Glucocorticoid-Regulated Stability of a Constitutively ...

Glucocorticoid-Regulated Stability of a Constitutively Expressed Mouse Mammary Tumor Virus Glycoprotein

Laurie J. Goodman* and Gary L. Firestone

Department of Molecular and Cell Biology The Cancer Research Laboratory University of California Berkeley, California 94720

Glucocorticoids regulate the transport and processing of mouse mammary tumor virus (MMTV) glycoproteins in viral-infected HTC rat hepatoma cells. To begin to determine the role of cellular components involved in this steroid-mediated response, a constitutively expressed MMTV glycoprotein gene containing a mutation in the endoproteolytic cleavage site was used to simplify the viral maturation products. Expression of the uncleavable MMTV glycoprotein gene in transfected HTC rat hepatoma cells demonstrated that treatment with the synthetic glucocorticoid dexamethasone resulted in a 5fold increase in the steady state level of the intracellular and cell surface MMTV glycoproteins. Under these conditions, dexamethasone did not alter the level of MMTV glycoprotein transcripts. Pulse-chase radiolabeling with [%]methionine demonstrated that dexamethasone did not affect the apparent rate of MMTV glycoprotein translation, and an analysis of oligosaccharide side-chain structure by endoglyco- sidase-H digestion revealed that glucocorticoids did not alter the 45min endoplasmic reticulum to Golgi transit time. Pulse-chase kinetic analysis of 4-h pulse-labeled cells revealed that the half-life of the mature glycosylated MMTV polyprotein, gp78, was 105 min in glucocorticoid-treated cells and 45 min in untreated cells. Taken together, our results suggest that glucocorticoids increase the stability of MMTV glycoproteins by a posttranslational mechanism and that this effect may be occurring relatively early in the exocytic pathway. (Molecular Endocrinology 7: 94-103,1993)

INTRODUCTION

Glucocorticoids as well as other steroid hormones elicit a broad spectrum of characteristic responses in a variety of eukaryotic cell types (1, 2). It has been well documented that glucocorticoids, after binding to their

0e88-8809/93/0094-0103$03.00/0 Molecular Endocrmology CopyrIght 0 1993 by The Endocrine Soaety

cognate cytoplasmic receptors, can stimulate or re- press the transcription of glucocorticoid-regulated genes (l-6). The primary effect of glucocorticoids on gene transcription can occur either by specific binding of steroid-receptor complexes to DNA at glucocorticoid regulated enhancers (1, 2, 4, 5) or by interfering with the action of other transcription factors via direct protein-protein interactions (4, 7-9). In addition to these primary effects on gene transcription, glucocorticoids have been shown to elicit a variety of secondary modulatory effects. These effects may be mediated by the steroid-receptor complex regulating the transcription of genes whose protein products modulate other gene products at transcriptional, posttranscriptional, translational, or posttranslational levels. For example, glucocorticoids have been shown to stabilize mRNAs of certain proteins by a posttranscriptional mechanism (10, 11). Although the exact mechanism has not been identified, it has been postulated that steroid-inducible gene products are directly involved in altering transcript turnover (10). Similarly, glucocorticoids and other steroid hormones have been shown to regulate a variety of posttranslational responses (12-l 7). A biological advantage to such a modulatory response is the capability to rapidly turn on or off many functionally related sets of proteins by altering the expression of only a few steroid-regulated genes.

We have previously shown that glucocotticoids regulate the intracellular trafficking of the mouse mammary tumor virus (MMTV) envelope glycoproteins in the viral- infected rat hepatoma cell line Ml 54 (12-l 4, 16, 18). In the presence or absence of the synthetic glucocorticoid dexamethasone, the MMTV glycoprotein precursor is initially synthesized as a 70-kilodalton (kDa) en- doglycosidase-H (endo-H)-sensitive polyprotein (gp70). During transport through the exocytic pathway, the carbohydrate side-chains undergo modification in the Golgi to form a 78-kDa endo-H resistant, terminally sialated form of the precursor (gp78) (16, 19). This modified form of the precursor is proteolytically cleaved after the dibasic residues in the Arg-Ala-Lys-Arg endoproteolytic cleavage site into the amino-terminal 50-kDa (gp50) product and the carboxy-terminal 32-kDa (gp32) maturation product (20). Using an endoproteolytic site

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Steroid-Regulated Stabilization of Membrane Glycoproteins 95

mutant, we have recently shown that the cleavage reaction occurs in the trans-Golgi after the formation of an endo-H-resistant intermediate (20). In the absence of the glucocorticoids, the only detectable MMTV glycoproteins in Ml 54 cells are the intracellular form of gp70 and a low level of the secreted amino-terminal gp50 product. In the presence of dexamethasone, previously undetectable transmembrane forms of the MMTV glycoprotein (gp70, gp78, and gp32) can be detected at the cell surface (12-14, 18). Also, in glucocorticoid-treated cells, gp78 is proteolytically proc- essed at the plasma membrane, resulting in the exter- nalization of a terminally sialylated gp70 (19). This posttranslational response appears to be regulated only by steroids with glucocorticoid biological activity and requires a functional glucocorticoid receptor as well as de nova RNA and protein synthesis (18).

