Figure 9.

ECE-1 does not regulate recycling of the B₂R. (A) Localization of Alexa-kallidin and ECE-1b-HSV in HEK cells expressing ECE-1b-HSV. Cells were incubated with Alexa-kallidin and FITC-HSV antibody for 1 h at 4°C, washed, and incubated at 37°C for 10 min. Kallidin colocalized with ECE-1b-HSV (arrows). (B–E) Effects of ECE-1 inhibition on BK-induced translocation of β-arrestin2-GFP in HEK cells. HEK-B₂R cells (B and C) or HEK-B₂R-V₂RCT cells (D and E) were incubated with BK (100 nM) for 0–10 min at 37°C, washed and recovered in BK-free medium for 1–2 h. (B and D) In unstimulated vehicle-treated cells, β-arrestin2 was cytosolic (arrows) and B₂R and B₂R-V₂RCT were at the plasma membrane (arrowheads). After 10 min with BK, β-arrestin2 colocalized in endosomes with B₂R and B₂R-V₂RCT (arrows). After 1 h (B₂R) or 2 h (B₂R-V₂RCT) recovery, β-arrestin2 was cytosolic and B₂R and B₂R-V₂RCT were at the plasma membrane. (C and E) SM-19712 did not affect trafficking of B₂R or β-arrestin2 (C), but caused retention of β-arrestin2 and B₂R-V₂RCT in endosomes (E) (arrows). Bar, 10 μm. (F) Quantification of the effect of SM-19712 on recycling of B₂R at 1 h recovery and B₂R-V₂RCT at 2 h recovery. SM-19712 caused an ∼50% reduction in B₂R-V₂RCT recycling. (G) Colocalization coefficient of β-arrestin2 and B₂R at 1 h recovery and B₂R-V₂RCT at 2 h recovery. SM-19712 did not affect colocalization with B₂R but caused an approximately twofold increase in colocalization with B₂R-V₂RCT. *, P < 0.05.