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Fig. 1. ß-arrestin2 accumulates in the nucleus and acquires a modulatory function in a GAL4-based transcription assay upon odorant receptor stimulation. (A) Confocal microscopy of HEK293 cells cotransfected with hOR17-4 and ß-arrestin2-GFP before (0) and after (20 min) bourgeonal stimulation. Bourgeonal treatment causes nuclear translocation of ß-arrestin2-GFP. (B) Quantification of the fluorescence intensities in the nucleus relative to intensities in the cytosol measured in single confocal sections. The averages of at least 100 HEK293 cells demonstrate a 
fourfold increase in the amount of nuclear ß-arrestin2 in responding cells. Error bars represent s.d. (**P<0.01 compared with levels in the control). (C) Pre-treatment of cells with concanavalin A (+conA) as inhibitor of clathrin-mediated endocytosis blocks ß-arrestin2-GFP nuclear translocation, pre-treatment with ß-cyclodextrin (+CD) as inhibitor of caveolae-mediated endocytosis has no effect. At least 5000 cells were investigated and the number of cells showing nuclear accumulation of ß-arrestin2 was counted and given as percentage of the total number of cells investigated. Error bars represent SD (**P<0.01). (D) Nuclear translocation of ß-arrestin2-GFP is specific to hOR17-4 activation. The upper panel shows changes in the Ca2+ concentration in Calcium-Orange-loaded individual cells that were cotransfected with hOR17-4 and ß-arrestin2-GFP in pseudocolors; the lower panel shows ß-arrestin2-GFP localization. Bourgeonal (5 µM) was present during the whole experiment. The ß-arrestin2-GFP expressing cell which show nuclear translocation of the ß-arrestin2 also showed an increase in the intracellular Ca2+ concentration in response bourgeonal (5 µm). The curve shows the mean change in the fluorescence intensities in response to Borgeonal application as a function of time for ten Calcium Orange loaded cells cotransfected with ß-arrestin2-GFP and hOR17-4. (E) Western blot analysis of fractionated HEK293 cells cotransfected with ß-arrestin2 and hOR17-4, nonstimulated (-) and stimulated (+) with bourgeonal for 20 minutes. The purity of the nuclear fraction was verified by staining with anti-ß-actin antibodies. Nuclear ß-arrestin2 signals were quantified by densitometry; the graph depicts the average from three independent experiments, error bars represent s.e.m. (F) HEK293 cells and Hana3A were cotransfected with a GAL4-regulated luciferase reporter construct and chimeric constructs encoding the GAL4 DNA-binding domain alone (gal4), or fused to ß-arrestin2. Luciferase activity was measured 48 hours after transfection using equal amounts of total cellular lysates from untreated cells (ß-arr2) and from HEK293 (ß-arr2+B) and Hana3A (ß-arr2+B hana) cells that were treated with bourgeonal for 6 hours. Luciferase activity significantly increased upon hOR17-4 stimulation. For each panel, results of five independent experiments performed in triplicate were averaged and plotted, error bars represent s.e.m. Bars, 10 µm.
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