Size and steroid-binding characterization of membraneassociated glucocorticoid receptor in S-49 lymphoma cells Bahiru Gametchu, Cheryl S. Watson,* and Dean Pasko Department

of Pediatrics,

Medical

College

of Wisconsin,

Milwaukee,

Wisconsin,

USA

The precise mechanism for glucocorticoid-mediated lymphocytolysis is not understood, although it is presumed to be receptor mediated. We have recently presented evidence that this response is mediated by a specialized form of the glucocorticoid receptor (GR) that resides in the plasma membrane (mGR). ConJirmation of the previous receptor identtjkation studies in a population of S-49 cells enriched for mGR is now made using another antibody speci$c for the rodent GR, BUGR-2. The membrane resident receptor could be labeled competitively with the affinity ligand dexamethasone 21 -mesylate, and Scatchard analysis of whole cell binding revealed that receptor number, but not the ajj5nity for hormone, varied between the mGR-enriched and -deficient cell populations. Steroid spectjkity displacement analyses showed an order of affinities asfollows: triamcinolone acetonide > progesterone > dexamethasane > testosterone = estrogen. Studies of mGR by one- and two-dimensional gel electrophoresis, immunoblot, autoradiography, and density gradients revealed a species with an equivalent size to cytosolic receptor as well as multiple higher molecular weight species, confirming earlier studies. To offer a possible explanation for the nucleic acid origins of the mGR, RNA from the mGR-enriched cells was probed with rat GR cDNA; mGR-enriched cells contained higher levels of GR mRNA. Possible molecular etiologies of larger receptor species in membrane are discussed. (Steroids 56:402-410, 1991)

Keywords:

membrane glucocorticoid receotor: BUGR monoclonal antibodies; lymphocytolysis; RNA; steroid

Introduction Glucocorticoid hormones elicit a number of cellular responses in lymphoid cells, including both anabolic and catabolic effects. Of particular interest for the development of clinical management strategies for certain leukemias and lymphomas’~* is the cytolytic response. However, the mechanism by which glucocorticoids induce cell lysis is still a matter of much debate. High numbers of receptor sites have been correlated with good clinical response to glucocorticoids3*4 and cytolytic responses of established lymphoid cell lines.5l6 However, the correlation is far from perfect, and the

*Present address: University of Texas Medical Branch, Galveston, Texas, 77550. Address reprint requests to Dr. Bahiru Gametchu at the Department of Pediatrics, Medical College of Wisconsin, Milwaukee, WI 53226, USA. Received January 11, 1991; accepted March 27, 1991.

402

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1991, vol. 56, August

hormone specificity;

existence of unresponsive patients’ cells and rodent cell lines with high receptor numbers has remained a puzzle.2,5,7 Several recent studies suggest that the lymphocytolytic effect of glucocorticoids is mediated by a lysis gene(s) product(s) that is induced by the steroid. One such putative gene product was reported by Compton and Cidlowski.8 However, their newly appearing endonucleases proved to be cleavage products of constitutive histone protein(s).9 Other recent studies implicated cyclic adenosine monophosphate (CAMP)-dependent protein kinase in the modulation of the lymphocytolytic effect of glucocorticoids.iO This study postulates that glucocorticoids (dexamethasone) and CAMP both induce a common set of lysis gene(s). In light of the lack of a complete correlation of the cytolytic response with intracellular receptor concentration,” we explored an alternative or additional explanation for the lytic effect of glucocorticoids. It is generally believed that steroids enter cells by passive diffusion through the plasma membrane by virtue of

0 1991 Butterworth-Heinemann

Size and steroid-binding of mGR in S-49 cells: Gametchu et al. their small size and lipophilic nature.‘* However, this does not rule out that receptors for steroids residing in the plasma membranes of cells could be responding via pathways not directly connected to the well-described gene expression mechanism. Alternate mechanisms may be responsible for functions involving plasma membrane transport, permeability, and integrity.13v14 Historically, several investigators have presented data in favor of a membrane localization of steroid receptors, including the glucocorticoid receptor (GR).“,“j One of us has (BG) recently reported a novel visualization of GR in the plasma membrane of S-49 lymphoma cells using an extensively characterized and frequently used monoclonal antibody to the rat intracellular glucocorticoid receptor (iGR).” In addition, using methods to enrich for cells ‘bearing the membrane receptor, the presence of this specialized receptor was shown to be highly correlated with the glucocorticoid-mediated lymphocytolysis function. I7 We present results further characterizing the size and hormone-binding aspects of the plasma membrane GR (mGR) from S-49 cells, and then corroborate these results using another anti-rat GR antibody (BUGR-2) not previously used for this purpose.r8 We present additional studies confirming the presence of the receptor in membrane preparations, the affinity and specificity of hormone binding to the receptor, and, finally, its possible molecular etiology.

