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

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

Christina Maier^1,*,, Dominik Rünzler^2,*, Julia Schindelar^1,2, 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

View larger version (21K): [in a new window]   Fig. 1. FCS analysis of F-dexa in a single AtT-20 cell. (A) The confocal volume was positioned above the center of a cell and a z-intensity scan was performed towards the glass surface. Representative positions, (a) outside of the cell (in the solution), (b) inside the cytoplasm and (c) at the cell membrane, are indicated by horizontal lines. (B) Intensity fluctuations were recorded for 30 seconds and are shown for the three representative positions together with their normalized autocorrelation curves (C). Data were fitted with the three component model.

View larger version (27K): [in a new window]   Fig. 2. F-dexa–binding to the membrane of single AtT-20 cells quantified by FCS. Intensity fluctuations and the measured and calculated corresponding autocorrelation curves G() at the cell membrane position for representative F-dexa concentrations of (a) 25, (b) 80 and (c)120 nM. Data were fitted with the three component model, with the fixed diffusion times of 1=0.05, 2=3.6 and 3=255 ms. The resulting fractions were f1=0.61, f2=0.26, and f3=0.13 for 25 nM, f1=0.55, f2=0.16, and f3=0.30 for 80 nM, and f1=0.23, f2=0.21, and f3=0.56 for 120 nM.

View larger version (16K): [in a new window]   Fig. 3. F-dexa–binding isotherm. (A) Membrane-bound F-dexa S(3) as a function of the F-dexa concentration in the binding medium for the average of measurements for each particular concentration (± s.d.). (B) Calculated molar concentrations of bound F-dexa for individual measurements () and for the average of measurements () for each particular concentration. Data were fitted to a Hill equation with variable slope (solid line), which yields a Bmax of 230±10 nM, an apparent Kd of 180±10 nM and a Hill coefficient of 2.1±0.2. Insert shows the average of measurements () for each particular concentration (± s.d.) on a linear F-dexa concentration scale. (C) Scatchard plot of transformed data (individual measurements () and for the average of measurements () together with the transformed nonlinear fit (solid line).

View larger version (28K): [in a new window]   Fig. 4. Specificity of F-dexa–binding to the cell membrane of AtT-20 cells. Examples of intensity fluctuations (normalized scale, measurement time: 30 seconds) and corresponding measured and calculated autocorrelation curves G() at the cell membrane position for F-dexa after preincubation with (A) binding buffer as a control, and after preincubation with 500-fold excess of unlabeled (B) dexamethasone, (C) corticosterone, (D) progesterone or (E) estradiol.

View larger version (13K): [in a new window]   Fig. 5. Overview of specificity experiments. Fraction S(3) (mean ± s.e.m.) of at least eight cells from three independent experiments are shown. Subsequent F-dexa binding is reduced from the control value 58±2% (a) by dexamethasone (8±1%) (b) and corticosterone (29±2%) (c), but unaltered by progesterone (50±4%) (d) and estradiol (56±4%) (e).

View larger version (17K): [in a new window]   Fig. 6. Characterization of F-dexa–binding to the cell membrane of AtT-20 cells. Examples of intensity fluctuations (normalized scale, measurement time: 30 seconds) and measured and calculated corresponding autocorrelation curves G() at the cell-membrane-position for F-dexa after preincubation with (A) binding buffer as a control, and after preincubation with (B) RU486 or (C) PTX.

View larger version (10K): [in a new window]   Fig. 7. Overview of characterization experiments. Fraction S(3) (mean ± s.e.m.) of at least seven cells from three independent experiments are shown. Subsequent F-dexa–binding is not reduced from the control value 58±2% (a) by RU486 (56±2%) (b) but is almost abolished by PTX (11±4%) (c).

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