Characterization of the AtT-20 cell's ‘repleting’ glucocorticoid receptor

Glucocorticoids deplete the AtT-20 mouse pituitary tumor cell of its glucocorticoid receptors. Once placed into steroid-free medium, however, these ‘receptor-deplete’ cells gradually regain their ability to bind glucocorticoids displaceably. The goal of this paper is to determine whether this replete binding-species is indeed a glucocorticoid receptor, and whether any precursor or modified forms are seen during repletion. Once depleted, cells take nearly 3–4 days to replete their full complement of cytosolic glucocorticoid-binding species. Scatchard analysis revealed only one binding site, of which the affinity for dexamethasone was identical to that of the native receptor. The steroid-binding specificity of the ‘replete’ binding species was exactly the same as that of the original receptor. The molecular size and surface charge density of the glucocorticoid-binding species obtained throughout the process of repletion were identical to those of the original receptor. Finally, studies in which replete cells were exposed to glucocorticoid agonists showed that the cell was able to decrease its production of ACTH, indicating that the regenerated binding species was able to function as a biologically active glucocorticoid receptor. We conclude that the replete species is a glucocorticoid receptor identical to the original, and that probably no precursor or modified forms of the repleting receptor exist.     

Activation of the dioxin and glucocorticoid receptors to a DNA binding state under cell-free conditions

The activation in vitro of dioxin and glucocorticoid receptors from a non-DNA binding to a DNA binding state was characterized. Ligand-free dioxin and glucocorticoid receptors were partially co-purified from rat liver cytosol, and both receptors sedimented at 9 S following labeling with the respective ligand. The 9 S forms of the dioxin and glucocorticoid receptors have previously been shown to represent heteromeric complexes containing the Mr approximately equal to 90,000 heat shock protein. The 9 S ligand-free or ligand-bound glucocorticoid receptor was converted to the monomeric 4-5 S form upon exposure to 0.4 M NaCl even in the presence of the stabilizing agent molybdate. Under identical conditions, the 9 S ligand-free and ligand-bound dioxin receptor forms remained essentially intact. However, in the absence of molybdate, the dioxin receptor could be converted to a 4-5 S form upon exposure to high concentrations of salt. These results indicate that the glucocorticoid receptor readily dissociates from the 9 S to the 4-5 S form even in the absence of hormone, whereas both the ligand-free and ligand-occupied 9 S dioxin receptor forms represent more stable species. Gel mobility shift experiments revealed that the 4-5 S glucocorticoid receptor interacted with a glucocorticoid response element both in the absence and presence of ligand. On the other hand, occupation of the dioxin receptor by ligand greatly enhanced the ability of the receptor to be activated to a form that binds to its target enhancer element. Once dissociated, the monomeric form of the dioxin receptor was also able to interact with its DNA target sequences even in the absence of ligand. Thus, ligand binding efficiently facilitates subunit dissociation of the dioxin receptor but is not a prerequisite for DNA binding per se. Given the apparent stability of its non-DNA binding 9 S form, the dioxin receptor system might be a useful model for the investigation of the mechanism of activation of soluble receptor proteins.     

Factor-assisted DNA binding as a possible general mechanism for steroid receptors. Functional heterogeneity among activated receptor-steroid complexes

