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.
Ko
J L
JL
Department of Pharmacology, Medical School, 3-249 Millard Hall, University of Minnesota, Minneapolis, MN 55455, USA. koxxx001@maroon.tc.umn.edu
Arvidsson
U
U
Williams
F G
FG
Law
P Y
PY
Elde
R
R
Loh
H H
HH
D000918:Antibody Specificity; D002478:Cells, Cultured; D016308:Enkephalin, Leucine-2-Alanine / Q000494:pharmacology*; D000939:Epitopes / Q000276:immunology; D006801:Humans; D009474:Neurons / Q000187:drug effects* / Q000378:metabolism; D017465:Receptors, Opioid, delta / Q000819:agonists* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013194:Staining and Labeling; D013997:Time Factors; D014162:Transfection; D014764:Viral Proteins / Q000276:immunology
D000939:Epitopes; D017465:Receptors, Opioid, delta; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D016308:Enkephalin, Leucine-2-Alanine