionation. First, at short time intervals, lo-20% of the total PR pool ‘translocates’ to the nuclear compartment. Second, between - 1 h and - 6 h after progesterone administration a decrease in the total cellular content of PR occurs; the minimum PR level is - 40% of the initial value (Mester and Baulieu, 1977). In view of the histological data mentioned above, ‘translocation’ from cytosol to the nucleus may be an artefact of experimental procedure. In support of this possibility, nuclear receptors for estrogens have also been detected in the absence of ligand in the chick liver (Mester and Baulieu, 1972), in the mammalian uterus (Sheridan et al., 1979; Levy et al., 1980) and in rat pituitary tumour cells in culture (GH, cells; Mester et al., 1973). Exclusively nuclear localization of the unoccupied estrogen receptor is indicated by the recent data of Ring and Greene (1984) and of Welshons et al. (1984). If the receptor is localized in the nucleus, prior to the administration of the hormone, then upon binding of the ligand it becomes ‘activated’ and therefore more tightly associated with the nuclear compartment. The reason why only a minority of all PR molecules are recovered with the nuclear fraction of the chick oviduct homogenate may be that the majority of the PR molecules, even after activation, are never tightly bound to the chro-

matin. Alternatively, not all PR molecules may become activated in vivo. Yet another possibility is that, simultaneously with PR activation, degradative processes could be triggered. This last possibility is supported by the relatively fast disappearance of - 2/3 of the total (cytoplasmic + nuclear) cellular PR following progesterone treatment. It would also account for the earlier observation that the ‘non-translocated’ PR is refractory to a second progesterone treatment (Fig. 5; Sutherland et al., 1977). Between - 6 h and - 40 h after progesterone administration the PR level in the tissue is replenished, and nearly all PR is again found in the cytosol fraction of the homogenate. This is in agreement with the fact that during the first 6 h nearly all (90%) progesterone has been eliminated from the plasma (Binart et al., 1982). Interestingly, in the estrogen-primed withdrawn chicks, replenishment of PR after P administration leads to an ‘overshoot’ of - 50% above the initial level (Mester and Baulieu, 1977). No studies have been done to discern whether this is due to an increased rate of synthesis of new PR molecules, or to another mechanism. All the data based on determination of P-binding capacity should be complemented by immunological detection of PR molecules, both in tissue slices and in subcellular fractions. Such detection