We previously reported that activated glucocorticoid receptor-steroid complexes from rat HTC cell cytosol exist as at least two sub-populations, one of which requires a low molecular weight (700–3000 Da) factor(s) for binding to DNA. This factor is removed by Sephadex G-50 chromatography and is found predominantly in extracts of crude HTC cell nuclei. We have now determined that factor is not limited to HTC cells since an apparently identical factor(s) was found in nuclear extracts of rat kidney and liver as well as human HeLa and MCF-7 cells. Furthermore, the DNA binding of a sub-population of human glucocorticoid receptors depends on factor. While these results were obtained with agonist (dexamethasone) bound receptors, a sub-population of HTC cell receptors covalently labeled by the antiglucocorticoid dexamethasone 21-mesylate also displayed factor-dependent DNA binding. This receptor heterogeneity was not an artifact of cell-free activation since the cell-free nuclear binding of dexamethasone mesylate labeled complexes was, as in intact cells, less than that for dexamethasone bound complexes. Earlier results suggested that the increased DNA binding with factor involved a direct interaction of receptor with factor(s). We now find that the factor-induced DNA binding is retained by amino terminal truncated (42 kDa) glucocorticoid receptors from HTC cells. Thus the ability of receptor to interact with factor(s) is encoded by the DNA and/or steroid binding domains. Two dimensional gel electrophoresis analysis of dexamethasone-mesylate labeled 98 kDa receptors revealed multiple charged isoforms for both sub-populations but no differences in the amount of the various isoforms in each sub-population. Finally, activated progesterone and estrogen receptor complexes were also found to be heterogeneous, with a similar, if not identical, small molecular weight factor(s) being required for the DNA binding of one sub-population. The observations that functional heterogeneity of receptors is not unique to glucocorticoid receptors, whether bound by an agonist or antagonist, and that the factor(s) is neither species nor tissue specific suggests that factor-assisted DNA binding may be a general mechanism for all steroid receptors.     

Assembly of a glucocorticoid receptor complex prior to DNA binding enhances its specific interaction with a glucocorticoid response element

Gel retardation analysis with full- and half-palindromic sequences using partially purified glucocorticoid receptor (GR) resulted in GR-glucocorticoid response element (GRE) species of identical mobilities, suggesting that formation of the dimeric GR protein complex is not catalyzed by DNA binding. These results are in contrast to the behavior of the isolated DNA binding domain of the glucocorticoid receptor where dimerization occurred on the GRE. Density gradient centrifugation of cytosolic GR resulted in two forms, a 4 S peak characteristic of the monomeric GR and a fraction which sediments at 6 S which is consistent with the observed size of the dimeric GR. These two forms were found to differ in their ability to bind to specific DNA sequences with the 6 S species having a higher affinity for a GRE. Taken together our results are consistent with a two-step model for hormone-induced transformation of GR: dissociation of the multimeric untransformed complex and dimerization of the GR to yield a high affinity DNA binding species.     

The glucocorticoid antagonist 17 alpha-methyltestosterone binds to the 10 S glucocorticoid receptor and blocks agonist-mediated dissociation of the 10 S oligomer to the 4 S deoxyribonucleic acid-binding subunit

The glucocorticoid antagonist 17 alpha-methyltestosterone inhibits binding of the agonist [3H]triamcinolone acetonide ot the glocucorticoid receptor in cytosol prepared from rat pituitary tumor GH1 cells. Competitive binding studies indicate that the dissociation constant for 17 alpha-methyltestosterone is about 1 microM. After incubation of intact GH1 cells with 10 nM [3H]triamcinolone acetonide at 37 C and subsequent cell fractionation at 4 C, three glucocorticoid receptor forms are observed: cytosolic 10 S receptor, cytosolic 4 S receptor, and nuclear receptor. Concurrent incubation with 17 alpha-methyltestosterone reduces the amount of [3H]triamcinolone acetonide bound to each of these receptor forms. Ligand-exchange assays performed at 0 C in intact cells using [3H]triamcinolone acetonide show that the exchangeable antagonist is associated predominantly with cytosolic 10 S receptor. Immunochemical analysis using monoclonal antibody BuGR2 indicates that 17 alpha-methyltestosterone does not cause substantial accumulation of glucocorticoid receptors in GH1 cell nuclei and, when present together with agonist, reduces nuclear accumulation of receptor seen with agonist alone. Results from dense amino acid labeling studies show that unlike [3H]triamcinolone acetonide, 17 alpha-methyltestosterone does not reduce the total amount of cellular glucocorticoid receptor and does not reduce receptor half-life. These results are consistent with a model for glucocorticoid receptor transformation in which binding of agonist promotes the dissociation of an oligomeric 10 S cytosolic receptor protein to its DNA-binding 4 S subunit. The antagonist 17 alpha-methyltestosterone competes with agonist for binding to the 10 S cytosolic receptor but does not appear to promote dissociation of the oligomer, thus inhibiting agonist-mediated nuclear actions of the glucocorticoid receptor.     

