Regulation of glucocorticoid receptor activity

0022-4731/89 $3.00 + 0.00 Copyright 0 1989 Pergamon Press plc

J. steroid Biochem. Vol. 34, Nos l-6, pp. 71-78, 1989 Printed in Great Britain. All rights reserved

REGULATION

WOLFGANG

H&K,’

OF GLUCOCORTICOID ACTIVITY FINIAN MARTIN,*

ROLF JAC&

RECEPTOR

and BERND GRONER’*

‘Friedrich Miescher Institute, P.O. Box 2543 CH-4002 Base], Switzerland; *University College Dublin, Department of Biochemistry, Belfield, Dublin 4, Ireland; and ‘Institut fiir klinisch-experimentelle Tumorforschung, UniversitLt Bern, Tiefenaustr. 120, CH-3004 Bern, Switzerland

Summary-Four levels of regulation of glucocorticoid receptor (GR) activity have been investigated. (1) Phosphorylation of the GR was studied in NIH 3T3 cells metabolically labeled with [32P]orthophosphate. A highly specific antiserum against the GR was used to immunoprecipitate 32P-labeled GR, and protein blotting was used to determine the GR concentration. Comparison of the relative specific activities of non-activated and activated receptor revealed a 34-fold increase in GR phosphorylation within 60 min upon hormone activation. (2) The affinity of the GR for its hormone response element (GRE) was quantitated in in vitro binding and gel shift experiments. The comparison of monomers, dimers and trimers of the GRE showed that GR binding affinity to multimers is much higher than the affinity for a GRE monomer. (3) The concentration of the GR was determined in quantitative protein blot assays as a function of time after hormone treatment of NIH 3T3 cells. A down-regulation of GR was observed. Only 30% of the maximal GR concentration observed in the absence of hormone remained after 24 h of hormone treatment. (4) The effect of the presence of hormone on the subcellular location of the GR was studied. Hormone treatment and withdrawal experiments indicated that the presence of hormone is not only required to initiate the cascade of events resulting in transcriptional trans-activation. GR translocated to the nucleus upon hormone addition returns rapidly to the cytoplasm upon hormone withdrawal. This indicated an active role for the hormone in the tight nuclear binding of GR.

confer receptor activity to the transcriptional process have led to a high-resolution picture of some of the early events in steroid hormone regulation of transcription [ 1 l-l 31. Additional levels of control, however, have to be postulated if we think of steroid hormone action as a dynamic process affecting a multitude of physiological events. Control over the activity of the GR is primarily exerted by the glucocorticoid hormones which form specific complexes with the cytoplasmic receptor molecules, and initiate a chain of events which includes the nuclear translocation and the assumption of the role of the GR as a transcription factor. We have identified additional levels at which the activity of the GR is regulated. Phosphorylation of the GR is an early event upon nuclear translocation. The specific phosphorylation of the GR is increased 4-fold within 60 min after hormone addition to the medium of NIH 3T3 cells[l4]. The importance of this secondary modification in the transactivation of gene transcription was emphasized in experiments with a glucocorticoid antagonist. RU38486 inhibited receptor phosphorylation and the transcriptional induction of responsive genes. The binding affinity of the activated receptor to monomers, dimers and trimers of the GRE was determined in vitro. Quantitative gel shift experiments were carried out and the complex formation was monitored [15]. GRE trimers formed complexes most efficiently, a lower affinity for the GR

INTRODUCTION

glucocorticoid receptor (GR) is a transcription factor which confers positive [l-3] or negative [4-61 regulation to genes which contain specific recognition sequences (glucocorticoid hormone response elements, GRE) in the vicinity of their transcriptional initiation sites. Like other members of the steroid hormone receptor gene family [7], the GR is organized in structurally distinct domains. The Nterminal domain shows the least sequence homology when different steroid receptors are compared, and its role is thought to be in the modulation of transactivation [8]. The centrally located cysteine-rich domain is essential for the specific DNA binding activity of the receptor. The C-terminal domain harbors a number of functional roles: the hormone binding ability, a nuclear translocation signal, and functions for dimerisation and transactivation have been assigned to this region of the molecule [9, lo]. Cloning of the steroid hormone receptor genes and a precise definition of the DNA sequences required to

The

-_ Proceedings of the 9th International Symposium of the Journal of Steroid Biochemistry, Recent Advances in Steroid Biochemistry, Las Palmas, Canary Islands, Spain, 28-31 May 1989. *To whom correspondence should be addressed. [Tel. (061) 697 6163; Fax (061) 697 66711. 71

