title: G-protein-coupled glucocorticoid receptors on the pituitary cell membrane.
abstract: 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 (tau3; 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 tau3, 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.
authors.affiliation: Department of Medicine III, Clinical Division of Endocrinology and Metabolism, Medical University of Vienna, Währinger Gürtel 18-20, 1090 Vienna, Austria. christiana.maier@meduniwien.ac.at
authors.forename: Christina
authors.initials: C
authors.lastname: Maier
authors.forename: Dominik
authors.initials: D
authors.lastname: Rünzler
authors.forename: Julia
authors.initials: J
authors.lastname: Schindelar
authors.forename: Gottfried
authors.initials: G
authors.lastname: Grabner
authors.forename: Werner
authors.initials: W
authors.lastname: Waldhäusl
authors.forename: Gottfried
authors.initials: G
authors.lastname: Köhler
authors.forename: Anton
authors.initials: A
authors.lastname: Luger
mesh_terms: D000818:Animals; D001667:Binding, Competitive / Q000187:drug effects; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism*; D005443:Flumethasone / Q000494:pharmacology; D005938:Glucocorticoids / Q000494:pharmacology; D008560:Membrane Fluidity / Q000187:drug effects / Q000502:physiology; D051379:Mice; D010902:Pituitary Gland / Q000166:cytology / Q000378:metabolism*; D011485:Protein Binding; D043562:Receptors, G-Protein-Coupled / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D011965:Receptors, Glucocorticoid / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence / Q000379:methods
chemical_list: D005938:Glucocorticoids; D043562:Receptors, G-Protein-Coupled; D011965:Receptors, Glucocorticoid; D005443:Flumethasone