| Autor: |
Maier, Christina, Rünzler, Dominik, Schindelar, Julia, Grabner, Gottfried, Waldhäusl, Werner, Köhler, Gottfried, Luger, Anton |
| Předmět: |
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| Zdroj: |
Journal of Cell Science; 8/1/2005, Vol. 118 Issue 15, p3353-3361, 9p, 17 Graphs |
| Abstrakt: |
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. [ABSTRACT FROM AUTHOR] |
| Databáze: |
Complementary Index |
| Externí odkaz: |
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