12

WOLFGANG

Hii~~et al.

was observed with dimers and an even lower affinity with monomers. Receptor concentration was quantitated in cells exposed to hormone for prolonged periods with a highly specific antiserum. Receptor down-regulation was observed [16]. Only about 30% of the GR present in NIH 3T3 cells grown in the absence of hormone remain after 24 h of hormone treatment. These observations suggest that the magnitude of transcriptional transactivation can be regulated in a time-dependent manner and suggest a mechanism for the differential extent of induction observed in individual responsive genes. Withdrawal of hormone shortly after nuclear translocation of the GR resulted in a return of GR to the cytoplasm. This observation suggests a hormonal control of tight nuclear binding of GR.

4-

RESULTS

AND DISCUSSION

Increase in GR phosphorylation upon ligand activation The multiple steps in the conversion of the GR from its latent state in the cytoplasm to its active state in transcriptional regulation are initiated by the ligand-receptor interaction. It is not clear if GR complex formation is a sufficient requirement or if other mechanisms of cellular control are important. Secondary modification of proteins by different classes of kinases and dephosphorylation by phosphatases is a basic means of cellular regulation of protein activity. It has been postulated that phosphorylation might play a role in the process of GR activation or function [ 171. We have measured the degree of GR phosphorylation in NIH 3T3 cells as a function of time after hormone addition to the tissue culture medium. In order to gain a precise determination of the relative degree of phosphorylation of GR, two measurements were made. First, total cellular GR was quantitated in a protein blot experiment. For this purpose a highly specific antiserum was used [14, 161. This antiserum was raised against a rat GR fragment (amino acid positions 440-795) in rabbits. The antiserum was shown to recognize GR of human, rat and mouse origin as well as nonactivated cytoplasmic and tightly bound nuclear receptor [ 141. Quantitation of GR at 0, 20, 40 and 60min after hormone addition showed that the receptor concentration changes only very little during the first hour after hormone activation. Down-regulation of the receptor was only observed after longer periods of induction. A second measurement concerned the incorporation of phosphate groups into the receptor molecule. Cells were cultured for 4 h in [“Plorthophosphate-containing medium. After this period dexamethasone was added for 0, 20, 40 and 60min and total cellular GR was immunoprecipitated with the specific antiserum. The autoradiographic signal obtained provided a measure of 32P-incorporation into the 100 kDa GR molecule. Figure 1 shows the relative specific activities (amount of phosphate per receptor) observed as a function of

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Fig. 1. Activation-dependent increase in glucocorticoid receptor phosphorylation. NIH 3T3 cells were grown in DMEM and 10% fetal calf serum. The medium was replaced for 1h with phosphate-free medium containing 1% dialyzed serum before fresh phosphate-free medium including lOOpCi/ml [32P]orthophosphate was added for 4 h. Dexamethasone was added to 10e6M and cells were harvested after 0, 20, 40 and 60min. GR concentration in cellular extracts was determined by immunoblotting. Equal amounts of protein were separated on a 10% polyacrylamide gel. The proteins were electroblotted onto nitrocellulose filters and reacted with a highly specific antiserum against the GR. Rabbit antibodies were visualized with ‘251-labeled protein A and autoradiography. The incorporation of phosphate groups into the GR was determined by immunoprecipitation. Labeled cells were lysed in RIPA buffer and the solubilized proteins were incubated with the GR-specific antiserum for 1 h on ice. Immunocomplexes were adsorbed to Pansorbin for 30 min on ice and washed four times with RIPA buffer. Bound GR was released in SDS sample buffer at 100°C for 1Omin. The GR was visualized by gel elemtrophoresis and autoradiography. The autoradiographic signals obtained in the blotting and in the immunoprecipitation procedures were quantitated by densitometry and a ratio of signals (phosphate incorporation: receptor concentration) was calculated. The time 0 value was set at 1. These ratios reflect the relative specific activities (phosphate groups per receptor molecule) in arbitrary units.

