Gordon M. Tomkins

Glucocorticoid receptors: relations between steroid binding and biological effects.

Abstract Steroid binding has been studied in cytoplasmic extracts of cultured rat hepatoma cells to investigate the mechanism of enzyme induction by glucocorticoids. The affinity of inducer steroids for the specific receptors contained in the extracts is directly related to the potency of these steroids as inducers of tyrosine amino-transferase. The ability of anti-inducer steroids to compete with inducers for binding is similar to their ability to inhibit induction. Furthermore, the affinity of an anti-inducer for the receptors can be predicted from its ability to inhibit inducer binding. These and other correlations allow distinction between the specific cytoplasmic receptors and a number of other molecules, includingplasma transcortin, which also bind glucocorticoid hormones. Further experiments were carried out to determine whether an allosteric model, proposed earlier, could explain the differential effects of inducer, suboptimal inducer and anti-inducer steroids. According to the model, steroids interact with either, or both, of two conformational states of the receptors; the uncomplexed receptors are predominantly in one (inactive) form and binding by inducers, but not by anti-inducers, increases the concentration of the other (active) conformation. Consistent with this idea, we find that receptors are less stable when they are free, or complexed by an anti-inducer, than when they are bound by an inducer. Furthermore, kinetic studies are in accordance with the proposal that binding by inducers, but not anti-inducers, is associated with conformational changes in the receptor molecules. Specific glucocorticoid receptors were also characterized in other tissues and found to be similar to those in hepatoma tissue culture cells.

Mechanisms of steroid resistance

Abstract A method is described for the rapid determination of binding capacity and subcellular distribution of radioactive dexamethasone by cultured lymphoma cells. Using it, a number of steroid-resistant lymphoma cell clones were shown to be deficient in steroid binding to specific cytoplasmic receptors (r-) in transfer of the receptor-steroid complex to nuclei (nt-), or in the reactions subsequent to nuclear localization of the complex (d-). The relative proportions of those types of variants were determined in a steady-state population. About 80% were r-, and the remainder were equally divided between nt- and d-. Quantitative steroid-binding experiments suggest that certain receptor-containing variants possess altered receptor molecules.

Dexamethasone-mediated induction of mouse mammary tumor virus RNA: a system for studying glucocorticoid action

We have investigated the mechanisms by which dexamethasone (a synthetic glucocorticoid) stimulates the production of mouse mammary tumor virus (MMTV) by cell cultures derived from mammary carcinomas of GR mice. Treatment of these cells with dexamethasone stimulates a rapid accumulation of intracellular virus-specific RNA which is dependent upon RNA synthesis but not upon DNA or protein synthesis. The effect of dexamethasone is probably mediated by a specific and saturable glucocorticoid receptor. We conclude that the accumulation of MMTV RNA is a primary response to dexamethasone and that the rate of synthesis of MMTV RNA is probably accelerated by treatment with dexamethasone.

Steroid-induced nuclear binding of glucocorticoid receptors in intact hepatoma cells.

Abstract Some of the early steps of steroid hormone action have been studied in cultured hepatoma cells, in which glucocorticoids induce tyrosine aminotransferase. The hypothesis that inducer steroids promote the binding of specific cytoplasmic receptors to the cell nucleus has been examined in intact cells. Binding of steroids such as dexamethasone and cortisol results in a loss of most of the receptor sites from the cytoplasm. This coincides with the binding of an equivalent number of steroid molecules in the nucleus. Both processes occur concomitantly, even when their kinetics are altered by reducing the temperature. When the inducer is removed from the culture, steroid dissociates from the nucleus while the level of cytoplasmic receptor returns to normal, even if protein or RNA synthesis is inhibited. These results suggest that nuclear binding of glucocorticoids is due to the association with the nucleus of the cytoplasmic receptor-steroid complex itself and make it unlikely that the receptor acts as a mere carrier for the intracellular transfer of the steroid. Steroids that differ in their effects on tyrosine aminotransferase induction were also studied. In contrast to those bound with inducer steroids, receptors complexed with the anti-inducer progesterone did not leave the cytosol. Further, a suboptimal inducer (deoxycorticosterone) produced an intermediate level of depletion. Thus, the biological effect of different classes of steroids can be related to their capacity to promote nuclear binding of the receptor. These data support a model proposed earlier, according to which the receptor is an allosteric regulatory protein directly involved in the hormone action, under the control of specific steroid ligands. They further suggest that the conformational state influenced by the inducer is such that a nuclear binding site on the receptor is exposed. Evidence is also presented that a distinct reaction takes place between the binding of the steroid to the receptor and the association of the complex with the nucleus. At 0 °C, this change is rate-limiting. It could correspond to the “activation” of receptor-steroid complexes known to be required for binding of the complexes by isolated nuclei, and thus represent an additional step in hormone action.