Experimental Cell culturelcell

selectionlantibody

preparation

S-49 cells were obtained from ATCC (Bethesda, MD, USA) and were grown in RPM1 1640 (Biofluids, Rockville, MD, USA) in the presence of 10% bovine calf serum (KC Biologic&, St. Louis, MO, USA). Using sequential cell-separation techniques previously described (immunopanning, I7 fluorescent cell sorting,r9 and, finally, soft agar cloning*‘) stable mGR-enriched and -deficient cell lines were produced. Unless otherwise stated, cells sequentially separated by all three methods, culminating in soft agar cloning, were used in all of the following experiments. The labeled steroids [3H]triamcinolone acetonide (45 Ci/mmol), [‘Hldexamethasone, and [3H]dexamethasone 21-mesylate (48 Ci/mmol) were obtained from New England Nuclear (Boston, MA, USA). Unlabeled steroids, Nonidet P-40 (NP-40), and RNase were obtained from Sigma Chemical Co. (St. Louis, MO, USA). BUGR-1 and BUGR-2 anti-rat GR monoclonal antibodiesI were produced by culturing the hybridoma-producing cells in RPM1 1640 media and purifying antibody on a protein A-Sepharose 4B affinity column from the medium supernatant.‘* Buffers The following buffers are referred to in the text by number: (no. 1) 10 mM Tris-HCl, 140 mM NaCl, and 10 mM sodium molybdate, containing enzyme inhibitors (83 pg/ml aprotinin, 100 pg/ml bacitracin, 125 pg/ ml trypsin inhibitor, 21 pg/ml pepstatin, 1 nM PMSF,

5 mM diisopropylfluorophosphate [DIP], pH 8.2); (no. 2) buffer no. 1 containing 0.5% NP-40, 1 pg/ml RNase, and 33 U/ml DNase; (no. 3) 0.9% NaCl, 0.5% bovine serum albumin, 20 mM Tris-HCl, 0.1 SDS, and 0.25% Triton X-100; (no. 4) 25 mM Tris-HCl, 192 mM glycine, and 20% methanol (v/v); (no. 5) 9.5 M urea, 2% NP-40, 1.6% Ampholines (pH 3-lo), 0.4% Ampholines (pH 5-10.5), and 2% @mercaptoethanol; (no. 6) 0.125 M Trizma base, 2% SDS, 10% glycerol, and 1% P-mercaptoethanol; and (no. 7) 10 mM Tris-HCl, 1 mM N+ EDTA, 0.3 M KCl, and 12 mM thioglycerol, pH 7.5. Membrane

receptor puri$cation

and labeling

From 2 to 5 x lo9 cells (mGR-enriched, mGR-deficient, or wild type) were harvested, washed, and resuspended in serum-free RPM1 1640, pH 8.2. Whole cell receptor labeling was accomplished by incubating cells for 2 hours on ice in 1 x lo-’ M [3H]dexamethasone 21mesylate or this affinity labeling agent plus a lOO-fold excess concentration of unlabeled competitor.21 This was done both to stabilize the receptor during membrane preparation and for use in affinity-labeling studies. The cells were resuspended in buffer no. 1 at a 1: 1 packed cell volume to buffer ratio and disrupted at 4 C using a Tissuemizer (Tekmar, Cincinnati, OH, USA) three times, 10 seconds each. A nuclear pellet was removed by low-speed centrifugation (600 x g for 10 minutes) of the resulting broken cell preparation. The supernatant was further centrifuged at 15,000 x g to pellet mitochondria and lysosomes. The remaining supematant was centrifuged at 120,000 x g for 2 hours, yielding a supernatant and a plasma membrane-containing pellet.” Membrane protein was extracted by incubating and gently mixing the pellet (with a magnetic stirring bar) for 3 hours at 4 C in approximately 750 ~1 of buffer no. 2. The solution was ultracentrifuged at 120,000 x g for 1 hour at 4 C, and the supernatant containing extracted mGR was recovered. Specific binding of extracted receptor was estimated by charcoal assay.** Sucrose