time after hormone addition. The GR is a phosphoprotein in the absence of hormone [18, 191. The addition of hormone resulted in a 4-fold increase in the specific activity within 60 min. The functional significance of these phosphorylation events was emphasized in an experiment with the glucocorticoid antagonist RU38486. This antagonist prevented the increase in GR phosphorylation and the transcriptional transactivation [14]. Although circumstantial, the evidence from this experiment could indicate a role for phosphorylation in the transactivation process. The close relationship of the members of the steroid hormone receptor gene family suggests that other steroid receptors might be subjected to a similar mode of regulation. It has indeed been observed that the mammalian and avian progesterone receptors as well as the avian vitamin D receptor are phosphorylated upon hormone induction [2&23]. The receptor domain in which phosphorylation was observed in the chicken oviduct progesterone receptor in viuo is limited to the N-terminal modulator domain [23]. This might indicate that phosphorylation is more important for the protein-protein interactions in transactivation than for the DNA or hormone binding ability. This observation is corroborated by the

13

Regulation of glucocorticoid receptor activity finding that bacterially expressed progesterone receptor [24] and glucocorticoid receptor [ 151 retain their hormone and DNA binding activity, but no transactivation has been observed so far. Estrogen receptor phosphorylation has been studied in the rat uterus [li’] and phosphorylation on tyrosine residues has been reported. This is in contrast to the observations on progesterone receptor (PR) and the GR where only serine and a little threonine phosphorylation was detected in the nonactivated and in the activated states [18,25].

Glucocorticoid receptors bind to oligomers of the GRE with increased aflnity

A large number of genes are steroid hormoneresponsive and the response elements have been detected in the vicinity of the RNA initiation site of these genes [2,26-301. The glucocorticoid hormone response element (GRE) has been defined as a short oligonucleotide of 15 bp. This sequence represents a palindrome which is able to specifically interact with the GR or the PR [31,32]. Differences in the extent

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Fig. 2. Glucocorticoid receptors bind to oligomers of the GRE with increased affinity. NIH 3T3 cells were grown in DMEM and 10% fetal calf serum. Dexamethasome was added to the cells for 1 h at 10m6M. Cells were lysed in hypertonic buffer [15] and whole cell protein extracts were prepared. Synthetic, double-stranded oligonucleotides comprising the sequence S-GATCCAGAACATGATGTTCTAGCTACGGATC-3’ were made and labeled with polynucleotide kinase and [‘*P]ATP. Dimeric and trimeric forms of the oligonucleotide were generated with T4 DNA kinase and purified after gel electrophoresis. Gel retardation assays were carried out by reacting whole cell protein extracts with radioactively labeled oligonucleotides. 8 pg of cellular protein were incubated in 20~11 with 0.1 ng of oligonucleotide (10,000 cpm, 2 pg poly dI-dC, lO-6 M dexamethasone, for 15 min at 0°C and for 15 min at 23°C. The mixture was then applied to a low ionic strength 4% acrylamide gel and electrophoresed in Tris-borate-EDTA buffer. (A) Gel retardation analysis of monomers, dimers and trimers of the GRE with whole cell extract (WCE) from NIH 3T3 cells. Lane 1: monomer, lane 2: monomer reacted with WCE, lane 3: dimer, lane 4: dimer reacted with WCE, lane 5: trimer, lane 6: trimer reacted with WCE. (B) Comparison of glucocorticoid receptor binding affinity of GRE monomers, dimers and trimers assayed by gel retardation analysis. Monomers, dimers and trimers of the GRE were mixed in equimolar ratios and incubated with increasing amounts of whole cell protein extract. Gel retardation assays were performed and the autoradiographic signals originating from the unshifted position of the oligonucleotides were quantitated by densitometry. The graph shows that trimers are shifted at lower protein concentration, i.e. that they bind GR with a higher affinity.