Corticosterone binding in AtT-20 pituitary tumor cell cytosol: evidence for one class of binding site for both natural and synthetic glucocorticoids.

The AtT-20 mouse pituitary cell is an established, cloned cell line which produced adrenocorticotrophic hormone in a glucocorticoid-suppressible manner. A receptor for glucocorticoids was identified in cytosol prepared from these cells using the natural mouse glucocorticoid, corticosterone, as the labeled ligand. The question of whether this binding component is identical to the one detectable using labeled triamcinolone acetonide was addressed by comparing their physicochemical characteristics and by detailed studied of binding specificity using both ligands. The corticosterone and triamcinolone acetonide binding components behaved similarly on sucrose density gradient analysis and DEAE-cellulose ion-exchange chromatography. Scatchard analysis with corticosterone detected 30% fewer binding sites than a similar analysis with triamcinolone acetonide, probably because corticosterone binding was of lower affinity (Kd = 8.6 . 10(-9)M vs. 1.4 . 10(-9)M) and hence less stable. The relative glucocorticoid binding affinities of thirteen unlabeled steroids were obtained using either labeled steroid as ligand. Both ligands yielded similar results, suggesting that they both detected a similar binding site. The results suggest that AtT-20 cell cytosol contains a single class of binding site which detects both natural and synthetic glucocorticoids.     

Nonactivated and activated glucocorticoid receptor complexes from human salivary gland adenocarcinoma cell line

[3H]Triamcinolone acetonide glucocorticoid receptor complexes from human salivary gland adenocarcinoma cells (HSG cells) were shown to be activated with an accompanying decrease in molecular weight in intact cells, as analyzed by gel filtration, DEAE chromatography, the mini-column method and glycerol gradient centrifugation. Glucocorticoid receptor complexes consist of steroid-binding protein (or glucocorticoid receptor) and non-steroid-binding factors such as the heat-shock protein of molecular weight 90,000. To determine whether the steroid-binding protein decreases in molecular weight upon activation, affinity labeling of glucocorticoid receptor in intact cells by incubation with [3H]dexamethasone 21-mesylate, which forms a covalent complex with glucocorticoid receptor, was performed. Analysis by gel filtration and a mini-column method indicated that [3H]dexamethasone 21-mesylate-labeled receptor complexes can be activated under culture conditions at 37 degrees C. SDS-polyacrylamide gel electrophoresis of [3H]dexamethasone 21-mesylate-labeled steroid-binding protein resolved only one specific 92 kDa form. Furthermore, only one specific band at 92 kDa was detected in the nuclear fraction which was extracted from the cells incubated at 37 degrees C. These results suggest that there is no change in the molecular weight of steroid-binding protein of HSG cell glucocorticoid receptor complexes upon activation and that the molecular weight of nuclear-binding receptor does not change, although the molecular weight of activated glucocorticoid receptor complexes does decrease. Triamcinolone acetonide induced an inhibitory effect on DNA synthesis in HSG cells. Dexamethasone 21-mesylate exerted no such effect and blocked the action of triamcinolone acetonide on DNA synthesis. These results suggests that dexamethasone 21-mesylate acts as antagonist of glucocorticoid in HSG cells. The fact that dexamethasone 21-mesylate-labeled receptor complexes could be activated and could bind to DNA or nuclei as well as triamcinolone acetonide-labeled complexes suggests that dexamethasone 21-mesylate-labeled complexes can not induce specific gene expression after their binding to DNA.     

Evidence for glucocorticoid transport into AtT-20/D-1 cells.

