Nuclear-bound Complexes Research Articles

Binding of cytoplasmic testosterone-receptor complex to nucleic from male rat livers.

A crude cytoplasmic androgen receptor preparation of rat livers or its partially purified one by a 30% ammonium sulfate precipitation was prelabeled with [3H] testosterone ([3H]T) and then incubated with nuclei from rat livers at 30 degrees C for 30 min. The formations of the [3H]T-cytosol receptor complex and its nuclear bound complex were determined. The formation of testosterone-cytosol receptor complex was a prerequisite for the nuclear binding of testosterone. Nuclear fraction, when incubated with the prelabeled crude cytosol, bound [3H]T-receptor complex as much as when incubated with the prelabeled partially purified cytosol. The addition of unlabeled crude cytosol to the prelabeled partially purified cytosol increased the nuclear binding of the receptor. The presence of a thermostable, low molecular inhibitor and a thermolabile, macromolecular activator in the crude cytosol preparation was suggested by dialysis, heating and gel chromatography.

Cyclic, nonequilibrium models of glucocorticoid antagonism: Role of activation, nuclear binding and receptor recycling

Quantitative models that have been proposed to date to explain mechanisms of glucocorticoid antagonism have generally been of the equilibrium type, involving hypothetical allosteric equilibria between active and inactive states of the receptor or the steroid-receptor complex. We describe here the agonist-antagonist relationships predicted by a nonequilibrium cyclic model that we have recently devised to account for the kinetic behavior of glucocorticoid-receptor complexes in intact rat thymus cells. This model simulates quantitatively most kinetic and steady state results that have been obtained so far. It postulates the existence of only well-established receptor species, and its kinetic parameters can in principle be determined by receptor measurements with intact cells. To calculate the steady state agonist-antagonist properties it is assumed that biological activity is proportional to the total amount of nuclear-bound complex, whether formed by agonist or antagonist. The agonist activity of a steroid is determined by the steady state ratio of nuclear-bound to total complexes it forms. This ratio varies from 0 for a pure antagonist to 1 for a pure agonist. It turns out to be independent of agonist and antagonist concentrations, and a function only of the rate constants for the reactions of the complexes formed by a steroid. Analysis of the dependence of the ratio on each rate constant shows quantitatively how each reaction in the cyclic model—activation of the nonactivated complex, nuclear binding of the activated complexes, and dissociation and recycling of activated and nuclear-bound complexes—affects antagonist properties. Steady state interactions of agonists with antagonists are found to be determined by equations that are identical to those for competition in simple equilibrium systems. Predicted dose-response relations agree qualitatively with experimentally observed relations. They are similar to those predicted by two-state allosteric models, although the cyclic model has no allosteric mechanisms, and is based on quite different assumptions. Present limitations of the model arise particularly from lack of information about the mechanisms by which nuclear-bound complexes generate biological activity; for lack of such information the model includes no steps to account for substances that have low agonist activity despite forming nuclear-bound complexes.

Steroid hormone antagonism and a cyclic model of receptor kinetics

Steady state agonist-antagonist relations have been derived for a general version of a cyclic model of glucocorticoid-receptor kinetics. The model was previously shown to account quantitatively for the transient and steady state distribution of hormone-receptor complexes formed in thymus cells by several glucocorticoids. Agonist-antagonist properties of a steroid in the model are expressed quantitatively by its “agonist activity” A, the steady state ratio of nuclear-bound to total complexes it forms. For a pure agonist A = 1, for a pure antagonist A = 0. This ratio is found to be independent of steroid concentration and a function only of the rate constants of reactions involving complexes formed by the steroid. Analysis of the dependence of A on each rate constant reveals how each reaction in the cyclic model—activation, nuclear binding, dissociation of activated and nuclear-bound complexes—influences antagonist properties. The steady state interaction of an antagonist with an agonist is shown to be governed by relations that are indistinguishable from competition relations for the simplest equilibrium system, and to yield dose-response curves that are very similar to those produced by two-state allosteric models of steroid hormone antagonism, despite the fact that the cyclic model includes no allosteric mechanisms. With steroids for which relevant rate constants can be measured, the model is directly testable. Limitations of the model arise from lack of information about the nuclear events that lead to biological activity following binding of activated complexes to the nucleus.