12

74

WOLFGANGHOCK et

of hormonal inducibility have been observed and the number as well as the sequence context of the response elements have been thought to be important [2, 33, 341. The well-studied promoter of the MMTV LTR contains four GREs in its immediate vicinity. This promoter is very strongly inducible when compared to a weakly inducible “minimal” construct of the tk gene promoter with a single GRE. Duplication of this single GRE caused an increase in the hormonal inducibility, suggesting a functional synergism between GREs [3 1, 351. We tested the possibility that the functional synergism observed at the level of transcriptional activity is reflected at the level of GR binding to its response element [ 151. For this purpose on oligonucleotide of 32 base pairs was synthesized which contained internally the perfect palindromic sequence .5’AGAACATGATGTTCT-3’. This oligonucleotide was multimerized by ligation to itself and dimers and trimers of this DNA fragment were isolated. Each fragment was radioactively labeled and reacted in vitro with a whole cell extract from NIH 3T3 cells, and specific DNA protein complexes between the GR and its GRE were formed. These complexes were subjected to gel electrophoretic analysis (Fig. 2A). A comparison between the free DNA of GRE monomer (lane 1), dimer (lane 3) and timer (lane 5) and the complexed DNA of GRE monomer (lane 2) dimer (lane 4) and trimer (lane 6) was carried out. No specific DNA-protein interaction between the GRE monomer and the GR (lane 2) was detected. A weak band of complexed DNA-protein was seen when GRE dimer was reacted with GR (lane 4). A strong band of GRE trimer-GR complex was found (lane 6). Since the same concentrations of DNA and protein were introduced in the binding reactions, the gel retardation assay shows that complex formation occurs preferentially with multimerized forms of the GRE. In a second experiment these observations have been corroborated. Monomers, dimers and trimers of the GRE were mixed before they were reacted with different amounts of cellular protein. The gel retardation assays were carried out and the autoradiograms were quantitated. Figure 2B shows the percentage of monomer, dimer and trimer which remained in the non-complexed, free DNA form upon incubation with various amounts of protein. It can be seen that the GRE trimer is shifted to a high molecular weight form nearly quantitatively at a protein and GR concentration, which leaves about 30% of the dimer and 90% of the monomer unshifted. This experiment confirms the increased affinity of the multimerized GRE and suggests that the differential affinity of the GR might be the basis for the differences in the functional properties. Since the gel retardation assays were carried out with whole cell extracts, it was important to ascertain the specificity of the observed DNA-protein interactions, i.e. it was necessary to show that the GR was

al.

indeed responsible for the complex formation. For this purpose three additional sources of GR were mobilized: (1) CV-1 cells contain very little CR. These cells were transfected with a plasmid encoding the rat GR [36] and extracts from control and transfected cells were compared in their ability to form GRE complexes. (2) GR was synthesized in vitro using a transcription-translation system. (3) A GR receptor fragment comprising the DNA binding and hormone binding domains was expressed in bacteria [16]. These receptor preparations or cell extracts containing GR were reacted with GRE monomer, dimer and trimer and in all cases a preferential complex formation with the oligomers was observed. In an additional control experiment no specific interactions with the estrogen response element (ERE) was found [15]. These experiments strongly suggest that the observed gel shifts are really due to GRE-GR interactions and that GRE oligomerisation might play a functional role in the regulation of receptor binding affinity. The concept that cooperative binding of receptors contributes to the strength of inducible enhancer elements was confirmed in studies with single and tandemly linked GREjPRE sequences. First, it was shown that two molecules of receptor bind to a single response element [32, 371. Second, a comparison of the progesterone inducibility of the tk promoter linked to a single and a duplicated GREjPRE showed a strong functional synergism of the duplicated sequences. Binding studies indicated that the association of one GREjPRE site with the PR increased the binding affinity of the second site for PR about loo-fold. This cooperativity in binding could explain the synergism in transcriptional strength [38]. Glucocorticoid receptor concentration regulated upon extended ligand exposure

is

down -

The molecular cloning of GR cDNA, its integration into a eukaryotic expression vector and transfer into cultured cells allowed the experimental manipulation of GR levels in vivo [39]. The presence of hormone-responsive reporter genes in GR cDNAtransfected rat hepatoma cells allowed the measurement of gene induction at different GR levels. Three responsive genes, the MMTV proviral gene, the tyrosine aminotransferase (TAT) gene and the a-l acid glycoprotein (APG) gene, were tested. For all three genes the GR concentration seems to limit the magnitude of the hormone response, i.e. transfected cells with a higher GR expression level also show a stronger inducibility [39]. We have determined the transcriptional rate of the MMTV LTR in NIH 3T3 cells as a function of time after hormone addition to the culture medium and observed a transient induction of the transcriptional rate [40]. Approximately 30 min after hormone addition the maximal transcription rate was reached. Thereafter a steady decrease in the transcriptional

Regulation

0

0.5

3

8

DEX treatment

of glucocorticoid

24

(h)