Glucocorticoid uptake by AtT-20/D-1 mouse pituitary adenocarcinoma cells grown in tissue culture was examined. The binding of triamcinolone acetonide, a potent synthetic glucocorticoid, by intact cells and by cell cytosol was studied at both 4 and 25 degrees. Specific binding of [3H]triamcinolone acetonide by intact cells was markedly different from cell-free cytosol binding at 4 degrees. Intact cells bound a relatively small amount of labeled steroid within 2 min, after which no further binding was observed. In contrast, the receptor in a cell-free cytosol preparation was capable of binding steroid progressively at 4 degrees, indicating that the limited binding by intact cells was not a consequence of receptor characteristics. At 25 degrees, uptake by intact cells and cytosol was nearly identical and appeared to be limited only by the binding kinetics of the cytosol receptor. Estradiol-17 beta, a nonglucocorticoid steroid, was not bound by the AtT-20/D-1 cell at 4 degrees. Triamcinolone was not bound significantly at 4 or 25 degrees by an adrenal carcinoma cell that does not appear to be a glucocorticoid target cell. An Arrhenius plot of cell steroid uptake vs. the reciprocal of absolute temperature revealed an abrupt change in slope at 16 degrees, which is compatible with the temperature-dependent mechanism involved in glucocortidoid uptake being associated with lipid constituents of the cell membrane. These data suggest that glucocorticoid uptake by this target cell involves a mechanism of specific, temperature-dependent transport through the cell membrane.     

Cross-reactivity of a monoclonal antiglucocorticoid receptor antibody BuGR1 with glucocorticoid and mineralocorticoid receptors of various species

The reactivity of a monoclonal antibody BuGR1, raised against glucocorticoid receptors of rat liver, with glucocorticoid and mineralocorticoid receptors of mammalian (rabbit) and amphibian (A6 cells) origin was examined. The glucocorticoid receptors of rabbit kidney and liver and of A6 cells were labeled with tritiated dexamethasone. The mineralocorticoid receptors were labeled with tritiated aldosterone in the presence or absence of RU26988, depending on whether aldosterone was bound to glucocorticoid receptors (A6 cells) or not (rabbit kidney), in addition to its binding to mineralocorticoid receptors. BuGR1 did not recognize mineralocorticoid receptors of A6 cells and rabbit kidney. BuGR1 cross-reacted with glucocorticoid receptors of rabbit liver and kidney but not of A6 cells, suggesting that the domain of glucocorticoid receptors recognized by BuRG1 could be present only in the mammalian species. The findings indicate that BuGR1 shows species differences as well as receptor class specificity.     

Corticosterone regulates the level of hepatic glucocorticoid receptors in mice

The effect of exogenous corticosterone on the level of mouse hepatic glucocorticoid receptor was monitored to ascertain whether agonist-induced glucocorticoid receptor regulation takes place in living animals as it does in isolated cell systems. Adrenalectomized male Swiss-Webster mice were given 1 mg of corticosterone ip and 24 hr later the glucocorticoid receptor binding capacity of a high-speed cytosolic extract of liver was measured. It was shown that at this time point the administered steroid had been totally cleared and thus, the decrease in binding capacity was a reflection of downregulation. Receptor binding capacity was decreased by 25%. Downregulation was not permanent; 48-72 hr after the injection receptor content returned to baseline. Multiple daily injections of corticosterone were no more effective at causing downregulation than a single injection. It is concluded that glucocorticoid agonists downregulate their own receptors in the glucocorticoid target organs of intact animals as they do in cloned cell models.     

Characterization of wild type and mutant glucocorticoid receptors from rat hepatoma and mouse lymphoma cells

