Quantitation of type-specific differences in opioid receptor internalization in transiently and stably transfected cells. a, receptor internalization was assayed in transiently transfected cells by visualizing receptor-mediated endocytosis of M1 monoclonal antibody, which recognizes the extracellular epitope tag sequence of mutant δ and κ opioid receptors, into endocytic vesicles visualized by fluorescence microscopy. Nonspecific uptake of monoclonal antibody from the culture medium was negligible (not shown), and the specificity of uptake was further confirmed by the lack of antibody uptake in cells expressing HA-tagged δ receptors that are not recognized by the M1 antibody (a, HA-delta bars). A small number of endocytic vesicles contained endocytosed M1 antibody in untreated cells expressing appropriately FLAG-tagged δ receptors, and a large increase in antibody uptake was induced by etorphine (5 μm × 30 min), consistent with a large amount of etorphine-induced internalization of δ receptors (FLAG-delta bars). In contrast, FLAG-tagged κ receptors mediated little endocytosis of monoclonal antibody, either in untreated or etorphine-treated cells, consistent with the failure of κ opioid receptors to undergo rapid internalization in the presence of etorphine (FLAG-kappa bars). The bars represent the mean number of antibody-positive vesicles (± S.E.) observed in 25 randomly selected cells expressing epitope-tagged receptors from a population of transiently transfected cells. b, receptor internalization was quantitated in stably transfected cells using fluorescence flow cytometry to measure the etorphine-dependent removal of epitope-tagged receptors from the plasma membrane. Stably transfected cells expressing similar amounts of FLAG-tagged δ (closed circles) or κ receptors (closed squares) were incubated at 37 °C for the indicated times with 10 μmetorphine, and then receptors present in the plasma membrane were labeled with fluorescein-conjugated M1 antibody and quantitated by flow cytometry, as described under “Experimental Procedures.” Points represent mean fluorescence values (± S.D.) for triplicate determinations made at each time point. Etorphine-induced internalization of δ receptors is indicated by the rapid, time-dependent reduction in antibody-labeled receptors present in the plasma membrane of etorphine-treated cells, whereas the failure of κ receptors to internalize is indicated by the constant amount of receptor immunoreactivity measure in the plasma membrane under the same conditions.
