Rapid, nongenomic actions of glucocorticoids (GCs) have been well documented, but information about putative membrane receptors that mediate them is scarce. We used fluorescence correlation spectroscopy to search for membrane GC-binding on the mouse pituitary cell line AtT-20. A slowly diffusing fraction (τ3; diffusion constant 3×10-10 cm2 s-1) of fluorescein-labeled dexamethasone on the cell membrane corresponds to fluorescein-dexamethasone binding. Preincubation experiments were performed to test binding specificity: a 500-fold excess of unlabeled dexamethasone abolished subsequent fluorescein-dexamethasone membrane binding from 58±2 (control) to 8±1 (% of τ3, mean ± s.e.m.), the natural ligand corticosterone prevented it partially (29±2), while the sex steroids estradiol (56±4) and progesterone (50±4) and the GC-receptor antagonist RU486 (56±2) had no effect. Preincubation with pertussis toxin resulted in disappearance of the slowest diffusion component (11±4) suggesting association of the receptor with a G-protein. Varying the concentration of fluorescein-dexamethasone showed that membrane binding is highly cooperative with an apparent Kd of 180 nM and Bmax of 230 nM. Taken together, these results demonstrate high-affinity GC-binding on the cell membrane of AtT-20 cells with characteristics distinct from intracellular binding.

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