The precise mechanism involved in the glucocorticoid regulation of MMTV glycoprotein trafficking in Ml .54 cells has not been elucidated; however, our recent evidence implicates several factors in this regulation, including both cellular and viral components. For example, constitutive expression of the viral glycoprotein in the absence of all other viral components resulted in efficient transport of mature MMTV glycoproteins to the cell surface in the presence or absence of hormone (19, 21). Dexamethasone induced an approximately 2- fold increase in the level of detectable cell surface- associated gp78, with no apparent increase in the levels of total MMTV proteins (21). The magnitude of this response is much lower than that observed in glucocorticoid-treated Ml 54 cells, suggesting the possibility that other viral components may be involved in mediating this response. Consistent with this notion, disruption of intracellular MMTV glycoprotein transport or the expression of a truncated form of the MMTV glycoprotein revealed a functional interaction between the membrane-associated MMTV glycoprotein and the cytoplasmic viral phosphoprotein, which appears to be important for the proteolytic maturation of the gag polyprotein (22). To begin to understand the cellular components involved in the glucocorticoid-regulated transport of MMTV glycoproteins, we have used a constitutively expressed MMTV glycoprotein gene containing a mutation in the endoproteolytic cleavage site. Cleavage of this mutant glycoprotein to the maturation products gp50 and gp32 was almost completely abolished in transfected cells. An advantage of this mutation is that it simplified the maturation products so that the steroid effect on only the MMTV-glycosylated precursor could be directly characterized. Implications are made as to the potential contributions of cellular and viral components in mediating this glucocorticoid-induced posttranslational response.

RESULTS

Glucocorticoids Increase the Steady State Accumulation of Constitutively Expressed Wild- Type (WT) and Endoproteolytic Mutant MMTV Glycoproteins

To examine the effects of glucocorticoids on the accumulation of intracellular, cell surface, and secreted

MMTV glycoproteins, viral protein production was ana- lyzed in HTC rat hepatoma cells constitutively expressing the WT or a cleavage site mutant of the MMTV glycoprotein gene. Dexamethasone-treated and untreated [35S]methionine-labeled cells were first incubated with anti-MMTV antibodies before detergent sol- ubilization to immunoprecipitate the cell surface-associated MMTV glycoproteins. Anti-MMTV antibodies were then added to the residual solubilized fraction to immunoprecipitate the intracellular MMTV-specific glycoproteins. Electrophoretic fractionation revealed that in the presence or absence of dexamethasone, WT cells express four stable intracellular MMTV glycoproteins: 78- and 70-kDa polyproteins (gp78 and gp70), the amino-terminal 50-kDa maturation product (gp50), and the carboxy-terminal 32-kDa maturation product (gp32; Fig. 1). Glucocorticoids stimulate the steady state intracellular accumulation of the gp50 and gp32 maturation products by approximately 2-fold, with little effect on the amount of the gp78 and gp70 polyproteins (Fig. 1, lanes A vs. B). Consistent with our previous results, dexamethasone induced a 2-fold increase in cell surface-associated gp78 in WT cells (Fig. 1, lanes E vs. F). Previous experiments have established that we cannot detect gp32 at the cell surface easily due to the ectodomain being hidden by a noncovalent interaction with gp50 (19).

To simplify the overall pattern of viral maturation products, HTC cells were transfected with a MMTV glycoprotein gene containing a mutation in the endoproteolytic site so that the effects of glucocorticoid on the

-+ --ODEX - + - + CJEX - + - + DEX

WT P-7 WT P-7 WT P-7

Fig. 1. Effects of Glucocorticoids on the Steady State Levels of Cleavable and Uncleavable MMTV Glycoproteins

Monolayers of WT cells transfected with the WT MMTV glycoprotein gene (lanes designed WT) and P-7 cells transfected with the proteolytic mutant (lanes designated P-7) were incubated with (lanes B, D, F, H, J, and L) or without (lanes A, C, E, G, I, and K) 1 PM dexamethasone (DEX) for 16 h and radiolabeled with [35S]methionine for 4 h. The secreted (lanes I-L) MMTV glycoproteins were immunoprecipitated from the culture medium using MMTV antibodies. The cell surface (lanes E-H) and intracellular fractions (lanes A-D) were immunoprecipitated from the remaining cells, as described in the text. The immunoprecipitated MMTV proteins were electrophoretically fractionated in SDS-polyacrylamide gels, and the radioactive bands were visualized by fluorography. The protein bands designated by arrows are the glycosylated MMTV polyproteins (gp78 and gp70) and the proteolytic maturation products (gp50 and gp32).

