To date, the visualization of δ-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. Copyright (C) 1999 Elsevier Science B.V.
Bibliographical noteFunding Information:
This research was supported by NIH research grants DA-00546, DA-01583, DA-05695, KO5-DA-70554, and A and F Stark fund of the Minnesota medical foundation. The Anti-LAMP2 monoclonal antibody, used in the experiments shown in Fig. 7 , was developed by Dr. J.T. August (Johns Hopkins University), and was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by The University of Iowa, Department of Biological Sciences, Iowa City, IA 52242. We also like to thank Dr. I.R. Nabi (University of Montreal, Quebec, Canada) for generously providing the anti-LAMP2 antibody (AC17 hybridoma supernatant against canine LAMP2) for the initial experiment involving 24 h agonist treatment, which gave results similar to those subsequently obtained, and shown in Fig. 7 E,F.
- Time course of δ-opioid receptor trafficking