Cyclic AMP-dependent Protein Kinase Promotes Glucocorticoid

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May 13, 1985 - We have exploited this behavior to ask if cyclic AMP- dependent protein kinase plays a role in regulating glucocorticoid receptor function.
Vol. 261, No. 11, Issue of April 15, pp. 49094914,1986 Printed in U.S.A.

THEJOURNALOF BIOLOGICAL CHEMISTRY 0 1986 by The American Society of Biological Chemists, Inc.

Cyclic AMP-dependent Protein Kinase Promotes Glucocorticoid Receptor Function* (Received for publication, May 13,1985, and in revised form, November 13,1985)

Donald J. Gruol, N. Faith Campbell, and Suzanne Bourgeois From The Salk Institute for Biological Studies, Regulatory Bwbgy Laboratory, Sun Diego, California 92138

Murine lymphomacell lines such as WEHI-7 exhibit cocorticoid receptor) play the pivotal role in their respective a cytolytic response to both cAMP andglucocorticoids. signal's transduction and are responsible for regulating the We have exploited this behavior to ask if cyclic AMP- subsequent "downstreap" effects. Thus, the failure to detect dependent protein kinase plays a role in regulating cross-resistance (10) does not rule out thepossibility that one glucocorticoid receptor function. We have found that signaling system could have effects on specific aspects of the CAMP-resistant cell lines containing a defective other. CAMP-dependent protein kinase activity give rise to In a number of instances, dexamethasone has been shown spontaneous steroid-resistant variants at a high fre- to alter cAMP regulation through effects on the levels of quency(approximately relative towildtype cells adenylate cyclase activity (13, 14), cAMP phosphodiesterase (less than 10-l'). Unlike previous results with wild activity (19, and autophosphorylation of the regulatory subtype cells, nearly complete loss of glucocorticoid receptor function was observed in a single selection using unit of the CAMP-dependentprotein kinase (16). On the other unmutagenized CAMP' derivatives of WEHI-7. Thus, hand, the glucocorticoid receptor has recently been shown to the initial selection of theCAMP' phenotypeserves as be a phosphoprotein (17-19) which may also be a substrate a permissive step toward the acquisition of glucocor- for CAMP-dependent protein kinase (20). Our approach has ticoid resistance in WEHI-7. In addition, cAMP was been to ask if murine lymphoma cells containing defective found to increase thelevels of steroid binding in these kinase activity would lead to variants with an additional cell lines, and the dose response was dependent upon defect in steroid responsiveness. We have chosen the WEHIthe phenotype of the cyclic AMP-dependent protein 7 (W7) cell line since it contains two functional glucocorticoid kinase. The results demonstrate an important role forreceptor alleles and will not yield totally dexamethasoneCAMP in regulating glucocorticoid receptor activity resistant variants due to loss of a single receptor gene as does and strongly suggest thatthis novel two-step selection the S49 cell line (21). Spontaneous loss of both W7 functional scheme leadsto the isolationof new forms of glucocor-receptor alleles does not occur at a measurable frequency. ticoid resistance. Thus, we have sought and identified a new class ofdex', variants from a CAMP'population of W7 cells. The decreased levels of steroid binding that we have observed inthe cAMP'dex' lines indicate that we have found a new basis for Lymphoid cell lines which exhibit a cytolytic response to steroid resistance possibly involving alterations in functions both cyclic AMP and glucocorticoids offer a unique opportu- which regulate receptor activity rather thanmutations in the nity to select resistant variantsthat reflect mutations ingenes receptor structural gene itself. involved in hormone regulation. Tomkinsand co-workers MATERIALS AND METHODS (1-3) were among the firstto appreciate this approach and to develop it in the murine line S49. Since then, several other Cell Growth and Selection-WEHI-7.1 is a thymoma cell line lymphoma cell lines have been utilized to great advantage (4- obtained from a female BALB/c mouse after x-irradiation (22). 9). This has led to the isolation of a considerable number of Derivatives of this line which are resistant to thioguanine (W7TG) cell lines resistant to either cAMP or the synthetic glucocor- or bromodeoxyuridine (W7TB) were used in all of these experiments ticoid dexamethasone (dexl). These studies have proceeded (21). The cells weregrown as previously described (8), and cell viability was evaluated using trypan blue exclusion. The selection for largely independent of one another since resistance to one cAMP resistance was accomplished by plating the cells (2 X 106/ml) agent was not found to convey resistance to the other (10). in multiwell dishes in medium containing 100 p~ dibutyryl cAMP The resistance to dibutyryl cyclic AMP (Bt2cAMP) hasbeen and 75 p~ methylisobutylxanthine. In a series of such experiments, shown to be predominantly associated with alterationsin the CAMP' phenotype was obtained at a frequency of1.5 X The cyclic AMP-dependent protein kinase function (11). Simi- individual colonies were picked, grown, and cloned by limiting dilularly, single-step selection for glucocorticoid resistance in S49 tion. Cyclic AMP-resistant derivatives of W7TG and W7TBare CXG-n and C1X-n, respectively. The CAMP' phenotypes yielded variants with altered steroid receptor function (12). designated have been stable for more than 2 years. The second step, a selection The basis for the parallelism resides in the fact that both for dex', was carried out in a similar fashion. The cells were plated in functional entities (CAMP-dependentprotein kinase and gluM dex, and theresulting colonies were picked and subcloned. In * This work was supported by Grant CA36146 from the National Institutes of Health anda grant from the Elsa U. Pardee Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This articlemusttherefore be hereby marked ''advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. The abbreviations used are: dex, dexamethasone; BtcAMP, dibutyryl cyclic AMP; W7, WEHI-7.