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precursor polyprotein could be followed. As also shown in Fig. 1, the effect of dexamethasone on accumulation of the uncleavable MMTV glycoprotein is more en- hanced than that observed with the cleavable MMTV polyprotein. Dexamethasone treatment consistently stimulated a 5-fold increase in the combined level of intracellular gp78 and gp70 expressed in P-7 cells (Fig. 1, lanes C vs. D). The levels of gp78 in glucocorticoid- treated P-7 cells were much higher than those in WT cells, since the proteolytic cleavage is almost completely abolished in this mutant, and the effect of the hormone is not diluted out by maturation into gp50 and gp32. To determine if the increased levels of the MMTV glycoproteins in the presence of glucocorticoids re- flected an increased amount of total MMTV proteins transported through the exocytic pathway or, possibly, a specific retention of MMTV glycoproteins, the cell surface localization of MMTV glycoproteins was ana- lyzed in dexamethasone-treated and untreated P-7 cells. Consistent with the intracellular results, dexamethasone-treated P-7 cells produced approximately 5fold higher levels of cell surface gp78 and gp70 polyproteins (Fig. 1, lanes G vs. H). In the presence or absence of glucocorticoids, there was no discernable difference in the MMTV glycoprotein secretion products in either P-7 or WT cells (Fig. 1, lanes K vs. L and I vs. J). This result suggests that the increased level of cell surface MMTV glycoproteins observed in dexamethasone-treated cells is not due to inhibition of the exter- nalization of gp70 or gp50.

Treatment of WT and Endoproteolytic Site Mutant Cells with Glucocorticoids Has no Effect on the Steady State Accumulation of MMTV Glycoprotein Transcripts

The MMTV glycoprotein genes transfected into HTC cells are driven by the constitutive Rous sarcoma virus (RSV) promoter (23). To confirm that expression of MMTV transcripts is constitutive, Northern blot analysis was performed on total RNA isolated from P-7 and WT cells treated in the presence or absence of dexamethasone. As shown in Fig. 2, dexamethasone treatment had no effect on the amount of MMTV mRNA in either of the transfected cell types (lanes A vs. B and C vs. D). Furthermore, as expected, the basepair substitu- tions in P-7 had no effect on the size of the mRNA relative to that of RNA isolated from WT cells, which is approximately 4.0 kb. Two smaller transcript bands were also observed in dexamethasone-treated and untreated P-7 cells. Possibly, these mRNA species non- specifically hybridize with the MMTV cDNA probe or represent specific MMTV transcript degradation products. We have, however, observed the same sized transcript bands in WT cells after longer autoradi- ographic exposure. The levels of actin mRNA were used to compare gel loading among hormonal conditions. These results demonstrate that glucocorticoids did not affect the levels of intracellular and cell surface MMTV glycoproteins by a transcriptional or posttran-

MMTV- Env

Actin -

r

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WT P-7 -II ABCD

kb -9.5 - 7.5

-4.4

- 2.37

- 2.37

- 1.35

- + - + DEX Fig. 2. Effects of Glucocorticoids on Expression of MMTV Transcripts in Transfected HTC Cells

WT (lanes A and B) or P-7 (lanes C and D) cells were treated with (lanes B and D) or without (lanes A and C) 1 PM dexamethasone (DEX) for 16 h, and the total RNA was isolated, fractionated on formaldehyde-agarose gels, and blotted onto Nytran filters. The blotted RNA was hybridized with either MMTV envelope (ENV)-specific (upper panel) or actin-specific (lower panel) 32P-labeled cDNA probes, and the individual RNA species were visualized by autoradiography. The size of the MMTV glycoprotein transcript is approximately 4.0 kilobases

W).

scriptional mechanism. P-7 cells were used for the remainder of the experiments so the glucocotticoid effect could be examined in a cell type with a more simplified pattern of MMTV glycoprotein expression.

Effects of Glucocorticoids on the Translation and Transport Kinetics of the MMTV Glycoproteins

To determine if glucocorticoids regulate the rate of translation or the transport kinetics of MMTV glycoproteins, the expression of viral glycoproteins in P-7 cells was examined by pulse-chase kinetics. Dexametha- sone-treated and untreated P-7 cells were pulse labeled for 10 min with [35S]methionine and then chased for the indicated times in medium containing excess unlabeled methionine. As shown in Fig. 3 (lanes A, panel A vs. B), the relative levels of the glycoprotein precursor gp70 synthesized during the pulse period were identical in

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A: -DEX ----- 0 0.5 1 2 3 Chase (hours)

