Study On The Mechanism Of Opioid Receptor Internalization

G protein-coupled receptors (GPCRs) are the largest superfamily of membrane receptors. Upon agonist challenge, cell surface GPCRs transduce various extracellular signals to the cell inside. However, long-term agonist exposure often leads to receptor internalization and desensitization. Once internalized from plasma membrane, GPCRs can either recycle back to the cell surface or be delivered to lysosomes for degradation. A prevailing paradigm of GPCR internalization is that β-arrestins, which bind to G protein-coupled receptor kinases (GRKs) phosphorylated cytoplasmic domain of GPCRs to trigger receptor internalization through clathrin-coated pits and thus GRK is a critical prerequisite for receptor endocytosis. However, emerging evidence suggests that an agonist-stimulated internalization mechanism that is independent of receptor phosphorylation may also be employed in some cases, although the molecular mechanism for the phosphorylation-independent GPCR internalization is not clear. In addition, little is known about whether cells exist some regulatory mechanisms that can modulate receptor postencytic fate.Opioid receptors, which play a very important role in regulating the function of nervous and immune systems, belong to the GPCR superfamily. The desensitization and internalizaiton of opioid receptors have been implicated as one of the possible molecular mechanisms underlying opioid tolerance and dependence. Consequently, understanding the molecular mechanisms underlying opioid receptor desensitization and internalizaiton is of great importance. Our previous studies have demonstrated that there exist GRK phosphorylation sites and GRK binding sites in the carboxyl-terminal domain of 5-opioid receptor (DOR) (Mol Pharmacol. 2000, 58: 1050-1056; J Biol Chem. 2003, 278: 30219-30226.). The current study investigated the role of receptor phosphorylation and the involvement of different β-arrestin subtypes in agonist-induced DOR internalization in HEK 293 cells. Results from flow cytometry, fluorescence microscopy, and surface biotin labeling experiments showed that elimination of agonist-induced DOR phosphorylation by mutation GRK binding or phosphorylation sites only partially blocked agonist-induced receptor internalization, indicating the presence of an agonist-induced, GRK-independent mechanism for DOR internalization. Fluorescence and coimmunoprecipitation studies indicated that both the wild type DOR and thephosphoryiation-deficient mutant receptor could bind and recruit β-arrestinl and β-arrestin2 to the plasma membrane in an agonist-stimulated manner. Furthermore, internalization of both the wild type and phosphoryiation-deficient receptors was increased by overexpression of either type of β-arrestins and blocked by β-arrestin-mediate internalization dominant negative mutants, demonstrating that both phosphorylation-dependent and -independent internalization require β-arrestin. Moreover, double-stranded RNA-mediated interference experiments showed that either β-arrestin 1 or β-arrestin2 subtype-specific RNAi only partially inhibited agonist-induced internalization of the wild type DOR. However, agonist-induced internalization of the phosphoryiation-deficient DOR was not affected by β-arrestinl-specific RNAi but was blocked by RNAi against β-arrestin2 subtype. These data indicate that endogenous β-arrestinl functions exclusively in the phosphorylation-dependent receptor internalization, whereas endogenous β-arrestin2, but not β-arrestinl, is required for the phosphorylation-independent receptor internalization. These results thus provide the first evidence that distinct β-arrestin isoforms are required in the agonist induced phosphorylation-dependent and -independent GPCR internalization.We next investigated whether both phosphorylation-dependent and -independent mechanisms function equally in DOR regulation, such as resensitizaiton or downregulation. Flow cytometry assay demonstrated that DPDPE administration caused a remarkable loss of cell surface wild type DOR. After agonist washout for 15 min, cell surface wild type DOR increased rapidly and at the time point of 60 min, about 40-50% internalized receptor reappeared. In contrast, the phosphoryiation-deficient DOR, M4/5/6 could not reappeared after internalizaiton. Surface biotinylation experiments demonstrated that about 50-60% internalized wild type DOR recycled to the cell surface, while 40-50% internalized receptor were degraded. Consistent with the flow cytometry assay that M4/5/6 could not reappeared after internalization, biotinylation experiments showed that internalized M4/5/6 was mostly degraded and few receptors were recycled. These data suggested that receptors internalized through phosphorylation-independent mechanism cannot recycle to the cell surface and are degraded. Receptor phosphorylation is critically required for receptor recycling. To further study the correlation between receptor phosphorlation and recycling, we observed receptor postendocytic fate through GRK2 overexpression or GRK2 RNAi.