four separate selections using the cell lines CXG-56, CXG-79, C1X46, and ClX-49 with a total of 9.9 X lo7 cells, 19 dex' variants were obtained for a frequency of 1.9 X Steroid Binding and Nuclear Translocation-Titration of the glucocorticoid-binding sites was carried out on whole cells as described by Pfahl et al. (23). Analysis of the data was made according to Scatchard (24). Single-point estimates of relative steroid binding were carried out by incubating cells with 3 X lo-' M [3H]triamcinolone acetonide for 1h a t 37 "C.The cells were then washed free of unbound

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hormone as described by Pfahl et ul. (23). Nuclear translocation of the receptor was evaluated as described by Gruol et ul. (25). Cyclic AMP-dependent Protein Kinase Activity-Extracts of the wild type and variant cell lines were prepared as described by Lemaire and Coffino (26). The capacity of the extract to transfer 32Pfrom [y32P]ATP to histones was measured as described by Roskoski (27). Each point was carried out in duplicate, and the datashown in Fig. 2 represent the average of two separate experiments. Reagents-N6,O2'-Dibutyryl cyclic AMP, dexamethasone, 8-bromo cyclic AMP, and methylisobutylxanthine were all purchased from Sigma. Tritiated dexamethasone and triamcinolone acetonide were purchased from Amersham Corp.

gene alleles occurs at a frequency less than 10-l'. Thus, the results suggest that theinitial selection of CAMP' contributes to theselection of a new form of dex' variants. Fig. 2 shows the results of an in vitro assay of the CAMPdependent protein kinase activity found in thecell lines from Fig. 1. For convenience, the data areexpressed as per cent of maximum activity since the measured levels of total activity did not differ between these cell lines. CXG-79, the CAMP' derivative of wild type W7, had a shifted CAMP-dependent protein kinase activity which presumably reflects an alteration in the affinity of the regulatory subunit for cAMP (11). CXG-79D1, the cAMP'dex' variant, containsa kinase activity RESULTS identical to the activity observed in CXG-79, shifted 2-fold The synthetic glucocorticoid dexamethasone elicits a cyto- from the wild type. Thus, the first step in the two-stage toxic response in wild type W7 cells as shown in Fig. 1. In the selection produced the expected phenotype, a cell line with absence of dex, the cells grow with a doubling time of approx- an altered CAMP-dependent protein kinase activity. The secimately 12 h, whereas in the presence of saturating levels of ond step (selection in dex) did not produce any further apthe drug, there is an initial increase in number followed by a parent modification in enzyme activity and suggests that the loss of viability over a period of several days. Derivatives of change which results inthe dex' component of the phenotype the wild type W7 line which were selected for resistance to resides in a function other than theCAMP-dependentprotein dibutyryl cAMP remain sensitive to dex, which confirms an kinase. To test thisfurther, we have used very high levels of observation made by Gehring and Coffino (10) with the S49 BbcAMP (1 mM) to select for variants from CXG-79 with lymphoma line. One example of a CAMP'derivative of W7TG, more resistance to cAMP and even greater shifts in their CXG-79, is illustrated by the open circles in Fig. 1.There was enzyme activity. None of these doubly selected lines showed a slight delay in the killing which did notappear to be a significant increased resistance to dex (not shown). significant, but which encouraged us to ask if further dex Previous studies with dex' variants of the W7 and S49 cell resistance could be obtained from this and similar lines. lines had shown that resistance to steroid was accompanied Unmutagenized cultures of CAMP' cells were grown up and by a decrease in glucocorticoid receptor function (28). Lower plated into multiwell dishes in medium containing M dex levels of hormone binding along with an altered capacity to from which resistant derivatives were obtained at a frequency translocate receptor to the nucleus were indicative of mutaof approximately The growth of one of the cAMP'dex' tionsin the receptor structural gene.We have measured variants (CXG-79D1) in M dex is shown by the closed steroid binding in the cell lines described above, and one set triangles in Fig. 1. The cell line is refractory to killing by the of the results in the form of a Scatchard analysis is shown steroid. This result is unique in thatselection of spontaneous in Fig. 3. The closed circles represent the wild type W7 line resistance to dex from wild type (relative to steroid sensitivity) and indicate 26,000 binding sites/cell and a dissociation conW7TG in a single step has notbeen observed previously (28). tent of 1.1X lo-' M.This is very similar to theresults obtained Acquisition of steroid resistance by mutations in both receptor previously with the wild type W7 lines. CXG-79, the CAMPdex" derivative, contains only slightly fewer binding sites (22,000/cell), consistent with its continued sensitivity to dexamethasone. CXG-79D1, the CAMP' dex' variant, was found to have a greatly reduced capacity to bind hormone. These loo\ data indicate 7,500 binding sites/cell. Table I lists a summary of the results of steroid binding studies carried out with the wild type and several of the variant cell lines that we have