ABCDEFGHI J kDa

97.4 -

68--

6: +DEX ,\

97.4-

88-

-9P78 \ - BP70

45-

29-

-+-+-+-+-+ Endo H

Fig. 3. Pulse-Chase Kinetic Analysis of MMTV Glycoproteins Expressed in Glucocorticoid-Treated or Untreated P-7 Cells P-7 cells were treated in the presence (+ DEX; Et) or absence (- DEX; A) of 1 PM dexamethasone for 16 h. Cells were pulse

labeled with [%]methionine for 10 min, washed three times with PBS, and chased for 0, 0.5, 1, 2, and 3 h in excess unlabeled methionine. At the indicated times, cells were harvested and solubilized, and the immunoprecipitated radiolabeled MMTV glycoproteins were digested with (lanes B, D, F, H, and J) or without (lanes A, C, E, G, and I) endo-H, as described in the text. The final immunoprecipitated proteins were fractionated on SDS-polyacrylamide gels, and the radioactive bands were visualized by fluorography. In lane D of panel B, the radiolabeled glycosidase-digested MMTV glycoprotein was apparently lost after recovery of the immunoprecipitated material. The molecular mass standards are phosphorylase-b (97.4 kDa), BSA (68 kDa), ovalbumin (45 kDa), and carbonic anhydrase (29 kDa).

dexamethasone-treated and untreated cells, as measured by densitometric analysis (data not shown). This result suggests that dexamethasone does not increase the overall translation rate of MMTV mRNA. In fact, the first indication of a hormonal effect on the accumulation of MMTV glycoproteins was observed at the 1 h chase time (Fig. 3, lanes 3, panel A vs. B). By the 3 h chase time, dexamethasone-treated cells showed an approximately 3-fold increase in the combined levels of gp70 and gp78 compared to those in untreated cells (Fig. 3, lanes I, panel A vs. B).

Endo-H cleaves simple or high mannose oligosaccharides, and the rate of acquisition of endo-H-resistant carbohydrate side-chains was used to monitor endoplasmic reticulum (ER) to Golgi transit times in dexamethasone-treated and untreated cells. Immunoprecip- itated MMTV glycoproteins were tested for their sensitivity to endo-H digestion by observing a shift in molecular mass on sodium dodecyl sulfate (SDS)-polyacrylamide gels. The glycosylated MMTV polyprotein synthesized during the pulse (gp70) was completely endo-H sensitive, since a lower molecular mass (62- kDa) band was detected after glycosidase digestion (Fig. 3, panels A and B, lanes A vs. B). The gp70

precursor remains endo-H sensitive throughout the en- tire chase period, and between 30 min to 1 h, a glycoprotein containing a mixture of endo-H-resistant and endo-H-sensitive oligosaccharides, gp78, was produced. As shown in Fig. 3 (lanes D, F, and H), in both steroid-treated and untreated cells, 70- to 74-kDa forms of this precursor are generated after glycosidase treatment. Based on the rate of acquisition of endo-H re- sistance, the ER to Golgi transit times for the glycosylated MMTV polyprotein was approximately 45 min in dexamethasone-treated and untreated P-7 cells, suggesting that glucocorticoids do not discernibly alter the rate of exocytic transport.

Glucocorticoids Affect the Steady State Accumulation and Subsequent Turnover of MMTV Glycoproteins

A long pulse radiolabeling was used to determine if the steady state turnover of MMTV glycoproteins varied during the chase period in dexamethasone-treated vs. untreated cells. P-7 cells were radiolabeled with [““S] methionine for 4 h, the labeled proteins were chased for 2 and 4 h, and the immunoprecipitated material from

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intracellular, cell surface, and secreted fractions cells was resolved on SDS-polyacrylamide gels. As shown in Fig. 4 (lanes A vs. 6 and G vs. H), after a 4-h pulse, the intracellular and cell surface levels of both gp70 and gp78 were higher in dexamethasone-treated cells than in untreated cells, which confirms the previous steady state result (see Fig. 1). Subsequently, at the 2 and 4 h chase points (Fig. 4, lanes C-F and I-L), the turnover of intracellular and cell surface MMTV glycoproteins was decreased by approximately 3-fold in dexamethasone-treated P-7 cells relative to that in untreated cells. By the 4 h chase point, most of the radiolabeled intracellular and cell surface MMTV glycoproteins were chased into the secreted fraction. Higher levels of MMTV glycoproteins were externalized at the later chase points in glucocorticoid-treated cells (Fig. 3, lanes Q vs. R), which most likely results from a decrease in the rate of protein turnover.

