The membrane glucocorticoid receptor on the pituitary is G protein coupled and interacts with the cytoskeleton

Background: While nongenomic glucocorticoid actions like fast feedback inhibition of ACTH release are well documented, only very little is known about possible receptors mediating them. We recently identified specific, positive-cooperative glucocorticoid binding sites on the cell membrane of the ACTH-producing pituitary cell line AtT-20. The bound dexamethasone molecules showed a high degree of mobility restriction suggesting tighter association e.g. with the cytoskeleton. Additionally, two fractions of fast (freely diffusing ligand) and intermediate mobility (diffusion on/inside the cell) were found. The aim of the present study was to further characterize these binding sites on living cells in relation to the intracellular glucocorticoid receptor (iGR), the actin cytoskeleton (which is involved in ACTH release) and the possible involvement of a G protein.

Methods: AtT-20 cells were incubated for 3h at 37°C with either the iGR antagonist RU486 or Pertussis toxin (PTX) or 15min. with Cytochalasin D, followed by 1h incubation with 120 nM fluorescein labelled dexamethasone (f-dexa). The diffusion behaviour of f-dexa on the cell membrane was studied using Fluorescence Correlation Spectroscopy (FCS) and autocorrelation analysis.

Results and conclusion: The pronounced mobility restriction associated with f-dexa binding was unaltered by RU486, but completely abolished by both PTX and Cytochalasin D. Instead the proportion of f-dexa molecules with intermediate mobility increased on the location of the cell membrane. These results suggest that the membrane glucocorticoid binding sites on the pituitary could be G protein coupled receptors interacting with the cytoskeleton and structurally unrelated to the iGR.