Using a combination of immunological blotting techniques and hormone affinity labeling, we have characterized the glucocorticoid receptors present in wild type and mutant rat hepatoma (HTC) and mouse thymoma (S49 and WEHI7) cells. Mutant HTC and WEHI7 cells of the receptorless phenotype, which contain greatly reduced amounts of glucocorticoid hormone binding activity, show parallel decreases in immunoreactive material using a monoclonal antibody raised against the rat liver glucocorticoid receptor. This indicates that these receptorless mutant cells harbor defects in either the production or accumulation of receptor protein. Quantitation of immunoreactivity and hormone binding activity present in wild type and mutant S49 cells indicates that these cells contain significantly more immunoreactive material than hormone binding activity. We conclude that S49 cells produce, in addition to their well characterized wild type or mutant receptors, a mutant receptor from a second allele which is of wild type size, is immunologically reactive, but is unable to bind hormone. The S49 mutant cell line nti (nuclear transfer increase) contains a glucocorticoid receptor which has a molecular weight of 40,000, while the wild type receptor has a molecular weight of 94,000. Affinity labeling of glucocorticoid receptors in nti cells with [3H]dexamethasone mesylate indicates that nti cells do not contain wild type sized precursor molecules which bind hormone, nor do they contain immunoreactive fragments of a molecular mass smaller than 94 kDa. It is proposed that the 40-kDa nti receptor is produced as a truncated protein most likely resulting from a nonsense mutation or from a truncated messenger RNA.     

Analysis of the relation between receptor binding affinity and antagonist efficacy of antiglucocorticoids

The biological potencies of four antiglucocorticoids, RU486 (RU), dexamethasone-oxetanone (DOX), R5020, and progesterone have been studied with respect to dexamethasone induction of tyrosine aminotransferase (TAT) in rat hepatoma tissue culture (HTC) cells. Their inhibitory effects in whole-cell competition binding studies (at 37 degrees C) and in TAT induction studies were analyzed by Dixon plots and Schild plots, respectively. We show that: In both cases, there is an actual competition of each antiglucocorticoid with the agonist dexamethasone for the same binding site; the two Kd values derived from the two plots are almost identical for each antiglucocorticoid; RU486 can be distinguished from the three other antiglucocorticoids by its high biological efficacy and its high affinity for the glucocorticoid receptor in whole cells at 37 degrees C (identical to its affinity in cytosol at 0 degree C). These results imply that: There is a linear correlation between the antagonist efficacies of antiglucocorticoids and their affinities for the glucocorticoid receptor in whole cells at 37 degrees C; the antagonistic action is solely mediated by competition with the agonist for the receptor binding site; this is verified by the fact that in all cases, in the presence or absence of antiglucocorticoids, a specific TAT induction level was always related to the same level of receptor saturation by the agonist in whole cells; the phenomena responsible for the high antagonist efficacy of RU486 are also responsible for its high affinity in whole cells at 37 degrees C.     

Evaluation of murine interleukin 4 (IL-4) receptor expression using anti-receptor monoclonal antibodies and S1 nuclease protection analyses

Anti-receptor antibodies have previously been used in two cytokine systems (IL-1 and TNF alpha) to identify the existence of different cytokine receptors on different cell types. In this study, we have similarly used two approaches to evaluate whether IL-4 receptors on different cell types are identical, or whether more than one species of IL-4 receptor exists. The first approach involved production of monoclonal antibodies specific for the IL-4 receptor expressed by the murine mast cell line, MC/9. Six anti-IL-4 receptor monoclonal antibodies were produced against the purified soluble extracellular domain of the recombinant IL-4 receptor derived from MC/9 cells. These antibodies were capable of binding to and specifically immunoprecipitating the soluble extracellular domain of the recombinant mast cell IL-4 receptor. Following biotinylation of the antibodies and addition of phycoerythrin-streptavidin, their binding to cell associated IL-4 receptors on MC/9 mast cells could be readily visualized by immunofluorescence. Using this approach, the anti-mast cell IL-4R antibodies were found to specifically bind IL-4 receptors expressed on a variety of other murine cell types, including T cells, B cells, macrophages, fibroblasts, and L cells. The antibodies did not bind to two human cell lines known to bind human but not murine IL-4. The intensity of staining was directly related to the number of IL-4 binding sites identified previously by receptor-ligand equilibrium binding analyses. As a second approach to evaluating potential receptor heterogeneity, we constructed S1 nuclease protection assay probes for two separate regions of the mast cell IL-4 receptor, one located in the extracellular domain and one in the intracellular domain. Subsequent S1 analyses showed that both regions are expressed by the following types of cells: T cells, B cells, macrophages, myeloid cells, L cells, and stromal cells. The two approaches used in this study therefore indicate that the same or highly similar IL-4 receptor species is expressed by a wide variety of hemopoietic and nonhemopoietic cells. Since the anti-IL-4 receptor antibodies produced in this study did not block binding of IL-4 to its receptor, we cannot exclude the possible existence of a second type of IL-4R coexpressed on the cells tested in this study, or expressed uniquely by other cell types that were not investigated.     