To more accurately quantitate the glucocorticoid- induced stabilization of MMTV glycoproteins, dexamethasone-treated and untreated P-7 cells were radiolabeled for 4 h and chased for 0.5, 1, 2, 3, and 4 h, and the immunoprecipitated MMTV glycoproteins from total cellular extracts were fractionated on SDS-polyacrylamide gels. A glucocorticoid-dependent accumulation of the MMTV glycoproteins was observed during the chase period (Fig. 5A). The level of MMTV glycoproteins displayed in this fluorograph during the pulse and various chase periods was quantitated by densitometric analysis. As shown in Fig. 58, exposure to dexamethasone increased the half-life of the MMTV glycoproteins from 45 to 105 min. Although this densi-

Intracellular Cell Surface

tometric analysis was performed only on the pulse- chase data shown in Fig. 5A, the glucocorticoid-induced decrease in the turnover rate of MMTV glycoproteins was reproduced in five independent experiments. Taken together, our results suggest that the increase in the steady state accumulation of MMTV glycoproteins in glucocorticoid-treated cells results from a stabilization of the viral glycoproteins during their exocytic transport.

DISCUSSION

We have documented previously that glucocorticoids regulate the maturation and cell surface localization of MMTV glycoproteins in virus-infected HTC rat hepatoma cells (12-14, 16, 18). This hormonal effect appears to involve both a functional interaction between the MMTV glycoproteins and phosphoproteins as well as the actions of cellular components (22). Part of the difficulty in understanding the mechanism of this process has been the presence of a complicated profile of MMTV maturation products that are normally produced in virus-infected cells. Therefore, to selectively distin- guish cell-mediated events under glucocorticoid control, HTC cells were transfected with a MMTV glycoprotein gene containing a mutated endoproteolytic cleavage site. This mutation eliminated the formation of the processing products and thereby produced a more simplied maturation pattern. Futhermore, the MMTV glycoprotein gene was driven by a constitutive promoter, so that

‘0 2 4’ -Ir- ABCD EF

0 2 4’ --II GHIJKL

Fig. 4. Effects of Glucocorticoids on the Steady State Accumulation and Subsequent Turnover of MMTV Glycoproteins P-7 cells were treated with (lanes B, D, F, H, J, L, N, P, and R) or without (lanes A, C, E, G, I, K, M, 0, and Q) 1 PM dexamethasone

(DEX) for 16 h. Cells were radiolabeled for 4 h with [?S]methionine and chased for 2 and 4 h in excess unlabeled methionine. In the appropriate incubations, 1 PM dexamethasone was included in the medium during the labeling and chase periods. The secreted (lanes M-R) MMTV proteins were immunoprecipitated from the culture medium using anti-MMTV antibodies. The cell surface (lanes G-L) and the intracellular fractions (lanes A-F) were immunoprecipitated from the remaining cells, as described in the text. The immunoprecipitated MMTV proteins were electrophoretically fractionated in SDS-polyacrylamide gels, and the radioactive bands were visualized by fluorography. The protein bands designated by arrows, gp78 and gp70, are the uncleaved forms of the glycosylated MMTV polyprotein.

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A

CHASE (hours)

I

L DEX - + - + - + _ + _ + _ + I 4-8P78 c-BP70

B

- DEX t 1,~. 45’

l DEX t 1/2- 105

I 2 3 4

TIME (hours)

Fig. 5. Quantitation of the Turnover of MMTV Glycoproteins Expressed in Glucocorticoid-Treated and Untreated P-7 Cells A, P-7 cells were treated with (lanes B, D, F, H, J, and L) or without (lanes A, C, E, G, I, and K) 1 PM dexamethasone (DEX) for

16 h before radiolabeling the cells with [%]methionine for 4 h. After the 4-h pulse, cells were chased for 0, 0.5, 1, 2, 3, and 4 h in excess unlabeled methionine, when appropriate in the continual presence of 1 PM dexamethasone. At the indicated times, cells were harvested and solubilized, the immunoprecipitated radiolabeled MMTV glycoproteins were fractionated on SDS-polyacrylamide gels, and the radioactive bands were visualized by fluorography. The protein bands indicated by arrows (gp78 and gp70) are the glycocosylated MMTV polyproteins. B, The fluorogram in A was scanned with a Microtex GS400 scanner using the NIH Image program to determine the relative levels of MMTV glycoproteins produced at each chase point in the presence or absence of dexamethasone. The values were plotted logarithmically as relative units vs. time.

equivalent levels of MMTV glycoproteins could be compared in glucocorticoid-treated and untreated cells. Dexamethasone induced an approximately 5-fold increase in the steady state levels of the cell surface MMTV glycoproteins, gp78 and gp70, without altering the levels of viral transcripts. Under these conditions, the translation rate of MMTV glycoproteins and the ER to Golgi transport kinetics remained unaffected. Pulse- chase kinetic analysis of 4-h pulse-labeled cells was used to monitor MMTV glycoprotein turnover in total cellular fractions and revealed that the half-life of gp78 was 105 min in dexamethasone-treated cells and 45 min in untreated cells. Thus, the glucocorticoid regulation of MMTV glycoproteins trafficking in virus-infected

HTC cells can be accounted for in part by a posttranslational stabilization of MMTV glycoproteins, which pre- sumably occurs through the actions of regulated cellular components.

