Visualization of time-dependent redistribution of delta-opioid receptors in neuronal cells during prolonged agonist exposure

To date, the visualization of delta-opioid receptor (DOR) internalization has been largely focused on the events of short-term agonist treatment in transfected non-neuronal cells. In this study, we followed DOR trafficking upon prolonged agonist exposure in the neuronally derived neuro2a cells, stably transfected with the fusion DOR (HA-DOR) cDNA. Internalization of surface DOR was clearly visualized in 5 min of exposure to agonist (100 nM DADLE), and the cell surface DOR remained low throughout the entire 24 h agonist exposure. Significant intracellular accumulation was visible at 20 min exposure, and increased to a maximum at 4 h, after which intracellular DOR staining gradually diminished. DOR intracellular staining was enhanced in the presence of agonist and chloroquine, a lysosomotropic agent, suggesting that internalized receptors were targeted to lysosomes and degraded upon prolonged treatment. Time-dependent colocalization of DOR with transferrin and LAMP-2 following short-term and prolonged agonist exposure further confirmed that receptor was distributed to early endosomes (sequestration) and subjected to lysosomes for degradation (down-regulation), respectively.