G Protein independent phosphorylation and internalization of the δ-opioid receptor

The δ-opioid receptor belongs to the family of 7-transmembrane domain receptors coupled to heterotrimeric G proteins (GPCRs). In addition to antinociceptive effects (Narita and Suzuki 2004), δ-opioid receptor agonists have been shown to have antidepressant-like (Broom et al. 2002a) and antiparkinson-like activities (Hudzik et al. 2000). However, the potential beneficial actions of δ-opioid agonists are hindered by their propensity to induce rapid and prolonged tolerance (Brandt et al. 2001; Broom et al. 2002c; Broom et al. 2002b; Jutkiewicz et al. 2005). Internalization of the δ-opioid receptor has long been thought to play a role in tolerance (Eisinger and Schulz 2005). After activation by agonist, the δ-opioid receptor undergoes phosphorylation by G protein coupled receptor kinase 2 (GRK) (Pei et al. 1995), followed by interaction with β-arrestin to block further coupling to G proteins leading to desensitization (Kovoor et al. 1997) and internalization (Zhang et al. 1999) via clathrin coated pits (Trapaidze et al. 1996). Recruitment of GRK2 to the membrane requires the βγ subunits of the G protein heterotrimer (Li et al. 2003). However, the necessity of G protein activation for δ-opioid receptor internalization is unclear. An early report (Law et al. 1985) showed that pretreatment of NG108-15 cells (rat neuroblastoma x mouse glioma hybridoma) with pertussis toxin (PTX), to inhibit δ-opioid receptor coupling to Gαi/o proteins, had no effect on the ability of the δ-opioid agonist DADLE ([D-Ala2,D-Leu5]enkephalin) to induce receptor internalization. Similarly, in mouse neuroblastoma Neuro2A cells, stably expressing a δ-opioid receptor, PTX failed to block internalization by the selective agonist DPDPE ([D-Pen2,D-Pen5]enkephalin) (Chakrabarti et al. 1997). In contrast, in human embryonic kidney (HEK) 293 cells stably expressing the mouse δ-opioid receptor, PTX pretreatment inhibited by 60 % the ability of the δ-opioid agonist DTLET ([DThr2]-Leu-enkephalin-Thr6) to mediate internalization (Kramer et al. 2000b). Whether G proteins are required or not, there is evidence that GRKs of some type, possibly including those not recruited by Gβγ such as GRK5 and GRK6 (Ferguson 2001; Lodowski et al. 2006) are involved in δ-opioid receptor desensitization (Willets and Kelly 2001) and internalization (Hasbi et al. 2000). Certainly, mutagenesis data from several groups suggests that preventing phosphorylation of serine and/or threonine residues in the C-terminus of the δ-opioid receptor blocks receptor internalization across a variety of cell lines, although this effect is not always complete (Trapaidze et al. 1996; Zhao et al. 1997; Murray et al. 1998; Kouhen et al. 2000; Whistler et al. 2001; Zhang et al. 2005; Navratilova et al. 2007). These data thus indicate that the δ-opioid receptor may be able to participate in both G protein dependent and independent phosphorylation and internalization. Several studies have suggested that δ-opioid receptor agonists are capable of stimulating distinct fates of the receptor. For example, preincubation of HEK 293 cells stably expressing the mouse δ-opioid receptor with the peptide agonists DPDPE or DSLET ([D-Ser2,D-Leu5]enkephalin-Thr6), or the alkaloid etorphine resulted in receptor desensitization and internalization, but the alkaloid levorphanol, which induced receptor desensitization failed to stimulate internalization (Bot et al. 1997). In human neuroblastoma SK-N-BE cells DPDPE and deltorphin I stimulated internalization and subsequent lysosomal degradation of the δ-opioid receptor, whereas etorphine stimulated internalization followed by recycling of receptor to the cell surface (Marie et al. 2003). However, activation of a pathway for degradation of the δ-opioid receptor is not specific to peptides, as the endogenous peptides, [Leu5]enkephalin and [Met5]enkephalin, caused recycling after internalization, but the synthetic δ-opioid, SNC80, led to degradation (Lecoq et al. 2004). Obviously, whether the δ-opioid receptor is recycled or degraded appears to be agonist and cell specific. In the present study we test the hypothesis that the δ-opioid receptor undergoes agonist specific Gi/o-protein dependent and independent phosphorylation and internalization. To answer this question we have studied these events in HEK 293 cells expressing a FLAG-tagged δ-opioid receptor (HEKδ) in the presence or absence of PTX and with a variety of δ-opioid agonists. PTX treatment had no effect on δ-opioid receptor phosphorylation or translocation of β-arrestin 2-GFP to the cell surface, but did reduce receptor internalization. There was agreement between the ability of different agonists to stimulate G protein and cause internalization with the exception of the non-peptide δ-opioid agonist, TAN-67, which caused a level of G protein activation equal to that of DPDPE, but did not cause internalization.