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FIG.1. Effect of dexamethasoneon WEHI-7 and two CAMPdependent derivatives. Cell cultures were plated out (5 X 104/ml) in medium containing 1O"j M dexamethasone and incubated for the times indicated. The number of living cells was determined by trypan exclusion. The closed circles represent the wild type W7 grown in the absence of dexamethasone, whereas the open triangles (W7), the open circles (CXG-79), and theclosed triangles (CXG-79D1) contained the steroid.

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FIG.2. Dose responseof CAMP-dependent protein kinase in extracts of wild type W7, CXG-79, and CXG-79D1. Cytosol samples were prepared and incubated with increasing concentrations of cAMP in the presence of [32P]ATPand histones. The levels of incorporation obtained in incubations without cAMP have been subtracted. The data have been normalized to themaximum of the wild type activity, and each curve represents the average two experiments. 0,W7; 0, CXG-79; A, CXG-79D1.

CAMPRegulation of the Glucocorticoid Receptor

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tors/cell. These values as well as the dissociation constants (Kd) were determined by Scatchard analysis. None of the Stage I variants shows a statistically significant change in the number or affinity of the steroid-binding sites. Thus, even the complete loss of CAMP-dependentprotein kinase activity (as in CXG-56) did not measurably affect glucocorticoid binding in these cells. Similarly, the receptor's ability to translocate to the nucleus was not appreciably altered in the Stage I variants. These data areconsistent with the continued steroid sensitivity observed in the Stage I variants (see Fig. 1) and demonstrate that CAMP-dependentprotein kinase activity is not absolutely required for glucocorticoid receptor function. The steroid-resistant Stage I1 variants, on the other hand, clearly show loss of receptor function. ClX-46Dl contains 0 5 10 15 20 less than 5000 binding sites anda greatly diminished capacity Bound Hormone (10"'M 1 of the receptor to translocate to the nucleus (14.7 versus FIG.3. Scatchard analysis of dexamethasone binding to wild type W7 and two CAMP-resistant derivatives. The whole 48.7%). Thus, ina single step andwithout mutagenesis, there cell binding of [3H]dexamethasone to cultures of W7TG (O),CXG- was an almost complete loss in receptor function. So, the 79 (0),and CXG-79D1 (A)was carried out as described by Pfahl et change which leads to steroid resistance must be effecting the al. (23). expression of functional receptor from both receptor structural gene alleles. The observation that a defective CAMPobtained. Cyclic AMP-resistant derivatives of W7TG and dependent proteinkinase activity can lead to a loss of receptor W7TB (see "Materials and Methods") are designated CXG-n function is confirmed by the datafrom CXG-56D2 and CXGand C1X-n, respectively. This phenotype (cAMPdex*)will be 79D1 which also have a large (more than half) reduction in referred to as a Stage I variant. The combined CAMP-dexa- binding and a partial decrease in nuclear translocation. C1Xmethasone-resistant phenotype (cAMP'dex') is called Stage 49D1 provides a valuable comparison, since it appears to I1 and contains the additional letter D to indicate steroid contain approximately half the number of binding sites and resistance. Thus, CXG-79 is a Stage I (cAMP'dex") derivative little change in nuclear translocation. However, the dex' comof W7TG and cXG-79D1 is a Stage I1 derivative (cAMP'dex') ponent of the mixed phenotype is unlikely to be the result of of CXG-79. The four Stage I variants listed in Table I (CXG- a simple loss of one receptor allele (r+/r+ to r+/r-) since such 56, CXG-79, C1X-46, C1X-49) all contain an altered CAMP- cell lines retaintheir sensitivity to dexamethasone (21), dependent protein kinase activity. As shown in Fig. 4, CXG- whereas ClX-49D1 is resistant. Therefore, in conjunction 46 is similar to CXG-79 in that it exhibits an increase (ap- with the changes in binding and translocation, the receptors proximately &fold) in the dose response to CAMP. C1X-49 lose the capacity to efficiently elicit a lytic response. The results show that the initial selection of the CAMP' also gives a similar pattern (data notshown). Thus, they are K,-type CAMP'variants. CXG-56 (open squares), on the other phenotype acts as a permissive step toward the selection of a hand, contains no measurable enzyme activity and is desig- form of steroid resistance which exhibits a concomitant loss nated as a kin--type variant (29) similar to those reported in in steroid binding function. The degree of the overall loss of the S49 cell line. The kin- phenotype of CXG-56 is also binding (in CXG-56D2, CXG-79D1,and ClX-46D1) is indicdominantly expressed in hybrids formed by cell fusion (not ative of more than the loss of a single functional receptor allele. Thus, thedefect(s) in theStage I1 variants may involve shown). Table I lists the dex binding capacity expressed as recep- functions which regulate receptor levels or activate the recepTABLE I Summary of steroid binding data from wild type, Stage I. and Stage II variants Cell Iine

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ClX-49 cAMP'dex" 21,700 t 4,800 1.47 f 0.48 (n= 4) 39.6 t 6.6 (n = 5 ) K, C1X-49D1 cAMP'dex' 13,700 f 1,900 1.82 t 0.87 (n = 3) 41.8 f 3.0 ( n = 3) K. Mean f S.D. kin- variants lack CAMP-dependent protein kinase activity. Ka variants have an increased dose response to CAMP. E Taken from Bourgeois and Newby (21).

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TABLE I1 Glucocorticoid binding to cells treated with CAMP-elevating drugs Cultures of CXG-79 and CXG-79Dl were grown for 18 h in the presence of derivatives of cAMP or the phosphodiesterase inhibitor methylisobutylxanthine (MIX). The cells were then incubated with 3 X lo-' M tritiated triamcinolone acetonide for 1h. Parallel cultures were also incubated with an excess of unlabeled steroid. The cells were washed four times in cold phosphate-buffered saline, and the specifically retained hormone was measured in a scintillationcounter. The results are the average S.E. of three separate experiments. Addition

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tor to itssteroid-binding form. If so, then theresults indicate that CAMP-dependent protein kinase plays a role in this process. To test if increased levels of cAMP could affect steroid binding in these cells, cultures of both CXG-79 and its dex' derivative CXG-79D1 were incubated overnight in the presence and absence of the phosphodiesterase inhibitor methylisobutylxanthine and two forms of CAMP. The glucocorticoid binding capacity was then measured using a saturating concentration of tritiated triamcinolone acetonide, and the results are shown in Table 11. Dibutyryl and 8-bromo cAMP were found to increase the levels of binding in thecAMP'dex' variant CXG-79D1 by up to 2.08-fold. The phosphodiesterase inhibitor methylisobutylxanthine also had a 1.56-fold effect on thelevel of bound hormone. With the cAMP'dex" variant CXG-79, all three conditions again raised the binding capacity above the control (upto 1.56-fold). Similar studiescarried out with the kin- variant CXG-56 found no increase inthe binding capacity (see below). Thus, the increase in binding illustrated in Table 11 appears dependent upon the presence of cyclic AMP-dependent protein kinase. We have extended these studies by using the drug forskolin which has been shown to activate adenylate cyclase activity and to result in increased levels of intracellular cAMP (30, 31). Fig. 5 shows the effect of forskolin on steroid binding