Fig. 3. Ligand dependent downregulation of cellular glucocorticoid receptors. NIH 3T3 cells were grown in DMEM and 10% fetal calf serum. Dexamethasone was added at time O-lo-‘M to the growth medium and total cellular

protein was extracted 0, 0.5, 3, 8 and 24 h after hormone addition. The GR concentration was determined by the immunoblotting procedure described in the legend to Fig. 1. The autoradiographic signals were quantitated by densitometry and the GR level observed at time 0 was used as a 100%. rate was observed and only about 50% of the maximal rate was achieved 24 h after hormone addition. We have investigated the possibility that a cellular regulation of the GR levels contributes to the temporal sequence of events, i.e. that the GR as the rate limiting component in the gene induction pathway is altered in its concentration and thus causes a maximal initial and a lower sustained response to hormonal stimulation. For this purpose we used a highly reactive and specific antiserum which recognized GR from the cytoplasm and the nuclei and which can be used to quantitate GR levels in protein blot experiments [14]. NIH 3T3 cells were treated with dexamethasone for increasing times and total cellular protein was extracted. The proteins were electrophoretically separated, transferred to a nitrocellulose membrane and reacted with the specific GR antiserum and iodine labeled protein A. The autoradiographs were quantitated by laser scanning densitometry and GR levels were plotted as a function of time (Fig. 3). It becomes apparent that GR levels decrease over a period of 24 h. Only about 30% of the maximal levels observed at time 0 remain after 24 h of hormone treatment. The extraction of total cellular protein from hormonally induced NIH 3T3 cells and GR quantitation with the specific antiserum clearly distinguishes between two possible molecular mechanisms of regulation of GR activity. The decline of GR levels seen in Fig. 3 indicates that the receptor is degraded upon nuclear translocation. Alternative considerations such as the loss of hormone binding ability due to secondary modification or sequestration of the receptor into an “inactive” form into a subcellular compartment, can be ruled out [14]. We also found that down-regulation of GR is enhanced by oncogene expression. The presence of the activated Ha-ras or v-mos oncoproteins and the simultaneous activation of the GR results in a very low level of GR after 24 h of hormone induction. This enhanced down-regulation is reflected by a low expression of glucocorticoidresponsive genes [16, 35,401.

receptor

activity

75

We have investigated the contributions of a decrease in receptor synthesis and an increase in receptor degradation to the down-regulation of the receptor levels and have found that both mechanisms participate [14]. GR mRNA levels were rapidly reduced upon hormone induction of NIH 3T3 cells, resulting in a decreased rate of receptor synthesis. The half-life of the receptor protein was decreased from 8 to 3 h, resulting in an increased rate of degradation. These results confirm observations in other cell types and tissues [4144]. Our results on the change in rate of the utilization of the MMTV LTR promoter and the change in cellular GR concentration indicate that the extent of hormone inducibility is regulated via the GR concentration. An initial strong inducibility mediated by high cytoplasmic GR levels declines over a period of 24 h. This “immediate early response” might allow induction of “downstream” cellular genes required for maintenance of the physiological action of the hormone. The down-regulation of the receptor allows a transient response to increased hormone levels even in the continued presence of the ligand.

The glucocorticoid hormone has a role in the tight nuclear binding of the receptor The conventional model proposed for the action of steroid hormones involves the binding of the steroid ligand to the “inactive” cytoplasmic receptor. This binding causes activation or transformation of the receptor, its nuclear translocation and its tight nuclear binding. The model, initially based on observations using radioactive steroids [45], has been basically confirmed with more modern methods. Genomic footprinting studies of the tyrosine aminotransferase gene promoter have shown that the hormone response element is only occupied by the GR complex after hormone administration [46]. These in vivo studies have been complemented by in vitro binding experiments. Partially purified, hormone-free GR binds very specifically to the HRE [47; R. J. and F. M., unpublished observations]. These differences indicate that there is an important level of regulation which can be circumvented in vitro. It has been speculated that the association of the GR in the cytoplasm with the 90 kDa heat shock protein gives rise to an ohgomeric complex which prevents the nuclear translocation [48,49]. The GR-hormone complex formation might cause the dissociation of this complex and activate the nuclear translocation signal [50]. If this model is correct, one may assume that the principal role of the hormone lies in the initiation of the cascade of events leading to nuclear translocation and transcriptional transactivation. The specific binding ability of bacterially expressed or in vitro transcribed and translated receptors supports the concept that the hormonal ligand is not necessarily required for the GR-DNA

WOLFGANG H&K et al.

