Nancy C. Lan

Nuclear binding of glucocorticoid receptors: Relations between cytosol binding, activation and the biological response

Abstract To better understand the early steps in glucocorticoid hormone action from cytoplasmic receptor-steroid binding to nuclear binding of the resulting complexes, we have in the current studies examined in cultured hepatoma (HTC) cells the kinetics of the nuclear binding of glucocorticoid receptors, the activation of the receptor-steroid complex required for this binding and the nature of the nuclear binding sites. The quantitative relationships of these events have also been compared to the glucocorticoid response. When intact cells are incubated with dexamethasone at 0°C, cytosol binding of the steroid occurs readily but nuclear binding proceeds very slowly. After heating the cells at 37°C for 40s and then chilling them, the kinetics of the nuclear binding at 0°C are markedly increased. Thus, in the intact cell at 0°C. like cell-free systems, the heat-sensitive activation and not the nuclear binding itself appears to occur and be rate-limiting for nuclear binding of the complexes. Nuclear binding measured at 37°C was similar at 30–40 min, and 16 h, by which time induction of tyrosine aminotransferase is maximal. Thus, unlike the case with most other classes of hormones, in this system glucocorticoids do not affect the concentration of their receptors. Nuclear binding was linearly related to the content of cytoplasmic reccptor-glucocorticotd complexes and a Scatchard plot (nuclear-bound over cytosol-bound vs nuclear-bound dexamethasone) was parallel to the abscissa. These observations suggest that in the cell the nuclear acceptors are far from saturated with receptors. It has been proposed that there are multiple orders of affinity for binding receptors in the nucleus. Salt sensitivity of bound receptors has been used on one criterion. Indeed, whereas most of the nuclear-bound receptors can be extracted with 0.5 M KCl, about 20% resist this extraction. In the current studies, the proportion of salt-extractable and salt-resistant nuclear-bound receptors was found to be constant with time (to 16 h) and cytosol receptor-steroid complex concentration. Thus, the thermodynamics of receptor association with the salt-labile and salt-resistant nuclear-bound receptors appear similar and the evidence does not support the idea that these receptors have fundamental differences; the incomplete extraction of the receptors with salt may reflect some property of the solubilization process rather than two types of nuclear acceptors. The content of nuclear-bound receptors was found to be linearly related to the induction of tyrosine aminotransferase; this suggests that the number of glucocorticoid receptors, and not some distal event, is the limiting factor in the glucocorticoid hormone response and that “spare receptors” are not present. These data also do not support the idea that in these cells, a physiological “acceptor” that binds receptors with an affinity much higher than the observed nuclear-binding (“high-affinity operators”) mediates glucocorticoid action; these hypothetical sites would have been more saturated at the receptor concentrations achieved. Instead, the data are consistent with the possibility that a nuclear acceptor population with an affinity that does not vary greatly from the observed nuclear binding is responsible for the glucocorticoid response. The data are discussed in terms of a model in which receptors bind similarly to acceptors present in excess, many of which are located at sites where a physiological response is not elicited. By this model, the probability of obtaining a glucocorticoid response is proportional to the chance association of receptors with those acceptors located at important loci.

Interaction of sodium molybdate with highly purified glucocorticoid receptor

Sodium molybdate can affect the properties of the glucocorticoid receptor in relatively crude preparations. To obtain more information as to whether these effects are due to direct interactions of the ion with the receptor or with other components present in the receptor-containing mixtures, the effects were examined of sodium molybdate on glucocorticoid receptors purified 3000-5000-fold to about 10% homogeneity from rat liver cytosol. The ion was found to: (1) increase the stability of the purified receptor at either 0 or 20 degrees C, although the effect was more pronounced at 20 degrees C (2) induce an apparent dimerization of the receptors as judged by sephadex G-150 gel filtration and sucrose density gradient sedimentation and (3) decrease the ionic strength required for elution of the purified receptor from DEAE-cellulose columns. Although, it is conceivable that each of these observed effects is due to indirect actions of the ion on contaminants in the preparations, it is more likely that the ion exerts its effects through direct interactions with the receptor.

Monoclonal antibodies against human DNA polymerase-α do not cross-react with glucocorticoid receptors☆

None of 16 independent monoclonal antibodies against human (KB cell) DNA polymerase-alpha recognizes epitopes on cytoplasmic glucocorticoid receptors prepared from the same cells. Consistently negative results are obtained with separate assays that measure antibody binding to uncharged receptors or to charged receptor complexes that have been preloaded with a specific steroid ligand. These results must qualify the interpretation of possible immunological relations between polymerase-alpha and glucocorticoid receptors that were inferred from studies with polyclonal antisera of poorly defined specificity.