Internalization and recycling of delta-opioid receptor are dependent on a phosphorylation-dephosphorylation mechanism

Internalization, recycling, and resensitization of the human delta-opioid r eceptor (hDOR) were studied in the neuroblastoma cell line SK-N-BE, endogen ously expressing this receptor. Conventional and confocal fluorescence micr oscopy observations, corroborated by Scatchard analysis, indicated that aft er a 100 nM Eto treatment, 60 to 70% of hDOR were rapidly internalized (t(1 /2) < 15 min). This agonist-triggered internalization was reversible for a treatment not exceeding 1 h and became irreversible for prolonged treatment (4 h), leading probably to the degradation and/or down-regulation of the r eceptor. The rapid internalization of hDOR was totally blocked in the prese nce of heparin, known as an inhibitor of G protein-coupled receptor kinases (Benovic et al., 1989), a result indicating that phosphorylation by these kinases is a critical step in desensitization (Hasbi et al., 1998) and inte rnalization of hDOR (present study) in SK-N-BE cell line. Blockade of inter nalization by agents not interferring with phosphorylation, as hypertonic s ucrose or concanavalin A, also blocked the resensitization (receptor functi onal recovering) process. Furthermore, blockade of dephosphorylation of the internalized hDOR by okadaic acid totally suppressed its recycling to the plasma membrane and its subsequent resensitization. These results indicate that regulatory events leading to desensitization, internalization, and rec ycling in a functional state of hDOR involve phosphorylation by a G protein -coupled receptor kinase, internalization via clathrin-coated vesicles, and dephosphorylation by acid phosphatases.