Studies on the antiglucocorticoid action of progesterone in rat thymocytes: Early in vitro effects

Progesterone was found to exhibit antiglucocorticoid properties in rat thymocytes, as indicated by its ability to compete effectively for triamcinolone acetonide (TA) binding sites as well as to inhibit the biochemical effects of TA. In a cell free system, progesterone competitively inhibited the binding of TA to glucocorticoid-specific binding sites and was more effective than the antiglucocorticoid cortexolone in this respect. Progesterone at 10 −7−10 −6M antagonized the inhibitory effect of 5 × 10 −8TA on RNA metabolism, although it was inactive by itself as an inhibitor of [ 3H]-uricline uptake and incorporation into thymocyte RNA; this antagonism was not due to competition by progesterone for TA uptake into the cells. Progesterone was less efficient than cortisol or TA in depleting the cytosol receptors. However, unlike the glucocorticoid agonists which formed a temperature-dependent, salt extractable nuclear bound complex, progesterone was bound tightly to a fraction in the 27,000 g pellet which was unaffected by changes in the incubation temperature or salt extraction conditions. Additional binding of progesterone was observed in cells pre-saturated with TA. suggesting the presence in rat thymocytes of progesterone binding components other than the glucocorticoid receptors.

Activation of steroid hormone–receptor complexes in intact target cells in physiological conditions

AT physiological temperatures glucocorticoids1–5, in common with other steroid hormones6, initially interact with normal target cells such as the rat thymocyte by forming hormone–receptor complexes that seem first to be located in the cytoplasm and then rapidly become bound to the nucleus. At low temperatures (0–4 °C) the hormones, when incubated either with target cells or with cytosols from such cells, form ‘non-activated’ complexes that do not bind to nuclei; subsequent warming of the cells1–5, or of the cytosols together with nuclei2,7–11, leads to the formation of nuclear-bound complexes. As shown originally with oestrogens12 and later with glucocorticoids2,7–10,13, if cytosols with non-activated complexes formed at 0–4 °C are warmed to 20–37 °C in the absence of nuclei, the complexes become ‘activated’, and will then bind to nuclei even at low temperatures. Activation, which can also be brought about by increased ionic strength7–9,13, gel filtration, dilution and other treatments14, has been studied extensively and is known to be accompanied by several changes other than enhanced affinity for nuclei. For glucocorticoid–receptor complexes the changes include enhanced affinity for DNA7,10,13–15 and altered mobility on phosphocellulose16, DEAE-Sephadex17 and DEAE-cellulose18 columns. This last property is the one we have used in our experiments. Despite the many published studies on activation, formation of non-activated complexes and subsequent activation have apparently never been demonstrated in cells or cytosols exposed to steroids at normal body temperatures only. Consequently, it is not known whether non-activated complexes and activation have any significant role in physiological conditions, and at least one well known scheme of steroid hormone action19 does not include the activation step. It is therefore possible that when steroid hormones bind to cytoplasmic receptors at physiological temperatures they immediately form activated complexes. The results described here show that for glucocorticoids this alternative can be excluded: when these steroids interact with receptors in rat thymus cells at 37 °C they initially form non-activated complexes, and subsequently give rise to activated complexes.

Kinetics of glucocorticoid-receptor complexes in rat thymus cells

Using cortisol and dexamethasone, we have studied the kinetic behavior of glucocorticoid receptors in intact rat thymus cells at 37°C. As reported previously, translocation of the cortisol-receptor complexes from “cytoplasmic” to nuclear-bound form takes about 1 min. At 26°C this time is doubled. Consistent with these observations and with the current view that formation of nuclear-bound complex must be preceded by formation of cytoplasmic complex, on addition of hormone to cells at 37°C the initial time-course of formation of cytoplasmic complex precedes that of nuclear complex by about 30 s. After cells have achieved a steady state with [ 3H]-dexamethasone, however, a “chase” of unlabelled dexamethasone lowers nuclear levels of [ 3H]-dexamethasone several minutes before cytoplasmic levels, suggesting formation of nuclear complex by direct reaction of hormone with nuclear-bound receptors. Following depletion of cytoplasmic receptor by incubation with a high concentration of dexamethasone and then sudden lowering of steroid concentration by dilution, the time-course of replenishment of cytoplasmic receptors appears to lag significantly behind the time-course of removal of nuclear-bound steroid. The lag is roughly the same magnitude as the time-constant of dissociation of the glucocorticoid used, and is much shorter with cortisol. The pathway of replenishment thus may be different from simple reversal of the pathway of depletion of cytoplasmic receptor, and may depend on the dissociation rate or affinity constant of the steroid.