G-protein-coupled glucocorticoid receptors on the pituitary cell membrane

• Published in: Journal of cell science Vol. 118; no. 15; pp. 3353 - 3361
• Main Authors: Maier, Christina, Růnzler, Dominik, Schindelar, Julia, Grabner, Gottfried, Waldhäusl, Werner, Köhler, Gottfried, Luger, Anton
• Format: Journal Article
• Language: English
• Published: England 01-08-2005
• Subjects: Animals; Binding, Competitive - drug effects; Cell Line; Cell Membrane - drug effects; Cell Membrane - metabolism; Flumethasone - pharmacology; Glucocorticoids - pharmacology; Membrane Fluidity - drug effects; Membrane Fluidity - physiology; Mice; Pituitary Gland - cytology; Pituitary Gland - metabolism; Protein Binding; Receptors, G-Protein-Coupled - chemistry; Receptors, G-Protein-Coupled - drug effects; Receptors, G-Protein-Coupled - metabolism; Receptors, Glucocorticoid - chemistry; Receptors, Glucocorticoid - drug effects; Receptors, Glucocorticoid - metabolism; Sensitivity and Specificity; Spectrometry, Fluorescence - methods
• ISSN: 0021-9533; 1477-9137
• DOI: 10.1242/jcs.02462

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 ([tau]₃; diffusion constant 3x10⁻¹⁰ 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 [tau]₃, 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 K[subscript d] of 180 nM and B[subscript max] 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.