Intracellular targeting of DOR is induced rapidly by receptor-Tyr kinase activation specifically in neurosecretory cells.A, Stably transfected PC12 cells expressing epitope-tagged DOR or MOR were maintained in normal culture medium and then incubated for 60 min in the absence (Control) or presence (NGF) of 100 ng/ml added NGF. NGF effects on DOR localization were visualized by epifluorescence microscopy (top row, main panels) and was confirmed at higher magnification in confocal optical sections imaged through the center of cells (bottom right inset; arrow indicates the intracellular membrane pool, and arrowhead indicates plasma membrane). NGF caused no detectable change in the plasma membrane localization of MOR (bottom row, main panels) visualized by epifluorescence (main panels) and in confocal optical sections (bottom right inset; arrowheadindicates plasma membrane). B, Quantification of the acute NGF effect was conducted by scoring ∼100 cells (selected at random in coded specimens) for each condition in each experiment. The proportion of cells characterized by pronounced intracellular localization of DOR or MOR (using the criteria described in Materials and Methods) is presented as mean ± SEM, compiled from three independent experiments. C, Wild-type (PC12 wt) or TrkA-deficient (nnr) PC12 cells were transiently transfected with HA-tagged DOR and analyzed for NGF-induced intracellular targeting of DOR using same method as that applied to stably transfected cells (B). Data represent means ± SEM from three independent experiments. D, Localization of HA-tagged DOR in stably transfected PC12 neurosecretory cells or HEK293 cells (selected for comparable levels of DOR expression) was examined in cells maintained in normal culture medium and then serum-starved for 120 min (Control) and compared with that in serum-deprived cells in which NGF (100 ng/ml) or epidermal growth factor (EGF; 100 ng/ml) was added to the culture medium for 60 min before fixation. Both NGF and EGF caused pronounced intracellular targeting of DOR in PC12 cells (left panels), whereas neither NGF nor EGF caused a detectable effect on DOR localization in HEK293 cells (right panels).E, Quantification of experiments illustrated inD. Randomly selected images were scored blindly as inB (∼100 cells per condition per experiment). Results are presented as mean fraction of cells displaying pronounced intracellular DOR immunoreactivity ± SEM, compiled from three independent experiments.