Differential Sorting of Human δ-Opioid Receptors after Internalization by Peptide and Alkaloid Agonists

• Published in: The Journal of biological chemistry Vol. 278; no. 25; p. 22795
• Main Authors: Nicolas Marie, Isabelle Lecoq, Philippe Jauzac, Stéphane Allouche
• Format: Journal Article
• Language: English
• Published: American Society for Biochemistry and Molecular Biology 20-06-2003
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M300084200

Desensitization and internalization of G protein-coupled receptors observed after agonist activation are considered two important
regulatory processes of receptor transduction. Endogenous human δ-opioid receptors (hDOR) are differentially regulated in
terms of desensitization by peptide ([ d -Pen 2,5 ]enkephalin (DPDPE) and Deltorphin I) and alkaloid (etorphine) agonists in the neuroblastoma cell line SK-N-BE (Allouche,
S., Roussel, M., Marie, N., and Jauzac, P. (1999) Eur. J. Pharmacol. 371, 235–240). In the present study, we examined the role of hDOR internalization and down-regulation in this differential
desensitization. Sustained activation by peptides for 30 min caused a marked decrease of both [ 3 H]diprenorphine binding sites and hDOR immunoreactivity, observed in a Western blot, whereas a moderate reduction by 30%
was observed after a 30- and 60-min etorphine exposure in binding experiments without opioid receptor degradation. Using
fluorescence microscopy, we visualized hDOR internalization promoted by different agonists in SK-N-BE cells expressing FLAG-tagged
hDOR. Agonist withdrawal results in a greater recycling process correlated with a stronger hDOR resensitization after etorphine
treatment compared with DPDPE or Deltorphin I, as shown in binding, immunocytochemical, and functional experiments. This
suggests a distinct sorting of opioid receptors after their internalization. We demonstrated a lysosomal hDOR targeting
upon peptides by using chloroquine in binding, Western blot, and immunocytochemical experiments and by colocalization of
this receptor with a late endosome marker. In contrast, when the recycling endosome blocker monensin was used, acceleration
of desensitization associated with a strong intracellular immunostaining was observed upon etorphine treatment. The possibility
of separate endocytic pathways responsible for the differential sorting of hDOR upon peptide and alkaloid ligand exposure
was ruled out by binding and immunocytochemical experiments using sucrose hypertonic solution. First, these results showed
complex relationships between hDOR internalization/down-regulation and desensitization. Second, we demonstrated for the first
time that the same receptor could undergo a distinct sorting after internalization by peptide and alkaloid agonists.