Fig. 6.
Agonist-induced Gαs internalization requires lipid microdomains and caveolin-1. C6 glioma cells were transiently transfected with Gαs-GFP, and trafficking was assessed using digital fluorescence microscopy. Living cells were imaged in real time, and individual frames are shown before and after 10 μM isoproterenol (ISO) treatment. A and B, Gαs-GFP localization in wild-type C6 cells before agonist treatment (A) or after 20 min of isoproterenol stimulation (B). C to E, wild-type C6 cells expressing Gαs-GFP were preincubated with 10 mM methyl-β-cyclodextrin (CD) for 30 min and imaged (C), and cells were subsequently treated with isoproterenol for 30 min (D). These cells were washed and incubated for 90 min with cyclodextrin-cholesterol complexes (CHOL) to restore cellular cholesterol and then were treated again with isoproterenol for 30 min (E). F to H, C6 cells in which caveolin-1 was stably knocked-down by RNAi were transfected with Gαs-GFP and imaged before agonist exposure (F) or after isoproterenol treatment at 15 min (G) and 30 min (H). I, Gαs-GFP internalization in response to agonist was quantified by determining the mean fluorescence intensity in the cytoplasm of cells and is expressed as a percentage control. More than 50 cells in each treatment group were quantified from six or more independent experiments (n = 6). *, p < 0.05 versus CON. Scale bars, 10 μm (all panels).