FIG. 1.

V460Z CB₁ receptors do not undergo agonist-induced internalization and fail to recruit βarrestin-2 to the membrane in AtT20 cells. A, Immunocytochemical detection of wild-type and V460Z CB₁ receptors stably expressed in AtT20 cells. Cells were treated with either vehicle (a, f), 1 µM WIN 55,212-2 alone (b, g), 1 µM WIN 55,212-2 in the presence of 1 µM SR141716A (c, h), 100 nM CP 55,940 alone (d, i) or, 100 nM CP 55,940 in the presence of 1 µM SR141716A (e, j) for the indicated times. CB₁ receptors were detected immunocytochemically (see Experimental Procedures). Agonist stimulation resulted in the intracellular accumulation of wild-type CB₁ receptors (b, d). Co-application of SR1 prevented agonist-induced internalization (c, e). In contrast, V460Z CB₁ receptors remained highly enriched at the cell surface in the presence of agonist (g, i). B, Confocal images of AtT20 cells transiently expressing βarrestin-2-GFP and either wild-type or V460Z CB₁ receptors. βarrestin-2-GFP was detected by its green fluorescence. In unstimulated cells, βarrestin-2-GFP was diffusely present throughout the cell (a, c). CB₁ receptor activation with 100 nM WIN 55,212-2 for 5 min caused a dramatic translocation of βarrestin-2-GFP to the surface with a concomitant decrease in cytosolic fluorescence (b). No translocation was evident in cells expressing V460Z CB₁ receptors (d). Scale bar, 20 µm (A) or 10 µm (B). Images are representative of three independent experiments.