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First published online August 3, 2005
doi: 10.1242/10.1242/jcs.02462

Journal of Cell Science 118, 3353-3361 (2005)
Published by The Company of Biologists 2005
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Research Article

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

Christina Maier1,*,{ddagger}, Dominik Rünzler2,*, Julia Schindelar1,2, Gottfried Grabner2, Werner Waldhäusl1, Gottfried Köhler2 and Anton Luger1

1 Department of Medicine III, Clinical Division of Endocrinology and Metabolism, Medical University of Vienna, Währinger Gürtel 18-20, 1090 Vienna, Austria
2 Max F. Perutz Laboratories, Department of Chemistry, University of Vienna, Campus Vienna Biocenter 5/1, 1030 Vienna, Austria

{ddagger} Author for correspondence (e-mail: christina.maier{at}meduniwien.ac.at)

Accepted 28 April 2005

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 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.

Key words: Nongenomic, Pituitary, ACTH release, Glucocorticoid receptor, Fluorescence correlation spectroscopy


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