Evidence for Specific Glucocorticoid Binding Sites on the Cell Membrane by Fluorescence Correlation Spectroscopy

Fluorescence correlation spectroscopy (FCS) was used to study the diffusion behavior of the glucocorticoid dexamethasone on the cell membrane of the mouse pituitary cell line AtT-20 to investigate the putative membrane glucocorticoid receptor (mGR). This putative membrane receptor is thought to mediate glucocorticoid effects independent from protein synthesis and the well-known intracellular glucocorticoid receptor (iGR), the so-called “nongenomic” effects. The high spatial resolution of the FCS technique allowed for focusing exclusively on the cell membrane; measurements were taken on living cells in solution after incubation with 60 nM fluoresceindexamethasone for 1 h without manipulation of the cells. FCS measurements were performed by moving the position of the laser focus in 1 μm steps through a single cell. In a scan through the cell a component typical for a receptor-bound ligand (diffusion constant 3 × 10-10 cm2 sec-1) becomes apparent in the autocorrelation function (ACF) at the position of the cell membrane whereas another component (diffusion constant 2 × 10-8 cm2 sec-1) typical for a ligand diffusing in the cytosol becomes predominant in the ACF curves inside the cell. After pre-incubation with unlabeled dexamethasone in 1000-fold excess the “receptor-typical” component on the cell membrane was absent. This result is the first direct evidence for the existence of a mGR on a living cell.