Binding studies of the antiglucocorticoid RU38486 in Daudi and Raji lymphoma cells

The activity of RU38486 has been studied in Burkitt's lymphoma cells which are Epstein-Barr virus (EBV) positive. The early antigens (EA) of the virus are induced by dexamethasone (DXM) in Daudi but not in Raji cells, whereas a growth factor (transforming growth factor-beta, TGF-beta) induces the EA in both cell lines. RU38486 blocks the EA induction obtained by DXM or by TGF-beta in either cell line. In order to understand the interaction of RU38486, we considered its binding to specific receptors. We first investigated the binding of the antagonist in whole cells at 22 degrees C. A number of specific binding sites higher for RU38486 than for DXM was found, suggesting that RU38486 may bind to the glucocorticoid receptor and also to other cellular structures which we called the antiglucocorticoid binding sites ("AGBS"). To support this hypothesis, competition experiments have been conducted between RU38486 and other steroid hormones (progesterone and testosterone) since it is known that RU38486 is also able to interact with their cognate receptors. Binding studies of RU38486 in vitro at 4 degrees C in the presence of cytosolic extracts from Daudi and Raji cells led to conclusions similar to those drawn from the whole cell experiments: more complexes were formed with RU38486 than with DXM. Finally, the steroid-receptor complexes were incubated with DNA-cellulose. Since the binding measured for RU38486 was higher than for DXM, we suspect that sites different from the classical glucocorticoid receptor sites are also able to interact with DNA. The blockage exerted by RU38486 on the EA induced by glucocorticoids or by non-steroidal molecules and the lack of responsiveness to glucocorticoids in Raji cells are discussed in the light of the present findings.     

Nonactivated and activated glucocorticoid receptor complexes from human salivary gland adenocarcinoma cell line

[³H]Triamcinolone acetonide glucocorticoid receptor complexes from human salivary gland adenocarcinoma cells (HSG cells) were shown to be activated with an accompanying decrease in molecular weight in intact cells, as analyzed by gel filtration, DEAE chromatography, the mini-column method and glycerol gradient centrifugation. Glucocorticoid receptor complexes consist of steroid-binding protein (or glucocorticoid receptor) and non-steroid-binding factors such as the heat-shock protein of molecular weight 90 000. To determine whether the steroid-binding protein decreases in molecular weight upon activation, affinity labeling of glucocorticoid receptor in intact cells by incubation with [³H]dexamethasone 21-mesylate, which forms a covalent complex with glucocorticoid receptor, was performed. Analysis by gel filtration and a mini-column method indicated that [³H]dexamethasone 21-mesylate-labeled receptor complexes can be activated under culture conditions at 37°C. SDS-polyacrylamide gel electrophoresis of [³H]dexamethasone 21-mesylate-labeled steroid-binding protein resolved only one specific 92 kDa form. Furthermore, only one specific band at 92 kDa was detected in the nuclear fraction which was extracted from the cells incubated at 37°C. These results suggest that there is no change in the molecular weight of steroid-binding protein of HSG cell glucocorticoid receptor complexes upon activation and that the molecular weight of nuclear-binding receptor does not change, although the molecular weight of activated glucocorticoid receptor complexes does decrease. Triamcinolone acetonide induced an inhibitory effect on DNA synthesis in HSG cells. Dexamethasone 21-mesylate exerted no such effect and blocked the action of triamcinolone acetonide on DNA synthesis. These results suggests that dexamethasone 21-mesylate acts as antagonist of glucocorticoid in HSG cells. The fact that dexamethasone 21-mesylate-labeled receptor complexes could be activated and could bind to DNA or nuclei aas well as triamcinolone acetonide-labeled complexes suggests that dexamethasone 21-mesylate-labeled complexes can not induce specific gene expression after their binding to DNA.     