1

150 I

7

0

I 2100

/ \

~~~_.~/.

0

20

30

-

..4O 1

Fig. 5(a). Effect of progesterone (3 mg/kg) on the progesterone receptor level in the cytosol(0) and nuclei (0) of the withdrawn chicken magnum. 100% corresponds to -14000 progesterone binding sites/cell.

11

0

1

2

3

4

5

6

Time

[IL]

Fig. 5(b). Effect of a second injection of progesterone (3 mg/kg) at 1, 2, 3 and 6 h on progesterone-induced concentration of nuclear PR. The nuclear PR levels were measured 1 h after the second progesterone administration (0); 0 represents the nuclear progesterone levels in the chicken given a single progesterone dose at time 0 (from Sutherland et al., 1977).

is now possible since the anti-PR antibodies are available, and work in our laboratory is in progress. V. Function A. Transcription of specific genes Hitherto only indirect evidence has been available pointing to an action of PR at the DNA level to induce gene expression. This evidence is becoming more and more convincing, although many problems remain unresolved. Experiments performed in the early 1960s using metabolic inhibitors (cycloheximide, actinomycin D, ar-amanitin) have indicated that, to obtain final effects of estrogens in mammalian uterus, continuous RNA and protein synthesis is required (Mueller et al., 1958). In the chick oviduct, the relative rate of synthesis of the major egg-white protein is increased in parallel with the respective mRNA concentration (Palmiter et al., 1976). Increased rate of transcription is the most probable (but perhaps not the only - see below) mechanism that leads to accumulation of specific mRNAs in the cell (Palmiter et al., 1981). We do not know as yet how progesterone increases the rate of specific gene transcription, and this is hardly surprising since transcription of eukaryotic DNA by RNA

polymerase II is a complex and not yet elucidated process. However, binding of one or more molecule(s) of PR to more or less specific nucleotide sequences within the gene and adjoining DNA stretches seems to be involved. Such sequences may be those which bind preferentially to the A subunit and which have been identified close to the 5’ end of the transcribed portion (Mulvihill et al., 1982). This hypothesis is also consistent with data concerning glucocorticosteroid effects of MMTV DNA transcription where the region of preferential receptor binding has been found to correlate well with the region necessary for the maintenance of glucocorticosteroid regulation of the transcription (Ringold, 1982) and also with the very recent data of Renkawitz et al. (1982) on transcription of chicken lysozyme gene (one of those regulated by progesterone in the oviduct). There still remains a question mark about the role of the B subunit which has also been known to become associated with the nuclear fraction after exposure of the oviduct to P (review in Schrader et al., 1981; Spelsberg et al., 1983). The next unresolved problem is the lack of correlation between the nuclear PR levels and accumulation of the specific mRNAs in the cytoplasm (Mester et al., 1979). This problem may be only an apparent one if one realizes that the rate of transcription correlates better than the amount of total accumulated mRNA with the nuclear PR levels (Palmiter et al., 1981). Another possibility remains that the PR, after having lost its hormone-binding capacity, may still be able to induce its effects on mRNA synthesis. B. Post-transcriptional effects Beside increased transcription, progesterone treatment also has other consequences leading to specific mRNA accumulation and translation. One of these is the accelerated initiation of translation as seen by polysome assembly (Palmiter, 1972; Pennequin et al., 1978; Robins and Schimke, 1978). Slower mRNA degradation (Schimke, 1977) is probably a consequence of the fact that a greater proportion of all mRNA molecules coding for egg-white proteins is engaged in polysomes. Finally, slower catabolism of proteins was also reported to be a consequence of progesterone treatment (Cox and Sauerwein, 1970). There is no

evidence as to whether PR is directly implicated in any of these effects of progesterone observed in the chick oviduct, and if so, its mechanism of action is totally obscure. A recent observation (Garcia et al., 1983) that the purified B subunit possesses or is associated with a protein kinase activity should also be considered in future experiments aiming at elucidation of the biological activity of PR. It is interesting that in fact both A and B subunits have long been known to be ATP-binding proteins (see review in Moudgil, 1983). References Baulieu, E.E., Atger, M., Best-Belpomme, M., Corvol, P., Courvalin, J.C., Mester, J., Milgrom, E., Robel, P., Rochefort, H. and De Catalogne, D. (1975) Vitamins Hormones 33, 649-736. Baulieu, E.E., Binart, N., Buchou, T., Catelli, M.G., Garcia, T., Gasc, J.M., Groyer, A., Joab, I., Montcharmont, B., Radanyi, C., Renoir, J.M., Tuohimaa, P. and Mester J. (1984) In: Nobel Symposium on Steroid Hormone Receptors: Structure and Function, Eds.: J.A. Gustafsson and H. Eriksson (Elsevier, Amsterdam) in press. Binart, N., Mester, J., Baulieu, E.E. and Catelli, M.G. (1982) Endocrinology 111, 7-16. Bimbaumer, M.E., Schrader, W.T. and G’Malley, B.W. (1979) B&hem. J. 181, 201-213. Bimbaumer, M.E., Schrader, W.T. and O’Malley, B.W. (1983a) J. Biol. Chem. 258, 1637-1644. Bimbaumer, M.E., Schrader, W.T. and O’Malley, B.W. (1983b) J. Biol. Chem. 258, 7331-7337. Bimbaumer, M.E., Weigel, N.L., Grody, W.W., Minghetti, P., Schrader, W.T. and O’Malley, B.W. (1983~) In: Progesterone and Progestins, Eds.: C.W. Bardin, E. Milgrom and P. Mauvais-Jarvis (Raven Press, New York) pp. 19-32. Boyd, P.A. and Spelsberg, T.C. (1979) Biochemistry 18, 3685-3690. Buchou, T., Mester, J., Renoir, J.M. and Baulieu, E.E. (1983) B&hem. Biophys. Res. Commun. 114,479-487. Buller, R.E., Schrader, W.T. and O’Malley, B.W. (1975) J. Biol. Chem. 250, 809-818. Coty, W.A., Schrader, W.T. and O’Malley, B.W. (1979) J. Steroid B&hem. 10, 12. Cox, R.E. and Sauerwein, H. (1970) Exp. Cell Res. 61, 79-90. Dicker, P.D., Tsai, S.Y., Tsai, M.J., Weigel, N.L., Schrader, W.T. and O’MaIley, B.W. (1982) American Society for Cell Biology, 22nd Annual Meeting, Baltimore, 30 Nov.-4 Dec., 95(2), Part 2. Dougherty, J.J. and Toft, D.O. (1982) J. Biol. Chem. 257, 3113-3120. Dougherty, J.J., Puri, R.K. and Toft, D.O. (1982) J. Biol. Chem. 257, 14226-14231. Dure IV, L.S., Schrader, W.T. and O’Malley, B.W. (1980) Nature (London) 283, 784-786. Edwards, D.P., Weigel, N.L. and McGuire, W.L. (1983) 65th

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