76

z ‘““r

m

Cytoplosmic GR

@

Nuclaor

GR

experiment demonstrates that the role of the hormone goes beyond the initiation step, i.e. unmasking the DNA binding domain from the hsp90 complex. The maintenance of tight nuclear binding is dependent upon the continuous presence of hormone. Outlook

DEX

treatment

and withdrawal

(h 1

Fig. 4. Subcellular distribution of glucocorticoid receptors after hormone treatment and hormone withdrawal. NIH 3T3 cells were grown in DMEM and 10% fetal calf serum. Dexamethasone was added at time 0 to 10m6M of the growth medium. After 1 h of hormone treatment the cells were washed twice with prewarmed growth medium without hormone and incubated for 1, 2 and 3 h in hormone-free medium. The cells were harvested at the times indicated and suspended in hypotonic buffer on ice [14] and homogenized

with a Potter homogenizer. Nuclei were pelleted and re-extracted in hypertonic buffer containing 0.5 M NaCl. The cytoplasmic and nuclear proteins were separated by gel electrophoresis and blotted onto nitrocellulose filters. The GR content was visualized by the immunoblot procedure and then quantitated from the autoradiograms by densitometry. The distribution of nuclear vs cytoplasmic receptor is shown. interaction [ 15,241. However, differences in the kinetics of binding of GR and progesterone receptor (PR) to DNA have been observed when hormone-free and hormone-complexed receptors were compared [.51]. Although both receptors recognize preferentially the HRE of the MMTV LTR in the absence of hormone, hormone addition accelerates the on-and-off rates of receptor binding to DNA. We have carried out shortterm hormone treatment and withdrawal experiments on NIH 3T3 cells and combined them with GR quantitations in the cytoplasmic and nuclear fractions to determine if the nuclear translocation step is irreversible and if the presence of hormone is required to maintain the nuclear association. The treatment times were chosen so as to distinguish between receptor relocalisation and de nova synthesis. For this purpose cells were treated for 0 or 1 h with dexamethasone and subsequently hormone was removed and the cells were further cultured for 1, 2 and 3 h in hormone-free medium. Cytoplasmic and nuclear fractions were prepared and GR levels were quantitated by the protein blot procedure (Fig. 4). In this experiment a relatively high percentage (20%) was found in the nucleus in the absence of added hormone. This might be due to residual glucocorticoids in the fetal calf serum or an imperfect subcellular fractionation. One hour after hormone addition about 70% of the GR was found in the nucleus. Withdrawal for 1, 2 and 3 h caused a decrease of nuclear receptor and an increase of cytoplasmic receptor. Since the half-life of the receptor is about 8 h in the absence of hormone, the accumulation of GR in the cytoplasm upon hormone withdrawal is most likely not due to de nova synthesis, but represents an outflux of nuclear receptor. This

The rapid and dramatic advances in the molecular description of steroid hormone action have provided us with a detailed view of receptor structure and DNA response elements. The domain structure of the receptor has been clearly described and the specificity of DNA-protein interactions has been elucidated. A few of the open questions should be mentioned. (1) Although receptor-DNA interactions have been observed in vitro and in vivo, it is not yet clear what constitutes a “productive” interaction. DNA binding to the HRE seems to be a necessary but not a sufficient requirement for transactivation. The facts that the ER can bind to the GRE, the thyroid hormone receptor to the ERE [52] and the steroid hormone-free GR to the GRE [15,_51] might all be reflections of the same requirement for transactivation not met in the above examples. (2) Protein phosphorylation is an important regulatory mechanism for protein activity. The multitude of events and interactions following the activation of cytoplasmic steroid receptors by ligand makes it extremely difficult to assign a particular function to the increased phosphorylation which we observed upon nuclear translocation [14]. Possible aspects of the receptor biology involved could include the nuclear translocation process, the interaction of receptor with auxilliary transcription factors or the down-regulation process. The localisation of the phosphorylation sites, amino acid specific mutagenesis and a precise description of the mutated receptor with respect to all aspects of its life cycle might bestow reality upon these possibilities. (3) The regulation of receptor concentration reflects the dynamic aspects of gene regulation by steroid hormones. Early and sustained responses to hormones become possible, i.e. direct effects and effects on gene expression mediated via the involvement of “downstream” cellular genes can be envisaged. It will be interesting to investigate the cell and tissue specificity of the downregulation process, especially since the differential expression of hormone receptors has been shown to determine the cell type specific effects [53]. (4) Finally, the role of the hormone ligand remains to be defined in its entirety in the transactivation process. Conventionally accepted as the “initiator” of the activation or receptor transformation step, its role seems to extend beyond the unmasking of the nuclear translocation signal and the DNA binding domain. The necessity for the presence of hormone to retain the nuclear location of the receptor might indicate that a hormone occupancy masks a cytoplasmic localisation signal, i.e. that a negative signal for action is counterbalanced by hormone binding. These and many other

Regulation potential out shortly bination

interpretations

will undoubtedly

by the potent and

gene

methods

of glucocorticoid be sorted

of in vitro recom-

transfer. REFERENCES

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