In P-7 cells expressing the cleavage site mutant, dexamethasone induced a 5-fold increase in accumulation of cell surface gp78, whereas in WT cells expressing the WT MMTV glycoprotein, a 2-fold increase in accumulated gp78 was observed. Most likely, this difference is due to the generation of endoproteolytic maturation products in WT cells which may be inher- ently less stable than the uncleaved polyprotein. Also, the aminoterminal fragment gp50 is rapidly externalized, which lowers its cellular concentration. Steady

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state radiolabeling and the long pulse-chase experiments revealed that the increase in glucocorticoid-sta- bilized MMTV glycoproteins in the intracellular fraction resulted in a corresponding increase in the amount of viral glycosylated polyproteins localized at the cell surface. The steroid-dependent difference in MMTV glycoprotein stability was first detectable between the 0.5 to l-h chase times. This time frame approximates the ER to Golgi transit time for both the endoproteolytic site mutant and WT MMTV glycoproteins (19, 20) suggesting that the differential stability induced by glucocorticoids may be occurring early in the exocytic pathway. Consistent with this notion, recent evidence has uncovered an ER degradive pathway that appears to function as an early quality control step to eliminate misfolded proteins as well as proteins that have not oligomerized properly and are not competent for exocytic transport (24-30). Also, ER to Golgi transit is an important rate-limiting step in exocytic transport (31) and it seems biologically relevant that glucocorticoids may be exerting their stabilizing effects early in this pathway. Further evidence for the involvement of a nonlysosomal pathway mediating MMTV glycoprotein turnover is that the lysosomal inhibitor monensin had no effect on either the rate of tranport of MMTV glycoproteins or the stability of the protein (16).

Given its known positive or negative transcriptional effects (2, 4-6, 32) glucocorticoids may be regulating the expression and/or activity of a variety of cellular factors involved in stabilizing the MMTV glycoproteins. Conceivably, glucocorticoids may be inducing a specific inhibitor of one of the proteases associated with the ER degradative pathway. Other investigators have re- ported that thiol-oxidizing reagents are potent inhibitors of ER degradation, suggesting that cysteine proteases are associated with this process (33). Similarily, glucocorticoids may inhibit the synthesis of crucial components of the degradative machinery, such as a protease or a factor(s) that recognizes misassembled proteins, It is also tempting to speculate that glucocorticoids induce assembly protein(s) or molecular chaperones (34) which may aid in the folding and/or oligomerization of the MMTV glycoprotein. However, this possibility seems unlikely, since we have not been able to demonstrate ER oligomerization of the MMTV glycoproteins in the virus-infected or transfected HTC rat hepatoma cells (unpublished result), although others have shown that the MMTV glycoproteins oligomerize in the intact virus (35). Perhaps oligomerization of the MMTV glycoprotein occurs in a different compartment or during assembly of the virus at the plasma membrane.

Glucocorticoids have been shown to induce several types of regulatory responses, involving the posttranscriptional, translational, and posttranslational regulation of cellular gene products. For example, in conjunc- tion with insulin and PRL, glucocorticoids are required to maximally stabilize mRNAs of the casein gene family and a mRNA encoding the whey acidic protein in a process that requires ongoing protein synthesis (36, 37). Glucocorticoids have also been shown to facilitate

protein synthesis by inducing cellular components involved in translation (38). For example, in the mammary gland, PRL and glucocorticoids induce the synthesis of certain transfer RNAs (38) 28s ribosomal RNA (39) and the formation of rough ER (40). Besides the glucocorticoid regulation of MMTV glycoprotein trafficking (12-14, 16, 18), posttranslational events, such as proteolytic processing, oligosaccharide maturation, and/or transport of specific membrane-associated and secreted proteins, have been shown to be under the control of steroid hormones (15, 17). For example, particular steroid hormones alter the expression of certain oligosaccharide-processing enzymes (41, 42) and members of the kallikrein protease family (43). By these regulatory pathways in combination with gene products under direct transcriptional control, steroid hormones could induce pleiotropic responses in a wide variety of target tissues.

In our current study, a MMTV glycoprotein gene containing a mutation in the endoproteolytic site was used as a unique tool to characterize the cellular effects of glucocorticoids on turnover of the glycosylated viral precursor polyprotein. This study provided direct evidence that glucocorticoids can induce the stabilization of MMTV glycoproteins and suggests that steroid hormones may regulate the turnover of a wide variety of cellular proteins in response to appropriate biological signals. We are currently attempting to determine the cell type specificity of this response and uncover cellular encoded substrates in hepatic-derived tissue. Further studies will be aimed at determining the mechanism of this glucocorticoid-induced posttranslational response and the potential contributions of cellular as well as viral components to mediation of MMTV glycoprotein trafficking in virus-infected HTC cells.