Binding of glucocorticoid antagonists to androgen and glucocorticoid hormone receptors in rat skeletal muscle

The binding of ten steroids possessing antiglucocorticoid activity has been studied in rat skeletal muscle cytosol. The affinity of these steroids for both the androgen and the glucocorticoid receptors was determined by competition with radioactive R1881 (methyltrienolone, metribolone) and dexamethasone, respectively. The antiglucocorticoid activity of these compounds was assessed in rat hepatoma (HTC) cells by measuring their inhibitory effect on the glucocorticoid-induced tyrosine aminotransferase activity. This led to identification of five novel in vitro glucocorticoid antagonists. All the steroids tested bound to both the glucocorticoid and the androgen receptors in muscle. Four steroids had an affinity for the glucocorticoid receptor higher than for the androgen receptor. The assumption is made that the steroids tested also behave as antagonists when binding to the glucocorticoid receptor in muscle and behave as agonists when binding to the androgen receptor. On this basis, the data allow one to compute a potential anticatabolic (PAG) and a potential anabolic (PAA) index for each compound. These indices might be of predictive value to determine whether these steroids exert their anabolic action in muscle through the glucocorticoid receptor or through the androgen receptor. The data also make it unlikely that satellite cells are a preferential target for anabolic steroids in muscle.     

Two forms of precursors of a heparin-binding EGF-like growth factor-diphtheria toxin receptor

A new mRNA coding for the heparin-binding EGF-like growth factor (HB-EGF) was found in Vero cells. The corresponding cDNA had C-156 in place of T, which resulted in a loss of the NheI site and a substitution of Leu-33 with Pro in the HB-EGF precursor. The known and new forms of the precursor were accordingly termed L and P. A possible conformational change in the corresponding propeptide region were assumed to affect processing of soluble secreted HB-EGF. The L and P mRNAs are differently expressed in various cell lines and have the identical 5'-untranslated sequences. Possibly, they are transcribed from one promoter and then alternatively spliced. Stimulation of resting Vero cells with tetraphorbol ester (TPA) substantially increased production of the L form, decreased production of the P form, and did not affect expression of the total HB-EGF mRNA. This was associated with an increase in binding of the diphtheria toxin, suggesting that the L HB-EGF precursor acts as its receptor.     

Differences in nuclear retention characteristics of agonist-activated glucocorticoid receptor may determine specific responses

We studied the glucocorticoid response to the synthetic steroid pregna-1,4-diene-11beta-ol-3,20-dione (DeltaHOP) in several cell types and correlated its biological effect with the ability of the glucocorticoid receptor (GR) to be retained in the nuclear compartment. We observed that the DeltaHOP-transformed GR was diffusely distributed in the nucleus compared to the discrete structures observed for the dexamethasone (DEX)-transformed GR. Despite the fact that the receptor was entirely nuclear upon binding of each steroid and exhibited identical nuclear export rates, a greater amount of DeltaHOP-transformed GR was recovered in the cytoplasmic fraction after hypotonic cell lysis. Furthermore, accelerated nuclear export of GR was evidenced in digitonin-permeabilized cells treated with ATP and molybdate. Inasmuch as limited trypsinization of DEX-GR and DeltaHOP-GR complexes yielded different proteolytic products, we conclude that GR undergoes a differential conformational change upon binding of each ligand. We propose that these conformational differences may consequently lead to changes of stability in the interaction of the GR with chromatin. Therefore, the dynamic exchange of liganded GR with chromatin is likely to have significant consequences for the observed pleiotropic physiological responses triggered by glucocorticoid ligands, not only in different tissues but also in the same cell type.