density gradient analysis

Membrane extracts obtained from mGR-enriched cells were incubated overnight with monoclonal antibody BUGR-2 (1:80 dilution) at 4 C. The following morning the mixture was incubated with protein-A Sepharose4B beads (150 ~1 of a 1 g/2 ml suspension in buffer no. 1) for 1 hour. The receptor-BUGR-2-protein A complex was then washed three times with buffer no. 1 by centrifugation at 1,000 x g for 10 minutes at 4 C. To release mGR from the complex, 50 ~1 of 4 M NaSCN, pH 5, was added and the mixture was incubated for 20 minutes at 4 C. The beads were then removed by centrifugation at 12,000 x g for 10 minutes in a table-top microfuge (Beckman Instruments, Palo Alto, CA, USA). To estimate receptor recovery, 5 ~1 of the supematant containing competed and uncompeted mGR was assessed by liquid scintillation counting. BUGR-2 antibody was removed from mGR by adsorption over a protein-A Sepharose-4B column.

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1991, vol. 56, August

403

Papers A portion of this preparation containing 25,000 dpm of specific binding (of the 400,000 dpm of specific binding recovered from 5 x lo9 cells) was layered on a 5ml 5% to 20% (w/w, in buffer no. 7) sucrose gradient. After centrifugation in an SW 50.1 rotor (Beckman Instruments) at 120,000 x g for 18 hours, the gradients were analyzed by puncturing the tube bottom and collecting 23 0.2-ml fractions. Radioactivity in each fraction and that associated with the tube bottom were estimated by liquid scintillation counting. Cytosolic GR was labeled with 1 x lo-’ M [3H]triamcinolone acetonide and analyzed in the same fashion.” All gradients shown were centrifuged and analyzed simultaneously. One- and two-dimensional

gel analysis

Membrane GR preparations were made from mGRenriched S-49 cells and processed for one- and twodimensional analysis as described by Laemmli23 and Garrels,24 respectively. For gel slice and immunoblot experiments, membrane preparations containing 3 x 105and 1 x lo5 specific dpm (see above), respectively, were resolved on 7.5% SDS-PAGE. The samples used to demonstrate specific labeling and part of the preparations used in the immunoblot analysis had been concentrated by an overnight immunoprecipitation with BUGR-2 prior to analysis by SDS-PAGE. After electrophoresis, a portion of the slab gel (lanes containing samples labeled in the presence and absence of lOOfold excess unlabeled dexamethasone) was sliced into 0.3-cm segments. The gel slices were dissolved in TS1 tissue solubilizer (RPE Corp., Mount Propsect, IL, USA) and assayed for radioactivity to indicate specific binding. The remaining portion of the gel was transferred to a nitrocellulose filter by applying 60 V for 3 hours (in buffer no. 4) in an electrophoretic transfer apparatus.25 The filters were then blocked by soaking in 20% bovine serum albumin in phosphate-buffered saline for 30 minutes at 37 C followed by incubation for 16 hours at 4 C with BUGR-2. Filters were then processed for Western blotting. l8 For electrophoresis in the second dimension, dexamethasone 21-mesylate affinity-labeled samples were diluted in buffer no. 5. Preparation containing 90,000 dpm of specific binding (mGR) in 20 ~1 volume was focused in 7.5% acrylamide tube gels at 14,000 V hours at 4 C overnight. The pH gradient was determined immediately after electrophoresis by pH measurement of the 6-mm gel slices, which had been incubated in 0.5 ml distilled water for 30 minutes. For the second dimensional analysis, the tube gels were placed on slab polyacrylamide gels and electrophoresed overnight at 50 mV/slab in a gel containing 0.1% SDS prepared as described by Laemmli.23 Western analysis of this preparation was carried out as described above. Scatchard

analysis

Membrane GR-enriched and mGR-deficient as well as wild-type S-49 cells were harvested and washed in fresh RPM1 1640 medium. Approximately 1 ml of suspension containing 6 x IO6cells was added (in triplicate) to IO404