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FIG.6. Scatchard analysis of dexamethasone binding to CXG-79. Cell cultures were incubated for 18h in the presence (0) or absence (0)of20 p~ forskolin. After the incubation, samples of the cells were incubated at 37 "C for 2.5 h with increasing concentrations of [3H]triamcinolone acetonide. The retained hormone was measured after three washes in cold (0 "C) phosphate-buffered saline. The nonspecific binding was determined by samples containing an additional 200-fold excess of unlabeled hormone. Each point was carried out in duplicate. The data were normalized to reflect binding to 10'' cells. levels as a function of time. The closed circles represent wild type W7 and are theresult of four independent experiments. After an apparent lag of 60 min, forskolin caused a %fold increase in glucocorticoid binding over the following 2 h. Overnight incubation (18 h) resulted in an increase of 2.4-fold and was accompanied by a decrease in viability (to approximately 60%). Similar results have been obtained with the Stage I1 variant CXG-79D1, but without the large loss of viability after 18 h (not shown). The results of a single experiment with the kin- variant CXG-56 are shown by the open circles. There was no increase in steroid binding (even after 18 h), which illustrates the dependence upon CAMP-dependent protein kinase for promoting the change. The effect of forskolin on the binding can be further characterized by Scatchard analysis, as is shown in Fig. 6. CXG-79 cells were grown overnight in thepresence of forskolin, and thebinding sites were titrated by incubations with [3H]triamcinolone acetonide. In two such experiments, forskolin caused an av-

