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. 2017 Jan;91(1):65–73. doi: 10.1124/mol.116.106252

Fig. 2.

Fig. 2.

Vps34 is required for efficient β2AR recycling, but not for endocytosis. (A) Representative images of HEK293 cells expressing GFP-2xFYVE. Cells were treated with PIK-III or DMSO for 1 hour, fixed, and imaged by confocal microscopy. Scale bar, 10 µm. (B) Effects of PI3K inhibitors on basal surface β2AR levels. Cells stably expressing Flag-tagged β2AR were treated with the indicated inhibitors for 1 hour. Surface receptor levels were then determined by flow cytometry and were expressed as a percentage of the levels in DMSO-treated cells (n = 4 independent experiments). (C) Effects of PI3K inhibitors on agonist-induced reduction of surface β2AR levels. After 1 hour of pretreatment of inhibitors, cells were treated with isoproterenol for the indicated periods, and surface receptor levels were determined. Basal receptor levels shown in (B) were included in a graph as “0 minutes” for comparison, and data were expressed as a percentage of the basal receptor levels in DMSO-treated cells. Data are from n = 3 (5–40 minutes) or n = 4 (0 minutes) independent experiments. PIK-III or WM treatment significantly reduced surface β2AR levels compared with DMSO control at 10, 20, and 40 minutes after isoproterenol treatment (*P < 0.05, **P < 0.01, ***P < 0.001 by two-way ANOVA and Tukey’s post hoc tests). (D) The efficiency of β2AR endocytosis was estimated from the percentage reduction in surface receptor levels after the shortest (5 minutes) isoproterenol application in (C). Data were from n = 3 independent experiments. (E) Direct measurement of β2AR recycling after agonist removal and antagonist treatment. After 1 hour of treatment of the indicated inhibitor, cells were treated with isoproterenol for 25 minutes, and then with alprenolol for 45 minutes. The percentage of recycled receptors was calculated as described in Materials and Methods and is shown in bar graphs (n = 3 independent experiments, ***P < 0.001 compared with DMSO control by one-way ANOVA and Tukey’s post hoc tests). (F) Recycling defect in PIK-III–treated cells was verified by fluorescence microscopy. After 1 hour treatment with PIK-III or DMSO, cells were treated with isoproterenol for 25 minutes (in the agonist condition) and then with alprenolol for 45 minutes (in the agonist-to-antagonist condition). After fixation, cells were stained for the Flag epitope. Representative confocal images are shown. Scale bars, 10 µm. (G) β2AR recycling in the continuous presence of agonist. Surface receptors were labeled with Alexa Fluor 647–conjugated anti-Flag antibody, and then internalized by 1 µM isoproterenol for 25 minutes. After stripping antibodies bound to residual surface receptors by calcium-depleted medium, cells were further incubated for 5 minutes in calcium-depleted medium. β2AR recycling was then estimated by measuring the antibody efflux in this 5-minute period (n = 3 samples from one experiment, **P < 0.01 by two-tailed t test. Similar results were obtained in two other independent experiments). Error bars indicate the S.D.