Steroids,

1991, vol. 56, August

ml centrifuge tubes containing [3H]dexamethasone at concentrations ranging from 1.25 to 100 nM (for estimation of total binding). To estimate nonspecific binding, a lOO-fold excess concentration of unlabeled dexamethasone was included in another set of triplicate tubes. These cell suspensions were incubated at 21 C for 2 hours with shaking. Cells were then washed with cold RPM1 1640 (three times, 1 ml each, by centrifugation at 800 x g in a Beckman GPR centrifuge). The final cell pellet was resuspended in 1 ml RPM1 1640, and 50~1 aliquots were used to estimate final cell number and radioactivity. Surviving cells numbered 2.80 to 4.27 x lo6 (from an original 6 x lo6 cells); this experimental loss in cell number was considered when calculating number of binding sites per cell. All the binding data have been normalized to represent 3 x lo6 cells. Specifically bound steroid was calculated by subtracting the nonspecific binding from total binding. The steroid affinity and receptor numbers were estimated according to the method of Scatchard2’j using the personal computer version of the program Ligand.27 One-way analysis of variance was used to compare the three types of S-49 cells after using a square root transformation to stabilize the variance.28 Specificity of binding for different steroid hormones Membrane-associated GR was prepared from mGRenriched S-49 cells and labeled for 2 hours at 4 C with 2 x lo-’ M [3H]dexamethasone 21-mesylate in the presence and absence of IOO-fold excess concentration of the following unlabeled hormones: dexamethasone, estradiol, progesterone, testosterone, and triamcinolone acetonide. After labeling, the preparations were treated with charcoal,22 and specific binding of the postcharcoal supernatant was estimated by subtracting the values of radioactivity in competed samples from those of uncompeted preparations. Northern

analysis

RNA was isolated from mGR-enriched and -deficient S-49 cells produced by fluorescent cell sorting using the LiCl-urea precipitation technique of Auffray and Rougeon. 29Messenger RNA was isolated on an oligoDT cellulose column3’ and 10 pg of each sample was electrophoresed on a 1% formaldehyde gel in MOPS buffer 31 followed by capillary blot transfer to Gene Screen Plus (DuPont, Boston, MA, USA). The gel was stained with ethidium bromide to view markers (both Escherichia coli and rabbit reticulocyte poly(A-) RNA ribosomal bands). The blot was prehybridized and then hybridized according to the Gene Screen Plus manufacturer’s directions in a solution containing 47% formamide and 10% dextran sulfate at 42 C for 12 to 18 hours with nick-translated32 rat GR cDNA prepared from a plasmid containing the complete coding sequence.33 Quantitative RNA loading irregularities (of which none were evident) were monitored by ethidium bromide staining of residual ribosomal RNA and by subsequent hybridization of a probe to rat a-tubulin.34

Size and steroid-binding

7 BSA A

fi 3z

3000

0

mGR

0

mGR/
progesterone > dexamethasone > testosterone = estrogen. With regard to the triamcinolone acetonide binding, our data are at variance with those reported by Trueba et al.,49 who showed that triamcinolone acetonide did not bind to mGR. We do not know whether this discrepancy is attributed to tissue specificity, since the other investigators used liver cell plasma membranes, or to the steroid with which the initial labeling was accomplished. In addition, it is known that dexamethasone mesylate is a less specific ligand for the glucocorticoid receptor than other glucocorticoids and that many other proteins bind to this affinity ligand.‘O Therefore, large displacements of other steroids could be attributed as well to displacement from those other bound proteins. It was interesting to note that progesterone competed for dexamethasone 21-mesylate binding better than did dexamethasone in our preparation of mGR. Unusual hierarchies and broad steroid specificities seem to be characteristics of other membrane-resident steroid receptors that have been described.51-54 This may have to do with our present lack of understanding about which naturally occurring steroids are the primary ligands for these receptors” or from some conformational alternatives that such a receptor would experience in a lipid rather than an aqueous environment. Whole cell receptor binding studies of the mGRenriched, mGR-deficient, and wild-type S-49 cells showed a single class of glucocorticoid binding site and no significant variation of receptor affinity for steroid between the three cell groups. The 34% higher number of total binding sites per cell displayed by the mGRenriched cells versus the mGR-deficient cell population corroborates other data suggesting that receptor number correlates positively with hormonal response. Our immunosorting method of producing mGR-enriched cells and our other data suggest that the additional sites in the mGR-enriched cells consist of receptors predominantly associated with the plasma membrane. Although the intermediate number of binding sites for the wild-type population did not differ significantly in these experiments from the other cell populations, previous dataI suggest that these cells do have functional differences associated with intermediate number of plasma membrane receptors. Other studies from our laboratory support this finding. Northern analysis revealed higher mRNA concentration in the mGR-enriched cells than in the mGRdeficient cells, and immunocytochemical studies revealed a more intense GR antigen-specific extracellular fluorescence in the mGR-enriched cells than in either the wild-type or mGR-deficient cell groups.17 The two most frequently used methods for measuring glucocorticoid binding are the cytosolic and whole cell assays,