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erage 1.8-fold increase in total binding sites, from 22,500 to DISCUSSION 40,40O/cell. The dissociation constants remained essentially The results presented here demonstrate that: 1)a decrease the same, 3.62 X lo-' and 4.61 X lo-' M, respectively. of CAMP-dependentprotein kinase activity acts as a permisThe data presented above indicate that CAMP-dependent sive step toward the acquisition of glucocorticoid resistance protein kinase may play a role in regulating the capacity of in unmutagenized cultures of the murinelymphomaline murine lymphoma cellsto bind glucocorticoid hormone.If so, WEHI-7, 2) nearly complete loss of glucocorticoid receptor then theobserved increase in binding should be dose-depend- function can be obtained in a single-stepselectionfrom ent over cAMP concentrations that are found to affect the CAMP' variants ofW7; and 3) exogenouslyaddedcAMP proliferation of these cells. K-type variants require higher caused an increased level of steroid binding in the murine concentrations to induce a lytic response due to a reduced lymphomaW7 andits derivativeswhich contain CAMPaffinity of enzyme for cAMP (3). Therefore, it is reasonable dependent protein kinase activity. to expect that higher concentrations of cAMP would be reThe cytolytic response elicitedby CAMPin WEHI-7 (and quired to change steroid binding in Stage I variants than in S49) had previouslybeenused in the selection of CAMP' the wild type cells. This is shown in Fig. 7. With wild type variants containing altered CAMP-dependent protein kinase cells, dibutyryl cAMPwas found to cause a 2-fold increasein activity. The mostcommonly obtained form has a shifted steroid binding over a range of 0-100 p ~ which , is the same dose response to CAMP, as is illustrated in Fig. 2. Similar range of concentration which results in arrest of cell growth variants have been shownto contain alterations in the type I and loss of viability (seeFig. 8). In theK.-type variant CXG- enzyme regulatory subunit (32). We have tested a series of 79, Bt2cAMP caused a similar increase in binding, but over such CAMP' variants ofW7 for their ability to generate much higher concentrations. As shown in Fig.8, the dose spontaneous steroid-resistant derivatives and found that a response of the lytic effect was shifted to a similar degree. majority (40 out of 49) are capable of doing so. The CAMP' Again, Figs. 7 and 8 illustrate that the kin- variant CXG-56 Stage I cell lines and their cAMP'dex' derivatives are examwas refractory to the changes promoted by CAMP. ples of the type of results that we have obtained. The spontaneous frequency with which the cAMP'dex' variants were 2.5 obtained (approximatelyfrom the CAMP' lines suggests an alteration in a single gene. Thus, one possibleexplanation a, C for the success of the multistep selection schemeis the existence of another activity which overlaps withor is redundant to CAMP-dependent protein kinase in affecting the receptor. Interestingly, there are two forms of this enzyme whichdiffer in their regulatory subunit (33), but we doubt that this form of redundancy is the basis of our findings since CAMP' W7 lines such as CXG-56 totally lacking both forms of CAMPdependent protein kinase (kin-) were also found to possess CXG-56 normal levels of receptor and steroid sensitivity. 0 The severe lossin steroid binding capacity observedin the I I I I Stage I1 variants has several possible explanations, none of 0 1 2 3 4 5 6 which can be excluded at this time. These include: 1)loss of dbcAMP Concentration (IOe4M) receptor gene expression at the transcriptional or translaFIG. 