Figure 2.

Agonist-dependent internalization and lysosomal degradation of CB₁ in a stable cell line expressing N-terminally EGFP-tagged CB₁ demonstrated with confocal imaging. A, HEK293 cells stably expressing EGFP–CB₁ untreated cells (left) and cells treated for 1 h with 40 μm cycloheximide (middle, right). 0 min indicates basal state before agonist exposure. Scale bars, 10 μm. B, Treatment with a synthetic cannabinoid agonist, WIN 55,212-2 (WIN; 100 nm) for varying periods (5–90 min) leads to internalization of EGFP–CB₁ (INT; indicated by arrowheads). Additional incubation of cells for 1 h after termination of agonist exposure to allow recycling to the cell surface (RC) leads to recycling after agonist exposure over 5–10 min (arrows) but not after agonist exposure over 60–90 min (arrows). White dashed lines indicate the position of the cell membrane. C, Endocytic vesicles containing EGFP–CB₁ after an agonist exposure of 60 min colocalize with a lysosomal marker, Lyso-tracker dye (yellow puncta in overlay). Scale bars, 3 μm.