and receptor levels are usually much higher when whole-cell assays are applied.55*s6The presence of more receptor sites in the mGR-enriched cell group suggests that this assay system measures both the intracellular and plasma membrane-associated GR. In conclusion, we have shown that mGR and iGR differ in our characterizations only by size and a slightly altered ability of other steroids to compete for glucocorticoid binding. However, mGR and iGR share immunoreactivity and characteristics of binding to glucocorticoids. Further investigations will attempt to determine the degree of identity between these two receptors and their molecular etiology.

Acknowledgments This work was supported by grants from the NC1 (CA49297) and the MACC FUND (2-25oS-0). We thank Dr. Keith Yamamoto from the University of California-San Francisco for providing the rat glucocorticoid receptor cDNA. We also thank Dr. Raymond Hoffmann from the Medical College of Wisconsin for assistance with our statistical analysis.

References 1. 2. 3. 4. 5.

6. I. 8. 9.

10.

11.

12. 13. 14.

Claman HN (1972). Corticosteroids and lymphocytolysis. N Engl J Med 287:388-397. Goldin A, Sandberg J, Hendersonn E, Newman J (1976). The chemotherapy of human and animal acute leukemia. Cancer Chemother Rep 55:309-335. Lippman ME, Halterman RH, Leventhal BG, Perry S, Thompson EB (1973). Glucocorticoid-binding in human acute lvmphobla& leukemic cells. J Clin Znveit 52~1715-1725. . Lippman ME, Yarbro GK, Leventhal BG (1978). Clinical implications of ghxocorticoid receptor in human leukemia. Cancer Res 38:425 l-4256. Norman MR, Thompson EB (1977). Characterization of a glucocorticoid-sensitive human lymphoid cell line. Cancer Res 37~3785-3791. Bourgeois S, Newby RF (1977). Glucocorticoid resistance in murine lymphoma and thymoma lines. Cancer Res X4279-4284. Horibato D, Harris AW (1970). Mouse myelomas and lymphomas in culture. Exp Cell Res 60~61-77. Compton MM, Cidlowski FA (1987). Identification of plucocorticoid-induced nuclease in thymocytes. J Biol Chem 262~8288-8292. Alnemri ES, Litwack G (1989). Glucocorticoid-induced lymphocytolysis not mediated by an induced nuclease. J Biol Chem 264:4104-4111. Gruol DF, Rajah FM, Bourgeois S (1989). Cyclic AMP-dependent protein kinase modulation of glucocorticoid-induced cytolytic response in murine T-lymphoma cells. Mol Endocrinol 3~2119-2127. Crabtree GR, Smith FA, Munk A (1978). Glucocorticoid receptors and sensitivity of isolated human leukemia and lymphoma cells. Cancer Res 38~4268-4272. Peck EJ, Burgner J, Clark J (1973). Estrophylic binding sites of the uterus. Relationship to uptake and retention of estradiol in vitro. Biochemistry l2:45%-4603. Garman D, Center MS (1982). Alterations in cell surface membranes in Chinese hamster lung cells resistant to Adriamycin. Biochem Biophys Res Commun 105: 157-163. Touchette N (1990). Man bites dogma: a new role for steroid hormones. Steroid hormones, classical transcription regulators, may also bind to cell membrane receptors. J NZH Res 2:71-14.

