FIGURE 1.

Evaluation of arrestin-dependent agonist-stimulated α_2AAR endocytosis in native neurons. All immunostaining experiments detect endogenously expressed HA-α_2AARs. A, neurons were subjected to primary antibody prelabeling of surface α_2AARs prior to stimulation, followed by saturation of labeled surface receptors with AlexaFluor488-conjugated secondary labeling, and then permeabilization and cytosolic AlexaFluor594-conjugated secondary labeling. Cytosolic labeling was seen only in stimulated cells, indicating endocytosis of prelabeled surface α_2AARs. B, endocytosis was assayed in arrestin3-null neurons, transduced with either arrestin2 shRNA (to achieve complete ablation of arrestins) or control shRNA. Analysis revealed that arrestin2 shRNA resulted in a 69 ± 5.8% reduction in arrestin2 immunoreactivity. α_2AARs were detected by primary antibody prelabeling followed by permeabilization and AlexaFluor488-conjugated secondary labeling. In control but not arrestin2 shRNA cells, agonist stimulation resulted in significant α_2AAR endocytosis, indicated by the appearance of intracellular punctae containing internalized receptors (arrows), as well as a partial co-localization of α_2AARs with arrestin2. C, endocytosis was additionally observed by monitoring α_2AAR co-localization with the late endolysosomal pathway marker LAMP1; prelabeling of α_2AARs was done as in A, with the addition of co-immunostaining for LAMP1. In agonist-stimulated but not unstimulated control neurons, endocytosis was seen as indicated by the appearance of intracellular punctae which exhibited co-localization with LAMP1 (arrows). Confocal images are representative of at least three independent samples.