Agonist-induced β-arrestin2-GFP translocation and BRS-3 internalization in HEK293 cells. (A) HEK293
Adrenorphin were transiently transfected with HA-tagged rBRS-3 and GFP-tagged β-arrestin2 (βarr2-GFP). Confocal microscopy was used to obtain real-time imaging of the cells before (first two panels at time 0 (T = 0)) and after 1 μM agonist treatment at room temperature (times indicated in minutes). The first panel represents cell surface staining of Alexafluor 568-conjugated anti-HA antibody to demonstrate that the receptor is co-expressed with the βarr2-GFP. The top row demonstrates how some
cells show puncta formation prior to drug treatment, while other cells (inset enlarged 2× below) reveal translocation after compound 16a (1 μM) treatment within 5 min (second row, white arrow, punctuate accumulation at membrane). (B) Overexpression of GRK2 improves the detection of βarr2-GFP translocation in HEK293 cells expressing untagged BRS-3 and βarr2-GFP. (C) Treatment with compound 16a (1 μM, up to 2 h) does not lead to internalization of the N-terminally HA-tagged rBRS-3 as visualized by Alexafluor 488-conjugated anti-HA antibody labeling of live cells.