Steroids,

1991, vol. 56, August

409

Papers 15.

16. 17.

18. 19. 20.

21.

22. 23. 24.

25.

26. 27. 28.

29. 30. 31. 32.

33.

34.

35. 36. 37.

410

Szego CM, Pietras RJ (1981). Membrane recognition and effector sites in steroid hormone action. In: Litwack G (ed), Biochemical Action of Hormones, ed. 8., Academic Press, San Diego, pp. 307-363. Hua SY, Chen YZ (1989). Membrane receptor-mediated electrophysiological effects of glucocorticoids on mammalian neurons. Endocrinology W:687-691. Gametchu B (1987). Glucocorticoid receptor-like antigen in lymphoma cell membranes: correlation to cell lysis. Science 236:456-461. Gametchu B, Harrison RW (1984). Characterization of monoclonal antibody to the rat glucocorticoid receptor. Endocrinology 114~274-279. Shapiro HM (1985). Flow sorting. In: PracticalNow Cytomefry. Alan R. Liss, New York, pp. 78-84. Koprowski H, Gerhard W, Croce CM (1977). Production of antibodies against influenza virus by somatic cell hybrids between mouse myeloma and primed spleen cells. Proc Nat1 Acad Sci USA 74:2985-2988. Simons SS, Thompson EB (1981). Dexamethasone 21-mesylate: an affinity label of glucocorticoids from rat hepatoma tissue culture cells. Proc Nat1 Acad Sci USA 78: 3541-3545. Korrenmann SG (1970). Relationship between estrogen inhibitory activity and binding of cytosol of rabbit and human uterus. Endocrinology 821119-l 123. Laemmli UK (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227~680-685. Garrels JI (1979). Two dimensional gel electrophoresis and computer analysis of proteins synthesized by clonal cell lines. J Biol Chem 245:7961-7977. Towbin H, Staehelin T, Gordon J (1979). Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Nat1 Acad Sci USA 76~4350-4354. Scatchard G (1949). The attractions of proteins for small molecules and ions. Ann NY Acad Sci 51:660-672. Munson PJ, Rodbard D (1980). A versatile computerized approach for characterization of ligand binding systems. Anal Biochem 107:220-239. Kirk RE (1982). Completely randomized design. In: Experimental Design: Procedure for Behavioral Scientists, 2d ed. Brooks/Cole Publishing, Monterey, CA, pp. 79-148. Auffray C, Rougeon F (1980). Purification of mouse immunoglobulin heavy-chain messenger RNAs from total myeloma tumor RNA. Eur J Biochem lW:303-314. Aviv H, Leder P (1972). Purification of biologically active globin messenger RNA by chromatography of oligoth;midylic acid-cellulose. Proc Nat1 Acad Sci USA 69:1408-1412. Thomas P (1980). Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc Nat1 Acad Sci USA-77~5201-5205. Wahl G. Stern M. Stark G (1979). Efficient transfer of DNA fragmenis from agarose gel‘to diazobenzylmethyl-paper and rapid hybridization by using dextran sulfate. Proc Nat1 Acad Sci USA 76~3683-3687. Miesfield R, Ocret S, Wilkstrom AC, Wrange 0, Gustafsson JA, Yamamoto KR (1984). Characterization of a steroid hormone receptor gene and mRNA in wild-type and mutant cells. Nature 3l2:779-781. Lemischka IR, Rarmer S, Racaniello VR, Phillip AS (1981). Nucleotide sequence and evolution of a mammalian a-tubulin messenger RNA. J Mel Biol151:101-120. Simons SS, Jr., Schleenbaker RE, Eisen HJ (1983). Activation of covalent affinity labeled glucocorticoid receptor-steroid complexes. J Biol Chem 258~2229-2238. Porter RR (1950). Human rhabdomyosarcoma cells induced in vitro by antineoplastic drugs. Biochem J 46~479-484. Northrop JP, Danielson M, Ringold GM (1986). Analysis of glucocorticoid unresponsive cell variants using a mouse glucocorticoid receptor complementary DNA clone. J Biol Chem 261:11064-l 1070.

Steroids,

1991, vol. 56, August

38.

39.

40.

41.

42.

43.

44.

45.

46.

47.

48. 49.

50.

51.