Glucocorticoid and mineralocorticoid receptors for aldosterone

Abstract Specific binding of aldosterone and dexamethasone by rat kidney and hepatoma tissue culture (HTC) cell cytosol has been studied. In cytosol of HTC cells, aldosterone and dexamethasone bind to a single class of sites with affinities that correspond with their potencies as inducers of tyrosine aminotransferase. Kidney cytosol, however, contains two classes of specific aldosterone binding sites. The higher affinity sites bind aldosterone with an affinity (equilibrium, dissociation, constant) which is similar to the plasma concentration of aldosterone required for antinatriuresis. The lower affinity aldosterone-binding sites are present at a higher concentration than the higher affinity sites and also bind dexamethasone with a high affinity. We have tentatively identified these two classes of binding sites in renal cytosol as “mineraloeonicoid” and “glucocorticoid” receptors, respectively.

Isolation of lymphoma cell variants resistant to killing by glucocorticoids

Abstract Pseudo-diploid (2s) and pseudo-tetraploid (4s) cultured mouse lymphoma cells are killed by physiological concentrations of adrenal glucocorticoid hormones. A method is described for the single-step isolation of 2s and 4s variants resistant to dexamethasone, a synthetic adrenal hormone. The transition from steroid sensitivity to resistance is stochastic and occurs at a rate of 3.5 × 10 −6 /cell/generation. Several types of mutagens significantly increase this rate. The chromosome number, growth rate, gross morphology, and cloning efficiency of the steroid-resistant variants are not detectably different from those of their steroid-sensitive parents.

Cyclic AMP-dependent protein kinase: pivotal role in regulation of enzyme induction and growth

Dibutyryl cyclic adenosine 3,5-monophosphate (cyclic AMP) produces phosphodiesterase induction, growth arrest, and cytolysis in S49 lymphoma cells. The striking parallelism between protein kinase activity that is dependent on cytosol cyclic AMP and cellular responses to dibutyryl cyclic AMP in wild-type cells and three classes of clones resistant to cyclic AMP indicates that protein kinase mediates cyclic AMP regulation of growth and enzyme induction in S49 cells.

A new mechanism for steroid unresponsiveness: Loss of nuclear binding activity of a steroid hormone receptor

Abstract The glucocorticoid, dexamethasone, binds to the specific cytosol receptors of a steroid-resistant mouse lymphoma cell line with the same affinity as to the receptors of the steroid-responsive parental cells. In the sensitive cells, the receptor-steroid complex translocates to the nucleus, whereas in the resistant cells nuclear transfer is greatly diminished. Activated receptor-dexamethasone complex from sensitive cells binds to isolated nuclei from both sensitive and resistant cell types, whereas the complex from the resistant cells binds to neither nuclei. Furthermore, the activated complex from sensitive cells binds to isolated homologous and heterologous DNA, whereas the complex from the resistant cells displays greatly reduced binding activity, implying that DNA plays a significant role in nuclear binding. These results suggest that the normal glucocorticoid receptor has two active domains: one for steroid binding, and the other for interaction with nuclear acceptor sites. The resistant cells described in this paper contain a receptor apparently defective in the latter activity.

Characterization of a hormone receptor defect in the androgen-insensitivity mutant

Abstract Mouse kidney cytosol contains specific receptors that reversibly bind dihydrotestosterone at a concentration of 43 f moles/mg protein. [Nonstandard abbreviation: DHT, dihydrotestosterone, 17 β-hydroxy-5 α-androstan-3-one.] The equilibrium dissociation constant of the receptor-dihydrotestosterone complex is 1.3 × 10 −9 M for females and 1.7 × 10 −9 M for castrated males. The complex sediments at 8–9S in glycerol gradients. In males bearing the androgen-insensitivity mutation (analogous to human testicular feminization), the specific dihydrotestosterone receptor activity is decreased about 8 fold. The residual binding activity has wild type affinity (K D = 1.5 × 10 −9 M) for dihydrotestosterone and also sediments at 8–9S. Kidney cytosol from castrated mutant mice displays a new binding component with low affinity and high capacity for dihydrotestosterone.

“Superinduction” of tyrosine aminotransferase by actinomycin D: a reevaluation

Reexamination of the effects of actinomycin D (AMD) on the intracellular level and rate of synthesis of tyrosine aminotransferase (TAT) in hepatoma tissue culture (HTC) cells reveals that much apparent controversy can be resolved with acknowledgment of the multi-faceted nature of this inhibitors action. AMD can slow overall protein synthesis and inhibit the degradation of both TAT and its mRNA as well as block the synthesis of RNA. The extent of these secondary actions of the inhibitor depend somewhat upon the growth condition of the cells. The effects of cordycepin (3-deoxyadenosine) on the metabolism of TAT and its mRNA are also complex, but differ in several respects from those of AMD.