Figure 6. AGAP2 regulated β₂AR recycling.

(A–D) Distribution of FLAG–β₂AR in control HEK-293 cells. Cells were transfected with FLAG–β₂AR cDNA for 24 h and replated on fibronectin-coated coverslips in DMEM containing 0.2% FBS for 6 h. FLAG–β₂ARs, as stained using anti-FLAG antibody, were detected on the plasma membrane of resting cells (A and B) and were concentrated on the perinuclear recycling endosomes at 15 min of ISO stimulation (C and D, arrows). (E–H) Distribution of FLAG–β₂AR in HEK-293 cells with stable knockdown of AGAP2. Cells were transfected with FLAG–β₂AR cDNA and processed as described above. FLAG-tagged β₂ARs were distributed on the plasma membrane of resting cells (E and F), and were enriched on the perinuclear recycling endosomes at 15 min of ISO stimulation (G and H, arrows). (I–L) Recycling of FLAG-β₂AR in control HEK-293 cells. Cells were stimulated with ISO for 10 min, washed and incubated with DMEM containing 0.2% FBS for 15 min (I and J) or 30 min (K and L). At 15 min, FLAG-tagged β₂ARs were recycling from the perinuclear region to the periphery and by 30 min they reached the plasma membrane (K and L, arrowhead). (M–P) Recycling of FLAG–β₂ARs in HEK-293 cells with stable knockdown of AGAP2. At 15 min of recycling, most of the FLAG–β₂AR signals were still present in the perinuclear region (M and N, arrows). By 30 min, FLAG-tagged β₂ARs were present in perinuclear regions (P, arrows) or on endosomes evenly distributed throughout the cytosol (O). A small fraction of FLAG-tagged β₂ARs reached plasma membrane (O and P, arrowhead). Scale bars, 20 μm. (Q) AGAP2 affected recycling of endogenous β₂ARs. HEK-293 cells with or without stable AGAP2 knockdown were treated, or not, with ISO (1 μM, 10 min). Cells were washed and lysed to prepare plasma membrane, or washed and incubated with medium for an additional 30 min before plasma membrane preparation. The amount of β₂ARs was determined by ¹²⁵I-cyanopindolol binding and non-specific binding was determined using alprenolol. *P <0.05 compared with control.