Fig. 2.

PKA-mediated B2AR phosphorylation restricts the entry of B2AR into bulk recycling tubules. (A) Immunofluorescence images of B2AR-expressing HEK293 cells double-labeled with anti-FLAG and anti-phosphoSer346 B2AR, before and after 10 min of iso treatment. (B) The ratio of pB2AR/FLAG in B2AR cells treated with iso and vehicle (DMSO), RpcAMPS (10 μM), or KT-5720 (10 μM) for 10 min, and in B2ARS>A cells treated with iso and vehicle. P values are with respect to B2AR. (C) In vitro phosphorylation of GST-B2AR tail with or without the S > A mutation. Representative blots from three experiments with anti-pB2AR (Upper) and anti-GST (Lower) are shown. (D) Percentages of B2AR (white) and B2ARS>A (gray) endosomes with 0, 1, or >1 receptor tubules, calculated from paired experiments (n = 266 for B2AR, n = 336 for S > A), with P values. (E) Examples of dual-color confocal images of B2AR (Upper) and B2ARS>A (Lower) endosomes with cortactin, showing B2ARS>A tubules lacking cortactin. (F) Typical B2AR (Upper) and B2ARS>A (Lower) endosomes imaged with TfR, showing a subset of TfR tubules devoid of B2AR but accessed by B2ARS>A. Oval Profile plots, as in Fig 1F, are shown to the right (Movie S3). (G) Percentage of B2AR (Left) and B2ARS>A (Right) endosomes with 0, 1, or >1 receptor tubules from the same cells before (white) and 5 min after (gray) lat-A. (H) (Upper) Frames taken 1 s apart from a time-lapse movie showing FRAP of B2AR (Top) and B2ARS>A (Bottom) endosomes, demonstrating slower recovery for B2AR. (Lower) Fluorescence values from the bleached region (blue) and a control region on the same endosome (blue), from another B2ARS>A example (Movie S4). (I) Quantitation of recovery rates of B2AR and B2ARS>A from multiple endosomes fitted to a single-phase exponential curves showing slow recovery of B2AR (t_1/2 = 25.9 s, n = 10, R² = 0.813) and rapid recovery of B2ARS>A (t_1/2 = 5.9 s, n = 14, R² = 0.779). Error bars are SEM. (Scale bars: 1 µm.)