MATERIALS AND METHODS

Materials

All media and sera used for tissue culture were purchased from Whittaker Bioproducts (Walkersville, MD). L-[%]Methio- nine (1000 Ci/mmol) and cytidine 5’-[&*P]triphosphate (3000 Ci/mmol) were obtained from DuPont-New England Nuclear Research Products (Boston, MA). Dexamethasone was purchased from Sigma Chemical Co. (St. Louis, MO). Fixed Staph- y/ococcus aureaus (Pansorbin) was acquired from Calbiochem Corp. (San Diego, CA). Geneticin (G418) was obtained from Gibco (Santa Clara, CA). Total anti-MMTV (44) and preimmune sera used for immunoprecipitations were generously provided by D. Moore, L. J. T. Young, and R. D. Cardiff (Department of Pathology, University of California-Davis). All additional reagents were of the highest quality available.

Construction of an Endoproteolytic Site Mutant

The endoproteolytic cleavage site in the MMTV glycoprotein gene was altered by site-directed mutagenesis, as previously described (20). Briefly the Lys-Arg residues in the proteolytic cleavage site were mutated to the neutral polar amino acids Asn-Gln. The expression vector containing the mutant glycoprotein is driven by a RSV constitutive promoter and, except

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for the mutation in the proteolytic cleavage site, is identical to the WT MMTV glycoprotein gene.

Cells and Method of Culture

P-7 cells are an HTC cell line transfected with a constitutively expressed MMTV glycoprotein gene containing a mutated endoproteolytic site. WT cells are an HTC rat hepatoma cell line stably transfected with a constitutively expressed WT MMTV glycoprotein gene (19). All cell lines were propagated as monolayers on Corning Tissue Culture plates (Corning, NY) in Dulbecco’s Modified Eagle’s Medium supplemented with 10% horse serum in a humidified atmosphere of air-CO2 (95:5).

Transfection and Expression of Plasmids Bearing WT and Mutated MMTV Glycoprotein Genes

The WT and mutated MMTV glycoprotein expression plasmids were cotransfected with pSV2neo into rat HTC hepatoma cells using the lipofectin technique (45). Lipofection was performed according to the manufacturer’s recommendations, except for the following changes. DNA (100 ng pSV2-neo and 650 ng of each construct) and lipofectin reagent were each diluted into 0.5 ml Opti-mem medium (Bethesda Research Laboratories, Gaithersburg, MD), and the two were mixed and allowed to stand at room temperature for 10 min. The mixture was added to cells and allowed to incubate for 8 h at 37 C. The reaction was stopped by aspirating the mixture and then adding Dul- becco’s Modified Eagle’s Medium supplemented with 10% horse serum. After 36-48 h, cells were passaged into serum- supplemented medium containing 1.2 mg/ml G418. Single cell- derived colonies were picked and expanded in selective medium for 2 weeks. Cell clones were initially screened for MMTV glycoprotein gene expression by RNA cytoblot analysis (46). Clones expressing high amounts of MMTV-specific RNA were further screened for the production of MMTV glycoproteins by immunoprecipitation of [35S]methionine-labeled cell extracts with anti-MMTV antibodies. WT cells express the WT MMTV glycoprotein gene, and P-7 express the proteolytic site mutant.

Northern Analysis

Dexamethasone-treated and untreated WT and P-7 cells were grown to subconfluency, washed with PBS, harvested, and pelleted by 500 x g centrifugation. Cells were resuspended in ice-cold 10 mM Tris-HCI (pH 7.5) 1.5 mM MgCI,, 200 mM NaCI, 1% Nonidet P-40, and 200 pg/ml heparin and incubated on ice for 5 min. Nuclei were pelleted by low speed centrifugation, and the resulting supernatants were brought to a final concentration of 1.6% SDS and then extracted twice with equal volumes of phenol-chloroform. The RNA in the upper aqueous phase was precipitated with sodium acetate and ethanol, using standard procedures. Twenty micrograms of cytoplasmic RNA were first denatured by heating at 65 C for 5 min in the presence of 1 x MOPS containing 50% formamide and 6% formaldehyde and then electrophoretically fractionated in 1% agarose gels containing 1 x MOPS and 6% formaldehyde. The fractionated RNA was blotted onto nytran filters (Schleicher and Schuell, Keene, NH) and probed with MMTV- specific 32P-labeled DNA, and the blots were washed and autoradiographed, as previously described (47).