52.

53.

54.

55.

56.

Danielsen M, Northrop P, Ringold GM (1986). The mouse glucocorticoid receptor: mapping of domains by cloning, sequencing and expression of wild-type and mutant receptor proteins. EMBO J 5:2513-2522. Hollenberg SM. Weinberger C, Ong ES, Cerelli G, Oro A, Lebo R, Thompson EB, Rosenfeld MG, Evans RM (1985). Primary structure and expression of a functional human glucocorticoid receptor cDNA. Nature 318:635-641. Conneely OM, Sullivan WP, Toft DO, Bimbaumer M, Cook RG, Maxwell BL, Zarucki-Schulz T, Green GL, Schrader WT, O’Malley BW (1986). Molecular cloning of the chicken progesterone receptor. Science 233~767-770. Gaspar ML, Meo T, Tosi M (1990). Structure and size distribution of the androgen receptor mRNA in wild-type and Tfmly mutant mice. Mol Endocrinol4:1600-1610. Nohno T, Kasai Y, Saito T (1989). Novel cDNA sequence possibly generated by alternative splicing of a mouse glucocorticoid receptor gene transcript from Shionogi carcinoma 115. Nucleic Acids Res 17:445. Keaveney M, Klug J, Dawson MT, Nestor PV, Neilan JG, Forde RC, Gannon F (1991). Evidence for a previously unidentified upstream exon in the human oestrogen receptor gene. J Mol Endocrinol 6: 1 1 1- 115. Housley PR, Pratt WB (1983). Direct demonstration of glucocorticoid receptor phosphorylation by intact L-cells. J Biol Chem 258:4630-4635. Sanchez ER, Pratt WB (1986). Phosphorylation of L-cell glucocorticoid receptors in immune complexes: evidence that the receptor is not a protein kinase. Biochemistry 25:1378-1382. Rothman JE, Fine RE (1980). Coated vesicles transport newly synthesized membrane glycoprotein from endoplasmic reticulum to plasma membrane in two successive stages. Proc Nat1 Acad Sci USA 77~780-784. Dunbar BS, Timmons TM, Maresh GA (1990). Protein analysis using high-resolution two-dimensional polyacrylamide gel electrophoresis. In: Schrader WT, O’Malley B (eds), Laboratory Methods for Hormone Action and Molecular Biology, ed. 12. Baylor College of Medicine, Houston, pp. l-41. Kolata G (1985). Novel protein/membrane attachment. Science 229:850. Trueba M, Ballejo AI, Rodriguez I, Ibarrola I, Sancho MI, Marino A, Macarulla JM (1989). Evidence of presence of specific binding sites for corticoids in mouse liver plasma membranes. J Membr Biol108:115-124. Eisen HJ, Schleenbaker RE, Simons SS, Jr. (1981). Affinity labeling of the rat liver glucocorticoid receptor with dexamethasone-21-mesylate: identification of covalently labeled receptor by immunocytochemical methods. J Biol Chem 256:12920-12925. Towle AC, Sze PY (1983). Steroid binding to synaptic plasma membrane: differential binding of glucocorticoids and gonadal steroids. J Steroid Biochem l&135-143. Patino R, Thomas P (1990). Characterization of membrane receptor activity for 17a, 2Op, 21-trihydroxy-4-pregnen-3-one in ovaries of spotted seatrout (Cynoscion nebulosus). Gen Comp Endocrinol78:204-217. Smith LD, Ecker RE (1971). The interaction of steroids with Rana pipiens oocytes in the induction of maturation. Dev Biol 25~232-247. Sadler SE, Bower MA, Mailer JL (1985). Studies of a plasma membrane steroid receptor in Xenopus oocytes using the synthetic progestin RU 486. J Steroid Biochem 22:419-426. Iacobelli S, Natoli V, Longo P (1981). Glucocorticoid receptor determinations in leukemia patients using cytosol and whole cell assays. Cancer Res 41:3979-3984. Nanni P, Nocoletti G, Prodi G, Galli M, Giovanni C, Grilli S, Lollini P, Gobbi M, Cavo M, Tura S (1982). Glucocorticoid receptor and in vitro sensitivity to steroid hormones in human lymphoproliferative disorders and myeloid leukemia. Cancer 49~623-632.