William J. Hendry

An analysis of autologous glucocorticoid receptor protein regulation in AtT-20 cells also reveals differential specificity of the BuGR2 monoclonal antibody

When the anti-glucocorticoid receptor monoclonal antibody (BuGR2) was initially incorporated either into a new immunoassay strategy or into a traditional sedimentation analysis technique, both methods failed to reveal any change in the cellular content or distribution of BuGR2-reactive antigen following glucocorticoid treatment of AtT-20 cells. Furthermore, the immunoassay also generated strong positive signals with cytosol and nuclear extracts from a receptor-negative cell line (E8.2) derived from L929 cells. However, when the BuGR2 antibody was incorporated into a combined immunoprecipitation/Western blot analysis of AtT-20 cell extracts, only the glucocorticoid receptor protein produced a signal on the Western blot, even though other proteins had been specifically immunoprecipitated by BuGR2 antibody and were clearly present on the Western blot membrane. Applying the latter approach to AtT-20 cells chronically treated with glucocorticoid, we observed not only that the receptor protein rapidly and persistently (1-96 h) accumulated in the nucleus, but also that its total cellular content was first depleted (24 h) and then was progressively replenished (48-96 h). From these studies in AtT-20 cells we conclude: (i), the BuGR2 antibody can exhibit differential immunospecificity dependent upon whether antigen mixtures are denatured or not; (ii), glucocorticoid receptor protein resided almost exclusively in the nucleus during four days of glucocorticoid treatment and (iii), the same treatment regimen resulted in total receptor protein levels being regulated in a biphasic pattern. Together, these results suggest that receptor regulation in AtT-20 cells is a complex event, and that, since steroid was constantly present during our experiments, other factors are involved in regulation of receptor levels.

Synthetic Estrogen-Mediated Alterations in Uterine Cell Fate

Our experimental system is uniquely suited to the pursuit of two complementary objectives: first, delineation of the mechanisms whereby estrogens regulate uterine growth and morphogenesis, and second, identification of mechanistic alterations that cause degeneration of the normal growth process to the unregulated neoplastic state. These are biomedically important issues because: first, successful conception and gestation demands normal uterine form and function, and second, estrogen-dependent uterine neoplasms are responsible for considerable morbidity and mortality. Since estrogens normally elicit a striking, yet ultimately limited, growth response in the mature mammalian uterus, a well-integrated interplay of positive and negative regulatory pathways must be involved. To probe this topic, we have exploited a model of atypical estrogen responsiveness that reflects lesions in such regulatory pathways.

The mouse glucocorticoid receptor DNA-binding domain is not phosphorylated invivo

The glucocorticoid receptor is phosphorylated, but the precise location of the phosphorylated groups is unknown. We cultured AtT-20 cells in medium containing [32P]-orthophosphate and used immunoaffinity methods to isolate the intact receptor and a tryptic fragment containing the DNA binding domain. Analysis of the intact receptor, co-labeled with the affinity ligand dexamethasone-mesylate, confirmed that the receptor was phosphorylated. Isolation of the DNA binding domain by trypsinization and immunopurification showed that it was not phosphorylated. Interestingly, a non-immunoreactive phosphorylated fragment similar in size to the DNA-binding fragment was observed. Our results suggest that phosphorylation of the DNA binding domain of the glucocorticoid receptor is not essential for hormone action.