Functionality of the liver glucocorticoid receptor during the life cycle and development of a low-affinity membrane binding site

The ability of the liver glucocorticoid receptor to bind steroid and translocate to the nucleus in vivo was examined in order to elucidate possible developmental factors in the receptor system which block its action in fetal rat liver. Using optimal doses and uptake times of [3H]-dexamethasone established for the liver of adrenalectomized adult males, we found fetal livers to be capable of specific glucocorticoid binding by cytoplasmic receptors and of in vivo translocation of the bound steroid to nuclei. Although the specific binding in the cytosol was lower than 2nM in the late fetus, this could be accounted for by occupation of receptor binding sites by endogenous corticosterone present in extremely high concentrations in fetal plasma. The nuclear binding of dexamethasone, on the other hand, was as great in the late fetal liver as in adult liver, indicating that fetal liver cells were very efficient at nuclear translocation of receptor complex. These studies of in vivo nuclear binding of dexamethasone demonstrated that inhibition of glucocorticoid action before birth is due to some event other than the entry of glucocorticoid receptor into the nucleus. The receptor is present and functional in nuclear translocation at all developmental stages examined. When nuclei from adults were assayed for dexamethasone binding in vitro, a lower- affinity, but saturable, binding component was present in large amounts. This membrane binding site of glucocorticoids was absent in nuclei from fetuses and newborn rats, and was not correlated with the appearance of glucocorticoid receptor in liver nuclei. Adult microsomal membranes contained even more low affinity glucocorticoid binder than nuclei. The presence of a high-affinity nuclear receptor was not detectable by the in vitro exchange assay of adult nuclei

Involvement of a low molecular weight component(s) in the mechanism of action of the glucocorticoid receptor

[3H]Dexamethasone-receptor complexes from rat liver cytosol preincubated at 0° bind poorly to DNA-cellulose. However, if the steroid-receptor complex is subjected to gel filtration at 0–4° separating it from the low molecular weight components of cytosol, the steroid-receptor complex becomes “activated” enabling its binding to DNA-cellulose. This activation can be prevented if the gel filtration column is first equilibrated with the low molecular weight components of cytosol. In addition, if adrenalectomized rat liver cytosol, in the absence of exogeneous steroid, is subjected to gel filtration the macromolecular fractions separated from the “small molecules” of that cytosol have much reduced binding activity towards [3H]dexamethasone. These results suggest that rat liver cytosol contains a low molecular weight component(s) which maintains the glucocorticoid receptor in a conformational state that allows the binding of dexamethasone. Furthermore, this component must be removed from the steroid-receptor complex before binding to DNA can occur