Steady State Radiolabeling and Harvesting of Cellular and Secreted Fractions

Before radiolabeling, monolayer cultures were incubated for 30 min in 4 ml methionine-free labeling medium; during the last 4 h of appropriate hormonal treatment, the cells were radiolabeled with 100 &i [35S]methionine. The secreted fractions were harvested from radiolabeled cells by centrifugation at 2000 x g for 10 min. The supernatant fractions, containing the externalized MMTV glycoproteins, were brought to 1%

Triton X-l 00. 0.5% deoxycholate, and 5 mM EDTA just before immunoprecipitation. For the cellular fractions, radiolabeled monolayers were washed three times in ice-cold PBS and then solubilized in 500 PI immunoprecipitation buffer (1% Triton X- 100, 0.5% deoxycholate, 5 mM EDTA, 250 mM NaCI, and 25 mM Tris-HCI, pH 7.5). The solubilized material was then centrifuged at 15,000 rpm (Sorvall SE12 rotor, Norwalk, CT) for 15 min. The total level of radiolabeled protein in each sample was determined by trichloroacetic acid precipitation with 10% trichloroacetic acid and scintillation counting to normalize the amount of secreted and cellular fractions to be immunoprecipitated.

Pulse-Chase Radiolabeling

Cells were placed in 4 ml methionine-free labeling medium for 30 min before the addition of 200 &i/ml [35S]methionine. Twelve hours before and during the labeling period, the cells were treated with or without 1 PM dexamethasone. After a 1 O- min pulse, cells were washed three times in PBS, then chased in medium containing excess unlabeled methionine for the indicated time points. Cell extracts were harvested and prepared for immunoprecipitation, as described below. For the 4- h pulse radiolabeling, dexamethasone-treated and untreated cells were placed in 4 ml methionine-free labeling medium for 30 min before the addition of 100 &i/ml [35S]methionine. After the 4-h pulse, cells were chased with unlabeled methionine, and cell extracts were harvested, as described above.

Immunoprecipitation, SDS-Polyacrylamide Gel Electrophoresis, and Fluorography

lmmunoprecipitations of cellular and secreted MMTV glycoproteins using total anti-MMTV antibodies were performed, as described previously (12-14, 18). Briefly, radiolabeled cells were detergent solubilized in 1 ml 1% Triton X-100, 0.5% deoxycholate, 5 mM EDTA, 250 mM NaCI, and 25 mrv Tris- HCI, pH 7.5. Cell homogenates were centrifuged at 20,000 x g for 10 min, and postnuclear supernants were isolated as solubilized cellular fractions. Secreted fractions were brought to 1% Triton X-100, 0.5% deoxycholate, and 5 mM EDTA before immunoprecipitation. Radiolabeled fractions were precipitated with 10% tricholoroacetic acid to normalize the total amount of protein to be immunoprecipitated, so that equivalent amounts of protein could be compared between dexamethasone-treated and untreated samples. Using anti-MMTV antibodies, viral antigens from both cellular and secreted fractions were immunoprecipitated with a Staph A immunoadsorption precedure (12-l 4). The immunoprecipitation of cell surface [35S]methionine-labeled MMTV glycoproteins was accomplished by a modified method of Krangel and co-workers (48) as we have previously described (16, 18). Briefly, intact cells were incubated with anti-MMTV antibodies before solubili- zation. Antibody-bound cells were washed three times with PBS, harvested, and then detergent solubilized, as described for the cellular immunoprecipitations. Postnuclear supernant fractions were isolated from detergent-solublized cells and incubated with fixed Staph A to immunoadsorb the cell surface MMTV glycoproteins. After a 20,000 x g centrifugation, the immunoadorbed proteins pelleted with the Staph A. The intracellular MMTV glycoproteins remaining in the supernatant were then immunoprecipitated as described above. The SDS- polyacrylamide gel electrophoresis of immunoprecipitated material and subsequent fluorography were accomplished as previously described (16, 18). After electrophoresis, the gels were prepared for fluorography by soaking in 1 M salicylic acid for 1 h before drying. The dried gels were exposed to Kodak X-Omat AR film (Eastman Kodak, Rochester, NY) at -80 C. Relative protein levels were measured by densitometry of the fluorograms using a Microtek GS400 scanner (Microtek Lab, Inc., Torrance, CA) and the NIH Image 1.37 computer program.

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MOL ENDO. 1993 102

Vol7 No. 1

Acknowledgments

We express our appreciation to Steven Ft. Kain for his critical comments and helpful suggestions during the course of this work. We also thank Candice R. Gonzalez for her superb typing and preparation of this manuscript, John Underhill for his skillful photography, Phyllis Thompson for her excellent illustrations, and Charles Jackson, Helen Cha, and Marina Chin for their technical support,

Received September 1, 1992. Revision received October 14, 1992. Accepted October 14, 1992.

Address requests for reprints to: Dr. Gary L. Firestone, Department of Molecular and Cell Biology, Box 591 LSA, University of California, Berkeley, California 94720.

This work was supported by a grant from the NIH (DK- 42799) and in part by funds awarded from the Lucille P. Markey Program in Biomolecular Structure and Design.

* Predoctoral trainee supported by a National Research Service Grant (CA-09041) awarded by the NIH.

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