7. Dose response to dibutyrylcAMP (dbcA1MP)of stetional level, 2) loss of a cellular function that converts the roid binding. Cell cultures were incubated with increasing concentrations of BbcAMP for 18 h. The cells were collected and resus- receptor from an inactive (steroid non-binding) to an active pended in media containing 3 X 10%M [3H]triamcinolone acetonide. (steroid binding) form, 3) a dominantly expressed receptor The amount of specifically bound hormone was determined as for structural gene mutation, and 4) alteration of a hitherto Fig. 5 . Each pointwas carried out induplicate, and thedata represent unidentifiedreceptorcomponent. In regard to the second the average value of four independent experimentsfor each cell line. possibility, Pratt and co-workers have provided evidence that 0, W7TG; A, CXG-79; 0, CXG-56. the receptor is phosphorylated (17) and that conversion to an inactive form may be the result of phosphatase activity (34). CXG-56 n Furthermore, they have shownthat thioredoxin also playsan n n n 0 important role in maintaining the receptor in a state which is required forsteroid binding (35). There are several examples of cyclic nucleotides affecting the levels of steroid binding capacity. Consistent with our results, Oikarinen et al. (36) showed that cAMP stimulated glucocorticoid bindingby more than 2-fold in cultured human skin fibroblasts. Cyclic GMP was found to have a slight inhibitory effect, and the authors concluded that it was the ratio of cAMP to cGMP that provided the important regulatory parameter. In a similar vein, Fleminget al. (37,38) found cGMP to stimulate and cAMP to inhibit estradiol binding to 0 human endometrial cells. Both studies suggest that the level I I I 0 1 2 3 4 5 of steroid binding is responsive to proliferation and presumdbcAMP Concentration ( lOW4M) ably metabolic status of the cells. We have not been able to FIG. 8. Dose response to dibutyryl cAMP (dbcAMP) of cytolytic effect. Cell cultures were plated at lo3cells/ml and grown in detect an effect of either 8-bromo or dibutyrylcGMP on the the indicated concentrationsof BbcAMP for 10 days. The turbidities glucocorticoid binding levelsin the W7 cells. Our results demonstrate that CAMP-dependentprotein kiof the cultures were measured at 660 nm and expressed relative to cultures without BtzcAMP(8).0, W7TG; 0, CXG-56; A, CXG-79. nase plays an important, but apparently non-obligatory, role 1

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cAMP Regulation of the Glucocorticoid Receptor

in regulating the levels of functional glucocorticoid receptors in WEHI-7 cells. Loss ofCAMP-dependent protein kinase activity does not result in steroid resistance and, as shown with the Stage 1variants, did not led to a large lossin steroid binding capacity. On the other hand, increased levelsof CAMP produced an increased levelof binding in both the cAMP'dex' and cAMP'dexs lines.The studies carried out using forskolin detected changes within Or h*This observation was made with both wild type and K,-type variants. The rapid change rules out the possibility that the increase in bindingcapacity was dueto a CAMP-mediated cell cycle arrest in GI.It should also be noted that the increase inducedby CAMPappears to be relatively stable. That is, the presence of forskolin during the subsequent incubation with steroid made little difference in the results of experiments such as the Scatchard analysis shown in Fig. 6. This behavior andthe lack of absolute dependenceuponCAMP-dependent protein kinase (as in CXG-56) for steroid binding may implya redundant role for the enzyme in a putative receptor conversion process. H ~ ever, as shown in theS49 cell line, several substrates of CAMPdependent Protein kinase are PhosPhodated only as n ~ ~ chains (39). It will be interesting to see if the glucocorticoid receptor falls into this category. Alternatively, cAMP may regulate the thioredoxin system which has been shown to activate glucOcOrticOid receptors their steroid-binding form (35). Acknowledgments-We wish to thank Matthew Ashby for technical assistance and Gloria Laky Swart for typing the manuscript.

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