Specific high-affinity glucocorticoid binding sites on the membrane of the pituitary cell line AtT-20 demonstrated by Fluorescence Correlation Spectroscopy

Abstract

The fast feedback regulation of ACTH release from the pituitary by glucocorticoids is a physiological rapid steroid effect that has been well documented in vivo and in vitro. The mouse pituitary cell line AtT-20 is a model system for ACTH release and its fast (within 10 minutes) suppression by glucocorticoids. The existence of membrane Glucocorticoid Receptors on this cell line has been suggested by studies on isolated cell membranes. The aim of this study was to identify and characterize such binding sites on the membrane of single living cells using the single molecule technique Fluorescence Correlation Spectroscopy (FCS), a method that allows to monitor the diffusion behaviour of fluorescently labeled molecules with single-molecule sensitivity in a confocal volume measuring 0,35µm in the xy (lateral) and 2,4µm in the z (axial) dimension defined by a microscope objective epi-illuminated by a laser beam. Binding of a ligand to the cell membrane of single living cells can be studied in solution without the need of membrane isolation or other manipulations. We found that fluorescein-labeled dexamethasone binds indeed to the membrane of AtT-20 cells. The binding is specific, since it is washed out by unlabeled dexamethasone or the natural ligand corticosterone, but not estradiol or progesterone in 1000-fold excess. Incubation with different concentrations of dexamethasone resulted in a binding curve with a binding constant of 6 nM. Membrane binding is observed only in a subpopulation of the cells. The fraction of binding-positive cells is significantly reduced upon feeding of the cells. In summary we have shown the existence of specific glucocorticoid binding sites with high affinity on the membrane of living AtT-20 cells. Whether or not these binding sites are related to the rapid glucocorticoid effects on the pituitary remains to be elucidated, but the fact that they are downregulated upon feeding clearly points in the direction of their functionality.